Rapid identification and quantification of Campylobacter coli and Campylobacter jejuni by real-time PCR in pure cultures and in complex samples

PubMed Central

2011-01-01

Background Campylobacter spp., especially Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), are recognized as the leading human foodborne pathogens in developed countries. Livestock animals carrying Campylobacter pose an important risk for human contamination. Pigs are known to be frequently colonized with Campylobacter, especially C. coli, and to excrete high numbers of this pathogen in their faeces. Molecular tools, notably real-time PCR, provide an effective, rapid, and sensitive alternative to culture-based methods for the detection of C. coli and C. jejuni in various substrates. In order to serve as a diagnostic tool supporting Campylobacter epidemiology, we developed a quantitative real-time PCR method for species-specific detection and quantification of C. coli and C. jejuni directly in faecal, feed, and environmental samples. Results With a sensitivity of 10 genome copies and a linear range of seven to eight orders of magnitude, the C. coli and C. jejuni real-time PCR assays allowed a precise quantification of purified DNA from C. coli and C. jejuni. The assays were highly specific and showed a 6-log-linear dynamic range of quantification with a quantitative detection limit of approximately 2.5 × 102 CFU/g of faeces, 1.3 × 102 CFU/g of feed, and 1.0 × 103 CFU/m2 for the environmental samples. Compared to the results obtained by culture, both C. coli and C. jejuni real-time PCR assays exhibited a specificity of 96.2% with a kappa of 0.94 and 0.89 respectively. For faecal samples of experimentally infected pigs, the coefficients of correlation between the C. coli or C. jejuni real-time PCR assay and culture enumeration were R2 = 0.90 and R2 = 0.93 respectively. Conclusion The C. coli and C. jejuni real-time quantitative PCR assays developed in this study provide a method capable of directly detecting and quantifying C. coli and C. jejuni in faeces, feed, and environmental samples. These assays represent a new diagnostic tool for studying the epidemiology of Campylobacter by, for instance, investigating the carriage and excretion of C. coli and C. jejuni by pigs from conventional herds. PMID:21600037

Real-time PCR in virology.

PubMed

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

2002-03-15

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

Quantitative real-time imaging of glutathione

USDA-ARS?s Scientific Manuscript database

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

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

NASA Astrophysics Data System (ADS)

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

2015-11-01

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05839b

Comparative evaluation of the performance of the Abbott RealTime HIV-1 assay for measurement of HIV-1 plasma viral load on genetically diverse samples from Greece

PubMed Central

2011-01-01

Background HIV-1 is characterized by increased genetic heterogeneity which tends to hinder the reliability of detection and accuracy of HIV-1 RNA quantitation assays. Methods In this study, the Abbott RealTime HIV-1 (Abbott RealTime) assay was compared to the Roche Cobas TaqMan HIV-1 (Cobas TaqMan) and the Siemens Versant HIV-1 RNA 3.0 (bDNA 3.0) assays, using clinical samples of various viral load levels and subtypes from Greece, where the recent epidemiology of HIV-1 infection has been characterized by increasing genetic diversity and a marked increase in subtype A genetic strains among newly diagnosed infections. Results A high correlation was observed between the quantitative results obtained by the Abbott RealTime and the Cobas TaqMan assays. Viral load values quantified by the Abbott RealTime were on average lower than those obtained by the Cobas TaqMan, with a mean (SD) difference of -0.206 (0.298) log10 copies/ml. The mean differences according to HIV-1 subtypes between the two techniques for samples of subtype A, B, and non-A/non-B were 0.089, -0.262, and -0.298 log10 copies/ml, respectively. Overall, differences were less than 0.5 log10 for 85% of the samples, and >1 log10 in only one subtype B sample. Similarly, Abbott RealTime and bDNA 3.0 assays yielded a very good correlation of quantitative results, whereas viral load values assessed by the Abbott RealTime were on average higher (mean (SD) difference: 0.160 (0.287) log10 copies/ml). The mean differences according to HIV-1 subtypes between the two techniques for subtype A, B and non-A/non-B samples were 0.438, 0.105 and 0.191 log10 copies/ml, respectively. Overall, the majority of samples (86%) differed by less than 0.5 log10, while none of the samples showed a deviation of more than 1.0 log10. Conclusions In an area of changing HIV-1 subtype pattern, the Abbott RealTime assay showed a high correlation and good agreement of results when compared both to the Cobas TaqMan and bDNA 3.0 assays, for all HIV-1 subtypes tested. All three assays could determine viral load from samples of different HIV-1 subtypes adequately. However, assay variation should be taken into account when viral load monitoring of the same individual is assessed by different systems. PMID:21219667

Toward real-time quantification of fluorescence molecular probes using target/background ratio for guiding biopsy and endoscopic therapy of esophageal neoplasia.

PubMed

Jiang, Yang; Gong, Yuanzheng; Rubenstein, Joel H; Wang, Thomas D; Seibel, Eric J

2017-04-01

Multimodal endoscopy using fluorescence molecular probes is a promising method of surveying the entire esophagus to detect cancer progression. Using the fluorescence ratio of a target compared to a surrounding background, a quantitative value is diagnostic for progression from Barrett's esophagus to high-grade dysplasia (HGD) and esophageal adenocarcinoma (EAC). However, current quantification of fluorescent images is done only after the endoscopic procedure. We developed a Chan-Vese-based algorithm to segment fluorescence targets, and subsequent morphological operations to generate background, thus calculating target/background (T/B) ratios, potentially to provide real-time guidance for biopsy and endoscopic therapy. With an initial processing speed of 2 fps and by calculating the T/B ratio for each frame, our method provides quasireal-time quantification of the molecular probe labeling to the endoscopist. Furthermore, an automatic computer-aided diagnosis algorithm can be applied to the recorded endoscopic video, and the overall T/B ratio is calculated for each patient. The receiver operating characteristic curve was employed to determine the threshold for classification of HGD/EAC using leave-one-out cross-validation. With 92% sensitivity and 75% specificity to classify HGD/EAC, our automatic algorithm shows promising results for a surveillance procedure to help manage esophageal cancer and other cancers inspected by endoscopy.

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

PubMed

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

2015-12-14

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

Protocol for the use of light upon extension real-time PCR for the determination of viral load in HBV infection.

PubMed

Li, Guimin; Li, Wangfeng; Liu, Lixia

2012-01-01

Real-time PCR has engendered wide acceptance for quantitation of hepatitis B virus (HBV) DNA in the blood due to its improved rapidity, sensitivity, reproducibility, and reduced contamination. Here we describe a cost-effective and highly sensitive HBV real-time quantitative assay based on the light upon extension real-time PCR platform and a simple and reliable HBV DNA preparation method using silica-coated magnetic beads.

Monitoring of Viral Induced Cell Death Using Real Time Cell Analysis

DTIC Science & Technology

2016-11-01

studies have shown that real- time cell analysis (RTCA) platforms such as the xCELLigence can be used to gather quantitative measurements of viral...Teng, Z., Kuang, X., Wang, J., Zhang, X. Real- time cell analysis – A new method for dynamic, quantitative measurement of infectious viruses and...cytopathogenicity. A) Real- time monitoring of BSR cells infected with a 1:10 dilution series of Gan Gan virus. The curve is an average of eight

Targeting neuroendocrine differentiation for prostate cancer radiosensitization

DTIC Science & Technology

2017-12-01

Secondary HRP-conjugated antibodies were purchased fromGEHealthcare UK Ltd. (Buckinghamshire, UK). 2.5. RNA isolation and quantitative real- time PCR (qRT...gene expression data using real- time quantitative PCR and the 2(-Delta Delta C(T))Method,Methods 25 (2001) 402–408. [47] T.K. Kelly, T.B. Miranda, G...relative gene expression data using real- time quantitative PCR and the 2(−Delta Delta C(T)) method, Methods 25 (2001) 402–408. [37] J. Ren, L. Wen, X

A novel duplex real time quantitative reverse transcription polymerase chain reaction for rubella virus with armored RNA as a noncompetitive internal positive control.

PubMed

Zhao, Lihong; Li, Ruiying; Liu, Aihua; Zhao, Shuping

2015-07-01

The objective of this study was to build and apply a duplex real time quantitative reverse transcription-polymerase chain reaction (RT-PCR) for rubella virus. Firstly, a 60-bp-long armored RV RNA was constructed in the laboratory. Secondly, a duplex real time RT-PCR assay was established. Thirdly, the 60-bp-long armored RV RNA was used as an internal positive control (IPC) for the duplex real time RT-PCR. And finally the duplex real time RT-PCR assay was applied to detect RV RNA in clinical specimens. The in-house assay has a high amplification efficiency (0.99), a high analytical sensitivity (200 copies/mL), and a good reproducibility. The diagnostic specificity and sensitivity of the in-house assay were both 100%, due to the monitoring of the armored RV RNA IPC. Therefore, the in-house duplex real time quantitative RT-PCR assay is a specific, sensitive, reproducible and accurate assay for quantitation of RV RNA in clinical specimens. And noncompetitive armored RV RNA IPC can monitor RT-PCR inhibition and prevent false-negative and inaccurate results in the real time detection system. Copyright © 2015 Elsevier B.V. All rights reserved.

Rapid, cost-effective, sensitive and quantitative detection of Acinetobacter baumannii from pneumonia patients

PubMed Central

Nomanpour, B; Ghodousi, A; Babaei, A; Abtahi, HR; Tabrizi, M; Feizabadi, MM

2011-01-01

Background and Objectives Pneumonia with Acinetobacter baumannii has a major therapeutic problem in health care settings. Decision to initiate correct antibiotic therapy requires rapid identification and quantification of organism. The aim of this study was to develop a rapid and sensitive method for direct detection of A. baumannii from respiratory specimens. Materials and Methods A Taqman real time PCR based on the sequence of bla oxa-51 was designed and used for direct detection of A. baumannii from 361 respiratory specimens of patients with pneumonia. All specimens were checked by conventional bacteriology in parallel. Results The new real time PCR could detect less than 200 cfu per ml of bacteria in specimens. There was agreement between the results of real time PCR and culture (Kappa value 1.0, p value<0.001). The sensitivity, specificity and predictive values of real time PCR were 100%. The prevalence of A. baumannii in pneumonia patients was 10.53 % (n=38). Poly-microbial infections were detected in 65.71% of specimens. Conclusion Acinetobacter baumannii is the third causative agent in nosocomial pneumonia after Pseudomonas aeroginosa (16%) and Staphylococcus aureus (13%) at Tehran hospitals. We recommend that 104 CFU be the threshold for definition of infection with A. baumannii using real time PCR. PMID:22530083

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

PubMed Central

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

2009-01-01

Background Visual assessment of left ventricular ejection fraction (LVEF) is often used in clinical routine despite general recommendations to use quantitative biplane Simpsons (BPS) measurements. Even thou quantitative methods are well validated and from many reasons preferable, the feasibility of visual assessment (eyeballing) is superior. There is to date only sparse data comparing visual EF assessment in comparison to quantitative methods available. The aim of this study was to compare visual EF assessment by two-dimensional echocardiography (2DE) and triplane echocardiography (TPE) using quantitative real-time three-dimensional echocardiography (RT3DE) as the reference method. Methods Thirty patients were enrolled in the study. Eyeballing EF was assessed using apical 4-and 2 chamber views and TP mode by two experienced readers blinded to all clinical data. The measurements were compared to quantitative RT3DE. Results There were an excellent correlation between eyeballing EF by 2D and TP vs 3DE (r = 0.91 and 0.95 respectively) without any significant bias (-0.5 ± 3.7% and -0.2 ± 2.9% respectively). Intraobserver variability was 3.8% for eyeballing 2DE, 3.2% for eyeballing TP and 2.3% for quantitative 3D-EF. Interobserver variability was 7.5% for eyeballing 2D and 8.4% for eyeballing TP. Conclusion Visual estimation of LVEF both using 2D and TP by an experienced reader correlates well with quantitative EF determined by RT3DE. There is an apparent trend towards a smaller variability using TP in comparison to 2D, this was however not statistically significant. PMID:19706183

Real-time quantitative fluorescence imaging using a single snapshot optical properties technique for neurosurgical guidance

NASA Astrophysics Data System (ADS)

Valdes, Pablo A.; Angelo, Joseph; Gioux, Sylvain

2015-03-01

Fluorescence imaging has shown promise as an adjunct to improve the extent of resection in neurosurgery and oncologic surgery. Nevertheless, current fluorescence imaging techniques do not account for the heterogeneous attenuation effects of tissue optical properties. In this work, we present a novel imaging system that performs real time quantitative fluorescence imaging using Single Snapshot Optical Properties (SSOP) imaging. We developed the technique and performed initial phantom studies to validate the quantitative capabilities of the system for intraoperative feasibility. Overall, this work introduces a novel real-time quantitative fluorescence imaging method capable of being used intraoperatively for neurosurgical guidance.

Mutation screening of patients with Alzheimer disease identifies APP locus duplication in a Swedish patient

PubMed Central

2011-01-01

Background Missense mutations in three different genes encoding amyloid-β precursor protein, presenilin 1 and presenilin 2 are recognized to cause familial early-onset Alzheimer disease. Also duplications of the amyloid precursor protein gene have been shown to cause the disease. At the Dept. of Geriatric Medicine, Karolinska University Hospital, Sweden, patients are referred for mutation screening for the identification of nucleotide variations and for determining copy-number of the APP locus. Methods We combined the method of microsatellite marker genotyping with a quantitative real-time PCR analysis to detect duplications in patients with Alzheimer disease. Results In 22 DNA samples from individuals diagnosed with clinical Alzheimer disease, we identified one patient carrying a duplication on chromosome 21 which included the APP locus. Further mapping of the chromosomal region by array-comparative genome hybridization showed that the duplication spanned a maximal region of 1.09 Mb. Conclusions This is the first report of an APP duplication in a Swedish Alzheimer patient and describes the use of quantitative real-time PCR as a tool for determining copy-number of the APP locus. PMID:22044463

EVALUATION OF QUANTITATIVE REAL TIME PCR FOR THE MEASUREMENT OF HELICOBATER PYLORI AT LOW CONCENTRATIONS IN DRINKING WATER

EPA Science Inventory

Aims: To determine the performance of a rapid, real time polymerase chain reaction (PCR) method for the detection and quantitative analysis Helicobacter pylori at low concentrations in drinking water.

Methods and Results: A rapid DNA extraction and quantitative PCR (QPCR)...

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae

PubMed Central

Teste, Marie-Ange; Duquenne, Manon; François, Jean M; Parrou, Jean-Luc

2009-01-01

Background Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. Results From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. Conclusion In this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of ACT1 as well as other commonly used reference genes (PDA1, TDH3, RDN18, etc) as internal controls for quantitative gene expression analysis in yeast. PMID:19874630

Testing the interaction between analytical modules: an example with Roundup Ready® soybean line GTS 40-3-2

PubMed Central

2010-01-01

Background The modular approach to analysis of genetically modified organisms (GMOs) relies on the independence of the modules combined (i.e. DNA extraction and GM quantification). The validity of this assumption has to be proved on the basis of specific performance criteria. Results An experiment was conducted using, as a reference, the validated quantitative real-time polymerase chain reaction (PCR) module for detection of glyphosate-tolerant Roundup Ready® GM soybean (RRS). Different DNA extraction modules (CTAB, Wizard and Dellaporta), were used to extract DNA from different food/feed matrices (feed, biscuit and certified reference material [CRM 1%]) containing the target of the real-time PCR module used for validation. Purity and structural integrity (absence of inhibition) were used as basic criteria that a DNA extraction module must satisfy in order to provide suitable template DNA for quantitative real-time (RT) PCR-based GMO analysis. When performance criteria were applied (removal of non-compliant DNA extracts), the independence of GMO quantification from the extraction method and matrix was statistically proved, except in the case of Wizard applied to biscuit. A fuzzy logic-based procedure also confirmed the relatively poor performance of the Wizard/biscuit combination. Conclusions For RRS, this study recognises that modularity can be generally accepted, with the limitation of avoiding combining highly processed material (i.e. biscuit) with a magnetic-beads system (i.e. Wizard). PMID:20687918

Real-time cardiovascular magnetic resonance at 1.5 T using balanced SSFP and 40 ms resolution

PubMed Central

2013-01-01

Background While cardiovascular magnetic resonance (CMR) commonly employs ECG-synchronized cine acquisitions with balanced steady-state free precession (SSFP) contrast at 1.5 T, recent developments at 3 T demonstrate significant potential for T1-weighted real-time imaging at high spatiotemporal resolution using undersampled radial FLASH. The purpose of this work was to combine both ideas and to evaluate a corresponding real-time CMR method at 1.5 T with SSFP contrast. Methods Radial gradient-echo sequences with fully balanced gradients and at least 15-fold undersampling were implemented on two CMR systems with different gradient performance. Image reconstruction by regularized nonlinear inversion (NLINV) was performed offline and resulted in real-time SSFP CMR images at a nominal resolution of 1.8 mm and with acquisition times of 40 ms. Results Studies of healthy subjects demonstrated technical feasibility in terms of robustness and general image quality. Clinical applicability with access to quantitative evaluations (e.g., ejection fraction) was confirmed by preliminary applications to 27 patients with typical indications for CMR including arrhythmias and abnormal wall motion. Real-time image quality was slightly lower than for cine SSFP recordings, but considered diagnostic in all cases. Conclusions Extending conventional cine approaches, real-time radial SSFP CMR with NLINV reconstruction provides access to individual cardiac cycles and allows for studies of patients with irregular heartbeat. PMID:24028285

Real-time fluorescence target/background (T/B) ratio calculation in multimodal endoscopy for detecting GI tract cancer

NASA Astrophysics Data System (ADS)

Jiang, Yang; Gong, Yuanzheng; Wang, Thomas D.; Seibel, Eric J.

2017-02-01

Multimodal endoscopy, with fluorescence-labeled probes binding to overexpressed molecular targets, is a promising technology to visualize early-stage cancer. T/B ratio is the quantitative analysis used to correlate fluorescence regions to cancer. Currently, T/B ratio calculation is post-processing and does not provide real-time feedback to the endoscopist. To achieve real-time computer assisted diagnosis (CAD), we establish image processing protocols for calculating T/B ratio and locating high-risk fluorescence regions for guiding biopsy and therapy in Barrett's esophagus (BE) patients. Methods: Chan-Vese algorithm, an active contour model, is used to segment high-risk regions in fluorescence videos. A semi-implicit gradient descent method was applied to minimize the energy function of this algorithm and evolve the segmentation. The surrounding background was then identified using morphology operation. The average T/B ratio was computed and regions of interest were highlighted based on user-selected thresholding. Evaluation was conducted on 50 fluorescence videos acquired from clinical video recordings using a custom multimodal endoscope. Results: With a processing speed of 2 fps on a laptop computer, we obtained accurate segmentation of high-risk regions examined by experts. For each case, the clinical user could optimize target boundary by changing the penalty on area inside the contour. Conclusion: Automatic and real-time procedure of calculating T/B ratio and identifying high-risk regions of early esophageal cancer was developed. Future work will increase processing speed to <5 fps, refine the clinical interface, and apply to additional GI cancers and fluorescence peptides.

Real-time automated thickness measurement of the in vivo human tympanic membrane using optical coherence tomography

PubMed Central

Hubler, Zita; Shemonski, Nathan D.; Shelton, Ryan L.; Monroy, Guillermo L.; Nolan, Ryan M.

2015-01-01

Background Otitis media (OM), an infection in the middle ear, is extremely common in the pediatric population. Current gold-standard methods for diagnosis include otoscopy for visualizing the surface features of the tympanic membrane (TM) and making qualitative assessments to determine middle ear content. OM typically presents as an acute infection, but can progress to chronic OM, and after numerous infections and antibiotic treatments over the course of many months, this disease is often treated by surgically inserting small tubes in the TM to relieve pressure, enable drainage, and provide aeration to the middle ear. Diagnosis and monitoring of OM is critical for successful management, but remains largely qualitative. Methods We have developed an optical coherence tomography (OCT) system for high-resolution, depth-resolved, cross-sectional imaging of the TM and middle ear content, and for the quantitative assessment of in vivo TM thickness including the presence or absence of a middle ear biofilm. A novel algorithm was developed and demonstrated for automatic, real-time, and accurate measurement of TM thickness to aid in the diagnosis and monitoring of OM and other middle ear conditions. The segmentation algorithm applies a Hough transform to the OCT image data to determine the boundaries of the TM to calculate thickness. Results The use of OCT and this segmentation algorithm is demonstrated first on layered phantoms and then during real-time acquisition of in vivo OCT from humans. For the layered phantoms, measured thicknesses varied by approximately 5 µm over time in the presence of large axial and rotational motion. In vivo data also demonstrated differences in thicknesses both spatially on a single TM, and across normal, acute, and chronic OM cases. Conclusions Real-time segmentation and thickness measurements of image data from both healthy subjects and those with acute and chronic OM demonstrate the use of OCT and this algorithm as a robust, quantitative, and accurate method for use during real-time in vivo human imaging. PMID:25694956

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

PubMed

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

2006-01-01

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

An Alu-based, MGB Eclipse real-time PCR method for quantitation of human DNA in forensic samples.

PubMed

Nicklas, Janice A; Buel, Eric

2005-09-01

The forensic community needs quick, reliable methods to quantitate human DNA in crime scene samples to replace the laborious and imprecise slot blot method. A real-time PCR based method has the possibility of allowing development of a faster and more quantitative assay. Alu sequences are primate-specific and are found in many copies in the human genome, making these sequences an excellent target or marker for human DNA. This paper describes the development of a real-time Alu sequence-based assay using MGB Eclipse primers and probes. The advantages of this assay are simplicity, speed, less hands-on-time and automated quantitation, as well as a large dynamic range (128 ng/microL to 0.5 pg/microL).

A novel quantitative real-time polymerase chain reaction method for detecting toxigenic Pasteurella multocida in nasal swabs from swine.

PubMed

Scherrer, Simone; Frei, Daniel; Wittenbrink, Max Michael

2016-12-01

Progressive atrophic rhinitis (PAR) in pigs is caused by toxigenic Pasteurella multocida. In Switzerland, PAR is monitored by selective culture of nasal swabs and subsequent polymerase chain reaction (PCR) screening of bacterial colonies for the P. multocida toxA gene. A panel of 203 nasal swabs from a recent PAR outbreak were used to evaluate a novel quantitative real-time PCR for toxigenic P. multocida in porcine nasal swabs. In comparison to the conventional PCR with a limit of detection of 100 genome equivalents per PCR reaction, the real-time PCR had a limit of detection of 10 genome equivalents. The real-time PCR detected toxA-positive P. multocida in 101 samples (49.8%), whereas the conventional PCR was less sensitive with 90 toxA-positive samples (44.3%). In comparison to the real-time PCR, 5.4% of the toxA-positive samples revealed unevaluable results by conventional PCR. The approach of culture-coupled toxA PCR for the monitoring of PAR in pigs is substantially improved by a novel quantitative real-time PCR.

Human fecal source identification with real-time quantitative PCR

EPA Science Inventory

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

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

PubMed Central

Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

2011-01-01

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

Real-time quantitative PCR detection of genetically modified Maximizer maize and Roundup Ready soybean in some representative foods.

PubMed

Vaïtilingom, M; Pijnenburg, H; Gendre, F; Brignon, P

1999-12-01

A fast and quantitative method was developed to detect transgenic "Maximizer" maize "event 176" (Novartis) and "Roundup Ready" soybean (Monsanto) in food by real-time quantitative PCR. The use of the ABI Prism 7700 sequence detection system allowed the determination of the amplified product accumulation through a fluorogenic probe (TaqMan). Fluorescent dyes were chosen in such a way as to coamplify total and transgenic DNA in the same tube. Using real-time quantitative PCR, 2 pg of transgenic or total DNA per gram of starting sample was detected in 3 h after DNA extraction and the relative amounts of "Maximizer" maize and "Roundup Ready" soybean in some representative food products were quantified.

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

PubMed

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.

Real-time probabilistic covariance tracking with efficient model update.

PubMed

Wu, Yi; Cheng, Jian; Wang, Jinqiao; Lu, Hanqing; Wang, Jun; Ling, Haibin; Blasch, Erik; Bai, Li

2012-05-01

The recently proposed covariance region descriptor has been proven robust and versatile for a modest computational cost. The covariance matrix enables efficient fusion of different types of features, where the spatial and statistical properties, as well as their correlation, are characterized. The similarity between two covariance descriptors is measured on Riemannian manifolds. Based on the same metric but with a probabilistic framework, we propose a novel tracking approach on Riemannian manifolds with a novel incremental covariance tensor learning (ICTL). To address the appearance variations, ICTL incrementally learns a low-dimensional covariance tensor representation and efficiently adapts online to appearance changes of the target with only O(1) computational complexity, resulting in a real-time performance. The covariance-based representation and the ICTL are then combined with the particle filter framework to allow better handling of background clutter, as well as the temporary occlusions. We test the proposed probabilistic ICTL tracker on numerous benchmark sequences involving different types of challenges including occlusions and variations in illumination, scale, and pose. The proposed approach demonstrates excellent real-time performance, both qualitatively and quantitatively, in comparison with several previously proposed trackers.

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

PubMed

Thornton, Brenda; Basu, Chhandak

2011-01-01

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

Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing.

PubMed

Nong, Rachel Yuan; Wu, Di; Yan, Junhong; Hammond, Maria; Gu, Gucci Jijuan; Kamali-Moghaddam, Masood; Landegren, Ulf; Darmanis, Spyros

2013-06-01

Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes ~5 h. The multiplex version of the assay takes 7-8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.

Developing noninvasive diagnosis for single-gene disorders: the role of digital PCR.

PubMed

Barrett, Angela N; Chitty, Lyn S

2014-01-01

Cell-free fetal DNA constitutes approximately 10 % of the cell-free DNA found in maternal plasma and can be used as a reliable source of fetal genetic material for noninvasive prenatal diagnosis (NIPD) from early pregnancy. The relatively high levels of maternal background can make detection of paternally inherited point mutations challenging. Diagnosis of inheritance of autosomal recessive disorders using qPCR is even more challenging due to the high background of mutant maternal allele. Digital PCR is a very sensitive modified method of quantitative real-time PCR (qPCR), allowing absolute quantitation and rare allele detection without the need for standards or normalization. Samples are diluted and then partitioned into a large number of small qPCR reactions, some of which contain the target molecule and some which do not; the proportion of positive reactions can be used to calculate the concentration of targets in the initial sample. Here we discuss the use of digital PCR as an accurate approach to NIPD for single-gene disorders.

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

EPA Science Inventory

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

Quantitative Real-Time PCR Analysis of Total Propidium Monazide -Resistant Fecal Indicator Bacteria in Wastewater

EPA Science Inventory

A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. Thes...

Direct quantitative 13 C-filtered 1 H magnetic resonance imaging of PEGylated biomacromolecules in vivo.

PubMed

Alvares, Rohan D A; Lau, Justin Y C; Macdonald, Peter M; Cunningham, Charles H; Prosser, R Scott

2017-04-01

1 H MRI is an established diagnostic method that generally relies on detection of water. Imaging specific macromolecules is normally accomplished only indirectly through the use of paramagnetic tags, which alter the water signal in their vicinity. We demonstrate a new approach in which macromolecular constituents, such as proteins and drug delivery systems, are observed directly and quantitatively in vivo using 1 H MRI of 13 C-labeled poly(ethylene glycol) ( 13 C-PEG) tags. Molecular imaging of 13 C-PEG-labeled species was accomplished by incorporating a modified heteronuclear multiple quantum coherence filter into a gradient echo imaging sequence. We demonstrate the approach by monitoring the real-time distribution of 13 C-PEG and 13 C-PEGylated albumin injected into the hind leg of a mouse. Filtering the 1 H PEG signal through the directly coupled 13 C nuclei largely eliminates background water and fat signals, thus enabling the imaging of molecules using 1 H MRI. PEGylation is widely employed to enhance the performance of a multitude of macromolecular therapeutics and drug delivery systems, and 13 C-filtered 1 H MRI of 13 C-PEG thus offers the possibility of imaging and quantitating their distribution in living systems in real time. Magn Reson Med 77:1553-1561, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

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

EPA Science Inventory

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...

Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution

EPA Science Inventory

There are numerous quantitative real-time PCR (qPCR) methods available to detect and enumerate human fecal pollution in ambient waters. Each assay employs distinct primers and/or probes and many target different genes and microorganisms leading to potential variations in method ...

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

EPA Science Inventory

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

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

EPA Science Inventory

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...

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

EPA Science Inventory

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

Single Laboratory Comparison of Quantitative Real-time PCR Assays for the Detection of Fecal Pollution

EPA Science Inventory

There are numerous quantitative real-time PCR (qPCR) assays available to detect and enumerate fecal pollution in ambient waters. Each assay employs distinct primers and probes that target different rRNA genes and microorganisms leading to potential variations in concentration es...

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

EPA Science Inventory

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

EVALUATION OF RAPID DNA EXTRACTION PROCEDURES FOR THE QUANTITATIVE DETECTION OF FUNGAL CELLS USING REAL TIME PCR ANALYSIS

EPA Science Inventory

The ease and rapidity of quantitative DNA sequence detection by real-time PCR instruments promises to make their use increasingly common for the microbial analysis many different types of environmental samples. To fully exploit the capabilities of these instruments, correspondin...

Real-time porphyrin detection in plaque and caries: a case study

NASA Astrophysics Data System (ADS)

Timoshchuk, Mari-Alina I.; Ridge, Jeremy S.; Rugg, Amanda L.; Nelson, Leonard Y.; Kim, Amy S.; Seibel, Eric J.

2015-02-01

An ultrathin scanning fiber endoscope, originally developed for cancer diagnosis, was used in a case study to locate plaque and caries. The imaging system incorporated software mitigation of background auto-fluorescence (AF). In conventional fluorescence imaging, varying AF across a tooth surface can mask low-level porphyrin signals. Laser-induced auto-fluorescence signals of dental tissue excited using a 405-nm laser typically produce fluorescence over a wavelength range extending from 440-nm to 750-nm. Anaerobic bacterial metabolism produces various porphyrin species (eg. protoporphyrin IX) that are located in carious enamel, dentin, gingivitis sites, and plaque. In our case study, these porphyrin deposits remained as long as one day after prophylaxis. Imaging the tooth surface using 405-nm excitation and subtracting the natural AF enhances the image contrast of low-level porphyrin deposits, which would otherwise be masked by the high background AF. In a case study, healthy tissues as well as sites of early and advanced caries formations were scanned for visual and quantitative signs of red fluorescence associated with porphyrin species using a background mitigation algorithm. Initial findings show increasing amplitudes of red fluorescence as caries severity increases from early to late stages. Sites of plaque accumulation also displayed red fluorescence similar to that found in carious dental tissue. The use of real-time background mitigation of natural dental AF can enhance the detection of low porphyrin concentrations that are indicators of early stage caries formation.

Pinhole Micro-SPECT/CT for Noninvasive Monitoring and Quantitation of Oncolytic Virus Dispersion and Percent Infection in Solid Tumors

PubMed Central

Penheiter, Alan R.; Griesmann, Guy E.; Federspiel, Mark J.; Dingli, David; Russell, Stephen J.; Carlson, Stephanie K.

2011-01-01

The purpose of our study was to validate the ability of pinhole micro-single-photon emission computed tomography/computed tomography (SPECT/CT) to 1) accurately resolve the intratumoral dispersion pattern and 2) quantify the infection percentage in solid tumors of an oncolytic measles virus encoding the human sodium iodide symporter (MV-NIS). NIS RNA level and dispersion pattern were determined in control and MV-NIS infected BxPC-3 pancreatic tumor cells and mouse xenografts using quantitative, real-time, reverse transcriptase, polymerase chain reaction, autoradiography, and immunohistochemistry (IHC). Mice with BxPC-3 xenografts were imaged with 123I or 99TcO4 micro-SPECT/CT. Tumor dimensions and radionuclide localization were determined with imaging software. Linear regression and correlation analyses were performed to determine the relationship between tumor infection percentage and radionuclide uptake (% injected dose per gram) above background and a highly significant correlation was observed (r2 = 0.947). A detection threshold of 1.5-fold above the control tumor uptake (background) yielded a sensitivity of 2.7% MV-NIS infected tumor cells. We reliably resolved multiple distinct intratumoral zones of infection from noninfected regions. Pinhole micro-SPECT/CT imaging using the NIS reporter demonstrated precise localization and quantitation of oncolytic MV-NIS infection and can replace more time-consuming and expensive analyses (eg, autoradiography and IHC) that require animal sacrifice. PMID:21753796

Improved HF183 quantitative real-time PCR assay for characterization of human fecal pollution in ambient surface water samples

EPA Science Inventory

Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster are considered to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. In response, the United States Environmental Protectio...

Single Laboratory Comparison of Quantitative Real-Time PCR Assays for the Detection of Human Fecal Pollution - Poster

EPA Science Inventory

There are numerous quantitative real-time PCR (qPCR) methods available to detect and enumerate human fecal pollution in ambient waters. Each assay employs distinct primers and/or probes and many target different genes and microorganisms leading to potential variations in method p...

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

EPA Science Inventory

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

The applicability of TaqMan-based quantitative real-time PCR assays for detecting and enumeratIng Cryptosporidium spp. oocysts in the environment

EPA Science Inventory

Molecular detection methods such as PCR have been extensively used to type Cryptosporidium oocysts detected in the environment. More recently, studies have developed quantitative real-time PCR assays for detection and quantification of microbial contaminants in water as well as ...

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

EPA Science Inventory

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...

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

USDA-ARS?s Scientific Manuscript database

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

Analysis of Enterococci and Bacteriodales Fecal Indicator Bacteria in a Lake Michigan Tributary by Real-Time Quantitative PCR

EPA Science Inventory

The Salt Creek watershed in northwest Indiana drains into Lake Michigan near several heavily used recreational beaches. This study aimed to investigate the levels of fecal indicator bacteria, enterococci and Bacteroidales, in Salt Creek using real-time quantitative PCR (qPCR) an...

Real-time Visualization and Quantification of Retrograde Cardioplegia Delivery using Near Infrared Fluorescent Imaging

PubMed Central

Rangaraj, Aravind T.; Ghanta, Ravi K.; Umakanthan, Ramanan; Soltesz, Edward G.; Laurence, Rita G.; Fox, John; Cohn, Lawrence H.; Bolman, R. M.; Frangioni, John V.; Chen, Frederick Y.

2009-01-01

Background and Aim of the Study Homogeneous delivery of cardioplegia is essential for myocardial protection during cardiac surgery. Presently, there exist no established methods to quantitatively assess cardioplegia distribution intraoperatively and determine when retrograde cardioplegia is required. In this study, we evaluate the feasibility of near infrared (NIR) imaging for real-time visualization of cardioplegia distribution in a porcine model. Methods A portable, intraoperative, real-time NIR imaging system was utilized. NIR fluorescent cardioplegia solution was developed by incorporating indocyanine green (ICG) into crystalloid cardioplegia solution. Real-time NIR imaging was performed while the fluorescent cardioplegia solution was infused via the retrograde route in 5 ex-vivo normal porcine hearts and in 5 ex-vivo porcine hearts status post left anterior descending (LAD) coronary artery ligation. Horizontal cross-sections of the hearts were obtained at proximal, middle, and distal LAD levels. Videodensitometry was performed to quantify distribution of fluorophore content. Results The progressive distribution of cardioplegia was clearly visualized with NIR imaging. Complete visualization of retrograde distribution occurred within 4 minutes of infusion. Videodensitometry revealed that retrograde cardioplegia primarily distributed to the left ventricle and anterior septum. In hearts with LAD ligation, antegrade cardioplegia did not distribute to the anterior left ventricle. This deficiency was compensated for with retrograde cardioplegia supplementation. Conclusions Incorporation of ICG into cardioplegia allows real-time visualization of cardioplegia delivery via NIR imaging. This technology may prove useful in guiding intraoperative decisions pertaining to when retrograde cardioplegia is mandated. PMID:19016995

Detection and enumeration of Salmonella enteritidis in homemade ice cream associated with an outbreak: comparison of conventional and real-time PCR methods.

PubMed

Seo, K H; Valentin-Bon, I E; Brackett, R E

2006-03-01

Salmonellosis caused by Salmonella Enteritidis (SE) is a significant cause of foodborne illnesses in the United States. Consumption of undercooked eggs and egg-containing products has been the primary risk factor for the disease. The importance of the bacterial enumeration technique has been enormously stressed because of the quantitative risk analysis of SE in shell eggs. Traditional enumeration methods mainly depend on slow and tedious most-probable-number (MPN) methods. Therefore, specific, sensitive, and rapid methods for SE quantitation are needed to collect sufficient data for risk assessment and food safety policy development. We previously developed a real-time quantitative PCR assay for the direct detection and enumeration of SE and, in this study, applied it to naturally contaminated ice cream samples with and without enrichment. The detection limit of the real-time PCR assay was determined with artificially inoculated ice cream. When applied to the direct detection and quantification of SE in ice cream, the real-time PCR assay was as sensitive as the conventional plate count method in frequency of detection. However, populations of SE derived from real-time quantitative PCR were approximately 1 log higher than provided by MPN and CFU values obtained by conventional culture methods. The detection and enumeration of SE in naturally contaminated ice cream can be completed in 3 h by this real-time PCR method, whereas the cultural enrichment method requires 5 to 7 days. A commercial immunoassay for the specific detection of SE was also included in the study. The real-time PCR assay proved to be a valuable tool that may be useful to the food industry in monitoring its processes to improve product quality and safety.

Using dynamic mode decomposition for real-time background/foreground separation in video

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kutz, Jose Nathan; Grosek, Jacob; Brunton, Steven

The technique of dynamic mode decomposition (DMD) is disclosed herein for the purpose of robustly separating video frames into background (low-rank) and foreground (sparse) components in real-time. Foreground/background separation is achieved at the computational cost of just one singular value decomposition (SVD) and one linear equation solve, thus producing results orders of magnitude faster than robust principal component analysis (RPCA). Additional techniques, including techniques for analyzing the video for multi-resolution time-scale components, and techniques for reusing computations to allow processing of streaming video in real time, are also described herein.

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

NASA Astrophysics Data System (ADS)

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.

Evaluation of reference genes for quantitative RT-PCR in Lolium temulentum under abiotic stress

USDA-ARS?s Scientific Manuscript database

Lolium temulentum is a valuable model grass species for the study of stress in forage and turf grasses. Gene expression analysis by quantitative real time RT-PCR relies on the use of proper internal standards. The aim of this study was to identify and evaluate reference genes for use in real-time q...

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed

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

2012-03-01

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

77 FR 48060 - Real-Time Public Reporting of Swap Transaction Data; Correction

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-13

... COMMODITY FUTURES TRADING COMMISSION 17 CFR Part 43 RIN 3038-AD08 Real-Time Public Reporting of...'') published the Real-Time Public Reporting of Swap Transaction Data (``Real-Time Public Reporting'') rule and.... Background The Commission published the final rule entitled Real-Time Public Reporting of Swap Transaction...

Validation of Geno-Sen's Scrub Typhus Real Time Polymerase Chain Reaction Kit by its Comparison with a Serological ELISA Test

PubMed Central

Anitharaj, Velmurugan; Stephen, Selvaraj; Pradeep, Jothimani; Pooja, Pratheesh; Preethi, Sridharan

2017-01-01

Background: In the recent past, scrub typhus (ST) has been reported from different parts of India, based on Weil-Felix/enzyme-linked immunosorbent assay (ELISA)/indirect immunofluorescence assay (IFA). Molecular tests are applied only by a few researchers. Aims: Evaluation of a new commercial real time polymerase chain reaction (PCR) kit for molecular diagnosis of ST by comparing it with the commonly used IgM ELISA is our aim. Settings and Design: ST has been reported all over India including Puducherry and surrounding Tamil Nadu and identified as endemic for ST. This study was designed to correlate antibody detection by IgM ELISA and Orientia tsutsugamushi DNA in real time PCR. Materials and Methods: ST IgM ELISA (InBios Inc., USA) was carried out for 170 consecutive patients who presented with the symptoms of acute ST during 11 months (November, 2015– September, 2016). All 77 of these patients with IgM ELISA positivity and 49 of 93 IgM ELISA negative patients were subjected to real time PCR (Geno-Sen's ST real time PCR, Himachal Pradesh, India). Statistical Analysis: Statistical analysis for clinical and laboratory results was performed using IBM SPSS Statistics 17 for Windows (SPSS Inc., Chicago, USA). Chi-square test with Yates correction (Fisher's test) was employed for a small number of samples. Results and Conclusion: Among 77 suspected cases of acute ST with IgM ELISA positivity and 49 IgM negative patients, 42 and 7 were positive, respectively, for O. tsutsugamushi 56-kDa type-specific gene in real time PCR kit. Until ST IFA, the gold standard diagnostic test, is properly validated in India, diagnosis of acute ST will depend on both ELISA and quantitative PCR. PMID:28878522

Detection of tumor markers in prostate cancer and comparison of sensitivity between real time and nested PCR.

PubMed

Matsuoka, Takayuki; Shigemura, Katsumi; Yamamichi, Fukashi; Fujisawa, Masato; Kawabata, Masato; Shirakawa, Toshiro

2012-06-27

The objective of this study is to investigate and compare the sensitivity in conventional PCR, quantitative real time PCR, nested PCR and western blots for detection of prostate cancer tumor markers using prostate cancer (PCa) cells. We performed conventional PCR, quantitative real time PCR, nested PCR, and western blots using 5 kinds of PCa cells. Prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and androgen receptor (AR) were compared for their detection sensitivity by real time PCR and nested PCR. In real time PCR, there was a significant correlation between cell number and the RNA concentration obtained (R(2)=0.9944) for PSA, PSMA, and AR. We found it possible to detect these markers from a single LNCaP cell in both real time and nested PCR. By comparison, nested PCR reached a linear curve in fewer PCR cycles than real time PCR, suggesting that nested PCR may offer PCR results more quickly than real time PCR. In conclusion, nested PCR may offer tumor maker detection in PCa cells more quickly (with fewer PCR cycles) with the same high sensitivity as real time PCR. Further study is necessary to establish and evaluate the best tool for PCa tumor marker detection.

[Multiplex real-time PCR method for rapid detection of Marburg virus and Ebola virus].

PubMed

Yang, Yu; Bai, Lin; Hu, Kong-Xin; Yang, Zhi-Hong; Hu, Jian-Ping; Wang, Jing

2012-08-01

Marburg virus and Ebola virus are acute infections with high case fatality rates. A rapid, sensitive detection method was established to detect Marburg virus and Ebola virus by multiplex real-time fluorescence quantitative PCR. Designing primers and Taqman probes from highly conserved sequences of Marburg virus and Ebola virus through whole genome sequences alignment, Taqman probes labeled by FAM and Texas Red, the sensitivity of the multiplex real-time quantitative PCR assay was optimized by evaluating the different concentrations of primers and Probes. We have developed a real-time PCR method with the sensitivity of 30.5 copies/microl for Marburg virus positive plasmid and 28.6 copies/microl for Ebola virus positive plasmids, Japanese encephalitis virus, Yellow fever virus, Dengue virus were using to examine the specificity. The Multiplex real-time PCR assays provide a sensitive, reliable and efficient method to detect Marburg virus and Ebola virus simultaneously.

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

PubMed

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. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evaluation of a real-time quantitative PCR method with propidium monazide treatment for analyses of viable fecal indicator bacteria in wastewater samples

EPA Science Inventory

The U.S. EPA is currently evaluating rapid, real-time quantitative PCR (qPCR) methods for determining recreational water quality based on measurements of fecal indicator bacteria DNA sequences. In order to potentially use qPCR for other Clean Water Act needs, such as updating cri...

Development of duplex SYBR Green I-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses

USDA-ARS?s Scientific Manuscript database

A SYBR® Green-based real-time quantitative reverse transcription PCR (qRT-PCR) assay in combination with melt curve analysis (MCA) was developed for the detection of nine grapevine viruses. The detection limits for singleplex qRT-PCR for all nine grapevine viruses were determined to be in the range ...

[Experimental studies of using real-time fluorescence quantitative PCR and RT-PCR to detect E6 and E7 genes of human papillomavirus type 16 in cervical carcinoma cell lines].

PubMed

Chen, Yue-yue; Peng, Zhi-lan; Liu, Shan-ling; He, Bing; Hu, Min

2007-06-01

To establish a method of using real-time fluorescence quantitative PCR and RT-PCR to detect the E6 and E7 genes of human papillomavirus type 16 (HPV-16). Plasmids containing HPV-16 E6 or E7 were used to generate absolute standard curves. Three cervical carcinoma cell lines CaSki, SiHa and HeLa were tested by real-time fluorescence quantitative PCR and RT-PCR analyses for the expressions of HPV-16 E6 and E7. The correlation coefficients of standard curves were larger than 0. 99, and the PCR efficiency was more than 90%. The relative levels of HPV-16 E6 and E7 DNA and RNA were CaSki>SiHa>HeLa cell. HPV-16 E6 and E7 quantum by real-time fluorescence quantitative PCR and RT-PCR analyses may serve as a reliable and sensitive tool. This study provides the possibility of further researches on the relationship between HPV-16 E6 or E7 copy number and cervical carcinoma.

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

PubMed

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

2018-03-05

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

A novel quadruplex real-time PCR method for simultaneous detection of Cry2Ae and two genetically modified cotton events (GHB119 and T304-40)

PubMed Central

2014-01-01

Background To date, over 150 genetically modified (GM) crops are widely cultivated. To comply with regulations developed for genetically modified organisms (GMOs), including labeling policies, many detection methods for GMO identification and quantification have been developed. Results To detect the entrance and exit of unauthorized GM crop events in China, we developed a novel quadruplex real-time PCR method for simultaneous detection and quantification of GM cotton events GHB119 and T304-40 in cotton-derived products (based on the 5′-flanking sequence) and the insect-resistance gene Cry2Ae. The limit of detection was 10 copies for GHB119 and Cry2Ae and 25 copies for T304-40. The limit of quantification was 25 copies for GHB119 and Cry2Ae and 50 copies for T304-40. Moreover, low bias and acceptable standard deviation and relative standard deviation values were obtained in quantification analysis of six blind samples containing different GHB119 and T304-40 ingredients. Conclusions The developed quadruplex quantitative method could be used for quantitative detection of two GM cotton events (GHB119 and T304-40) and Cry2Ae gene ingredient in cotton derived products. PMID:24884946

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

PubMed

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

2014-01-01

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

Utilization of DIRSIG in support of real-time infrared scene generation

NASA Astrophysics Data System (ADS)

Sanders, Jeffrey S.; Brown, Scott D.

2000-07-01

Real-time infrared scene generation for hardware-in-the-loop has been a traditionally difficult challenge. Infrared scenes are usually generated using commercial hardware that was not designed to properly handle the thermal and environmental physics involved. Real-time infrared scenes typically lack details that are included in scenes rendered in no-real- time by ray-tracing programs such as the Digital Imaging and Remote Sensing Scene Generation (DIRSIG) program. However, executing DIRSIG in real-time while retaining all the physics is beyond current computational capabilities for many applications. DIRSIG is a first principles-based synthetic image generation model that produces multi- or hyper-spectral images in the 0.3 to 20 micron region of the electromagnetic spectrum. The DIRSIG model is an integrated collection of independent first principles based on sub-models, each of which works in conjunction to produce radiance field images with high radiometric fidelity. DIRSIG uses the MODTRAN radiation propagation model for exo-atmospheric irradiance, emitted and scattered radiances (upwelled and downwelled) and path transmission predictions. This radiometry submodel utilizes bidirectional reflectance data, accounts for specular and diffuse background contributions, and features path length dependent extinction and emission for transmissive bodies (plumes, clouds, etc.) which may be present in any target, background or solar path. This detailed environmental modeling greatly enhances the number of rendered features and hence, the fidelity of a rendered scene. While DIRSIG itself cannot currently be executed in real-time, its outputs can be used to provide scene inputs for real-time scene generators. These inputs can incorporate significant features such as target to background thermal interactions, static background object thermal shadowing, and partially transmissive countermeasures. All of these features represent significant improvements over the current state of the art in real-time IR scene generation.

Real-time airborne gamma-ray background estimation using NASVD with MLE and radiation transport for calibration

NASA Astrophysics Data System (ADS)

Kulisek, J. A.; Schweppe, J. E.; Stave, S. C.; Bernacki, B. E.; Jordan, D. V.; Stewart, T. N.; Seifert, C. E.; Kernan, W. J.

2015-06-01

Helicopter-mounted gamma-ray detectors can provide law enforcement officials the means to quickly and accurately detect, identify, and locate radiological threats over a wide geographical area. The ability to accurately distinguish radiological threat-generated gamma-ray signatures from background gamma radiation in real time is essential in order to realize this potential. This problem is non-trivial, especially in urban environments for which the background may change very rapidly during flight. This exacerbates the challenge of estimating background due to the poor counting statistics inherent in real-time airborne gamma-ray spectroscopy measurements. To address this challenge, we have developed a new technique for real-time estimation of background gamma radiation from aerial measurements without the need for human analyst intervention. The method can be calibrated using radiation transport simulations along with data from previous flights over areas for which the isotopic composition need not be known. Over the examined measured and simulated data sets, the method generated accurate background estimates even in the presence of a strong, 60Co source. The potential to track large and abrupt changes in background spectral shape and magnitude was demonstrated. The method can be implemented fairly easily in most modern computing languages and environments.

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

PubMed

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

2017-02-01

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

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

PubMed

Harris, S C; Walker, D S

2000-09-01

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

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

EPA Science Inventory

Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.

ABSTRACT

DNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or du...

Real-time contaminant detection and classification in a drinking water pipe using conventional water quality sensors: techniques and experimental results.

PubMed

Jeffrey Yang, Y; Haught, Roy C; Goodrich, James A

2009-06-01

Accurate detection and identification of natural or intentional contamination events in a drinking water pipe is critical to drinking water supply security and health risk management. To use conventional water quality sensors for the purpose, we have explored a real-time event adaptive detection, identification and warning (READiw) methodology and examined it using pilot-scale pipe flow experiments of 11 chemical and biological contaminants each at three concentration levels. The tested contaminants include pesticide and herbicides (aldicarb, glyphosate and dicamba), alkaloids (nicotine and colchicine), E. coli in terrific broth, biological growth media (nutrient broth, terrific broth, tryptic soy broth), and inorganic chemical compounds (mercuric chloride and potassium ferricyanide). First, through adaptive transformation of the sensor outputs, contaminant signals were enhanced and background noise was reduced in time-series plots leading to detection and identification of all simulated contamination events. The improved sensor detection threshold was 0.1% of the background for pH and oxidation-reduction potential (ORP), 0.9% for free chlorine, 1.6% for total chlorine, and 0.9% for chloride. Second, the relative changes calculated from adaptively transformed residual chlorine measurements were quantitatively related to contaminant-chlorine reactivity in drinking water. We have shown that based on these kinetic and chemical differences, the tested contaminants were distinguishable in forensic discrimination diagrams made of adaptively transformed sensor measurements.

[Clinical significance of monitoring BK polyomavirus in patients after hematopoietic stem cell transplantation].

PubMed

Yin, Chang-Xin; Jiang, Qian-Li; He, Han; Yu, Guo-Pan; Xu, Yue; Meng, Fan-Yi; Yang, Mo

2012-02-01

This study was aimed to establish a method for rapid detecting BK polyomavirus (BKV) and to investigate the feasibility and value used in leukemia patients undergoing hematopoietic stem cell transplantation. Primers were designed according to BKV gene sequence; the quantitative standards for BKV and a real-time fluorescent quantitative PCR for BKV were established. The BKV level in urine samples from 36 patients after hematopoietic stem cell transplantation were detected by established method. The results showed that the standard of reconstructed plasmid and real time fluorescent quantitative PCR method were successfully established, its good specificity, sensitivity and stability were confirmed by experiments. BKV was found in 55.56% of urine samples, and the BKV load in urine was 2.46 × 10(4) - 7.8 × 10(9) copy/ml. It is concluded that the establishment of real-time fluorescent quantitative PCR for BKV detection provides a method for early diagnosis of the patients with hemorrhagic cystitis after hematopoietic stem cell transplantation.

Non-interferometric quantitative phase imaging of yeast cells

NASA Astrophysics Data System (ADS)

Poola, Praveen K.; Pandiyan, Vimal Prabhu; John, Renu

2015-12-01

Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.

Quantitative EEG analysis using error reduction ratio-causality test; validation on simulated and real EEG data.

PubMed

Sarrigiannis, Ptolemaios G; Zhao, Yifan; Wei, Hua-Liang; Billings, Stephen A; Fotheringham, Jayne; Hadjivassiliou, Marios

2014-01-01

To introduce a new method of quantitative EEG analysis in the time domain, the error reduction ratio (ERR)-causality test. To compare performance against cross-correlation and coherence with phase measures. A simulation example was used as a gold standard to assess the performance of ERR-causality, against cross-correlation and coherence. The methods were then applied to real EEG data. Analysis of both simulated and real EEG data demonstrates that ERR-causality successfully detects dynamically evolving changes between two signals, with very high time resolution, dependent on the sampling rate of the data. Our method can properly detect both linear and non-linear effects, encountered during analysis of focal and generalised seizures. We introduce a new quantitative EEG method of analysis. It detects real time levels of synchronisation in the linear and non-linear domains. It computes directionality of information flow with corresponding time lags. This novel dynamic real time EEG signal analysis unveils hidden neural network interactions with a very high time resolution. These interactions cannot be adequately resolved by the traditional methods of coherence and cross-correlation, which provide limited results in the presence of non-linear effects and lack fidelity for changes appearing over small periods of time. Copyright © 2013 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

A preamplification approach to GMO detection in processed foods.

PubMed

Del Gaudio, S; Cirillo, A; Di Bernardo, G; Galderisi, U; Cipollaro, M

2010-03-01

DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.

[Application of recombinase polymerase amplification in the detection of Pseudomonas aeruginosa].

PubMed

Jin, X J; Gong, Y L; Yang, L; Mo, B H; Peng, Y Z; He, P; Zhao, J N; Li, X L

2018-04-20

Objective: To establish an optimized method of recombinase polymerase amplification (RPA) to rapidly detect Pseudomonas aeruginosa in clinic. Methods: (1) The DNA templates of one standard Pseudomonas aeruginosa strain was extracted and detected by polymerase chain reaction (PCR), real-time fluorescence quantitative PCR and RPA. Time of sample loading, time of amplification, and time of detection of the three methods were recorded. (2) One standard Pseudomonas aeruginosa strain was diluted in 7 concentrations of 1×10(7,) 1×10(6,) 1×10(5,) 1×10(4,) 1×10(3,) 1×10(2,) and 1×10(1) colony forming unit (CFU)/mL after recovery and cultivation. The DNA templates of Pseudomonas aeruginosa and negative control strain Pseudomonas putida were extracted and detected by PCR, real-time fluorescence quantitative PCR, and RPA separately. The sensitivity of the three methods in detecting Pseudomonas aeruginosa was analyzed. (3) The DNA templates of one standard Pseudomonas aeruginosa strain and four negative control strains ( Staphylococcus aureus, Acinetobacter baumanii, Candida albicans, and Pseudomonas putida ) were extracted separately, and then they were detected by PCR, real-time fluorescence quantitative PCR, and RPA. The specificity of the three methods in detecting Pseudomonas aeruginosa was analyzed. (4) The DNA templates of 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin, 1 clinical strain of which was taken by cotton swab, and negative control strain Pseudomonas putida were extracted separately, and then they were detected by RPA. Positive amplification signals of the clinical strains were observed, and the detection rate was calculated. All experiments were repeated for 3 times. Sensitivity results were analyzed by GraphPad Prism 5.01 statistical software. Results: (1) The loading time of RPA, PCR, and real-time fluorescence quantitative PCR for detecting Pseudomonas aeruginosa were all 20 minutes. In PCR, time of amplification was 98 minutes, time of gel detection was 20 minutes, and the total time was 138 minutes. In real-time fluorescence quantitative PCR, amplification and detection could be completed simultaneously, which took 90 minutes, and the total time was 110 minutes. In RPA, amplification and detection could also be completed simultaneously, which took 15 minutes, and the total time was 35 minutes. (2) Pseudomonas putida did not show positive amplification signals or gel positive results in any of the three detection methods. The detection limit of Pseudomonas aeruginosa in real-time fluorescence quantitative PCR and PCR was 1×10(1) CFU/mL, and that of Pseudomonas aeruginosa in RPA was 1×10(2) CFU/mL. In RPA and real-time fluorescence quantitative PCR, the higher the concentration of Pseudomonas aeruginosa, the shorter threshold time and smaller the number of cycles, namely shorter time for detecting the positive amplified signal. In real-time fluorescence quantitative PCR, all positive amplification signal could be detected when the concentration of Pseudomonas aeruginosa was 1×10(1)-1×10(7) CFU/mL. In RPA, the detection rate of positive amplification signal was 0 when the concentration of Pseudomonas aeruginosa was 1×10(1) CFU/mL, while the detection rate of positive amplification signal was 67% when the concentration of Pseudomonas aeruginosa was 1×10(2) CFU/mL, and the detection rate of positive amplification signal was 100% when the concentration of Pseudomonas aeruginosa was 1×10(3)-1×10(7) CFU/mL. (3) In RPA, PCR, and real-time fluorescence quantitative PCR, Pseudomonas aeruginosa showed positive amplification signals and gel positive results, but there were no positive amplification signals or gel positive results in four negative control strains of Acinetobacter baumannii, Staphylococcus aureus, Candida albicans, and Pseudomonas putida . (4) In RPA, 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin and 1 clinical strain of Pseudomonas aeruginosa taken by cotton swab showed positive amplification signals, while Pseudomonas putida did not show positive amplification signal. The detection rate of positive amplification signal of 29 clinical strains of Pseudomonas aeruginosa in RPA was 100%. Conclusions: The established optimized RPA technology for fast detection of Pseudomonas aeruginosa requires shorter time, with high sensitivity and specificity. It was of great value in fast detection of Pseudomonas aeruginosa infection in clinic.

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

PubMed Central

Yankson, Kweku K.; Steck, Todd R.

2009-01-01

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

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

PubMed Central

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

2016-01-01

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

Coating process optimization through in-line monitoring for coating weight gain using Raman spectroscopy and design of experiments.

PubMed

Kim, Byungsuk; Woo, Young-Ah

2018-05-30

In this study the authors developed a real-time Process Analytical Technology (PAT) of a coating process by applying in-line Raman spectroscopy to evaluate the coating weight gain, which is a quantitative analysis of the film coating layer. The wide area illumination (WAI) Raman probe was connected to the pan coater for real-time monitoring of changes in the weight gain of coating layers. Under the proposed in-line Raman scheme, a non-contact, non-destructive analysis was performed using WAI Raman probes with a spot size of 6 mm. The in-line Raman probe maintained a focal length of 250 mm, and a compressed air line was designed to protect the lens surface from spray droplets. The Design of Experiment (DOE) was applied to identify factors affecting the Raman spectra background of laser irradiation. The factors selected for DOE were the strength of compressed air connected to the probe, and the shielding of light by the transparent door connecting the probe to the pan coater. To develop a quantitative model, partial least squares (PLS) models as multivariate calibration were developed based on the three regions showing the specificity of TiO 2 individually or in combination. For the three single peaks (636 cm -1 , 512 cm -1 , 398 cm -1 ), least squares method (LSM) was applied to develop three univariate quantitative analysis models. One of best multivariate quantitative model having a factor of 1 gave the lowest RMSEP of 0.128, 0.129, and 0.125, respectively for prediction batches. When LSM was applied to the single peak at 636 cm -1 , the univariate quantitative model with an R 2 of 0.9863, slope of 0.5851, and y-intercept of 0.8066 had the lowest RMSEP of 0.138, 0.144, and 0.153, respectively for prediction batches. The in-line Raman spectroscopic method for the analysis of coating weight gain was verified by considering system suitability and parameters such as specificity, range, linearity, accuracy, and precision in accordance with ICH Q2 regarding method validation. The proposed in-line Raman spectroscopy can be utilized as a PAT for product quality assurance as it offers real-time monitoring of quantitative changes in coating weight gain and process end-points during the film coating process. Copyright © 2018 Elsevier B.V. All rights reserved.

A PCR primer bank for quantitative gene expression analysis.

PubMed

Wang, Xiaowei; Seed, Brian

2003-12-15

Although gene expression profiling by microarray analysis is a useful tool for assessing global levels of transcriptional activity, variability associated with the data sets usually requires that observed differences be validated by some other method, such as real-time quantitative polymerase chain reaction (real-time PCR). However, non-specific amplification of non-target genes is frequently observed in the latter, confounding the analysis in approximately 40% of real-time PCR attempts when primer-specific labels are not used. Here we present an experimentally validated algorithm for the identification of transcript-specific PCR primers on a genomic scale that can be applied to real-time PCR with sequence-independent detection methods. An online database, PrimerBank, has been created for researchers to retrieve primer information for their genes of interest. PrimerBank currently contains 147 404 primers encompassing most known human and mouse genes. The primer design algorithm has been tested by conventional and real-time PCR for a subset of 112 primer pairs with a success rate of 98.2%.

New design, development, and optimization of an in-house quantitative TaqMan Real-time PCR assay for HIV-1 viral load measurement.

PubMed

Noorbazargan, Hassan; Nadji, Seyed Alireza; Samiee, Siamak Mirab; Paryan, Mahdi; Mohammadi-Yeganeh, Samira

2018-04-01

Background Viral load measurement is commonly applicable to monitor HIV infection in patients to determine the number of HIV-RNA in serum samples of individuals. The aim of the present study was to set up a highly specific, sensitive, and reproducible home-brewed Real-time PCR assay based on TaqMan chemistry to quantify HIV-1 RNA genome. Methods In this study, three sets of primer pairs and a TaqMan probe were designed for HIV subtypes conserved sequences. An internal control was included in this assay to evaluate the presence of inhibition. Standard curve and threshold cycle values were determined using in vitro transcribed RNA from int region of HIV-1. A serial dilution of RNA standards was generated by in vitro transcription, from 10 to 10 9 copies/ml to find the sensitivity and the limit of detection (LOD) of the assay and to evaluate its performance in a quantitative RT-PCR assay. Results The assay has a low LOD equivalent to 33.13 copies/ml of HIV-1 RNA and a linear range of detection from 10 to 10 9 copies/ml. The coefficient of variation (CV) for Inter and Intra-assay precision of this in-house HIV Real-time RT-PCR ranged from 0.28 to 2.49% and 0.72 to 4.47%, respectively. The analytical and clinical specificity was 100%. Conclusions The results indicate that the developed method has a suitable specificity and sensitivity and is highly reproducible and cost-benefit. Therefore, it will be useful to monitor HIV infection in plasma samples of individuals.

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore.

PubMed

Rich, Ryan M; Stankowska, Dorota L; Maliwal, Badri P; Sørensen, Thomas Just; Laursen, Bo W; Krishnamoorthy, Raghu R; Gryczynski, Zygmunt; Borejdo, Julian; Gryczynski, Ignacy; Fudala, Rafal

2013-02-01

Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background.

Application of real-time PCR for total airborne bacterial assessment: Comparison with epifluorescence microscopy and culture-dependent methods

NASA Astrophysics Data System (ADS)

Rinsoz, Thomas; Duquenne, Philippe; Greff-Mirguet, Guylaine; Oppliger, Anne

Traditional culture-dependent methods to quantify and identify airborne microorganisms are limited by factors such as short-duration sampling times and inability to count non-culturable or non-viable bacteria. Consequently, the quantitative assessment of bioaerosols is often underestimated. Use of the real-time quantitative polymerase chain reaction (Q-PCR) to quantify bacteria in environmental samples presents an alternative method, which should overcome this problem. The aim of this study was to evaluate the performance of a real-time Q-PCR assay as a simple and reliable way to quantify the airborne bacterial load within poultry houses and sewage treatment plants, in comparison with epifluorescence microscopy and culture-dependent methods. The estimates of bacterial load that we obtained from real-time PCR and epifluorescence methods, are comparable, however, our analysis of sewage treatment plants indicate these methods give values 270-290 fold greater than those obtained by the "impaction on nutrient agar" method. The culture-dependent method of air impaction on nutrient agar was also inadequate in poultry houses, as was the impinger-culture method, which gave a bacterial load estimate 32-fold lower than obtained by Q-PCR. Real-time quantitative PCR thus proves to be a reliable, discerning, and simple method that could be used to estimate airborne bacterial load in a broad variety of other environments expected to carry high numbers of airborne bacteria.

High-sensitivity, real-time, ratiometric imaging of surface-enhanced Raman scattering nanoparticles with a clinically translatable Raman endoscope device.

PubMed

Garai, Ellis; Sensarn, Steven; Zavaleta, Cristina L; Van de Sompel, Dominique; Loewke, Nathan O; Mandella, Michael J; Gambhir, Sanjiv S; Contag, Christopher H

2013-09-01

Topical application and quantification of targeted, surface-enhanced Raman scattering (SERS) nanoparticles offer a new technique that has the potential for early detection of epithelial cancers of hollow organs. Although less toxic than intravenous delivery, the additional washing required to remove unbound nanoparticles cannot necessarily eliminate nonspecific pooling. Therefore, we developed a real-time, ratiometric imaging technique to determine the relative concentrations of at least two spectrally unique nanoparticle types, where one serves as a nontargeted control. This approach improves the specific detection of bound, targeted nanoparticles by adjusting for working distance and for any nonspecific accumulation following washing. We engineered hardware and software to acquire SERS signals and ratios in real time and display them via a graphical user interface. We report quantitative, ratiometric imaging with nanoparticles at pM and sub-pM concentrations and at varying working distances, up to 50 mm. Additionally, we discuss optimization of a Raman endoscope by evaluating the effects of lens material and fiber coating on background noise, and theoretically modeling and simulating collection efficiency at various working distances. This work will enable the development of a clinically translatable, noncontact Raman endoscope capable of rapidly scanning large, topographically complex tissue surfaces for small and otherwise hard to detect lesions.

Role of Service Learning in Water Quality Studies

ERIC Educational Resources Information Center

Richards, Wilfred Sugumar

2009-01-01

Background: Chemistry students often complain that they are unmotivated because they see no applications of chemical principles in "real life." It was thus decided to put into use the knowledge gained during the course on water quality and analysis. Learning the principles of quantitative chemical analysis requires innovative, hands-on…

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

NASA Technical Reports Server (NTRS)

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

2002-01-01

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

Ambient Stable Quantitative PCR Reagents for the Detection of Yersinia pestis

PubMed Central

Zhou, Lei; Guo, Zhaobiao; Zhou, Dongsheng; Zhai, Junhui; Yang, Ruifu

2010-01-01

Background Although assays for detecting Yersinia pestis using TaqMan probe-based real-time PCR have been developed for years, little is reported on room-temperature-stable PCR reagents, which will be invaluable for field epidemic surveillance, immediate response to public health emergencies, counter-bioterrorism investigation, etc. In this work, a set of real-time PCR reagents for rapid detection of Y. pestis was developed with extraordinary stability at 37°C. Methods/Principal Findings TaqMan-based real-time PCR assays were developed using the primers and probes targeting the 3a sequence in the chromosome and the F1 antigen gene caf1 in the plasmid pMT1of Y. pestis, respectively. Then, carbohydrate mixtures were added to the PCR reagents, which were later vacuum-dried for stability evaluation. The vacuum-dried reagents were stable at 37°C for at least 49 days for a lower concentration of template DNA (10 copies/µl), and up to 79 days for higher concentrations (≥102 copies/µl). The reagents were used subsequently to detect soil samples spiked with Y. pestis vaccine strain EV76, and 5×104 CFU per gram of soil could be detected by both 3a- and caf1-based PCR reagents. In addition, a simple and efficient method for soil sample processing is presented here. Conclusions/Significance The vacuum-dried reagents for real-time PCR maintain accuracy and reproducibility for at least 49 days at 37°C, indicating that they can be easily transported at room temperature for field application if the machine for performing real-time PCR is available. This dry reagent is of great significance for routine plague surveillance. PMID:20231881

A Novel High Throughput Assay for Anthelmintic Drug Screening and Resistance Diagnosis by Real-Time Monitoring of Parasite Motility

PubMed Central

Smout, Michael J.; Kotze, Andrew C.; McCarthy, James S.; Loukas, Alex

2010-01-01

Background Helminth parasites cause untold morbidity and mortality to billions of people and livestock. Anthelmintic drugs are available but resistance is a problem in livestock parasites, and is a looming threat for human helminths. Testing the efficacy of available anthelmintic drugs and development of new drugs is hindered by the lack of objective high-throughput screening methods. Currently, drug effect is assessed by observing motility or development of parasites using laborious, subjective, low-throughput methods. Methodology/Principal Findings Here we describe a novel application for a real-time cell monitoring device (xCELLigence) that can simply and objectively assess anthelmintic effects by measuring parasite motility in real time in a fully automated high-throughput fashion. We quantitatively assessed motility and determined real time IC50 values of different anthelmintic drugs against several developmental stages of major helminth pathogens of humans and livestock, including larval Haemonchus contortus and Strongyloides ratti, and adult hookworms and blood flukes. The assay enabled quantification of the onset of egg hatching in real time, and the impact of drugs on hatch rate, as well as discriminating between the effects of drugs on motility of drug-susceptible and –resistant isolates of H. contortus. Conclusions/Significance Our findings indicate that this technique will be suitable for discovery and development of new anthelmintic drugs as well as for detection of phenotypic resistance to existing drugs for the majority of helminths and other pathogens where motility is a measure of pathogen viability. The method is also amenable to use for other purposes where motility is assessed, such as gene silencing or antibody-mediated killing. PMID:21103363

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

PubMed

Ploemen, Ivo H J; Prudêncio, Miguel; Douradinha, Bruno G; Ramesar, Jai; Fonager, Jannik; van Gemert, Geert-Jan; Luty, Adrian J F; Hermsen, Cornelus C; Sauerwein, Robert W; Baptista, Fernanda G; Mota, Maria M; Waters, Andrew P; Que, Ivo; Lowik, Clemens W G M; Khan, Shahid M; Janse, Chris J; Franke-Fayard, Blandine M D

2009-11-18

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc(con), expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1-5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.

Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

PubMed Central

Douradinha, Bruno G.; Ramesar, Jai; Fonager, Jannik; van Gemert, Geert-Jan; Luty, Adrian J. F.; Hermsen, Cornelus C.; Sauerwein, Robert W.; Baptista, Fernanda G.; Mota, Maria M.; Waters, Andrew P.; Que, Ivo; Lowik, Clemens W. G. M.; Khan, Shahid M.; Janse, Chris J.; Franke-Fayard, Blandine M. D.

2009-01-01

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite's life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luccon, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium. PMID:19924309

Simplex and duplex event-specific analytical methods for functional biotech maize.

PubMed

Lee, Seong-Hun; Kim, Su-Jeong; Yi, Bu-Young

2009-08-26

Analytical methods are very important in the control of genetically modified organism (GMO) labeling systems or living modified organism (LMO) management for biotech crops. Event-specific primers and probes were developed for qualitative and quantitative analysis for biotech maize event 3272 and LY 038 on the basis of the 3' flanking regions, respectively. The qualitative primers confirmed the specificity by a single PCR product and sensitivity to 0.05% as a limit of detection (LOD). Simplex and duplex quantitative methods were also developed using TaqMan real-time PCR. One synthetic plasmid was constructed from two taxon-specific DNA sequences of maize and two event-specific 3' flanking DNA sequences of event 3272 and LY 038 as reference molecules. In-house validation of the quantitative methods was performed using six levels of mixing samples, from 0.1 to 10.0%. As a result, the biases from the true value and the relative deviations were all within the range of +/-30%. Limits of quantitation (LOQs) of the quantitative methods were all 0.1% for simplex real-time PCRs of event 3272 and LY 038 and 0.5% for duplex real-time PCR of LY 038. This study reports that event-specific analytical methods were applicable for qualitative and quantitative analysis for biotech maize event 3272 and LY 038.

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

PubMed

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

2015-01-01

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

Imatinib induces up-regulation of NM23, a metastasis suppressor gene, in human Hepatocarcinoma (HepG2) Cell Line

PubMed Central

Keshavarz-Pakseresht, Behta; Shandiz, Seyed Ataollah Sadat; Baghbani-arani, Fahimeh

2017-01-01

Aim: The present study investigated the anti-tumor activity of Imatinib mesylate through modulation of NM23 gene expression in human hepatocellular carcinoma (HepG2) cell line. Background: Hepatocellular carcinoma (HCC) is considered to be the third leading cause of cancer related death worldwide. Down regulation of NM23, a metastasis suppressor gene, has been associated with several types of malignant cancer. Recently, effects of Imatinib mesylate, a first member of tyrosine kinases inhibitors, were indicated in research and treatment of different malignant tumors. Methods: Cell viability was quantitated by MTT assay after HepG2 cells exposure to Imatinib mesylate at various concentrations of 0, 1.56, 3.125, 6.25, 12.5, 25,50μM for 24 hours. Also, quantitative real time PCR technique was applied for the detection of NM23 gene expression in HepG2 cell line. Results: There was a dose dependent increase in the cytotoxicity effect of imatinib. The real time PCR results demonstrated that inhibitory effect of Imatinib mesylate on viability via up regulation of NM23 gene expression compared to GAPDH gene (internal control gene) in cancer cells. Conclusion: According to our findings, imatinib can modulate metastasis by enhancing Nm23 gene expression in human hepatocellular carcinoma (HepG2) cell line. PMID:28331561

Self-Assembled Tb3+ Complex Probe for Quantitative Analysis of ATP during Its Enzymatic Hydrolysis via Time-Resolved Luminescence in Vitro and in Vivo.

PubMed

Jung, Sung Ho; Kim, Ka Young; Lee, Ji Ha; Moon, Cheol Joo; Han, Noh Soo; Park, Su-Jin; Kang, Dongmin; Song, Jae Kyu; Lee, Shim Sung; Choi, Myong Yong; Jaworski, Justyn; Jung, Jong Hwa

2017-01-11

To more accurately assess the pathways of biological systems, a probe is needed that may respond selectively to adenosine triphosphate (ATP) for both in vitro and in vivo detection modes. We have developed a luminescence probe that can provide real-time information on the extent of ATP, ADP, and AMP by virtue of the luminescence and luminescence lifetime observed from a supramolecular polymer based on a C 3 symmetrical terpyridine complex with Tb 3+ (S1-Tb). The probe shows remarkable selective luminescence enhancement in the presence of ATP compared to other phosphate-displaying nucleotides including adenosine diphosphate (ADP), adenosine monophosphate (AMP), guanosine triphosphate (GTP), thymidine triphosphate (TTP), H 2 PO 4 - (Pi), and pyrophosphate (PPi). In addition, the time-resolved luminescence lifetime and luminescence spectrum of S1-Tb could facilitate the quantitative measurement of the exact amount of ATP and similarly ADP and AMP within living cells. The time-resolved luminescence lifetime of S1-Tb could also be used to quantitatively monitor the amount of ATP, ADP, and AMP in vitro following the enzymatic hydrolysis of ATP. The long luminescence lifetime, which was observed into the millisecond range, makes this S1-Tb-based probe particularly attractive for monitoring biological ATP levels in vivo, because any short lifetime background fluorescence arising from the complex molecular environment may be easily eliminated.

Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean.

PubMed

Demeke, Tigst; Ratnayaka, Indira; Phan, Anh

2009-01-01

The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

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

DTIC Science & Technology

1996-08-12

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

Sequence Optimized Real-Time RT-PCR Assay for Detection of Crimean-Congo Hemorrhagic Fever Virus

DTIC Science & Technology

2017-03-21

19-23]. Real-56 time reverse-transcription PCR remains the gold standard for quantitative , sensitive, and specific 57 detection of CCHFV; however...five-fold in two different series , and samples were run by real- time RT-PCR 116 in triplicate. The preliminary LOD was the lowest RNA dilution where...1 Sequence optimized real- time RT-PCR assay for detection of Crimean-Congo hemorrhagic fever 1 virus 2 3 JW Koehler1, KL Delp1, AT Hall1, SP

Identification of Lactobacillus delbrueckii and Streptococcus thermophilus Strains Present in Artisanal Raw Cow Milk Cheese Using Real-time PCR and Classic Plate Count Methods.

PubMed

Stachelska, Milena A

2017-12-04

The aim of this paper was to detect Lactobacillus delbrueckii and Streptococcus thermophilus using real-time quantitative PCR assay in 7-day ripening cheese produced from unpasteurised milk. Real-time quantitative PCR assays were designed to identify and enumerate the chosen species of lactic acid bacteria (LAB) in ripened cheese. The results of molecular quantification and classic bacterial enumeration showed a high level of similarity proving that DNA extraction was carried out in a proper way and that genomic DNA solutions were free of PCR inhibitors. These methods revealed the presence of L. delbrueckii and S. thermophilus. The real-time PCR enabled quantification with a detection of 101-103 CFU/g of product. qPCR-standard curves were linear over seven log units down to 101 copies per reaction; efficiencies ranged from 77.9% to 93.6%. Cheese samples were analysed with plate count method and qPCR in parallel. Compared with the classic plate count method, the newly developed qPCR method provided faster and species specific identification of two dairy LAB and yielded comparable quantitative results.

Real-time polymerase chain reaction analysis of MDM2 and CDK4 expression using total RNA from core-needle biopsies is useful for diagnosing adipocytic tumors

PubMed Central

2014-01-01

Background Diagnosing adipocytic tumors can be challenging because it is often difficult to morphologically distinguish between benign, intermediate and malignant adipocytic tumors, and other sarcomas that are histologically similar. Recently, a number of tumor-specific chromosome translocations and associated fusion genes have been identified in adipocytic tumors and atypical lipomatous tumors/well-differentiated liposarcomas (ALT/WDL), which have a supernumerary ring and/or giant chromosome marker with amplified sequences of the MDM2 and CDK4 genes. The purpose of this study was to investigate whether quantitative real-time polymerase chain reaction (PCR) could be used to amplify MDM2 and CDK4 from total RNA samples obtained from core-needle biopsy sections for the diagnosis of ALT/WDL. Methods A series of lipoma (n = 124) and ALT/WDL (n = 44) cases were analyzed for cytogenetic analysis and lipoma fusion genes, as well as for MDM2 and CDK4 expression by real-time PCR. Moreover, the expression of MDM2 and CDK4 in whole tissue sections was compared with that in core-needle biopsy sections of the same tumor in order to determine whether real-time PCR could be used to distinguish ALT/WDL from lipoma at the preoperative stage. Results In whole tissue sections, the medians for MDM2 and CDK4 expression in ALT/WDL were higher than those in the lipomas (P < 0.05). Moreover, karyotype subdivisions with rings and/or giant chromosomes had higher MDM2 and CDK4 expression levels compared to karyotypes with 12q13-15 rearrangements, other abnormal karyotypes, and normal karyotypes (P < 0.05). On the other hand, MDM2 and CDK4 expression levels in core-needle biopsy sections were similar to those in whole-tissue sections (MDM2: P = 0.6, CDK4: P = 0.8, Wilcoxon signed-rank test). Conclusion Quantitative real-time PCR of total RNA can be used to evaluate the MDM2 and CDK4 expression levels in core-needle biopsies and may be useful for distinguishing ALT/WDL from adipocytic tumors. Thus, total RNA from core-needle biopsy sections may have potential as a routine diagnostic tool for other tumors where gene overexpression is a feature of the tumor. PMID:24965044

[A quantitative real time polymerase chain reaction for detection of HBV covalently closed circular DNA in livers of the HBV infected patients].

PubMed

Wang, Mei-Rong; Qiu, Ning; Lu, Shi-Chun; Xiu, Dian-Rong; Yu, Jian-Guo; Li, Tong; Liu, Xue-En; Zhuang, Hui

2011-05-01

To establish and optimize a sensitive and specific quantitative real-time polymerase chain reaction (PCR) method for detection of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in liver tissue. Specific primers and probes were designed to detect HBV DNA (tDNA) and cccDNA. A series of plasmids (3.44 × 10(0) - 3.44 × 10(9) copies/µl) containing a full double-stranded copies of HBV genome (genotype C) were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma (HCC), 13 Chronic hepatitis B patients (CHB) and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase (PSAD) for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount (copies/cell) of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. The standard curves of real-time PCR with a linear range of 3.44 × 10(0) to 3.44 × 10(9) copies/µl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 10(0) copies/µl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031 copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 10(2) times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficient of variations on cycle threshold (Ct) were between 0.224% - 0.609%. A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies.

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

PubMed

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

2016-01-01

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

A Pilot Study of the Noninvasive Assessment of the Lung Microbiota as a Potential Tool for the Early Diagnosis of Ventilator-Associated Pneumonia

PubMed Central

Brady, Jacob S.; Romano-Keeler, Joann; Drake, Wonder P.; Norris, Patrick R.; Jenkins, Judith M.; Isaacs, Richard J.; Boczko, Erik M.

2015-01-01

BACKGROUND: Ventilator-associated pneumonia (VAP) remains a common complication in critically ill surgical patients, and its diagnosis remains problematic. Exhaled breath contains aerosolized droplets that reflect the lung microbiota. We hypothesized that exhaled breath condensate fluid (EBCF) in hygroscopic condenser humidifier/heat and moisture exchanger (HCH/HME) filters would contain bacterial DNA that qualitatively and quantitatively correlate with pathogens isolated from quantitative BAL samples obtained for clinical suspicion of pneumonia. METHODS: Forty-eight adult patients who were mechanically ventilated and undergoing quantitative BAL (n = 51) for suspected pneumonia in the surgical ICU were enrolled. Per protocol, patients fulfilling VAP clinical criteria undergo quantitative BAL bacterial culture. Immediately prior to BAL, time-matched HCH/HME filters were collected for study of EBCF by real-time polymerase chain reaction. Additionally, convenience samples of serially collected filters in patients with BAL-diagnosed VAP were analyzed. RESULTS: Forty-nine of 51 time-matched EBCF/BAL fluid samples were fully concordant (concordance > 95% by κ statistic) relative to identified pathogens and strongly correlated with clinical cultures. Regression analysis of quantitative bacterial DNA in paired samples revealed a statistically significant positive correlation (r = 0.85). In a convenience sample, qualitative and quantitative polymerase chain reaction analysis of serial HCH/HME samples for bacterial DNA demonstrated an increase in load that preceded the suspicion of pneumonia. CONCLUSIONS: Bacterial DNA within EBCF demonstrates a high correlation with BAL fluid and clinical cultures. Bacterial DNA within EBCF increases prior to the suspicion of pneumonia. Further study of this novel approach may allow development of a noninvasive tool for the early diagnosis of VAP. PMID:25474571

Evaluation of the Abbott RealTime HCV assay for quantitative detection of hepatitis C virus RNA.

PubMed

Michelin, Birgit D A; Muller, Zsofia; Stelzl, Evelyn; Marth, Egon; Kessler, Harald H

2007-02-01

The Abbott RealTime HCV assay for quantitative detection of HCV RNA has recently been introduced. In this study, the performance of the Abbott RealTime HCV assay was evaluated and compared to the COBAS AmpliPrep/COBAS TaqMan HCV test. Accuracy, linearity, interassay and intra-assay variations were determined, and a total of 243 routine clinical samples were investigated. When accuracy of the new assay was tested, the majority of results were found to be within +/-0.5 log(10) unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve up to 1.0 x 10(6)IU/ml. The interassay variation ranged from 15% to 32%, and the intra-assay variation ranged from 5% to 8%. When clinical samples were tested by the Abbott RealTime HCV assay and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HCV test, the results for 93% of all samples with positive results by both tests were found to be within +/-1.0 log(10) unit. The viral loads for all patients measured by the Abbott and Roche assays showed a high correlation (R(2)=0.93); quantitative results obtained by the Abbott assay were found to be lower than those obtained by the Roche assay. The Abbott RealTime HCV assay proved to be suitable for use in the routine diagnostic laboratory. The time to results was similar for both of the assays.

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae.

PubMed

Teste, Marie-Ange; Duquenne, Manon; François, Jean M; Parrou, Jean-Luc

2009-10-30

Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions. Since this assumption is very unlikely, a geometric averaging of multiple, carefully selected internal control genes is now strongly recommended for normalization to avoid this problem of expression variation of single reference genes. The aim of this work was to search for a set of reference genes for reliable gene expression analysis in Saccharomyces cerevisiae. From public microarray datasets, we selected potential reference genes whose expression remained apparently invariable during long-term growth on glucose. Using the algorithm geNorm, ALG9, TAF10, TFC1 and UBC6 turned out to be genes whose expression remained stable, independent of the growth conditions and the strain backgrounds tested in this study. We then showed that the geometric averaging of any subset of three genes among the six most stable genes resulted in very similar normalized data, which contrasted with inconsistent results among various biological samples when the normalization was performed with ACT1. Normalization with multiple selected genes was therefore applied to transcriptional analysis of genes involved in glycogen metabolism. We determined an induction ratio of 100-fold for GPH1 and 20-fold for GSY2 between the exponential phase and the diauxic shift on glucose. There was no induction of these two genes at this transition phase on galactose, although in both cases, the kinetics of glycogen accumulation was similar. In contrast, SGA1 expression was independent of the carbon source and increased by 3-fold in stationary phase. In this work, we provided a set of genes that are suitable reference genes for quantitative gene expression analysis by real-time RT-PCR in yeast biological samples covering a large panel of physiological states. In contrast, we invalidated and discourage the use of ACT1 as well as other commonly used reference genes (PDA1, TDH3, RDN18, etc) as internal controls for quantitative gene expression analysis in yeast.

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

PubMed

Daniel, Hubert Darius J; Fletcher, John G; Chandy, George M; Abraham, Priya

2009-01-01

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

Teachers' and Parental Attribution for School Performance of Ethnic Majority and Minority Children

ERIC Educational Resources Information Center

Wissink, Inge B.; de Haan, Mariette

2013-01-01

This study examines whether teachers' and parental attributions for children's school performance differ depending on the ethnic background of the child. Using both quantitative and qualitative methods, real-life attributions within 54 teacher-parent conversations (15 ethnic majority; 39 minority) were examined. The results indicated that,…

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

PubMed

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

2008-09-01

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

Real-time isothermal RNA amplification of toxic marine microalgae using preserved reagents on an integrated microfluidic platform.

PubMed

Tsaloglou, Maria-Nefeli; Laouenan, Florian; Loukas, Christos-Moritz; Monsalve, Lisandro Gabriel; Thanner, Christine; Morgan, Hywel; Ruano-López, Jesus M; Mowlem, Matthew C

2013-01-21

Quantitation of specific RNA sequences is a useful technique in marine biology that can elucidate cell abundance, speciation and viability, especially for early detection of harmful algal blooms. We are thus developing an integrated microfluidic system for cell concentration and lysis, RNA extraction/purification and quantitative RNA detection for environmental applications. The portable system is based on a microfluidic cartridge, or "lab-card", using a low-cost injection moulded device, with a laminated lid. Here we present real-time isothermal RNA amplification using reagent master-mixes preserved on-chip in a gel at 4 °C for up to eight months. We demonstrate quantitation by reference to an internal control in a competitive assay with 500 cell equivalents of the toxic microalga Karenia brevis. Annealing of primers, amplification at 41 °C and real-time fluorescence detection of the internal control and target using sequence-specific molecular beacons were all performed on-chip.

Multichannel series piezoelectric quartz crystal cell sensor for real time and quantitative monitoring of the living cell and assessment of cytotoxicity.

PubMed

Tong, Feifei; Lian, Yan; Zhou, Huang; Shi, Xiaohong; He, Fengjiao

2014-10-21

A new multichannel series piezoelectric quartz crystal (MSPQC) cell sensor for real time monitoring of living cells in vitro was reported in this paper. The constructed sensor was used successfully to monitor adhesion, spreading, proliferation, and apoptosis of MG63 osteosarcoma cells and investigate the effects of different concentrations of cobalt chloride on MG63 cells. Quantitative real time and dynamic cell analyses data were conducted using the MSPQC cell sensor. Compared with methods such as fluorescence staining and morphology observation by microscopy, the MSPQC cell sensor is noninvasive, label free, simple, cheap, and capable of online monitoring. It can automatically record the growth status of cells and quantitatively evaluate cell proliferation and the apoptotic response to drugs. It will be a valuable detection and analysis tool for the acquisition of cellular level information and is anticipated to have application in the field of cell biology research or cytotoxicity testing in the future.

Elimination of autofluorescence background from fluorescence tissue images by use of time-gated detection and the AzaDiOxaTriAngulenium (ADOTA) fluorophore

PubMed Central

Rich, Ryan M.; Stankowska, Dorota L.; Maliwal, Badri P.; Sørensen, Thomas Just; Laursen, Bo W.; Krishnamoorthy, Raghu R.; Gryczynski, Zygmunt; Borejdo, Julian

2013-01-01

Sample autofluorescence (fluorescence of inherent components of tissue and fixative-induced fluorescence) is a significant problem in direct imaging of molecular processes in biological samples. A large variety of naturally occurring fluorescent components in tissue results in broad emission that overlaps the emission of typical fluorescent dyes used for tissue labeling. In addition, autofluorescence is characterized by complex fluorescence intensity decay composed of multiple components whose lifetimes range from sub-nanoseconds to a few nanoseconds. For these reasons, the real fluorescence signal of the probe is difficult to separate from the unwanted autofluorescence. Here we present a method for reducing the autofluorescence problem by utilizing an azadioxatriangulenium (ADOTA) dye with a fluorescence lifetime of approximately 15 ns, much longer than those of most of the components of autofluorescence. A probe with such a long lifetime enables us to use time-gated intensity imaging to separate the signal of the targeting dye from the autofluorescence. We have shown experimentally that by discarding photons detected within the first 20 ns of the excitation pulse, the signal-to-background ratio is improved fivefold. This time-gating eliminates over 96 % of autofluorescence. Analysis using a variable time-gate may enable quantitative determination of the bound probe without the contributions from the background. PMID:23254457

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

PubMed

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

2016-07-01

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

Validation of PCR methods for quantitation of genetically modified plants in food.

PubMed

Hübner, P; Waiblinger, H U; Pietsch, K; Brodmann, P

2001-01-01

For enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients, quantitative detection methods such as quantitative competitive (QC-PCR) and real-time PCR are applied by official food control laboratories. The experiences of 3 European food control laboratories in validating such methods were compared to describe realistic performance characteristics of quantitative PCR detection methods. The limit of quantitation (LOQ) of GMO-specific, real-time PCR was experimentally determined to reach 30-50 target molecules, which is close to theoretical prediction. Starting PCR with 200 ng genomic plant DNA, the LOQ depends primarily on the genome size of the target plant and ranges from 0.02% for rice to 0.7% for wheat. The precision of quantitative PCR detection methods, expressed as relative standard deviation (RSD), varied from 10 to 30%. Using Bt176 corn containing test samples and applying Bt176 specific QC-PCR, mean values deviated from true values by -7to 18%, with an average of 2+/-10%. Ruggedness of real-time PCR detection methods was assessed in an interlaboratory study analyzing commercial, homogeneous food samples. Roundup Ready soybean DNA contents were determined in the range of 0.3 to 36%, relative to soybean DNA, with RSDs of about 25%. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are suggested. Because quantitative GMO detection methods measure GMO contents of samples in relation to reference material (calibrants), high priority must be given to international agreements and standardization on certified reference materials.

Real-time detection of small and dim moving objects in IR video sequences using a robust background estimator and a noise-adaptive double thresholding

NASA Astrophysics Data System (ADS)

Zingoni, Andrea; Diani, Marco; Corsini, Giovanni

2016-10-01

We developed an algorithm for automatically detecting small and poorly contrasted (dim) moving objects in real-time, within video sequences acquired through a steady infrared camera. The algorithm is suitable for different situations since it is independent of the background characteristics and of changes in illumination. Unlike other solutions, small objects of any size (up to single-pixel), either hotter or colder than the background, can be successfully detected. The algorithm is based on accurately estimating the background at the pixel level and then rejecting it. A novel approach permits background estimation to be robust to changes in the scene illumination and to noise, and not to be biased by the transit of moving objects. Care was taken in avoiding computationally costly procedures, in order to ensure the real-time performance even using low-cost hardware. The algorithm was tested on a dataset of 12 video sequences acquired in different conditions, providing promising results in terms of detection rate and false alarm rate, independently of background and objects characteristics. In addition, the detection map was produced frame by frame in real-time, using cheap commercial hardware. The algorithm is particularly suitable for applications in the fields of video-surveillance and computer vision. Its reliability and speed permit it to be used also in critical situations, like in search and rescue, defence and disaster monitoring.

Real-Time Airborne Gamma-Ray Background Estimation Using NASVD with MLE and Radiation Transport for Calibration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kulisek, Jonathan A.; Schweppe, John E.; Stave, Sean C.

2015-06-01

Helicopter-mounted gamma-ray detectors can provide law enforcement officials the means to quickly and accurately detect, identify, and locate radiological threats over a wide geographical area. The ability to accurately distinguish radiological threat-generated gamma-ray signatures from background gamma radiation in real time is essential in order to realize this potential. This problem is non-trivial, especially in urban environments for which the background may change very rapidly during flight. This exacerbates the challenge of estimating background due to the poor counting statistics inherent in real-time airborne gamma-ray spectroscopy measurements. To address this, we have developed a new technique for real-time estimation ofmore » background gamma radiation from aerial measurements. This method is built upon on the noise-adjusted singular value decomposition (NASVD) technique that was previously developed for estimating the potassium (K), uranium (U), and thorium (T) concentrations in soil post-flight. The method can be calibrated using K, U, and T spectra determined from radiation transport simulations along with basis functions, which may be determined empirically by applying maximum likelihood estimation (MLE) to previously measured airborne gamma-ray spectra. The method was applied to both measured and simulated airborne gamma-ray spectra, with and without man-made radiological source injections. Compared to schemes based on simple averaging, this technique was less sensitive to background contamination from the injected man-made sources and may be particularly useful when the gamma-ray background frequently changes during the course of the flight.« less

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

PubMed

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

2011-01-01

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

genRE: A Method to Extend Gridded Precipitation Climatology Data Sets in Near Real-Time for Hydrological Forecasting Purposes

NASA Astrophysics Data System (ADS)

van Osnabrugge, B.; Weerts, A. H.; Uijlenhoet, R.

2017-11-01

To enable operational flood forecasting and drought monitoring, reliable and consistent methods for precipitation interpolation are needed. Such methods need to deal with the deficiencies of sparse operational real-time data compared to quality-controlled offline data sources used in historical analyses. In particular, often only a fraction of the measurement network reports in near real-time. For this purpose, we present an interpolation method, generalized REGNIE (genRE), which makes use of climatological monthly background grids derived from existing gridded precipitation climatology data sets. We show how genRE can be used to mimic and extend climatological precipitation data sets in near real-time using (sparse) real-time measurement networks in the Rhine basin upstream of the Netherlands (approximately 160,000 km2). In the process, we create a 1.2 × 1.2 km transnational gridded hourly precipitation data set for the Rhine basin. Precipitation gauge data are collected, spatially interpolated for the period 1996-2015 with genRE and inverse-distance squared weighting (IDW), and then evaluated on the yearly and daily time scale against the HYRAS and EOBS climatological data sets. Hourly fields are compared qualitatively with RADOLAN radar-based precipitation estimates. Two sources of uncertainty are evaluated: station density and the impact of different background grids (HYRAS versus EOBS). The results show that the genRE method successfully mimics climatological precipitation data sets (HYRAS/EOBS) over daily, monthly, and yearly time frames. We conclude that genRE is a good interpolation method of choice for real-time operational use. genRE has the largest added value over IDW for cases with a low real-time station density and a high-resolution background grid.

High-Sensitivity Real-Time Imaging of Dual Protein-Protein Interactions in Living Subjects Using Multicolor Luciferases

PubMed Central

Hida, Naoki; Awais, Muhammad; Takeuchi, Masaki; Ueno, Naoto; Tashiro, Mayuri; Takagi, Chiyo; Singh, Tanuja; Hayashi, Makoto; Ohmiya, Yoshihiro; Ozawa, Takeaki

2009-01-01

Networks of protein-protein interactions play key roles in numerous important biological processes in living subjects. An effective methodology to assess protein-protein interactions in living cells of interest is protein-fragment complement assay (PCA). Particularly the assays using fluorescent proteins are powerful techniques, but they do not directly track interactions because of its irreversibility or the time for chromophore formation. By contrast, PCAs using bioluminescent proteins can overcome these drawbacks. We herein describe an imaging method for real-time analysis of protein-protein interactions using multicolor luciferases with different spectral characteristics. The sensitivity and signal-to-background ratio were improved considerably by developing a carboxy-terminal fragment engineered from a click beetle luciferase. We demonstrate its utility in spatiotemporal characterization of Smad1–Smad4 and Smad2–Smad4 interactions in early developing stages of a single living Xenopus laevis embryo. We also describe the value of this method by application of specific protein-protein interactions in cell cultures and living mice. This technique supports quantitative analyses and imaging of versatile protein-protein interactions with a selective luminescence wavelength in opaque or strongly auto-fluorescent living subjects. PMID:19536355

Reviewing effectiveness of ankle assessment techniques for use in robot-assisted therapy.

PubMed

Zhang, Mingming; Davies, T Claire; Zhang, Yanxin; Xie, Shane

2014-01-01

This article provides a comprehensive review of studies that investigated ankle assessment techniques to better understand those that can be used in the real-time monitoring of rehabilitation progress for implementation in conjunction with robot-assisted therapy. Seventy-six publications published between January 1980 and August 2013 were selected based on eight databases. They were divided into two main categories (16 qualitative and 60 quantitative studies): 13 goniometer studies, 18 dynamometer studies, and 29 studies about innovative techniques. A total of 465 subjects participated in the 29 quantitative studies of innovative measurement techniques that may potentially be integrated in a real-time monitoring device, of which 19 studies included less than 10 participants. Results show that qualitative ankle assessment methods are not suitable for real-time monitoring in robot-assisted therapy, though they are reliable for certain patients, while the quantitative methods show great potential. The majority of quantitative techniques are reliable in measuring ankle kinematics and kinetics but are usually available only for use in the sagittal plane. Limited studies determine kinematics and kinetics in all three planes (sagittal, transverse, and frontal) where motions of the ankle joint and the subtalar joint actually occur.

Comparison of two quantitative fit-test methods using N95 filtering facepiece respirators.

PubMed

Sietsema, Margaret; Brosseau, Lisa M

2016-08-01

Current regulations require annual fit testing before an employee can wear a respirator during work activities. The goal of this research is to determine whether respirator fit measured with two TSI Portacount instruments simultaneously sampling ambient particle concentrations inside and outside of the respirator facepiece is similar to fit measured during an ambient aerosol condensation nuclei counter quantitative fit test. Sixteen subjects (ten female; six male) were recruited for a range of facial sizes. Each subject donned an N95 filtering facepiece respirator, completed two fit tests in random order (ambient aerosol condensation nuclei counter quantitative fit test and two-instrument real-time fit test) without removing or adjusting the respirator between tests. Fit tests were compared using Spearman's rank correlation coefficients. The real-time two-instrument method fit factors were similar to those measured with the single-instrument quantitative fit test. The first four exercises were highly correlated (r > 0.7) between the two protocols. Respirator fit was altered during the talking or grimace exercise, both of which involve facial movements that could dislodge the facepiece. Our analyses suggest that the new real-time two-instrument methodology can be used in future studies to evaluate fit before and during work activities.

Tendency for interlaboratory precision in the GMO analysis method based on real-time PCR.

PubMed

Kodama, Takashi; Kurosawa, Yasunori; Kitta, Kazumi; Naito, Shigehiro

2010-01-01

The Horwitz curve estimates interlaboratory precision as a function only of concentration, and is frequently used as a method performance criterion in food analysis with chemical methods. The quantitative biochemical methods based on real-time PCR require an analogous criterion to progressively promote method validation. We analyzed the tendency of precision using a simplex real-time PCR technique in 53 collaborative studies of seven genetically modified (GM) crops. Reproducibility standard deviation (SR) and repeatability standard deviation (Sr) of the genetically modified organism (GMO) amount (%) was more or less independent of GM crops (i.e., maize, soybean, cotton, oilseed rape, potato, sugar beet, and rice) and evaluation procedure steps. Some studies evaluated whole steps consisting of DNA extraction and PCR quantitation, whereas others focused only on the PCR quantitation step by using DNA extraction solutions. Therefore, SR and Sr for GMO amount (%) are functions only of concentration similar to the Horwitz curve. We proposed S(R) = 0.1971C 0.8685 and S(r) = 0.1478C 0.8424, where C is the GMO amount (%). We also proposed a method performance index in GMO quantitative methods that is analogous to the Horwitz Ratio.

Avian influenza virus detection and quantitation by real-time RT-PCR

USDA-ARS?s Scientific Manuscript database

Real-time RT-PCR (rRT-PCR) has been used for avian influenza virus (AIV) detection since the early 2000’s for routine surveillance, during outbreaks and for research. Some of the advantages of rRT-PCR are: high sensitivity, high specificity, rapid time-to-result, scalability, cost, and its inherentl...

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

PubMed Central

Cankar, Katarina; Štebih, Dejan; Dreo, Tanja; Žel, Jana; Gruden, Kristina

2006-01-01

Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. Conclusion The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR. PMID:16907967

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

PubMed

Provenzano, Maurizio; Mocellin, Simone

2007-01-01

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

Quantitative real-time in vivo detection of magnetic nanoparticles by their nonlinear magnetization

NASA Astrophysics Data System (ADS)

Nikitin, M. P.; Torno, M.; Chen, H.; Rosengart, A.; Nikitin, P. I.

2008-04-01

A novel method of highly sensitive quantitative detection of magnetic nanoparticles (MP) in biological tissues and blood system has been realized and tested in real time in vivo experiments. The detection method is based on nonlinear magnetic properties of MP and the related device can record a very small relative variation of nonlinear magnetic susceptibility up to 10-8 at room temperature, providing sensitivity of several nanograms of MP in 0.1ml volume. Real-time quantitative in vivo measurements of dynamics of MP concentration in blood flow have been performed. A catheter that carried the blood flow of a rat passed through the measuring device. After an MP injection, the quantity of MP in the circulating blood was continuously recorded. The method has also been used to evaluate the MP distribution between rat's organs. Its sensitivity was compared with detection of the radioactive MP based on isotope of Fe59. The comparison of magnetic and radioactive signals in the rat's blood and organ samples demonstrated similar sensitivity for both methods. However, the proposed magnetic method is much more convenient as it is safe, less expensive, and provides real-time measurements in vivo. Moreover, the sensitivity of the method can be further improved by optimization of the device geometry.

Linking Laboratory Experiences to the Real World: The Extraction of Octylphenoxyacetic Acid from Water

ERIC Educational Resources Information Center

Loyo-Rosales, Jorge E.; Torrents, Alba; Rosales-Rivera, Georgina C.; Rice, Clifford C.

2006-01-01

Several chemical concepts to the extraction of a water pollutant OPC (octylphenoxyacetic acid) is presented. As an introduction to the laboratory experiment, a discussion on endocrine disrupters is conducted to familiarize the student with the background of the experiment and to explain the need for the extraction and quantitation of the OPC which…

Surrogate analysis and index developer (SAID) tool and real-time data dissemination utilities

USGS Publications Warehouse

Domanski, Marian M.; Straub, Timothy D.; Wood, Molly S.; Landers, Mark N.; Wall, Gary R.; Brady, Steven J.

2015-01-01

The use of acoustic and other parameters as surrogates for suspended-sediment concentrations (SSC) in rivers has been successful in multiple applications across the Nation. Critical to advancing the operational use of surrogates are tools to process and evaluate the data along with the subsequent development of regression models from which real-time sediment concentrations can be made available to the public. Recent developments in both areas are having an immediate impact on surrogate research, and on surrogate monitoring sites currently in operation. The Surrogate Analysis and Index Developer (SAID) standalone tool, under development by the U.S. Geological Survey (USGS), assists in the creation of regression models that relate response and explanatory variables by providing visual and quantitative diagnostics to the user. SAID also processes acoustic parameters to be used as explanatory variables for suspended-sediment concentrations. The sediment acoustic method utilizes acoustic parameters from fixed-mount stationary equipment. The background theory and method used by the tool have been described in recent publications, and the tool also serves to support sediment-acoustic-index methods being drafted by the multi-agency Sediment Acoustic Leadership Team (SALT), and other surrogate guidelines like USGS Techniques and Methods 3-C4 for turbidity and SSC. The regression models in SAID can be used in utilities that have been developed to work with the USGS National Water Information System (NWIS) and for the USGS National Real-Time Water Quality (NRTWQ) Web site. The real-time dissemination of predicted SSC and prediction intervals for each time step has substantial potential to improve understanding of sediment-related water-quality and associated engineering and ecological management decisions.

Use of real-time quantitative PCR to detect Chlamydophila felis infection.

PubMed

Helps, C; Reeves, N; Tasker, S; Harbour, D

2001-07-01

A real-time PCR assay was developed to detect and quantify Chlamydophila felis infection of cats. The assay uses a molecular beacon to specifically identify the major outer membrane protein gene, is highly reproducible, and is able to detect fewer than 10 genomic copies.

Preliminary Cost Benefit Assessment of Systems for Detection of Hazardous Weather. Volume I,

DTIC Science & Technology

1981-07-01

not be sufficient for adequate stream flow forecasting , it has important potential for real - time flash flood warning. This was illustrated by the 1977...provide a finer spatial resolution of the gridded data. See Table 9. 42 The results of a demonstration of the real - time capabilities of a radar-man system ...detailed real time measurement capabilities and scope for quantitative forecasting is most likely to provide the degree of lead time required if maximum

Transportable and vibration-free full-field low-coherent quantitative phase microscope

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Yamada, Hidenao; Goto, Kentaro; Matsui, Hisayuki; Yasuhiko, Osamu; Ueda, Yukio

2018-02-01

We developed a transportable Linnik-type full-field low-coherent quantitative phase microscope that is able to compensate for optical path length (OPL) disturbance due to environmental mechanical noises. Though two-beam interferometers such as Linnik ones suffer from unstable OPL difference, we overcame this problem with a mechanical feedback system based on digital signal-processing that controls the OPL difference in sub-nanometer resolution precisely with a feedback bandwidth of 4 kHz. The developed setup has a footprint of 200 mm by 200 mm, a height of 500 mm, and a weight of 4.5 kilograms. In the transmission imaging mode, cells were cultured on a reflection-enhanced glass-bottom dish, and we obtained interference images sequentially while performing stepwise quarter-wavelength phase-shifting. Real-time image processing, including retrieval of the unwrapped phase from interference images and its background correction, along with the acquisition of interference images, was performed on a laptop computer. Emulation of the phase contrast (PhC) images and the differential interference contrast (DIC) images was also performed in real time. Moreover, our setup was applied for full-field cell membrane imaging in the reflection mode, where the cells were cultured on an anti-reflection (AR)-coated glass-bottom dish. The phase and intensity of the light reflected by the membrane revealed the outer shape of the cells independent of the refractive index. In this paper, we show imaging results on cultured cells in both transmission and reflection modes.

[Comparative analysis of real-time quantitative PCR-Sanger sequencing method and TaqMan probe method for detection of KRAS/BRAF mutation in colorectal carcinomas].

PubMed

Zhang, Xun; Wang, Yuehua; Gao, Ning; Wang, Jinfen

2014-02-01

To compare the application values of real-time quantitative PCR-Sanger sequencing and TaqMan probe method in the detection of KRAS and BRAF mutations, and to correlate KRAS/BRAF mutations with the clinicopathological characteristics in colorectal carcinomas. Genomic DNA of the tumor cells was extracted from formalin fixed paraffin embedded (FFPE) tissue samples of 344 colorectal carcinomas by microdissection. Real-time quantitative PCR-Sanger sequencing and TaqMan probe method were performed to detect the KRAS/BRAF mutations. The frequency and types of KRAS/BRAF mutations, clinicopathological characteristics and survival time were analyzed. KRAS mutations were detected in 39.8% (137/344) and 38.7% (133/344) of 344 colorectal carcinomas by using real-time quantitative PCR-Sanger sequencing and TaqMan probe method, respectively. BRAF mutation was detected in 4.7% (16/344) and 4.1% (14/344), respectively. There was no significant correlation between the two methods. The frequency of the KRAS mutation in female was higher than that in male (P < 0.05). The frequency of the BRAF mutation in colon was higher than that in rectum. The frequency of the BRAF mutation in stage III-IV cases was higher than that in stageI-II cases. The frequency of the BRAF mutation in signet ring cell carcinoma was higher than that in mucinous carcinoma and nonspecific adenocarcinoma had the lowest mutation rate. The frequency of the BRAF mutation in grade III cases was higher than that in grade II cases (P < 0.05). The overall concordance for the two methods of KRAS/BRAF mutation detection was 98.8% (kappa = 0.976). There was statistic significance between BRAF and KRAS mutations for the survival time of colorectal carcinomas (P = 0.039). There were no statistic significance between BRAF mutation type and BRAF/KRAS wild type (P = 0.058). (1) Compared with real-time quantitative PCR-Sanger sequencing, TaqMan probe method is better with regard to handling time, efficiency, repeatability, cost and equipment. (2) The frequency of the KRAS mutation is correlated with gender. BRAF mutation is correlated with primary tumor site, TNM stage, histological types and histological grades.(3) BRAF gene mutation is an independent prognostic marker for colorectal carcinomas.

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

PubMed

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.

Real-time PCR: Advanced technologies and applications

USDA-ARS?s Scientific Manuscript database

This book brings together contributions from 20 experts in the field of PCR, providing a broad perspective of the applications of quantitative real-time PCR (qPCR). The editors state in the preface that the aim is to provide detailed insight into underlying principles and methods of qPCR to provide ...

Inter-laboratory Comparison of Real-time PCR Methods for Quantification of General Fecal Indicator Bacteria

EPA Science Inventory

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized prot...

Interlaboratory Comparison of Real-time PCR Protocols for Quantification of General Fecal Indicator Bacteria

EPA Science Inventory

The application of quantitative real-time PCR (qPCR) technologies for the rapid identification of fecal bacteria in environmental waters is being considered for use as a national water quality metric in the United States. The transition from research tool to a standardized proto...

QUANTIFICATION OF ENTEROVIRUS AND HEPATITIS A VIRUSES IN WELLS AND SPRINGS IN EAST TENNESSEE USING REAL-TIME REVERSE TRANSCIPTION PCR

EPA Science Inventory

This project involves development, validation testing and application of a fast, efficient method of quantitatively measuring occurrence and concentration of common human viral pathogens, enterovirus and hepatitis A virus, in ground water samples using real-time reverse transcrip...

Use of the Genomic Subtractive Hybridization Technique To Develop a Real-Time PCR Assay for Quantitative Detection of Prevotella spp. in Oral Biofilm Samples

PubMed Central

Nagashima, Shiori; Yoshida, Akihiro; Suzuki, Nao; Ansai, Toshihiro; Takehara, Tadamichi

2005-01-01

Genomic subtractive hybridization was used to design Prevotella nigrescens-specific primers and TaqMan probes. Based on this technique, a TaqMan real-time PCR assay was developed for quantifying four oral black-pigmented Prevotella species. The combination of real-time PCR and genomic subtractive hybridization is useful for preparing species-specific primer-probe sets for closely related species. PMID:15956428

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

PubMed

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

2017-08-01

HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar ® HHV-6 PCR Kit. The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar ® HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log 10 copies/ml and a coefficient of determination (R 2 ) of 0.999 for the in-house assay. A dilution series demonstrated a limit of detection and a limit of quantification of 1.7 log 10 and 2 log 10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar ® HHV-6 PCR Kit (R 2 =0.926; P<0.0001), with an average bias of -0.24 log 10 copies/ml. The in-house developed HHV-6 qPCR method is a sensitive and reliable assay with lower cost for the detection and quantification of HHV-6 DNA when compared to the RealStar ® HHV-6 PCR Kit. Copyright © 2017 Elsevier B.V. All rights reserved.

Development and comparative evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of West Nile virus in human patients.

PubMed

Kumar, Jyoti S; Saxena, Divyasha; Parida, Manmohan

2014-01-01

The recent outbreaks of West Nile Virus (WNV) in the Northeastern American continents and other regions of the world have made it essential to develop an efficient protocol for surveillance of WN virus. Nucleic acid based techniques like, RT-PCR have the advantage of sensitivity, specificity and rapidity. A one step single tube Env gene specific real-time RT-PCR was developed for early and reliable clinical diagnosis of WNV infection in clinical samples. The applicability of this assay for clinical diagnosis was validated with 105 suspected acute-phase serum and plasma samples from the recent epidemic of mysterious fever in Tamil Nadu, India in 2009-10. The comparative evaluation revealed the higher sensitivity of real-time RT-PCR assay by picking up 4 additional samples with low copy number of template in comparison to conventional RT-PCR. All the real-time positive samples further confirmed by CDC reported TaqMan real-time RT-PCR and quantitative real-time RT-PCR assays for the simultaneous detection of WNV lineage 1 and 2 strains. The quantitation of the viral load samples was done using a standard curve. These findings demonstrated that the assay has the potential usefulness for clinical diagnosis due to detection and quantification of WNV in acute-phase patient serum samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

Prevalence of Propionibacterium acnes in Intervertebral Discs of Patients Undergoing Lumbar Microdiscectomy: A Prospective Cross-Sectional Study

PubMed Central

Capoor, Manu N.; Ruzicka, Filip; Machackova, Tana; Jancalek, Radim; Smrcka, Martin; Schmitz, Jonathan E.; Hermanova, Marketa; Sana, Jiri; Michu, Elleni; Baird, John C.; Ahmed, Fahad S.; Maca, Karel; Lipina, Radim; Alamin, Todd F.; Coscia, Michael F.; Stonemetz, Jerry L.; Witham, Timothy; Ehrlich, Garth D.; Gokaslan, Ziya L.; Mavrommatis, Konstantinos; Birkenmaier, Christof; Fischetti, Vincent A.; Slaby, Ondrej

2016-01-01

Background The relationship between intervertebral disc degeneration and chronic infection by Propionibacterium acnes is controversial with contradictory evidence available in the literature. Previous studies investigating these relationships were under-powered and fraught with methodical differences; moreover, they have not taken into consideration P. acnes’ ability to form biofilms or attempted to quantitate the bioburden with regard to determining bacterial counts/genome equivalents as criteria to differentiate true infection from contamination. The aim of this prospective cross-sectional study was to determine the prevalence of P. acnes in patients undergoing lumbar disc microdiscectomy. Methods and Findings The sample consisted of 290 adult patients undergoing lumbar microdiscectomy for symptomatic lumbar disc herniation. An intraoperative biopsy and pre-operative clinical data were taken in all cases. One biopsy fragment was homogenized and used for quantitative anaerobic culture and a second was frozen and used for real-time PCR-based quantification of P. acnes genomes. P. acnes was identified in 115 cases (40%), coagulase-negative staphylococci in 31 cases (11%) and alpha-hemolytic streptococci in 8 cases (3%). P. acnes counts ranged from 100 to 9000 CFU/ml with a median of 400 CFU/ml. The prevalence of intervertebral discs with abundant P. acnes (≥ 1x103 CFU/ml) was 11% (39 cases). There was significant correlation between the bacterial counts obtained by culture and the number of P. acnes genomes detected by real-time PCR (r = 0.4363, p<0.0001). Conclusions In a large series of patients, the prevalence of discs with abundant P. acnes was 11%. We believe, disc tissue homogenization releases P. acnes from the biofilm so that they can then potentially be cultured, reducing the rate of false-negative cultures. Further, quantification study revealing significant bioburden based on both culture and real-time PCR minimize the likelihood that observed findings are due to contamination and supports the hypothesis P. acnes acts as a pathogen in these cases of degenerative disc disease. PMID:27536784

Estimating travel reduction associated with the use of telemedicine by patients and healthcare professionals: proposal for quantitative synthesis in a systematic review

PubMed Central

2011-01-01

Background A major benefit offered by telemedicine is the avoidance of travel, by patients, their carers and health care professionals. Unfortunately, there is very little published information about the extent of avoided travel. We propose to undertake a systematic review of literature which reports credible data on the reductions in travel associated with the use of telemedicine. Method The conventional approach to quantitative synthesis of the results from multiple studies is to conduct a meta analysis. However, too much heterogeneity exists between available studies to allow a meaningful meta analysis of the avoided travel when telemedicine is used across all possible settings. We propose instead to consider all credible evidence on avoided travel through telemedicine by fitting a linear model which takes into account the relevant factors in the circumstances of the studies performed. We propose the use of stepwise multiple regression to identify which factors are significant. Discussion Our proposed approach is illustrated by the example of teledermatology. In a preliminary review of the literature we found 20 studies in which the percentage of avoided travel through telemedicine could be inferred (a total of 5199 patients). The mean percentage avoided travel reported in the 12 store-and-forward studies was 43%. In the 7 real-time studies and in a single study with a hybrid technique, 70% of the patients avoided travel. A simplified model based on the modality of telemedicine employed (i.e. real-time or store and forward) explained 29% of the variance. The use of store and forward teledermatology alone was associated with 43% of avoided travel. The increase in the proportion of patients who avoided travel (25%) when real-time telemedicine was employed was significant (P = 0.014). Service planners can use this information to weigh up the costs and benefits of the two approaches. PMID:21824388

BK virus has tropism for human salivary gland cells in vitro: Implications for transmission

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeffers, Liesl K.; Madden, Vicki; Webster-Cyriaque, Jennifer, E-mail: jennifer@med.unc.ed

Background: In this study, it was determined that BKV is shed in saliva and an in vitro model system was developed whereby BKV can productively infect both submandibular (HSG) and parotid (HSY) salivary gland cell lines. Results: BKV was detected in oral fluids using quantitative real-time PCR (QRTPCR). BKV infection was determined using quantitative RT-PCR, immunofluorescence and immunoblotting assays. The infectivity of BKV was inhibited by pre-incubation of the virus with gangliosides that saturated the major capsid protein, VP1, halting receptor mediated BKV entry into salivary gland cells. Examination of infected cultures by transmission electron microscopy revealed 45-50 nm BKmore » virions clearly visible within the cells. Subsequent to infection, encapsidated BK virus was detected in the supernatant. Conclusion: We thus demonstrated that BKV was detected in oral fluids and that BK infection and replication occur in vitro in salivary gland cells. These data collectively suggest the potential for BKV oral route of transmission and oral pathogenesis.« less

Real time non invasive imaging of fatty acid uptake in vivo

PubMed Central

Henkin, Amy H.; Cohen, Allison S.; Dubikovskaya, Elena A.; Park, Hyo Min; Nikitin, Gennady F.; Auzias, Mathieu G.; Kazantzis, Melissa; Bertozzi, Carolyn R.; Stahl, Andreas

2012-01-01

Detection and quantification of fatty acid fluxes in animal model systems following physiological, pathological, or pharmacological challenges is key to our understanding of complex metabolic networks as these macronutrients also activate transcription factors and modulate signaling cascades including insulin-sensitivity. To enable non-invasive, real-time, spatiotemporal quantitative imaging of fatty acid fluxes in animals, we created a bioactivatable molecular imaging probe based on long-chain fatty acids conjugated to a reporter molecule (luciferin). We show that this probe faithfully recapitulates cellular fatty acid uptake and can be used in animal systems as a valuable tool to localize and quantitate in real-time lipid fluxes such as intestinal fatty acid absorption and brown adipose tissue activation. This imaging approach should further our understanding of basic metabolic processes and pathological alterations in multiple disease models. PMID:22928772

An integrated microfluidic sensor for real-time detection of RNA in seawater using preserved reagents

NASA Astrophysics Data System (ADS)

Tsaloglou, M.-N.; Loukas, C. M.; Ruano-López, J. M.; Morgan, H.; Mowlem, M. C.

2012-04-01

Quantitation of RNA sequences coding either for key metabolic proteins or highly conserved ribosomal subunits can provide insight on cell abundance, speciation and viability. Nucleic sequence-based amplification (NASBA) is an isothermal alternative to traditional nucleic acid amplification methods, such as quantitative PCR. We present here an integrated microfluidic sensor for cell concentration and lysis, RNA extraction/purification and quantitative RNA detection for environmental applications. The portable system uses pre-loaded reagents, stored as a gel on a disposable microfluidic cartridge, which is manufactured using low-cost injection moulding. The NASBA reaction is monitored real-time using a bespoke control unit which includes: an external fluorescence detector, three peristaltic micro-pumps, two heaters and temperature sensors, a battery, seven pin actuated micro-motors (or valve actuators), and an automatic cartridge insertion mechanism. The system has USB connectivity and none of the expensive components require replacing between reactions. Long-term storage of reagents is critically important for any diagnostic tool that will be used in the field, whether for medical or environmental analysis and has not been previously demonstrated for NASBA reagents on-chip. We have shown effective amplification, for as little as 500 cells of the toxic microalga Karenia brevis using reagents which had been preserved as a gel for 45 days. This is the first reported real-time isothermal RNA amplification using with on-chip preservation. Annealing of primers, amplification at 41 °C and real-time fluorescence detection using, also for the first time, an internal control and sequence-specific molecular beacons was all performed on our microfluidic sensor. Our results show excellent promise as a future quantitative tool of in situ phytoplankton analysis and other environmental applications, where long-term reagent storage and low power consumption is essential.

Comparison of Two Commercial Automated Nucleic Acid Extraction and Integrated Quantitation Real-Time PCR Platforms for the Detection of Cytomegalovirus in Plasma

PubMed Central

Tsai, Huey-Pin; Tsai, You-Yuan; Lin, I-Ting; Kuo, Pin-Hwa; Chen, Tsai-Yun; Chang, Kung-Chao; Wang, Jen-Ren

2016-01-01

Quantitation of cytomegalovirus (CMV) viral load in the transplant patients has become a standard practice for monitoring the response to antiviral therapy. The cut-off values of CMV viral load assays for preemptive therapy are different due to the various assay designs employed. To establish a sensitive and reliable diagnostic assay for preemptive therapy of CMV infection, two commercial automated platforms including m2000sp extraction system integrated the Abbott RealTime (m2000rt) and the Roche COBAS AmpliPrep for extraction integrated COBAS Taqman (CAP/CTM) were evaluated using WHO international CMV standards and 110 plasma specimens from transplant patients. The performance characteristics, correlation, and workflow of the two platforms were investigated. The Abbott RealTime assay correlated well with the Roche CAP/CTM assay (R2 = 0.9379, P<0.01). The Abbott RealTime assay exhibited higher sensitivity for the detection of CMV viral load, and viral load values measured with Abbott RealTime assay were on average 0.76 log10 IU/mL higher than those measured with the Roche CAP/CTM assay (P<0.0001). Workflow analysis on a small batch size at one time, using the Roche CAP/CTM platform had a shorter hands-on time than the Abbott RealTime platform. In conclusion, these two assays can provide reliable data for different purpose in a clinical virology laboratory setting. PMID:27494707

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

PubMed

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

2011-08-01

Analysis of changes in recipient and donor hematopoietic cell origin is extremely useful to monitor the effect of hematopoietic stem cell transplantation (HSCT) and sequential adoptive immunotherapy by donor lymphocyte infusions. We developed a sensitive, reliable and rapid real-time PCR method based on sequence polymorphism systems to quantitatively assess the hematopoietic chimerism after HSCT. A panel of 29 selected sequence polymorphism (SP) markers was screened by real-time PCR in 101 HSCT patients with leukemia and other hematological diseases. The chimerism kinetics of bone marrow samples of 8 HSCT patients in remission and relapse situations were followed longitudinally. Recipient genotype discrimination was possible in 97.0% (98 of 101) with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Using serial dilutions of plasmids containing specific SP markers, the linear correlation (r) of 0.99, the slope between -3.2 and -3.7 and the sensitivity of 0.1% were proved reproducible. By this method, it was possible to very accurately detect autologous signals in the range from 0.1% to 30%. The accuracy of the method in the very important range of autologous signals below 5% was extraordinarily high (standard deviation <1.85%), which might significantly improve detection accuracy of changes in autologous signals early in the post-transplantation course of follow-up. The main advantage of the real-time PCR method over short tandem repeat PCR chimerism assays is the absence of PCR competition and plateau biases, with demonstrated greater sensitivity and linearity. Finally, we prospectively analyzed bone marrow samples of 8 patients who received allografts and presented the chimerism kinetics of remission and relapse situations that illustrated the sensitivity level and the promising clinical application of this method. This SP-based real-time PCR assay provides a rapid, sensitive, and accurate quantitative assessment of mixed chimerism that can be useful in predicting graft rejection and early relapse.

Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize.

PubMed

Xu, Xiaoli; Peng, Cheng; Wang, Xiaofu; Chen, Xiaoyun; Wang, Qiang; Xu, Junfeng

2016-12-01

This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.

Comparison of 2 real-time PCR assays for diagnosis of Pneumocystis jirovecii pneumonia in human immunodeficiency virus (HIV) and non-HIV immunocompromised patients.

PubMed

Montesinos, Isabel; Brancart, Françoise; Schepers, Kinda; Jacobs, Frederique; Denis, Olivier; Delforge, Marie-Luce

2015-06-01

A total of 120 bronchoalveolar lavage specimens from HIV and non-HIV immunocompromised patients, positive for Pneumocystis jirovecii by an "in house" real-time polymerase chain reaction (PCR), were evaluated by the Bio-Evolution Pneumocystis real-time PCR, a commercial quantitative assay. Patients were classified in 2 categories based on clinical and radiological findings: definite and unlikely Pneumocystis pneumonia (PCP). For the "in house" PCR, cycle threshold 34 was established as cut-off value to discriminate definite PCP from unlikely PCP with 65% and 85% of sensitivity and specificity, respectively. For the Bio-Evolution quantitative PCR, a cut-off value of 2.8×10(5)copies/mL was defined with 72% and 82% of sensitivity and specificity, respectively. Overlapped zones of results for definite and unlikely PCP were observed. Quantitative PCR is probably a useful tool for PCP diagnosis. However, for optimal management of PCP in non-HIV immunocompromised patients, operational thresholds should be assessed according to underlying diseases and other clinical and radiological parameters. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantitative analysis of dengue-2 virus RNA during the extrinsic incubation period in individual Aedes aegypti.

PubMed

Richardson, Jason; Molina-Cruz, Alvaro; Salazar, Ma Isabel; Black, William

2006-01-01

Dengue virus-2 (DENV-2) RNA was quantified from the midgut and legs of individual Aedes aegypti at each of 14 days postinfectious blood meal (dpi) in a DENV-2 susceptible strain from Chetumal, Mexico. A SYBR Green I based strand-specific, quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed. The lower detection and quantitation limits were 20 and 200 copies per reaction, respectively. Amounts of positive and negative strand viral RNA strands were correlated. Numbers of plaque-forming units (PFU) were correlated with DENV-2 RNA copy number in both C6/36 cell cultures and mosquitoes. PFU were consistently lower than RNA copy number by 2-3 log(10). Midgut levels of DENV-2 RNA peaked 8 dpi and fluctuated erratically between 6 and 9 dpi. Copies of DENV-2 RNA varied significantly among infected mosquitoes at each time point. Quantitative real-time RT-PCR is a convenient and reliable method that provides new insights into virus-vector interactions.

Identification of suitable reference genes for hepatic microRNA quantitation.

PubMed

Lamba, Vishal; Ghodke-Puranik, Yogita; Guan, Weihua; Lamba, Jatinder K

2014-03-07

MicroRNAs (miRNAs) are short (~22 nt) endogenous RNAs that play important roles in regulating expression of a wide variety of genes involved in different cellular processes. Alterations in microRNA expression patterns have been associated with a number of human diseases. Accurate quantitation of microRNA levels is important for their use as biomarkers and in determining their functions. Real time PCR is the gold standard and the most frequently used technique for miRNA quantitation. Real time PCR data analysis includes normalizing the amplification data to suitable endogenous control/s to ensure that microRNA quantitation is not affected by the variability that is potentially introduced at different experimental steps. U6 (RNU6A) and RNU6B are two commonly used endogenous controls in microRNA quantitation. The present study was designed to investigate inter-individual variability and gender differences in hepatic microRNA expression as well as to identify the best endogenous control/s that could be used for normalization of real-time expression data in liver samples. We used Taqman based real time PCR to quantitate hepatic expression levels of 22 microRNAs along with U6 and RNU6B in 50 human livers samples (25 M, 25 F). To identify the best endogenous controls for use in data analysis, we evaluated the amplified candidates for their stability (least variability) in expression using two commonly used software programs: Normfinder and GeNormplus, Both Normfinder and GeNormplus identified U6 to be among the least stable of all the candidates analyzed, and RNU6B was also not among the top genes in stability. mir-152 and mir-23b were identified to be the two most stable candidates by both Normfinder and GeNormplus in our analysis, and were used as endogenous controls for normalization of hepatic miRNA levels. Measurements of microRNA stability indicate that U6 and RNU6B are not suitable for use as endogenous controls for normalizing microRNA relative quantitation data in hepatic tissue, and their use can led to possibly erroneous conclusions.

Performance evaluation of the Aptima® HCV Quant Dx assay for hepatitis C virus (HCV) RNA detection and quantification in comparison to the Abbott RealTime HCV assay.

PubMed

Garbuglia, Anna Rosa; Bibbò, Angela; Sciamanna, Roberta; Pisciotta, Marina; Capobianchi, Maria Rosaria

2017-07-01

The Aptima HCV Quant Dx assay (Aptima) is a real-time transcription-mediated amplification assay CE-approved for the diagnosis and monitoring of hepatitis C virus (HCV) infection. Aptima's analytical performance was compared to the Abbott RealTime HCV assay (RealTime) in a clinical routine setting. Overall 295 clinical plasma samples (117 prospective/fresh; 178 retrospective/frozen) from HCV-infected patients were tested in Aptima and RealTime to determine concordance on qualitative and quantitative results. Linearity and precision at low viral loads (VLs; 0.8-3.3LogIU/mL) was tested using dilutions of the 5th WHO standard, in 10 and 20 replicates in the two assays, respectively. The ability to measure different HCV genotypes and accuracy were analyzed using the Seracare EQA panel. Inter-assay agreement for qualitative results (prospective samples) was 88% (kappa=0.78). For the 127 samples with quantitative results in both assays, Aptima yielded on average slightly higher values (by 0.24LogIU/mL; Bland-Altman method) than RealTime. Concordance between assay results was excellent (R=0.98). At low VLs (0.8-3.3LogIU/mL), Aptima demonstrated good linearity and precision, similar to RealTime. Aptima detected and accurately quantified all main HCV genotypes. Aptima demonstrated excellent precision, linearity, and accuracy in all genotypes tested. Good concordance was observed between Aptima and RealTime assays in clinical samples. The performance of the Aptima assay, on the fully automated Panther platform, makes it an excellent candidate for the detection and monitoring of HCV RNA in plasma and serum samples. Copyright © 2017 Elsevier B.V. All rights reserved.

A Sensitive Detection Method for MPLW515L or MPLW515K Mutation in Chronic Myeloproliferative Disorders with Locked Nucleic Acid-Modified Probes and Real-Time Polymerase Chain Reaction

PubMed Central

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

2008-01-01

Acquired mutations in the juxtamembrane region of MPL (W515K or W515L), the receptor for thrombopoietin, have been described in patients with primary myelofibrosis or essential thrombocythemia, which are chronic myeloproliferative disorders. We have developed a real-time polymerase chain reaction assay for the detection and quantification of MPL mutations that is based on locked nucleic acid fluorescent probes. Mutational analysis was performed using DNA from granulocytes. Reference curves were obtained using cloned fragments of MPL containing either the wild-type or mutated sequence; the predicted sensitivity level was at least 0.1% mutant allele in a wild-type background. None of the 60 control subjects presented with a MPLW515L/K mutation. Of 217 patients with myelofibrosis, 19 (8.7%) harbored the MPLW515 mutation, 10 (52.6%) with the W515L allele. In one case, both the W515L and W515K alleles were detected by real-time polymerase chain reaction. By comparing results obtained with conventional sequencing, no erroneous genotype attribution using real-time polymerase chain reaction was found, whereas one patient considered wild type according to sequence analysis actually harbored a low W515L allele burden. This is a simple, sensitive, and cost-effective procedure for large-scale screening of the MPLW515L/K mutation in patients suspected to have a myeloproliferative disorder. It can also provide a quantitative estimate of mutant allele burden that might be useful for both patient prognosis and monitoring response to therapy. PMID:18669880

Impact of left ventricular outflow tract area on systolic outflow velocity in hypertrophic cardiomyopathy: a real-time three-dimensional echocardiographic study

NASA Technical Reports Server (NTRS)

Qin, Jian Xin; Shiota, Takahiro; Lever, Harry M.; Rubin, David N.; Bauer, Fabrice; Kim, Yong Jin; Sitges, Marta; Greenberg, Neil L.; Drinko, Jeanne K.; Martin, Maureen;

Expansion of the Real-time Sport-land Information System for NOAA / National Weather Service Situational Awareness and Local Modeling Applications

NASA Technical Reports Server (NTRS)

Case, Jonathan L.; White, Kristopher D.

2014-01-01

The NASA Short-term Prediction Research and Transition (SPoRT) Center in Huntsville, AL (Jedlovec 2013; Ralph et al. 2013; Merceret et al. 2013) is running a real-time configuration of the Noah land surface model (LSM) within the NASA Land Information System (LIS) framework (hereafter referred to as the "SPoRT-LIS"). Output from the real-time SPoRT-LIS is used for (1) initializing land surface variables for local modeling applications, and (2) displaying in decision support systems for situational awareness and drought monitoring at select NOAA/National Weather Service (NWS) partner offices. The SPoRT-LIS is currently run over a domain covering the southeastern half of the Continental United States (CONUS), with an additional experimental real-time run over the entire CONUS and surrounding portions of southern Canada and northern Mexico. The experimental CONUS run incorporates hourly quantitative precipitation estimation (QPE) from the National Severe Storms Laboratory Multi- Radar Multi-Sensor (MRMS) product (Zhang et al. 2011, 2014), which will be transitioned into operations at the National Centers for Environmental Prediction (NCEP) in Fall 2014. This paper describes the current and experimental SPoRT-LIS configurations, and documents some of the limitations still remaining through the advent of MRMS precipitation analyses in the SPoRT-LIS land surface model (LSM) simulations. Section 2 gives background information on the NASA LIS and describes the realtime SPoRT-LIS configurations being compared. Section 3 presents recent work done to develop a training module on situational awareness applications of real-time SPoRT-LIS output. Comparisons between output from the two SPoRT-LIS runs are shown in Section 4, including a documentation of issues encountered in using the MRMS precipitation dataset. A summary and future work in given in Section 5, followed by acknowledgements and references.

Analysis of carbohydrates in Fusarium verticillioides using size-exclusion HPLC – DRI and direct analysis in real time ionization – time-of-flight – mass spectrometry (DART-MS)

USDA-ARS?s Scientific Manuscript database

Direct analysis in real time ionization – time-of-flight – mass spectrometry (DART-MS) and size-exclusion HPLC – DRI are used, respectively, to qualitatively and quantitatively determine the carbohydrates extracted from the corn rot fungus Fusarium verticillioides. In situ permethylation in the DART...

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

PubMed

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

2014-12-10

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

Incremental principal component pursuit for video background modeling

DOEpatents

Rodriquez-Valderrama, Paul A.; Wohlberg, Brendt

2017-03-14

An incremental Principal Component Pursuit (PCP) algorithm for video background modeling that is able to process one frame at a time while adapting to changes in background, with a computational complexity that allows for real-time processing, having a low memory footprint and is robust to translational and rotational jitter.

Molecular identification and real-time quantitative PCR (qPCR) for rapid detection of Thelohanellus kitauei, a Myxozoan parasite causing intestinal giant cystic disease in the Israel carp.

PubMed

Seo, Jung Soo; Jeon, Eun Ji; Kim, Moo Sang; Woo, Sung Ho; Kim, Jin Do; Jung, Sung Hee; Park, Myoung Ae; Jee, Bo Young; Kim, Jin Woo; Kim, Yi-Cheong; Lee, Eun Hye

2012-06-01

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water.

Trace-Level Volatile Quantitation by Direct Analysis in Real Time Mass Spectrometry following Headspace Extraction: Optimization and Validation in Grapes.

PubMed

Jastrzembski, Jillian A; Bee, Madeleine Y; Sacks, Gavin L

2017-10-25

Ambient ionization mass spectrometric (AI-MS) techniques like direct analysis in real time (DART) offer the potential for rapid quantitative analyses of trace volatiles in food matrices, but performance is generally limited by the lack of preconcentration and extraction steps. The sensitivity and selectivity of AI-MS approaches can be improved through solid-phase microextraction (SPME) with appropriate thin-film geometries, for example, solid-phase mesh-enhanced sorption from headspace (SPMESH). This work improves the SPMESH-DART-MS approach for use in food analyses and validates the approach for trace volatile analysis for two compounds in real samples (grape macerates). SPMESH units prepared with different sorbent coatings were evaluated for their ability to extract a range of odor-active volatiles, with poly(dimethylsiloxane)/divinylbenzene giving the most satisfactory results. In combination with high-resolution mass spectrometry (HRMS), detection limits for SPMESH-DART-MS under 4 ng/L in less than 30 s acquisition times could be achieved for some volatiles [3-isobutyl-2-methoxypyrazine (IBMP) and β-damascenone]. A comparison of SPMESH-DART-MS and SPME-GC-MS quantitation of linalool and IBMP demonstrates excellent agreement between the two methods for real grape samples (r 2 ≥ 0.90), although linalool measurements appeared to also include isobaric interference.

One step screening of retroviral producer clones by real time quantitative PCR.

PubMed

Towers, G J; Stockholm, D; Labrousse-Najburg, V; Carlier, F; Danos, O; Pagès, J C

1999-01-01

Recombinant retroviruses are obtained from either stably or transiently transfected retrovirus producer cells. In the case of stably producing lines, a large number of clones must be screened in order to select the one with the highest titre. The multi-step selection of high titre producing clones is time consuming and expensive. We have taken advantage of retroviral endogenous reverse transcription to develop a quantitative PCR assay on crude supernatant from producing clones. We used Taqman PCR technology, which, by using fluorescence measurement at each cycle of amplification, allows PCR product quantification. Fluorescence results from specific degradation of a probe oligonucleotide by the Taq polymerase 3'-5' exonuclease activity. Primers and probe sequences were chosen to anneal to the viral strong stop species, which is the first DNA molecule synthesised during reverse transcription. The protocol consists of a single real time PCR, using as template filtered viral supernatant without any other pre-treatment. We show that the primers and probe described allow quantitation of serially diluted plasmid to as few as 15 plasmid molecules. We then test 200 GFP-expressing retroviral-producing clones either by FACS analysis of infected cells or by using the quantitative PCR. We confirm that the Taqman protocol allows the detection of virus in supernatant and selection of high titre clones. Furthermore, we can determine infectious titre by quantitative PCR on genomic DNA from infected cells, using an additional set of primers and probe to albumin to normalise for the genomic copy number. We demonstrate that real time quantitative PCR can be used as a powerful and reliable single step, high throughput screen for high titre retroviral producer clones.

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

PubMed

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

2017-01-01

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

Protein Analysis Using Real-Time PCR Instrumentation: Incorporation in an Integrated, Inquiry-Based Project

ERIC Educational Resources Information Center

Southard, Jonathan N.

2014-01-01

Instrumentation for real-time PCR is used primarily for amplification and quantitation of nucleic acids. The capability to measure fluorescence while controlling temperature in multiple samples can also be applied to the analysis of proteins. Conformational stability and changes in stability due to ligand binding are easily assessed. Protein…

Real-Time PCR (qPCR) Primer Design Using Free Online Software

ERIC Educational Resources Information Center

Thornton, Brenda; Basu, Chhandak

2011-01-01

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

Analysis of the aflatoxin AFB1 from corn by direct analysis in real time - mass spectrometry (DART-MS)

USDA-ARS?s Scientific Manuscript database

Direct analysis in real time (DART) ionization coupled to a high resolution mass spectrometer (MS) was used for screening of aflatoxins from a variety of surfaces and the rapid quantitative analysis of aflatoxins extracted from corn. Sample preparation procedure and instrument parameter settings wer...

A multiplex real-time polymerase chain reaction assay differentiates between Bolbphorus damnificus and Bolbophorus type II sp

USDA-ARS?s Scientific Manuscript database

A duplex quantitative real-time polymerase chain reaction (qPCR) assay was developed to differentiate between Bolbophorus damnificus and Bolbophorus type II species cercariae. Both trematode species are prevalent throughout the commercial catfish industry,.as both infect the ram’s horn snail, Plano...

Taqman real-time quantitative PCR for identification of western flower thrip (Frankliniella occidentalis) for plant quarantine

PubMed Central

Huang, K. S.; Lee, S. E.; Yeh, Y.; Shen, G. S.; Mei, E.; Chang, C. M.

2010-01-01

Western flower thrip (Frankliniella occidentalis) is a major global pest of agricultural products. It directly damages crops through feeding, oviposition activity or transmission of several plant viruses. We describe a Taqman real-time quantitative PCR detection system, which can rapidly identify F. occidentalis from thrips larvae to complement the traditional morphological identification. The data showed that our detection system targeted on the ribosomal RNA gene regions of F. occidentalis has high sensitivity and specificity. The rapid method can be used for on-site testing of samples at ports-of-entry in the future. PMID:20129946

Taqman real-time quantitative PCR for identification of western flower thrip (Frankliniella occidentalis) for plant quarantine.

PubMed

Huang, K S; Lee, S E; Yeh, Y; Shen, G S; Mei, E; Chang, C M

2010-08-23

Western flower thrip (Frankliniella occidentalis) is a major global pest of agricultural products. It directly damages crops through feeding, oviposition activity or transmission of several plant viruses. We describe a Taqman real-time quantitative PCR detection system, which can rapidly identify F. occidentalis from thrips larvae to complement the traditional morphological identification. The data showed that our detection system targeted on the ribosomal RNA gene regions of F. occidentalis has high sensitivity and specificity. The rapid method can be used for on-site testing of samples at ports-of-entry in the future.

Real-time quasi-3D tomographic reconstruction

NASA Astrophysics Data System (ADS)

Buurlage, Jan-Willem; Kohr, Holger; Palenstijn, Willem Jan; Joost Batenburg, K.

2018-06-01

Developments in acquisition technology and a growing need for time-resolved experiments pose great computational challenges in tomography. In addition, access to reconstructions in real time is a highly demanded feature but has so far been out of reach. We show that by exploiting the mathematical properties of filtered backprojection-type methods, having access to real-time reconstructions of arbitrarily oriented slices becomes feasible. Furthermore, we present , software for visualization and on-demand reconstruction of slices. A user of can interactively shift and rotate slices in a GUI, while the software updates the slice in real time. For certain use cases, the possibility to study arbitrarily oriented slices in real time directly from the measured data provides sufficient visual and quantitative insight. Two such applications are discussed in this article.

Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods.

PubMed

Yang, Litao; Pan, Aihu; Zhang, Kewei; Guo, Jinchao; Yin, Changsong; Chen, Jianxiu; Huang, Cheng; Zhang, Dabing

2005-08-10

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.

A quantitative risk-based model for reasoning over critical system properties

NASA Technical Reports Server (NTRS)

Feather, M. S.

2002-01-01

This position paper suggests the use of a quantitative risk-based model to help support reeasoning and decision making that spans many of the critical properties such as security, safety, survivability, fault tolerance, and real-time.

Study on real-time images compounded using spatial light modulator

NASA Astrophysics Data System (ADS)

Xu, Jin; Chen, Zhebo; Ni, Xuxiang; Lu, Zukang

2007-01-01

Image compounded technology is often used on film and its facture. In common, image compounded use image processing arithmetic, get useful object, details, background or some other things from the images firstly, then compounding all these information into one image. When using this method, the film system needs a powerful processor, for the process function is very complex, we get the compounded image for a few time delay. In this paper, we introduce a new method of image real-time compounded, use this method, we can do image composite at the same time with movie shot. The whole system is made up of two camera-lens, spatial light modulator array and image sensor. In system, the spatial light modulator could be liquid crystal display (LCD), liquid crystal on silicon (LCoS), thin film transistor liquid crystal display (TFTLCD), Deformable Micro-mirror Device (DMD), and so on. Firstly, one camera-lens images the object on the spatial light modulator's panel, we call this camera-lens as first image lens. Secondly, we output an image to the panel of spatial light modulator. Then, the image of the object and image that output by spatial light modulator will be spatial compounded on the panel of spatial light modulator. Thirdly, the other camera-lens images the compounded image to the image sensor, and we call this camera-lens as second image lens. After these three steps, we will gain the compound images by image sensor. For the spatial light modulator could output the image continuously, then the image will be compounding continuously too, and the compounding procedure is completed in real-time. When using this method to compounding image, if we will put real object into invented background, we can output the invented background scene on the spatial light modulator, and the real object will be imaged by first image lens. Then, we get the compounded images by image sensor in real time. The same way, if we will put real background to an invented object, we can output the invented object on the spatial light modulator and the real background will be imaged by first image lens. Then, we can also get the compounded images by image sensor real time. Commonly, most spatial light modulator only can do modulate light intensity, so we can only do compounding BW images if use only one panel which without color filter. If we will get colorful compounded image, we need use the system like three spatial light modulator panel projection. In the paper, the system's optical system framework we will give out. In all experiment, the spatial light modulator used liquid crystal on silicon (LCoS). At the end of the paper, some original pictures and compounded pictures will be given on it. Although the system has a few shortcomings, we can conclude that, using this system to compounding images has no delay to do mathematic compounding process, it is a really real time images compounding system.

Development and Evaluation of Event-Specific Quantitative PCR Method for Genetically Modified Soybean MON87701.

PubMed

Tsukahara, Keita; Takabatake, Reona; Masubuchi, Tomoko; Futo, Satoshi; Minegishi, Yasutaka; Noguchi, Akio; Kondo, Kazunari; Nishimaki-Mogami, Tomoko; Kurashima, Takeyo; Mano, Junichi; Kitta, Kazumi

2016-01-01

A real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event, MON87701. First, a standard plasmid for MON87701 quantification was constructed. The conversion factor (C f ) required to calculate the amount of genetically modified organism (GMO) was experimentally determined for a real-time PCR instrument. The determined C f for the real-time PCR instrument was 1.24. For the evaluation of the developed method, a blind test was carried out in an inter-laboratory trial. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSDr), respectively. The determined biases and the RSDr values were less than 30 and 13%, respectively, at all evaluated concentrations. The limit of quantitation of the method was 0.5%, and the developed method would thus be applicable for practical analyses for the detection and quantification of MON87701.

DNA microsatellite region for a reliable quantification of soft wheat adulteration in durum wheat-based foodstuffs by real-time PCR.

PubMed

Sonnante, Gabriella; Montemurro, Cinzia; Morgese, Anita; Sabetta, Wilma; Blanco, Antonio; Pasqualone, Antonella

2009-11-11

Italian industrial pasta and durum wheat typical breads must be prepared using exclusively durum wheat semolina. Previously, a microsatellite sequence specific of the wheat D-genome had been chosen for traceability of soft wheat in semolina and bread samples, using qualitative and quantitative Sybr green-based real-time experiments. In this work, we describe an improved method based on the same soft wheat genomic region by means of a quantitative real-time PCR using a dual-labeled probe. Standard curves based on dilutions of 100% soft wheat flour, pasta, or bread were constructed. Durum wheat semolina, pasta, and bread samples were prepared with increasing amounts of soft wheat to verify the accuracy of the method. Results show that reliable quantifications were obtained especially for the samples containing a lower amount of soft wheat DNA, fulfilling the need to verify labeling of pasta and typical durum wheat breads.

Parallel algorithm of real-time infrared image restoration based on total variation theory

NASA Astrophysics Data System (ADS)

Zhu, Ran; Li, Miao; Long, Yunli; Zeng, Yaoyuan; An, Wei

2015-10-01

Image restoration is a necessary preprocessing step for infrared remote sensing applications. Traditional methods allow us to remove the noise but penalize too much the gradients corresponding to edges. Image restoration techniques based on variational approaches can solve this over-smoothing problem for the merits of their well-defined mathematical modeling of the restore procedure. The total variation (TV) of infrared image is introduced as a L1 regularization term added to the objective energy functional. It converts the restoration process to an optimization problem of functional involving a fidelity term to the image data plus a regularization term. Infrared image restoration technology with TV-L1 model exploits the remote sensing data obtained sufficiently and preserves information at edges caused by clouds. Numerical implementation algorithm is presented in detail. Analysis indicates that the structure of this algorithm can be easily implemented in parallelization. Therefore a parallel implementation of the TV-L1 filter based on multicore architecture with shared memory is proposed for infrared real-time remote sensing systems. Massive computation of image data is performed in parallel by cooperating threads running simultaneously on multiple cores. Several groups of synthetic infrared image data are used to validate the feasibility and effectiveness of the proposed parallel algorithm. Quantitative analysis of measuring the restored image quality compared to input image is presented. Experiment results show that the TV-L1 filter can restore the varying background image reasonably, and that its performance can achieve the requirement of real-time image processing.

Oxidative stress provokes distinct transcriptional responses in the stress-tolerant atr7 and stress-sensitive loh2 Arabidopsis thaliana mutants as revealed by multi-parallel quantitative real-time PCR analysis of ROS marker and antioxidant genes.

PubMed

Mehterov, Nikolay; Balazadeh, Salma; Hille, Jacques; Toneva, Valentina; Mueller-Roeber, Bernd; Gechev, Tsanko

2012-10-01

The Arabidopsis thaliana atr7 mutant is tolerant to oxidative stress induced by paraquat (PQ) or the catalase inhibitor aminotriazole (AT), while its original background loh2 and wild-type plants are sensitive. Both, AT and PQ, which stimulate the intracellular formation of H₂O₂ or superoxide anions, respectively, trigger cell death in loh2 but do not lead to visible damage in atr7. To study gene expression during oxidative stress and ROS-induced programmed cell death, two platforms for multi-parallel quantitative real-time PCR (qRT-PCR) analysis of 217 antioxidant and 180 ROS marker genes were employed. The qRT-PCR analyses revealed AT- and PQ-induced expression of many ROS-responsive genes mainly in loh2, confirming that an oxidative burst plays a role in the activation of the cell death in this mutant. Some of the genes were specifically regulated by either AT or PQ, serving as markers for particular types of ROS. Genes significantly induced by both AT and PQ in loh2 included transcription factors (ANAC042/JUB1, ANAC102, DREB19, HSFA2, RRTF1, ZAT10, ZAT12, ethylene-responsive factors), signaling compounds, ferritins, alternative oxidases, and antioxidant enzymes. Many of these genes were upregulated in atr7 compared to loh2 under non-stress conditions at the first time point, indicating that higher basal levels of ROS and higher antioxidant capacity in atr7 are responsible for the enhanced tolerance to oxidative stress and suggesting a possible tolerance against multiple stresses of this mutant. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants

PubMed Central

Safeukui, Innocent; Millet, Pascal; Boucher, Sébastien; Melinard, Laurence; Fregeville, Frédéric; Receveur, Marie-Catherine; Pistone, Thierry; Fialon, Pierre; Vincendeau, Philippe; Fleury, Hervé; Malvy, Denis

2008-01-01

Background A simple real-time PCR assay using one set of primer and probe for rapid, sensitive and quantitative detection of Plasmodium species, with simultaneous differentiation of Plasmodium falciparum from the three other Plasmodium species (Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) in febrile returning travellers and migrants was developed and evaluated. Methods Consensus primers were used to amplify a species-specific region of the multicopy 18S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be perfect matches to the 18S rRNA gene of the fourth Plasmodium species, while the acceptor probe sequence was designed for P. falciparum over a region containing one mismatched, which allowed differentiation of the three other Plasmodium species. The performance characteristics of the real-time PCR assay were compared with those of conventional PCR and microscopy-based diagnosis from 119 individuals with a suspected clinical diagnostic of imported malaria. Results Blood samples with parasite densities less than 0.01% were all detected, and analytical sensitivity was 0.5 parasite per PCR reaction. The melt curve means Tms (standard deviation) in clinical isolates were 60.5°C (0.6°C) for P. falciparum infection and 64.6°C (1.8°C) for non-P. falciparum species. These Tms values of the P. falciparum or non-P. falciparum species did not vary with the geographic origin of the parasite. The real-time PCR results correlated with conventional PCR using both genus-specific (Kappa coefficient: 0.95, 95% confidence interval: 0.9 – 1) or P. falciparum-specific (0.91, 0.8 – 1) primers, or with the microscopy results (0.70, 0.6 – 0.8). The real-time assay was 100% sensitive and specific for differentiation of P. falciparum to non-P. falciparum species, compared with conventional PCR or microscopy. The real-time PCR assay can also detect individuals with mixed infections (P. falciparum and non-P. falciparum sp.) in the same sample. Conclusion This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of P. falciparum to other Plasmodium species. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed. PMID:18442362

Correlation of immune activation with HIV-1 RNA levels assayed by real-time RT-PCR in HIV-1 Subtype C infected patients in Northern India

PubMed Central

Agarwal, Atima; Sankaran, Sumathi; Vajpayee, Madhu; Sreenivas, V; Seth, Pradeep; Dandekar, Satya

2014-01-01

Background Assays with specificity and cost effectiveness are needed for the measurement of HIV-1 burden to monitor disease progression or response to anti-retroviral therapy (ART) in HIV-1 subtype C infected patients. Objectives The objective of this study was to develop and validate an affordable; one step Real-Time RT-PCR assay with high specificity and sensitivity to measure plasma HIV-1 loads in HIV-1 subtype C infected patients. Results We developed an RT-PCR assay to detect and quantitate plasma HIV-1 levels in HIV-1 subtype C infected patients. An inverse correlation between plasma viral loads (PVL) and CD4+ T-cell numbers was detected at all CDC stages. Significant correlations were found between CD8+ T-cell activation and PVL, as well as with the clinical and immunological status of the patients. Conclusions The RT-PCR assay provides a sensitive method to measure PVL in HIV-1 subtype C infected patients. Viral loads correlated with immune activation and can be used to monitor HIV care in India. PMID:17962068

Detection of Human Cytomegalovirus DNA by Real-Time Quantitative PCR

PubMed Central

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. PMID:10878073

Determination of the aflatoxin AFB1 from corn by direct analysis in real time-mass spectrometry (DART-MS)

USDA-ARS?s Scientific Manuscript database

Direct analysis in real time (DART) ionization coupled to a high resolution mass spectrometer (MS) was used for screening of aflatoxins from a variety of surfaces and the rapid quantitative analysis of a common form of aflatoxin, AFB1, extracted from corn. Sample preparation procedure and instrument...

Determination of T-2 and HT-2 toxins from maize by direct analysis in real time mass spectrometry

USDA-ARS?s Scientific Manuscript database

Direct analysis in real time (DART) ionization coupled to mass spectrometry (MS) was used for the rapid quantitative analysis of T-2 toxin, and the related HT-2 toxin, extracted from corn. Sample preparation procedures and instrument parameters were optimized to obtain sensitive and accurate determi...

The design of real time infrared image generation software based on Creator and Vega

NASA Astrophysics Data System (ADS)

Wang, Rui-feng; Wu, Wei-dong; Huo, Jun-xiu

2013-09-01

Considering the requirement of high reality and real-time quality dynamic infrared image of an infrared image simulation, a method to design real-time infrared image simulation application on the platform of VC++ is proposed. This is based on visual simulation software Creator and Vega. The functions of Creator are introduced simply, and the main features of Vega developing environment are analyzed. The methods of infrared modeling and background are offered, the designing flow chart of the developing process of IR image real-time generation software and the functions of TMM Tool and MAT Tool and sensor module are explained, at the same time, the real-time of software is designed.

Polymeric LabChip Real-Time PCR as a Point-of-Care-Potential Diagnostic Tool for Rapid Detection of Influenza A/H1N1 Virus in Human Clinical Specimens

PubMed Central

Song, Hyun-Ok; Kim, Je-Hyoung; Ryu, Ho-Sun; Lee, Dong-Hoon; Kim, Sun-Jin; Kim, Deog-Joong; Suh, In Bum; Choi, Du Young; In, Kwang-Ho; Kim, Sung-Woo; Park, Hyun

2012-01-01

It is clinically important to be able to detect influenza A/H1N1 virus using a fast, portable, and accurate system that has high specificity and sensitivity. To achieve this goal, it is necessary to develop a highly specific primer set that recognizes only influenza A viral genes and a rapid real-time PCR system that can detect even a single copy of the viral gene. In this study, we developed and validated a novel fluidic chip-type real-time PCR (LabChip real-time PCR) system that is sensitive and specific for the detection of influenza A/H1N1, including the pandemic influenza strain A/H1N1 of 2009. This LabChip real-time PCR system has several remarkable features: (1) It allows rapid quantitative analysis, requiring only 15 min to perform 30 cycles of real-time PCR. (2) It is portable, with a weight of only 5.5 kg. (3) The reaction cost is low, since it uses disposable plastic chips. (4) Its high efficiency is equivalent to that of commercially available tube-type real-time PCR systems. The developed disposable LabChip is an economic, heat-transferable, light-transparent, and easy-to-fabricate polymeric chip compared to conventional silicon- or glass-based labchip. In addition, our LabChip has large surface-to-volume ratios in micro channels that are required for overcoming time consumed for temperature control during real-time PCR. The efficiency of the LabChip real-time PCR system was confirmed using novel primer sets specifically targeted to the hemagglutinin (HA) gene of influenza A/H1N1 and clinical specimens. Eighty-five human clinical swab samples were tested using the LabChip real-time PCR. The results demonstrated 100% sensitivity and specificity, showing 72 positive and 13 negative cases. These results were identical to those from a tube-type real-time PCR system. This indicates that the novel LabChip real-time PCR may be an ultra-fast, quantitative, point-of-care-potential diagnostic tool for influenza A/H1N1 with a high sensitivity and specificity. PMID:23285281

Multimarker Quantitative Real-Time PCR Detection of Circulating Melanoma Cells in Peripheral Blood: Relation to Disease Stage in Melanoma Patients

PubMed Central

Koyanagi, Kazuo; Kuo, Christine; Nakagawa, Taku; Mori, Takuji; Ueno, Hideaki; Lorico, Arnulfo R.; Wang, He-Jing; Hseuh, Eddie; O’Day, Steven J.; Hoon, Dave S.B.

2010-01-01

Background Detection of melanoma cells in circulation may be important in assessing tumor progression. The objective of this study was to develop a specific, reliable, multimarker quantitative real-time reverse transcription-PCR (qRT) assay for detecting melanoma cells in patients’ blood. Methods We developed qRT assays for the mRNA of four melanoma-associated markers: MART-1, GalNAc-T, PAX-3, and MAGE-A3. In optimization studies, we tested 17 melanoma cell lines and 49 peripheral blood leukocyte (PBL) samples from volunteers. We performed RNA and melanoma cell dilution studies to assess the detection limits and imprecision of the assays. We measured the mRNAs in blood specimens from 94 melanoma patients [American Joint Committee on Cancer (AJCC) stage I, n = 20; II, n = 20; III, n = 32; IV, n = 22]. Results All markers were frequently detected in melanoma cell lines, whereas none of the markers was detected in PBLs from volunteers. The qRT assay could detect 1 melanoma cell in 107 PBLs in the melanoma cell-dilution studies. Markers were detected in 15%, 30%, 75%, and 86% of melanoma patients with AJCC stage I, II, III, and IV disease, respectively. The number of positive markers and AJCC stage were significantly correlated (Spearman correlation coefficient = 0.58; P <0.0001). Conclusions Multimarker qRT can detect circulating melanoma cells in blood. Measurement of the studied molecular markers in blood may be useful in detection of metastasis and monitoring treatment response of melanoma patients. PMID:15817820

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

PubMed Central

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

Inhibition of hepatitis B virus gene expression & replication by crude destruxins from Metarhizium anisopliae var. dcjhyium

PubMed Central

Dong, Cong; Yu, Jiuru; Zhu, Ying; Dong, Changjin

2013-01-01

Background & objectives: Destruxin A, destruxin B and destruxin E isolated from entomopathogenic fungus Metarhizium anisopliae showed a strong suppressive effect on the replication of hepatitis B virus (HBV) in human hepatoma cells. In this study, the anti-HBV effects of the crude destruxins extracted from M. anisopliae var. dcjhyium were detected both in vitro and in vivo. Methods: HepG2.2.15 cells were cultured to observe the inhibitory effects of the crude destruxins on the gene expression and replication of HBV by radioimmunoassay detection and real-time quantitative PCR. In vivo, duck HBV (DHBV)-infected ducks were treated with the crude destruxins at 2.0, 4.0, 6.0 μg/kg once a day for 15 days, DHBV DNA was examined by real-time quantitative PCR. Results: The crude destruxins suppressed the replication of HBV-DNA and the production of HBsAg and HBeAg with IC50 of about 1.2 and 1.4 μg/ml. Transcript of viral mRNA was significantly suppressed by the crude destruxins in HepG2.2.15 cells. In vivo, the duck serum DHBV-DNA levels were markedly reduced in the group of the crude destruxins. Interpretation & conclusions: The crude destruxins inhibited the gene expression and replication of HBV both in vitro and in vivo, and their anti-HBV effect was stronger than that with destruxin B. Our results indicate that the crude destruxins from M.anisopliae var. dcjhyium may be potential antivirus agents. Further studies need to be done to confirm these findings. PMID:24521644

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

PubMed

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

2015-10-19

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

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

PubMed Central

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

Rapid Quantitative Detection of Lactobacillus sakei in Meat and Fermented Sausages by Real-Time PCR

PubMed Central

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

2006-01-01

A quick and simple method for quantitative detection of Lactobacillus sakei in fermented sausages was successfully developed. It is based on Chelex-100-based DNA purification and real-time PCR enumeration using a TaqMan fluorescence probe. Primers and probes were designed in the L. sakei 16S-23S rRNA intergenic transcribed spacer region, and the assay was evaluated using L. sakei genomic DNA and an artificially inoculated sausage model. The detection limit of this technique was approximately 3 cells per reaction mixture using both purified DNA and the inoculated sausage model. The quantification limit was established at 30 cells per reaction mixture in both models. The assay was then applied to enumerate L. sakei in real samples, and the results were compared to the MRS agar count method followed by confirmation of the percentage of L. sakei colonies. The results obtained by real-time PCR were not statistically significantly different than those obtained by plate count on MRS agar (P > 0.05), showing a satisfactory agreement between both methods. Therefore, the real-time PCR assay developed can be considered a promising rapid alternative method for the quantification of L. sakei and evaluation of the implantation of starter strains of L. sakei in fermented sausages. PMID:16957227

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

PubMed

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

2006-09-01

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

A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens.

PubMed

Wang, Tiantian; Kim, Sanghyo; An, Jeong Ho

2017-02-01

Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids integrated with a disposable complementary-metal-oxide semiconductor (CMOS) image sensor. This novel system works as an amplification-coupled detection platform, relying on a CMOS image sensor, with the aid of a computerized circuitry controller for the temperature and light sources. The CMOS image sensor captures the light which is passing through the sensor surface and converts into digital units using an analog-to-digital converter (ADC). This new system monitors the real-time photon variation, caused by the color changes during amplification. Escherichia coli O157 was used as a proof-of-concept target for quantitative analysis, and compared with the results for Staphylococcus aureus and Salmonella enterica to confirm the efficiency of the system. The system detected various DNA concentrations of E. coli O157 in a short time (45min), with a detection limit of 10fg/μL. The low-cost, simple, and compact design, with low power consumption, represents a significant advance in the development of a portable, sensitive, user-friendly, real-time, and quantitative analytic tools for point-of-care diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Limitations of commonly used internal controls for real-time RT-PCR analysis of renal epithelial-mesenchymal cell transition.

PubMed

Elberg, Gerard; Elberg, Dorit; Logan, Charlotte J; Chen, Lijuan; Turman, Martin A

2006-01-01

Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by alpha smooth muscle actin (alphaSMA) expression. We quantitated gene expression using real-time RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor-beta1 (TGF-beta1). We report herein the limitations of commonly used reference genes for mRNA quantitation. We determined the expression of alphaSMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of alphaSMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-beta1. Thus, depending on which reference gene was used, TGF-beta1 appeared to have different effects on expression of MKL1 and alphaSMA. RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-beta1 treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression. 2006 S. Karger AG, Basel.

Validating internal controls for quantitative plant gene expression studies.

PubMed

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-08-18

Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.

Real-time PCR assays using internal controls for quantitation of HPV-16 and beta-globin DNA in cervicovaginal lavages.

PubMed

Lefevre, Jonas; Hankins, Catherine; Pourreaux, Karina; Voyer, Hélène; Coutlée, François

2003-12-01

High-risk human papillomavirus 16 (HPV-16) DNA viral load has been measured with real-time PCR assays by amplifying HPV-16 and a human gene. However, these assays have not used internal controls (ICs) to screen for the presence of inhibitors contained in samples. To quantitate HPV-16 DNA and cell content with real-time PCR, ICs for HPV-16 DNA and beta-globin were synthesised and used to control for inhibition. The assays were sensitive and linear over 5 logs. Good reproducibility was achieved with inter-run coefficients of variation of 23% (10(2) HPV-16 copies), 12% (10(4) HPV-16 copies), 17% (274 beta-globin DNA copies) and 7% (27,400 beta-globin DNA copies). Samples containing 56,800,000, 306,000, 18,000, and 4,070 HPV-16 copies/microg of cellular DNA were tested blindly and estimated to contain 48,800,000, 479,000, 20,300, and 6,620 HPV-16 copies/microg of DNA (mean ratio of measured to expected viral load of 1.27+/-0.32). Inhibition of amplification of HPV-16 and beta-globin ICs by six samples known to contain PCR inhibitors was variable: four inhibited both ICs while two inhibited only the HPV-16 IC. The use of internal controls with real-time PCR for HPV-16 quantitation allows to screen for the presence of inhibitors that do not affect equally primer-driven genomic amplification.

Propagating Qualitative Values Through Quantitative Equations

NASA Technical Reports Server (NTRS)

Kulkarni, Deepak

1992-01-01

In most practical problems where traditional numeric simulation is not adequate, one need to reason about a system with both qualitative and quantitative equations. In this paper, we address the problem of propagating qualitative values represented as interval values through quantitative equations. Previous research has produced exponential-time algorithms for approximate solution of the problem. These may not meet the stringent requirements of many real time applications. This paper advances the state of art by producing a linear-time algorithm that can propagate a qualitative value through a class of complex quantitative equations exactly and through arbitrary algebraic expressions approximately. The algorithm was found applicable to Space Shuttle Reaction Control System model.

Towards real-time quantitative optical imaging for surgery

NASA Astrophysics Data System (ADS)

Gioux, Sylvain

2017-07-01

There is a pressing clinical need to provide image guidance during surgery. Currently, assessment of tissue that needs to be resected or avoided is performed subjectively leading to a large number of failures, patient morbidity and increased healthcare cost. Because near-infrared (NIR) optical imaging is safe, does not require contact, and can provide relatively deep information (several mm), it offers unparalleled capabilities for providing image guidance during surgery. In this work, we introduce a novel concept that enables the quantitative imaging of endogenous molecular information over large fields-of-view. Because this concept can be implemented in real-time, it is amenable to provide video-rate endogenous information during surgery.

Diagnosis of feline leukaemia virus infection by semi-quantitative real-time polymerase chain reaction.

PubMed

Pinches, Mark D G; Helps, Christopher R; Gruffydd-Jones, Tim J; Egan, Kathy; Jarrett, Oswald; Tasker, Séverine

2007-02-01

In this paper the design and use of a semi-quantitative real-time polymerase chain reaction assay (RT-PCR) for feline leukaemia virus (FeLV) provirus is described. Its performance is evaluated against established methods of FeLV diagnosis, including virus isolation and enzyme-linked immunoassay (ELISA) in a population of naturally infected cats. The RT-PCR assay is found to have both a high sensitivity (0.92) and specificity (0.99) when examined by expectation maximisation methods and is also able to detect a large number of cats with low FeLV proviral loads that were negative by other conventional test methods.

Competitive RT-PCR Strategy for Quantitative Evaluation of the Expression of Tilapia (Oreochromis niloticus) Growth Hormone Receptor Type I

PubMed Central

2009-01-01

Quantization of gene expression requires that an accurate measurement of a specific transcript is made. In this paper, a quantitative reverse transcription-polymerase chain reaction (RT-PCR) by competition for tilapia growth hormone receptor type I is designed and validated. This experimental procedure was used to determine the abundance of growth hormone receptor type I transcript in different tilapia tissues. The results obtained with this developed competitive RT-PCR were similar to real-time PCR results reported recently. This protocol provides a reliable alternative, but less expensive than real-time PCR to quantify specific genes. PMID:19495916

[The validation of kit of reagents for quantitative detection of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode].

PubMed

Sil'veĭstrova, O Iu; Domonova, É A; Shipulina, O Iu

2014-04-01

The validation of kit of reagents destined to detection and quantitative evaluation of DNA of human cytomegalovirus in biological material using polymerase chain reaction technique in real time operation mode was implemented. The comparison was made against international WHO standard--The first WHO international standard for human cytomegalovirus to implement measures the kit of reagents "AmpliSens CMV-screen/monitor-FL" and standard sample of enterprise DNA HCMV (The central research institute of epidemiology of Rospotrebnadzor) was applied. The fivefold dilution of international WHO standard and standard sample of enterprise were carried out in concentrations of DNA HCMV from 106 to 102. The arrangement of polymerase chain reaction and analysis of results were implemented using programed amplifier with system of detection of fluorescent signal in real-time mode "Rotor-Gene Q" ("Qiagen", Germany). In the total of three series of experiments, all stages of polymerase chain reaction study included, the coefficient of translation of quantitative evaluation of DNA HCMV from copy/ml to ME/ml equal to 0.6 was introduced for this kit of reagents.

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

PubMed Central

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

2011-01-01

Quantitative real-time PCR (qPCR) is the gold standard for the quantification of specific nucleic acid sequences. However, a serious concern has been revealed in a recent report: supercoiled plasmid standards cause significant over-estimation in qPCR quantification. In this study, we investigated the effect of plasmid DNA conformation on the quantification of DNA and the efficiency of qPCR. Our results suggest that plasmid DNA conformation has significant impact on the accuracy of absolute quantification by qPCR. DNA standard curves shifted significantly among plasmid standards with different DNA conformations. Moreover, the choice of DNA measurement method and plasmid DNA conformation may also contribute to the measurement error of DNA standard curves. Due to the multiple effects of plasmid DNA conformation on the accuracy of qPCR, efforts should be made to assure the highest consistency of plasmid standards for qPCR. Thus, we suggest that the conformation, preparation, quantification, purification, handling, and storage of standard plasmid DNA should be described and defined in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) to assure the reproducibility and accuracy of qPCR absolute quantification. PMID:22194997

Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.

PubMed

Shewale, Jaiprakash G; Schneida, Elaine; Wilson, Jonathan; Walker, Jerilyn A; Batzer, Mark A; Sinha, Sudhir K

2007-03-01

The human DNA quantification (H-Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real-time amplification of AluYb8 insertions in hominoid primates. The relatively high copy number of subfamily-specific Alu repeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H-Quant are specific for human DNA and closely related great apes. During the real-time PCR, the SYBR Green I dye binds to the DNA that is synthesized by the human-specific AluYb8 oligonucleotide primers. The fluorescence of the bound SYBR Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H-Quant system is 7.6 pg/microL of human DNA. The amplicon generated in the H-Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real-time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.

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

PubMed

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

2013-04-10

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

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

PubMed

Kaliyaperumal, Subadra; Sankarapandian, Sathasivasubramanian

2016-01-01

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

Quantitative analysis of periodontal pathogens by ELISA and real-time polymerase chain reaction.

PubMed

Hamlet, Stephen M

2010-01-01

The development of analytical methods enabling the accurate identification and enumeration of bacterial species colonizing the oral cavity has led to the identification of a small number of bacterial pathogens that are major factors in the etiology of periodontal disease. Further, these methods also underpin more recent epidemiological analyses of the impact of periodontal disease on general health. Given the complex milieu of over 700 species of microorganisms known to exist within the complex biofilms found in the oral cavity, the identification and enumeration of oral periodontopathogens has not been an easy task. In recent years however, some of the intrinsic limitations of the more traditional microbiological analyses previously used have been overcome with the advent of immunological and molecular analytical methods. Of the plethora of methodologies reported in the literature, the enzyme-linked immunosorbent assay (ELISA), which combines the specificity of antibody with the sensitivity of simple enzyme assays and the polymerase chain reaction (PCR), has been widely utilized in both laboratory and clinical applications. Although conventional PCR does not allow quantitation of the target organism, real-time PCR (rtPCR) has the ability to detect amplicons as they accumulate in "real time" allowing subsequent quantitation. These methods enable the accurate quantitation of as few as 10(2) (using rtPCR) to 10(4) (using ELISA) periodontopathogens in dental plaque samples.

Insights from two industrial hygiene pilot e-cigarette passive vaping studies.

PubMed

Maloney, John C; Thompson, Michael K; Oldham, Michael J; Stiff, Charles L; Lilly, Patrick D; Patskan, George J; Shafer, Kenneth H; Sarkar, Mohamadi A

2016-01-01

While several reports have been published using research methods of estimating exposure risk to e-cigarette vapors in nonusers, only two have directly measured indoor air concentrations from vaping using validated industrial hygiene sampling methodology. Our first study was designed to measure indoor air concentrations of nicotine, menthol, propylene glycol, glycerol, and total particulates during the use of multiple e-cigarettes in a well-characterized room over a period of time. Our second study was a repeat of the first study, and it also evaluated levels of formaldehyde. Measurements were collected using active sampling, near real-time and direct measurement techniques. Air sampling incorporated industrial hygiene sampling methodology using analytical methods established by the National Institute of Occupational Safety and Health and the Occupational Safety and Health Administration. Active samples were collected over a 12-hr period, for 4 days. Background measurements were taken in the same room the day before and the day after vaping. Panelists (n = 185 Study 1; n = 145 Study 2) used menthol and non-menthol MarkTen prototype e-cigarettes. Vaping sessions (six, 1-hr) included 3 prototypes, with total number of puffs ranging from 36-216 per session. Results of the active samples were below the limit of quantitation of the analytical methods. Near real-time data were below the lowest concentration on the established calibration curves. Data from this study indicate that the majority of chemical constituents sampled were below quantifiable levels. Formaldehyde was detected at consistent levels during all sampling periods. These two studies found that indoor vaping of MarkTen prototype e-cigarette does not produce chemical constituents at quantifiable levels or background levels using standard industrial hygiene collection techniques and analytical methods.

Evaluation of Legionella pneumophila contamination in Italian hotel water systems by quantitative real-time PCR and culture methods.

PubMed

Bonetta, Sa; Bonetta, Si; Ferretti, E; Balocco, F; Carraro, E

2010-05-01

This study was designed to define the extent of water contamination by Legionella pneumophila of certain Italian hotels and to compare quantitative real-time PCR with the conventional culture method. Nineteen Italian hotels of different sizes were investigated. In each hotel three hot water samples (boiler, room showers, recycling) and one cold water sample (inlet) were collected. Physico-chemical parameters were also analysed. Legionella pneumophila was detected in 42% and 74% of the hotels investigated by the culture method and by real-time PCR, respectively. In 21% of samples analysed by the culture method, a concentration of >10(4) CFU l(-1) was found, and Leg. pneumophila serogroup 1 was isolated from 10.5% of the hotels. The presence of Leg. pneumophila was significantly influenced by water sample temperature, while no association with water hardness or residual-free chlorine was found. This study showed a high percentage of buildings colonized by Leg. pneumophila. Moreover, real-time PCR proved to be sensitive enough to detect lower levels of contamination than the culture method. This study indicates that the Italian hotels represent a possible source of risk for Legionnaires' disease and confirms the sensitivity of the molecular method. To our knowledge, this is the first report to demonstrate Legionella contamination in Italian hotels using real-time PCR and culture methods.

Lyme Borreliosis--the Utility of Improved Real-Time PCR Assay in the Detection of Borrelia burgdorferi Infections.

PubMed

Bil-Lula, Iwona; Matuszek, Patryk; Pfeiffer, Thomas; Woźniak, Mieczysław

2015-01-01

Infections of Borrelia burgdorferi sensu lato reveal clinical manifestations affecting numerous organs and tissues. The standard diagnostic procedure of these infections is quite simple if a positive history of tick exposure or typical erythema migrans appears. Lack of unequivocal clinical symptoms creates the necessity for further evaluation with laboratory tests. This study discusses the utility of a novel, improved, well-optimized, sensitive and highly specific quantitative real-time PCR assay for the diagnostics of infections caused by Borrelia burgdorferi sensu lato. We designed an improved, specific, highly sensitive real-time quantitative polymerase chain reaction (RQ-PCR) assay for the detection and quantification of all Borrelia burgdorferi genotypes. A wide validation effort was undertaken to ensure confidence in the highly sensitive and specific detection of B. burgdorferi. Due to high sensitivity and great specificity, as low as 1.6×10² copies of Borrelia per mL of whole blood could be detected. As much as 12 (3%) negative ELISA IgM results, 14 (2.8%) negative results of Line blot IgM, 11 (3.1%) and 7 (2.7%) of negative ELISA IgG and Line blot IgG results, respectively, were positive in real-time PCR. The data in this study confirms the high positive predictive value of real-time PCR test in the detection of Borrelia infections.

Interdyad Differences in Early Mother-Infant Face-to-Face Communication: Real-Time Dynamics and Developmental Pathways

ERIC Educational Resources Information Center

Lavelli, Manuela; Fogel, Alan

2013-01-01

A microgenetic research design with a multiple case study method and a combination of quantitative and qualitative analyses was used to investigate interdyad differences in real-time dynamics and developmental change processes in mother-infant face-to-face communication over the first 3 months of life. Weekly observations of 24 mother-infant dyads…

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

ERIC Educational Resources Information Center

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

2015-01-01

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

Strand-specific real-time RT-PCR quantitation of Maize fine streak virus genomic and positive-sense RNAs using high temperature reverse transcription

USDA-ARS?s Scientific Manuscript database

Efforts to analyze the replicative RNA produced by Maize fine streak virus (MVSF) within maize tissue was complicated by the lack of specificity during cDNA generation using standard reverse transcriptase protocols. Real-time qRT-PCR using cDNA generated by priming with random hexamers does not dist...

BK virus DNA detection by real-time polymerase chain reaction in clinical specimens.

PubMed

Marchetti, Simona; Graffeo, Rosalia; Siddu, Alessia; Santangelo, Rosaria; Ciotti, Marco; Picardi, Alessandra; Favalli, Cartesio; Fadda, Giovanni; Cattani, Paola

2007-04-01

The BK polyomavirus (BKV) is widespread in the general population. In transplant recipients, the patients' weakened immune response may encourage reactivation of latent infection, leading to BKV-related diseases. Rapid and quantitative detection might help to delineate viral reactivation patterns and could thus play an important role in their clinical management. In our study we developed an "in-house" quantitative real-time PCR to detect BKV DNA. The effectiveness of this assay was evaluated by a retrospective analysis of 118 plasma specimens from 22 bone marrow transplant (BMT) recipients and 107 samples from immunocompetent subjects. Eight (36.3%) of the 22 bone marrow transplant recipients tested positive for BKV. The viral load varied from specimen to specimen (10 to 10(5) copies/ml). BKV related disease like hemorrhagic cystitis (HC) was diagnosed in three patients. Specimens from the control group all tested negative. Our results showed the high sensitivity of the real-time PCR, allowing accurate and reproducible measuring of the viral load in order to identify patients at risk for BKV-related diseases. With due caution in interpreting threshold values, the real-time PCR could provide a rapid, sensitive and specific tool for detecting BKV and distinguishing latent and active infection.

Diagnosis of ocular toxoplasmosis by two polymerase chain reaction (PCR) examinations: qualitative multiplex and quantitative real-time.

PubMed

Sugita, Sunao; Ogawa, Manabu; Inoue, Shizu; Shimizu, Norio; Mochizuki, Manabu

2011-09-01

To establish a two-step polymerase chain reaction (PCR) diagnostic system for ocular toxoplasmosis. A total of 13 ocular fluid samples (11 aqueous humor and 2 vitreous fluid) were collected from 13 patients with clinically suspected ocular toxoplasmosis. Ten ocular samples from other uveitis patients and 20 samples from subjects without ocular inflammation were used as controls. Two polymerase chain reaction (PCR) methods, i.e., qualitative multiplex PCR and quantitative real-time PCR, were used to measure the toxoplasma genome (T. gondii B1 gene). Qualitative multiplex PCR detected T. gondii B1 gene in the ocular fluids of 11 out of 13 patients with clinically suspected ocular toxoplasmosis. In real-time PCR, we detected high copy numbers of T. gondii DNA (5.1 × 10(2)-2.1 × 10(6) copies/mL) in a total of 10 patients (10/13, 77%). Only ocular toxoplasmosis scar lesions were observed in the three real-time PCR-negative patients. PCR assay results for the samples from the two control groups were all negative. The two-step PCR examination to detect toxoplasma DNA is a useful tool for diagnosing ocular toxoplasmosis.

A goggle navigation system for cancer resection surgery

NASA Astrophysics Data System (ADS)

Xu, Junbin; Shao, Pengfei; Yue, Ting; Zhang, Shiwu; Ding, Houzhu; Wang, Jinkun; Xu, Ronald

2014-02-01

We describe a portable fluorescence goggle navigation system for cancer margin assessment during oncologic surgeries. The system consists of a computer, a head mount display (HMD) device, a near infrared (NIR) CCD camera, a miniature CMOS camera, and a 780 nm laser diode excitation light source. The fluorescence and the background images of the surgical scene are acquired by the CCD camera and the CMOS camera respectively, co-registered, and displayed on the HMD device in real-time. The spatial resolution and the co-registration deviation of the goggle navigation system are evaluated quantitatively. The technical feasibility of the proposed goggle system is tested in an ex vivo tumor model. Our experiments demonstrate the feasibility of using a goggle navigation system for intraoperative margin detection and surgical guidance.

Development and application of the RE-AIM QuEST mixed methods framework for program evaluation.

PubMed

Forman, Jane; Heisler, Michele; Damschroder, Laura J; Kaselitz, Elizabeth; Kerr, Eve A

2017-06-01

To increase the likelihood of successful implementation of interventions and promote dissemination across real-world settings, it is essential to evaluate outcomes related to dimensions other than Effectiveness alone. Glasgow and colleagues' RE-AIM framework specifies four additional types of outcomes that are important to decision-makers: Reach, Adoption, Implementation (including cost), and Maintenance. To further strengthen RE-AIM, we propose integrating qualitative assessments in an expanded framework: RE-AIM Qualitative Evaluation for Systematic Translation (RE-AIM QuEST), a mixed methods framework. RE-AIM QuEST guides formative evaluation to identify real-time implementation barriers and explain how implementation context may influence translation to additional settings. RE-AIM QuEST was used to evaluate a pharmacist-led hypertension management intervention at 3 VA facilities in 2008-2009. We systematically reviewed each of the five RE-AIM dimensions and created open-ended companion questions to quantitative measures and identified qualitative and quantitative data sources, measures, and analyses. To illustrate use of the RE-AIM QuEST framework, we provide examples of real-time, coordinated use of quantitative process measures and qualitative methods to identify site-specific issues, and retrospective use of these data sources and analyses to understand variation across sites and explain outcomes. For example, in the Reach dimension, we conducted real-time measurement of enrollment across sites and used qualitative data to better understand and address barriers at a low-enrollment site. The RE-AIM QuEST framework may be a useful tool for improving interventions in real-time, for understanding retrospectively why an intervention did or did not work, and for enhancing its sustainability and translation to other settings.

Optimization of a shorter variable-acquisition time for legs to achieve true whole-body PET/CT images.

PubMed

Umeda, Takuro; Miwa, Kenta; Murata, Taisuke; Miyaji, Noriaki; Wagatsuma, Kei; Motegi, Kazuki; Terauchi, Takashi; Koizumi, Mitsuru

2017-12-01

The present study aimed to qualitatively and quantitatively evaluate PET images as a function of acquisition time for various leg sizes, and to optimize a shorter variable-acquisition time protocol for legs to achieve better qualitative and quantitative accuracy of true whole-body PET/CT images. The diameters of legs to be modeled as phantoms were defined based on data derived from 53 patients. This study analyzed PET images of a NEMA phantom and three plastic bottle phantoms (diameter, 5.68, 8.54 and 10.7 cm) that simulated the human body and legs, respectively. The phantoms comprised two spheres (diameters, 10 and 17 mm) containing fluorine-18 fluorodeoxyglucose solution with sphere-to-background ratios of 4 at a background radioactivity level of 2.65 kBq/mL. All PET data were reconstructed with acquisition times ranging from 10 to 180, and 1200 s. We visually evaluated image quality and determined the coefficient of variance (CV) of the background, contrast and the quantitative %error of the hot spheres, and then determined two shorter variable-acquisition protocols for legs. Lesion detectability and quantitative accuracy determined based on maximum standardized uptake values (SUV max ) in PET images of a patient using the proposed protocols were also evaluated. A larger phantom and a shorter acquisition time resulted in increased background noise on images and decreased the contrast in hot spheres. A visual score of ≥ 1.5 was obtained when the acquisition time was ≥ 30 s for three leg phantoms, and ≥ 120 s for the NEMA phantom. The quantitative %errors of the 10- and 17-mm spheres in the leg phantoms were ± 15 and ± 10%, respectively, in PET images with a high CV (scan < 30 s). The mean SUV max of three lesions using the current fixed-acquisition and two proposed variable-acquisition time protocols in the clinical study were 3.1, 3.1 and 3.2, respectively, which did not significantly differ. Leg acquisition time per bed position of even 30-90 s allows axial equalization, uniform image noise and a maximum ± 15% quantitative accuracy for the smallest lesion. The overall acquisition time was reduced by 23-42% using the proposed shorter variable than the current fixed-acquisition time for imaging legs, indicating that this is a useful and practical protocol for routine qualitative and quantitative PET/CT assessment in the clinical setting.

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

PubMed

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

2014-01-01

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

Two-Photon Flow Cytometry

NASA Technical Reports Server (NTRS)

Zhog, Cheng Frank; Ye, Jing Yong; Norris, Theodore B.; Myc, Andrzej; Cao, Zhengyl; Bielinska, Anna; Thomas, Thommey; Baker, James R., Jr.

2004-01-01

Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantitation is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two- channel detection and two-photon excitation flow cytometry (T(sup 3)FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or in vivo. Real-time detection of fluorescent microbeads in the vasculature of mouse ear demonstrates the ability to do flow cytometry in vivo. The conditions required to perform quantitative in vivo cytometry on labeled cells will be presented.

[Difference of three standard curves of real-time reverse-transcriptase PCR in viable Vibrio parahaemolyticus quantification].

PubMed

Jin, Mengtong; Sun, Wenshuo; Li, Qin; Sun, Xiaohong; Pan, Yingjie; Zhao, Yong

2014-04-04

We evaluated the difference of three standard curves in quantifying viable Vibrio parahaemolyticus in samples by real-time reverse-transcriptase PCR (Real-time RT-PCR). The standard curve A was established by 10-fold diluted cDNA. The cDNA was reverse transcripted after RNA synthesized in vitro. The standard curve B and C were established by 10-fold diluted cDNA. The cDNA was synthesized after RNA isolated from Vibrio parahaemolyticus in pure cultures (10(8) CFU/mL) and shrimp samples (10(6) CFU/g) (Standard curve A and C were proposed for the first time). Three standard curves were performed to quantitatively detect V. parahaemolyticus in six samples, respectively (Two pure cultured V. parahaemolyticus samples, two artificially contaminated cooked Litopenaeus vannamei samples and two artificially contaminated Litopenaeus vannamei samples). Then we evaluated the quantitative results of standard curve and the plate counting results and then analysed the differences. The three standard curves all show a strong linear relationship between the fractional cycle number and V. parahaemolyticus concentration (R2 > 0.99); The quantitative results of Real-time PCR were significantly (p < 0.05) lower than the results of plate counting. The relative errors compared with the results of plate counting ranked standard curve A (30.0%) > standard curve C (18.8%) > standard curve B (6.9%); The average differences between standard curve A and standard curve B and C were - 2.25 Lg CFU/mL and - 0.75 Lg CFU/mL, respectively, and the mean relative errors were 48.2% and 15.9%, respectively; The average difference between standard curve B and C was among (1.47 -1.53) Lg CFU/mL and the average relative errors were among 19.0% - 23.8%. Standard curve B could be applied to Real-time RT-PCR when quantify the number of viable microorganisms in samples.

Comprehensive Multiplex One-Step Real-Time TaqMan qRT-PCR Assays for Detection and Quantification of Hemorrhagic Fever Viruses

PubMed Central

Li, Jiandong; Qu, Jing; He, Chengcheng; Zhang, Shuo; Li, Chuan; Zhang, Quanfu; Liang, Mifang; Li, Dexin

2014-01-01

Background Viral hemorrhagic fevers (VHFs) are a group of animal and human illnesses that are mostly caused by several distinct families of viruses including bunyaviruses, flaviviruses, filoviruses and arenaviruses. Although specific signs and symptoms vary by the type of VHF, initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of hemorrhagic fever viruses (HFVs) are important for effective case management and control of the spread of VHFs. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay is one of the reliable and desirable methods for specific detection and quantification of virus load. Multiplex PCR assay has the potential to produce considerable savings in time and resources in the laboratory detection. Results Primers/probe sets were designed based on appropriate specific genes for each of 28 HFVs which nearly covered all the HFVs, and identified with good specificity and sensitivity using monoplex assays. Seven groups of multiplex one-step real-time qRT-PCR assays in a universal experimental system were then developed by combining all primers/probe sets into 4-plex reactions and evaluated with serial dilutions of synthesized viral RNAs. For all the multiplex assays, no cross-reactivity with other HFVs was observed, and the limits of detection were mainly between 45 and 150 copies/PCR. The reproducibility was satisfactory, since the coefficient of variation of Ct values were all less than 5% in each dilution of synthesized viral RNAs for both intra-assays and inter-assays. Evaluation of the method with available clinical serum samples collected from HFRS patients, SFTS patients and Dengue fever patients showed high sensitivity and specificity of the related multiplex assays on the clinical specimens. Conclusions Overall, the comprehensive multiplex one-step real-time qRT-PCR assays were established in this study, and proved to be specific, sensitive, stable and easy to serve as a useful tool for rapid detection of HFVs. PMID:24752452

Guidelines for Implementing a Real Estate Cooperative Education Program.

ERIC Educational Resources Information Center

Pearson, Thomas R.

Background information and guidelines are provided for the development of cooperative education programs for real estate industry personnel. The first section outlines the operation of cooperative education programs and presents two organizational plans: the alternating plan, where students attend class full-time and work full-time during…

Continuous, real time microwave plasma element sensor

DOEpatents

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

1995-01-01

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

Development of a real-time PCR assay for the detection and identification of Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri.

PubMed

Iwase, Tadayuki; Seki, Keiko; Shinji, Hitomi; Mizunoe, Yoshimitsu; Masuda, Shogo

2007-10-01

Staphylococcus capitis, Staphylococcus haemolyticus and Staphylococcus warneri are coagulase-negative staphylococci. Each species has different characteristics, and a difference in pathology is also seen in compromised hosts. Therefore, the development of a species-specific simple detection method for the identification of these staphylococci is important. Here, a species-specific real-time PCR assay is reported that targets the superoxide dismutase A-encoding gene of these bacteria. Primers were designed with a base that was non-complementary with regard to the other bacteria. This base was at the 3' end of the primer (3' mismatch primer) and conferred high specificity. These primers were then evaluated using real-time PCR. They reacted only with the target bacterium. In addition, stable quantitative reactions were observed when experiments were performed using genomic DNA extracted from varying numbers of staphylococci cells (10(1)-10(7) cells). These results indicate that this method is useful for the identification and quantitative analysis of S. capitis, S. haemolyticus and S. warneri.

NAIMA as a solution for future GMO diagnostics challenges.

PubMed

Dobnik, David; Morisset, Dany; Gruden, Kristina

2010-03-01

In the field of genetically modified organism (GMO) diagnostics, real-time PCR has been the method of choice for target detection and quantification in most laboratories. Despite its numerous advantages, however, the lack of a true multiplexing option may render real-time PCR less practical in the face of future GMO detection challenges such as the multiplicity and increasing complexity of new transgenic events, as well as the repeated occurrence of unauthorized GMOs on the market. In this context, we recently reported the development of a novel multiplex quantitative DNA-based target amplification method, named NASBA implemented microarray analysis (NAIMA), which is suitable for sensitive, specific and quantitative detection of GMOs on a microarray. In this article, the performance of NAIMA is compared with that of real-time PCR, the focus being their performances in view of the upcoming challenge to detect/quantify an increasing number of possible GMOs at a sustainable cost and affordable staff effort. Finally, we present our conclusions concerning the applicability of NAIMA for future use in GMO diagnostics.

Detection of Leishmania infantum by real-time PCR in a canine blood bank.

PubMed

Tabar, M D; Roura, X; Francino, O; Altet, L; Ruiz de Gopegui, R

2008-07-01

Risk for transmission of Leishmania infantum from blood products has been largely demonstrated in human and veterinary literature. Appropriate screening of canine blood donors is important especially in an endemic area such as Barcelona (Spain). The purpose of this study was to evaluate the presence of L infantum DNA parasites by real-time quantitative PCR in our canine blood bank. Samples from blood products obtained from 92 canine blood donors were assayed for L infantum by means of real-time PCR amplification and quantification. The prevalence of quantitative PCR-positive blood samples among healthy seronegative blood donors was 19.6 per cent. The results of this study show that L infantum infection is common in canine blood donors and their blood products in an endemic area, despite a negative commercial serological screening for infectious diseases. Therefore, screening by PCR should be included in an integrated approach to evaluate L infantum infection among potential blood donors.

Integrated payload and mission planning, phase 3. Volume 3: Ground real-time mission operations

NASA Technical Reports Server (NTRS)

White, W. J.

1977-01-01

The payloads tentatively planned to fly on the first two Spacelab missions were analyzed to examine the cost relationships of providing mission operations support from onboard vs the ground-based Payload Operations Control Center (POCC). The quantitative results indicate that use of a POCC, with data processing capability, to support real-time mission operations is the most cost effective case.

RFLP Analysis and Allelic Discrimination with Real-Time PCR Using the Human Lactase Persistence Trait: A Pair of Molecular Genetic Investigations

ERIC Educational Resources Information Center

Weinlander, Kenneth M.; Hall, David J.; De Stasio, Elizabeth A.

2010-01-01

We describe here two open-ended laboratory investigations for an undergraduate laboratory course that uses students' DNA as templates for quantitative real-time PCR and for traditional PCR followed by RFLP analysis. Students are captivated by the immediacy of the application and the relevance of the genotypes and traits, lactase persistence or…

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

PubMed Central

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

2005-01-01

In this study, an immunomagnetic capture method and a real-time reverse transcription-PCR assay were used to quantify hepatitis A virus (HAV) in green onion and strawberry rinses. This combined protocol detected as low as 0.5 PFU HAV in produce rinses and concentrated HAV levels up to 20-fold. PMID:16151164

Development and comparison of TaqMan-based real-time PCR assays for detection and differentiation of Ralstonia solanacearum strains

USDA-ARS?s Scientific Manuscript database

Bacterial wilt caused by Ralstonia solanacearum is destructive to many plant species worldwide. The race 3 biovar 2 (r3b2) strains of R. solanacearum infect potatoes in temperature climates and are listed as select agents by the U.S. government. TaqMan-based real-time quantitative PCR (qPCR) is comm...

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

PubMed

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

2016-09-01

The Hologic Aptima(®) HIV-1 Quant Dx assay (Aptima HIV) is a real-time transcription-mediated amplification method CE-approved for use in diagnosis and monitoring of HIV-1 infection. The analytical performance of this new assay was compared to the FDA-approved Abbott RealTime HIV-1 (RealTime). The evaluation was performed using 220 clinical plasma samples, the WHO 3rd HIV-1 International Standard, and the QCMD HIV-1 RNA EQA. Concordance on qualitative results, correlation between quantitative results, accuracy, and reproducibility of viral load data were analyzed. The ability to measure HIV-1 subtypes was assessed on the second WHO International Reference Preparation Panel for HIV-1 Subtypes. With clinical samples, inter-assay agreement for qualitative results was high (91.8%) with Cohen's kappa statistic equal to 0.836. For samples with quantitative results in both assays (n = 93), Lin's concordance correlation coefficient was 0.980 (P < 0.0001) and mean differences of measurement, conducted according to Bland-Altman method, was low (0.115 log10  copies/ml). The Aptima HIV quantified the WHO 3rd HIV-1 International Standard diluted from 2000 to 31 cp/ml (5,700-88 IU/ml) at expected values with excellent linearity (R(2)  > 0.970) and showed higher sensitivity compared to RealTime being able to detect HIV-1 RNA in 10 out of 10 replicates containing down to 7 cp/ml (20 IU/ml). Reproducibility was very high, even at low HIV-1 RNA values. The Aptima HIV was able to detect and accurately quantify all the main HIV-1 subtypes in both reference panels and clinical samples. Besides excellent performance, Aptima HIV shows full automation, ease of use, and improved workflow compared to RealTime. J. Med. Virol. 88:1535-1544, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Real-Time Mapping Spectroscopy on the Ground, in the Air, and in Space

NASA Astrophysics Data System (ADS)

Thompson, D. R.; Allwood, A.; Chien, S.; Green, R. O.; Wettergreen, D. S.

2016-12-01

Real-time data interpretation can benefit both remote in situ exploration and remote sensing. Basic analyses at the sensor can monitor instrument performance and reveal invisible science phenomena in real time. This promotes situational awareness for remote robotic explorers or campaign decision makers, enabling adaptive data collection, reduced downlink requirements, and coordinated multi-instrument observations. Fast analysis is ideal for mapping spectrometers providing unambiguous, quantitative geophysical measurements. This presentation surveys recent computational advances in real-time spectroscopic analysis for Earth science and planetary exploration. Spectral analysis at the sensor enables new operations concepts that significantly improve science yield. Applications include real-time detection of fugitive greenhouse emissions by airborne monitoring, real-time cloud screening and mineralogical mapping by orbital spectrometers, and adaptive measurement by the PIXL instrument on the Mars 2020 rover. Copyright 2016 California Institute of Technology. All Rights Reserved. We acknowledge support of the US Government, NASA, the Earth Science Division and Terrestrial Ecology program.

Development of an on-site rapid real-time polymerase chain reaction system and the characterization of suitable DNA polymerases for TaqMan probe technology.

PubMed

Furutani, Shunsuke; Naruishi, Nahoko; Hagihara, Yoshihisa; Nagai, Hidenori

2016-08-01

On-site quantitative analyses of microorganisms (including viruses) by the polymerase chain reaction (PCR) system are significantly influencing medical and biological research. We have developed a remarkably rapid and portable real-time PCR system that is based on microfluidic approaches. Real-time PCR using TaqMan probes consists of a complex reaction. Therefore, in a rapid real-time PCR, the optimum DNA polymerase must be estimated by using actual real-time PCR conditions. In this study, we compared the performance of three DNA polymerases in actual PCR conditions using our rapid real-time PCR system. Although KAPA2G Fast HS DNA Polymerase has the highest enzymatic activity among them, SpeedSTAR HS DNA Polymerase exhibited better performance to rapidly increase the fluorescence signal in an actual real-time PCR using TaqMan probes. Furthermore, we achieved rapid detection of Escherichia coli in 7 min by using SpeedSTAR HS DNA Polymerase with the same sensitivity as that of a conventional thermal cycler.

Versatile utilization of real-time intraoperative contrast-enhanced ultrasound in cranial neurosurgery: technical note and retrospective case series.

PubMed

Lekht, Ilya; Brauner, Noah; Bakhsheshian, Joshua; Chang, Ki-Eun; Gulati, Mittul; Shiroishi, Mark S; Grant, Edward G; Christian, Eisha; Zada, Gabriel

2016-03-01

Intraoperative contrast-enhanced ultrasound (iCEUS) offers dynamic imaging and provides functional data in real time. However, no standardized protocols or validated quantitative data exist to guide its routine use in neurosurgery. The authors aimed to provide further clinical data on the versatile application of iCEUS through a technical note and illustrative case series. Five patients undergoing craniotomies for suspected tumors were included. iCEUS was performed using a contrast agent composed of lipid shell microspheres enclosing perflutren (octafluoropropane) gas. Perfusion data were acquired through a time-intensity curve analysis protocol obtained using iCEUS prior to biopsy and/or resection of all lesions. Three primary tumors (gemistocytic astrocytoma, glioblastoma multiforme, and meningioma), 1 metastatic lesion (melanoma), and 1 tumefactive demyelinating lesion (multiple sclerosis) were assessed using real-time iCEUS. No intraoperative complications occurred following multiple administrations of contrast agent in all cases. In all neoplastic cases, iCEUS replicated enhancement patterns observed on preoperative Gd-enhanced MRI, facilitated safe tumor debulking by differentiating neoplastic tissue from normal brain parenchyma, and helped identify arterial feeders and draining veins in and around the surgical cavity. Intraoperative CEUS was also useful in guiding a successful intraoperative needle biopsy of a cerebellar tumefactive demyelinating lesion obtained during real-time perfusion analysis. Intraoperative CEUS has potential for safe, real-time, dynamic contrast-based imaging for routine use in neurooncological surgery and image-guided biopsy. Intraoperative CEUS eliminates the effect of anatomical distortions associated with standard neuronavigation and provides quantitative perfusion data in real time, which may hold major implications for intraoperative diagnosis, tissue differentiation, and quantification of extent of resection. Further prospective studies will help standardize the role of iCEUS in neurosurgery.

Real-time reverse transcription polymerase chain reaction method for detection of Canine distemper virus modified live vaccine shedding for differentiation from infection with wild-type strains.

PubMed

Wilkes, Rebecca P; Sanchez, Elena; Riley, Matthew C; Kennedy, Melissa A

2014-01-01

Canine distemper virus (CDV) remains a common cause of infectious disease in dogs, particularly in high-density housing situations such as shelters. Vaccination of all dogs against CDV is recommended at the time of admission to animal shelters and many use a modified live virus (MLV) vaccine. From a diagnostic standpoint for dogs with suspected CDV infection, this is problematic because highly sensitive diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) tests are able to detect MLV virus in clinical samples. Real-time PCR can be used to quantitate amount of virus shedding and can differentiate vaccine strains from wild-type strains when shedding is high. However, differentiation by quantitation is not possible in vaccinated animals during acute infection, when shedding is low and could be mistaken for low level vaccine virus shedding. While there are gel-based RT-PCR assays for differentiation of vaccine strains from field strains based on sequence differences, the sensitivity of these assays is unable to match that of the real-time RT-PCR assay currently used in the authors' laboratory. Therefore, a real-time RT-PCR assay was developed that detects CDV MLV vaccine strains and distinguishes them from wild-type strains based on nucleotide sequence differences, rather than the amount of viral RNA in the sample. The test is highly sensitive, with detection of as few as 5 virus genomic copies (corresponding to 10(-1) TCID(50)). Sequencing of the DNA real-time products also allows phylogenetic differentiation of the wild-type strains. This test will aid diagnosis during outbreaks of CDV in recently vaccinated animals.

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

PubMed

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

2016-12-20

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

Development of fluorescent glucose bioprobes and their application on real-time and quantitative monitoring of glucose uptake in living cells.

PubMed

Lee, Hyang Yeon; Lee, Jae Jeong; Park, Jongmin; Park, Seung Bum

2011-01-03

We developed a novel fluorescent glucose bioprobe, GB2-Cy3, for the real-time and quantitative monitoring of glucose uptake in living cells. We synthesized a series of fluorescent glucose analogues by adding Cy3 fluorophores to the α-anomeric position of D-glucose through various linkers. Systematic and quantitative analysis of these Cy3-labeled glucose analogues revealed that GB2-Cy3 was the ideal fluorescent glucose bioprobe. The cellular uptake of this probe competed with the cellular uptake of D-glucose in the media and was mediated by a glucose-specific transport system, and not by passive diffusion. Flow cytometry and fluorescence microscopy analyses revealed that GB2-Cy3 is ten times more sensitive than 2-NBDG, a leading fluorescent glucose bioprobe. GB2-Cy3 can also be utilized for the quantitative flow cytometry monitoring of glucose uptake in metabolically active C2C12 myocytes under various treatment conditions. As opposed to a glucose uptake assay performed by using radioisotope-labeled deoxy-D-glucose and a scintillation counter, GB2-Cy3 allows the real-time monitoring of glucose uptake in living cells under various experimental conditions by using fluorescence microscopy or confocal laser scanning microscopy (CLSM). Therefore, we believe that GB2-Cy3 can be utilized in high-content screening (HCS) for the discovery of novel therapeutic agents and for making significant advances in biomedical studies and diagnosis of various diseases, especially metabolic diseases. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

2013-01-01

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

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

PubMed Central

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

2013-01-01

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

qF-SSOP: real-time optical property corrected fluorescence imaging

PubMed Central

Valdes, Pablo A.; Angelo, Joseph P.; Choi, Hak Soo; Gioux, Sylvain

2017-01-01

Fluorescence imaging is well suited to provide image guidance during resections in oncologic and vascular surgery. However, the distorting effects of tissue optical properties on the emitted fluorescence are poorly compensated for on even the most advanced fluorescence image guidance systems, leading to subjective and inaccurate estimates of tissue fluorophore concentrations. Here we present a novel fluorescence imaging technique that performs real-time (i.e., video rate) optical property corrected fluorescence imaging. We perform full field of view simultaneous imaging of tissue optical properties using Single Snapshot of Optical Properties (SSOP) and fluorescence detection. The estimated optical properties are used to correct the emitted fluorescence with a quantitative fluorescence model to provide quantitative fluorescence-Single Snapshot of Optical Properties (qF-SSOP) images with less than 5% error. The technique is rigorous, fast, and quantitative, enabling ease of integration into the surgical workflow with the potential to improve molecular guidance intraoperatively. PMID:28856038

SNSMIL, a real-time single molecule identification and localization algorithm for super-resolution fluorescence microscopy

PubMed Central

Tang, Yunqing; Dai, Luru; Zhang, Xiaoming; Li, Junbai; Hendriks, Johnny; Fan, Xiaoming; Gruteser, Nadine; Meisenberg, Annika; Baumann, Arnd; Katranidis, Alexandros; Gensch, Thomas

2015-01-01

Single molecule localization based super-resolution fluorescence microscopy offers significantly higher spatial resolution than predicted by Abbe’s resolution limit for far field optical microscopy. Such super-resolution images are reconstructed from wide-field or total internal reflection single molecule fluorescence recordings. Discrimination between emission of single fluorescent molecules and background noise fluctuations remains a great challenge in current data analysis. Here we present a real-time, and robust single molecule identification and localization algorithm, SNSMIL (Shot Noise based Single Molecule Identification and Localization). This algorithm is based on the intrinsic nature of noise, i.e., its Poisson or shot noise characteristics and a new identification criterion, QSNSMIL, is defined. SNSMIL improves the identification accuracy of single fluorescent molecules in experimental or simulated datasets with high and inhomogeneous background. The implementation of SNSMIL relies on a graphics processing unit (GPU), making real-time analysis feasible as shown for real experimental and simulated datasets. PMID:26098742

MOLD SPECIFIC QUANTITATIVE PCR: THE EMERGING STANDARD IN MOLD ANALYSIS

EPA Science Inventory

Today I will talk about the use of quantitative or Real time PCR for the standardized identification and quantification of molds. There are probably at least 100,000 species of molds or fungi. But there are actually about 100 typically found indoors. Some pose a threat to human...

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

PubMed

Walsh, Adrian A

2017-01-01

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

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

NASA Astrophysics Data System (ADS)

Liu, P.

2013-12-01

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

Calibration-free assays on standard real-time PCR devices

PubMed Central

Debski, Pawel R.; Gewartowski, Kamil; Bajer, Seweryn; Garstecki, Piotr

2017-01-01

Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration. PMID:28327545

Calibration-free assays on standard real-time PCR devices

NASA Astrophysics Data System (ADS)

Debski, Pawel R.; Gewartowski, Kamil; Bajer, Seweryn; Garstecki, Piotr

2017-03-01

Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration.

Quantitative Evaluation of PET Respiratory Motion Correction Using MR Derived Simulated Data

NASA Astrophysics Data System (ADS)

Polycarpou, Irene; Tsoumpas, Charalampos; King, Andrew P.; Marsden, Paul K.

2015-12-01

The impact of respiratory motion correction on quantitative accuracy in PET imaging is evaluated using simulations for variable patient specific characteristics such as tumor uptake and respiratory pattern. Respiratory patterns from real patients were acquired, with long quiescent motion periods (type-1) as commonly observed in most patients and with long-term amplitude variability as is expected under conditions of difficult breathing (type-2). The respiratory patterns were combined with an MR-derived motion model to simulate real-time 4-D PET-MR datasets. Lung and liver tumors were simulated with diameters of 10 and 12 mm and tumor-to-background ratio ranging from 3:1 to 6:1. Projection data for 6- and 3-mm PET resolution were generated for the Philips Gemini scanner and reconstructed without and with motion correction using OSEM (2 iterations, 23 subsets). Motion correction was incorporated into the reconstruction process based on MR-derived motion fields. Tumor peak standardized uptake values (SUVpeak) were calculated from 30 noise realizations. Respiratory motion correction improves the quantitative performance with the greatest benefit observed for patients of breathing type-2. For breathing type-1 after applying motion correction, SUVpeak of 12-mm liver tumor with 6:1 contrast was increased by 46% for a current PET resolution (i.e., 6 mm) and by 47% for a higher PET resolution (i.e., 3 mm). Furthermore, the results of this study indicate that the benefit of higher scanner resolution is small unless motion correction is applied. In particular, for large liver tumor (12 mm) with low contrast (3:1) after motion correction, the SUVpeak was increased by 34% for 6-mm resolution and by 50% for a higher PET resolution (i.e., 3-mm resolution. This investigation indicates that there is a high impact of respiratory motion correction on tumor quantitative accuracy and that motion correction is important in order to benefit from the increased resolution of future PET scanners.

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

USGS Publications Warehouse

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

2002-01-01

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

Toward real-time tumor margin identification in image-guided robotic brain tumor resection

NASA Astrophysics Data System (ADS)

Hu, Danying; Jiang, Yang; Belykh, Evgenii; Gong, Yuanzheng; Preul, Mark C.; Hannaford, Blake; Seibel, Eric J.

2017-03-01

For patients with malignant brain tumors (glioblastomas), a safe maximal resection of tumor is critical for an increased survival rate. However, complete resection of the cancer is hard to achieve due to the invasive nature of these tumors, where the margins of the tumors become blurred from frank tumor to more normal brain tissue, but in which single cells or clusters of malignant cells may have invaded. Recent developments in fluorescence imaging techniques have shown great potential for improved surgical outcomes by providing surgeons intraoperative contrast-enhanced visual information of tumor in neurosurgery. The current near-infrared (NIR) fluorophores, such as indocyanine green (ICG), cyanine5.5 (Cy5.5), 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX), are showing clinical potential to be useful in targeting and guiding resections of such tumors. Real-time tumor margin identification in NIR imaging could be helpful to both surgeons and patients by reducing the operation time and space required by other imaging modalities such as intraoperative MRI, and has the potential to integrate with robotically assisted surgery. In this paper, a segmentation method based on the Chan-Vese model was developed for identifying the tumor boundaries in an ex-vivo mouse brain from relatively noisy fluorescence images acquired by a multimodal scanning fiber endoscope (mmSFE). Tumor contours were achieved iteratively by minimizing an energy function formed by a level set function and the segmentation model. Quantitative segmentation metrics based on tumor-to-background (T/B) ratio were evaluated. Results demonstrated feasibility in detecting the brain tumor margins at quasi-real-time and has the potential to yield improved precision brain tumor resection techniques or even robotic interventions in the future.

Real-time understanding of lignocellulosic bioethanol fermentation by Raman spectroscopy

PubMed Central

2013-01-01

Background A substantial barrier to commercialization of lignocellulosic ethanol production is a lack of process specific sensors and associated control strategies that are essential for economic viability. Current sensors and analytical techniques require lengthy offline analysis or are easily fouled in situ. Raman spectroscopy has the potential to continuously monitor fermentation reactants and products, maximizing efficiency and allowing for improved process control. Results In this paper we show that glucose and ethanol in a lignocellulosic fermentation can be accurately monitored by a 785 nm Raman spectroscopy instrument and novel immersion probe, even in the presence of an elevated background thought to be caused by lignin-derived compounds. Chemometric techniques were used to reduce the background before generating calibration models for glucose and ethanol concentration. The models show very good correlation between the real-time Raman spectra and the offline HPLC validation. Conclusions Our results show that the changing ethanol and glucose concentrations during lignocellulosic fermentation processes can be monitored in real-time, allowing for optimization and control of large scale bioconversion processes. PMID:23425590

Processor core for real time background identification of HD video based on OpenCV Gaussian mixture model algorithm

NASA Astrophysics Data System (ADS)

Genovese, Mariangela; Napoli, Ettore

2013-05-01

The identification of moving objects is a fundamental step in computer vision processing chains. The development of low cost and lightweight smart cameras steadily increases the request of efficient and high performance circuits able to process high definition video in real time. The paper proposes two processor cores aimed to perform the real time background identification on High Definition (HD, 1920 1080 pixel) video streams. The implemented algorithm is the OpenCV version of the Gaussian Mixture Model (GMM), an high performance probabilistic algorithm for the segmentation of the background that is however computationally intensive and impossible to implement on general purpose CPU with the constraint of real time processing. In the proposed paper, the equations of the OpenCV GMM algorithm are optimized in such a way that a lightweight and low power implementation of the algorithm is obtained. The reported performances are also the result of the use of state of the art truncated binary multipliers and ROM compression techniques for the implementation of the non-linear functions. The first circuit has commercial FPGA devices as a target and provides speed and logic resource occupation that overcome previously proposed implementations. The second circuit is oriented to an ASIC (UMC-90nm) standard cell implementation. Both implementations are able to process more than 60 frames per second in 1080p format, a frame rate compatible with HD television.

Real-time fluorescence ligase chain reaction for sensitive detection of single nucleotide polymorphism based on fluorescence resonance energy transfer.

PubMed

Sun, Yueying; Lu, Xiaohui; Su, Fengxia; Wang, Limei; Liu, Chenghui; Duan, Xinrui; Li, Zhengping

2015-12-15

Most of practical methods for detection of single nucleotide polymorphism (SNP) need at least two steps: amplification (usually by PCR) and detection of SNP by using the amplification products. Ligase chain reaction (LCR) can integrate the amplification and allele discrimination in one step. However, the detection of LCR products still remains a great challenge for highly sensitive and quantitative SNP detection. Herein, a simple but robust strategy for real-time fluorescence LCR has been developed for highly sensitive and quantitative SNP detection. A pair of LCR probes are firstly labeled with a fluorophore and a quencher, respectively. When the pair of LCR probes are ligated in LCR, the fluorophore will be brought close to the quencher, and thus, the fluorescence will be specifically quenched by fluorescence resonance energy transfer (FRET). The decrease of fluorescence intensity resulted from FRET can be real-time monitored in the LCR process. With the proposed real-time fluorescence LCR assay, 10 aM DNA targets or 100 pg genomic DNA can be accurately determined and as low as 0.1% mutant DNA can be detected in the presence of a large excess of wild-type DNA, indicating the high sensitivity and specificity. The real-time measuring does not require the detection step after LCR and gives a wide dynamic range for detection of DNA targets (from 10 aM to 1 pM). As LCR has been widely used for detection of SNP, DNA methylation, mRNA and microRNA, the real-time fluorescence LCR assay shows great potential for various genetic analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Enzyme-modified carbon-fiber microelectrode for the quantification of dynamic fluctuations of nonelectroactive analytes using fast-scan cyclic voltammetry.

PubMed

Lugo-Morales, Leyda Z; Loziuk, Philip L; Corder, Amanda K; Toups, J Vincent; Roberts, James G; McCaffrey, Katherine A; Sombers, Leslie A

2013-09-17

Neurotransmission occurs on a millisecond time scale, but conventional methods for monitoring nonelectroactive neurochemicals are limited by slow sampling rates. Despite a significant global market, a sensor capable of measuring the dynamics of rapidly fluctuating, nonelectroactive molecules at a single recording site with high sensitivity, electrochemical selectivity, and a subsecond response time is still lacking. To address this need, we have enabled the real-time detection of dynamic glucose fluctuations in live brain tissue using background-subtracted, fast-scan cyclic voltammetry. The novel microbiosensor consists of a simple carbon fiber surface modified with an electrodeposited chitosan hydrogel encapsulating glucose oxidase. The selectivity afforded by voltammetry enables quantitative and qualitative measurements of enzymatically generated H2O2 without the need for additional strategies to eliminate interfering agents. The microbiosensors possess a sensitivity and limit of detection for glucose of 19.4 ± 0.2 nA mM(-1) and 13.1 ± 0.7 μM, respectively. They are stable, even under deviations from physiological normoxic conditions, and show minimal interference from endogenous electroactive substances. Using this approach, we have quantitatively and selectively monitored pharmacologically evoked glucose fluctuations with unprecedented chemical and spatial resolution. Furthermore, this novel biosensing strategy is widely applicable to the immobilization of any H2O2 producing enzyme, enabling rapid monitoring of many nonelectroactive enzyme substrates.

A video-based real-time adaptive vehicle-counting system for urban roads.

PubMed

Liu, Fei; Zeng, Zhiyuan; Jiang, Rong

2017-01-01

In developing nations, many expanding cities are facing challenges that result from the overwhelming numbers of people and vehicles. Collecting real-time, reliable and precise traffic flow information is crucial for urban traffic management. The main purpose of this paper is to develop an adaptive model that can assess the real-time vehicle counts on urban roads using computer vision technologies. This paper proposes an automatic real-time background update algorithm for vehicle detection and an adaptive pattern for vehicle counting based on the virtual loop and detection line methods. In addition, a new robust detection method is introduced to monitor the real-time traffic congestion state of road section. A prototype system has been developed and installed on an urban road for testing. The results show that the system is robust, with a real-time counting accuracy exceeding 99% in most field scenarios.

A video-based real-time adaptive vehicle-counting system for urban roads

PubMed Central

2017-01-01

In developing nations, many expanding cities are facing challenges that result from the overwhelming numbers of people and vehicles. Collecting real-time, reliable and precise traffic flow information is crucial for urban traffic management. The main purpose of this paper is to develop an adaptive model that can assess the real-time vehicle counts on urban roads using computer vision technologies. This paper proposes an automatic real-time background update algorithm for vehicle detection and an adaptive pattern for vehicle counting based on the virtual loop and detection line methods. In addition, a new robust detection method is introduced to monitor the real-time traffic congestion state of road section. A prototype system has been developed and installed on an urban road for testing. The results show that the system is robust, with a real-time counting accuracy exceeding 99% in most field scenarios. PMID:29135984

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

PubMed

Yang, Litao; Liang, Wanqi; Jiang, Lingxi; Li, Wenquan; Cao, Wei; Wilson, Zoe A; Zhang, Dabing

2008-06-04

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. 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. 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.

Molecular Mechanisms of Increased Heart Rate in Shenxianshengmai-treated Bradycardia Rabbits

PubMed Central

Liu, Zhou-Ying; Huang, Jian; Liu, Na-Na; Zheng, Min; Zhao, Tao; Zhao, Bu-Chang; Wang, Yi-Min; Pu, Jie-Lin

2017-01-01

Background: The molecular mechanisms of Shenxianshengmai (SXSM), a traditional Chinese medicine, on bradycardia have been incompletely understood. The study tried to investigate the gene expression profile and proteomics of bradycardia rabbits’ hearts after SXSM treatment. Methods: Twenty-four adult rabbits were randomly assigned in four groups: sham, model, model plus SXSM treatment, and sham plus SXSM treatment groups. Heart rate was recorded in all rabbits. Then, total RNA of atria and proteins of ventricle were isolated and quantified, respectively. Gene expression profiling was conducted by gene expression chip, and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to confirm the results of gene expression chip. We used isobaric tags for elative and absolute quantitation and Western blotting to identify altered proteins after SXSM treatment. Results: There was a constant decrease in the mean heart rate (32%, from 238 ± 6 beats/min to 149 ± 12 beats/min) after six weeks in model compared with that in sham group. This effect was partially reversed by 4-week SXSM treatment. Complementary DNA microarray demonstrated that the increased acetylcholinesterase and reduced nicotinic receptor were take responsibility for the increased heart rate. In addition, proteins involved in calcium handling and signaling were affected by SXSM treatment. Real-time RT-PCR verified the results from gene chip. Results from proteomics demonstrated that SXSM enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle in ventricular myocardium to improve ATP generation. Conclusions: Long-term SXSM stimulates sympathetic transmission by increasing the expression of acetylcholinesterase and reduces the expression of nicotinic receptor to increase heart rate. SXSM also restored the calcium handling genes and altered genes involved in signaling. In addition, SXSM improves the ATP supply of ventricular myocardium by increasing proteins involved in TCA cycle and oxidation-respiratory chain. PMID:28091410

Effects of Pistacia atlantica subsp. kurdica on Growth and Aflatoxin Production by Aspergillus parasiticus

PubMed Central

Khodavaisy, Sadegh; Rezaie, Sassan; Noorbakhsh, Fatemeh; Baghdadi, Elham; Sharifynia, Somayeh; Aala, Farzad

2016-01-01

Background Aflatoxins are highly toxic secondary metabolites mainly produced by Aspergillus parasiticus. This species can contaminate a wide range of agricultural commodities, including cereals, peanuts, and crops in the field. In recent years, research on medicinal herbs, such as Pistacia atlantica subsp. kurdica, have led to reduced microbial growth, and these herbs also have a particular effect on the production of aflatoxins as carcinogenic compounds. Objectives In this study, we to examine P. atlantica subsp. kurdica as a natural compound used to inhibit the growth of A. parasiticus and to act as an anti-mycotoxin. Materials and Methods In vitro antifungal susceptibility testing of P. atlantica subsp. kurdica for A. parasiticus was performed according to CLSI document M38-A2. The rate of aflatoxin production was determined using the HPLC technique after exposure to different concentrations (62.5 - 125 mg/mL) of the gum. The changes in expression levels of the aflR gene were analyzed with a quantitative real-time PCR assay. Results The results showed that P. atlantica subsp. kurdica can inhibit A. parasiticus growth at a concentration of 125 mg/mL. HPLC results revealed a significant decrease in aflatoxin production with 125 mg/mL of P. atlantica subsp. kurdica, and AFL-B1 production was entirely inhibited. Based on quantitative real-time PCR results, the rate of aflR gene expression was significantly decreased after treatment with P. atlantica subsp. kurdica. Conclusions Pistacia atlantica subsp. kurdica has anti-toxic properties in addition to an inhibitory effect on A. parasiticus growth, and is able to decrease aflatoxin production effectively in a dose-dependent manner. Therefore, this herbal extract maybe considered a potential anti-mycotoxin agent in medicine or industrial agriculture. PMID:27800127

Comparative analysis of detection limits and specificity of molecular diagnostic markers for three pathogens (Microsporidia, Nosema spp.) in the key pollinators Apis mellifera and Bombus terrestris.

PubMed

Erler, Silvio; Lommatzsch, Stefanie; Lattorff, H Michael G

2012-04-01

Global pollinator decline has recently been discussed in the context of honey and bumble bee infections from various pathogens including viruses, bacteria, microsporidia and mites. The microsporidian pathogens Nosema apis, Nosema ceranae and Nosema bombi may in fact be major candidates contributing to this decline. Different molecular and non-molecular detection methods have been developed; however, a comparison, especially of the highly sensitive PCR based methods, is currently lacking. Here, we present the first comparative quantitative real-time PCR study of nine Nosema spp. primers within the framework of primer specificity and sensitivity. With the help of dilution series of defined numbers of spores, we reveal six primer pairs amplifying N. apis, six for N. bombi and four for N. ceranae. All appropriate primer pairs detected an amount of at least 10(4) spores, the majority of which were even as sensitive to detect such low amounts as 10(3) to ten spores. Species specificity of primers was observed for N. apis and N. bombi, but not for N. ceranae. Additionally, we did not find any significant correlation for the amplified fragments with PCR efficiency or the limit of detection. We discuss our findings on the background of false positive and negative results using quantitative real-time PCR. On the basis of these results, future research might be based on appropriate primer selection depending on the experimental needs. Primers may be selected on the basis of specificity or sensitivity. Pathogen species and load may be determined with higher precision enhancing all kinds of diagnostic studies.

Tissue Inhibitor of Metalloproteinase-2 Suppresses Collagen Synthesis in Cultured Keloid Fibroblasts

PubMed Central

Dohi, Teruyuki; Aoki, Masayo; Ogawa, Rei; Akaishi, Satoshi; Shimada, Takashi; Okada, Takashi; Hyakusoku, Hiko

2015-01-01

Background: Keloids are defined as a kind of dermal fibroproliferative disorder resulting from the accumulation of collagen. In the remodeling of extracellular matrix, the balance between matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is as critical as the proper production of extracellular matrix. We investigate the role of TIMPs and MMPs in the pathogenesis of keloids and examine the therapeutic potential of TIMP-2. Methods: The expression of TIMPs and MMPs in most inflamed parts of cultured keloid fibroblasts (KFs) and peripheral normal skin fibroblasts (PNFs) in the same individuals and the reactivity of KFs to cyclic mechanical stretch were analyzed by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay (n = 7). To evaluate the effect of treating KFs with TIMP-2, collagen synthesis was investigated by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, and microscopic analysis was used to examine the treatment effects of TIMP-2 on ex vivo cultures of keloid tissue (n = 6). Results: TIMP-2 was downregulated in cultured KFs compared with PNFs in the same individuals, and the reduction in TIMP-2 was exacerbated by cyclic mechanical stretch. Administration of TIMP-2 (200 or 300 ng/mL) significantly suppressed expression of Col1A2 and Col3A1 mRNA and collagen type I protein in KFs. TIMP-2 also significantly reduced the skin dermal and collagen bundle thickness in ex vivo cultures of keloid tissue. Conclusion: These results indicated that downregulation of TIMP-2 in KFs is a crucial event in the pathogenesis of keloids, and the TIMP-2 would be a promising candidate for the treatment of keloids. PMID:26495233

Point-of-Care Autofluorescence Imaging for Real-Time Sampling and Treatment Guidance of Bioburden in Chronic Wounds: First-in-Human Results

PubMed Central

DaCosta, Ralph S.; Kulbatski, Iris; Lindvere-Teene, Liis; Starr, Danielle; Blackmore, Kristina; Silver, Jason I.; Opoku, Julie; Wu, Yichao Charlie; Medeiros, Philip J.; Xu, Wei; Xu, Lizhen; Wilson, Brian C.; Rosen, Cheryl; Linden, Ron

2015-01-01

Background Traditionally, chronic wound infection is diagnosed by visual inspection under white light and microbiological sampling, which are subjective and suboptimal, respectively, thereby delaying diagnosis and treatment. To address this, we developed a novel handheld, fluorescence imaging device (PRODIGI) that enables non-contact, real-time, high-resolution visualization and differentiation of key pathogenic bacteria through their endogenous autofluorescence, as well as connective tissues in wounds. Methods and Findings This was a two-part Phase I, single center, non-randomized trial of chronic wound patients (male and female, ≥18 years; UHN REB #09-0015-A for part 1; UHN REB #12-5003 for part 2; clinicaltrials.gov Identifier: NCT01378728 for part 1 and NCT01651845 for part 2). Part 1 (28 patients; 54% diabetic foot ulcers, 46% non-diabetic wounds) established the feasibility of autofluorescence imaging to accurately guide wound sampling, validated against blinded, gold standard swab-based microbiology. Part 2 (12 patients; 83.3% diabetic foot ulcers, 16.7% non-diabetic wounds) established the feasibility of autofluorescence imaging to guide wound treatment and quantitatively assess treatment response. We showed that PRODIGI can be used to guide and improve microbiological sampling and debridement of wounds in situ, enabling diagnosis, treatment guidance and response assessment in patients with chronic wounds. PRODIGI is safe, easy to use and integrates into the clinical workflow. Clinically significant bacterial burden can be detected in seconds, quantitatively tracked over days-to-months and their biodistribution mapped within the wound bed, periphery, and other remote areas. Conclusions PRODIGI represents a technological advancement in wound sampling and treatment guidance for clinical wound care at the point-of-care. Trial Registration ClinicalTrials.gov NCT01651845; ClinicalTrials.gov NCT01378728 PMID:25790480

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

PubMed

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.

Genetic Modifiers of Cardiovascular Phenotype Caused by Elastin Haploinsufficiency Act by Extrinsic Noncomplementation*

PubMed Central

Kozel, Beth A.; Knutsen, Russell H.; Ye, Li; Ciliberto, Christopher H.; Broekelmann, Thomas J.; Mecham, Robert P.

2011-01-01

Elastin haploinsufficiency causes the cardiovascular complications associated with Williams-Beuren syndrome and isolated supravalvular aortic stenosis. Significant variability exists in the vascular pathology in these individuals. Using the Eln+/− mouse, we sought to identify the source of this variability. Following outcrossing of C57Bl/6J Eln+/−, two backgrounds were identified whose cardiovascular parameters deviated significantly from the parental strain. F1 progeny of the C57Bl/6J; Eln+/−x129X1/SvJ were more hypertensive and their arteries less compliant. In contrast, Eln+/− animals crossed to DBA/2J were protected from the pathologic changes associated with elastin insufficiency. Among the crosses, aortic elastin and collagen content did not correlate with quantitative vasculopathy traits. Quantitative trait locus analysis performed on F2 C57; Eln+/−x129 intercrosses identified highly significant peaks on chromosome 1 (LOD 9.7) for systolic blood pressure and on chromosome 9 (LOD 8.7) for aortic diameter. Additional peaks were identified that affect only Eln+/−, including a region upstream of Eln on chromosome 5 (LOD 4.5). Bioinformatic analysis of the quantitative trait locus peaks revealed several interesting candidates, including Ren1, Ncf1, and Nos1; genes whose functions are unrelated to elastic fiber assembly, but whose effects may synergize with elastin insufficiency to predispose to hypertension and stiffer blood vessels. Real time RT-PCR studies show background-specific increased expression of Ncf1 (a subunit of the NOX2 NAPDH oxidase) that parallel the presence of increased oxidative stress in Eln+/− aortas. This finding raises the possibility that polymorphisms in genes affecting the generation of reactive oxygen species alter cardiovascular function in individuals with elastin haploinsufficiency through extrinsic noncomplementation. PMID:22049077

Continuous, real time microwave plasma element sensor

DOEpatents

Woskov, P.P.; Smatlak, D.L.; Cohn, D.R.; Wittle, J.K.; Titus, C.H.; Surma, J.E.

1995-12-26

Microwave-induced plasma is described for continuous, real time trace element monitoring under harsh and variable conditions. The sensor includes a source of high power microwave energy and a shorted waveguide made of a microwave conductive, refractory material communicating with the source of the microwave energy to generate a plasma. The high power waveguide is constructed to be robust in a hot, hostile environment. It includes an aperture for the passage of gases to be analyzed and a spectrometer is connected to receive light from the plasma. Provision is made for real time in situ calibration. The spectrometer disperses the light, which is then analyzed by a computer. The sensor is capable of making continuous, real time quantitative measurements of desired elements, such as the heavy metals lead and mercury. 3 figs.

Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR

PubMed Central

Chemidlin Prévost-Bouré, Nicolas; Christen, Richard; Dequiedt, Samuel; Mougel, Christophe; Lelièvre, Mélanie; Jolivet, Claudy; Shahbazkia, Hamid Reza; Guillou, Laure; Arrouays, Dominique; Ranjard, Lionel

2011-01-01

Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1 / FF390. This in silico analysis of the specificity of FR1 / FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1 / FF390 for Fungi was validated in vitro by cloning - sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils. PMID:21931659

Determination of left ventricular volume, ejection fraction, and myocardial mass by real-time three-dimensional echocardiography

NASA Technical Reports Server (NTRS)

Qin, J. X.; Shiota, T.; Thomas, J. D.

2000-01-01

Reconstructed three-dimensional (3-D) echocardiography is an accurate and reproducible method of assessing left ventricular (LV) functions. However, it has limitations for clinical study due to the requirement of complex computer and echocardiographic analysis systems, electrocardiographic/respiratory gating, and prolonged imaging times. Real-time 3-D echocardiography has a major advantage of conveniently visualizing the entire cardiac anatomy in three dimensions and of potentially accurately quantifying LV volumes, ejection fractions, and myocardial mass in patients even in the presence of an LV aneurysm. Although the image quality of the current real-time 3-D echocardiographic methods is not optimal, its widespread clinical application is possible because of the convenient and fast image acquisition. We review real-time 3-D echocardiographic image acquisition and quantitative analysis for the evaluation of LV function and LV mass.

Determination of left ventricular volume, ejection fraction, and myocardial mass by real-time three-dimensional echocardiography.

PubMed

Qin, J X; Shiota, T; Thomas, J D

2000-11-01

Reconstructed three-dimensional (3-D) echocardiography is an accurate and reproducible method of assessing left ventricular (LV) functions. However, it has limitations for clinical study due to the requirement of complex computer and echocardiographic analysis systems, electrocardiographic/respiratory gating, and prolonged imaging times. Real-time 3-D echocardiography has a major advantage of conveniently visualizing the entire cardiac anatomy in three dimensions and of potentially accurately quantifying LV volumes, ejection fractions, and myocardial mass in patients even in the presence of an LV aneurysm. Although the image quality of the current real-time 3-D echocardiographic methods is not optimal, its widespread clinical application is possible because of the convenient and fast image acquisition. We review real-time 3-D echocardiographic image acquisition and quantitative analysis for the evaluation of LV function and LV mass.

Performance of NucliSens HIV-1 EasyQ Version 2.0 compared with six commercially available quantitative nucleic acid assays for detection of HIV-1 in China.

PubMed

Xu, Sihong; Song, Aijing; Nie, Jianhui; Li, Xiuhua; Wang, Youchun

2010-10-01

Six HIV-1 viral load assays have been widely used in China. These include the Cobas Amplicor HIV-1 Monitor Version 1.5 ('Amplicor'), Cobas AmpliPrep/Cobas TaqMan HIV-1 test Version 1.0 ('CAP/CTM'), Versant HIV-1 RNA Version 3.0 (branched DNA [bDNA]-based assay; 'Versant bDNA'), Abbott RealTime HIV-1 assay ('Abbott RealTime'), NucliSens HIV-1 QT (nucleic acid sequence-based amplification assay; 'NucliSens NASBA'), and NucliSens EasyQ HIV-1 Version 1.1 ('EasyQ V1.1'). Recently, an updated version of EasyQ V1.1, NucliSens EasyQ HIV-1 Version 2.0 ('EasyQ V2.0') was introduced into China. It is important to evaluate the impact of HIV-1 genotypes on the updated assay compared with the other commercial available assays in China. A total of 175 plasma samples with different HIV-1 clades prevalent in China were collected from treatment-naïve patients. The viral loads of those samples were determined with the seven HIV-1 viral load assays, and the quantitative differences between them were evaluated. Overall, EasyQ V2.0 exhibited a significant correlation (R = 0.769-0.850, p ≤ 0.001) and high agreement (94.77-97.13%, using the Bland-Altman model) with the other six assays. Although no significant differences between EasyQ V2.0 and the other six assays were observed when quantifying clade B' samples, there were statistically significant differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays when quantifying clade BC samples, and between EasyQ V2.0 and the Versant bDNA and Abbott RealTime assays when quantifying clade AE samples. For clade BC samples, the quantitative differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log(10) IU/mL in approximately 50% of samples and exceeded 1 log(10) IU/mL in approximately 15% of samples. For clade AE samples, the quantitative differences between EasyQ V2.0 and the CAP/CTM, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log(10) IU/mL in approximately 50% of samples, and the differences between EasyQ V2.0 and CAP/CTM exceeded 1 log(10) IU/mL in approximately 15% of samples. Genotypes may affect the quantification of HIV-1 RNA, especially in clade BC samples with respect to EasyQ V2.0 and the Amplicor, Versant bDNA, or Abbott RealTime assays, and in clade AE samples with respect to EasyQ V2.0 and the Versant bDNA or Abbott RealTime assays. It is therefore strongly suggested that, where possible, the HIV-1 viral load in infected patients be quantified at follow-up by the same version of the same assay that was used initially.

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

USDA-ARS?s Scientific Manuscript database

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

Sexing chick mRNA: A protocol based on quantitative real-time polymerase chain reaction.

PubMed

Wan, Z; Lu, Y; Rui, L; Yu, X; Li, Z

2017-03-01

The accurate identification of sex in birds is important for research on avian sex determination and differentiation. Polymerase chain reaction (PCR)-based methods have been widely applied for the molecular sexing of birds. However, these methods have used genomic DNA. Here, we present the first sexing protocol for chick mRNA based on real-time quantitative PCR. We demonstrate that this method can accurately determine sex using mRNA from chick gonads and other tissues, such as heart, liver, spleen, lung, and muscle. The strategy of this protocol also may be suitable for other species in which sex is determined by the inheritance of sex chromosomes (ZZ male and ZW female). © 2016 Poultry Science Association Inc.

[Breeding of transgenic mice expressing human tau isoform with P301L mutation and identification of homozygous transgenic mice].

PubMed

Wang, Yan-yan; Chen, Ru-zhui; Zhu, Xiao-nani; Liu, Jing; Li, Zhi-hui; Liu, Xiu-juan; Li, Zhi-hui; Na, Xin; Liang, Shan-shan; Qiu, Guo-guang; Zhang, Wei; Wang, Hai; Wang, Xue-lan

2012-05-01

To establish homozygous transgenic mouse strain expressing human tau isoform with P301L mutation. Five transgenic mice expressing human tau isoform with P301L mutation were obtained by microinjection into male nuclei. Homozygote and hemizygote were identified by PCR and real-time fluorescent quantitative PCR. Ninety five homozygous transgenic mice were selected, and the results indicated that homozygous transgenic mice were superior to hemizygote in simulating the changes of biological characteristics. Exogenous gene tau is able to stably transmit to next generation and the combination of SYBR Green real-time fluorescent quantitative PCR with the traditional mating is a fast, reliable and economical way to screen homozygous and hemizygous transgenic mice.

Quantitation of Mycotoxins Using Direct Analysis in Real Time Mass Spectrometry (DART-MS).

PubMed

Busman, Mark

2018-05-01

Ambient ionization represents a new generation of MS ion sources and is used for the rapid ionization of small molecules under ambient conditions. The combination of ambient ionization and MS allows the analysis of multiple food samples with simple or no sample treatment or in conjunction with prevailing sample preparation methods. Two ambient ionization methods, desorptive electrospray ionization (DESI) and direct analysis in real time (DART) have been adapted for food safety application. Both ionization techniques provide unique advantages and capabilities. DART has been used for a variety of qualitative and quantitative applications. In particular, mycotoxin contamination of food and feed materials has been addressed by DART-MS. Applications to mycotoxin analysis by ambient ionization MS and particularly DART-MS are summarized.

A case of Pitt-Hopkins syndrome with absence of hyperventilation.

PubMed

Inati, Adlette; Abbas, Hussein A; Korjian, Serge; Daaboul, Yazan; Harajeily, Mohamad; Saab, Raya

2013-12-01

Pitt-Hopkins syndrome is characterized by mental retardation, hyperventilation, and dysmorphic features due to TCF4 mutations. We report a case of Pitt-Hopkins syndrome in a 2½-year-old boy presenting with psychomotor retardation, recurrent respiratory tract infections, and dysmorphic features with absence of hyperventilation or other breathing abnormalities. Comparative genomic hybridization and quantitative real-time polymerase chain reaction were used to confirm TCF4 haploinsufficiency. Pitt-Hopkins syndrome is a rare debilitating disease that should be in the differential diagnosis of other neurodevelopmental disorders characterized by mental retardation and hypotonicity despite the absence of hyperapnea and seizures. Quantitative real-time polymerase chain reaction is another method to identify TCF4 and to confirm Pitt-Hopkins syndrome diagnosis.

Development of a real-time quantitative PCR assay to enumerate Yersinia pestis in fleas.

PubMed

Gabitzsch, Elizabeth S; Vera-Tudela, Rommelle; Eisen, Rebecca J; Bearden, Scott W; Gage, Kenneth L; Zeidner, Nordin S

2008-07-01

A real-time quantitative polymerase chain reaction (qPCR) assay was developed for Yersina pestis. The qPCR assay was developed utilizing a conserved region of the Y. pestis ferric iron uptake regulator gene (fur) to design primers and a fluorescent (FAM-labeled) TaqMan probe. The assay was optimized using cultured Y. pestis (UG05-0454) and was confirmed to work with strains from 3 Y. pestis biovars. The optimized assay was capable of detecting a single organism of cultured Y. pestis and as little as 300 bacteria in infected flea triturates. This qPCR assay enables rapid enumeration of Y. pestis bacterium in laboratory-infected fleas when compared with conventional serial dilution plating.

The state of RT-quantitative PCR: firsthand observations of implementation of minimum information for the publication of quantitative real-time PCR experiments (MIQE).

PubMed

Taylor, Sean C; Mrkusich, Eli M

2014-01-01

In the past decade, the techniques of quantitative PCR (qPCR) and reverse transcription (RT)-qPCR have become accessible to virtually all research labs, producing valuable data for peer-reviewed publications and supporting exciting research conclusions. However, the experimental design and validation processes applied to the associated projects are the result of historical biases adopted by individual labs that have evolved and changed since the inception of the techniques and associated technologies. This has resulted in wide variability in the quality, reproducibility and interpretability of published data as a direct result of how each lab has designed their RT-qPCR experiments. The 'minimum information for the publication of quantitative real-time PCR experiments' (MIQE) was published to provide the scientific community with a consistent workflow and key considerations to perform qPCR experiments. We use specific examples to highlight the serious negative ramifications for data quality when the MIQE guidelines are not applied and include a summary of good and poor practices for RT-qPCR. © 2013 S. Karger AG, Basel.

A multisite assessment of the quantitative capabilities of the Xpert MTB/RIF assay.

PubMed

Blakemore, Robert; Nabeta, Pamela; Davidow, Amy L; Vadwai, Viral; Tahirli, Rasim; Munsamy, Vanisha; Nicol, Mark; Jones, Martin; Persing, David H; Hillemann, Doris; Ruesch-Gerdes, Sabine; Leisegang, Felicity; Zamudio, Carlos; Rodrigues, Camilla; Boehme, Catharina C; Perkins, Mark D; Alland, David

2011-11-01

The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy. Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods. Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results. Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r(s) = -0.77) and directly from sputum (r(s) =-0.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r(s) = 0.68) and semiquantitative colony counts (r(s) = -0.56) was weaker than smear. Tests of paired same-patient sputum showed that high viscosity sputum samples contained ×32 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy. Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.

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

PubMed

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

2015-07-01

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

Image visualization of hyperspectral spectrum for LWIR

NASA Astrophysics Data System (ADS)

Chong, Eugene; Jeong, Young-Su; Lee, Jai-Hoon; Park, Dong Jo; Kim, Ju Hyun

2015-07-01

The image visualization of a real-time hyperspectral spectrum in the long-wave infrared (LWIR) range of 900-1450 cm-1 by a color-matching function is addressed. It is well known that the absorption spectra of main toxic industrial chemical (TIC) and chemical warfare agent (CWA) clouds are detected in this spectral region. Furthermore, a significant spectral peak due to various background species and unknown targets are also present. However, those are dismissed as noise, resulting in utilization limit. Herein, we applied a color-matching function that uses the information from hyperspectral data, which is emitted from the materials and surfaces of artificial or natural backgrounds in the LWIR region. This information was used to classify and differentiate the background signals from the targeted substances, and the results were visualized as image data without additional visual equipment. The tristimulus value based visualization information can quickly identify the background species and target in real-time detection in LWIR.

A Real-Time Ultraviolet Radiation Imaging System Using an Organic Photoconductive Image Sensor†

PubMed Central

Okino, Toru; Yamahira, Seiji; Yamada, Shota; Hirose, Yutaka; Odagawa, Akihiro; Kato, Yoshihisa; Tanaka, Tsuyoshi

2018-01-01

We have developed a real time ultraviolet (UV) imaging system that can visualize both invisible UV light and a visible (VIS) background scene in an outdoor environment. As a UV/VIS image sensor, an organic photoconductive film (OPF) imager is employed. The OPF has an intrinsically higher sensitivity in the UV wavelength region than those of conventional consumer Complementary Metal Oxide Semiconductor (CMOS) image sensors (CIS) or Charge Coupled Devices (CCD). As particular examples, imaging of hydrogen flame and of corona discharge is demonstrated. UV images overlapped on background scenes are simply made by on-board background subtraction. The system is capable of imaging weaker UV signals by four orders of magnitude than that of VIS background. It is applicable not only to future hydrogen supply stations but also to other UV/VIS monitor systems requiring UV sensitivity under strong visible radiation environment such as power supply substations. PMID:29361742

Real-Time Language Processing in School-Age Children with Specific Language Impairment

ERIC Educational Resources Information Center

Montgomery, James W.

2006-01-01

Background:School-age children with specific language impairment (SLI) exhibit slower real-time (i.e. immediate) language processing relative to same-age peers and younger, language-matched peers. Results of the few studies that have been done seem to indicate that the slower language processing of children with SLI is due to inefficient…

A Real-Time Plagiarism Detection Tool for Computer-Based Assessments

ERIC Educational Resources Information Center

Jeske, Heimo J.; Lall, Manoj; Kogeda, Okuthe P.

2018-01-01

Aim/Purpose: The aim of this article is to develop a tool to detect plagiarism in real time amongst students being evaluated for learning in a computer-based assessment setting. Background: Cheating or copying all or part of source code of a program is a serious concern to academic institutions. Many academic institutions apply a combination of…

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

PubMed

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

2008-12-01

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

Forecasting seasonal outbreaks of influenza.

PubMed

Shaman, Jeffrey; Karspeck, Alicia

2012-12-11

Influenza recurs seasonally in temperate regions of the world; however, our ability to predict the timing, duration, and magnitude of local seasonal outbreaks of influenza remains limited. Here we develop a framework for initializing real-time forecasts of seasonal influenza outbreaks, using a data assimilation technique commonly applied in numerical weather prediction. The availability of real-time, web-based estimates of local influenza infection rates makes this type of quantitative forecasting possible. Retrospective ensemble forecasts are generated on a weekly basis following assimilation of these web-based estimates for the 2003-2008 influenza seasons in New York City. The findings indicate that real-time skillful predictions of peak timing can be made more than 7 wk in advance of the actual peak. In addition, confidence in those predictions can be inferred from the spread of the forecast ensemble. This work represents an initial step in the development of a statistically rigorous system for real-time forecast of seasonal influenza.

Forecasting seasonal outbreaks of influenza

PubMed Central

Shaman, Jeffrey; Karspeck, Alicia

2012-01-01

Influenza recurs seasonally in temperate regions of the world; however, our ability to predict the timing, duration, and magnitude of local seasonal outbreaks of influenza remains limited. Here we develop a framework for initializing real-time forecasts of seasonal influenza outbreaks, using a data assimilation technique commonly applied in numerical weather prediction. The availability of real-time, web-based estimates of local influenza infection rates makes this type of quantitative forecasting possible. Retrospective ensemble forecasts are generated on a weekly basis following assimilation of these web-based estimates for the 2003–2008 influenza seasons in New York City. The findings indicate that real-time skillful predictions of peak timing can be made more than 7 wk in advance of the actual peak. In addition, confidence in those predictions can be inferred from the spread of the forecast ensemble. This work represents an initial step in the development of a statistically rigorous system for real-time forecast of seasonal influenza. PMID:23184969

Real-time attitude determination and gyro calibration

NASA Technical Reports Server (NTRS)

Challa, M.; Filla, O.; Sedlak, J.; Chu, D.

1993-01-01

We present results for two real-time filters prototyped for the Compton Gamma Ray Observatory (GRO), the Extreme Ultraviolet Explorer (EUVE), the Cosmic Background Explorer (COBE), and the next generation of Geostationary Operational Environmental Satellites (GOES). Both real and simulated data were used to solve for attitude and gyro biases. These filters promise advantages over single-frame and batch methods for missions like GOES, where startup and transfer-orbit operations require quick knowledge of attitude and gyro biases.

Real-Time Quantitative Analysis of Valproic Acid in Exhaled Breath by Low Temperature Plasma Ionization Mass Spectrometry

NASA Astrophysics Data System (ADS)

Gong, Xiaoxia; Shi, Songyue; Gamez, Gerardo

2017-04-01

Real-time analysis of exhaled human breath is a rapidly growing field in analytical science and has great potential for rapid and noninvasive clinical diagnosis and drug monitoring. In the present study, an LTP-MS method was developed for real-time, in-vivo and quantitative analysis of γ-valprolactone, a metabolite of valproic acid (VPA), in exhaled breath without any sample pretreatment. In particular, the effect of working conditions and geometry of the LTP source on the ions of interest, protonated molecular ion at m/z 143 and ammonium adduct ion at m/z 160, were systematically characterized. Tandem mass spectrometry (MS/MS) with collision-induced dissociation (CID) was carried out in order to identify γ-valprolactone molecular ions ( m/z 143), and the key fragment ion ( m/z 97) was used for quantitation. In addition, the fragmentation of ammonium adduct ions to protonated molecular ions was performed in-source to improve the signal-to-noise ratio. At optimum conditions, signal reproducibility with an RSD of 8% was achieved. The concentration of γ-valprolactone in exhaled breath was determined for the first time to be 4.83 (±0.32) ng/L by using standard addition method. Also, a calibration curve was obtained with a linear range from 0.7 to 22.5 ng/L, and the limit of detection was 0.18 ng/L for γ-valprolactone in standard gas samples. Our results show that LTP-MS is a powerful analytical platform with high sensitivity for quantitative analysis of volatile organic compounds in human breath, and can have potential applications in pharmacokinetics or for patient monitoring and treatment.

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

PubMed

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

2014-05-01

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

Real-time quantitative fluorescence measurement of microscale cell culture analog systems

NASA Astrophysics Data System (ADS)

Oh, Taek-il; Kim, Donghyun; Tatosian, Daniel; Sung, Jong Hwan; Shuler, Michael

2007-02-01

A microscale cell culture analog (μCCA) is a cell-based lab-on-a-chip assay that, as an animal surrogate, is applied to pharmacological studies for toxicology tests. A μCCA typically comprises multiple chambers and microfluidics that connect the chambers, which represent animal organs and blood flow to mimic animal metabolism more realistically. A μCCA is expected to provide a tool for high-throughput drug discovery. Previously, a portable fluorescence detection system was investigated for a single μCCA device in real-time. In this study, we present a fluorescence-based imaging system that provides quantitative real-time data of the metabolic interactions in μCCAs with an emphasis on measuring multiple μCCA samples simultaneously for high-throughput screening. The detection system is based on discrete optics components, with a high-power LED and a charge-coupled device (CCD) camera as a light source and a detector, for monitoring cellular status on the chambers of each μCCA sample. Multiple samples are characterized mechanically on a motorized linear stage, which is fully-automated. Each μCCA sample has four chambers, where cell lines MES-SA/DX- 5, and MES-SA (tumor cells of human uterus) have been cultured. All cell-lines have been transfected to express the fusion protein H2B-GFP, which is a human histone protein fused at the amino terminus to EGFP. As a model cytotoxic drug, 10 μM doxorubicin (DOX) was used. Real-time quantitative data of the intensity loss of enhanced green fluorescent protein (EGFP) during cell death of target cells have been collected over several minutes to 40 hours. Design issues and improvements are also discussed.

Visual in vivo degradation of injectable hydrogel by real-time and non-invasive tracking using carbon nanodots as fluorescent indicator.

PubMed

Wang, Lei; Li, Baoqiang; Xu, Feng; Li, Ying; Xu, Zheheng; Wei, Daqing; Feng, Yujie; Wang, Yaming; Jia, Dechang; Zhou, Yu

2017-11-01

Visual in vivo degradation of hydrogel by fluorescence-related tracking and monitoring is crucial for quantitatively depicting the degradation profile of hydrogel in a real-time and non-invasive manner. However, the commonly used fluorescent imaging usually encounters limitations, such as intrinsic photobleaching of organic fluorophores and uncertain perturbation of degradation induced by the change in molecular structure of hydrogel. To address these problems, we employed photoluminescent carbon nanodots (CNDs) with low photobleaching, red emission and good biocompatibility as fluorescent indicator for real-time and non-invasive visual in vitro/in vivo degradation of injectable hydrogels that are mixed with CNDs. The in vitro/in vivo toxicity results suggested that CNDs were nontoxic. The embedded CNDs in hydrogels did not diffuse outside in the absence of hydrogel degradation. We had acquired similar degradation kinetics (PBS-Enzyme) between gravimetric and visual determination, and established mathematical equation to quantitatively depict in vitro degradation profile of hydrogels for the predication of in vivo hydrogel degradation. Based on the in vitro data, we developed a visual platform that could quantitatively depict in vivo degradation behavior of new injectable biomaterials by real-time and non-invasive fluorescence tracking. This fluorescence-related visual imaging methodology could be applied to subcutaneous degradation of injectable hydrogel with down to 7 mm depth in small animal trials so far. This fluorescence-related visual imaging methodology holds great potentials for rational design and convenient in vivo screening of biocompatible and biodegradable injectable hydrogels in tissue engineering. Copyright © 2017 Elsevier Ltd. All rights reserved.

Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence.

PubMed

Yang, Litao; Xu, Songci; Pan, Aihu; Yin, Changsong; Zhang, Kewei; Wang, Zhenying; Zhou, Zhigang; Zhang, Dabing

2005-11-30

Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.

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

PubMed

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

2013-06-01

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

Adaptive microfluidic gradient generator for quantitative chemotaxis experiments.

PubMed

Anielski, Alexander; Pfannes, Eva K B; Beta, Carsten

2017-03-01

Chemotactic motion in a chemical gradient is an essential cellular function that controls many processes in the living world. For a better understanding and more detailed modelling of the underlying mechanisms of chemotaxis, quantitative investigations in controlled environments are needed. We developed a setup that allows us to separately address the dependencies of the chemotactic motion on the average background concentration and on the gradient steepness of the chemoattractant. In particular, both the background concentration and the gradient steepness can be kept constant at the position of the cell while it moves along in the gradient direction. This is achieved by generating a well-defined chemoattractant gradient using flow photolysis. In this approach, the chemoattractant is released by a light-induced reaction from a caged precursor in a microfluidic flow chamber upstream of the cell. The flow photolysis approach is combined with an automated real-time cell tracker that determines changes in the cell position and triggers movement of the microscope stage such that the cell motion is compensated and the cell remains at the same position in the gradient profile. The gradient profile can be either determined experimentally using a caged fluorescent dye or may be alternatively determined by numerical solutions of the corresponding physical model. To demonstrate the function of this adaptive microfluidic gradient generator, we compare the chemotactic motion of Dictyostelium discoideum cells in a static gradient and in a gradient that adapts to the position of the moving cell.

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.).

PubMed

Vera-Jimenez, N I; Pietretti, D; Wiegertjes, G F; Nielsen, M E

2013-05-01

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development and evaluation of event-specific quantitative PCR method for genetically modified soybean A2704-12.

PubMed

Takabatake, Reona; Akiyama, Hiroshi; Sakata, Kozue; Onishi, Mari; Koiwa, Tomohiro; Futo, Satoshi; Minegishi, Yasutaka; Teshima, Reiko; Mano, Junichi; Furui, Satoshi; Kitta, Kazumi

2011-01-01

A novel real-time PCR-based analytical method was developed for the event-specific quantification of a genetically modified (GM) soybean event; A2704-12. During the plant transformation, DNA fragments derived from pUC19 plasmid were integrated in A2704-12, and the region was found to be A2704-12 specific. The pUC19-derived DNA sequences were used as primers for the specific detection of A2704-12. We first tried to construct a standard plasmid for A2704-12 quantification using pUC19. However, non-specific signals appeared with both qualitative and quantitative PCR analyses using the specific primers with pUC19 as a template, and we then constructed a plasmid using pBR322. The conversion factor (C(f)), which is required to calculate the amount of the genetically modified organism (GMO), was experimentally determined with two real-time PCR instruments, the Applied Biosystems 7900HT and the Applied Biosystems 7500. The determined C(f) values were both 0.98. The quantitative method was evaluated by means of blind tests in multi-laboratory trials using the two real-time PCR instruments. The limit of quantitation for the method was estimated to be 0.1%. The trueness and precision were evaluated as the bias and reproducibility of relative standard deviation (RSD(R)), and the determined bias and RSD(R) values for the method were each less than 20%. These results suggest that the developed method would be suitable for practical analyses for the detection and quantification of A2704-12.

Quantitative detection of Streptococcus mutans in the dental plaque of Japanese preschool children by real-time PCR.

PubMed

Hata, S; Hata, H; Miyasawa-Hori, H; Kudo, A; Mayanagi, H

2006-02-01

To detect quantitatively the total bacteria and Streptococcus mutans in dental plaque by real-time PCR with prbac, Sm and GTF-B primers, and to compare their presence with the prevalence of dental caries in Japanese preschool children. Human dental plaque samples were collected from the labial surfaces of the upper primary central incisors of 107 children. The dental status was recorded as dft by WHO caries diagnostic criteria. Positive dt and dft scores by the Sm or GTF-B primer were significantly higher than negative scores (P < 0.01). The proportions of Strep. mutans to the total bacteria from sound, and sound and/or filled upper primary incisors were significantly lower than those from decayed or filled, and decayed incisors, respectively (P < 0.01). The ratios of Strep. mutans to total bacteria in plaque detected by real-time PCR with Sm and GTF-B primers were closely associated with the prevalence of dental caries in Japanese preschool children. These assays may be useful for the assessment of an individual's risk of dental caries.

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

USGS Publications Warehouse

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

2006-01-01

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

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

PubMed

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.

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

PubMed Central

Richards-Kortum, Rebecca

2015-01-01

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest. PMID:25867513

Development of a quantitative recombinase polymerase amplification assay with an internal positive control.

PubMed

Crannell, Zachary A; Rohrman, Brittany; Richards-Kortum, Rebecca

2015-03-30

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

Impact of scatterometer wind (ASCAT-A/B) data assimilation on semi real-time forecast system at KIAPS

NASA Astrophysics Data System (ADS)

Han, H. J.; Kang, J. H.

2016-12-01

Since Jul. 2015, KIAPS (Korea Institute of Atmospheric Prediction Systems) has been performing the semi real-time forecast system to assess the performance of their forecast system as a NWP model. KPOP (KIAPS Protocol for Observation Processing) is a part of KIAPS data assimilation system and has been performing well in KIAPS semi real-time forecast system. In this study, due to the fact that KPOP would be able to treat the scatterometer wind data, we analyze the effect of scatterometer wind (ASCAT-A/B) on KIAPS semi real-time forecast system. O-B global distribution and statistics of scatterometer wind give use two information which are the difference between background field and observation is not too large and KPOP processed the scatterometer wind data well. The changes of analysis increment because of O-B global distribution appear remarkably at the bottom of atmospheric field. It also shows that scatterometer wind data cover wide ocean where data would be able to short. Performance of scatterometer wind data can be checked through the vertical error reduction against IFS between background and analysis field and vertical statistics of O-A. By these analysis result, we can notice that scatterometer wind data will influence the positive effect on lower level performance of semi real-time forecast system at KIAPS. After, long-term result based on effect of scatterometer wind data will be analyzed.

Clinical characteristics of children with viral single- and co-infections and a petechial rash.

PubMed

Schneider, Henriette; Adams, Ortwin; Weiss, Christel; Merz, Ulrich; Schroten, Horst; Tenenbaum, Tobias

2013-05-01

Children with petechial rash are more likely to undergo invasive diagnostics, to be treated with antibiotics for potential bacterial infection and to be hospitalized. However, viruses have also been associated with petechial rash. Nonetheless, a systematic analysis of viral infections with modern available techniques as quantitative real-time polymerase chain reaction in the context of petechial rash is lacking. The purpose of this pediatric study was to prospectively uncover viral pathogens that may promote the emergence of petechiae and to analyze the correlation with the clinical characteristics and course. We conducted a prospective study in children (0 to 18 years) presenting with petechiae and signs or symptoms of infection at the emergency department between November 2009 and March 2012. In nasopharyngeal aspirates the following viruses were analyzed by quantitative real-time polymerase chain reaction: cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B, parainfluenza viruses, human respiratory syncytial virus A and B, human metapneumovirus, rhinovirus, enterovirus, adenovirus, human coronavirus OC43, 229E, NL63 and human bocavirus. A viral pathogen was identified in 67% of the analyzed 58 cases with petechial rash. Virus positive patients showed a significantly higher incidence of lower respiratory tract infections. Forty-one percent were viral coinfections, which were significantly younger than virus negative patients, had a higher leukocyte count and were hospitalized for a longer time. A petechial rash is frequently associated viral single- and coinfections and can rapidly be identified via quantitative real-time polymerase chain reaction.

A Quantitative Real-Time PCR-Based Strategy for Molecular Evaluation of Nicotine Conversion in Burley Tobacco.

PubMed

Sun, Bo; Xue, Sheng-Ling; Zhang, Fen; Luo, Zhao-Peng; Wu, Ming-Zhu; Chen, Qing; Tang, Hao-Ru; Lin, Fu-Cheng; Yang, Jun

2015-11-17

Nornicotine production in Nicotiana tabacum is undesirable because it is the precursor of the carcinogen N'-nitrosonornicotine. In some individual burley tobacco plants, a large proportion of the nicotine can be converted to nornicotine, and this process of nicotine conversion is mediated primarily by enzymatic N-demethylation of nicotine which is controlled mainly by CYP82E4. Here we report a novel strategy based on quantitative real-time polymerase chain reaction (qPCR) method, which analyzed the ratio of nicotine conversion through examining the transcript level of CYP82E4 in burley leaves and do not need ethylene induction before detected. The assay was linear in a range from 1 × 10¹ to 1 × 10⁵ copies/mL of serially diluted standards, and also showed high specificity and reproducibility (93%-99%). To assess its applicability, 55 plants of burley cultivar Ky8959 at leaf maturing stage were analyzed, and the results were in accordance with those from gas chromatograph-mass spectrometry (GC-MS) method. Moreover, a linear correlation existed between conversion level and CYP82E4 transcript abundance. Taken together, the quantitative real-time PCR assay is standardized, rapid and reproducible for estimation of nicotine conversion level in vivo, which is expected to shed new light on monitoring of burley tobacco converter.

Quantitative analysis of SMN1 gene and estimation of SMN1 deletion carrier frequency in Korean population based on real-time PCR.

PubMed

Lee, Tae-Mi; Kim, Sang-Wun; Lee, Kwang-Soo; Jin, Hyun-Seok; Koo, Soo Kyung; Jo, Inho; Kang, Seongman; Jung, Sung-Chul

2004-12-01

Spinal muscular atrophy (SMA) is an autosomal recessive disorder, caused by homozygous absence of the survival motor neuron gene (SMN1) in approximately 94% of patients. Since most carriers have only one SMN1 gene copy, several SMN1 quantitative analyses have been used for the SMA carrier detection. We developed a reliable quantitative real-time PCR with SYBR Green I dye and studied 13 patients with SMA and their 24 parents, as well as 326 healthy normal individuals. The copy number of the SMN1 gene was determined by the comparative threshold cycle (Ct) method and albumin was used as a reference gene. The homozygous SMN1 deletion ratio of patients was 0.00 and the hemizygous SMN1 deletion ratio of parents ranged from 0.39 to 0.59. The deltadelta Ct ratios of 7 persons among 326 normal individuals were within the carrier range, 0.41-0.57. According to these data, we estimated the carrier and disease prevalence of SMA at 1/47 and 1/8,496 in Korean population, respectively. These data indicated that there would be no much difference in disease prevalence of SMA compared with western countries. Since the prevalence of SMA is higher than other autosomal recessive disorders, the carrier detection method using real-time PCR could be a useful tool for genetic counseling.

Development of a SYBR Green I real-time PCR for detection and quantitation of orthopoxvirus by using Ectromelia virus.

PubMed

Cheng, Wenyu; He, Xiaobing; Jia, Huaijie; Chen, Guohua; Wang, Cong; Zhang, Jun; Jing, Zhizhong

2018-04-01

Ectromelia virus (ECTV) is the causative agent of mousepox, which has devastating effects in laboratory-mouse colonies and causes economic loss in biomedical research. More importantly, ECTV has been extensively used as an excellent model for studies of the pathogenesis and immunobiology of human smallpox. A rapid and sensitive SYBR Green I-based real-time PCR assay was developed and used for the detection and quantitation of orthopoxvirus by using ECTV in this study. Primers targeted to the highly conserved region of major core protein P4b gene of orthopoxvirus were designed and the standard plasmid was constructed. This assay was able to detect a minimum of 10 copies of standard DNA and 5 TCID 50 units of ECTV. In addition, no cross-reactions were observed with two DNA viruses, such as herpes simplex virus and swine pseudorabies virus, and one RNA virus, vesicular stomatitis virus. Furthermore, intra- and inter-assay variability data showed that this method had a highly reproducibility and reliability. Moreover, the current assay was faster and had a higher sensitivity for detection of ECTV genomic DNA in cell cultured and clinical test samples. Therefore, the high sensitivity and reproducibility of this SYBR Green real-time PCR approach is a more effective method than the conventional PCR for ECTV diagnosis and quantitation. Copyright © 2017. Published by Elsevier Ltd.

Quantitative real-time RT-PCR assay for research studies on enterovirus infections in the central nervous system.

PubMed

Volle, Romain; Nourrisson, Céline; Mirand, Audrey; Regagnon, Christel; Chambon, Martine; Henquell, Cécile; Bailly, Jean-Luc; Peigue-Lafeuille, Hélène; Archimbaud, Christine

2012-10-01

Human enteroviruses are the most frequent cause of aseptic meningitis and are involved in other neurological infections. Qualitative detection of enterovirus genomes in cerebrospinal fluid is a prerequisite in diagnosing neurological diseases. The pathogenesis of these infections is not well understood and research in this domain would benefit from the availability of a quantitative technique to determine viral load in clinical specimens. This study describes the development of a real-time RT-qPCR assay using hydrolysis TaqMan probe and a competitive RNA internal control. The assay has high specificity and can be used for a large sample of distinct enterovirus strains and serotypes. The reproducible limit of detection was estimated at 1875 copies/ml of quantitative standards composed of RNA transcripts obtained from a cloned echovirus 30 genome. Technical performance was unaffected by the introduction of a competitive RNA internal control before RNA extraction. The mean enterovirus RNA concentration in an evaluation series of 15 archived cerebrospinal fluid specimens was determined at 4.78 log(10)copies/ml for the overall sample. The sensitivity and reproducibility of the real time RT-qPCR assay used in combination with the internal control to monitor the overall specimen process make it a valuable tool with applied research into enterovirus infections. Copyright © 2012 Elsevier B.V. All rights reserved.

Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay.

PubMed

Wu, Qingqing; Xiang, Shengnan; Wang, Wenjun; Zhao, Jinyan; Xia, Jinhua; Zhen, Yueran; Liu, Bang

2018-05-01

Various detection methods have been developed to date for identification of animal species. New techniques based on PCR approach have raised the hope of developing better identification methods, which can overcome the limitations of the existing methods. PCR-based methods used the mitochondrial DNA (mtDNA) as well as nuclear DNA sequences. In this study, by targeting nuclear DNA, multiplex PCR and real-time PCR methods were developed to assist with qualitative and quantitative analysis. The multiplex PCR was found to simultaneously and effectively distinguish four species (fox, dog, mink, and rabbit) ingredients by the different sizes of electrophoretic bands: 480, 317, 220, and 209 bp. Real-time fluorescent PCR's amplification profiles and standard curves showed good quantitative measurement responses and linearity, as indicated by good repeatability and coefficient of determination R 2  > 0.99. The quantitative results of quaternary DNA mixtures including mink, fox, dog, and rabbit DNA are in line with our expectations: R.D. (relative deviation) varied between 1.98 and 12.23% and R.S.D. (relative standard deviation) varied between 3.06 and 11.51%, both of which are well within the acceptance criterion of ≤ 25%. Combining the two methods is suitable for the rapid identification and accurate quantification of fox-, dog-, mink-, and rabbit-derived ingredients in the animal products.

PAVENET OS: A Compact Hard Real-Time Operating System for Precise Sampling in Wireless Sensor Networks

NASA Astrophysics Data System (ADS)

Saruwatari, Shunsuke; Suzuki, Makoto; Morikawa, Hiroyuki

The paper shows a compact hard real-time operating system for wireless sensor nodes called PAVENET OS. PAVENET OS provides hybrid multithreading: preemptive multithreading and cooperative multithreading. Both of the multithreading are optimized for two kinds of tasks on wireless sensor networks, and those are real-time tasks and best-effort ones. PAVENET OS can efficiently perform hard real-time tasks that cannot be performed by TinyOS. The paper demonstrates the hybrid multithreading realizes compactness and low overheads, which are comparable to those of TinyOS, through quantitative evaluation. The evaluation results show PAVENET OS performs 100 Hz sensor sampling with 0.01% jitter while performing wireless communication tasks, whereas optimized TinyOS has 0.62% jitter. In addition, PAVENET OS has a small footprint and low overheads (minimum RAM size: 29 bytes, minimum ROM size: 490 bytes, minimum task switch time: 23 cycles).

Portable real-time fluorescence cytometry of microscale cell culture analog devices

NASA Astrophysics Data System (ADS)

Kim, Donghyun; Tatosian, Daniel A.; Shuler, Michael L.

2006-02-01

A portable fluorescence cytometric system that provides a modular platform for quantitative real-time image measurements has been used to explore the applicability to investigating cellular events on multiple time scales. For a short time scale, we investigated the real-time dynamics of uptake of daunorubicin, a chemotherapeutic agent, in cultured mouse L-cells in a micro cell culture analog compartment using the fluorescent cytometric system. The green fluorescent protein (GFP) expression to monitor induction of pre-specified genes, which occurs on a much longer time scale, has also been measured. Here GFP fluorescence from a doxycycline inducible promoter in a mouse L-cell line was determined. Additionally, a system based on inexpensive LEDs showed performance comparable to a broadband light source based system and reduced photobleaching compared to microscopic examination.

Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti

PubMed Central

2013-01-01

Background The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection with tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been increasing in endemic regions of the USA and Europe. The currently used serological diagnostic tests are often difficult to interpret and, moreover, antibodies against the pathogens persist for a long time making it difficult to confirm the cure of the disease. In addition, these tests cannot be used for diagnosis of early disease state before the adaptive immune response is established. Since nucleic acids of the pathogens do not persist after the cure, DNA-based diagnostic tests are becoming highly useful for detecting infectious diseases. Results In this study, we describe a real-time multiplex PCR assay to detect the presence of B. burgdorferi, B. microti and A. phagocytophilum simultaneously even when they are present in very low copy numbers. Interestingly, this quantitative PCR technique is also able to differentiate all three major Lyme spirochete species, B. burgdorferi, B. afzelii, and B. garinii by utilizing a post-PCR denaturation profile analysis and a single molecular beacon probe. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in European countries where all three Lyme spirochete species are prevalent. As proof of the principle for patient samples, we detected the presence of low number of Lyme spirochetes spiked in the human blood using our assay. Finally, our multiplex assay can detect all three tick-borne pathogens in a sensitive and specific manner irrespective of the level of each pathogen present in the sample. We anticipate that this novel diagnostic method will be able to simultaneously diagnose early to chronic stages of Lyme disease, babesiosis and anaplasmosis using the patients’ blood samples. Conclusion Real-time quantitative PCR using specific primers and molecular beacon probes for the selected amplicon described in this study can detect three tick-borne pathogens simultaneously in an accurate manner. PMID:24359556

A RAPID METHOD FOR THE EXTRACTION OF FUNGAL DNA FROM ENVIRONMENTAL SAMPLES: EVALUATION IN THE QUANTITATIVE ANALYSIS OF MEMNONIELLA ECHINATA CONIDIA USING REAL TIME DETECTION OF PCR PRODUCTS

EPA Science Inventory

New technologies are creating the potential for using nucleic acid sequence detection to perform routine microbiological analyses of environmental samples. Our laboratory has recently reported on the development of a method for the quantitative detection of Stachybotrys chartarum...

Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

PubMed Central

Abate, Teresa; Cayo, Nelly M.; Parrado, Rudy; Bello, Zoraida Diaz; Velazquez, Elsa; Muñoz-Calderon, Arturo; Juiz, Natalia A.; Basile, Joaquín; Garcia, Lineth; Riarte, Adelina; Nasser, Julio R.; Ocampo, Susana B.; Yadon, Zaida E.; Torrico, Faustino; de Noya, Belkisyole Alarcón; Ribeiro, Isabela; Schijman, Alejandro G.

2013-01-01

Background The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment. PMID:23350002

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

PubMed Central

2010-01-01

Background The existence of circulating tumor cells (CTCs) in peripheral blood as an indicator of tumor recurrence has not been clearly established, particularly for gastric cancer patients. We conducted a retrospective analysis of the relationship between CTCs in peripheral blood at initial diagnosis and clinicopathologic findings in patients with gastric carcinoma. Methods Blood samples were obtained from 123 gastric carcinoma patients at initial diagnosis. mRNA was extracted and amplified for carcinoembryonic antigen (CEA) mRNA detection using real-time RT-PCR. Periodic 3-month follow-up examinations included serum CEA measurements and imaging. Results The minimum threshold for corrected CEA mRNA score [(CEA mRNA/GAPDH mRNA) × 106] was set at 100. Forty-five of 123 patients (36.6%) were positive for CEA mRNA expression. CEA mRNA expression significantly correlated with T stage and postoperative recurrence status (P = 0.001). Recurrent disease was found in 44 of 123 cases (35.8%), and 25 of these (56.8%) were positive for CEA mRNA. Of these patients, CEA mRNA was more sensitive than serum CEA in indicating recurrence. Three-year disease-free survival of patients positive for CEA mRNA was significantly poorer than of patients negative for CEA mRNA (P < 0.001). Only histological grade and CEA mRNA positivity were independent factors for disease-free survival using multivariate analysis. Conclusions CEA mRNA copy number in peripheral blood at initial diagnosis was significantly associated with disease recurrence in gastric adenocarcinoma patients. Real-time RT-PCR detection of CEA mRNA levels at initial diagnosis appears to be a promising predictor for disease recurrence in gastric adenocarcinoma patients. PMID:21040522

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

PubMed Central

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

2014-01-01

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

A Java Program for LRE-Based Real-Time qPCR that Enables Large-Scale Absolute Quantification

PubMed Central

Rutledge, Robert G.

2011-01-01

Background Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Findings Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. Conclusions The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples. PMID:21407812

Real-Time Intracellular Measurements of ROS and RNS in Living Cells with Single Core-Shell Nanowire Electrodes.

PubMed

Zhang, Xin-Wei; Qiu, Quan-Fa; Jiang, Hong; Zhang, Fu-Li; Liu, Yan-Lin; Amatore, Christian; Huang, Wei-Hua

2017-10-09

Nanoelectrodes allow precise and quantitative measurements of important biological processes at the single living-cell level in real time. Cylindrical nanowire electrodes (NWEs) required for intracellular measurements create a great challenge for achieving excellent electrochemical and mechanical performances. Herein, we present a facile and robust solution to this problem based on a unique SiC-core-shell design to produce cylindrical NWEs with superior mechanical toughness provided by the SiC nano-core and an excellent electrochemical performance provided by the ultrathin carbon shell that can be used as such or platinized. The use of such NWEs for biological applications is illustrated by the first quantitative measurements of ROS/RNS in individual phagolysosomes of living macrophages. As the shell material can be varied to meet any specific detection purpose, this work opens up new opportunities to monitor quantitatively biological functions occurring inside cells and their organelles. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Portable low-coherence interferometry for quantitatively imaging fast dynamics with extended field of view

NASA Astrophysics Data System (ADS)

Shaked, Natan T.; Girshovitz, Pinhas; Frenklach, Irena

2014-06-01

We present our recent advances in the development of compact, highly portable and inexpensive wide-field interferometric modules. By a smart design of the interferometric system, including the usage of low-coherence illumination sources and common-path off-axis geometry of the interferometers, spatial and temporal noise levels of the resulting quantitative thickness profile can be sub-nanometric, while processing the phase profile in real time. In addition, due to novel experimentally-implemented multiplexing methods, we can capture low-coherence off-axis interferograms with significantly extended field of view and in faster acquisition rates. Using these techniques, we quantitatively imaged rapid dynamics of live biological cells including sperm cells and unicellular microorganisms. Then, we demonstrated dynamic profiling during lithography processes of microscopic elements, with thicknesses that may vary from several nanometers to hundreds of microns. Finally, we present new algorithms for fast reconstruction (including digital phase unwrapping) of off-axis interferograms, which allow real-time processing in more than video rate on regular single-core computers.

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

PubMed Central

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

2017-01-01

We developed an optical thrombus sensor for a monopivot extracorporeal centrifugal blood pump. In this study, we investigated its quantitative performance for thrombus detection in acute animal experiments of left ventricular assist using the pump on pathogen-free pigs. Optical fibers were set in the driver unit of the pump. The incident light at the near-infrared wavelength of 810 nm was aimed at the pivot bearing, and the resulting scattered light was guided to the optical fibers. The detected signal was analyzed to obtain the thrombus formation level. As a result, real-time and quantitative monitoring of the thrombus surface area on the pivot bearing was achieved with an accuracy of 3.6 ± 2.3 mm2. In addition, the sensing method using the near-infrared light was not influenced by changes in the oxygen saturation and the hematocrit. It is expected that the developed sensor will be useful for optimal anticoagulation management for long-term extracorporeal circulation therapies. PMID:29359096

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

PubMed

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

2018-01-01

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

Feasibility of quantitative diffuse reflectance spectroscopy for targeted measurement of renal ischemia during laparoscopic partial nephrectomy.

PubMed

Goel, Utsav O; Maddox, Michael M; Elfer, Katherine N; Dorsey, Philip J; Wang, Mei; McCaslin, Ian Ross; Brown, J Quincy; Lee, Benjamin R

2014-01-01

Reduction of warm ischemia time during partial nephrectomy (PN) is critical to minimizing ischemic damage and improving postoperative kidney function, while maintaining tumor resection efficacy. Recently, methods for localizing the effects of warm ischemia to the region of the tumor via selective clamping of higher-order segmental artery branches have been shown to have superior outcomes compared with clamping the main renal artery. However, artery identification can prolong operative time and increase the blood loss and reduce the positive effects of selective ischemia. Quantitative diffuse reflectance spectroscopy (DRS) can provide a convenient, real-time means to aid in artery identification during laparoscopic PN. The feasibility of quantitative DRS for real-time longitudinal measurement of tissue perfusion and vascular oxygenation in laparoscopic nephrectomy was investigated in vivo in six Yorkshire swine kidneys (n=three animals ). DRS allowed for rapid identification of ischemic areas after selective vessel occlusion. In addition, the rates of ischemia induction and recovery were compared for main renal artery versus tertiary segmental artery occlusion, and it was found that the tertiary segmental artery occlusion trends toward faster recovery after ischemia, which suggests a potential benefit of selective ischemia. Quantitative DRS could provide a convenient and fast tool for artery identification and evaluation of the depth, spatial extent, and duration of selective tissue ischemia in laparoscopic PN.

Feasibility of quantitative diffuse reflectance spectroscopy for targeted measurement of renal ischemia during laparoscopic partial nephrectomy

NASA Astrophysics Data System (ADS)

Goel, Utsav O.; Maddox, Michael M.; Elfer, Katherine N.; Dorsey, Philip J.; Wang, Mei; McCaslin, Ian Ross; Brown, J. Quincy; Lee, Benjamin R.

2014-10-01

Reduction of warm ischemia time during partial nephrectomy (PN) is critical to minimizing ischemic damage and improving postoperative kidney function, while maintaining tumor resection efficacy. Recently, methods for localizing the effects of warm ischemia to the region of the tumor via selective clamping of higher-order segmental artery branches have been shown to have superior outcomes compared with clamping the main renal artery. However, artery identification can prolong operative time and increase the blood loss and reduce the positive effects of selective ischemia. Quantitative diffuse reflectance spectroscopy (DRS) can provide a convenient, real-time means to aid in artery identification during laparoscopic PN. The feasibility of quantitative DRS for real-time longitudinal measurement of tissue perfusion and vascular oxygenation in laparoscopic nephrectomy was investigated in vivo in six Yorkshire swine kidneys (n=three animals). DRS allowed for rapid identification of ischemic areas after selective vessel occlusion. In addition, the rates of ischemia induction and recovery were compared for main renal artery versus tertiary segmental artery occlusion, and it was found that the tertiary segmental artery occlusion trends toward faster recovery after ischemia, which suggests a potential benefit of selective ischemia. Quantitative DRS could provide a convenient and fast tool for artery identification and evaluation of the depth, spatial extent, and duration of selective tissue ischemia in laparoscopic PN.

Acceptability and Feasibility of Real-Time Antiretroviral Therapy Adherence Interventions in Rural Uganda: Mixed-Method Pilot Randomized Controlled Trial

PubMed Central

Atukunda, Esther C; Tumuhimbise, Wilson; Pisarski, Emily E; Tam, Melanie; Wyatt, Monique A; Ware, Norma C; Haberer, Jessica E

2018-01-01

Background Wireless electronic adherence monitors can detect antiretroviral therapy (ART) adherence lapses and trigger interventions in real time, thus potentially avoiding unnecessary HIV viremia. Evidence about the acceptability and feasibility of these monitors and associated interventions, however, is limited. Objective The aim of this study was to assess the acceptability and feasibility of real-time adherence monitoring linked to text messaging (short message service, SMS) reminders and notifications to support adherence among individuals living with HIV who are taking ART in rural southwestern Uganda. Methods Individuals living with HIV who were initiating ART were enrolled in a pilot randomized controlled trial and followed up for 9 months. Participants received a real-time adherence monitor and were randomized to one of the following study arms: (1) scheduled SMS, (2) SMS triggered by missed or delayed doses, or (3) no SMS. SMS notifications were also sent to 45 patient-identified social supporters for sustained adherence lapses in the scheduled SMS and triggered SMS arms. Study participants and social supporters participated in qualitative semistructured in-depth interviews on acceptability and feasibility of this technology. An inductive, content analytic approach, framed by the unified theory of acceptance and use of technology model, was used to analyze qualitative data. Quantitative feasibility data, including device functionality and SMS tracking data, were recorded based upon device metrics collected electronically and summarized descriptively. Results A total of 63 participants participated in the study. Participants reported that real-time monitoring intervention linked to SMS reminders and notifications are generally acceptable; the predominant feedback was perceived utility—the intervention was beneficial in motivating and reminding patients to take medication, as well as enabling provision of social support. The intervention was found to be technically feasible, as data were obtained from most participants as expected most of the time. Potential challenges included the impact of the technology on confidentiality, shared phone ownership, usability skills, and availability of electricity. Conclusions Real-time adherence monitoring integrated with SMS reminders and social support notifications is a generally acceptable (based primarily on perceived utility) and feasible intervention in a resource-limited country. Future efforts should focus on optimized device design, user training to overcome the challenges we encountered, cost effectiveness studies, as well as studying the monitoring aspect of the device without accompanying interventions. Trial Registration ClinicalTrials.gov NCT01957865; https://clinicaltrials.gov/ct2/show/NCT01957865 (Archived by WebCite at http://www.webcitation.org/6zFiDlXDa) PMID:29773527

Performance of a New HPV Cervi-Collect Collection and Transportation Kit

PubMed Central

Chernesky, M.; Huang, S.; Jang, D.; Erickson, B.; Salituro, J.; Engel, H.; Gilchrist, J.; Neuscheler, P.; Mak, W. B.; Abravaya, K.

2012-01-01

Background. Liquid-based Pap (L-Pap) media are used for Pap and human papillomavirus (HPV) testing. Objectives. To compare RealTime High Risk (HR) HPV testing of a new collection kit (Cervi-Collect) and PreservCyt L-Pap specimens. To determine ease of use and safety of Cervi-Collect. Methods. L-Pap samples (n = 203) were tested with HC2 and RealTime HR HPV and Cervi-Collect with RealTime HR HPV. Discordant samples were genotyped. Results. L-Pap and Cervi-Collect specimens tested by RealTime HR HPV showed 93.1% agreement (Kappa 0.86). RealTime HR HPV and HC2 on L-Pap had 90.3% agreement (Kappa 0.80). RealTime HR HPV on Cervi-Collect and HC2 on L-Pap showed 88.2% agreement (Kappa 0.76). Sixteen of 21 samples which were HC2 negative and RealTime HR HPV positive on L-Pap or Cervi-Collect contained HR HPV genotypes. Eleven healthcare collectors were in strong agreement on a usability and safety questionnaire. Conclusion. Cervi-Collect samples were easy to collect and showed strong agreement with L-Pap samples tested with RealTime HR HPV or HC2. PMID:22174716

Evaluation of a shortened QIAsymphony DNA extraction protocol for stool samples using a multiplex real-time PCR for the detection of enteric pathogens.

PubMed

van Zanten, E; Wisselink, G J; Stoll, S; Alvarez, R; Kooistra-Smid, A M D

2011-02-01

A shortened DNA extraction protocol for the QIAsymphony SP was evaluated by quantitative and qualitative comparison of real-time PCR results of 150 co-extracted stool samples. The average ∆Cycle threshold value for positive pathogenic targets was 0.28 Ct. A consensus of 96.91%, with a correlation coefficient of 0.9880 was recorded. Copyright © 2010 Elsevier B.V. All rights reserved.

Quantitative measurement of permeabilization of living cells by terahertz attenuated total reflection

NASA Astrophysics Data System (ADS)

Grognot, Marianne; Gallot, Guilhem

2015-09-01

Using Attenuated Total Reflection imaging technique in the terahertz domain, we demonstrate non-invasive, non-staining real time measurements of cytoplasm leakage during permeabilization of epithelial cells by saponin. The terahertz signal is mostly sensitive to the intracellular protein concentration in the cells, in a very good agreement with standard bicinchoninic acid protein measurements. It opens the way to in situ real time dynamics of protein content and permeabilization in live cells.

The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting Rotavirus A and Norovirus

PubMed Central

Dung, Tran Thi Ngoc; Phat, Voong Vinh; Nga, Tran Vu Thieu; My, Phan Vu Tra; Duy, Pham Thanh; Campbell, James I.; Thuy, Cao Thu; Hoang, Nguyen Van Minh; Van Minh, Pham; Le Phuc, Hoang; Tuyet, Pham Thi Ngoc; Vinh, Ha; Kien, Duong Thi Hue; Huy, Huynh Le Anh; Vinh, Nguyen Thanh; Nga, Tran Thi Thu; Hau, Nguyen Thi Thu; Chinh, Nguyen Tran; Thuong, Tang Chi; Tuan, Ha Manh; Simmons, Cameron; Farrar, Jeremy J.; Baker, Stephen

2013-01-01

Rotavirus (RoV) and Norovirus (NoV) are the main causes of viral gastroenteritis. Currently, there is no validated multiplex real-time PCR that can detect and quantify RoV and NoV simultaneously. The aim of the study was to develop, validate, and internally control a multiplex one-step RT real-time PCR to detect and quantify RoV and NoV in stool samples. PCR sensitivity was assessed by comparing amplification against the current gold standard, enzyme immunoassay (EIA), on stool samples from 94 individuals with diarrhea and 94 individuals without diarrhea. PCR detected 10% more RoV positive samples than EIA in stools samples from patients with diarrhea. PCR detected 23% more NoV genogroup II positive samples from individuals with diarrhea and 9% more from individuals without diarrhea than EIA, respectively. Genotyping of the PCR positive/EIA negative samples suggested the higher rate of PCR positivity, in comparison to EIA, was due to increased sensitivity, rather than nonspecific hybridization. Quantitation demonstrated that the viral loads of RoV and NoV in the stools of diarrheal patients were an order of magnitude greater than in individuals without diarrhea. This internally controlled real-time PCR method is robust, exhibits a high degree of reproducibility, and may have a greater utility and sensitivity than commercial EIA kits. PMID:23046990

Portable Dew Point Mass Spectrometry System for Real-Time Gas and Moisture Analysis

NASA Technical Reports Server (NTRS)

Arkin, C.; Gillespie, Stacey; Ratzel, Christopher

2010-01-01

A portable instrument incorporates both mass spectrometry and dew point measurement to provide real-time, quantitative gas measurements of helium, nitrogen, oxygen, argon, and carbon dioxide, along with real-time, quantitative moisture analysis. The Portable Dew Point Mass Spectrometry (PDP-MS) system comprises a single quadrupole mass spectrometer and a high vacuum system consisting of a turbopump and a diaphragm-backing pump. A capacitive membrane dew point sensor was placed upstream of the MS, but still within the pressure-flow control pneumatic region. Pressure-flow control was achieved with an upstream precision metering valve, a capacitance diaphragm gauge, and a downstream mass flow controller. User configurable LabVIEW software was developed to provide real-time concentration data for the MS, dew point monitor, and sample delivery system pressure control, pressure and flow monitoring, and recording. The system has been designed to include in situ, NIST-traceable calibration. Certain sample tubing retains sufficient water that even if the sample is dry, the sample tube will desorb water to an amount resulting in moisture concentration errors up to 500 ppm for as long as 10 minutes. It was determined that Bev-A-Line IV was the best sample line to use. As a result of this issue, it is prudent to add a high-level humidity sensor to PDP-MS so such events can be prevented in the future.

Quantitative Comparison of Enzyme Immobilization Strategies for Glucose Biosensing in Real-Time Using Fast-Scan Cyclic Voltammetry Coupled with Carbon-Fiber Microelectrodes.

PubMed

Smith, Samantha K; Lugo-Morales, Leyda Z; Tang, C; Gosrani, Saahj P; Lee, Christie A; Roberts, James G; Morton, Stephen W; McCarty, Gregory S; Khan, Saad A; Sombers, Leslie A

2018-05-22

Electrochemical monitoring of non-electroactive species requires a biosensor that is stable and selective, with sensitivity to physiological concentrations of targeted analytes. We have combined glucose oxidase-modified carbon-fiber microelectrodes with fast-scan cyclic voltammetry for real-time measurements of glucose fluctuations in brain tissue. Work presented herein quantitatively compares three approaches to enzyme immobilization on the microelectrode surface-physical adsorption, hydrogel entrapment, and entrapment in electrospun nanofibers. The data suggest that each of these methods can be used to create functional microbiosensors. Immobilization of glucose oxidase by physical adsorption generates a biosensor with poor sensitivity to glucose and unstable performance. Entrapment of glucose oxidase in poly(vinyl alcohol) nanofibers generates microbiosensors that are effective for glucose measurements over a large linear range, and that may be particularly useful when targeting glucose concentrations in excess of 3 mm, such as in blood. Hydrogel entrapment is the most effective in terms of sensitivity and stability. These microbiosensors can be used for simultaneous monitoring of glucose and dopamine in real time. The findings outlined herein should be applicable to other oxidase enzymes, and thus they are broadly important for the development of new tools for real-time measurements of fluctuating molecules that are not inherently electroactive. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quality of Learners' Time and Learning Performance beyond Quantitative Time-on-Task

ERIC Educational Resources Information Center

Romero, Margarida; Barbera, Elena

2011-01-01

Along with the amount of time spent learning (or time-on-task), the quality of learning time has a real influence on learning performance. Quality of time in online learning depends on students' time availability and their willingness to devote quality cognitive time to learning activities. However, the quantity and quality of the time spent by…

External optical imaging of freely moving mice with green fluorescent protein-expressing metastatic tumors

NASA Astrophysics Data System (ADS)

Yang, Meng; Baranov, Eugene; Shimada, Hiroshi; Moossa, A. R.; Hoffman, Robert M.

2000-04-01

We report here a new approach to genetically engineering tumors to become fluorescence such that they can be imaged externally in freely-moving animals. We describe here external high-resolution real-time fluorescent optical imaging of metastatic tumors in live mice. Stable high-level green flourescent protein (GFP)-expressing human and rodent cell lines enable tumors and metastasis is formed from them to be externally imaged from freely-moving mice. Real-time tumor and metastatic growth were quantitated from whole-body real-time imaging in GFP-expressing melanoma and colon carcinoma models. This GFP optical imaging system is highly appropriate for high throughput in vivo drug screening.

Real-time Social Internet Data to Guide Forecasting Models

DOE Office of Scientific and Technical Information (OSTI.GOV)

Del Valle, Sara Y.

Our goal is to improve decision support by monitoring and forecasting events using social media, mathematical models, and quantifying model uncertainty. Our approach is real-time, data-driven forecasts with quantified uncertainty: Not just for weather anymore. Information flow from human observations of events through an Internet system and classification algorithms is used to produce quantitatively uncertain forecast. In summary, we want to develop new tools to extract useful information from Internet data streams, develop new approaches to assimilate real-time information into predictive models, validate approaches by forecasting events, and our ultimate goal is to develop an event forecasting system using mathematicalmore » approaches and heterogeneous data streams.« less

Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design.

PubMed

Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M

2016-05-05

Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared - non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents.

Quantitative laser speckle flowmetry of the in vivo microcirculation using sidestream dark field microscopy

PubMed Central

Nadort, Annemarie; Woolthuis, Rutger G.; van Leeuwen, Ton G.; Faber, Dirk J.

2013-01-01

We present integrated Laser Speckle Contrast Imaging (LSCI) and Sidestream Dark Field (SDF) flowmetry to provide real-time, non-invasive and quantitative measurements of speckle decorrelation times related to microcirculatory flow. Using a multi exposure acquisition scheme, precise speckle decorrelation times were obtained. Applying SDF-LSCI in vitro and in vivo allows direct comparison between speckle contrast decorrelation and flow velocities, while imaging the phantom and microcirculation architecture. This resulted in a novel analysis approach that distinguishes decorrelation due to flow from other additive decorrelation sources. PMID:24298399

Real-time biscuit tile image segmentation method based on edge detection.

PubMed

Matić, Tomislav; Aleksi, Ivan; Hocenski, Željko; Kraus, Dieter

2018-05-01

In this paper we propose a novel real-time Biscuit Tile Segmentation (BTS) method for images from ceramic tile production line. BTS method is based on signal change detection and contour tracing with a main goal of separating tile pixels from background in images captured on the production line. Usually, human operators are visually inspecting and classifying produced ceramic tiles. Computer vision and image processing techniques can automate visual inspection process if they fulfill real-time requirements. Important step in this process is a real-time tile pixels segmentation. BTS method is implemented for parallel execution on a GPU device to satisfy the real-time constraints of tile production line. BTS method outperforms 2D threshold-based methods, 1D edge detection methods and contour-based methods. Proposed BTS method is in use in the biscuit tile production line. Copyright © 2018 ISA. Published by Elsevier Ltd. All rights reserved.

Real-Time, Single-Step Bioassay Using Nanoplasmonic Resonator With Ultra-High Sensitivity

NASA Technical Reports Server (NTRS)

Zhang, Xiang (Inventor); Chen, Fanqing Frank (Inventor); Su, Kai-Hang (Inventor); Wei, Qi-Huo (Inventor); Ellman, Jonathan A. (Inventor); Sun, Cheng (Inventor)

2014-01-01

A nanoplasmonic resonator (NPR) comprising a metallic nanodisk with alternating shielding layer(s), having a tagged biomolecule conjugated or tethered to the surface of the nanoplasmonic resonator for highly sensitive measurement of enzymatic activity. NPRs enhance Raman signals in a highly reproducible manner, enabling fast detection of protease and enzyme activity, such as Prostate Specific Antigen (paPSA), in real-time, at picomolar sensitivity levels. Experiments on extracellular fluid (ECF) from paPSA-positive cells demonstrate specific detection in a complex bio-fluid background in real-time single-step detection in very small sample volumes.

Real-time, single-step bioassay using nanoplasmonic resonator with ultra-high sensitivity

DOEpatents

Zhang, Xiang; Ellman, Jonathan A; Chen, Fanqing Frank; Su, Kai-Hang; Wei, Qi-Huo; Sun, Cheng

2014-04-01

A nanoplasmonic resonator (NPR) comprising a metallic nanodisk with alternating shielding layer(s), having a tagged biomolecule conjugated or tethered to the surface of the nanoplasmonic resonator for highly sensitive measurement of enzymatic activity. NPRs enhance Raman signals in a highly reproducible manner, enabling fast detection of protease and enzyme activity, such as Prostate Specific Antigen (paPSA), in real-time, at picomolar sensitivity levels. Experiments on extracellular fluid (ECF) from paPSA-positive cells demonstrate specific detection in a complex bio-fluid background in real-time single-step detection in very small sample volumes.

Real-time Stack Monitoring at the BaTek Medical Isotope Production Facility

DOE Office of Scientific and Technical Information (OSTI.GOV)

McIntyre, Justin I.; Agusbudiman, A.; Cameron, Ian M.

2016-04-01

Radioxenon emissions from radiopharmaceutical production are a major source of background concentrations affecting the radioxenon detection systems of the International Monitoring System (IMS). Collection of real-time emissions data from production facilities makes it possible to screen out some medical isotope signatures from the IMS radioxenon data sets. This paper describes an effort to obtain and analyze real-time stack emissions data with the design, construction and installation of a small stack monitoring system developed by a joint CTBTO-IDC, BATAN, and PNNL team at the BaTek medical isotope production facility near Jakarta, Indonesia.

An updated assessment of Symbiodinium spp. that associate with common scleractinian corals from Moorea (French Polynesia) reveals high diversity among background symbionts and a novel finding of clade B.

PubMed

Rouzé, Héloïse; Lecellier, Gaël J; Saulnier, Denis; Planes, Serge; Gueguen, Yannick; Wirshing, Herman H; Berteaux-Lecellier, Véronique

2017-01-01

The adaptative bleaching hypothesis (ABH) states that, depending on the symbiotic flexibility of coral hosts (i.e., the ability of corals to "switch" or "shuffle" their algal symbionts), coral bleaching can lead to a change in the composition of their associated Symbiodinium community and, thus, contribute to the coral's overall survival. In order to determine the flexibility of corals, molecular tools are required to provide accurate species delineations and to detect low levels of coral-associated Symbiodinium . Here, we used highly sensitive quantitative (real-time) PCR (qPCR) technology to analyse five common coral species from Moorea (French Polynesia), previously screened using only traditional molecular methods, to assess the presence of low-abundance (background) Symbiodinium spp. Similar to other studies, each coral species exhibited a strong specificity to a particular clade, irrespective of the environment. In addition, however, each of the five species harboured at least one additional Symbiodinium clade, among clades A-D, at background levels. Unexpectedly, and for the first time in French Polynesia, clade B was detected as a coral symbiont. These results increase the number of known coral- Symbiodinium associations from corals found in French Polynesia, and likely indicate an underestimation of the ability of the corals in this region to associate with and/or "shuffle" different Symbiodinium clades. Altogether our data suggest that corals from French Polynesia may favor a trade-off between optimizing symbioses with a specific Symbiodinium clade(s), maintaining associations with particular background clades that may play a role in the ability of corals to respond to environmental change.

EASY: a simple tool for simultaneously removing background, deadtime and acoustic ringing in quantitative NMR spectroscopy--part I: basic principle and applications.

PubMed

Jaeger, Christian; Hemmann, Felix

2014-01-01

Elimination of Artifacts in NMR SpectroscopY (EASY) is a simple but very effective tool to remove simultaneously any real NMR probe background signal, any spectral distortions due to deadtime ringdown effects and -specifically- severe acoustic ringing artifacts in NMR spectra of low-gamma nuclei. EASY enables and maintains quantitative NMR (qNMR) as only a single pulse (preferably 90°) is used for data acquisition. After the acquisition of the first scan (it contains the wanted NMR signal and the background/deadtime/ringing artifacts) the same experiment is repeated immediately afterwards before the T1 waiting delay. This second scan contains only the background/deadtime/ringing parts. Hence, the simple difference of both yields clean NMR line shapes free of artefacts. In this Part I various examples for complete (1)H, (11)B, (13)C, (19)F probe background removal due to construction parts of the NMR probes are presented. Furthermore, (25)Mg EASY of Mg(OH)2 is presented and this example shows how extremely strong acoustic ringing can be suppressed (more than a factor of 200) such that phase and baseline correction for spectra acquired with a single pulse is no longer a problem. EASY is also a step towards deadtime-free data acquisition as these effects are also canceled completely. EASY can be combined with any other NMR experiment, including 2D NMR, if baseline distortions are a big problem. © 2013 Published by Elsevier Inc.

Platform for Automated Real-Time High Performance Analytics on Medical Image Data.

PubMed

Allen, William J; Gabr, Refaat E; Tefera, Getaneh B; Pednekar, Amol S; Vaughn, Matthew W; Narayana, Ponnada A

2018-03-01

Biomedical data are quickly growing in volume and in variety, providing clinicians an opportunity for better clinical decision support. Here, we demonstrate a robust platform that uses software automation and high performance computing (HPC) resources to achieve real-time analytics of clinical data, specifically magnetic resonance imaging (MRI) data. We used the Agave application programming interface to facilitate communication, data transfer, and job control between an MRI scanner and an off-site HPC resource. In this use case, Agave executed the graphical pipeline tool GRAphical Pipeline Environment (GRAPE) to perform automated, real-time, quantitative analysis of MRI scans. Same-session image processing will open the door for adaptive scanning and real-time quality control, potentially accelerating the discovery of pathologies and minimizing patient callbacks. We envision this platform can be adapted to other medical instruments, HPC resources, and analytics tools.

Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs.

PubMed

Sehata, Go; Sato, Hiroaki; Ito, Toshihiro; Imaizumi, Yoshitaka; Noro, Taichi; Oishi, Eiji

2015-07-01

We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication.

Use of quantitative real-time RT-PCR to investigate the correlation between viremia and viral shedding of canine distemper virus, and infection outcomes in experimentally infected dogs

PubMed Central

SEHATA, Go; SATO, Hiroaki; ITO, Toshihiro; IMAIZUMI, Yoshitaka; NORO, Taichi; OISHI, Eiji

2015-01-01

We used real-time RT-PCR and virus titration to examine canine distemper virus (CDV) kinetics in peripheral blood and rectal and nasal secretions from 12 experimentally infected dogs. Real-time RT-PCR proved extremely sensitive, and the correlation between the two methods for rectal and nasal (r=0.78, 0.80) samples on the peak day of viral RNA was good. Although the dogs showed diverse symptoms, viral RNA kinetics were similar; the peak of viral RNA in the symptomatic dogs was consistent with the onset of symptoms. These results indicate that real-time RT-PCR is sufficiently sensitive to monitor CDV replication in experimentally infected dogs regardless of the degree of clinical manifestation and suggest that the peak of viral RNA reflects active CDV replication. PMID:25728411

On the multifractal effects generated by monofractal signals

NASA Astrophysics Data System (ADS)

Grech, Dariusz; Pamuła, Grzegorz

2013-12-01

We study quantitatively the level of false multifractal signal one may encounter while analyzing multifractal phenomena in time series within multifractal detrended fluctuation analysis (MF-DFA). The investigated effect appears as a result of finite length of used data series and is additionally amplified by the long-term memory the data eventually may contain. We provide the detailed quantitative description of such apparent multifractal background signal as a threshold in spread of generalized Hurst exponent values Δh or a threshold in the width of multifractal spectrum Δα below which multifractal properties of the system are only apparent, i.e. do not exist, despite Δα≠0 or Δh≠0. We find this effect quite important for shorter or persistent series and we argue it is linear with respect to autocorrelation exponent γ. Its strength decays according to power law with respect to the length of time series. The influence of basic linear and nonlinear transformations applied to initial data in finite time series with various levels of long memory is also investigated. This provides additional set of semi-analytical results. The obtained formulas are significant in any interdisciplinary application of multifractality, including physics, financial data analysis or physiology, because they allow to separate the ‘true’ multifractal phenomena from the apparent (artificial) multifractal effects. They should be a helpful tool of the first choice to decide whether we do in particular case with the signal with real multiscaling properties or not.

Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection

PubMed Central

Paudel, Damodar; Jarman, Richard; Limkittikul, Kriengsak; Klungthong, Chonticha; Chamnanchanunt, Supat; Nisalak, Ananda; Gibbons, Robert; Chokejindachai, Watcharee

2011-01-01

Background: Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman® assay and conventional nested PCR assay. Aims: To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti). Materials and Methods: Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR® Green assay, real time Taqman® assay to compare the sensitivity and specificity. Results: Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR® green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay. Conclusion: We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus. PMID:22363089

Detection of the downy mildew pathogens of spinach (Peronospora effusa) and beet (P. schachtii) using spore traps and quantitative PCR assays

USDA-ARS?s Scientific Manuscript database

Downy mildew of spinach, caused by Peronospora effusa, is a disease constraint on spinach production worldwide. The aim of this study was to develop a real-time quantitative PCR assay for detection of airborne inoculum of P. effusa in California. This type of assay may, in combination with disease-...

Coupling spore traps and quantitative PCR assays for detection of the downy mildew pathogens of spinach (Peronospora effusa) and beet (Peronospora schachtii)

USDA-ARS?s Scientific Manuscript database

Downy mildew of spinach (Spinacia oleracea L.), caused by Peronospora effusa, is a disease constraint on production worldwide, including in California where the majority of United States spinach is grown. The aim of this study was to develop a real-time quantitative PCR (qPCR) assay for detection o...

Label-Free Quantitative Immunoassay of Fibrinogen in Alzheimer Disease Patient Plasma Using Fiber Optical Surface Plasmon Resonance

NASA Astrophysics Data System (ADS)

Kim, Jisoo; Kim, SeJin; Nguyen, Tan Tai; Lee, Renee; Li, Tiehua; Yun, Changhyun; Ham, Youngeun; An, Seong Soo A.; Ju, Heongkyu

2016-05-01

We present a real-time quantitative immunoassay to detect fibrinogen in the blood plasma of Alzheimer's disease patients using multimode fiber optical sensors in which surface plasmon resonance (SPR) was employed. Nanometer-thick bimetals including silver and aluminum were coated onto the core surface of the clad-free part (5 cm long) of the fiber for SPR excitation at the He-Ne laser wavelength of 632.8 nm. The histidine-tagged peptide was then coated on the metal surface to immobilize the fibrinogen antibody for the selective capture of fibrinogen among the proteins in the patient blood plasma. The SPR fiber optical sensor enabled quantitative detection of concentrations of fibrinogen from the different human patient blood at a detection limit of ˜20 ng/ml. We also observed a correlation in the fibrinogen concentration measurement between enzyme-linked immunosorbent assay and our SPR fiber-based sensors. This suggests that the presented SPR fiber-based sensors that do not rely on the use of labels such as fluorophores can be used for a real-time quantitative assay of a specific protein such as fibrinogen in a human blood that is known to contain many other kinds of proteins together.

Apparatus and method for quantitative determination of materials contained in fluids

DOEpatents

Radziemski, Leon J.; Cremers, David A.

1985-01-01

Apparatus and method for near real-time in-situ monitoring of particulates and vapors contained in fluids. Initial filtration of a known volume of the fluid sample is combined with laser-induced dielectric breakdown spectroscopy of the filter employed to obtain qualitative and quantitative information with high sensitivity. Application of the invention to monitoring of beryllium, beryllium oxide, or other beryllium-alloy dusts is demonstrated. Significant shortening of analysis time is achieved from those of the usual chemical techniques of analysis.

Apparatus and method for quantitative determination of materials contained in fluids

DOEpatents

Radziemski, L.J.; Cremers, D.A.

1982-09-07

Apparatus and method for near real-time in-situ monitoring of particulates and vapors contained in fluids are described. Initial filtration of a known volume of the fluid sample is combined with laser-induced dielectric breakdown spectroscopy of the filter employed to obtain qualitative and quantitative information with high sensitivity. Application of the invention to monitoring of beryllium, beryllium oxide, or other beryllium-alloy dusts is shown. Significant shortening of analysis time is achieved from the usual chemical techniques of analysis.

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

NASA Astrophysics Data System (ADS)

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

2012-10-01

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

Evaluation of Hologic Aptima HIV-1 Quant Dx Assay on the Panther System on HIV Subtypes

PubMed Central

Hack, Holly R.; Nair, Sangeetha V.; Worlock, Andrew; Malia, Jennifer A.; Peel, Sheila A.; Jagodzinski, Linda L.

2016-01-01

Quantitation of the HIV-1 viral load in plasma is the current standard of care for clinical monitoring of HIV-infected individuals undergoing antiretroviral therapy. This study evaluated the analytical and clinical performances of the Aptima HIV-1 Quant Dx assay (Hologic, San Diego, CA) for monitoring viral load by using 277 well-characterized subtype samples, including 171 cultured virus isolates and 106 plasma samples from 35 countries, representing all major HIV subtypes, recombinants, and circulating recombinant forms (CRFs) currently in circulation worldwide. Linearity of the Aptima assay was tested on each of 6 major HIV-1 subtypes (A, B, C, D, CRF01_AE, and CRF02_AG) and demonstrated an R2 value of ≥0.996. The performance of the Aptima assay was also compared to those of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 v.2 (CAP/CTM) and Abbott m2000 RealTime HIV-1 (RealTime) assays on all subtype samples. The Aptima assay values averaged 0.21 log higher than the CAP/CTM values and 0.30 log higher than the RealTime values, and the values were >0.4 log higher than CAP/CTM values for subtypes F and G and than RealTime values for subtypes C, F, and G and CRF02_AG. Two samples demonstrated results with >1-log differences from RealTime results. When the data were adjusted by the average difference, 94.9% and 87.0% of Aptima results fell within 0.5 log of the CAP/CTM and RealTime results, respectively. The linearity and accuracy of the Aptima assay in correctly quantitating all major HIV-1 subtypes, coupled with the completely automated format and high throughput of the Panther system, make this system well suited for reliable measurement of viral load in the clinical laboratory. PMID:27510829

Comparative performance of the new Aptima HIV-1 Quant Dx assay with three commercial PCR-based HIV-1 RNA quantitation assays.

PubMed

Hopkins, Mark; Hau, Sarah; Tiernan, Caroline; Papadimitropoulos, Athanasios; Chawla, Anu; Beloukas, Apostolos; Geretti, Anna Maria

2015-08-01

Quantitative measurement of HIV-1 RNA levels in plasma ('viral load') plays a central role in clinical management. The choice of assay platform can influence results and treatment decisions. To compare the analytical performance of the new TMA-based Hologic Aptima(®) HIV-1 Quant Dx assay with that of three PCR-based assays: Abbott RealTime HIV-1, Qiagen Artus(®) HI Virus-1 QS-RGQ, and Roche CAP/CTM HIV-1 Test v2. Assay performance was evaluated using Acrometrix HIV-1 RNA Standard panels; the 3rd WHO HIV-1 RNA International Standard (12-500 copies/ml; 6 dilutions; 9 replicates); and plasma samples from 191 HIV-positive patients. Aptima showed high (>0.99) precision, accuracy and concordance with the Acrometrix Standards across a wide dynamic range (2.0-6.7 log10copies/ml). Variance caused up to 2.1 (Aptima), 1.7 (RealTime), 7.5 (Artus), and 1.9 (CAP/CTM) fold changes in the International Standard quantifications at 50-500 copies/ml. HIV-1 RNA detection rates in plasma samples were 141/191 (74%), 119/191 (62%), 108/191 (57%), and 145/191 (76%) for Aptima, RealTime, Artus and CAP/CTM, respectively. For categorising samples either side of 50 copies/ml, Aptima had excellent agreement with RealTime (kappa 0.92; 95% CI 0.87-0.98); lowest agreement was with Artus (kappa 0.79; 95%CI 0.70-0.88). Aptima quantifications were mean 0.12 and 0.06 log10copies/ml higher compared with RealTime and CAP/CTM, respectively, and 0.05 log10copies/ml lower compared with Artus. Limits of agreement were narrowest when comparing Aptima to RealTime. The new Aptima HIV assay is sensitive, precise, and accurate. HIV assays exhibit discordance at low HIV-1 RNA copy numbers. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Evaluation of Hologic Aptima HIV-1 Quant Dx Assay on the Panther System on HIV Subtypes.

PubMed

Manak, Mark M; Hack, Holly R; Nair, Sangeetha V; Worlock, Andrew; Malia, Jennifer A; Peel, Sheila A; Jagodzinski, Linda L

2016-10-01

Quantitation of the HIV-1 viral load in plasma is the current standard of care for clinical monitoring of HIV-infected individuals undergoing antiretroviral therapy. This study evaluated the analytical and clinical performances of the Aptima HIV-1 Quant Dx assay (Hologic, San Diego, CA) for monitoring viral load by using 277 well-characterized subtype samples, including 171 cultured virus isolates and 106 plasma samples from 35 countries, representing all major HIV subtypes, recombinants, and circulating recombinant forms (CRFs) currently in circulation worldwide. Linearity of the Aptima assay was tested on each of 6 major HIV-1 subtypes (A, B, C, D, CRF01_AE, and CRF02_AG) and demonstrated an R(2) value of ≥0.996. The performance of the Aptima assay was also compared to those of the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 v.2 (CAP/CTM) and Abbott m2000 RealTime HIV-1 (RealTime) assays on all subtype samples. The Aptima assay values averaged 0.21 log higher than the CAP/CTM values and 0.30 log higher than the RealTime values, and the values were >0.4 log higher than CAP/CTM values for subtypes F and G and than RealTime values for subtypes C, F, and G and CRF02_AG. Two samples demonstrated results with >1-log differences from RealTime results. When the data were adjusted by the average difference, 94.9% and 87.0% of Aptima results fell within 0.5 log of the CAP/CTM and RealTime results, respectively. The linearity and accuracy of the Aptima assay in correctly quantitating all major HIV-1 subtypes, coupled with the completely automated format and high throughput of the Panther system, make this system well suited for reliable measurement of viral load in the clinical laboratory. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Estimation of Discontinuous Displacement Vector Fields with the Minimum Description Length Criterion.

DTIC Science & Technology

1990-10-01

type of approach for finding a dense displacement vector field has a time complexity that allows a real - time implementation when an appropriate control...hardly vector fields as they appear in Stereo or motion. The reason for this is the fact that local displacement vector field ( DVF ) esti- mates bave...2 objects’ motion, but that the quantitative optical flow is not a reliable measure of the real motion [VP87, SU87]. This applies even more to the

Incremental value of live/real time three-dimensional transesophageal echocardiography over the two-dimensional technique in the assessment of primary cardiac malignant fibrous histiocytoma.

PubMed

Gok, Gulay; Elsayed, Mahmoud; Thind, Munveer; Uygur, Begum; Abtahi, Firoozeh; Chahwala, Jugal R; Yıldırımtürk, Özlem; Kayacıoğlu, İlyas; Pehlivanoğlu, Seçkin; Nanda, Navin C

2015-07-01

We describe a case of primary cardiac malignant fibrous histiocytoma where live/real time three-dimensional transesophageal echocardiography added incremental value to the two-dimensional modalities. Specifically, the three-dimensional technique allowed us to delineate the true extent and infiltration of the tumor, to identify characteristics of the tumor mass suggestive of its malignant nature, and to quantitatively assess the total tumor burden. © 2015, Wiley Periodicals, Inc.

Determination of drugs and drug-like compounds in different samples with direct analysis in real time mass spectrometry.

PubMed

Chernetsova, Elena S; Morlock, Gertrud E

2011-01-01

Direct analysis in real time (DART), a relatively new ionization source for mass spectrometry, ionizes small-molecule components from different kinds of samples without any sample preparation and chromatographic separation. The current paper reviews the published data available on the determination of drugs and drug-like compounds in different matrices with DART-MS, including identification and quantitation issues. Parameters that affect ionization efficiency and mass spectra composition are also discussed. Copyright © 2011 Wiley Periodicals, Inc.

Detection of Alicyclobacillus spp. in Fruit Juice by Combination of Immunomagnetic Separation and a SYBR Green I Real-Time PCR Assay

PubMed Central

Yuan, Yahong; Liu, Bin; Wang, Ling; Yue, Tianli

2015-01-01

An approach based on immunomagnetic separation (IMS) and SYBR Green I real-time PCR (real-time PCR) with species-specific primers and melting curve analysis was proposed as a rapid and effective method for detecting Alicyclobacillus spp. in fruit juices. Specific primers targeting the 16S rDNA sequences of Alicyclobacillus spp. were designed and then confirmed by the amplification of DNA extracted from standard strains and isolates. Spiked samples containing known amounts of target bacteria were used to obtain standard curves; the correlation coefficient was greater than 0.986 and the real-time PCR amplification efficiencies were 98.9%- 101.8%. The detection limit of the testing system was 2.8×101 CFU/mL. The coefficient of variation for intra-assay and inter-assay variability were all within the acceptable limit of 5%. Besides, the performance of the IMS-real-time PCR assay was further investigated by detecting naturally contaminated kiwi fruit juice; the sensitivity, specificity and accuracy were 91.7%, 95.9% and 95.3%, respectively. The established IMS-real-time PCR procedure provides a new method for identification and quantitative detection of Alicyclobacillus spp. in fruit juice. PMID:26488469

[Quantitative real-time PCR for target periodontal bacteria in subgingival plaque before and after local delivery of periocline, scaling and root planning].

PubMed

Deng, Shu-li; Wang, Ying; He, Jia-yan; Chen, Zhuo; Chen, Hui

2013-06-01

To compare the copy number of Porphyromonas gingivalis (Pg) and Prevotella intermedia (Pi) in subgingival plaque before and after local delivery of periocline (2% minocycline hydrochloride ointment, MO), scaling and root planning (SRP) by quantitative real-time PCR (qRT-PCR) and evaluate the efficacy. Sixty-two adults with moderate to severe chronic periodontitis were selected in the study. Microbial samples were taken from pocket before and after MO and SRP(7d). The samples were evaluated by qRT-PCR for Pg and Pi. Microbiological effectiveness of treatments was assessed using Kruskal-Wallis and Wilcoxon rank-sum test. All tests were two-sided with a significance level of 0.05. All analyses were conducted with SAS 9.1.3 software package. The copy number of Pg and Pi in subgingival plaque was 10(3)-10(6) and 10(2)-10(6). Bacterial loads of Pg were reduced in SPR+ MO, SRP and MO site. The counts of Pi decreased in SRP+ MO sites compared with those in the MO or SRP alone sites significantly (P<0.05). Quantitative real-time PCR (qRT-PCR) is used as a powerful tool with high sensitivity and specificity to quantitatively assess target periodontal bacteria. The results show that subgingival administration of MO and SRP was effective for reducing pathogenic bacteria and improving clinical outcome. Supported by 2011 National Clinical Specialist Construction Project; Natural Science Foundation of Zhejiang Province(LY13H140002); Education Department Funds of Zhejiang Province(20061258) and Medical General Research Project of Zhejiang Province(2012KYB121).

Fast-tracking determination of homozygous transgenic lines and transgene stacking using a reliable quantitative real-time PCR assay.

PubMed

Wang, Xianghong; Jiang, Daiming; Yang, Daichang

2015-01-01

The selection of homozygous lines is a crucial step in the characterization of newly generated transgenic plants. This is particularly time- and labor-consuming when transgenic stacking is required. Here, we report a fast and accurate method based on quantitative real-time PCR with a rice gene RBE4 as a reference gene for selection of homozygous lines when using multiple transgenic stacking in rice. Use of this method allowed can be used to determine the stacking of up to three transgenes within four generations. Selection accuracy reached 100 % for a single locus and 92.3 % for two loci. This method confers distinct advantages over current transgenic research methodologies, as it is more accurate, rapid, and reliable. Therefore, this protocol could be used to efficiently select homozygous plants and to expedite time- and labor-consuming processes normally required for multiple transgene stacking. This protocol was standardized for determination of multiple gene stacking in molecular breeding via marker-assisted selection.

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

PubMed

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

2009-08-25

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

Smartphone-based quantitative measurements on holographic sensors.

PubMed

Khalili Moghaddam, Gita; Lowe, Christopher Robin

2017-01-01

The research reported herein integrates a generic holographic sensor platform and a smartphone-based colour quantification algorithm in order to standardise and improve the determination of the concentration of analytes of interest. The utility of this approach has been exemplified by analysing the replay colour of the captured image of a holographic pH sensor in near real-time. Personalised image encryption followed by a wavelet-based image compression method were applied to secure the image transfer across a bandwidth-limited network to the cloud. The decrypted and decompressed image was processed through four principal steps: Recognition of the hologram in the image with a complex background using a template-based approach, conversion of device-dependent RGB values to device-independent CIEXYZ values using a polynomial model of the camera and computation of the CIEL*a*b* values, use of the colour coordinates of the captured image to segment the image, select the appropriate colour descriptors and, ultimately, locate the region of interest (ROI), i.e. the hologram in this case, and finally, application of a machine learning-based algorithm to correlate the colour coordinates of the ROI to the analyte concentration. Integrating holographic sensors and the colour image processing algorithm potentially offers a cost-effective platform for the remote monitoring of analytes in real time in readily accessible body fluids by minimally trained individuals.

Real-time PCR as an effective technique to assess the impact of phoresy by Paenibacillus sp. bacteria on Steinernema diaprepesi nematodes in nature.

PubMed

Campos-Herrera, R; El-Borai, F E; Duncan, L W

2012-09-01

Quantitative real-time PCR (qPCR) is a powerful tool to study species of cryptic organisms in complex food webs. This technique was recently developed to detect and quantify several species of entomopathogenic nematodes (EPNs), which are widely used for biological control of insects, and some natural enemies of EPNs such as nematophagous fungi and the phoretic bacteria Paenibacillus sp. and Paenibacillus nematophilus. A drawback to the use of primers and TaqMan probes designed for Paenibacillus sp. is that the qPCR also amplified Paenibacillus thiaminolyticus and Paenibacillus popilliae, two closely related species that are not phoretically associated with EPNs. Here, we report that the detection of Paenibacillus sp. DNA in nematode samples was two orders of magnitude greater (P < 0.001) when the bacterium was added to soil together with its EPN species-specific host Steinernema diaprepesi than when it was added concomitantly with other EPNs or with species of bacterial-feeding nematodes. Just 6% of samples detected trace amounts of P. thiaminolyticus and P. popilliae exposed to the same experimental conditions. Thus, although the molecular assay detects Paenibacillus spp. DNA in nonphoretic associations, the levels are essentially background compared to the detection of Paenibacillus sp. in association with its nematode host. © 2012 Blackwell Publishing Ltd.

Smartphone-based quantitative measurements on holographic sensors

PubMed Central

Khalili Moghaddam, Gita

2017-01-01

The research reported herein integrates a generic holographic sensor platform and a smartphone-based colour quantification algorithm in order to standardise and improve the determination of the concentration of analytes of interest. The utility of this approach has been exemplified by analysing the replay colour of the captured image of a holographic pH sensor in near real-time. Personalised image encryption followed by a wavelet-based image compression method were applied to secure the image transfer across a bandwidth-limited network to the cloud. The decrypted and decompressed image was processed through four principal steps: Recognition of the hologram in the image with a complex background using a template-based approach, conversion of device-dependent RGB values to device-independent CIEXYZ values using a polynomial model of the camera and computation of the CIEL*a*b* values, use of the colour coordinates of the captured image to segment the image, select the appropriate colour descriptors and, ultimately, locate the region of interest (ROI), i.e. the hologram in this case, and finally, application of a machine learning-based algorithm to correlate the colour coordinates of the ROI to the analyte concentration. Integrating holographic sensors and the colour image processing algorithm potentially offers a cost-effective platform for the remote monitoring of analytes in real time in readily accessible body fluids by minimally trained individuals. PMID:29141008

A Study on Micropipetting Detection Technology of Automatic Enzyme Immunoassay Analyzer.

PubMed

Shang, Zhiwu; Zhou, Xiangping; Li, Cheng; Tsai, Sang-Bing

2018-04-10

In order to improve the accuracy and reliability of micropipetting, a method of micro-pipette detection and calibration combining the dynamic pressure monitoring in pipetting process and quantitative identification of pipette volume in image processing was proposed. Firstly, the normalized pressure model for the pipetting process was established with the kinematic model of the pipetting operation, and the pressure model is corrected by the experimental method. Through the pipetting process pressure and pressure of the first derivative of real-time monitoring, the use of segmentation of the double threshold method as pipetting fault evaluation criteria, and the pressure sensor data are processed by Kalman filtering, the accuracy of fault diagnosis is improved. When there is a fault, the pipette tip image is collected through the camera, extract the boundary of the liquid region by the background contrast method, and obtain the liquid volume in the tip according to the geometric characteristics of the pipette tip. The pipette deviation feedback to the automatic pipetting module and deviation correction is carried out. The titration test results show that the combination of the segmented pipetting kinematic model of the double threshold method of pressure monitoring, can effectively real-time judgment and classification of the pipette fault. The method of closed-loop adjustment of pipetting volume can effectively improve the accuracy and reliability of the pipetting system.

High-throughput quantitative analysis by desorption electrospray ionization mass spectrometry.

PubMed

Manicke, Nicholas E; Kistler, Thomas; Ifa, Demian R; Cooks, R Graham; Ouyang, Zheng

2009-02-01

A newly developed high-throughput desorption electrospray ionization (DESI) source was characterized in terms of its performance in quantitative analysis. A 96-sample array, containing pharmaceuticals in various matrices, was analyzed in a single run with a total analysis time of 3 min. These solution-phase samples were examined from a hydrophobic PTFE ink printed on glass. The quantitative accuracy, precision, and limit of detection (LOD) were characterized. Chemical background-free samples of propranolol (PRN) with PRN-d(7) as internal standard (IS) and carbamazepine (CBZ) with CBZ-d(10) as IS were examined. So were two other sample sets consisting of PRN/PRN-d(7) at varying concentration in a biological milieu of 10% urine or porcine brain total lipid extract, total lipid concentration 250 ng/microL. The background-free samples, examined in a total analysis time of 1.5 s/sample, showed good quantitative accuracy and precision, with a relative error (RE) and relative standard deviation (RSD) generally less than 3% and 5%, respectively. The samples in urine and the lipid extract required a longer analysis time (2.5 s/sample) and showed RSD values of around 10% for the samples in urine and 4% for the lipid extract samples and RE values of less than 3% for both sets. The LOD for PRN and CBZ when analyzed without chemical background was 10 and 30 fmol, respectively. The LOD of PRN increased to 400 fmol analyzed in 10% urine, and 200 fmol when analyzed in the brain lipid extract.

Volcanology curricula development aided by online educational resource

USGS Publications Warehouse

Poland, Michael P.; van der Hoeven Kraft, Katrien J.; Teasdale, Rachel

2011-01-01

Volcanic activity is an excellent hook for engaging college and university students in geoscience classes. An increasing number of Internet-accessible real-time and near–real time volcano monitoring data are now available and constitute an important resource for geoscience education; however, relatively few data sets are comprehensive, and many lack background information to aid in interpretation. In response to the need for organized, accessible, and well-documented volcano education resources, the U.S. Geological Survey's Hawaiian Volcano Observatory (HVO), in collaboration with NASA and the University of Hawai`i at Manoa, established the Volcanoes Exploration Project: Pu`u `Ō`ō (VEPP). The VEPP Web site (http://vepp.wr.usgs.gov) is an educational resource that provides access, in near real time, to geodetic, seismic, and geologic data from the active Pu`u `Ō`ō eruptive vent on Kilauea volcano, Hawaii, along with background and context information. A strength of the VEPP site is the common theme of the Pu`u `Ō`ō eruption, which allows the site to be revisited multiple times to demonstrate different principles and integrate many aspects of volcanology.

Detection and quantitation of HPV in genital and oral tissues and fluids by real time PCR

PubMed Central

2010-01-01

Background Human papillomaviruses (HPVs) remain a serious world health problem due to their association with anogenital/oral cancers and warts. While over 100 HPV types have been identified, a subset is associated with malignancy. HPV16 and 18 are the most prevalent oncogenic types, while HPV6 and 11 are most commonly responsible for anogenital warts. While other quantitative PCR (qPCR) assays detect oncogenic HPV, there is no single tube assay distinguishing the most frequent oncogenic types and the most common types found in warts. Results A Sybr Green-based qPCR assay was developed utilizing degenerate primers to the highly conserved HPV E1 theoretically detecting any HPV type. A single tube multiplex qPCR assay was also developed using type-specific primer pairs and TaqMan probes that allowed for detection and quantitation of HPV6,11,16,18. Each HPV type was detected over a range from 2 × 101 to 2 × 106copies/reaction providing a reliable method of quantitating type-specific HPV in 140 anogenital/cutaneous/oral benign and malignant specimens. 35 oncogenic and low risk alpha genus HPV types were detected. Concordance was detected in previously typed specimens. Comparisons to the gold standard detected an overall sensitivity of 89% (95% CI: 77% - 96%) and specificity of 90% (95%CI: 52% - 98%). Conclusion There was good agreement between the ability of the qPCR assays described here to identify HPV types in malignancies previously typed using standard methods. These novel qPCR assays will allow rapid detection and quantitation of HPVs to assess their role in viral pathogenesis. PMID:20723234

Digital video timing analyzer for the evaluation of PC-based real-time simulation systems

NASA Astrophysics Data System (ADS)

Jones, Shawn R.; Crosby, Jay L.; Terry, John E., Jr.

2009-05-01

Due to the rapid acceleration in technology and the drop in costs, the use of commercial off-the-shelf (COTS) PC-based hardware and software components for digital and hardware-in-the-loop (HWIL) simulations has increased. However, the increase in PC-based components creates new challenges for HWIL test facilities such as cost-effective hardware and software selection, system configuration and integration, performance testing, and simulation verification/validation. This paper will discuss how the Digital Video Timing Analyzer (DiViTA) installed in the Aviation and Missile Research, Development and Engineering Center (AMRDEC) provides quantitative characterization data for PC-based real-time scene generation systems. An overview of the DiViTA is provided followed by details on measurement techniques, applications, and real-world examples of system benefits.

The use of comparative duplex PCR in monitoring of patients with non-Hodgkin's lymphoma and chronic lymphocytic leukaemia.

PubMed

Slavícková, A; Forsterová, K; Ivánek, R; Cerný, J; Klener, P

2005-01-01

Various quantitative PCR approaches have been utilized during the last years to provide information about the treatment efficacy and the risk of recurrent disease in haematological malignancies. Apart from the frequently used real-time PCR, cost-saving modified standard PCR methods may be applied as well. This report evaluates the utility of the end-point comparative duplex PCR. We have used this method for monitoring of 35 patients with either NHL or CLL and observed a good correlation between quantitative molecular results and clinical outcome. There was also an agreement between comparative duplex PCR and real-time PCR in patients who were monitored by both methods. We therefore believe that use of this technique should be strongly considered instead of simple qualitative detection in monitoring of therapeutic outcome in NHL or CLL patients.

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

PubMed

Kitta, Kazumi; Takabatake, Reona; Mano, Junichi

2016-01-01

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

Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy.

PubMed

Bergallo, Massimiliano; Galliano, Ilaria; Montanari, Paola; Brusin, Martina Rosa; Finotti, Serena; Paderi, Giulia; Gabiano, Clara

2017-04-01

Gastroenteritis is a common disease in children. It is characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis, but it causes milder illness than do rotavirus and norovirus. There is high variability in the analytical performance of quantitative PCR-based assays among clinical laboratories. This study developed a reverse transcription real-time PCR method to detect SaV in fecal specimens collected from children under 5-years-old with acute gastroenteritis. Of 137 episodes of acute gastroenteritis, 15 (10.9%) were associated with SaV genomic detection, with a median viral load of 6.6(log 10 ) ± 7.1(log 10 ) genomes/mg fecal specimens. There was a significant difference in detection rate between males and females (9.48% (13/15) vs. 1.46% (2/15), p = 0.0232). Among the 15 SaV-positive cases, 6 were also positive for rotavirus. Viral RNA recovery rate ranged from 46% to 77% in the manual RNAzol protocol and from 31% to 90% in the automated Maxwell protocol. We also studied whether human genomic DNA influences the sensitivity of the assay: its presence caused a decrease in PCR sensitivity. The development of a laboratory-designed real-time PCR TaqMan assay for quantitative detection of SaV and the optimization and standardization of this assay, using stools of children with acute gastroenteritis, are described.

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

PubMed

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

2008-09-01

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

Monitoring the Single-Cell Stress Response of the Diatom Thalassiosira pseudonana by Quantitative Real-Time Reverse Transcription-PCR

PubMed Central

Shi, Xu; Gao, Weimin; Chao, Shih-hui

2013-01-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition. PMID:23315741

Monitoring the single-cell stress response of the diatom Thalassiosira pseudonana by quantitative real-time reverse transcription-PCR.

PubMed

Shi, Xu; Gao, Weimin; Chao, Shih-hui; Zhang, Weiwen; Meldrum, Deirdre R

2013-03-01

Directly monitoring the stress response of microbes to their environments could be one way to inspect the health of microorganisms themselves, as well as the environments in which the microorganisms live. The ultimate resolution for such an endeavor could be down to a single-cell level. In this study, using the diatom Thalassiosira pseudonana as a model species, we aimed to measure gene expression responses of this organism to various stresses at a single-cell level. We developed a single-cell quantitative real-time reverse transcription-PCR (RT-qPCR) protocol and applied it to determine the expression levels of multiple selected genes under nitrogen, phosphate, and iron depletion stress conditions. The results, for the first time, provided a quantitative measurement of gene expression at single-cell levels in T. pseudonana and demonstrated that significant gene expression heterogeneity was present within the cell population. In addition, different expression patterns between single-cell- and bulk-cell-based analyses were also observed for all genes assayed in this study, suggesting that cell response heterogeneity needs to be taken into consideration in order to obtain accurate information that indicates the environmental stress condition.

Detection of Legionella by cultivation and quantitative real-time polymerase chain reaction in biological waste water treatment plants in Norway.

PubMed

Lund, Vidar; Fonahn, Wenche; Pettersen, Jens Erik; Caugant, Dominique A; Ask, Eirik; Nysaeter, Ase

2014-09-01

Cases of Legionnaires' disease associated with biological treatment plants (BTPs) have been reported in six countries between 1997 and 2010. However, knowledge about the occurrence of Legionella in BTPs is scarce. Hence, we undertook a qualitative and quantitative screening for Legionella in BTPs treating waste water from municipalities and industries in Norway, to assess the transmission potential of Legionella from these installations. Thirty-three plants from different industries were sampled four times within 1 year. By cultivation, 21 (16%) of 130 analyses were positive for Legionella species and 12 (9%) of 130 analyses were positive for Legionella pneumophila. By quantitative real-time polymerase chain reaction (PCR), 433 (99%) of 437 analyses were positive for Legionella species and 218 (46%) of 470 analyses were positive for L. pneumophila. This survey indicates that PCR could be the preferable method for detection of Legionella in samples from BTPs. Sequence types of L. pneumophila associated with outbreaks in Norway were not identified from the BTPs. We showed that a waste water treatment plant with an aeration basin can produce high concentrations of Legionella. Therefore, these plants should be considered as a possible source of community-acquired Legionella infections.

Gene expression in developing watermelon fruit

PubMed Central

Wechter, W Patrick; Levi, Amnon; Harris, Karen R; Davis, Angela R; Fei, Zhangjun; Katzir, Nurit; Giovannoni, James J; Salman-Minkov, Ayelet; Hernandez, Alvaro; Thimmapuram, Jyothi; Tadmor, Yaakov; Portnoy, Vitaly; Trebitsh, Tova

2008-01-01

Background Cultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR. Results High-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon genotype with a similar phenotype, i.e. seeded, bright red flesh, dark green rind, etc., determined that ethylene levels were highest during the green fruit stage followed by a decrease during the white and pink fruit stages. Additionally, quantitative Real-Time PCR was used to validate modulation of 127 ESTs that were differentially expressed in developing and ripening fruits based on array analysis. Conclusion This study identified numerous ESTs with putative involvement in the watermelon fruit developmental and ripening process, in particular the involvement of the vascular system and ethylene. The production of ethylene during fruit development in watermelon gives further support to the role of ethylene in fruit development in non-climacteric fruits. PMID:18534026

Preliminary results of real-time in-vitro electronic speckle pattern interferometry (ESPI) measurements in otolaryngology

NASA Astrophysics Data System (ADS)

Conerty, Michelle D.; Castracane, James; Cacace, Anthony T.; Parnes, Steven M.; Gardner, Glendon M.; Miller, Mitchell B.

1995-05-01

Electronic Speckle Pattern Interferometry (ESPI) is a nondestructive optical evaluation technique that is capable of determining surface and subsurface integrity through the quantitative evaluation of static or vibratory motion. By utilizing state of the art developments in the areas of lasers, fiber optics and solid state detector technology, this technique has become applicable in medical research and diagnostics. Based on initial support from NIDCD and continued support from InterScience, Inc., we have been developing a range of instruments for improved diagnostic evaluation in otolaryngological applications based on the technique of ESPI. These compact fiber optic instruments are capable of making real time interferometric measurements of the target tissue. Ongoing development of image post- processing software is currently capable of extracting the desired quantitative results from the acquired interferometric images. The goal of the research is to develop a fully automated system in which the image processing and quantification will be performed in hardware in near real-time. Subsurface details of both the tympanic membrane and vocal cord dynamics could speed the diagnosis of otosclerosis, laryngeal tumors, and aid in the evaluation of surgical procedures.

Effect of food matrix and thermal processing on the performance of a normalised quantitative real-time PCR approach for lupine (Lupinus albus) detection as a potential allergenic food.

PubMed

Villa, Caterina; Costa, Joana; Gondar, Cristina; Oliveira, M Beatriz P P; Mafra, Isabel

2018-10-01

Lupine is widely used as an ingredient in diverse food products, but it is also a source of allergens. This work aimed at proposing a method to detect/quantify lupine as an allergen in processed foods based on a normalised real-time PCR assay targeting the Lup a 4 allergen-encoding gene of Lupinus albus. Sensitivities down to 0.0005%, 0.01% and 0.05% (w/w) of lupine in rice flour, wheat flour and bread, respectively, and 1 pg of L. albus DNA were obtained, with adequate real-time PCR performance parameters using the ΔCt method. Both food matrix and processing affected negatively the quantitative performance of the assay. The method was successfully validated with blind samples and applied to processed foods. Lupine was estimated between 4.12 and 22.9% in foods, with some results suggesting the common practice of precautionary labelling. In this work, useful and effective tools were proposed for the detection/quantification of lupine in food products. Copyright © 2018 Elsevier Ltd. All rights reserved.

A haptic sensor-actor-system based on ultrasound elastography and electrorheological fluids for virtual reality applications in medicine.

PubMed

Khaled, W; Ermert, H; Bruhns, O; Boese, H; Baumann, M; Monkman, G J; Egersdoerfer, S; Meier, A; Klein, D; Freimuth, H

2003-01-01

Mechanical properties of biological tissue represent important diagnostic information and are of histological relevance (hard lesions, "nodes" in organs: tumors; calcifications in vessels: arteriosclerosis). The problem is, that such information is usually obtained by digital palpation only, which is limited with respect to sensitivity. It requires intuitive assessment and does not allow quantitative documentation. A suitable sensor is required for quantitative detection of mechanical tissue properties. On the other hand, there is also some need for a realistic mechanical display of those tissue properties. Suitable actuator arrays with high spatial resolution and real-time capabilities are required operating in a haptic sensor actuator system with different applications. The sensor system uses real time ultrasonic elastography whereas the tactile actuator is based on electrorheological fluids. Due to their small size the actuator array elements have to be manufactured by micro-mechanical production methods. In order to supply the actuator elements with individual high voltages a sophisticated switching and control concept have been designed. This haptic system has the potential of inducing real time substantial forces, using a compact lightweight mechanism which can be applied to numerous areas including intraoperative navigation, telemedicine, teaching, space and telecommunication.

Design, Construction and Evaluation of 1a/JFH1 HCV Chimera by Replacing the Intergenotypic Variable Region

PubMed Central

Ghasemi, Faezeh; Ghayour-Mobarhan, Majid; Pasdar, Alireza; Pourianfar, Hamid; Reza Aghasadeghi, Mohammad; Gouklani, Hamed; Meshkat, Zahra

2016-01-01

Background The E2 glycoprotein is an important encoded hepatitis C virus (HCV) protein that contains three different variable regions. Objectives The aim of the present study was to construct an HCV 1a/JFH1 chimeric virus by replacing the intergenotypic variable region (igVR) fragment of the highly variable region of the E2 gene of the Japanese Fulminant hepatitis genotype 2a JFH1 virus with a similar region of HCV genotype 1a. This chimera was produced as a model virus with the ability to be cultured. We analyzed the adapted virus and the variations of nucleic acids within it. Methods Specific primers were designed for the igVR of HCV genotype 1a followed by the overlap-PCR method for the synthesis of the desired DNA fragment. The amplified igVR-1a chimera gene and pFL-J6/JFH were digested by KpnI and BsiWI restriction enzymes, and the fragment was ligated into pFL-J6/JFH. The recombinant vector was transformed into Escherichia coli JM109 strain competent cells. All clones were confirmed by colony PCR using specific primers, and the confirmed recombinant vector was sequenced. The recombinant vector was targeted for RNA synthesis by T7 RNA polymerase enzyme. RNA transfection was performed in the Huh7.5 cell line. Virus production in several passages and the evaluated viral load were studied using quantitative real-time PCR and ELISA methods. After 30 passages, the RNA virus was extracted and cloned in PCDNA3.1 vector, and was then sequenced Results Quantitative real-time PCR results showed 11,292,514 copies/mL of chimeric virus production in cell culture. The virus production was confirmed using ELISA, which showed a virus core production of 808.2 pg/mL. The results of cloning and sequencing showed that some of the nucleic acids in the chimera virus were changed, affecting the viral behavior in the cell culture. Conclusions Real-time PCR and ELISA showed high levels of production of 1a/JFH1 chimeric HCV in the Huh7.5 cell culture. The constructed virus can be used for future studies, including the development of new HCV drugs and vaccines. PMID:27882063

Safety of real-time convection-enhanced delivery of liposomes to primate brain: a long-term retrospective.

PubMed

Krauze, Michal T; Vandenberg, Scott R; Yamashita, Yoji; Saito, Ryuta; Forsayeth, John; Noble, Charles; Park, John; Bankiewicz, Krystof S

2008-04-01

Convection-enhanced delivery (CED) is gaining popularity in direct brain infusions. Our group has pioneered the use of liposomes loaded with the MRI contrast reagent as a means to track and quantitate CED in the primate brain through real-time MRI. When co-infused with therapeutic nanoparticles, these tracking liposomes provide us with unprecedented precision in the management of infusions into discrete brain regions. In order to translate real-time CED into clinical application, several important parameters must be defined. In this study, we have analyzed all our cumulative animal data to answer a number of questions as to whether real-time CED in primates depends on concentration of infusate, is reproducible, allows prediction of distribution in a given anatomic structure, and whether it has long term pathological consequences. Our retrospective analysis indicates that real-time CED is highly predictable; repeated procedures yielded identical results, and no long-term brain pathologies were found. We conclude that introduction of our technique to clinical application would enhance accuracy and patient safety when compared to current non-monitored delivery trials.

Quantification of Wilms' tumor 1 mRNA by digital polymerase chain reaction.

PubMed

Koizumi, Yuki; Furuya, Daisuke; Endo, Teruo; Asanuma, Kouichi; Yanagihara, Nozomi; Takahashi, Satoshi

2018-02-01

Wilms' tumor 1 (WT1) is overexpressed in various hematopoietic tumors and widely used as a marker of minimal residual disease. WT1 mRNA has been analyzed using quantitative real-time polymerase chain reaction (real-time PCR). In the present study, we analyzed 40 peripheral blood and bone marrow samples obtained from cases of acute myeloid leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome at Sapporo Medical University Hospital from April 2012 to January 2015. We performed quantification of WT1 was performed using QuantStudio 3D Digital PCR System (Thermo Fisher Scientific‎) and compared the results between digital PCR and real-time PCR technology. The correlation between digital PCR and real-time PCR was very strong (R = 0.99), and the detection limits of the two methods were equivalent. Digital PCR was able to accurately detect lower WT levels compared with real-time PCR. Digital PCR technology can thus be utilized to predict WT1/ABL1 expression level accurately and should thus be useful for diagnosis or the evaluation of drug efficiency in patients with leukemia.

Application of clone library analysis and real-time PCR for comparison of microbial communities in a low-grade copper sulfide ore bioheap leachate.

PubMed

Bowei, Chen; Xingyu, Liu; Wenyan, Liu; Jiankang, Wen

2009-11-01

The microbial communities of leachate from a bioleaching heap located in China were analyzed using the 16S rRNA gene clone library and real-time quantitative PCR. Both methods showed that Leptospirillum spp. were the dominant bacteria, and Ferroplasma acidiphilum were the only archaea detected in the leachate. Clone library results indicated that nine operational taxonomic units (OTUs) were obtained, which fell into four divisions, the Nitrospirae (74%), the gamma-Proteobacteria (14%), the Actinobacteria (6%) and the Euryarchaeota (6%). The results obtained by real-time PCR in some ways were the same as clone library analysis. Furthermore, Sulfobacillus spp., detected only by real-time PCR, suggests that real-time PCR was a reliable technology to study the microbial communities in bioleaching environments. It is a useful tool to assist clone library analysis, to further understand microbial consortia and to have comprehensive and exact microbiological information about bioleaching environments. Finally, the interactions among the microorganisms detected in the leachate were summarized according to the characteristics of these species.

8 -+

NASA Astrophysics Data System (ADS)

Li, Bo; Charan, Kriti; Wang, Ke; Sinefeld, David; Xu, Chris

2017-02-01

We demonstrate a robust, all-fiber, two-wavelength time-lens source for background-free coherent anti-Stokes Raman scattering (CARS) imaging. The time-lens source generates two picosecond pulse trains simultaneously: one at 1064 nm and the other tunable between 1040 nm and 1075 nm ( 400 mW for each wavelength). When synchronized to a modelocked Ti:Sa laser, the two wavelengths are used to obtain on- and off-resonance CARS images. Real-time subtraction of the nonresonant background in the CARS image is achieved by the synchronization of the pixel clock and the time-lens source. Background-free CARS imaging of sebaceous glands in ex vivo mouse tissue is demonstrated.

Far-field photostable optical nanoscopy (PHOTON) for real-time super-resolution single-molecular imaging of signaling pathways of single live cells

NASA Astrophysics Data System (ADS)

Huang, Tao; Browning, Lauren M.; Xu, Xiao-Hong Nancy

2012-04-01

Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions.Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11739h

Comparison Between One-Point Calibration and Two-Point Calibration Approaches in a Continuous Glucose Monitoring Algorithm

PubMed Central

Mahmoudi, Zeinab; Johansen, Mette Dencker; Christiansen, Jens Sandahl

2014-01-01

Background: The purpose of this study was to investigate the effect of using a 1-point calibration approach instead of a 2-point calibration approach on the accuracy of a continuous glucose monitoring (CGM) algorithm. Method: A previously published real-time CGM algorithm was compared with its updated version, which used a 1-point calibration instead of a 2-point calibration. In addition, the contribution of the corrective intercept (CI) to the calibration performance was assessed. Finally, the sensor background current was estimated real-time and retrospectively. The study was performed on 132 type 1 diabetes patients. Results: Replacing the 2-point calibration with the 1-point calibration improved the CGM accuracy, with the greatest improvement achieved in hypoglycemia (18.4% median absolute relative differences [MARD] in hypoglycemia for the 2-point calibration, and 12.1% MARD in hypoglycemia for the 1-point calibration). Using 1-point calibration increased the percentage of sensor readings in zone A+B of the Clarke error grid analysis (EGA) in the full glycemic range, and also enhanced hypoglycemia sensitivity. Exclusion of CI from calibration reduced hypoglycemia accuracy, while slightly increased euglycemia accuracy. Both real-time and retrospective estimation of the sensor background current suggest that the background current can be considered zero in the calibration of the SCGM1 sensor. Conclusions: The sensor readings calibrated with the 1-point calibration approach indicated to have higher accuracy than those calibrated with the 2-point calibration approach. PMID:24876420

Digital PCR analysis of circulating nucleic acids.

PubMed

Hudecova, Irena

2015-10-01

Detection of plasma circulating nucleic acids (CNAs) requires the use of extremely sensitive and precise methods. The commonly used quantitative real-time polymerase chain reaction (PCR) poses certain technical limitations in relation to the precise measurement of CNAs whereas the costs of massively parallel sequencing are still relatively high. Digital PCR (dPCR) now represents an affordable and powerful single molecule counting strategy to detect minute amounts of genetic material with performance surpassing many quantitative methods. Microfluidic (chip) and emulsion (droplet)-based technologies have already been integrated into platforms offering hundreds to millions of nanoliter- or even picoliter-scale reaction partitions. The compelling observations reported in the field of cancer research, prenatal testing, transplantation medicine and virology support translation of this technology into routine use. Extremely sensitive plasma detection of rare mutations originating from tumor or placental cells among a large background of homologous sequences facilitates unraveling of the early stages of cancer or the detection of fetal mutations. Digital measurement of quantitative changes in plasma CNAs associated with cancer or graft rejection provides valuable information on the monitoring of disease burden or the recipient's immune response and subsequent therapy treatment. Furthermore, careful quantitative assessment of the viral load offers great value for effective monitoring of antiviral therapy for immunosuppressed or transplant patients. The present review describes the inherent features of dPCR that make it exceptionally robust in precise and sensitive quantification of CNAs. Moreover, I provide an insight into the types of potential clinical applications that have been developed by researchers to date. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

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

PubMed

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

2018-05-02

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

Direct real-time measurement of intra-oocyte nitric oxide concentration in vivo.

PubMed

Goud, Pravin T; Goud, Anuradha P; Najafi, Tohid; Gonik, Bernard; Diamond, Michael P; Saed, Ghassan M; Zhang, Xueji; Abu-Soud, Husam M

2014-01-01

Nitric oxide (NO) is reported to play significant a role in oocyte activation and maturation, implantation, and early embryonic development. Previously we have shown that NO forms an important component of the oocyte microenvironment, and functions effectively to delay oocyte aging. Thus, precise information about intra-oocyte NO concentrations [NO] will result in designing more accurate treatment plans in assisted reproduction. In this work, the direct, real-time and quantitative intra-oocyte [NO] was measured utilizing an L-shaped amperometric integrated NO-selective electrode. This method not only provides an elegant and convenient approach to real-time the measurement of NO in physiological environments, but also mimics the loss of NO caused by rapid NO diffusion combined with its reactivity in the biological milieu. This experiment suggests that the NO levels of oocytes obtained from young animals are significantly higher than those of oocytes obtained from old animals. Additionally the NO levels stay constant during the measurements; however, the intra-oocyte [NO] is reduced significantly (70-75% reduction) in response to L-NAME incubation, suggesting that NO measurements are truly NOS based rather than caused by an unknown interfering substance in our system. We believe this first demonstration of the direct quantitative measurement of [NO] in situ in an intact cellular complex should be useful in tracking real-time and rapid changes at nanomolar levels. Moreover, this finding confirms and extends our previous work showing that supplementation with NO delays the oocyte aging process.

Direct Real-Time Measurement of Intra-Oocyte Nitric Oxide Concentration In Vivo

PubMed Central

Goud, Pravin T.; Goud, Anuradha P.; Najafi, Tohid; Gonik, Bernard; Diamond, Michael P.; Saed, Ghassan M.; Zhang, Xueji; Abu-Soud, Husam M.

2014-01-01

Nitric oxide (NO) is reported to play significant a role in oocyte activation and maturation, implantation, and early embryonic development. Previously we have shown that NO forms an important component of the oocyte microenvironment, and functions effectively to delay oocyte aging. Thus, precise information about intra-oocyte NO concentrations [NO] will result in designing more accurate treatment plans in assisted reproduction. In this work, the direct, real-time and quantitative intra-oocyte [NO] was measured utilizing an L-shaped amperometric integrated NO-selective electrode. This method not only provides an elegant and convenient approach to real-time the measurement of NO in physiological environments, but also mimics the loss of NO caused by rapid NO diffusion combined with its reactivity in the biological milieu. This experiment suggests that the NO levels of oocytes obtained from young animals are significantly higher than those of oocytes obtained from old animals. Additionally the NO levels stay constant during the measurements; however, the intra-oocyte [NO] is reduced significantly (70–75% reduction) in response to L-NAME incubation, suggesting that NO measurements are truly NOS based rather than caused by an unknown interfering substance in our system. We believe this first demonstration of the direct quantitative measurement of [NO] in situ in an intact cellular complex should be useful in tracking real-time and rapid changes at nanomolar levels. Moreover, this finding confirms and extends our previous work showing that supplementation with NO delays the oocyte aging process. PMID:24887331

Sensitive detection of porcine DNA in processed animal proteins using a TaqMan real-time PCR assay.

PubMed

Pegels, N; González, I; Fernández, S; García, T; Martín, R

2012-01-01

A TaqMan real-time PCR method was developed for specific detection of porcine-prohibited material in industrial feeds. The assay combines the use of a porcine-specific primer pair, which amplifies a 79 bp fragment of the mitochondrial (mt) 12 S rRNA gene, and a locked nucleic acid (LNA) TaqMan probe complementary to a target sequence lying between the porcine-specific primers. The nuclear 18 S rRNA gene system, yielding a 77 bp amplicon, was employed as a positive amplification control to monitor the total content of amplifiable DNA in the samples. The specificity of the porcine primers-probe system was verified against different animal and plant species, including mammals, birds and fish. The applicability of the real-time PCR protocol to detect the presence of porcine mt DNA in feeds was determined through the analysis of 190 industrial feeds (19 known reference and 171 blind samples) subjected to stringent processing treatments. The performance of the method allows qualitative and highly sensitive detection of short fragments from porcine DNA in all the industrial feeds declared to contain porcine material. Although the method has quantitative potential, the real quantitative capability of the assay is limited by the existing variability in terms of composition and processing conditions of the feeds, which affect the amount and quality of amplifiable DNA.

Detection and correction of laser induced breakdown spectroscopy spectral background based on spline interpolation method

NASA Astrophysics Data System (ADS)

Tan, Bing; Huang, Min; Zhu, Qibing; Guo, Ya; Qin, Jianwei

2017-12-01

Laser-induced breakdown spectroscopy (LIBS) is an analytical technique that has gained increasing attention because of many applications. The production of continuous background in LIBS is inevitable because of factors associated with laser energy, gate width, time delay, and experimental environment. The continuous background significantly influences the analysis of the spectrum. Researchers have proposed several background correction methods, such as polynomial fitting, Lorenz fitting and model-free methods. However, less of them apply these methods in the field of LIBS Technology, particularly in qualitative and quantitative analyses. This study proposes a method based on spline interpolation for detecting and estimating the continuous background spectrum according to its smooth property characteristic. Experiment on the background correction simulation indicated that, the spline interpolation method acquired the largest signal-to-background ratio (SBR) over polynomial fitting, Lorenz fitting and model-free method after background correction. These background correction methods all acquire larger SBR values than that acquired before background correction (The SBR value before background correction is 10.0992, whereas the SBR values after background correction by spline interpolation, polynomial fitting, Lorentz fitting, and model-free methods are 26.9576, 24.6828, 18.9770, and 25.6273 respectively). After adding random noise with different kinds of signal-to-noise ratio to the spectrum, spline interpolation method acquires large SBR value, whereas polynomial fitting and model-free method obtain low SBR values. All of the background correction methods exhibit improved quantitative results of Cu than those acquired before background correction (The linear correlation coefficient value before background correction is 0.9776. Moreover, the linear correlation coefficient values after background correction using spline interpolation, polynomial fitting, Lorentz fitting, and model-free methods are 0.9998, 0.9915, 0.9895, and 0.9940 respectively). The proposed spline interpolation method exhibits better linear correlation and smaller error in the results of the quantitative analysis of Cu compared with polynomial fitting, Lorentz fitting and model-free methods, The simulation and quantitative experimental results show that the spline interpolation method can effectively detect and correct the continuous background.

Corporate Social Responsibility: A Real Options Approach to the Challenge of Financial Sustainability

PubMed Central

2015-01-01

Background In contemporary complex societies, social values like ethics, corporate social responsibility, and being respectful with the environment, among others, are becoming social requirements. Corporations are expected to fulfill them and, according to empirical evidence, an overwhelming majority aspires to good social valuation. At the same time, the maximization of market share value in the long run continues to be the central corporate goal. Making environmental and social expenses compatible with value creation is a central challenge for corporations since it implies the financial sustainability of Corporate Social Responsibility (CSR). Methods and Results The value creation capacity of CSR projects, mainly through innovation, is widely acknowledged in economic literature and corporate practice. This fact arouses the need of having a quantitative framework capable of summarizing the value creation capacity of the variables involved in CSR projects. With this aim we build up a sensitivity analysis of real option ratios that studies and quantifies the value creation capacity of CSR projects connected with innovation. Ratio analysis has the advantage of being scale independent. Hence, it furnishes a homogeneous framework to express the interaction of value creation variables and, thus, supports strategic thinking quantitatively. Often, CSR expenses can be regarded as preliminary projects that create the opportunity to undertake a full future project. For them, we obtain the minimum expectations scenario that makes financially sustainable a preliminary project that can be interpreted as a call option. We propose a classification of CSR projects from the decision analysis perspective following a two-fold approach: Their relationship with value creation and their links with existing corporate activities. This classification of CSR projects aims at contributing to choose the best capital budgeting method to study the financial sustainability of the project and identifying those CSR projects that fulfill the required features to be studied from the real options perspective. PMID:25938410

Purification of infectious human herpesvirus 6A virions and association of host cell proteins

PubMed Central

Hammarstedt, Maria; Ahlqvist, Jenny; Jacobson, Steven; Garoff, Henrik; Fogdell-Hahn, Anna

2007-01-01

Background Viruses that are incorporating host cell proteins might trigger autoimmune diseases. It is therefore of interest to identify possible host proteins associated with viruses, especially for enveloped viruses that have been suggested to play a role in autoimmune diseases, like human herpesvirus 6A (HHV-6A) in multiple sclerosis (MS). Results We have established a method for rapid and morphology preserving purification of HHV-6A virions, which in combination with parallel analyses with background control material released from mock-infected cells facilitates qualitative and quantitative investigations of the protein content of HHV-6A virions. In our iodixanol gradient purified preparation, we detected high levels of viral DNA by real-time PCR and viral proteins by metabolic labelling, silver staining and western blots. In contrast, the background level of cellular contamination was low in the purified samples as demonstrated by the silver staining and metabolic labelling analyses. Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions. Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions. Conclusion Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated. PMID:17949490

Real-time forecasts of tomorrow's earthquakes in California: a new mapping tool

USGS Publications Warehouse

Gerstenberger, Matt; Wiemer, Stefan; Jones, Lucy

2004-01-01

We have derived a multi-model approach to calculate time-dependent earthquake hazard resulting from earthquake clustering. This file report explains the theoretical background behind the approach, the specific details that are used in applying the method to California, as well as the statistical testing to validate the technique. We have implemented our algorithm as a real-time tool that has been automatically generating short-term hazard maps for California since May of 2002, at http://step.wr.usgs.gov

Real-time PCR Demonstrates Ancylostoma duodenale Is a Key Factor in the Etiology of Severe Anemia and Iron Deficiency in Malawian Pre-school Children

PubMed Central

Jonker, Femkje A. M.; Calis, Job C. J.; Phiri, Kamija; Brienen, Eric A. T.; Khoffi, Harriet; Brabin, Bernard J.; Verweij, Jaco J.; van Hensbroek, Michael Boele; van Lieshout, Lisette

2012-01-01

Background Hookworm infections are an important cause of (severe) anemia and iron deficiency in children in the tropics. Type of hookworm species (Ancylostoma duodenale or Necator americanus) and infection load are considered associated with disease burden, although these parameters are rarely assessed due to limitations of currently used diagnostic methods. Using multiplex real-time PCR, we evaluated hookworm species-specific prevalence, infection load and their contribution towards severe anemia and iron deficiency in pre-school children in Malawi. Methodology and Findings A. duodenale and N. americanus DNA loads were determined in 830 fecal samples of pre-school children participating in a case control study investigating severe anemia. Using multiplex real-time PCR, hookworm infections were found in 34.1% of the severely anemic cases and in 27.0% of the non-severely anemic controls (p<0.05) whereas a 5.6% hookworm prevalence was detected by microscopy. Prevalence of A. duodenale and N. americanus was 26.1% and 4.9% respectively. Moderate and high load A. duodenale infections were positively associated with severe anemia (adjusted odds ratio: 2.49 (95%CI 1.16–5.33) and 9.04 (95%CI 2.52–32.47) respectively). Iron deficiency (assessed through bone marrow examination) was positively associated with intensity of A. duodenale infection (adjusted odds ratio: 3.63 (95%CI 1.18–11.20); 16.98 (95%CI 3.88–74.35) and 44.91 (95%CI 5.23–385.77) for low, moderate and high load respectively). Conclusions/Significance This is the first report assessing the association of hookworm load and species differentiation with severe anemia and bone marrow iron deficiency. By revealing a much higher than expected prevalence of A. duodenale and its significant and load-dependent association with severe anemia and iron deficiency in pre-school children in Malawi, we demonstrated the need for quantitative and species-specific screening of hookworm infections. Multiplex real-time PCR is a powerful diagnostic tool for public health research to combat (severe) anemia and iron deficiency in children living in resource poor settings. PMID:22514750

Quantitative Real-Time PCR Fecal Source Identification in the Tillamook Basin (abstract)

EPA Science Inventory

Rivers in the Tillamook Basin play a vital role in supporting a thriving dairy and cheese-making industry, as well as providing a safe water resource for local human and wildlife populations. Historical concentrations of fecal bacteria in these waters are at times too high to all...

Time management situation assessment (TMSA)

NASA Technical Reports Server (NTRS)

Richardson, Michael B.; Ricci, Mark J.

1992-01-01

TMSA is a concept prototype developed to support NASA Test Directors (NTDs) in schedule execution monitoring during the later stages of a Shuttle countdown. The program detects qualitative and quantitative constraint violations in near real-time. The next version will support incremental rescheduling and reason over a substantially larger number of scheduled events.

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

PubMed

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

2014-01-01

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

Processing of next generation weather radar-multisensor precipitation estimates and quantitative precipitation forecast data for the DuPage County streamflow simulation system

USGS Publications Warehouse

Bera, Maitreyee; Ortel, Terry W.

2018-01-12

The U.S. Geological Survey, in cooperation with DuPage County Stormwater Management Department, is testing a near real-time streamflow simulation system that assists in the management and operation of reservoirs and other flood-control structures in the Salt Creek and West Branch DuPage River drainage basins in DuPage County, Illinois. As part of this effort, the U.S. Geological Survey maintains a database of hourly meteorological and hydrologic data for use in this near real-time streamflow simulation system. Among these data are next generation weather radar-multisensor precipitation estimates and quantitative precipitation forecast data, which are retrieved from the North Central River Forecasting Center of the National Weather Service. The DuPage County streamflow simulation system uses these quantitative precipitation forecast data to create streamflow predictions for the two simulated drainage basins. This report discusses in detail how these data are processed for inclusion in the Watershed Data Management files used in the streamflow simulation system for the Salt Creek and West Branch DuPage River drainage basins.

A real-time RT-PCR assay for molecular identification and quantitation of feline morbillivirus RNA from biological specimens.

PubMed

De Luca, Eliana; Crisi, Paolo Emidio; Di Domenico, Marco; Malatesta, Daniela; Vincifori, Giacomo; Di Tommaso, Morena; Di Guardo, Giovanni; Di Francesco, Gabriella; Petrini, Antonio; Savini, Giovanni; Boari, Andrea; Lorusso, Alessio

2018-05-03

The aim of this study was to develop a real-time RT-PCR to detect and quantitate feline morbillivirus (FeMV) RNA in biological samples. Primers and probe were targeted on a conserved region of FeMV P/V/C gene. To validate the assay with field samples, a total number of specimens of cats have been recruited including 264 urine and blood samples and compared with a generic RT-PCR targeting the L protein encoding gene of morbilliviruses. In addition, 385 tissue samples from 35 carcasses of cats have been also employed. RNA titres were low in all tested samples. Results also indicated the absence of cross-reaction with related morbilliviruses and existing pathogens of cats. In tissues with low levels of FeMV RNA, the presence of viral antigen was also evidenced by immunohistochemistry targeting the N viral protein. This newly described assay allows for a rapid, accurate and reliable quantitative detection of FeMV RNA that can be applied for diagnostics and research studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Applicability of the chymopapain gene used as endogenous reference gene for transgenic huanong no. 1 papaya detection.

PubMed

Guo, Jinchao; Yang, Litao; Liu, Xin; Zhang, Haibo; Qian, Bingjun; Zhang, Dabing

2009-08-12

The virus-resistant papaya (Carica papaya L.), Huanong no. 1, was the genetically modified (GM) fruit approved for growing in China in 2006. To implement the labeling regulation of GM papaya and its derivates, the development of papaya endogenous reference gene is very necessary for GM papaya detection. Herein, we reported one papaya specific gene, Chymopapain (CHY), as one suitable endogenous reference gene, used for GM papaya identification. Thereafter, we established the conventional and real-time quantitative PCR assays of the CHY gene. In the CHY conventional PCR assay, the limit of detection (LOD) was 25 copies of haploid papaya genome. In the CHY real-time quantitative PCR assay, both the LOD and the limit of quantification (LOQ) were as low as 12.5 copies of haploid papaya genome. Furthermore, we revealed the construct-specific sequence of Chinese GM papaya Huanong no. 1 and developed its conventional and quantitative PCR systems employing the CHY gene as endogenous reference gene. This work is useful for papaya specific identification and GM papaya detection.

Real-time PCR to determine transgene copy number and to quantitate the biolocalization of adoptively transferred cells from EGFP-transgenic mice.

PubMed

Joshi, Molishree; Keith Pittman, H; Haisch, Carl; Verbanac, Kathryn

2008-09-01

Quantitative real-time PCR (qPCR) is a sensitive technique for the detection and quantitation of specific DNA sequences. Here we describe a Taqman qPCR assay for quantification of tissue-localized, adoptively transferred enhanced green fluorescent protein (EGFP)-transgenic cells. A standard curve constructed from serial dilutions of a plasmid containing the EGFP transgene was (i) highly reproducible, (ii) detected as few as two copies, and (iii) was included in each qPCR assay. qPCR analysis of genomic DNA was used to determine transgene copy number in several mouse strains. Fluorescent microscopy of tissue sections showed that adoptively transferred vascular endothelial cells (VEC) from EGFP-transgenic mice specifically localized to tissue with metastatic tumors in syngeneic recipients. VEC microscopic enumeration of liver metastases strongly correlated with qPCR analysis of identical sections (Pearson correlation 0.81). EGFP was undetectable in tissue from control mice by qPCR. In another study using intra-tumor EGFP-VEC delivery to subcutaneous tumors, manual cell count and qPCR analysis of alternating sections also strongly correlated (Pearson correlation 0.82). Confocal microscopy of the subcutaneous tumor sections determined that visual fluorescent signals were frequently tissue artifacts. This qPCR methodology offers specific, objective, and rapid quantitation, uncomplicated by tissue autofluorescence, and should be readily transferable to other in vivo models to quantitate the biolocalization of transplanted cells.

High preservation of DNA standards diluted in 50% glycerol.

PubMed

Schaudien, Dirk; Baumgärtner, Wolfgang; Herden, Christiane

2007-09-01

Standard curves are important tools in real-time quantitative polymerase chain reaction (PCR) to precisely analyze gene expression patterns under physiologic and pathologic conditions. Handling of DNA standards often implies multiple cycles of freezing and thawing that might affect DNA stability and integrity. This in turn might influence the reliability and reproducibility of quantitative measurements in real-time PCR assays. In this study, 3 DNA standards such as murine tumor necrosis factor (TNF) alpha, interferon (IFN) gamma, and kainat-1 receptor were diluted in 50% glycerol or water after 1, 4, and 16 cycles of freezing and thawing and amplified copy numbers after real-time PCR were compared. The standards diluted in water showed a reduction to 83%, 55%, and 50% after 4 cycles, to 24%, 5%, and 4% after 16 cycles for kainat-1 receptor, TNFalpha, and IFNgamma standards, respectively, when compared with a single cycle of freezing and thawing. Interestingly, all cDNA samples diluted in 50% glycerol were amplified in comparable copy numbers even after 16 cycles of freezing and thawing. The effect of the standards undergoing different cycles of freezing and thawing on sample values was demonstrated by amplifying cDNA obtained from Borna disease virus infected and noninfected TNF-transgenic mice brain. This revealed significant differences of measured cDNA copy numbers using water-diluted DNA standards. In contrast, sample values did not vary using glycerol-diluted standards that were frozen and thawed for 16 times. In conclusion, glycerol storage of DNA standards represents a suitable tool for the accurate and reproducible quantification of cDNA samples in real-time PCR analysis.

Disposable MoS2-Arrayed MALDI MS Chip for High-Throughput and Rapid Quantification of Sulfonamides in Multiple Real Samples.

PubMed

Zhao, Yaju; Tang, Minmin; Liao, Qiaobo; Li, Zhoumin; Li, Hui; Xi, Kai; Tan, Li; Zhang, Mei; Xu, Danke; Chen, Hong-Yuan

2018-04-27

In this work, we demonstrate, for the first time, the development of a disposable MoS 2 -arrayed matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) chip combined with an immunoaffinity enrichment method for high-throughput, rapid, and simultaneous quantitation of multiple sulfonamides (SAs). The disposable MALDI MS chip was designed and fabricated by MoS 2 array formation on a commercial indium tin oxide (ITO) glass slide. A series of SAs were analyzed, and clear deprotonated signals were obtained in negative-ion mode. Compared with MoS 2 -arrayed commercial steel plate, the prepared MALDI MS chip exhibited comparable LDI efficiency, providing a good alternative and disposable substrate for MALDI MS analysis. Furthermore, internal standard (IS) was previously deposited onto the MoS 2 array to simplify the experimental process for MALDI MS quantitation. 96 sample spots could be analyzed within 10 min in one single chip to perform quantitative analysis, recovery studies, and real foodstuff detection. Upon targeted extraction and enrichment by antibody conjugated magnetic beads, five SAs were quantitatively determined by the IS-first method with the linear range of 0.5-10 ng/mL ( R 2 > 0.990). Good recoveries and repeatability were obtained for spiked pork, egg, and milk samples. SAs in several real foodstuffs were successfully identified and quantified. The developed method may provide a promising tool for the routine analysis of antibiotic residues in real samples.

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

PubMed

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

2014-11-01

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

Scientist/AMPS equipment interface study

NASA Technical Reports Server (NTRS)

Anderson, H. R.

1977-01-01

The principal objective was to determine for each experiment how the operating procedures and modes of equipment onboard shuttle can be managed in real-time or near-real-time to enhance the quality of results. As part of this determination the data and display devices that a man will need for real-time management are defined. The secondary objectives, as listed in the RFQ and technical proposal, were to: (1) determine what quantities are to be measured (2) determine permissible background levels (3) decide in what portions of space measurements are to be made (4) estimate bit rates (5) establish time-lines for operating the experiments on a mission or set of missions and (6) determine the minimum set of hardware needed for real-time display. Experiment descriptions and requirements were written. The requirements of the various experiments are combined and a minimal set of joint requirements are defined.

Quantitative 3-D imaging topogrammetry for telemedicine applications

NASA Technical Reports Server (NTRS)

Altschuler, Bruce R.

1994-01-01

The technology to reliably transmit high-resolution visual imagery over short to medium distances in real time has led to the serious considerations of the use of telemedicine, telepresence, and telerobotics in the delivery of health care. These concepts may involve, and evolve toward: consultation from remote expert teaching centers; diagnosis; triage; real-time remote advice to the surgeon; and real-time remote surgical instrument manipulation (telerobotics with virtual reality). Further extrapolation leads to teledesign and telereplication of spare surgical parts through quantitative teleimaging of 3-D surfaces tied to CAD/CAM devices and an artificially intelligent archival data base of 'normal' shapes. The ability to generate 'topogrames' or 3-D surface numerical tables of coordinate values capable of creating computer-generated virtual holographic-like displays, machine part replication, and statistical diagnostic shape assessment is critical to the progression of telemedicine. Any virtual reality simulation will remain in 'video-game' realm until realistic dimensional and spatial relational inputs from real measurements in vivo during surgeries are added to an ever-growing statistical data archive. The challenges of managing and interpreting this 3-D data base, which would include radiographic and surface quantitative data, are considerable. As technology drives toward dynamic and continuous 3-D surface measurements, presenting millions of X, Y, Z data points per second of flexing, stretching, moving human organs, the knowledge base and interpretive capabilities of 'brilliant robots' to work as a surgeon's tireless assistants becomes imaginable. The brilliant robot would 'see' what the surgeon sees--and more, for the robot could quantify its 3-D sensing and would 'see' in a wider spectral range than humans, and could zoom its 'eyes' from the macro world to long-distance microscopy. Unerring robot hands could rapidly perform machine-aided suturing with precision micro-sewing machines, splice neural connections with laser welds, micro-bore through constricted vessels, and computer combine ultrasound, microradiography, and 3-D mini-borescopes to quickly assess and trace vascular problems in situ. The spatial relationships between organs, robotic arms, and end-effector diagnostic, manipulative, and surgical instruments would be constantly monitored by the robot 'brain' using inputs from its multiple 3-D quantitative 'eyes' remote sensing, as well as by contact and proximity force measuring devices. Methods to create accurate and quantitative 3-D topograms at continuous video data rates are described.

A Comparison of Two Delivery Modalities of a Mobile Phone-Based Assessment for Serious Mental Illness: Native Smartphone Application vs Text-Messaging Only Implementations

PubMed Central

Palmier-Claus, Jasper E; Machin, Matthew; Barrowclough, Christine; Dunn, Graham; Rogers, Anne; Buchan, Iain; Barkus, Emma; Kapur, Shitij; Wykes, Til; Hopkins, Richard S; Lewis, Shôn

2013-01-01

Background Mobile phone–based assessment may represent a cost-effective and clinically effective method of monitoring psychotic symptoms in real-time. There are several software options, including the use of native smartphone applications and text messages (short message service, SMS). Little is known about the strengths and limitations of these two approaches in monitoring symptoms in individuals with serious mental illness. Objective The objective of this study was to compare two different delivery modalities of the same diagnostic assessment for individuals with non-affective psychosis—a native smartphone application employing a graphical, touch user interface against an SMS text-only implementation. The overall hypothesis of the study was that patient participants with sewrious mental illness would find both delivery modalities feasible and acceptable to use, measured by the quantitative post-assessment feedback questionnaire scores, the number of data points completed, and the time taken to complete the assessment. It was also predicted that a native smartphone application would (1) yield a greater number of data points, (2) take less time, and (3) be more positively appraised by patient participant users than the text-based system. Methods A randomized repeated measures crossover design was employed. Participants with currently treated Diagnostic and Statistical Manual (Fourth Edition) schizophrenia or related disorders (n=24) were randomly allocated to completing 6 days of assessment (four sets of questions per day) with a native smartphone application or the SMS text-only implementation. There was then a 1-week break before completing a further 6 days with the alternative delivery modality. Quantitative feedback questionnaires were administered at the end of each period of sampling. Results A greater proportion of data points were completed with the native smartphone application in comparison to the SMS text-only implementation (β = -.25, SE=.11, P=.02), which also took significantly less time to complete (β =.78, SE= .09, P<.001). Although there were no significant differences in participants’ quantitative feedback for the two delivery modalities, most participants reported preferring the native smartphone application (67%; n=16) and found it easier to use (71%; n=16). 33% of participants reported that they would be willing to complete mobile phone assessment for 5 weeks or longer. Conclusions Native smartphone applications and SMS text are both valuable methods of delivering real-time assessment in individuals with schizophrenia. However, a more streamlined graphical user interface may lead to better compliance and shorter entry times. Further research is needed to test the efficacy of this technology within clinical services, to assess validity over longer periods of time and when delivered on patients’ own phones. PMID:23563184

Quantitatively in Situ Imaging Silver Nanowire Hollowing Kinetics

DOE PAGES

Yu, Le; Yan, Zhongying; Cai, Zhonghou; ...

2016-09-28

We report the in-situ investigation of the morphological evolution of silver nanowires to hollow silver oxide nanotubes using transmission x-ray microscopy (TXM). Complex silver diffusion kinetics and hollowing process via the Kirkendall effect have been captured in real time. Further quantitative x-ray absorption analysis reveals the difference between the longitudinal and radial diffusions. In conclusion, the diffusion coefficient of silver in its oxide nanoshell is, for the first time, calculated to be 1.2 × 10 -13 cm 2/s from the geometrical parameters extracted from the TXM images.

Data forwarding mechanism for supporting real-time services during relocations in UMTS systems

NASA Astrophysics Data System (ADS)

Cai, Wei; Liao, Xianglong; Zheng, Liang; Liu, Zehong

2004-04-01

To minimize the interruption during the handovers or relocations invoked by subscribers moving is a very critical factor to enhance the performance of the UMTS systems. We know that the 2G systems have been optimized to minimize the interruption of speech during handovers by two main technologies: one is the bi-casting for the DL traffic and the other is the fast radio resynchronization by the UE for the UL traffic. In the UMTS systems, we have also implemented lossless relocations for non real-time services with high reliability by data buffering in the source RNC and target RNC for the UE. However, the UMTS systems support four QoS classes traffic flow: conversational class, streaming class, interactive class and background class. The main distinguishing factor between these QoS classes is how delay sensitive the traffic is: Conversational and Streaming classes are mainly used to carry real-time traffic flows, like video telephony, interactive and background classes are mainly used by traditional Internet applications like WWW, E-mail and FTP. It"s essential to provide the solutions for supporting real-time services to meet the requirement for QoS in UMTS systems. Apparently, the Data buffering mechanism is not adapted to real-time services because of it"s delay may exceed the basic requirement for real-time services. Under this background, the paper discussed two data forwarding solutions for real-time services from the PS domain in the UMTS systems: packet duplication and Core Network bi-casting. The former mechanism does not require any new procedures, messages nor information elements. The later mechanism requires that the GGSN or SGSN is able to bi-cast the DL traffic to the target RNC according to the relocations involving two SGSNs or just involving one SGSN. It also implicitly shows that we need change procedures at the nodes SGSN, GGSN and RNC which are involved in the relocation procedure based on existing procedures that we have already designed if adopt the later solution. In a detail way, the paper analyzed the characteristic for these two solutions respectively, concentrated on the packet flows and the message flows in those nodes involved in relocations. Additionally, also gave out the impact on present transport technologies in the wireless communication systems. However we shall minimize the impact of evolution of transport mechanism and utilize the resource efficiently according to the general requirements for QoS in UMTS systems.

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

PubMed

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

2015-02-14

An "off-the-shelf" fluorescence "turn-on" Mg(2+) chemosensor 3,5-dichlorosalicylaldehyde (BCSA) was rationally designed and developed. This proposed sensor works based on Mg(2+)-induced formation of the 2 : 1 BCSA-Mg(2+) complex. The coordination of BSCA to Mg(2+) increases its structural rigidity generating a chelation-enhanced fluorescence (CHEF) effect which was confirmed by single crystal XRD studies of the BSCA-Mg(2+) complex and TD/DFT calculations. This sensor exhibits high sensitivity and selectivity for the quantitative monitoring of Mg(2+) with a wide detection range (0-40 μM), a low detection limit (2.89 × 10(-7) mol L(-1)) and a short response time (<0.5 s). It can also resist the interference from the other co-existing metal ions, especially Ca(2+). Consequently, this fluorescent sensor can be utilized to monitor Mg(2+) in real time within actual samples from drinking water.

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

PubMed

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

2014-12-01

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

Structural models used in real-time biosurveillance outbreak detection and outbreak curve isolation from noisy background morbidity levels

PubMed Central

Cheng, Karen Elizabeth; Crary, David J; Ray, Jaideep; Safta, Cosmin

2013-01-01

Objective We discuss the use of structural models for the analysis of biosurveillance related data. Methods and results Using a combination of real and simulated data, we have constructed a data set that represents a plausible time series resulting from surveillance of a large scale bioterrorist anthrax attack in Miami. We discuss the performance of anomaly detection with structural models for these data using receiver operating characteristic (ROC) and activity monitoring operating characteristic (AMOC) analysis. In addition, we show that these techniques provide a method for predicting the level of the outbreak valid for approximately 2 weeks, post-alarm. Conclusions Structural models provide an effective tool for the analysis of biosurveillance data, in particular for time series with noisy, non-stationary background and missing data. PMID:23037798

Securing Real-Time Sessions in an IMS-Based Architecture

NASA Astrophysics Data System (ADS)

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

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

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

PubMed

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

2017-04-04

The family Enterobacteriaceae, members of which are widely distributed in the environment, includes many important human pathogens. In this study, a rapid real-time PCR method targeting rplP, coding for L16 protein, a component of the ribosome large subunit, was developed for enumerating Enterobacteriaceae strains, and its efficiency was evaluated using naturally contaminated food products. The rplP-targeted real-time PCR amplified Enterobacteriaceae species with Ct values of 14.0-22.8, whereas the Ct values for non-Enterobacteriaceae species were >30, indicating the specificity of this method for the Enterobacteriaceae. Using a calibration curve of Ct=-3.025 (log CFU/g)+37.35, which was calculated from individual plots of the cell numbers in different concentrations of 5 Enterobacteriaceae species, the rplP-targeted real-time PCR was applied to 51 food samples. A <1log difference between the real-time PCR and culture methods was obtained in a majority of the food samples (81.8%), with good correlation (r 2 =0.8285). This study demonstrated that the rplP-targeted real-time PCR method could detect and enumerate Enterobacteriaceae species in foods rapidly and accurately, and therefore, it can be used for the microbiological risk analysis of foods. Copyright © 2017 Elsevier B.V. All rights reserved.

Real-time, wide-area hyperspectral imaging sensors for standoff detection of explosives and chemical warfare agents

NASA Astrophysics Data System (ADS)

Gomer, Nathaniel R.; Tazik, Shawna; Gardner, Charles W.; Nelson, Matthew P.

2017-05-01

Hyperspectral imaging (HSI) is a valuable tool for the detection and analysis of targets located within complex backgrounds. HSI can detect threat materials on environmental surfaces, where the concentration of the target of interest is often very low and is typically found within complex scenery. Unfortunately, current generation HSI systems have size, weight, and power limitations that prohibit their use for field-portable and/or real-time applications. Current generation systems commonly provide an inefficient area search rate, require close proximity to the target for screening, and/or are not capable of making real-time measurements. ChemImage Sensor Systems (CISS) is developing a variety of real-time, wide-field hyperspectral imaging systems that utilize shortwave infrared (SWIR) absorption and Raman spectroscopy. SWIR HSI sensors provide wide-area imagery with at or near real time detection speeds. Raman HSI sensors are being developed to overcome two obstacles present in standard Raman detection systems: slow area search rate (due to small laser spot sizes) and lack of eye-safety. SWIR HSI sensors have been integrated into mobile, robot based platforms and handheld variants for the detection of explosives and chemical warfare agents (CWAs). In addition, the fusion of these two technologies into a single system has shown the feasibility of using both techniques concurrently to provide higher probability of detection and lower false alarm rates. This paper will provide background on Raman and SWIR HSI, discuss the applications for these techniques, and provide an overview of novel CISS HSI sensors focusing on sensor design and detection results.

Psychovisual masks and intelligent streaming RTP techniques for the MPEG-4 standard

NASA Astrophysics Data System (ADS)

Mecocci, Alessandro; Falconi, Francesco

2003-06-01

In today multimedia audio-video communication systems, data compression plays a fundamental role by reducing the bandwidth waste and the costs of the infrastructures and equipments. Among the different compression standards, the MPEG-4 is becoming more and more accepted and widespread. Even if one of the fundamental aspects of this standard is the possibility of separately coding video objects (i.e. to separate moving objects from the background and adapt the coding strategy to the video content), currently implemented codecs work only at the full-frame level. In this way, many advantages of the flexible MPEG-4 syntax are missed. This lack is due both to the difficulties in properly segmenting moving objects in real scenes (featuring an arbitrary motion of the objects and of the acquisition sensor), and to the current use of these codecs, that are mainly oriented towards the market of DVD backups (a full-frame approach is enough for these applications). In this paper we propose a codec for MPEG-4 real-time object streaming, that codes separately the moving objects and the scene background. The proposed codec is capable of adapting its strategy during the transmission, by analysing the video currently transmitted and setting the coder parameters and modalities accordingly. For example, the background can be transmitted as a whole or by dividing it into "slightly-detailed" and "highly detailed" zones that are coded in different ways to reduce the bit-rate while preserving the perceived quality. The coder can automatically switch in real-time, from one modality to the other during the transmission, depending on the current video content. Psychovisual masks and other video-content based measurements have been used as inputs for a Self Learning Intelligent Controller (SLIC) that changes the parameters and the transmission modalities. The current implementation is based on the ISO 14496 standard code that allows Video Objects (VO) transmission (other Open Source Codes like: DivX, Xvid, and Cisco"s Mpeg-4IP, have been analyzed but, as for today, they do not support VO). The original code has been deeply modified to integrate the SLIC and to adapt it for real-time streaming. A personal RTP (Real Time Protocol) has been defined and a Client-Server application has been developed. The viewer can decode and demultiplex the stream in real-time, while adapting to the changing modalities adopted by the Server according to the current video content. The proposed codec works as follows: the image background is separated by means of a segmentation module and it is transmitted by means of a wavelet compression scheme similar to that used in the JPEG2000. The VO are coded separately and multiplexed with the background stream. At the receiver the stream is demultiplexed to obtain the background and the VO that are subsequently pasted together. The final quality depends on many factors, in particular: the quantization parameters, the Group Of Video Object (GOV) length, the GOV structure (i.e. the number of I-P-B VOP), the search area for motion compensation. These factors are strongly related to the following measurement parameters (that have been defined during the development): the Objects Apparent Size (OAS) in the scene, the Video Object Incidence factor (VOI), the temporal correlation (measured through the Normalized Mean SAD, NMSAD). The SLIC module analyzes the currently transmitted video and selects the most appropriate settings by choosing from a predefined set of transmission modalities. For example, in the case of a highly temporal correlated sequence, the number of B-VOP is increased to improve the compression ratio. The strategy for the selection of the number of B-VOP turns out to be very different from those reported in the literature for B-frames (adopted for MPEG-1 and MPEG-2), due to the different behaviour of the temporal correlation when limited only to moving objects. The SLIC module also decides how to transmit the background. In our implementation we adopted the Visual Brain theory i.e. the study of what the "psychic eye" can get from a scene. According to this theory, a Psychomask Image Analysis (PIA) module has been developed to extract the visually homogeneous regions of the background. The PIA module produces two complementary masks one for the visually low variance zones and one for the higly variable zones; these zones are compressed with different strategies and encoded into two multiplexed streams. From practical experiments it turned out that the separate coding is advantageous only if the low variance zones exceed 50% of the whole background area (due to the overhead given by the need of transmitting the zone masks). The SLIC module takes care of deciding the appropriate transmission modality by analyzing the results produced by the PIA module. The main features of this codec are: low bitrate, good image quality and coding speed. The current implementation runs in real-time on standard PC platforms, the major limitation being the fixed position of the acquisition sensor. This limitation is due to the difficulties in separating moving objects from the background when the acquisition sensor moves. Our current real-time segmentation module does not produce suitable results if the acquisition sensor moves (only slight oscillatory movements are tolerated). In any case, the system is particularly suitable for tele surveillance applications at low bit-rates, where the camera is usually fixed or alternates among some predetermined positions (our segmentation module is capable of accurately separate moving objects from the static background when the acquisition sensor stops, even if different scenes are seen as a result of the sensor displacements). Moreover, the proposed architecture is general, in the sense that when real-time, robust segmentation systems (capable of separating objects in real-time from the background while the sensor itself is moving) will be available, they can be easily integrated while leaving the rest of the system unchanged. Experimental results related to real sequences for traffic monitoring and for people tracking and afety control are reported and deeply discussed in the paper. The whole system has been implemented in standard ANSI C code and currently runs on standard PCs under Microsoft Windows operating system (Windows 2000 pro and Windows XP).

Performance Assessment of Human and Cattle Associated Quantitative Real-time PCR Assays - slides

EPA Science Inventory

The presentation overview is (1) Single laboratory performance assessment of human- and cattle associated PCR assays and (2) A Field Study: Evaluation of two human fecal waste management practices in Ohio watershed.

When Should Zero Be Included on a Scale Showing Magnitude?

ERIC Educational Resources Information Center

Kozak, Marcin

2011-01-01

This article addresses an important problem of graphing quantitative data: should one include zero on the scale showing magnitude? Based on a real time series example, the problem is discussed and some recommendations are proposed.

A real-time monitoring platform of myogenesis regulators using double fluorescent labeling

PubMed Central

Sapoznik, Etai; Niu, Guoguang; Zhou, Yu; Prim, Peter M.; Criswell, Tracy L.

2018-01-01

Real-time, quantitative measurement of muscle progenitor cell (myoblast) differentiation is an important tool for skeletal muscle research and identification of drugs that support skeletal muscle regeneration. While most quantitative tools rely on sacrificial approach, we developed a double fluorescent tagging approach, which allows for dynamic monitoring of myoblast differentiation through assessment of fusion index and nuclei count. Fluorescent tagging of both the cell cytoplasm and nucleus enables monitoring of cell fusion and the formation of new myotube fibers, similar to immunostaining results. This labeling approach allowed monitoring the effects of Myf5 overexpression, TNFα, and Wnt agonist on myoblast differentiation. It also enabled testing the effects of surface coating on the fusion levels of scaffold-seeded myoblasts. The double fluorescent labeling of myoblasts is a promising technique to visualize even minor changes in myogenesis of myoblasts in order to support applications such as tissue engineering and drug screening. PMID:29444187

Using digital inline holographic microscopy and quantitative phase contrast imaging to assess viability of cultured mammalian cells

NASA Astrophysics Data System (ADS)

Missan, Sergey; Hrytsenko, Olga

2015-03-01

Digital inline holographic microscopy was used to record holograms of mammalian cells (HEK293, B16, and E0771) in culture. The holograms have been reconstructed using Octopus software (4Deep inwater imaging) and phase shift maps were unwrapped using the FFT-based phase unwrapping algorithm. The unwrapped phase shifts were used to determine the maximum phase shifts in individual cells. Addition of 0.5 mM H2O2 to cell media produced rapid rounding of cultured cells, followed by cell membrane rupture. The cell morphology changes and cell membrane ruptures were detected in real time and were apparent in the unwrapped phase shift images. The results indicate that quantitative phase contrast imaging produced by the digital inline holographic microscope can be used for the label-free real time automated determination of cell viability and confluence in mammalian cell cultures.

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

PubMed

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

2015-10-01

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

High-Throughput RT-PCR for small-molecule screening assays

PubMed Central

Bittker, Joshua A.

2012-01-01

Quantitative measurement of the levels of mRNA expression using real-time reverse transcription polymerase chain reaction (RT-PCR) has long been used for analyzing expression differences in tissue or cell lines of interest. This method has been used somewhat less frequently to measure the changes in gene expression due to perturbagens such as small molecules or siRNA. The availability of new instrumentation for liquid handling and real-time PCR analysis as well as the commercial availability of start-to-finish kits for RT-PCR has enabled the use of this method for high-throughput small-molecule screening on a scale comparable to traditional high-throughput screening (HTS) assays. This protocol focuses on the special considerations necessary for using quantitative RT-PCR as a primary small-molecule screening assay, including the different methods available for mRNA isolation and analysis. PMID:23487248

Real time quantitative imaging for semiconductor crystal growth, control and characterization

NASA Technical Reports Server (NTRS)

Wargo, Michael J.

1991-01-01

A quantitative real time image processing system has been developed which can be software-reconfigured for semiconductor processing and characterization tasks. In thermal imager mode, 2D temperature distributions of semiconductor melt surfaces (900-1600 C) can be obtained with temperature and spatial resolutions better than 0.5 C and 0.5 mm, respectively, as demonstrated by analysis of melt surface thermal distributions. Temporal and spatial image processing techniques and multitasking computational capabilities convert such thermal imaging into a multimode sensor for crystal growth control. A second configuration of the image processing engine in conjunction with bright and dark field transmission optics is used to nonintrusively determine the microdistribution of free charge carriers and submicron sized crystalline defects in semiconductors. The IR absorption characteristics of wafers are determined with 10-micron spatial resolution and, after calibration, are converted into charge carrier density.

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

NASA Astrophysics Data System (ADS)

Pi, Shiqiang; Liu, Wenzhong; Jiang, Tao

2018-03-01

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

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

PubMed

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.

Repertoire of novel sequence signatures for the detection of Candidatus Liberibacter asiaticus by quantitative real-time PCR

PubMed Central

2014-01-01

Background Huanglongbing (HLB) or citrus greening is a devastating disease of citrus. The gram-negative bacterium Candidatus Liberibacter asiaticus (Las) belonging to the α-proteobacteria is responsible for HLB in North America as well as in Asia. Currently, there is no cure for this disease. Early detection and quarantine of Las-infected trees are important management strategies used to prevent HLB from invading HLB-free citrus producing regions. Quantitative real-time PCR (qRT-PCR) based molecular diagnostic assays have been routinely used in the detection and diagnosis of Las. The oligonucleotide primer pairs based on conserved genes or regions, which include 16S rDNA and the β-operon, have been widely employed in the detection of Las by qRT-PCR. The availability of whole genome sequence of Las now allows the design of primers beyond the conserved regions for the detection of Las explicitly. Results We took a complimentary approach by systematically screening the genes in a genome-wide fashion, to identify the unique signatures that are only present in Las by an exhaustive sequence based similarity search against the nucleotide sequence database. Our search resulted in 34 probable unique signatures. Furthermore, by designing the primer pair specific to the identified signatures, we showed that most of our primer sets are able to detect Las from the infected plant and psyllid materials collected from the USA and China by qRT-PCR. Overall, 18 primer pairs of the 34 are found to be highly specific to Las with no cross reactivity to the closely related species Ca. L. americanus (Lam) and Ca. L. africanus (Laf). Conclusions We have designed qRT-PCR primers based on Las specific genes. Among them, 18 are suitable for the detection of Las from Las-infected plant and psyllid samples. The repertoire of primers that we have developed and characterized in this study enhanced the qRT-PCR based molecular diagnosis of HLB. PMID:24533511

CAPN 7 promotes the migration and invasion of human endometrial stromal cell by regulating matrix metalloproteinase 2 activity

PubMed Central

2013-01-01

Background Matrix metalloproteinase 2 (MMP-2) has been reported to be an important regulator of cell migration and invasion through degradation of the extracellular matrix (ECM) in many diseases, such as cancer and endometriosis. Here, we found calcium-activated neutral protease 7 (CAPN 7) expression was markedly upregulated in the eutopic endometrium and endometrial stromal cells of women diagnosed with endometriosis. Our studies were carried out to detect the effects of CAPN 7 on human endometrial stromal cell (hESC) migration and invasion. Methods Western blotting and quantitative real-time PCR were used to detect the expression of CAPN 7 in endometriosis patients and normal fertile women. Scratch-wound-healing and invasion chamber assay were used to investigate the role of CAPN 7 in hESC migration and invasion. Western blotting, quantitative real-time PCR and zymography were carried out to detect the effect of CAPN 7 on the expressions and activity of MMP-2. Results CAPN 7 was markedly up-regulated in endometriosis, thereby promoting the migration and invasion of hESC. CAPN 7 overexpression led to increased expression of MMP-2 and tissue inhibitor of metalloproteinases 2 (TIMP-2); CAPN 7 knockdown reversed these changes. CAPN 7 increased MMP-2 activity by increasing the ratio of MMP-2 to TIMP-2. We also found that OA-Hy (an MMP-2 inhibitor) decreased the effects of CAPN 7 overexpression on hESC migration and invasion by approximately 50% and 55%, respectively. Additionally, a coimmunoprecipitation assay demonstrated that CAPN 7 interacted with activator protein 2α (AP-2α): an important transcription factor of MMP-2. Conclusions CAPN 7 promotes hESC migration and invasion by increasing the activity of MMP-2 via an increased ratio of MMP-2 to TIMP-2. PMID:23855590

Expression of the Long Intergenic Non-Protein Coding RNA 665 (LINC00665) Gene and the Cell Cycle in Hepatocellular Carcinoma Using The Cancer Genome Atlas, the Gene Expression Omnibus, and Quantitative Real-Time Polymerase Chain Reaction.

PubMed

Wen, Dong-Yue; Lin, Peng; Pang, Yu-Yan; Chen, Gang; He, Yun; Dang, Yi-Wu; Yang, Hong

2018-05-05

BACKGROUND Long non-coding RNAs (lncRNAs) have a role in physiological and pathological processes, including cancer. The aim of this study was to investigate the expression of the long intergenic non-protein coding RNA 665 (LINC00665) gene and the cell cycle in hepatocellular carcinoma (HCC) using database analysis including The Cancer Genome Atlas (TCGA), the Gene Expression Omnibus (GEO), and quantitative real-time polymerase chain reaction (qPCR). MATERIAL AND METHODS Expression levels of LINC00665 were compared between human tissue samples of HCC and adjacent normal liver, clinicopathological correlations were made using TCGA and the GEO, and qPCR was performed to validate the findings. Other public databases were searched for other genes associated with LINC00665 expression, including The Atlas of Noncoding RNAs in Cancer (TANRIC), the Multi Experiment Matrix (MEM), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) networks. RESULTS Overexpression of LINC00665 in patients with HCC was significantly associated with gender, tumor grade, stage, and tumor cell type. Overexpression of LINC00665 in patients with HCC was significantly associated with overall survival (OS) (HR=1.47795%; CI: 1.046-2.086). Bioinformatics analysis identified 469 related genes and further analysis supported a hypothesis that LINC00665 regulates pathways in the cell cycle to facilitate the development and progression of HCC through ten identified core genes: CDK1, BUB1B, BUB1, PLK1, CCNB2, CCNB1, CDC20, ESPL1, MAD2L1, and CCNA2. CONCLUSIONS Overexpression of the lncRNA, LINC00665 may be involved in the regulation of cell cycle pathways in HCC through ten identified hub genes.

Real-time quantitative analysis of H2, He, O2, and Ar by quadrupole ion trap mass spectrometry.

PubMed

Ottens, Andrew K; Harrison, W W; Griffin, Timothy P; Helms, William R

2002-09-01

The use of a quadrupole ion trap mass spectrometer (QITMS) for quantitative analysis of hydrogen and helium as well as of other permanent gases is demonstrated. Like commercial instruments, the customized QITMS uses mass selective instability; however, this instrument operates at a greater trapping frequency and without a buffer gas. Thus, a useable mass range from 2 to over 50 daltons (Da) is achieved. The performance of the ion trap is evaluated using part-per-million (ppm) concentrations of hydrogen, helium, oxygen, and argon mixed into a nitrogen gas stream, as outlined by the National Aeronautics and Space Administration (NASA), which is interested in monitoring for cryogenic fuel leaks within the Space Shuttle during launch preparations. When quantitating the four analytes, relative accuracy and precision were better than the NASA-required minimum of 10% error and 5% deviation, respectively. Limits of detection were below the NASA requirement of 25-ppm hydrogen and 100-ppm helium; those for oxygen and argon were within the same order of magnitude as the requirements. These results were achieved at a fast data recording rate, and demonstrate the utility of the QITMS as a real-time quantitative monitoring device for permanent gas analysis. c. 2002 American Society for Mass Spectrometry.

The Advanced Gamma-ray Imaging System (AGIS): A Nanosecond Time Scale Stereoscopic Array Trigger System.

NASA Astrophysics Data System (ADS)

Krennrich, Frank; Buckley, J.; Byrum, K.; Dawson, J.; Drake, G.; Horan, D.; Krawzcynski, H.; Schroedter, M.

2008-04-01

Imaging atmospheric Cherenkov telescope arrays (VERITAS, HESS) have shown unprecedented background suppression capabilities for reducing cosmic-ray induced air showers, muons and night sky background fluctuations. Next-generation arrays with on the order of 100 telescopes offer larger collection areas, provide the possibility to see the air shower from more view points on the ground, have the potential to improve the sensitivity and give additional background suppression. Here we discuss the design of a fast array trigger system that has the potential to perform a real time image analysis allowing substantially improved background rate suppression at the trigger level.

Quantitative echocardiographic measures in the assessment of single ventricle function post-Fontan: Incorporation into routine clinical practice.

PubMed

Rios, Rodrigo; Ginde, Salil; Saudek, David; Loomba, Rohit S; Stelter, Jessica; Frommelt, Peter

2017-01-01

Quantitative echocardiographic measurements of single ventricular (SV) function have not been incorporated into routine clinical practice. A clinical protocol, which included quantitative measurements of SV deformation (global circumferential and longitudinal strain and strain rate), standard deviation of time to peak systolic strain, myocardial performance index (MPI), dP/dT from an atrioventricular valve regurgitant jet, and superior mesenteric artery resistance index, was instituted for all patients with a history of Fontan procedure undergoing echocardiography. All measures were performed real time during clinically indicated studies and were included in clinical reports. A total of 100 consecutive patients (mean age = 11.95±6.8 years, range 17 months-31.3 years) completed the protocol between September 1, 2014 to April 29, 2015. Deformation measures were completed in 100% of the studies, MPI in 93%, dP/dT in 55%, and superior mesenteric artery Doppler in 82%. The studies were reviewed to assess for efficiency in completing the protocol. The average time for image acquisition was 27.4±8.8 (range 10-62 minutes). The average time to perform deformation measures was 10.8±5.5 minutes (range 5-35 minutes) and time from beginning of imaging to report completion was 53.4±13.7 minutes (range 27-107 minutes). There was excellent inter-observer reliability when deformation indices were blindly repeated. Patients with a single left ventricle had significantly higher circumferential strain and strain rate, longitudinal strain and strain rate, and dP/dT compared to a single right ventricle. There were no differences in quantitative indices of ventricular function between patients <10 vs. >10 years post-Fontan. Advanced quantitative assessment of SV function post-Fontan can be consistently and efficiently performed real time during clinically indicated echocardiograms with excellent reliability. © 2016, Wiley Periodicals, Inc.

An Integrated Hot-Stage Microscope-Direct Analysis in Real Time-Mass Spectrometry System for Studying the Thermal Behavior of Materials.

PubMed

Ashton, Gage P; Harding, Lindsay P; Parkes, Gareth M B

2017-12-19

This paper describes a new analytical instrument that combines a precisely temperature-controlled hot-stage with digital microscopy and Direct Analysis in Real Time-mass spectrometry (DART-MS) detection. The novelty of the instrument lies in its ability to monitor processes as a function of temperature through the simultaneous recording of images, quantitative color changes, and mass spectra. The capability of the instrument was demonstrated through successful application to four very varied systems including profiling an organic reaction, decomposition of silicone polymers, and the desorption of rhodamine B from an alumina surface. The multidimensional, real-time analytical data provided by this instrument allow for a much greater insight into thermal processes than could be achieved previously.

Kurtosis-Based Blind Source Extraction of Complex Non-Circular Signals with Application in EEG Artifact Removal in Real-Time

PubMed Central

Javidi, Soroush; Mandic, Danilo P.; Took, Clive Cheong; Cichocki, Andrzej

2011-01-01

A new class of complex domain blind source extraction algorithms suitable for the extraction of both circular and non-circular complex signals is proposed. This is achieved through sequential extraction based on the degree of kurtosis and in the presence of non-circular measurement noise. The existence and uniqueness analysis of the solution is followed by a study of fast converging variants of the algorithm. The performance is first assessed through simulations on well understood benchmark signals, followed by a case study on real-time artifact removal from EEG signals, verified using both qualitative and quantitative metrics. The results illustrate the power of the proposed approach in real-time blind extraction of general complex-valued sources. PMID:22319461

Event-specific real-time detection and quantification of genetically modified Roundup Ready soybean.

PubMed

Huang, Chia-Chia; Pan, Tzu-Ming

2005-05-18

The event-specific real-time detection and quantification of Roundup Ready soybean (RRS) using an ABI PRISM 7700 sequence detection system with light upon extension (LUX) primer was developed in this study. The event-specific primers were designed, targeting the junction of the RRS 5' integration site and the endogenous gene lectin1. Then, a standard reference plasmid was constructed that carried both of the targeted sequences for quantitative analysis. The detection limit of the LUX real-time PCR system was 0.05 ng of 100% RRS genomic DNA, which was equal to 20.5 copies. The range of quantification was from 0.1 to 100%. The sensitivity and range of quantification successfully met the requirement of the labeling rules in the European Union and Taiwan.

Assessment of Spectroscopic, Real-time Ion Thruster Grid Erosion-rate Measurements

NASA Technical Reports Server (NTRS)

Domonkos, Matthew T.; Stevens, Richard E.

2000-01-01

The success of the ion thruster on the Deep Space One mission has opened the gate to the use of primary ion propulsion. Many of the projected planetary missions require throughput and specific impulse beyond those qualified to date. Spectroscopic, real-time ion thruster grid erosion-rate measurements are currently in development at the NASA Glenn Research Center. A preliminary investigation of the emission spectra from an NSTAR derivative thruster with titanium grid was conducted. Some titanium lines were observed in the discharge chamber; however, the signals were too weak to estimate the erosion of the screen grid. Nevertheless, this technique appears to be the only non-intrusive real-time means to evaluate screen grid erosion, and improvement of the collection optics is proposed. Direct examination of the erosion species using laser-induced fluorescence (LIF) was determined to be the best method for a real-time accelerator grid erosion diagnostic. An approach for a quantitative LIF diagnostic was presented.

Real-time automated thickness measurement of the in vivo human tympanic membrane using optical coherence tomography.

PubMed

Hubler, Zita; Shemonski, Nathan D; Shelton, Ryan L; Monroy, Guillermo L; Nolan, Ryan M; Boppart, Stephen A

2015-02-01

Otitis media (OM), an infection in the middle ear, is extremely common in the pediatric population. Current gold-standard methods for diagnosis include otoscopy for visualizing the surface features of the tympanic membrane (TM) and making qualitative assessments to determine middle ear content. OM typically presents as an acute infection, but can progress to chronic OM, and after numerous infections and antibiotic treatments over the course of many months, this disease is often treated by surgically inserting small tubes in the TM to relieve pressure, enable drainage, and provide aeration to the middle ear. Diagnosis and monitoring of OM is critical for successful management, but remains largely qualitative. We have developed an optical coherence tomography (OCT) system for high-resolution, depth-resolved, cross-sectional imaging of the TM and middle ear content, and for the quantitative assessment of in vivo TM thickness including the presence or absence of a middle ear biofilm. A novel algorithm was developed and demonstrated for automatic, real-time, and accurate measurement of TM thickness to aid in the diagnosis and monitoring of OM and other middle ear conditions. The segmentation algorithm applies a Hough transform to the OCT image data to determine the boundaries of the TM to calculate thickness. The use of OCT and this segmentation algorithm is demonstrated first on layered phantoms and then during real-time acquisition of in vivo OCT from humans. For the layered phantoms, measured thicknesses varied by approximately 5 µm over time in the presence of large axial and rotational motion. In vivo data also demonstrated differences in thicknesses both spatially on a single TM, and across normal, acute, and chronic OM cases. Real-time segmentation and thickness measurements of image data from both healthy subjects and those with acute and chronic OM demonstrate the use of OCT and this algorithm as a robust, quantitative, and accurate method for use during real-time in vivo human imaging.

Quantification of Agrobacterium tumefaciens C58 attachment to Arabidopsis thaliana roots.

PubMed

Petrovicheva, Anna; Joyner, Jessica; Muth, Theodore R

2017-10-02

Agrobacterium tumefaciens is the causal agent of crown gall disease and is a vector for DNA transfer in transgenic plants. The transformation process by A. tumefaciens has been widely studied, but the attachment stage has not been well characterized. Most measurements of attachment have used microscopy and colony counting, both of which are labor and time intensive. To reduce the time and effort required to analyze bacteria attaching to plant tissues, we developed a quantitative real-time PCR (qPCR) assay to quantify attached A. tumefaciens using the chvE gene as marker for the presence of the bacteria. The qPCR detection threshold of A. tumefaciens from pure culture was 104 cell equivalents/ml. The A. tumefaciens minimum threshold concentration from root-bound populations was determined to be 105 cell equivalents/ml inoculum to detect attachment above background. The qPCR assay can be used for measuring A. tumefaciens attachment in applications such as testing the effects of mutations on bacterial adhesion molecules or biofilm formation, comparing attachment across various plant species and ecotypes, and detecting mutations in putative attachment receptors expressed in plant roots. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy.

PubMed

Remans, Tony; Keunen, Els; Bex, Geert Jan; Smeets, Karen; Vangronsveld, Jaco; Cuypers, Ann

2014-10-01

Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data. © 2014 American Society of Plant Biologists. All rights reserved.

Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.

PubMed

Lueders, Tillmann; Manefield, Mike; Friedrich, Michael W

2004-01-01

Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.

Real-time detection of solar neutrinos with Borexino

NASA Astrophysics Data System (ADS)

Marcocci, S.; Agostini, M.; Altenmüller, K.; Appel, S.; Bellini, G.; Benziger, J.; Bick, D.; Bonfini, G.; Bravo, D.; Caccianiga, B.; Calaprice, F.; Caminata, A.; Carlini, M.; Cavalcante, P.; Chepurnov, A.; D'Angelo, D.; Davini, S.; Derbin, A.; Di Noto, L.; Drachnev, I.; Etenko, A.; Fomenko, K.; Formozov, A.; Franco, D.; Gabriele, F.; Galbiati, C.; Ghiano, C.; Giammarchi, M.; Goeger-Neff, M.; Goretti, A.; Gromov, M.; Hagner, C.; Hungerford, E.; Ianni, Aldo; Ianni, Andrea; Jedrzejczak, K.; Jeschke, D.; Kaiser, M.; Kobychev, V.; Korablev, D.; Korga, G.; Kryn, D.; Laubenstein, M.; Lehnert, B.; Litvinovich, E.; Lombardi, F.; Lombardi, P.; Ludhova, L.; Lukyanchenko, G.; Machulin, I.; Manecki, S.; Maneschg, W.; Meroni, E.; Meyer, M.; Miramonti, L.; Misiaszek, M.; Montuschi, M.; Mosteiro, P.; Muratova, V.; Neumair, B.; Oberauer, L.; Obolensky, M.; Ortica, F.; Pallavicini, M.; Papp, L.; Perasso, L.; Pocar, A.; Ranucci, G.; Razeto, A.; Re, A.; Romani, A.; Roncin, R.; Rossi, N.; Schönert, S.; Semenov, D.; Simgen, H.; Skorokhvatov, M.; Smirnov, O.; Sotnikov, A.; Sukhotin, S.; Suvorov, Y.; Tartaglia, R.; Testera, G.; Thurn, J.; Toropova, M.; Unzhakov, E.; Vishneva, A.; B. Vogelaar, R.; von Feilitzsch, F.; Wang, H.; Weinz, S.; Winter, J.; Wojcik, M.; Wurm, M.; Yokley, Z.; Zaimidoroga, O.; Zavatarelli, S.; Zuber, K.; Zuzel, G.; Borexino Collaboration

2017-01-01

Solar neutrinos have been fundamental in the discovery of neutrino flavor oscillations and are a unique tool to probe the nuclear reactions that fuel the Sun. The Borexino experiment, located in the Gran Sasso National Laboratory, is an ultra-pure liquid scintillator detector conceived for the real time spectroscopy of low energy solar neutrinos. Thanks to its unprecedented background levels, Borexino could measure in real time the fluxes of different components of the solar neutrino spectrum, thus probing both solar neutrino oscillations and the Standard Solar Model. We review these fundamental results and also discuss the prospects for the Phase-II of Borexino, which is entering the precision era of solar neutrino measurements.

Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis.

PubMed

Menard, J-P; Mazouni, C; Fenollar, F; Raoult, D; Boubli, L; Bretelle, F

2010-12-01

The purpose of this investigation was to determine the diagnostic accuracy of quantitative real-time polymerase chain reaction (PCR) assay in diagnosing bacterial vaginosis versus the standard methods, the Amsel criteria and the Nugent score. The Amsel criteria, the Nugent score, and results from the molecular tool were obtained independently from vaginal samples of 163 pregnant women who reported abnormal vaginal symptoms before 20 weeks gestation. To determine the performance of the molecular tool, we calculated the kappa value, sensitivity, specificity, and positive and negative predictive values. Either or both of the Amsel criteria (≥3 criteria) and the Nugent score (score ≥7) indicated that 25 women (15%) had bacterial vaginosis, and the remaining 138 women did not. DNA levels of Gardnerella vaginalis or Atopobium vaginae exceeded 10(9) copies/mL or 10(8) copies/mL, respectively, in 34 (21%) of the 163 samples. Complete agreement between both reference methods and high concentrations of G. vaginalis and A. vaginae was found in 94.5% of women (154/163 samples, kappa value = 0.81, 95% confidence interval 0.70-0.81). The nine samples with discordant results were categorized as intermediate flora by the Nugent score. The molecular tool predicted bacterial vaginosis with a sensitivity of 100%, a specificity of 93%, a positive predictive value of 73%, and a negative predictive value of 100%. The quantitative real-time PCR assay shows excellent agreement with the results of both reference methods for the diagnosis of bacterial vaginosis.

Successful Validation of RNA Purification and Quantitative Real-Time PCR Analysis of Gene Expression on the International Space Station

NASA Technical Reports Server (NTRS)

Tran, L.; Parra, Macarena P.; Jung, J.; Boone, T.; Schonfeld, Julie; Almeida, Eduardo

2017-01-01

The NASA Ames WetLab-2 system was developed to offer new on-orbit gene expression analysis capabilities to ISS researchers and can be used to conduct on-orbit RNA isolation and quantitative real time PCR (RT-qPCR) analysis of gene expression from a wide range of biological samples ranging from microbes to mammalian tissues. On orbit validation included three quantitative PCR (qPCR) runs using an E. coli genomic DNA template pre-loaded at three different concentrations. The flight Ct values for the DNA standards showed no statistically significant differences relative to ground controls although there was increased noise in Ct curves, likely due to microgravity-related bubble retention in the optical windows. RNA was successfully purified from both E. coli and mouse liver samples and successfully generated singleplex, duplex and triplex data although with higher standard deviations than ground controls, also likely due to bubbles. Using volunteer science activities, a potential bubble reduction strategy was tested and resulted in smooth amplification curves and tighter Cts between replicates. The WetLab-2 validation experiment demonstrates a novel molecular biology workbench on ISS which allows scientists to purify and stabilize RNA, and to conduct RT-qPCR analyses on-orbit with rapid results. This novel ability is an important step towards utilizing ISS as a National Laboratory facility with the capability to conduct and adjust science experiments in real time without sample return, and opens new possibilities for rapid medical diagnostics and biological environmental monitoring on ISS.

Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

PubMed Central

Ghelardini, Luisa; Belbahri, Lassaâd; Quartier, Marion; Santini, Alberto

2013-01-01

Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10−2 to 1.4 × 10−2 pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management. PMID:23811499

Characterization of neural development in zebrafish embryos using real-time quantitative PCR.

EPA Science Inventory

Chemicals adversely affecting the developing nervous system may cause long-term consequences on human health. Little information exists on a large number of environmental compounds to guide developmental neurotoxicity risk assessments. Because developmental neurotoxicity studies ...

Human Fecal Source Identification: Real-Time Quantitative PCR Method Standardization

EPA Science Inventory

Method standardization or the formal development of a protocol that establishes uniform performance benchmarks and practices is necessary for widespread adoption of a fecal source identification approach. Standardization of a human-associated fecal identification method has been...

Fecal Source Identification with Real-Time Quantitative PCR

EPA Science Inventory

Waterborne diseases that originate from fecal pollution remain a significant public health issue. Current fecal indicator technologies recommended by the U.S. Environmental Protection Agency for water quality testing do not discriminate between different animal sources of fecal ...

Overview of EPA Research on Drinking Water Distribution System Nitrification

EPA Science Inventory

Results from USEPA research investigating drinking water distribution system nitrification will be presented. The two research areas include: (1) monochloramine disinfection kinetics of Nitrosomonas europaea using Propidium Monoazide Quantitative Real-time PCR (PMA-qPCR) and (2...

A QUANTITATIVE REAL-TIME PCR ASSAY FOR DETECTION OF CYCLOSPORA CAYETANESIS

EPA Science Inventory

Cyclospora cayetanensis, a coccidian parasite with a direct fecal-oral life cycle, has become recognized world-wide as an emerging pathogen in both immunocompromised and immunocompetent individuals. Clinical manifestations include prolonged diarrhea, nausea, abdominal cramps, an...

Integrating real-time GIS and social media for qualitative transportation data collection.

DOT National Transportation Integrated Search

2016-12-26

New technologies such as global positioning system, smartphone, and social media are changing the way we move around. Traditional : transportation research has overwhelmingly emphasized the collection of quantitative data for modeling, without much c...

Breaking through the false coincidence barrier in electron–ion coincidence experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osborn, David L.; Hayden, Carl C.; Hemberger, Patrick

Photoelectron Photoion Coincidence (PEPICO) spectroscopy holds the promise of a universal, isomer-selective, and sensitive analytical technique for time-resolved quantitative analysis of bimolecular chemical reactions. Unfortunately, its low dynamic range of ~10 3 has largely precluded its use for this purpose, where a dynamic range of at least 10 5 is generally required. This limitation is due to the false coincidence background common to all coincidence experiments, especially at high count rates. Electron/ion pairs emanating from separate ionization events but arriving within the ion time of flight (TOF) range of interest constitute the false coincidence background. Although this background has uniformmore » intensity at every m/z value, the Poisson scatter in the false coincidence background obscures small signals. In this paper, temporal ion deflection coupled with a position-sensitive ion detector enables suppression of the false coincidence background, increasing the dynamic range in the PEPICO TOF mass spectrum by 2–3 orders of magnitude. The ions experience a time-dependent electric deflection field at a well-defined fraction of their time of flight. This deflection defines an m/z- and ionization-time dependent ion impact position for true coincidences, whereas false coincidences appear randomly outside this region and can be efficiently suppressed. When cold argon clusters are ionized, false coincidence suppression allows us to observe species up to Ar 9 +, whereas Ar 4 + is the largest observable cluster under traditional operation. As a result, this advance provides mass-selected photoelectron spectra for fast, high sensitivity quantitative analysis of reacting systems.« less

Breaking through the false coincidence barrier in electron–ion coincidence experiments

DOE PAGES

Osborn, David L.; Hayden, Carl C.; Hemberger, Patrick; ...

2016-10-31

Photoelectron Photoion Coincidence (PEPICO) spectroscopy holds the promise of a universal, isomer-selective, and sensitive analytical technique for time-resolved quantitative analysis of bimolecular chemical reactions. Unfortunately, its low dynamic range of ~10 3 has largely precluded its use for this purpose, where a dynamic range of at least 10 5 is generally required. This limitation is due to the false coincidence background common to all coincidence experiments, especially at high count rates. Electron/ion pairs emanating from separate ionization events but arriving within the ion time of flight (TOF) range of interest constitute the false coincidence background. Although this background has uniformmore » intensity at every m/z value, the Poisson scatter in the false coincidence background obscures small signals. In this paper, temporal ion deflection coupled with a position-sensitive ion detector enables suppression of the false coincidence background, increasing the dynamic range in the PEPICO TOF mass spectrum by 2–3 orders of magnitude. The ions experience a time-dependent electric deflection field at a well-defined fraction of their time of flight. This deflection defines an m/z- and ionization-time dependent ion impact position for true coincidences, whereas false coincidences appear randomly outside this region and can be efficiently suppressed. When cold argon clusters are ionized, false coincidence suppression allows us to observe species up to Ar 9 +, whereas Ar 4 + is the largest observable cluster under traditional operation. As a result, this advance provides mass-selected photoelectron spectra for fast, high sensitivity quantitative analysis of reacting systems.« less

Real-time quantitative polymerase chain reaction analysis of patients with refractory chronic periodontitis.

PubMed

Marconcini, Simone; Covani, Ugo; Barone, Antonio; Vittorio, Orazio; Curcio, Michele; Barbuti, Serena; Scatena, Fabrizio; Felli, Lamberto; Nicolini, Claudio

2011-07-01

Periodontitis is a complex multifactorial disease and is typically polygenic in origin. Genes play a fundamental part in each biologic process forming complex networks of interactions. However, only some genes have a high number of interactions with other genes in the network and may, therefore, be considered to play an important role. In a preliminary bioinformatic analysis, five genes that showed a higher number of interactions were identified and termed leader genes. In the present study, we use real-time quantitative polymerase chain reaction (PCR) technology to evaluate the expression levels of leader genes in the leukocytes of 10 patients with refractory chronic periodontitis and compare the expression levels with those of the same genes in 24 healthy patients. Blood was collected from 24 healthy human subjects and 10 patients with refractory chronic periodontitis and placed into heparinized blood collection tubes by personnel trained in phlebotomy using a sterile technique. Blood leukocyte cells were immediately lysed by using a kit for total RNA purification from human whole blood. Complementary DNA (cDNA) synthesis was obtained from total RNA and then real-time quantitative PCR was performed. PCR efficiencies were calculated with a relative standard curve derived from a five cDNA dilution series in triplicate that gave regression coefficients >0.98 and efficiencies >96%. The standard curves were obtained using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and growth factor receptor binding protein 2 (GRB2), casitas B-lineage lymphoma (CBL), nuclear factor-KB1 (NFKB1), and REL-A (gene for transcription factor p65) gene primers and amplified with 1.6, 8, 40, 200, and 1,000 ng/μL total cDNA. Curves obtained for each sample showed a linear relationship between RNA concentrations and the cycle threshold value of real-time quantitative PCR for all genes. Data were expressed as mean ± SE (SEM). The groups were compared to the analysis of variance. A probability value <0.01 was considered statistically significant. The present study agrees with the preliminary bioinformatics analysis. In our experiments, the association of pathology with the genes was statistically significant for GRB2 and CBL (P <0.01), and it was not statistically significant for REL-A and NFKB1. This article lends support to our preliminary hypothesis that assigned an important role in refractory aggressive periodontitis to leader genes.

Real-Time Tracking of Knee Adduction Moment in Patients with Knee Osteoarthritis

PubMed Central

Kang, Sang Hoon; Lee, Song Joo; Zhang, Li-Qun

2014-01-01

Background The external knee adduction moment (EKAM) is closely associated with the presence, progression, and severity of knee osteoarthritis (OA). However, there is a lack of convenient and practical method to estimate and track in real-time the EKAM of patients with knee OA for clinical evaluation and gait training, especially outside of gait laboratories. New Method A real-time EKAM estimation method was developed and applied to track and investigate the EKAM and other knee moments during stepping on an elliptical trainer in both healthy subjects and a patient with knee OA. Results Substantial changes were observed in the EKAM and other knee moments during stepping in the patient with knee OA. Comparison with Existing Method(s) This is the first study to develop and test feasibility of real-time tracking method of the EKAM on patients with knee OA using 3-D inverse dynamics. Conclusions The study provides us an accurate and practical method to evaluate in real-time the critical EKAM associated with knee OA, which is expected to help us to diagnose and evaluate patients with knee OA and provide the patients with real-time EKAM feedback rehabilitation training. PMID:24361759

Real-time people counting system using a single video camera

NASA Astrophysics Data System (ADS)

Lefloch, Damien; Cheikh, Faouzi A.; Hardeberg, Jon Y.; Gouton, Pierre; Picot-Clemente, Romain

2008-02-01

There is growing interest in video-based solutions for people monitoring and counting in business and security applications. Compared to classic sensor-based solutions the video-based ones allow for more versatile functionalities, improved performance with lower costs. In this paper, we propose a real-time system for people counting based on single low-end non-calibrated video camera. The two main challenges addressed in this paper are: robust estimation of the scene background and the number of real persons in merge-split scenarios. The latter is likely to occur whenever multiple persons move closely, e.g. in shopping centers. Several persons may be considered to be a single person by automatic segmentation algorithms, due to occlusions or shadows, leading to under-counting. Therefore, to account for noises, illumination and static objects changes, a background substraction is performed using an adaptive background model (updated over time based on motion information) and automatic thresholding. Furthermore, post-processing of the segmentation results is performed, in the HSV color space, to remove shadows. Moving objects are tracked using an adaptive Kalman filter, allowing a robust estimation of the objects future positions even under heavy occlusion. The system is implemented in Matlab, and gives encouraging results even at high frame rates. Experimental results obtained based on the PETS2006 datasets are presented at the end of the paper.

Real-time visual tracking of less textured three-dimensional objects on mobile platforms

NASA Astrophysics Data System (ADS)

Seo, Byung-Kuk; Park, Jungsik; Park, Hanhoon; Park, Jong-Il

2012-12-01

Natural feature-based approaches are still challenging for mobile applications (e.g., mobile augmented reality), because they are feasible only in limited environments such as highly textured and planar scenes/objects, and they need powerful mobile hardware for fast and reliable tracking. In many cases where conventional approaches are not effective, three-dimensional (3-D) knowledge of target scenes would be beneficial. We present a well-established framework for real-time visual tracking of less textured 3-D objects on mobile platforms. Our framework is based on model-based tracking that efficiently exploits partially known 3-D scene knowledge such as object models and a background's distinctive geometric or photometric knowledge. Moreover, we elaborate on implementation in order to make it suitable for real-time vision processing on mobile hardware. The performance of the framework is tested and evaluated on recent commercially available smartphones, and its feasibility is shown by real-time demonstrations.

Objective speech quality evaluation of real-time speech coders

NASA Astrophysics Data System (ADS)

Viswanathan, V. R.; Russell, W. H.; Huggins, A. W. F.

1984-02-01

This report describes the work performed in two areas: subjective testing of a real-time 16 kbit/s adaptive predictive coder (APC) and objective speech quality evaluation of real-time coders. The speech intelligibility of the APC coder was tested using the Diagnostic Rhyme Test (DRT), and the speech quality was tested using the Diagnostic Acceptability Measure (DAM) test, under eight operating conditions involving channel error, acoustic background noise, and tandem link with two other coders. The test results showed that the DRT and DAM scores of the APC coder equalled or exceeded the corresponding test scores fo the 32 kbit/s CVSD coder. In the area of objective speech quality evaluation, the report describes the development, testing, and validation of a procedure for automatically computing several objective speech quality measures, given only the tape-recordings of the input speech and the corresponding output speech of a real-time speech coder.

Quantitative comparison between crowd models for evacuation planning and evaluation

NASA Astrophysics Data System (ADS)

Viswanathan, Vaisagh; Lee, Chong Eu; Lees, Michael Harold; Cheong, Siew Ann; Sloot, Peter M. A.

2014-02-01

Crowd simulation is rapidly becoming a standard tool for evacuation planning and evaluation. However, the many crowd models in the literature are structurally different, and few have been rigorously calibrated against real-world egress data, especially in emergency situations. In this paper we describe a procedure to quantitatively compare different crowd models or between models and real-world data. We simulated three models: (1) the lattice gas model, (2) the social force model, and (3) the RVO2 model, and obtained the distributions of six observables: (1) evacuation time, (2) zoned evacuation time, (3) passage density, (4) total distance traveled, (5) inconvenience, and (6) flow rate. We then used the DISTATIS procedure to compute the compromise matrix of statistical distances between the three models. Projecting the three models onto the first two principal components of the compromise matrix, we find the lattice gas and RVO2 models are similar in terms of the evacuation time, passage density, and flow rates, whereas the social force and RVO2 models are similar in terms of the total distance traveled. Most importantly, we find that the zoned evacuation times of the three models to be very different from each other. Thus we propose to use this variable, if it can be measured, as the key test between different models, and also between models and the real world. Finally, we compared the model flow rates against the flow rate of an emergency evacuation during the May 2008 Sichuan earthquake, and found the social force model agrees best with this real data.

Development of a real-time wave field reconstruction TEM system (II): correction of coma aberration and 3-fold astigmatism, and real-time correction of 2-fold astigmatism.

PubMed

Tamura, Takahiro; Kimura, Yoshihide; Takai, Yoshizo

2018-02-01

In this study, a function for the correction of coma aberration, 3-fold astigmatism and real-time correction of 2-fold astigmatism was newly incorporated into a recently developed real-time wave field reconstruction TEM system. The aberration correction function was developed by modifying the image-processing software previously designed for auto focus tracking, as described in the first article of this series. Using the newly developed system, the coma aberration and 3-fold astigmatism were corrected using the aberration coefficients obtained experimentally before the processing was carried out. In this study, these aberration coefficients were estimated from an apparent 2-fold astigmatism induced under tilted-illumination conditions. In contrast, 2-fold astigmatism could be measured and corrected in real time from the reconstructed wave field. Here, the measurement precision for 2-fold astigmatism was found to be ±0.4 nm and ±2°. All of these aberration corrections, as well as auto focus tracking, were performed at a video frame rate of 1/30 s. Thus, the proposed novel system is promising for quantitative and reliable in situ observations, particularly in environmental TEM applications.

Real-Time Safety Risk Assessment Based on a Real-Time Location System for Hydropower Construction Sites

PubMed Central

Fan, Qixiang; Qiang, Maoshan

2014-01-01

The concern for workers' safety in construction industry is reflected in many studies focusing on static safety risk identification and assessment. However, studies on real-time safety risk assessment aimed at reducing uncertainty and supporting quick response are rare. A method for real-time safety risk assessment (RTSRA) to implement a dynamic evaluation of worker safety states on construction site has been proposed in this paper. The method provides construction managers who are in charge of safety with more abundant information to reduce the uncertainty of the site. A quantitative calculation formula, integrating the influence of static and dynamic hazards and that of safety supervisors, is established to link the safety risk of workers with the locations of on-site assets. By employing the hidden Markov model (HMM), the RTSRA provides a mechanism for processing location data provided by the real-time location system (RTLS) and analyzing the probability distributions of different states in terms of false positives and negatives. Simulation analysis demonstrated the logic of the proposed method and how it works. Application case shows that the proposed RTSRA is both feasible and effective in managing construction project safety concerns. PMID:25114958

Real-time safety risk assessment based on a real-time location system for hydropower construction sites.

PubMed

Jiang, Hanchen; Lin, Peng; Fan, Qixiang; Qiang, Maoshan

2014-01-01

The concern for workers' safety in construction industry is reflected in many studies focusing on static safety risk identification and assessment. However, studies on real-time safety risk assessment aimed at reducing uncertainty and supporting quick response are rare. A method for real-time safety risk assessment (RTSRA) to implement a dynamic evaluation of worker safety states on construction site has been proposed in this paper. The method provides construction managers who are in charge of safety with more abundant information to reduce the uncertainty of the site. A quantitative calculation formula, integrating the influence of static and dynamic hazards and that of safety supervisors, is established to link the safety risk of workers with the locations of on-site assets. By employing the hidden Markov model (HMM), the RTSRA provides a mechanism for processing location data provided by the real-time location system (RTLS) and analyzing the probability distributions of different states in terms of false positives and negatives. Simulation analysis demonstrated the logic of the proposed method and how it works. Application case shows that the proposed RTSRA is both feasible and effective in managing construction project safety concerns.

Ultrafast Screening and Quantitation of Pesticides in Food and Environmental Matrices by Solid-Phase Microextraction-Transmission Mode (SPME-TM) and Direct Analysis in Real Time (DART).

PubMed

Gómez-Ríos, Germán Augusto; Gionfriddo, Emanuela; Poole, Justen; Pawliszyn, Janusz

2017-07-05

The direct interface of microextraction technologies to mass spectrometry (MS) has unquestionably revolutionized the speed and efficacy at which complex matrices are analyzed. Solid Phase Micro Extraction-Transmission Mode (SPME-TM) is a technology conceived as an effective synergy between sample preparation and ambient ionization. Succinctly, the device consists of a mesh coated with polymeric particles that extracts analytes of interest present in a given sample matrix. This coated mesh acts as a transmission-mode substrate for Direct Analysis in Real Time (DART), allowing for rapid and efficient thermal desorption/ionization of analytes previously concentrated on the coating, and dramatically lowering the limits of detection attained by sole DART analysis. In this study, we present SPME-TM as a novel tool for the ultrafast enrichment of pesticides present in food and environmental matrices and their quantitative determination by MS via DART ionization. Limits of quantitation in the subnanogram per milliliter range can be attained, while total analysis time does not exceed 2 min per sample. In addition to target information obtained via tandem MS, retrospective studies of the same sample via high-resolution mass spectrometry (HRMS) were accomplished by thermally desorbing a different segment of the microextraction device.

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

PubMed

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

2005-04-01

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

Habitat associations of two entomopathogenic nematodes: a quantitative study using real-time quantitative polymerase chain reactions.

PubMed

Torr, Peter; Spiridonov, Sergei E; Heritage, Stuart; Wilson, Michael J

2007-03-01

1. Despite nematodes being the most abundant animals on earth, very few animal ecologists study them, probably because of the difficulties of identifying them to species by morphological methods. 2. A group of nematodes that are important both ecologically and economically is the entomopathogenic nematodes, which play a key role in regulating soil food webs and are sold throughout the world as biological insecticides, yet for which very little is known of their population ecology. 3. A novel detection and quantification method was developed for soil nematodes using real-time polymerase chain reaction (PCR), and the technique was used to estimate numbers of two closely related species of entomopathogenic nematodes, Steinernema kraussei and S. affine in 50 soil samples from 10 sites in Scotland representing two distinct habitats (woodland and grassland). 4. There was a high degree of correlation between our molecular and traditional morphological estimates of population size and our data clearly showed that Steinernema affine occurred only in grassland areas, whereas S. kraussei was found in grassland and woodland samples to a similar degree. 5. Real-time PCR offers a rapid and accurate method of detecting individual nematode species from soil samples without the need for a specialist taxonomist, and has much potential for use in studies of nematode population ecology.

Correlative atomic force microscopy quantitative imaging-laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time.

PubMed

Bhat, Supriya V; Sultana, Taranum; Körnig, André; McGrath, Seamus; Shahina, Zinnat; Dahms, Tanya E S

2018-05-29

There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.

Identification and quantitative detection of Legionella spp. in various aquatic environments by real-time PCR assay.

PubMed

Kao, Po-Min; Tung, Min-Che; Hsu, Bing-Mu; Chiu, Yi-Chou; She, Cheng-Yu; Shen, Shu-Min; Huang, Yu-Li; Huang, Wen-Chien

2013-09-01

In this study, a SYBR green quantitative real-time PCR was developed to quantify and detect the Legionella spp. in various environmental water samples. The water samples were taken from watershed, water treatment plant, and thermal spring area in Taiwan. Legionella was detected in 13.6 % (24/176), and the detection rate for river water, raw drinking water, and thermal spring water was 10, 21.4, and 16.6 %, respectively. Using real-time PCR, concentration of Legionella spp. in detected samples ranged between 9.75 × 10(4) and 3.47 × 10(5) cells/L in river water, 6.92 × 10(4) and 4.29 × 10(5) cells/L in raw drinking water, and 5.71 × 10(4) and 2.12 × 10(6) cells/L for thermal spring water samples. The identified species included Legionella pneumophila (20.8 %), Legionella jordanis (4.2 %), Legionella nautarum (4.2 %), Legionella sp. (4.2 %), and uncultured Legionella sp. (66.6 %). The presence of L. pneumophila in aquatic environments suggested a potential public health threat that must be further examined.

Comparative evaluation of performance measures for shading correction in time-lapse fluorescence microscopy.

PubMed

Liu, L; Kan, A; Leckie, C; Hodgkin, P D

2017-04-01

Time-lapse fluorescence microscopy is a valuable technology in cell biology, but it suffers from the inherent problem of intensity inhomogeneity due to uneven illumination or camera nonlinearity, known as shading artefacts. This will lead to inaccurate estimates of single-cell features such as average and total intensity. Numerous shading correction methods have been proposed to remove this effect. In order to compare the performance of different methods, many quantitative performance measures have been developed. However, there is little discussion about which performance measure should be generally applied for evaluation on real data, where the ground truth is absent. In this paper, the state-of-the-art shading correction methods and performance evaluation methods are reviewed. We implement 10 popular shading correction methods on two artificial datasets and four real ones. In order to make an objective comparison between those methods, we employ a number of quantitative performance measures. Extensive validation demonstrates that the coefficient of joint variation (CJV) is the most applicable measure in time-lapse fluorescence images. Based on this measure, we have proposed a novel shading correction method that performs better compared to well-established methods for a range of real data tested. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Real-Time Imaging System for the OpenPET

NASA Astrophysics Data System (ADS)

Tashima, Hideaki; Yoshida, Eiji; Kinouchi, Shoko; Nishikido, Fumihiko; Inadama, Naoko; Murayama, Hideo; Suga, Mikio; Haneishi, Hideaki; Yamaya, Taiga

2012-02-01

The OpenPET and its real-time imaging capability have great potential for real-time tumor tracking in medical procedures such as biopsy and radiation therapy. For the real-time imaging system, we intend to use the one-pass list-mode dynamic row-action maximum likelihood algorithm (DRAMA) and implement it using general-purpose computing on graphics processing units (GPGPU) techniques. However, it is difficult to make consistent reconstructions in real-time because the amount of list-mode data acquired in PET scans may be large depending on the level of radioactivity, and the reconstruction speed depends on the amount of the list-mode data. In this study, we developed a system to control the data used in the reconstruction step while retaining quantitative performance. In the proposed system, the data transfer control system limits the event counts to be used in the reconstruction step according to the reconstruction speed, and the reconstructed images are properly intensified by using the ratio of the used counts to the total counts. We implemented the system on a small OpenPET prototype system and evaluated the performance in terms of the real-time tracking ability by displaying reconstructed images in which the intensity was compensated. The intensity of the displayed images correlated properly with the original count rate and a frame rate of 2 frames per second was achieved with average delay time of 2.1 s.

Method and apparatus for non-invasive evaluation of diaphragmatic function

NASA Technical Reports Server (NTRS)

Yost, William T. (Inventor); Wait, Juliette L. (Inventor); Nahormek, Patricia A. (Inventor); Cantrell, John H. (Inventor); Hanna-Hawver, Pamela D. (Inventor)

1995-01-01

A method for non-invasive evaluation of diaphragmatic function in humans measures the thickness of the diaphragm in real time with an ultrasonic device, and displays the variations of diaphragm thickness versus time. Formulae are given for calculating a quantitative value for the reserve fatigue capacity of a patient's diaphragm from data obtained by measuring the time limits for maintaining a constant breathing pattern on the display at two different pressure differentials in series with the patient's airways. An apparatus for displaying the diaphragm thickness in real time is also described. The method can be used both on healthy patients and on patients with so severe breathing dysfunctions that they require breathing support from respirators.

Method and apparatus for non-invasive evaluation of diaphragmatic function

NASA Technical Reports Server (NTRS)

Yost, William T. (Inventor); Wait, Juliette L. (Inventor); Nahormek, Patricia A. (Inventor); Cantrell, John H. (Inventor); Hanna-Hawver, Pamela D. (Inventor)

1994-01-01

A method for non-invasive evaluation of diaphragmatic function in humans measures the thickness of the diaphragm in real time with an ultrasonic device, and displays the variations of diaphragm thickness versus time. Formulae are given for calculating a quantitative value for the reserve fatigue capacity of a patient's diaphragm from data obtained by measuring the time limits for maintaining a constant breathing pattern on the display at two different pressure differentials in series with the patient's airways. An apparatus for displaying the diaphragm thickness in real time is also described. The method can be used both on healthy patients and on patients with so severe breathing dysfunctions that they require breathing support from respirators.

Stability of gametocyte-specific Pfs25-mRNA in dried blood spots on filter paper subjected to different storage conditions

PubMed Central

2012-01-01

Background Real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) is a sensitive method for detection of sub-microscopic gametocytaemia by measuring gametocyte-specific mRNA. Performing analysis on fresh whole blood samples is often not feasible in remote and resource-poor areas. Convenient methods for sample storage and transport are urgently needed. Methods Real-time QT-NASBA was performed on whole blood spiked with a dilution series of purified in-vitro cultivated gametocytes. The blood was either freshly processed or spotted on filter papers. Gametocyte detection sensitivity for QT-NASBA was determined and controlled by microscopy. Dried blood spot (DBS) samples were subjected to five different storage conditions and the loss of sensitivity over time was investigated. A formula to approximate the loss of Pfs25-mRNA due to different storage conditions and time was developed. Results Pfs25-mRNA was measured in time to positivity (TTP) and correlated well with the microscopic counts and the theoretical concentrations of the dilution series. TTP results constantly indicated higher amounts of RNA in filter paper samples extracted after 24 hours than in immediately extracted fresh blood. Among investigated storage conditions freezing at −20°C performed best with 98.7% of the Pfs25-mRNA still detectable at day 28 compared to fresh blood samples. After 92 days, the RNA detection rate was only slightly decreased to 92.9%. Samples stored at 37°C showed most decay with only 64.5% of Pfs25-mRNA detectable after one month. The calculated theoretical detection limit for 24 h-old DBS filter paper samples was 0.0095 (95% CI: 0.0025 to 0.0380) per μl. Conclusions The results suggest that the application of DBS filter papers for quantification of Plasmodium falciparum gametocytes with real-time QT-NASBA is practical and recommendable. This method proved sensitive enough for detection of sub-microscopic densities even after prolonged storage. Decay rates can be predicted for different storage conditions as well as durations. PMID:22545954

Monitoring airborne molecular contamination: a quantitative and qualitative comparison of real-time and grab-sampling techniques

NASA Astrophysics Data System (ADS)

Shupp, Aaron M.; Rodier, Dan; Rowley, Steven

2007-03-01

Monitoring and controlling Airborne Molecular Contamination (AMC) has become essential in deep ultraviolet (DUV) photolithography for both optimizing yields and protecting tool optics. A variety of technologies have been employed for both real-time and grab-sample monitoring. Real-time monitoring has the advantage of quickly identifying "spikes" and upset conditions, while 2 - 24 hour plus grab sampling allows for extremely low detection limits by concentrating the mass of the target contaminant over a period of time. Employing a combination of both monitoring techniques affords the highest degree of control, lowest detection limits, and the most detailed data possible in terms of speciation. As happens with many technologies, there can be concern regarding the accuracy and agreement between real-time and grab-sample methods. This study utilizes side by side comparisons of two different real-time monitors operating in parallel with both liquid impingers and dry sorbent tubes to measure NIST traceable gas standards as well as real world samples. By measuring in parallel, a truly valid comparison is made between methods while verifying the results against a certified standard. The final outcome for this investigation is that a dry sorbent tube grab-sample technique produced results that agreed in terms of accuracy with NIST traceable standards as well as the two real-time techniques Ion Mobility Spectrometry (IMS) and Pulsed Fluorescence Detection (PFD) while a traditional liquid impinger technique showed discrepancies.

Non-contact rapid optical coherence elastography by high-speed 4D imaging of elastic waves

NASA Astrophysics Data System (ADS)

Song, Shaozhen; Yoon, Soon Joon; Ambroziński, Łukasz; Pelivanov, Ivan; Li, David; Gao, Liang; Shen, Tueng T.; O'Donnell, Matthew; Wang, Ruikang K.

2017-02-01

Shear wave OCE (SW-OCE) uses an OCT system to track propagating mechanical waves, providing the information needed to map the elasticity of the target sample. In this study we demonstrate high speed, 4D imaging to capture transient mechanical wave propagation. Using a high-speed Fourier domain mode-locked (FDML) swept-source OCT (SS-OCT) system operating at 1.62 MHz A-line rate, the equivalent volume rate of mechanical wave imaging is 16 kvps (kilo-volumes per second), and total imaging time for a 6 x 6 x 3 mm volume is only 0.32 s. With a displacement sensitivity of 10 nanometers, the proposed 4D imaging technique provides sufficient temporal and spatial resolution for real-time optical coherence elastography (OCE). Combined with a new air-coupled, high-frequency focused ultrasound stimulator requiring no contact or coupling media, this near real-time system can provide quantitative information on localized viscoelastic properties. SW-OCE measurements are demonstrated on tissue-mimicking phantoms and porcine cornea under various intra-ocular pressures. In addition, elasticity anisotropy in the cornea is observed. Images of the mechanical wave group velocity, which correlates with tissue elasticity, show velocities ranging from 4-20 m/s depending on pressure and propagation direction. These initial results strong suggest that 4D imaging for real-time OCE may enable high-resolution quantitative mapping of tissue biomechanical properties in clinical applications.

Fully automated, internally controlled quantification of hepatitis B Virus DNA by real-time PCR by use of the MagNA Pure LC and LightCycler instruments.

PubMed

Leb, Victoria; Stöcher, Markus; Valentine-Thon, Elizabeth; Hölzl, Gabriele; Kessler, Harald; Stekel, Herbert; Berg, Jörg

2004-02-01

We report on the development of a fully automated real-time PCR assay for the quantitative detection of hepatitis B virus (HBV) DNA in plasma with EDTA (EDTA plasma). The MagNA Pure LC instrument was used for automated DNA purification and automated preparation of PCR mixtures. Real-time PCR was performed on the LightCycler instrument. An internal amplification control was devised as a PCR competitor and was introduced into the assay at the stage of DNA purification to permit monitoring for sample adequacy. The detection limit of the assay was found to be 200 HBV DNA copies/ml, with a linear dynamic range of 8 orders of magnitude. When samples from the European Union Quality Control Concerted Action HBV Proficiency Panel 1999 were examined, the results were found to be in acceptable agreement with the HBV DNA concentrations of the panel members. In a clinical laboratory evaluation of 123 EDTA plasma samples, a significant correlation was found with the results obtained by the Roche HBV Monitor test on the Cobas Amplicor analyzer within the dynamic range of that system. In conclusion, the newly developed assay has a markedly reduced hands-on time, permits monitoring for sample adequacy, and is suitable for the quantitative detection of HBV DNA in plasma in a routine clinical laboratory.

Quantification of Fusarium oxysporum in fumigated soils by a newly developed real-time PCR assay to assess the efficacy of fumigants for Fusarium wilt disease in strawberry plants.

PubMed

Li, Yuan; Mao, Liangang; Yan, Dongdong; Ma, Taotao; Shen, Jin; Guo, Meixia; Wang, Qiuxia; Ouyang, Canbin; Cao, Aocheng

2014-11-01

Two soil fumigants, chloropicrin (CP) and dimethyl disulfide (DMDS), were used to control Fusarium wilt disease (FWD) which caused large economic losses in strawberries. The fumigants were evaluated alone and in combination in a laboratory study and in strawberry greenhouses. Laboratory tests found that combinations of CP and DMDS indicated a positive synergistic activity on Fusarium oxysporum. A newly developed quantitative assay for F. oxysporum involving real-time PCR was used successfully to evaluate F. oxysporum control by the fumigants; it provided similar results to the selective medium but was less time-consuming and less labor intensive. Greenhouse trials revealed that the combination of CP and DMDS successfully suppressed the incidence of FWD and sharply reduced the population density of F. oxysporum, which significantly increased fruit branch number and maintained a good strawberry yield, higher than methyl bromide (MB) treatment. All of the treatments provided significantly better results than the non-treated control. This study confirms that the newly developed real-time PCR quantitative assay for F. oxysporum was suitable for the control efficacy evaluation of soil fumigants and that the novel fumigant combination of CP and DMDS offers a promising effective alternative to MB for the control of F. oxysporum in strawberry greenhouses. © 2013 Society of Chemical Industry.

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

PubMed

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

2013-01-01

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

Capturing patient experience: a qualitative study of implementing real-time feedback in primary care

PubMed Central

Carter, Mary; Davey, Antoinette; Wright, Christine; Elmore, Natasha; Newbould, Jenny; Roland, Martin; Campbell, John; Burt, Jenni

2016-01-01

Background In recent years, hospitals have made use of new technologies, such as real-time feedback, to collect patient experience information. This approach is currently rarely used in primary care settings, but may provide practices with a useful tool that enables them to take prompt, focused action to improve their services. Aim To identify the factors inhibiting and enabling the implementation of real-time feedback in general practices. Design and setting Qualitative study embedded within an exploratory trial (July 2014 to February 2015) of a real-time feedback intervention targeting patient experience in general practices in south-west England and Cambridgeshire. Method Semi-structured interviews (n = 22) and focus groups (n = 4, total of 28 attendees) with practice staff were audiorecorded, transcribed, and analysed thematically, using a framework based on constructs from normalisation process theory. Results Staff engagement with real-time feedback varied considerably, and staff made sense of real-time feedback by comparing it with more familiar feedback modalities. Effective within-team communication was associated with positive attitudes towards real-time feedback. Timing of requests for feedback was important in relation to patient engagement. Real-time feedback may offer potential as a means of informing practice development, perhaps as a component of a wider programme of capturing and responding to patients’ comments. Conclusion Successful implementation of real-time feedback requires effective communication across the practice team to engender thorough engagement. Feedback processes should be carefully introduced to fit with existing patient and practice routines. Future studies should consider making real-time feedback content relevant to specific practice needs, and support participation by all patient groups. PMID:27621292

Development of a high-throughput detection system for HIV-1 using real-time NASBA based on molecular beacons

NASA Astrophysics Data System (ADS)

van Beuningen, Rinie; Marras, Salvatore A.; Kramer, Fred R.; Oosterlaken, Tom; Weusten, Jos; Borst, G.; van de Wiel, Paul

2001-04-01

HIV-1 viral load assays require accuracy and sensitivity at low RNA levels with the capability to detect all subtypes. Furthermore, the assay should be easy to perform and fast to be useful for routine diagnostics. In order to meet these demands we have combined isothermal NASBA amplification with molecular beacon probes for real-time detection and quantitation of HIV-1 RNA. Quantitation is based on co-amplification of the HIV-1 RNA in the clinical sample and a synthetic calibrator RNA which is amplified by the same primer set but detected with a differently labeled molecular beacon. The entire procedure is simple and analysis of 48 samples requires less than 1» hours with minimal hands-on time. A fluorescent plate reader is used for real-time detection and isothermal amplification. The linearity and precision of the assay was determined with the VQC HIV-1 type B standard of the Central Laboratory of the Dutch Red Cross Blood Banks, The Netherlands. Sensitivity was shown to be 50 copies per ml (cps/ml). The average assay precision was 0,19 log10 over a range of 100-300,000 cps/ml tested at nine concentrations. The linearity of dilution series of 15 cultured HIV-1 gag clades A-H was shown. The specificity was 100% on non HIV-1 samples HIV-2, HTLV-1 and HTLV-2. The assay robustness in terms of valid results was 99%. In conclusion, the new real-time NASBA assay meets state-of-the-art HIV-1 viral load performance requirements combined with a high level of user convenience.

Elevated expression of MDR1 associated with Line-1 hypomethylation in esophageal squamous cell carcinoma

PubMed Central

Zhu, Jing; Ling, Yang; Xu, Yun; Lu, Ming-Zhu; Liu, Yong-Ping; Zhang, Chang-Song

2015-01-01

Background: The aim is to discuss the relationship of Line-1 methylation and the MDR1 expression in esophageal squamous cell carcinoma (ESCC). Methods: We analyzed the methylation level of Line-1 by quantitative real-time MSP, and the expression of MDR1 by real-time RT-PCR in 310 ESCC and corresponding non-tumor tissues. Results: We found that the methylation index (MI) of Line-1 decreased from 0.90 in non-tumor tissues toward 0.78 in ESCC. The cumulative survival was significantly shorter in ESCC patients with MI ≤ 0.78 (34 months) than that in patients with MI > 0.78 (43 months). There was a statistical difference between MI ≤ 0.78 and MI > 0.78 cases with these clinicopathologic parameters (age, AJCC stage, differentiation; P = 0.010, P < 0.0001, P = 0.015, respectively). These results implied that Line-1 hypomethylation could be more in ESCC patients with older, advanced tumor and poor differentiation group. Meanwhile, ESCC with demethylation of Line-1 were shown elevated MDR1 expression in tumor (Mean-∆∆Ct = 0.21), but ESCC with hypermethylation of Line-1 were considered to be decreased MDR1 expression in tumor (Mean-∆∆Ct = -0.86). Conclusions: Line-1 hypomethylation could be as a biomarker of poor prognosis in ESCC patients. MDR1 gene could be activated via epigenetic mechanisms with demethylation of Line-1 in ESCC, and enhance tumor progression. PMID:26823755