Imaging systems and algorithms to analyze biological samples in real-time using mobile phone microscopy

PubMed Central

Mayberry, Addison; Perkins, David L.; Holcomb, Daniel E.

2018-01-01

Miniaturized imaging devices have pushed the boundaries of point-of-care imaging, but existing mobile-phone-based imaging systems do not exploit the full potential of smart phones. This work demonstrates the use of simple imaging configurations to deliver superior image quality and the ability to handle a wide range of biological samples. Results presented in this work are from analysis of fluorescent beads under fluorescence imaging, as well as helminth eggs and freshwater mussel larvae under white light imaging. To demonstrate versatility of the systems, real time analysis and post-processing results of the sample count and sample size are presented in both still images and videos of flowing samples. PMID:29509786

Clinical evaluation of the Abbott RealTime MTB Assay for direct detection of Mycobacterium tuberculosis-complex from respiratory and non-respiratory samples.

PubMed

Hinić, Vladimira; Feuz, Kinga; Turan, Selda; Berini, Andrea; Frei, Reno; Pfeifer, Karin; Goldenberger, Daniel

2017-05-01

Rapid and reliable diagnosis is crucial for correct management of tuberculosis. The Abbott RealTime MTB Assay represents a novel qualitative real-time PCR assay for direct detection of M. tuberculosis-complex (MTB) DNA from respiratory samples. The test targets two highly conserved sequences, the multi-copy insertion element IS6110 and the protein antigen B (PAB) gene of MTB, allowing even the detection of IS6610-deficient strains. We evaluated this commercial diagnostic test by analyzing 200 respiratory and, for the first time, 87 non-respiratory clinical specimens from our tertiary care institution and compared its results to our IS6110-based in-house real-time PCR for MTB as well as MTB culture. Overall sensitivity for Abbott RealTime MTB was 100% (19/19) in smear positive and 87.5% (7/8) in smear negative specimens, while the specificity of the assay was 100% (260/260). For both non-respiratory smear positive and smear negative specimens Abbott RealTime MTB tests showed 100% (8/8) sensitivity and 100% (8/8) specificity. Cycle threshold (Ct) value analysis of 16 MTB positive samples showed a slightly higher Ct value of the Abbott RealTime MTB test compared to our in-house MTB assay (mean delta Ct = 2.55). In conclusion, the performance of the new Abbott RealTime MTB Assay was highly similar to culture and in-house MTB PCR. We document successful analysis of 87 non-respiratory samples with the highly automated Abbott RealTime MTB test with no inhibition observed. Copyright © 2017 Elsevier Ltd. All rights reserved.

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...

A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis.

PubMed

Marin, M S; Quintana, S; Leunda, M R; Recavarren, M; Pagnuco, I; Späth, E; Pérez, S; Odeón, A

2016-01-01

Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative analysis of detection methods for congenital cytomegalovirus infection in a Guinea pig model.

PubMed

Park, Albert H; Mann, David; Error, Marc E; Miller, Matthew; Firpo, Matthew A; Wang, Yong; Alder, Stephen C; Schleiss, Mark R

2013-01-01

To assess the validity of the guinea pig as a model for congenital cytomegalovirus (CMV) infection by comparing the effectiveness of detecting the virus by real-time polymerase chain reaction (PCR) in blood, urine, and saliva. Case-control study. Academic research. Eleven pregnant Hartley guinea pigs. Blood, urine, and saliva samples were collected from guinea pig pups delivered from pregnant dams inoculated with guinea pig CMV. These samples were then evaluated for the presence of guinea pig CMV by real-time PCR assuming 100% transmission. Thirty-one pups delivered from 9 inoculated pregnant dams and 8 uninfected control pups underwent testing for guinea pig CMV and for auditory brainstem response hearing loss. Repeated-measures analysis of variance demonstrated no statistically significantly lower weight for the infected pups compared with the noninfected control pups. Six infected pups demonstrated auditory brainstem response hearing loss. The sensitivity and specificity of the real-time PCR assay on saliva samples were 74.2% and 100.0%, respectively. The sensitivity of the real-time PCR on blood and urine samples was significantly lower than that on saliva samples. Real-time PCR assays of blood, urine, and saliva revealed that saliva samples show high sensitivity and specificity for detecting congenital CMV infection in guinea pigs. This finding is consistent with recent screening studies in human newborns. The guinea pig may be a good animal model in which to compare different diagnostic assays for congenital CMV infection.

Performance Evaluation of the Operational Air Quality Monitor for Water Testing Aboard the International Space Station

NASA Technical Reports Server (NTRS)

Wallace, William T.; Limero, Thomas F.; Gazda, Daniel B.; Minton, John M.; Macatangay, Ariel V.; Dwivedi, Prabha; Fernandez, Facundo M.

2014-01-01

Real-time environmental monitoring on ISS is necessary to provide data in a timely fashion and to help ensure astronaut health. Current real-time water TOC monitoring provides high-quality trending information, but compound-specific data is needed. The combination of ETV with the AQM showed that compounds of interest could be liberated from water and analyzed in the same manner as air sampling. Calibration of the AQM using water samples allowed for the quantitative analysis of ISS archival samples. Some calibration issues remain, but the excellent accuracy of DMSD indicates that ETV holds promise for as a sample introduction method for water analysis in spaceflight.

Fast analysis of glycosides based on HKUST-1-coated monolith solid-phase microextraction and direct analysis in real-time mass spectrometry.

PubMed

Li, Xianjiang; Wang, Xin; Ma, Wen; Ai, Wanpeng; Bai, Yu; Ding, Li; Liu, Huwei

2017-04-01

Glycosides are a kind of highly important natural aromatic precursors in tobacco leaves. In this study, a novel HKUST-1-coated monolith dip-it sampler was designed for the fast and sensitive analysis of trace glycosides using direct analysis in real-time mass spectrometry. This device was prepared in two steps: in situ polymerization of monolith in a glass capillary of dip-it and layer-by-layer growth of HKUST-1 on the surface of monolith. Sufficient extraction was realized by immersing the tip to solution and in situ desorption was carried out by plasma direct analysis in real time. Compared with traditional solid-phase microextraction protocols, sample desorption was not needed anymore, and only extraction conditions were needed to be optimized in this method, including the gas temperature of direct analysis in real time, extraction time, and CH 3 COONH 4 additive concentration. This method enabled the simultaneous detection of six kinds of glycosides with the limits of detection of 0.02-0.05 μg/mL and the linear ranges covering two orders of magnitude with the limits of quantitation of 0.05-0.1 μg/mL. Moreover, the developed method was applied for the glycosides analysis of three tobacco samples, which only took about 2 s for every sample. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Application of real-time PCR and melting curve analysis in rapid Diego blood group genotyping.

PubMed

Novaretti, M C Z; Ruiz, A S; Dorlhiac-Llacer, P E; Chamone, D A F

2010-01-01

The paucity of appropriate reagents for serologic typing of the Diego blood group antigens has prompted the development of a real-time PCR and melting curve analysis for Diego blood group genotyping. In this study, we phenotyped 4326 donor blood samples for Di(a) using semiautomated equipment. All 157 Di(a+) samples were then genotyped by PCR using sequence-specific primers (PCR-SSP) for DI*02 because of anti-Di(b) scarcity. Of the 4326 samples, we simultaneously tested 160 samples for Di(a) and Di(b) serology, and DI*01 and DI*02 by PCR-SSP and by real-time PCR. We used the same primers for Diego genotyping by real-time PCR and PCR-SSP. Melting curve profiles obtained using the dissociation software of the real-time PCR apparatus enabled the discrimination of Diego alleles. Of the total samples tested, 4169 blood donors, 96.4 percent (95% confidence interval [CI], 95.8-96.9%), were homozygous for DI*02 and 157, 3.6 percent (95% CI, 3.1%-4.2%), were heterozygous DI*01/02. No blood donor was found to be homozygous for DI*01 in this study. The calculated DI*01 and DI*02 allele frequencies were 0.0181 (95% CI, 0.0173-0.0189) and 0.9819 (95% CI, 0.9791-0.9847), respectively, showing a good fit for the Hardy-Weinberg equilibrium. There was full concordance among Diego phenotype results by PCR-SSP and real-time PCR. DI*01 and DI*02 allele determination with SYBR Green I and thermal cycler technology are useful methods for Diego determination. The real-time PCR with SYBR Green I melting temperature protocol can be used as a rapid screening tool for DI*01 and DI*02 blood group genotyping.

Method for analysis of psychopharmaceuticals in real industrial wastewater and groundwater with suspended organic particulate matter using solid phase extraction disks extraction and ultra-high performance liquid chromatography/time-of-flight mass spectrometry.

PubMed

Křesinová, Zdena; Linhartová, Lucie; Petrů, Klára; Krejčová, Lucie; Šrédlová, Kamila; Lhotský, Ondřej; Kameník, Zdeněk; Cajthaml, Tomáš

2016-04-01

A rapid and reliable analytical method was developed for the quantitative determination of psychopharmaceuticals, their precursors and by-products in real contaminated samples from a pharmaceutical company in Olomouc (Czech Republic), based on SPE disk extraction and detection by ultra performance liquid chromatography, combined with time-of-flight mass spectrometry. The target compounds were quantified in the real whole-water samples (water including suspended particles), both in the presence of suspended particulate matter (SPM) and high concentrations of other organic pollutants. A total of nine compounds were analyzed which consisted of three commonly used antidepressants (tricyclic antidepressants and antipsychotics), one antitussive agent and five by-products or precursors. At first, the SPE disk method was developed for the extraction of water samples (dissolved analytes, recovery 84-104%) and pressurised liquid extraction technique was verified for solid matrices (sludge samples, recovery 81-95%). In order to evaluate the SPE disk technique for whole water samples containing SPM, non contaminated groundwater samples were also loaded with different amounts (100 and 300mgL(-1)) of real contaminated sludge originating from the same locality. The recoveries from the whole-water samples obtained by SPE disk method ranged between 67 and 119% after the addition of the most contaminated sludge. The final method was applied to several real groundwater (whole-water) samples from the industrial area and high concentrations (up to 10(3)μgL(-1)) of the target compounds were detected. The results of this study document and indicate the feasibility of the SPE disk method for analysis of groundwater. Copyright © 2016 Elsevier B.V. All rights reserved.

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…

Comparison of nested-multiplex, Taqman & SYBR Green real-time PCR in diagnosis of amoebic liver abscess in a tertiary health care institute in India

PubMed Central

Dinoop, K.P.; Parija, Subhash Chandra; Mandal, Jharna; Swaminathan, R.P.; Narayanan, P.

2016-01-01

Background & objectives: Amoebiasis is a common parasitic infection caused by Entamoeba histolytica and amoebic liver abscess (ALA) is the most common extraintestinal manifestation of amoebiasis. The aim of this study was to standardise real-time PCR assays (Taqman and SYBR Green) to detect E. histolytica from liver abscess pus and stool samples and compare its results with nested-multiplex PCR. Methods: Liver abscess pus specimens were subjected to DNA extraction. The extracted DNA samples were subjected to amplification by nested-multiplex PCR, Taqman (18S rRNA) and SYBR Green real-time PCR (16S-like rRNA assays to detect E. histolytica/E. dispar/E. moshkovskii). The amplification products were further confirmed by DNA sequence analysis. Receiver operator characteristic (ROC) curve analysis was done for nested-multiplex and SYBR Green real-time PCR and the area under the curve was calculated for evaluating the accuracy of the tests to dignose ALA. Results: In all, 17, 19 and 25 liver abscess samples were positive for E. histolytica by nested-multiplex PCR, SYBR Green and Taqman real-time PCR assays, respectively. Significant differences in detection of E. histolytica were noted in the real-time PCR assays evaluated (P<0.0001). The nested-multiplex PCR, SYBR Green real-time PCR and Taqman real-time PCR evaluated showed a positivity rate of 34, 38 and 50 per cent, respectively. Based on ROC curve analysis (considering Taqman real-time PCR as the gold standard), it was observed that SYBR Green real-time PCR was better than conventional nested-multiplex PCR for the diagnosis of ALA. Interpretation & conclusions: Taqman real-time PCR targeting the 18S rRNA had the highest positivity rate evaluated in this study. Both nested multiplex and SYBR Green real-time PCR assays utilized were evaluated to give accurate results. Real-time PCR assays can be used as the gold standard in rapid and reliable diagnosis, and appropriate management of amoebiasis, replacing the conventional molecular methods. PMID:26997014

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

PubMed

Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

2011-01-01

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

QC-ART: A tool for real-time quality control assessment of mass spectrometry-based proteomics data.

PubMed

Stanfill, Bryan A; Nakayasu, Ernesto S; Bramer, Lisa M; Thompson, Allison M; Ansong, Charles K; Clauss, Therese; Gritsenko, Marina A; Monroe, Matthew E; Moore, Ronald J; Orton, Daniel J; Piehowski, Paul D; Schepmoes, Athena A; Smith, Richard D; Webb-Robertson, Bobbie-Jo; Metz, Thomas O

2018-04-17

Liquid chromatography-mass spectrometry (LC-MS)-based proteomics studies of large sample cohorts can easily require from months to years to complete. Acquiring consistent, high-quality data in such large-scale studies is challenging because of normal variations in instrumentation performance over time, as well as artifacts introduced by the samples themselves, such as those due to collection, storage and processing. Existing quality control methods for proteomics data primarily focus on post-hoc analysis to remove low-quality data that would degrade downstream statistics; they are not designed to evaluate the data in near real-time, which would allow for interventions as soon as deviations in data quality are detected.  In addition to flagging analyses that demonstrate outlier behavior, evaluating how the data structure changes over time can aide in understanding typical instrument performance or identify issues such as a degradation in data quality due to the need for instrument cleaning and/or re-calibration.  To address this gap for proteomics, we developed Quality Control Analysis in Real-Time (QC-ART), a tool for evaluating data as they are acquired in order to dynamically flag potential issues with instrument performance or sample quality.  QC-ART has similar accuracy as standard post-hoc analysis methods with the additional benefit of real-time analysis.  We demonstrate the utility and performance of QC-ART in identifying deviations in data quality due to both instrument and sample issues in near real-time for LC-MS-based plasma proteomics analyses of a sample subset of The Environmental Determinants of Diabetes in the Young cohort. We also present a case where QC-ART facilitated the identification of oxidative modifications, which are often underappreciated in proteomic experiments. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of Iron in Lawn Fertilizer: A Sampling Study

ERIC Educational Resources Information Center

Jeannot, Michael A.

2006-01-01

An experiment is described which uses a real-world sample of lawn fertilizer in a simple exercise to illustrate problems associated with the sampling step of a chemical analysis. A mixed-particle fertilizer containing discrete particles of iron oxide (magnetite, Fe[subscript 3]O[subscript 4]) mixed with other particles provides an excellent…

Software algorithm and hardware design for real-time implementation of new spectral estimator

PubMed Central

2014-01-01

Background Real-time spectral analyzers can be difficult to implement for PC computer-based systems because of the potential for high computational cost, and algorithm complexity. In this work a new spectral estimator (NSE) is developed for real-time analysis, and compared with the discrete Fourier transform (DFT). Method Clinical data in the form of 216 fractionated atrial electrogram sequences were used as inputs. The sample rate for acquisition was 977 Hz, or approximately 1 millisecond between digital samples. Real-time NSE power spectra were generated for 16,384 consecutive data points. The same data sequences were used for spectral calculation using a radix-2 implementation of the DFT. The NSE algorithm was also developed for implementation as a real-time spectral analyzer electronic circuit board. Results The average interval for a single real-time spectral calculation in software was 3.29 μs for NSE versus 504.5 μs for DFT. Thus for real-time spectral analysis, the NSE algorithm is approximately 150× faster than the DFT. Over a 1 millisecond sampling period, the NSE algorithm had the capability to spectrally analyze a maximum of 303 data channels, while the DFT algorithm could only analyze a single channel. Moreover, for the 8 second sequences, the NSE spectral resolution in the 3-12 Hz range was 0.037 Hz while the DFT spectral resolution was only 0.122 Hz. The NSE was also found to be implementable as a standalone spectral analyzer board using approximately 26 integrated circuits at a cost of approximately $500. The software files used for analysis are included as a supplement, please see the Additional files 1 and 2. Conclusions The NSE real-time algorithm has low computational cost and complexity, and is implementable in both software and hardware for 1 millisecond updates of multichannel spectra. The algorithm may be helpful to guide radiofrequency catheter ablation in real time. PMID:24886214

[Rapid screening the alkaloids of poppy shell in hot pot condiment, beef noodle soup and seasoning by direct analysis in real time-tandem mass spectrometry].

PubMed

Zhang, Baile; Gao, Lihong; Xie, Yingshuang; Zhou, Wei; Chen, Xiaofeng; Lei, Chunni; Zhang, Huan

2017-07-08

A direct analysis in real time tandem mass spectrometry (DART-MS/MS) method was established for quickly screening five illegally added alkaloids of poppy shell from the hot pot condiment, beef noodle soup and seasoning. The samples were extracted and purified by acetonitrile, and then injected under the conditions of ionization temperature of 300℃, grid electrode voltage of 150 V and sampling rate of 0.8 mm/s using DART in the positive ion mode. The determination was conducted by tandem mass spectrometry in positive ESI mode under multiple reaction monitoring (MRM) mode. The method is simple and rapid, and can meet the requirement of rapid screening and analysis of large quantities of samples.

Soft Ionization of Saturated Hydrocarbons, Alcohols and Nonpolar Compounds by Negative-Ion Direct Analysis in Real-Time Mass Spectrometry

NASA Astrophysics Data System (ADS)

Cody, Robert B.; Dane, A. John

2013-03-01

Large polarizable n-alkanes (approximately C18 and larger), alcohols, and other nonpolar compounds can be detected as negative ions when sample solutions are injected directly into the sampling orifice of the atmospheric pressure interface of the time-of-flight mass spectrometer with the direct analysis in real time (DART) ion source operating in negative-ion mode. The mass spectra are dominated by peaks corresponding to [M + O2]‾•. No fragmentation is observed, making this a very soft ionization technique for samples that are otherwise difficult to analyze by DART. Detection limits for cholesterol were determined to be in the low nanogram range.

Soft ionization of saturated hydrocarbons, alcohols and nonpolar compounds by negative-ion direct analysis in real-time mass spectrometry.

PubMed

Cody, Robert B; Dane, A John

2013-03-01

Large polarizable n-alkanes (approximately C18 and larger), alcohols, and other nonpolar compounds can be detected as negative ions when sample solutions are injected directly into the sampling orifice of the atmospheric pressure interface of the time-of-flight mass spectrometer with the direct analysis in real time (DART) ion source operating in negative-ion mode. The mass spectra are dominated by peaks corresponding to [M + O2]‾(•). No fragmentation is observed, making this a very soft ionization technique for samples that are otherwise difficult to analyze by DART. Detection limits for cholesterol were determined to be in the low nanogram range.

Automated high-throughput flow-through real-time diagnostic system

DOEpatents

Regan, John Frederick

2012-10-30

An automated real-time flow-through system capable of processing multiple samples in an asynchronous, simultaneous, and parallel fashion for nucleic acid extraction and purification, followed by assay assembly, genetic amplification, multiplex detection, analysis, and decontamination. The system is able to hold and access an unlimited number of fluorescent reagents that may be used to screen samples for the presence of specific sequences. The apparatus works by associating extracted and purified sample with a series of reagent plugs that have been formed in a flow channel and delivered to a flow-through real-time amplification detector that has a multiplicity of optical windows, to which the sample-reagent plugs are placed in an operative position. The diagnostic apparatus includes sample multi-position valves, a master sample multi-position valve, a master reagent multi-position valve, reagent multi-position valves, and an optical amplification/detection system.

Detecting spatial patterns of rivermouth processes using a geostatistical framework for near-real-time analysis

USGS Publications Warehouse

Xu, Wenzhao; Collingsworth, Paris D.; Bailey, Barbara; Carlson Mazur, Martha L.; Schaeffer, Jeff; Minsker, Barbara

2017-01-01

This paper proposes a geospatial analysis framework and software to interpret water-quality sampling data from towed undulating vehicles in near-real time. The framework includes data quality assurance and quality control processes, automated kriging interpolation along undulating paths, and local hotspot and cluster analyses. These methods are implemented in an interactive Web application developed using the Shiny package in the R programming environment to support near-real time analysis along with 2- and 3-D visualizations. The approach is demonstrated using historical sampling data from an undulating vehicle deployed at three rivermouth sites in Lake Michigan during 2011. The normalized root-mean-square error (NRMSE) of the interpolation averages approximately 10% in 3-fold cross validation. The results show that the framework can be used to track river plume dynamics and provide insights on mixing, which could be related to wind and seiche events.

In vivo Real-Time Mass Spectrometry for Guided Surgery Application

NASA Astrophysics Data System (ADS)

Fatou, Benoit; Saudemont, Philippe; Leblanc, Eric; Vinatier, Denis; Mesdag, Violette; Wisztorski, Maxence; Focsa, Cristian; Salzet, Michel; Ziskind, Michael; Fournier, Isabelle

2016-05-01

Here we describe a new instrument (SpiderMass) designed for in vivo and real-time analysis. In this instrument ion production is performed remotely from the MS instrument and the generated ions are transported in real-time to the MS analyzer. Ion production is promoted by Resonant Infrared Laser Ablation (RIR-LA) based on the highly effective excitation of O-H bonds in water molecules naturally present in most biological samples. The retrieved molecular patterns are specific to the cell phenotypes and benign versus cancer regions of patient biopsies can be easily differentiated. We also demonstrate by analysis of human skin that SpiderMass can be used under in vivo conditions with minimal damage and pain. Furthermore SpiderMass can also be used for real-time drug metabolism and pharmacokinetic (DMPK) analysis or food safety topics. SpiderMass is thus the first MS based system designed for in vivo real-time analysis under minimally invasive conditions.

A system architecture for online data interpretation and reduction in fluorescence microscopy

NASA Astrophysics Data System (ADS)

Röder, Thorsten; Geisbauer, Matthias; Chen, Yang; Knoll, Alois; Uhl, Rainer

2010-01-01

In this paper we present a high-throughput sample screening system that enables real-time data analysis and reduction for live cell analysis using fluorescence microscopy. We propose a novel system architecture capable of analyzing a large amount of samples during the experiment and thus greatly minimizing the post-analysis phase that is the common practice today. By utilizing data reduction algorithms, relevant information of the target cells is extracted from the online collected data stream, and then used to adjust the experiment parameters in real-time, allowing the system to dynamically react on changing sample properties and to control the microscope setup accordingly. The proposed system consists of an integrated DSP-FPGA hybrid solution to ensure the required real-time constraints, to execute efficiently the underlying computer vision algorithms and to close the perception-action loop. We demonstrate our approach by addressing the selective imaging of cells with a particular combination of markers. With this novel closed-loop system the amount of superfluous collected data is minimized, while at the same time the information entropy increases.

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.

Real-time PCR detection of Plasmodium directly from whole blood and filter paper samples

PubMed Central

2011-01-01

Background Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products. Methods Reagents designed to specifically overcome PCR inhibition and quenching of fluorescence were evaluated for real-time PCR amplification of Plasmodium DNA directly from blood. Whole blood from clinical samples and dried blood spots collected in the field in Colombia were tested. Results Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBR® Green dye and 5% blood volume in the PCR reaction. Plasmodium DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93-100% compared with PCR performed on purified Plasmodium DNA. Conclusions The methodology described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs and time-savings. PMID:21851640

Near-Real-Time and Laboratory Data by State

EPA Pesticide Factsheets

RadNet data from individual near-real-time air monitors is available on a clickable map and is also listed by state and city. Sampling locations with drinking water, precipitation and air filter analysis results in 2016 are also listed.

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.

LIBS: a potential tool for industrial/agricultural waste water analysis

NASA Astrophysics Data System (ADS)

Karpate, Tanvi; K. M., Muhammed Shameem; Nayak, Rajesh; V. K., Unnikrishnan; Santhosh, C.

2016-04-01

Laser Induced Breakdown Spectroscopy (LIBS) is a multi-elemental analysis technique with various advantages and has the ability to detect any element in real time. This technique holds a potential for environmental monitoring and various such analysis has been done in soil, glass, paint, water, plastic etc confirms the robustness of this technique for such applications. Compared to the currently available water quality monitoring methods and techniques, LIBS has several advantages, viz. no need for sample preparation, fast and easy operation, and chemical free during the process. In LIBS, powerful pulsed laser generates plasma which is then analyzed to get quantitative and qualitative details of the elements present in the sample. Another main advantage of LIBS technique is that it can perform in standoff mode for real time analysis. Water samples from industries and agricultural strata tend to have a lot of pollutants making it harmful for consumption. The emphasis of this project is to determine such harmful pollutants present in trace amounts in industrial and agricultural wastewater. When high intensity laser is made incident on the sample, a plasma is generated which gives a multielemental emission spectra. LIBS analysis has shown outstanding success for solids samples. For liquid samples, the analysis is challenging as the liquid sample has the chances of splashing due to the high energy of laser and thus making it difficult to generate plasma. This project also deals with determining the most efficient method for testing of water sample for qualitative as well as quantitative analysis using LIBS.

A Field Investigation of Bacillus anthracis Contamination of U.S. Department of Agriculture and Other Washington, D.C., Buildings during the Anthrax Attack of October 2001

PubMed Central

Higgins, James A.; Cooper, Mary; Schroeder-Tucker, Linda; Black, Scott; Miller, David; Karns, Jeffrey S.; Manthey, Erlynn; Breeze, Roger; Perdue, Michael L.

2003-01-01

In response to a bioterrorism attack in the Washington, D.C., area in October 2001, a mobile laboratory (ML) was set up in the city to conduct rapid molecular tests on environmental samples for the presence of Bacillus anthracis spores and to route samples for further culture analysis. The ML contained class I laminar-flow hoods, a portable autoclave, two portable real-time PCR devices (Ruggedized Advanced Pathogen Identification Device [RAPID]), and miscellaneous supplies and equipment to process samples. Envelopes and swab and air samples collected from 30 locations in the metropolitan area once every three days were subjected to visual examination and DNA extraction, followed by real-time PCR using freeze-dried, fluorescent-probe-based reagents. Surface swabs and air samples were also cultured for B. anthracis at the National Veterinary Service Laboratory (NVSL) in Ames, Iowa. From 24 October 2001 to 15 September 2002, 2,092 pieces of mail were examined, 405 real-time PCR assays were performed (comprising 4,639 samples), and at the NVSL 6,275 samples were subjected to over 18,000 platings. None of the PCR assays on DNA extracted from swab and air samples were positive, but viable spores were cultured from surface swabs taken from six locations in the metropolitan area in October, November, and December 2001 and February, March, and May 2002. DNA extracted from these suspected B. anthracis colonies was positive by real-time and conventional PCRs for the lethal factor, pXO1, and for capA and vrr genes; sequence analysis of the latter amplicons indicated >99% homology with the Ames, vollum, B6273-93, C93022281, and W-21 strains of B. anthracis, suggesting they arose from cross-contamination during the attack through the mail. The RAPID-based PCR analysis provided fast confirmation of suspect colonies from an overnight incubation on agar plates. PMID:12514046

Open Probe fast GC-MS - combining ambient sampling ultra-fast separation and in-vacuum ionization for real-time analysis.

PubMed

Keshet, U; Alon, T; Fialkov, A B; Amirav, A

2017-07-01

An Open Probe inlet was combined with a low thermal mass ultra-fast gas chromatograph (GC), in-vacuum electron ionization ion source and a mass spectrometer (MS) of GC-MS for obtaining real-time analysis with separation. The Open Probe enables ambient sampling via sample vaporization in an oven that is open to room air, and the ultra-fast GC provides ~30-s separation, while if no separation is required, it can act as a transfer line with 2 to 3-s sample transfer time. Sample analysis is as simple as touching the sample, pushing the sample holder into the Open Probe oven and obtaining the results in 30 s. The Open Probe fast GC was mounted on a standard Agilent 7890 GC that was coupled with an Agilent 5977A MS. Open Probe fast GC-MS provides real-time analysis combined with GC separation and library identification, and it uses the low-cost MS of GC-MS. The operation of Open Probe fast GC-MS is demonstrated in the 30-s separation and 50-s full analysis cycle time of tetrahydrocannabinol and cannabinol in Cannabis flower, sub 1-min analysis of trace trinitrotoluene transferred from a finger onto a glass surface, vitamin E in canola oil, sterols in olive oil, polybrominated flame retardants in plastics, alprazolam in Xanax drug pill and free fatty acids and cholesterol in human blood. The extrapolated limit of detection for pyrene is <1 fg, but the concentration is too high and the software noise calculation is untrustworthy. The broad range of compounds amenable for analysis is demonstrated in the analysis of reserpine. The possible use with alternate standard GC-MS and Open Probe fast GC-MS is demonstrated in the analysis of heroin in its street drug powder. The use of Open Probe with the fast GC acting as a transfer line is demonstrated in <10-s analysis without separation of ibuprofen and estradiol. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Electro-thermal vaporization direct analysis in real time-mass spectrometry for water contaminant analysis during space missions.

PubMed

Dwivedi, Prabha; Gazda, Daniel B; Keelor, Joel D; Limero, Thomas F; Wallace, William T; Macatangay, Ariel V; Fernández, Facundo M

2013-10-15

The development of a direct analysis in real time-mass spectrometry (DART-MS) method and first prototype vaporizer for the detection of low molecular weight (∼30-100 Da) contaminants representative of those detected in water samples from the International Space Station is reported. A temperature-programmable, electro-thermal vaporizer (ETV) was designed, constructed, and evaluated as a sampling interface for DART-MS. The ETV facilitates analysis of water samples with minimum user intervention while maximizing analytical sensitivity and sample throughput. The integrated DART-ETV-MS methodology was evaluated in both positive and negative ion modes to (1) determine experimental conditions suitable for coupling DART with ETV as a sample inlet and ionization platform for time-of-flight MS, (2) to identify analyte response ions, (3) to determine the detection limit and dynamic range for target analyte measurement, and (4) to determine the reproducibility of measurements made with the method when using manual sample introduction into the vaporizer. Nitrogen was used as the DART working gas, and the target analytes chosen for the study were ethyl acetate, acetone, acetaldehyde, ethanol, ethylene glycol, dimethylsilanediol, formaldehyde, isopropanol, methanol, methylethyl ketone, methylsulfone, propylene glycol, and trimethylsilanol.

EPA Method 3031 (SW-846): Acid Digestion of Oils for Metals Analysis by Atomic Absorption or ICP Spectrometry

EPA Pesticide Factsheets

Procedures are described for analysis of water samples and may be adapted for assessment of solid, particulate and liquid samples. The method uses real-time PCR assay for detecting Toxoplasma gondii DNA using gene-specific primers and probe.

High resolution DNA melting analysis: an application for prenatal control of alpha-thalassemia.

PubMed

Sirichotiyakul, Supatra; Wanapirak, Chanane; Saetung, Rattika; Sanguansermsri, Torpong

2010-04-01

To report the use of real-time gap-PCR using SYTO9 with high-resolution melting analysis (HRMA) in prenatal diagnosis of alpha-thalassemia 1. Real-time gap-PCR using SYTO9 with HRMA was performed in 33 DNA samples from chorionic villi sampling (8 normal, 16 heterozygous, and 9 homozygous) to determine the alpha-thalassemia 1 gene [normal and Southeast Asia (-SEA) allele]. The dissociation curve analysis in normal and - SEA allele gave a peak of T(m) at 91.80 +/- 0.14 degrees C and 88.67 +/- 0.08 degrees C, respectively. Normal genotype and homozygous alpha-thalassemia 1 showed a single peak of T(m) that corresponded to their alleles. The heterozygotes gave both peaks with higher normal peak and smaller - SEA peak. Thirty one samples showed consistent results with the conventional gap-PCR. Two samples with ambiguous results were confirmed to be maternal DNA contamination on real-time quantitative PCR and microsatellite assay. HRMA from both samples showed similar pattern to that of heterozygotes. However, they showed much smaller normal peak compared with the - SEA peak, which is in contrast to those of heterozygotes and can readily be distinguished. HRMA with SYTO9 is feasible for prenatal diagnosis of alpha-thalassemia. It had potential advantage of prompt detection maternal DNA contamination. Copyright (c) 2010 John Wiley & Sons, Ltd.

Pushing the limits: Quantification of chromophores in real-world paper samples by GC-ECD and EI-GC-MS.

PubMed

Schedl, A; Zweckmair, T; Kikul, F; Bacher, M; Rosenau, T; Potthast, A

2018-03-01

Widening the methodology of chromophore analysis in pulp and paper science, a sensitive gas-chromatographic approach with electron-capture detection is presented and applied to model samples and real-world historic paper material. Trifluoroacetic anhydride was used for derivatization of the chromophore target compounds. The derivative formation was confirmed by NMR and accurate mass analysis. The method successfully detects and quantifies hydroxyquinones which are key chromophores in cellulosic matrices. The analytical figures of merit appeared to be in an acceptable range with an LOD down to approx. 60ng/g for each key chromophore, which allows for their successful detection in historic sample material. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of a micropulverized extraction method for rapid toxicological analysis of methamphetamine in hair.

PubMed

Miyaguchi, Hajime; Kakuta, Masaya; Iwata, Yuko T; Matsuda, Hideaki; Tazawa, Hidekatsu; Kimura, Hiroko; Inoue, Hiroyuki

2007-09-07

We developed a rapid sample preparation method for the toxicological analysis of methamphetamine and amphetamine (the major metabolite of methamphetamine) in human hair by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), to facilitate fast screening and quantitation. Two milligrams of hair were mechanically micropulverized for 5 min in a 2-ml plastic tube together with 100 microl of an aqueous solvent containing 10% acetonitrile, 100 mM trifluoroacetic acid and the corresponding deuterium analogues as internal standards. The pulverizing highly disintegrated the hair components, simultaneously allowing the extraction of any drugs present in the hair. After filtering the suspension with a membrane-filter unit, the clear filtrate was directly analyzed by HPLC-MS/MS. No evaporation processes were required for sample preparation. Method optimization and validation study were carried out using real-case specimens and fortified samples in which the drugs had been artificially absorbed, respectively. Concentration ranges for quantitation were 0.040-125 and 0.040-25 ng/mg for methamphetamine and amphetamine, respectively. Real-case specimens were analyzed by the method presented here and by conventional ones to verify the applicability of our method to real-world analysis. Our method took less than 30 min for a set of chromatograms to be obtained from a washed hair sample.

Large-volume constant-concentration sampling technique coupling with surface-enhanced Raman spectroscopy for rapid on-site gas analysis

NASA Astrophysics Data System (ADS)

Zhang, Zhuomin; Zhan, Yisen; Huang, Yichun; Li, Gongke

2017-08-01

In this work, a portable large-volume constant-concentration (LVCC) sampling technique coupling with surface-enhanced Raman spectroscopy (SERS) was developed for the rapid on-site gas analysis based on suitable derivatization methods. LVCC sampling technique mainly consisted of a specially designed sampling cell including the rigid sample container and flexible sampling bag, and an absorption-derivatization module with a portable pump and a gas flowmeter. LVCC sampling technique allowed large, alterable and well-controlled sampling volume, which kept the concentration of gas target in headspace phase constant during the entire sampling process and made the sampling result more representative. Moreover, absorption and derivatization of gas target during LVCC sampling process were efficiently merged in one step using bromine-thiourea and OPA-NH4+ strategy for ethylene and SO2 respectively, which made LVCC sampling technique conveniently adapted to consequent SERS analysis. Finally, a new LVCC sampling-SERS method was developed and successfully applied for rapid analysis of trace ethylene and SO2 from fruits. It was satisfied that trace ethylene and SO2 from real fruit samples could be actually and accurately quantified by this method. The minor concentration fluctuations of ethylene and SO2 during the entire LVCC sampling process were proved to be < 4.3% and 2.1% respectively. Good recoveries for ethylene and sulfur dioxide from fruit samples were achieved in range of 95.0-101% and 97.0-104% respectively. It is expected that portable LVCC sampling technique would pave the way for rapid on-site analysis of accurate concentrations of trace gas targets from real samples by SERS.

Large-volume constant-concentration sampling technique coupling with surface-enhanced Raman spectroscopy for rapid on-site gas analysis.

PubMed

Zhang, Zhuomin; Zhan, Yisen; Huang, Yichun; Li, Gongke

2017-08-05

In this work, a portable large-volume constant-concentration (LVCC) sampling technique coupling with surface-enhanced Raman spectroscopy (SERS) was developed for the rapid on-site gas analysis based on suitable derivatization methods. LVCC sampling technique mainly consisted of a specially designed sampling cell including the rigid sample container and flexible sampling bag, and an absorption-derivatization module with a portable pump and a gas flowmeter. LVCC sampling technique allowed large, alterable and well-controlled sampling volume, which kept the concentration of gas target in headspace phase constant during the entire sampling process and made the sampling result more representative. Moreover, absorption and derivatization of gas target during LVCC sampling process were efficiently merged in one step using bromine-thiourea and OPA-NH 4 + strategy for ethylene and SO 2 respectively, which made LVCC sampling technique conveniently adapted to consequent SERS analysis. Finally, a new LVCC sampling-SERS method was developed and successfully applied for rapid analysis of trace ethylene and SO 2 from fruits. It was satisfied that trace ethylene and SO 2 from real fruit samples could be actually and accurately quantified by this method. The minor concentration fluctuations of ethylene and SO 2 during the entire LVCC sampling process were proved to be <4.3% and 2.1% respectively. Good recoveries for ethylene and sulfur dioxide from fruit samples were achieved in range of 95.0-101% and 97.0-104% respectively. It is expected that portable LVCC sampling technique would pave the way for rapid on-site analysis of accurate concentrations of trace gas targets from real samples by SERS. Copyright © 2017 Elsevier B.V. All rights reserved.

Analysis of select Dalbergia and trade timber using direct analysis in real time and time-of-flight mass spectrometry for CITES enforcement.

PubMed

Lancaster, Cady; Espinoza, Edgard

2012-05-15

International trade of several Dalbergia wood species is regulated by The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). In order to supplement morphological identification of these species, a rapid chemical method of analysis was developed. Using Direct Analysis in Real Time (DART) ionization coupled with Time-of-Flight (TOF) Mass Spectrometry (MS), selected Dalbergia and common trade species were analyzed. Each of the 13 wood species was classified using principal component analysis and linear discriminant analysis (LDA). These statistical data clusters served as reliable anchors for species identification of unknowns. Analysis of 20 or more samples from the 13 species studied in this research indicates that the DART-TOFMS results are reproducible. Statistical analysis of the most abundant ions gave good classifications that were useful for identifying unknown wood samples. DART-TOFMS and LDA analysis of 13 species of selected timber samples and the statistical classification allowed for the correct assignment of unknown wood samples. This method is rapid and can be useful when anatomical identification is difficult but needed in order to support CITES enforcement. Published 2012. This article is a US Government work and is in the public domain in the USA.

Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis.

PubMed

Janwan, Penchom; Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Anamnart, Witthaya; Maleewong, Wanchai

2011-12-01

Human opisthorchiasis caused by the liver fluke Opisthorchis viverrini is an endemic disease in Southeast Asian countries including the Lao People's Democratic Republic, Cambodia, Vietnam, and Thailand. Infection with the soil-transmitted roundworm Strongyloides stercoralis is an important problem worldwide. In some areas, both parasitic infections are reported as co-infections. A duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis was developed for the rapid detection of O. viverrini and S. stercoralis in human fecal samples. Duplex real-time FRET PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two genera of DNA elements: the 162 bp pOV-A6 DNA sequence specific to O. viverrini and the 244 bp 18S rRNA sequence specific to S. stercoralis, and two pairs of specific fluorophore-labeled probes. Both O. viverrini and S. stercoralis can be differentially detected in infected human fecal samples by this process through their different fluorescence channels and melting temperatures. Detection limit of the method was as little as two O. viverrini eggs and four S. stercoralis larvae in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasite materials, as well as from the DNA of human leukocytes and other control parasites. The technique showed 100% sensitivity and specificity. The introduced duplex real-time FRET PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The method is important for simultaneous detection especially in areas where both parasites overlap incidence and is useful as the screening tool in the returning travelers and immigrants to industrialized countries where number of samples in the diagnostic units will become increasing.

Detection of central nervous system leukemia in children with acute lymphoblastic leukemia by real-time polymerase chain reaction.

PubMed

Pine, Sharon R; Yin, Changhong; Matloub, Yousif H; Sabaawy, Hatem E; Sandoval, Claudio; Levendoglu-Tugal, Oya; Ozkaynak, M Fevzi; Jayabose, Somasundaram

2005-02-01

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. We examined various cerebrospinal fluid (CSF) preservation methods to yield adequate DNA stability for polymerase chain reaction (PCR) analysis and developed a quantitative real-time PCR assay to detect occult CNS leukemia. Sixty CSF specimens were maintained in several storage conditions for varying amounts of time, and we found that preserving CSF in 1:1 serum-free RPMI tissue culture medium offers the best stability of DNA for PCR analysis. Sixty CSF samples (30 at diagnosis and 30 at the end of induction therapy) from 30 children with ALL were tested for CNS leukemic involvement by real-time PCR using patient-specific antigen receptor gene rearrangement primers. Six of thirty patient diagnosis samples were PCR-positive at levels ranging from 0.5 to 66% leukemic blasts in the CSF. Four of these patients had no clinical or cytomorphological evidence of CNS leukemia involvement at that time. All 30 CSF samples drawn at the end of induction therapy were PCR-negative. The data indicate that real-time PCR analysis of CSF is an excellent tool to assess occult CNS leukemia involvement in patients with ALL and can possibly be used to refine CNS status classification.

Detection of Central Nervous System Leukemia in Children with Acute Lymphoblastic Leukemia by Real-Time Polymerase Chain Reaction

PubMed Central

Pine, Sharon R.; Yin, Changhong; Matloub, Yousif H.; Sabaawy, Hatem E.; Sandoval, Claudio; Levendoglu-Tugal, Oya; Ozkaynak, M. Fevzi; Jayabose, Somasundaram

2005-01-01

Accurate detection of central nervous system (CNS) involvement in children with newly diagnosed acute lymphoblastic leukemia (ALL) could have profound prognostic and therapeutic implications. We examined various cerebrospinal fluid (CSF) preservation methods to yield adequate DNA stability for polymerase chain reaction (PCR) analysis and developed a quantitative real-time PCR assay to detect occult CNS leukemia. Sixty CSF specimens were maintained in several storage conditions for varying amounts of time, and we found that preserving CSF in 1:1 serum-free RPMI tissue culture medium offers the best stability of DNA for PCR analysis. Sixty CSF samples (30 at diagnosis and 30 at the end of induction therapy) from 30 children with ALL were tested for CNS leukemic involvement by real-time PCR using patient-specific antigen receptor gene rearrangement primers. Six of thirty patient diagnosis samples were PCR-positive at levels ranging from 0.5 to 66% leukemic blasts in the CSF. Four of these patients had no clinical or cytomorphological evidence of CNS leukemia involvement at that time. All 30 CSF samples drawn at the end of induction therapy were PCR-negative. The data indicate that real-time PCR analysis of CSF is an excellent tool to assess occult CNS leukemia involvement in patients with ALL and can possibly be used to refine CNS status classification. PMID:15681484

Rapid DNA extraction protocol for detection of alpha-1 antitrypsin deficiency from dried blood spots by real-time PCR.

PubMed

Struniawski, R; Szpechcinski, A; Poplawska, B; Skronski, M; Chorostowska-Wynimko, J

2013-01-01

The dried blood spot (DBS) specimens have been successfully employed for the large-scale diagnostics of α1-antitrypsin (AAT) deficiency as an easy to collect and transport alternative to plasma/serum. In the present study we propose a fast, efficient, and cost effective protocol of DNA extraction from dried blood spot (DBS) samples that provides sufficient quantity and quality of DNA and effectively eliminates any natural PCR inhibitors, allowing for successful AAT genotyping by real-time PCR and direct sequencing. DNA extracted from 84 DBS samples from chronic obstructive pulmonary disease patients was genotyped for AAT deficiency variants by real-time PCR. The results of DBS AAT genotyping were validated by serum IEF phenotyping and AAT concentration measurement. The proposed protocol allowed successful DNA extraction from all analyzed DBS samples. Both quantity and quality of DNA were sufficient for further real-time PCR and, if necessary, for genetic sequence analysis. A 100% concordance between AAT DBS genotypes and serum phenotypes in positive detection of two major deficiency S- and Z- alleles was achieved. Both assays, DBS AAT genotyping by real-time PCR and serum AAT phenotyping by IEF, positively identified PI*S and PI*Z allele in 8 out of the 84 (9.5%) and 16 out of 84 (19.0%) patients, respectively. In conclusion, the proposed protocol noticeably reduces the costs and the hand-on-time of DBS samples preparation providing genomic DNA of sufficient quantity and quality for further real-time PCR or genetic sequence analysis. Consequently, it is ideally suited for large-scale AAT deficiency screening programs and should be method of choice.

Detection of airborne genetically modified maize pollen by real-time PCR.

PubMed

Folloni, Silvia; Kagkli, Dafni-Maria; Rajcevic, Bojan; Guimarães, Nilson C C; Van Droogenbroeck, Bart; Valicente, Fernando H; Van den Eede, Guy; Van den Bulcke, Marc

2012-09-01

The cultivation of genetically modified (GM) crops has raised numerous concerns in the European Union and other parts of the world about their environmental and economic impact. Especially outcrossing of genetically modified organisms (GMO) was from the beginning a critical issue as airborne pollen has been considered an important way of GMO dispersal. Here, we investigate the use of airborne pollen sampling combined with microscopic analysis and molecular PCR analysis as an approach to monitor GM maize cultivations in a specific area. Field trial experiments in the European Union and South America demonstrated the applicability of the approach under different climate conditions, in rural and semi-urban environment, even at very low levels of airborne pollen. The study documents in detail the sampling of GM pollen, sample DNA extraction and real-time PCR analysis. Our results suggest that this 'GM pollen monitoring by bioaerosol sampling and PCR screening' approach might represent an useful aid in the surveillance of GM-free areas, centres of origin and natural reserves. © 2012 Blackwell Publishing Ltd.

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...

Description of a Portable Wireless Device for High-Frequency Body Temperature Acquisition and Analysis

PubMed Central

Cuesta-Frau, David; Varela, Manuel; Aboy, Mateo; Miró-Martínez, Pau

2009-01-01

We describe a device for dual channel body temperature monitoring. The device can operate as a real time monitor or as a data logger, and has Bluetooth capabilities to enable for wireless data download to the computer used for data analysis. The proposed device is capable of sampling temperature at a rate of 1 sample per minute with a resolution of 0.01 °C . The internal memory allows for stand-alone data logging of up to 10 days. The device has a battery life of 50 hours in continuous real-time mode. In addition to describing the proposed device in detail, we report the results of a statistical analysis conducted to assess its accuracy and reproducibility. PMID:22408473

Description of a portable wireless device for high-frequency body temperature acquisition and analysis.

PubMed

Cuesta-Frau, David; Varela, Manuel; Aboy, Mateo; Miró-Martínez, Pau

2009-01-01

We describe a device for dual channel body temperature monitoring. The device can operate as a real time monitor or as a data logger, and has Bluetooth capabilities to enable for wireless data download to the computer used for data analysis. The proposed device is capable of sampling temperature at a rate of 1 sample per minute with a resolution of 0.01 °C . The internal memory allows for stand-alone data logging of up to 10 days. The device has a battery life of 50 hours in continuous real-time mode. In addition to describing the proposed device in detail, we report the results of a statistical analysis conducted to assess its accuracy and reproducibility.

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

PubMed

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

2010-06-01

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

Rapid quantification and sex determination of forensic evidence materials.

PubMed

Andréasson, Hanna; Allen, Marie

2003-11-01

DNA quantification of forensic evidence is very valuable for an optimal use of the available biological material. Moreover, sex determination is of great importance as additional information in criminal investigations as well as in identification of missing persons, no suspect cases, and ancient DNA studies. While routine forensic DNA analysis based on short tandem repeat markers includes a marker for sex determination, analysis of samples containing scarce amounts of DNA is often based on mitochondrial DNA, and sex determination is not performed. In order to allow quantification and simultaneous sex determination on minute amounts of DNA, an assay based on real-time PCR analysis of a marker within the human amelogenin gene has been developed. The sex determination is based on melting curve analysis, while an externally standardized kinetic analysis allows quantification of the nuclear DNA copy number in the sample. This real-time DNA quantification assay has proven to be highly sensitive, enabling quantification of single DNA copies. Although certain limitations were apparent, the system is a rapid, cost-effective, and flexible assay for analysis of forensic casework samples.

Real Time Text Analysis

NASA Astrophysics Data System (ADS)

Senthilkumar, K.; Ruchika Mehra Vijayan, E.

2017-11-01

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

Implementation guide for turbidity threshold sampling: principles, procedures, and analysis

Treesearch

Jack Lewis; Rand Eads

2009-01-01

Turbidity Threshold Sampling uses real-time turbidity and river stage information to automatically collect water quality samples for estimating suspended sediment loads. The system uses a programmable data logger in conjunction with a stage measurement device, a turbidity sensor, and a pumping sampler. Specialized software enables the user to control the sampling...

Molecular diagnostics and ITS-based phylogenic analysis of Streptococcus suis serotype 2 in central Vietnam.

PubMed

Nguyen, Bach Hoang; Phan, Dieu Hong Nu; Nguyen, Hien Xuan; Le, An Van; Alberti, Alberto

2015-07-04

Streptococcus suis (S. suis) serotype 2 has recently become the most prevalent cause of meningitis in adults in many areas of Vietnam. This study provides data on S. suis molecular diagnosis in central Vietnam using a real-time polymerase chain reaction (PCR) assay targeting the S. suis serotype 2 cps2J gene. Additionally, 16S-23S rDNA intragenic spacer (ITS)-based phylogenic analysis of strains isolated from cerebrospinal fluid (CSF) in Thua Thien Hue Province, Vietnam, is presented and discussed. Pathogenic bacteria were isolated from 40 CSF samples, and 18 were identified as S. suis by culture-dependent methods. Capsular serotyping was assessed by real-time PCR. ITS sequences were obtained after traditional PCR and were used in phylogenic analyses. Pathogenic bacteria were isolated from 36 out of 40 CSF samples. A total of 18 S. suis strains were isolated and assigned to serotype 2 by real-time PCR. One CSF sample, negative when tested by culture-dependent methods, was positive to S. suis serotype 2 by real-time PCR. Pairwise alignments of the 18 ITS sequences did not reveal any variable nucleotide position, and resulted in a single sequence type. Sequences were similar to S. suis serotype 2 reference ITS sequences (> 98.1%), and there was no lack of an ITS spacer region in the isolates. S. suis serotype 2 is the most prevalent serotype in central Vietnam. Real-time PCR assay proved to be a reliable diagnostic method for early detection of S. suis 2 in CSF samples.

Evaluation of two commercial real-time PCR assays for detecting Campylobacter in broiler carcass rinses.

USDA-ARS?s Scientific Manuscript database

Traditional plating methods are reliable means for Campylobacter identification from poultry samples but automated gene-based detection systems now available can reduce assay time, data collection and analysis. Bio-Rad and DuPont Qualicon recently introduced Campylobacter assays for their real-time ...

Rapid and specific detection of Salmonella in water samples using real-time PCR and High Resolution Melt (HRM) curve analysis.

PubMed

van Blerk, G N; Leibach, L; Mabunda, A; Chapman, A; Louw, D

2011-01-01

A real-time PCR assay combined with a pre-enrichment step for the specific and rapid detection of Salmonella in water samples is described. Following amplification of the invA gene target, High Resolution Melt (HRM) curve analysis was used to discriminate between products formed and to positively identify invA amplification. The real-time PCR assay was evaluated for specificity and sensitivity. The assay displayed 100% specificity for Salmonella and combined with a 16-18 h non-selective pre-enrichment step, the assay proved to be highly sensitive with a detection limit of 1.0 CFU/ml for surface water samples. The detection assay also demonstrated a high intra-run and inter-run repeatability with very little variation in invA amplicon melting temperature. When applied to water samples received routinely by the laboratory, the assay showed the presence of Salmonella in particularly surface water and treated effluent samples. Using the HRM based assay, the time required for Salmonella detection was drastically shortened to less than 24 h compared to several days when using standard culturing methods. This assay provides a useful tool for routine water quality monitoring as well as for quick screening during disease outbreaks.

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

PubMed

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

2018-04-01

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

High-throughput real-time quantitative reverse transcription PCR.

PubMed

Bookout, Angie L; Cummins, Carolyn L; Mangelsdorf, David J; Pesola, Jean M; Kramer, Martha F

2006-02-01

Extensive detail on the application of the real-time quantitative polymerase chain reaction (QPCR) for the analysis of gene expression is provided in this unit. The protocols are designed for high-throughput, 384-well-format instruments, such as the Applied Biosystems 7900HT, but may be modified to suit any real-time PCR instrument. QPCR primer and probe design and validation are discussed, and three relative quantitation methods are described: the standard curve method, the efficiency-corrected DeltaCt method, and the comparative cycle time, or DeltaDeltaCt method. In addition, a method is provided for absolute quantification of RNA in unknown samples. RNA standards are subjected to RT-PCR in the same manner as the experimental samples, thus accounting for the reaction efficiencies of both procedures. This protocol describes the production and quantitation of synthetic RNA molecules for real-time and non-real-time RT-PCR applications.

Extraction of trace tilmicosin in real water samples using ionic liquid-based aqueous two-phase systems.

PubMed

Pan, Ru; Shao, Dejia; Qi, Xueyong; Wu, Yun; Fu, Wenyan; Ge, Yanru; Fu, Haizhen

2013-01-01

The effective method of ionic liquid-based aqueous two-phase extraction, which involves ionic liquid (IL) (1-butyl-3-methyllimidazolium chloride, [C4mim]Cl) and inorganic salt (K2HPO4) coupled with high-performance liquid chromatography (HPLC), has been used to extract trace tilmicosin in real water samples which were passed through a 0.45 μm filter. The effects of the different types of salts, the concentration of K2HPO4 and of ILs, the pH value and temperature of the systems on the extraction efficiencies have all been investigated. Under the optimum conditions, the average extraction efficiency is up to 95.8%. This method was feasible when applied to the analysis of tilmicosin in real water samples within the range 0.5-40 μg mL(-1). The limit of detection was found to be 0.05 μg mL(-1). The recovery rate of tilmicosin was 92.0-99.0% from the real water samples by the proposed method. This process is suggested to have important applications for the extraction of tilmicosin.

Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR.

PubMed

Kamau, Everlyn; Agoti, Charles N; Lewa, Clement S; Oketch, John; Owor, Betty E; Otieno, Grieven P; Bett, Anne; Cane, Patricia A; Nokes, D James

2017-03-01

Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Micromagnetic Cancer Cell Immobilization and Release for Real-Time Single Cell Analysis

NASA Astrophysics Data System (ADS)

Jaiswal, Devina; Rad, Armin Tahmasbi; Nieh, Mu-Ping; Claffey, Kevin P.; Hoshino, Kazunori

2017-04-01

Understanding the interaction of live cells with macromolecules is crucial for designing efficient therapies. Considering the functional heterogeneity found in cancer cells, real-time single cell analysis is necessary to characterize responses. In this study, we have designed and fabricated a microfluidic channel with patterned micromagnets which can temporarily immobilize the cells during analysis and release them after measurements. The microchannel is composed of plain coverslip top and bottom panels to facilitate easy microscopic observation and undisturbed application of analytes to the cells. Cells labeled with functionalized magnetic beads were immobilized in the device with an efficiency of 90.8±3.6%. Since the micromagnets are made of soft magnetic material (Ni), they released cells when external magnetic field was turned off from the channel. This allows the reuse of the channel for a new sample. As a model drug analysis, the immobilized breast cancer cells (MCF7) were exposed to fluorescent lipid nanoparticles and association and dissociation were measured through fluorescence analysis. Two concentrations of nanoparticles, 0.06 μg/ml and 0.08 μg/ml were tested and time lapse images were recorded and analyzed. The microfluidic device was able to provide a microenvironment for sample analysis, making it an efficient platform for real-time analysis.

Molecular detection of Toxoplasma gondii in water samples from Scotland and a comparison between the 529bp real-time PCR and ITS1 nested PCR.

PubMed

Wells, Beth; Shaw, Hannah; Innocent, Giles; Guido, Stefano; Hotchkiss, Emily; Parigi, Maria; Opsteegh, Marieke; Green, James; Gillespie, Simon; Innes, Elisabeth A; Katzer, Frank

2015-12-15

Waterborne transmission of Toxoplasma gondii is a potential public health risk and there are currently no agreed optimised methods for the recovery, processing and detection of T. gondii oocysts in water samples. In this study modified methods of T. gondii oocyst recovery and DNA extraction were applied to 1427 samples collected from 147 public water supplies throughout Scotland. T. gondii DNA was detected, using real time PCR (qPCR) targeting the 529bp repeat element, in 8.79% of interpretable samples (124 out of 1411 samples). The samples which were positive for T. gondii DNA originated from a third of the sampled water sources. The samples which were positive by qPCR and some of the negative samples were reanalysed using ITS1 nested PCR (nPCR) and results compared. The 529bp qPCR was the more sensitive technique and a full analysis of assay performance, by Bayesian analysis using a Markov Chain Monte Carlo method, was completed which demonstrated the efficacy of this method for the detection of T. gondii in water samples. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

PubMed

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

2016-06-21

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

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.

Analytical quality assurance in veterinary drug residue analysis methods: matrix effects determination and monitoring for sulfonamides analysis.

PubMed

Hoff, Rodrigo Barcellos; Rübensam, Gabriel; Jank, Louise; Barreto, Fabiano; Peralba, Maria do Carmo Ruaro; Pizzolato, Tânia Mara; Silvia Díaz-Cruz, M; Barceló, Damià

2015-01-01

In residue analysis of veterinary drugs in foodstuff, matrix effects are one of the most critical points. This work present a discuss considering approaches used to estimate, minimize and monitoring matrix effects in bioanalytical methods. Qualitative and quantitative methods for estimation of matrix effects such as post-column infusion, slopes ratios analysis, calibration curves (mathematical and statistical analysis) and control chart monitoring are discussed using real data. Matrix effects varying in a wide range depending of the analyte and the sample preparation method: pressurized liquid extraction for liver samples show matrix effects from 15.5 to 59.2% while a ultrasound-assisted extraction provide values from 21.7 to 64.3%. The matrix influence was also evaluated: for sulfamethazine analysis, losses of signal were varying from -37 to -96% for fish and eggs, respectively. Advantages and drawbacks are also discussed considering a workflow for matrix effects assessment proposed and applied to real data from sulfonamides residues analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

Web-Based Statistical Sampling and Analysis

ERIC Educational Resources Information Center

Quinn, Anne; Larson, Karen

2016-01-01

Consistent with the Common Core State Standards for Mathematics (CCSSI 2010), the authors write that they have asked students to do statistics projects with real data. To obtain real data, their students use the free Web-based app, Census at School, created by the American Statistical Association (ASA) to help promote civic awareness among school…

[Development of sample pretreatment techniques-rapid detection coupling methods for food security analysis].

PubMed

Huang, Yichun; Ding, Weiwei; Zhang, Zhuomin; Li, Gongke

2013-07-01

This paper summarizes the recent developments of the rapid detection methods for food security, such as sensors, optical techniques, portable spectral analysis, enzyme-linked immunosorbent assay, portable gas chromatograph, etc. Additionally, the applications of these rapid detection methods coupled with sample pretreatment techniques in real food security analysis are reviewed. The coupling technique has the potential to provide references to establish the selective, precise and quantitative rapid detection methods in food security analysis.

Pesticide analysis in teas and chamomile by liquid chromatography and gas chromatography tandem mass spectrometry using a modified QuEChERS method: validation and pilot survey in real samples.

PubMed

Lozano, Ana; Rajski, Łukasz; Belmonte-Valles, Noelia; Uclés, Ana; Uclés, Samanta; Mezcua, Milagros; Fernández-Alba, Amadeo R

2012-12-14

This paper presents the validation of a modified QuEChERS method in four matrices - green tea, red tea, black tea and chamomile. The experiments were carried out using blank samples spiked with a solution of 86 pesticides (insecticides, fungicides and herbicides) at four levels - 10, 25, 50 and 100 μg/kg. The samples were extracted according to the citrate QuEChERS protocol; however, to reduce the amount of coextracted matrix compounds, calcium chloride was employed instead of magnesium sulphate in the clean-up step. The samples were analysed by LC-MS/MS and GC-MS/MS. Included in the scope of validation were: recovery, linearity, matrix effects, limits of detection and quantitation as well as intra-day and inter-day precision. The validated method was used in a real sample survey carried out on 75 samples purchased in ten different countries. In all matrices, recoveries of the majority of compounds were in the 70-120% range and were characterised by precision lower than 20%. In 85% of pesticide/matrix combinations the analytes can be detected quantitatively by the proposed method at the European Union Maximum Residue Level. The analysis of the real samples revealed that large number of teas and chamomiles sold in the European Union contain pesticides whose usage is not approved and also pesticides in concentrations above the EU MRLs. Copyright © 2012 Elsevier B.V. All rights reserved.

Literature Reference for Cryptosporidium spp. (Applied and Environmental Microbiology. 2007. 73(13): 4218–4225)

EPA Pesticide Factsheets

Procedures are described for analysis of drinking water samples and may be adapted for assessment of solid, particulate, aerosol, and liquid samples. The method uses real-time PCR for identification of Cryptosporidium spp.

X-Ray Imaging Applied to Problems in Planetary Materials

NASA Technical Reports Server (NTRS)

Jurewicz, A. J. G.; Mih, D. T.; Jones, S. M.; Connolly, H.

2000-01-01

Real-time radiography (X-ray imaging) can be a useful tool for tasks such as (1) the non-destructive, preliminary examination of opaque samples and (2) optimizing how to section opaque samples for more traditional microscopy and chemical analysis.

Smart Sampling and HPC-based Probabilistic Look-ahead Contingency Analysis Implementation and its Evaluation with Real-world Data

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

Chen, Yousu; Etingov, Pavel V.; Ren, Huiying

This paper describes a probabilistic look-ahead contingency analysis application that incorporates smart sampling and high-performance computing (HPC) techniques. Smart sampling techniques are implemented to effectively represent the structure and statistical characteristics of uncertainty introduced by different sources in the power system. They can significantly reduce the data set size required for multiple look-ahead contingency analyses, and therefore reduce the time required to compute them. High-performance-computing (HPC) techniques are used to further reduce computational time. These two techniques enable a predictive capability that forecasts the impact of various uncertainties on potential transmission limit violations. The developed package has been tested withmore » real world data from the Bonneville Power Administration. Case study results are presented to demonstrate the performance of the applications developed.« less

Evaluation of micro-organism-detaching efficacy from meat samples by spindle or stomacher treatment and quality analysis of suspensions.

PubMed

Kim, S-J; Kim, D-K; Kang, D-H

2016-04-01

We investigated and compared the efficacy of a new apparatus for detaching micro-organisms from meat samples. The efficacy of Spindle and stomacher in detaching micro-organisms from meat samples was evaluated. Also, evaluation of appropriateness of suspensions generated by both methods for carrying out molecular biological analysis was implemented. A nearly identical correlation and high R(2) were obtained between Spindle and stomacher in Aerobic Plate Count (APC), and no significant differences were observed in detachment of three major foodborne pathogens. The suspension generated by the Spindle showed lower turbidity and total protein concentration. Also, significantly different threshold cycles were observed in Real-time PCR analysis using suspensions generated by both methods. The Spindle shows nearly identical efficacy with stomacher treatment in detaching micro-organisms from meat samples. Furthermore, the high quality of suspensions generated by the Spindle, in terms of turbidity and total protein assay, allows for a lower threshold cycle than stomached suspension in Real-time PCR. The Spindle could be an alternative method for detaching micro-organisms, yielding a higher quality of suspensions which may be better suited for further molecular microbiological analysis. © 2016 The Society for Applied Microbiology.

Sampling from complex networks using distributed learning automata

NASA Astrophysics Data System (ADS)

Rezvanian, Alireza; Rahmati, Mohammad; Meybodi, Mohammad Reza

2014-02-01

A complex network provides a framework for modeling many real-world phenomena in the form of a network. In general, a complex network is considered as a graph of real world phenomena such as biological networks, ecological networks, technological networks, information networks and particularly social networks. Recently, major studies are reported for the characterization of social networks due to a growing trend in analysis of online social networks as dynamic complex large-scale graphs. Due to the large scale and limited access of real networks, the network model is characterized using an appropriate part of a network by sampling approaches. In this paper, a new sampling algorithm based on distributed learning automata has been proposed for sampling from complex networks. In the proposed algorithm, a set of distributed learning automata cooperate with each other in order to take appropriate samples from the given network. To investigate the performance of the proposed algorithm, several simulation experiments are conducted on well-known complex networks. Experimental results are compared with several sampling methods in terms of different measures. The experimental results demonstrate the superiority of the proposed algorithm over the others.

Sample-based engine noise synthesis using an enhanced pitch-synchronous overlap-and-add method.

PubMed

Jagla, Jan; Maillard, Julien; Martin, Nadine

2012-11-01

An algorithm for the real time synthesis of internal combustion engine noise is presented. Through the analysis of a recorded engine noise signal of continuously varying engine speed, a dataset of sound samples is extracted allowing the real time synthesis of the noise induced by arbitrary evolutions of engine speed. The sound samples are extracted from a recording spanning the entire engine speed range. Each sample is delimitated such as to contain the sound emitted during one cycle of the engine plus the necessary overlap to ensure smooth transitions during the synthesis. The proposed approach, an extension of the PSOLA method introduced for speech processing, takes advantage of the specific periodicity of engine noise signals to locate the extraction instants of the sound samples. During the synthesis stage, the sound samples corresponding to the target engine speed evolution are concatenated with an overlap and add algorithm. It is shown that this method produces high quality audio restitution with a low computational load. It is therefore well suited for real time applications.

Proficiency program for real-time PCR diagnosis of Bordetella pertussis infections in French hospital laboratories and at the French National Reference Center for Whooping Cough and other Bordetelloses.

PubMed

Caro, Valérie; Guiso, Nicole; Alberti, Corinne; Liguori, Sandrine; Burucoa, Christophe; Couetdic, Gérard; Doucet-Populaire, Florence; Ferroni, Agnès; Papin-Gibaud, Sophie; Grattard, Florence; Réglier-Poupet, Hélène; Raymond, Josette; Soler, Catherine; Bouchet, Sylvie; Charreau, Sandrine; Couzon, Brigitte; Leymarie, Isabelle; Tavares, Nicole; Choux, Mathilde; Bingen, Edouard; Bonacorsi, Stéphane

2009-10-01

With the support of a ministerial program for innovative and expensive technologies, dedicated to the economic evaluation of laboratory diagnosis of pertussis by real-time PCR, external quality assessment for real-time IS481 PCR was carried out. Coordinated by the National Centre of Reference of Pertussis and other Bordetelloses (NCR), this study aimed to harmonize and to assess the performances of eight participating microbiology hospital laboratories throughout the French territory. Between January 2006 and February 2007, 10 proficiency panels were sent by the NCR (ascending proficiency program), representing a total of 49 samples and including eight panels to analyze and evaluate the global sensitivity and specificity of real-time PCR, one to assess the limit of detection, and one to evaluate nucleic acid extraction methods. As part of the descending proficiency program, extracted DNA from clinical samples was sent by the eight participating laboratories in different panels and analyzed by the NCR. In the ascending proficiency analysis, the sensitivity and specificity of the real-time PCR methods were 92.2% and 94.3%, respectively. The limit of detection of the different methods ranged between 0.1 and 1 fg/microl (0.2 to 2 CFU/microl). The nucleic acid extraction methods showed similar performances. During the descending proficiency analysis, performed with 126 samples, the result of the NCR for 15 samples (11.9%) was discordant with the result obtained by the source laboratory. Despite several initial differences, harmonization was easy and performances were homogeneous. However, the risk of false-positive results remains quite high, and we strongly recommend establishment of uniform quality control procedures performed regularly.

Direct Analysis in Real Time-Mass Spectrometry for the Rapid Detection of Metabolites of Aconite Alkaloids in Intestinal Bacteria

NASA Astrophysics Data System (ADS)

Li, Xue; Hou, Guangyue; Xing, Junpeng; Song, Fengrui; Liu, Zhiqiang; Liu, Shuying

2014-12-01

In the present work, direct analysis of real time ionization combined with multi-stage tandem mass spectrometry (DART-MSn) was used to investigate the metabolic profile of aconite alkaloids in rat intestinal bacteria. A total of 36 metabolites from three aconite alkaloids were identified by using DART-MSn, and the feasibility of quantitative analysis of these analytes was examined. Key parameters of the DART ion source, such as helium gas temperature and pressure, the source-to-MS distance, and the speed of the autosampler, were optimized to achieve high sensitivity, enhance reproducibility, and reduce the occurrence of fragmentation. The instrument analysis time for one sample can be less than 10 s for this method. Compared with ESI-MS and UPLC-MS, the DART-MS is more efficient for directly detecting metabolic samples, and has the advantage of being a simple, high-speed, high-throughput method.

Direct analysis in real time-mass spectrometry for the rapid detection of metabolites of aconite alkaloids in intestinal bacteria.

PubMed

Li, Xue; Hou, Guangyue; Xing, Junpeng; Song, Fengrui; Liu, Zhiqiang; Liu, Shuying

2014-12-01

In the present work, direct analysis of real time ionization combined with multi-stage tandem mass spectrometry (DART-MS(n)) was used to investigate the metabolic profile of aconite alkaloids in rat intestinal bacteria. A total of 36 metabolites from three aconite alkaloids were identified by using DART-MS(n), and the feasibility of quantitative analysis of these analytes was examined. Key parameters of the DART ion source, such as helium gas temperature and pressure, the source-to-MS distance, and the speed of the autosampler, were optimized to achieve high sensitivity, enhance reproducibility, and reduce the occurrence of fragmentation. The instrument analysis time for one sample can be less than 10 s for this method. Compared with ESI-MS and UPLC-MS, the DART-MS is more efficient for directly detecting metabolic samples, and has the advantage of being a simple, high-speed, high-throughput method.

Development of a new screening method for the detection of antibiotic residues in muscle tissues using liquid chromatography and high resolution mass spectrometry with a LC-LTQ-Orbitrap instrument.

PubMed

Hurtaud-Pessel, D; Jagadeshwar-Reddy, T; Verdon, E

2011-10-01

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed for screening meat for a wide range of antibiotics used in veterinary medicine. Full-scan mode under high resolution mass spectral conditions using an LTQ-Orbitrap mass spectrometer with resolving power 60,000 full width at half maximum (FWHM) was applied for analysis of the samples. Samples were prepared using two extraction protocols prior to LC-HRMS analysis. The scope of the method focuses on screening the following main families of antibacterial veterinary drugs: penicillins, cephalosporins, sulfonamides, macrolides, tetracyclines, aminoglucosides and quinolones. Compounds were successfully identified in spiked samples from their accurate mass and LC retention times from the acquired full-scan chromatogram. Automated data processing using ToxId software allowed rapid treatment of the data. Analyses of muscle tissues from real samples collected from antibiotic-treated animals was carried out using the above methodology and antibiotic residues were identified unambiguously. Further analysis of the data for real samples allowed the identification of the targeted antibiotic residues but also non-targeted compounds, such as some of their metabolites.

DART - LTQ ORBITRAP as an expedient tool for the identification of synthetic cannabinoids.

PubMed

Habala, Ladislav; Valentová, Jindra; Pechová, Iveta; Fuknová, Mária; Devínsky, Ferdinand

2016-05-01

Synthetic cannabinoids as designer drugs constitute a major problem due to their rapid increase in number and the difficulties connected with their identification in complex mixtures. DART (Direct Analysis in Real Time) has emerged as an advantageous tool for the direct and rapid analysis of complex samples by mass spectrometry. Here we report on the identification of six synthetic cannabinoids originating from seized material in various matrices, employing the combination of ambient pressure ion source DART and hybrid ion trap - LTQ ORBITRAP mass spectrometer. This report also describes the sampling techniques for the provided herbal material containing the cannabinoids, either directly as plant parts or as an extract in methanol and their influence on the outcome of the analysis. The high resolution mass spectra supplied by the LTQ ORBITRAP instrument allowed for an unambiguous assignment of target compounds. The utilized instrumental coupling proved to be a convenient way for the identification of synthetic cannabinoids in real-world samples. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Literature Reference for Noroviruses (Journal of Clinical Microbiology. 2004. 42(10): 4679–4685)

EPA Pesticide Factsheets

Procedures are described for analysis of clinical samples and may be adapted for of solid, particulate, aerosol, and water samples. This method is an assay for detection and quantitation of norovirus using real-time reverse transcription-PCR.

Literature Reference for Influenza H5N1 (Emerging Infectious Diseases. 2005. 11(8): 1303–1305)

EPA Pesticide Factsheets

Procedures are described for analysis of clinical samples and may be adapted for assessment of solid, particulate, aerosol, liquid and water samples. This is a two-step, real-time reverse transcriptase-PCR multiplex assay.

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.

Real-Time Analysis of Isoprene in Breath by Using Ultraviolet-Absorption Spectroscopy with a Hollow Optical Fiber Gas Cell

PubMed Central

Iwata, Takuro; Katagiri, Takashi; Matsuura, Yuji

2016-01-01

A breath analysis system based on ultraviolet-absorption spectroscopy was developed by using a hollow optical fiber as a gas cell for real-time monitoring of isoprene in breath. The hollow optical fiber functions as an ultra-small-volume gas cell with a long path. The measurement sensitivity of the system was evaluated by using nitric-oxide gas as a gas sample. The evaluation result showed that the developed system, using a laser-driven, high-intensity light source and a 3-m-long, aluminum-coated hollow optical fiber, could successfully measure nitric-oxide gas with a 50 ppb concentration. An absorption spectrum of a breath sample in the wavelength region of around 200–300 nm was measured, and the measured spectrum revealed the main absorbing components in breath as water vapor, isoprene, and ozone converted from oxygen by radiation of ultraviolet light. The concentration of isoprene in breath was estimated by multiple linear regression. The regression analysis results showed that the proposed analysis system enables real-time monitoring of isoprene during the exhaling of breath. Accordingly, it is suitable for measuring the circadian variation of isoprene. PMID:27929387

Real-Time Analysis of Isoprene in Breath by Using Ultraviolet-Absorption Spectroscopy with a Hollow Optical Fiber Gas Cell.

PubMed

Iwata, Takuro; Katagiri, Takashi; Matsuura, Yuji

2016-12-05

A breath analysis system based on ultraviolet-absorption spectroscopy was developed by using a hollow optical fiber as a gas cell for real-time monitoring of isoprene in breath. The hollow optical fiber functions as an ultra-small-volume gas cell with a long path. The measurement sensitivity of the system was evaluated by using nitric-oxide gas as a gas sample. The evaluation result showed that the developed system, using a laser-driven, high-intensity light source and a 3-m-long, aluminum-coated hollow optical fiber, could successfully measure nitric-oxide gas with a 50 ppb concentration. An absorption spectrum of a breath sample in the wavelength region of around 200-300 nm was measured, and the measured spectrum revealed the main absorbing components in breath as water vapor, isoprene, and ozone converted from oxygen by radiation of ultraviolet light. The concentration of isoprene in breath was estimated by multiple linear regression. The regression analysis results showed that the proposed analysis system enables real-time monitoring of isoprene during the exhaling of breath. Accordingly, it is suitable for measuring the circadian variation of isoprene.

Population clustering based on copy number variations detected from next generation sequencing data.

PubMed

Duan, Junbo; Zhang, Ji-Gang; Wan, Mingxi; Deng, Hong-Wen; Wang, Yu-Ping

2014-08-01

Copy number variations (CNVs) can be used as significant bio-markers and next generation sequencing (NGS) provides a high resolution detection of these CNVs. But how to extract features from CNVs and further apply them to genomic studies such as population clustering have become a big challenge. In this paper, we propose a novel method for population clustering based on CNVs from NGS. First, CNVs are extracted from each sample to form a feature matrix. Then, this feature matrix is decomposed into the source matrix and weight matrix with non-negative matrix factorization (NMF). The source matrix consists of common CNVs that are shared by all the samples from the same group, and the weight matrix indicates the corresponding level of CNVs from each sample. Therefore, using NMF of CNVs one can differentiate samples from different ethnic groups, i.e. population clustering. To validate the approach, we applied it to the analysis of both simulation data and two real data set from the 1000 Genomes Project. The results on simulation data demonstrate that the proposed method can recover the true common CNVs with high quality. The results on the first real data analysis show that the proposed method can cluster two family trio with different ancestries into two ethnic groups and the results on the second real data analysis show that the proposed method can be applied to the whole-genome with large sample size consisting of multiple groups. Both results demonstrate the potential of the proposed method for population clustering.

Simulation of mixture microstructures via particle packing models and their direct comparison with real mixtures

NASA Astrophysics Data System (ADS)

Gulliver, Eric A.

The objective of this thesis to identify and develop techniques providing direct comparison between simulated and real packed particle mixture microstructures containing submicron-sized particles. This entailed devising techniques for simulating powder mixtures, producing real mixtures with known powder characteristics, sectioning real mixtures, interrogating mixture cross-sections, evaluating and quantifying the mixture interrogation process and for comparing interrogation results between mixtures. A drop and roll-type particle-packing model was used to generate simulations of random mixtures. The simulated mixtures were then evaluated to establish that they were not segregated and free from gross defects. A powder processing protocol was established to provide real mixtures for direct comparison and for use in evaluating the simulation. The powder processing protocol was designed to minimize differences between measured particle size distributions and the particle size distributions in the mixture. A sectioning technique was developed that was capable of producing distortion free cross-sections of fine scale particulate mixtures. Tessellation analysis was used to interrogate mixture cross sections and statistical quality control charts were used to evaluate different types of tessellation analysis and to establish the importance of differences between simulated and real mixtures. The particle-packing program generated crescent shaped pores below large particles but realistic looking mixture microstructures otherwise. Focused ion beam milling was the only technique capable of sectioning particle compacts in a manner suitable for stereological analysis. Johnson-Mehl and Voronoi tessellation of the same cross-sections produced tessellation tiles with different the-area populations. Control charts analysis showed Johnson-Mehl tessellation measurements are superior to Voronoi tessellation measurements for detecting variations in mixture microstructure, such as altered particle-size distributions or mixture composition. Control charts based on tessellation measurements were used for direct, quantitative comparisons between real and simulated mixtures. Four sets of simulated and real mixtures were examined. Data from real mixture was matched with simulated data when the samples were well mixed and the particle size distributions and volume fractions of the components were identical. Analysis of mixture components that occupied less than approximately 10 vol% of the mixture was not practical unless the particle size of the component was extremely small and excellent quality high-resolution compositional micrographs of the real sample are available. These methods of analysis should allow future researchers to systematically evaluate and predict the impact and importance of variables such as component volume fraction and component particle size distribution as they pertain to the uniformity of powder mixture microstructures.

Algorithm-based arterial blood sampling recognition increasing safety in point-of-care diagnostics.

PubMed

Peter, Jörg; Klingert, Wilfried; Klingert, Kathrin; Thiel, Karolin; Wulff, Daniel; Königsrainer, Alfred; Rosenstiel, Wolfgang; Schenk, Martin

2017-08-04

To detect blood withdrawal for patients with arterial blood pressure monitoring to increase patient safety and provide better sample dating. Blood pressure information obtained from a patient monitor was fed as a real-time data stream to an experimental medical framework. This framework was connected to an analytical application which observes changes in systolic, diastolic and mean pressure to determine anomalies in the continuous data stream. Detection was based on an increased mean blood pressure caused by the closing of the withdrawal three-way tap and an absence of systolic and diastolic measurements during this manipulation. For evaluation of the proposed algorithm, measured data from animal studies in healthy pigs were used. Using this novel approach for processing real-time measurement data of arterial pressure monitoring, the exact time of blood withdrawal could be successfully detected retrospectively and in real-time. The algorithm was able to detect 422 of 434 (97%) blood withdrawals for blood gas analysis in the retrospective analysis of 7 study trials. Additionally, 64 sampling events for other procedures like laboratory and activated clotting time analyses were detected. The proposed algorithm achieved a sensitivity of 0.97, a precision of 0.96 and an F1 score of 0.97. Arterial blood pressure monitoring data can be used to perform an accurate identification of individual blood samplings in order to reduce sample mix-ups and thereby increase patient safety.

In-line real time air monitor

DOEpatents

Wise, Marcus B.; Thompson, Cyril V.

1998-01-01

An in-line gas monitor capable of accurate gas composition analysis in a continuous real time manner even under strong applied vacuum conditions operates by mixing an air sample with helium forming a sample gas in two complementary sample loops embedded in a manifold which includes two pairs of 3-way solenoid valves. The sample gas is then analyzed in an ion trap mass spectrometer on a continuous basis. Two valve drivers actuate the two pairs of 3-way valves in a reciprocating fashion, so that there is always flow through the in-line gas monitor via one or the other of the sample loops. The duty cycle for the two pairs of 3-way valves is varied by tuning the two valve drivers to a duty cycle typically between 0.2 to 0.7 seconds.

Confronting Analytical Dilemmas for Understanding Complex Human Interactions in Design-Based Research from a Cultural-Historical Activity Theory (CHAT) Framework

ERIC Educational Resources Information Center

Yamagata-Lynch, Lisa C.

2007-01-01

Understanding human activity in real-world situations often involves complicated data collection, analysis, and presentation methods. This article discusses how Cultural-Historical Activity Theory (CHAT) can inform design-based research practices that focus on understanding activity in real-world situations. I provide a sample data set with…

Evaluation of performance across the dynamic range of the Abbott RealTime HIV-1 assay as compared to VERSANT HIV-1 RNA 3.0 and AMPLICOR HIV-1 MONITOR v1.5 using serial dilutions of 39 group M and O viruses.

PubMed

Swanson, Priscilla; Huang, Shihai; Abravaya, Klara; de Mendoza, Carmen; Soriano, Vincent; Devare, Sushil G; Hackett, John

2007-04-01

Performance of the Abbott m2000 instrument system and the Abbott RealTime HIV-1 assay was evaluated using a panel of 37 group M (subtypes A-D, F, G, CRF01_AE, CRF02_AG and unique recombinant forms) and 2 group O virus isolates. Testing was performed on 273 sample dilutions and compared to VERSANT HIV-1 RNA 3.0 (bDNA) and AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5) test results. RealTime HIV-1, bDNA, and Monitor v1.5 tests quantified 87%, 78%, and 81% of samples, respectively. RealTime HIV-1 detected an additional 31 samples at < 40 copies/mL. For group M, RealTime HIV-1 dilution profiles and viral loads were highly correlated with bDNA and Monitor v1.5 values; 87% and 89% of values were within 0.5 log(10) copies/mL. In contrast, the group O viruses were not detected by Monitor v1.5 and were substantially underquantified by approximately 2 log(10) copies/mL in bDNA relative to the RealTime HIV-1 assay. Sequence analysis revealed that RealTime HIV-1 primer/probe binding sites are highly conserved and exhibit fewer nucleotide mismatches relative to Monitor v1.5. The automated m2000 system and RealTime HIV-1 assay offer the advantages of efficient sample processing and throughput with reduced "hands-on" time while providing improved sensitivity, expanded dynamic range and reliable quantification of genetically diverse HIV-1 strains.

Literature Reference for Toxoplasma gondii (Applied and Environmental Microbiology. 2004. 70(7): 4035–4039)

EPA Pesticide Factsheets

Procedures are described for analysis of water samples and may be adapted for assessment of solid, particulate and liquid samples. The method uses real-time PCR assay for detecting Toxoplasma gondii DNA using gene-specific primers and probe.

Identification of feline polycystic kidney disease mutation using fret probes and melting curve analysis.

PubMed

Criado-Fornelio, A; Buling, A; Barba-Carretero, J C

2009-02-01

We developed and validated a real-time polymerase chain reaction (PCR) assay using fluorescent hybridization probes and melting curve analysis to identify the PKD1 exon 29 (C-->A) mutation, which is implicated in polycystic kidney disease of cats. DNA was isolated from peripheral blood of 20 Persian cats. The employ of the new real-time PCR and melting curve analysis in these samples indicated that 13 cats (65%) were wild type homozygotes and seven cats (35%) were heterozygotes. Both PCR-RFLP and sequencing procedures were in full agreement with real-time PCR test results. Sequence analysis showed that the mutant gene had the expected base change compared to the wild type gene. The new procedure is not only very reliable but also faster than the techniques currently applied for diagnosis of the mutation.

Robust gene selection methods using weighting schemes for microarray data analysis.

PubMed

Kang, Suyeon; Song, Jongwoo

2017-09-02

A common task in microarray data analysis is to identify informative genes that are differentially expressed between two different states. Owing to the high-dimensional nature of microarray data, identification of significant genes has been essential in analyzing the data. However, the performances of many gene selection techniques are highly dependent on the experimental conditions, such as the presence of measurement error or a limited number of sample replicates. We have proposed new filter-based gene selection techniques, by applying a simple modification to significance analysis of microarrays (SAM). To prove the effectiveness of the proposed method, we considered a series of synthetic datasets with different noise levels and sample sizes along with two real datasets. The following findings were made. First, our proposed methods outperform conventional methods for all simulation set-ups. In particular, our methods are much better when the given data are noisy and sample size is small. They showed relatively robust performance regardless of noise level and sample size, whereas the performance of SAM became significantly worse as the noise level became high or sample size decreased. When sufficient sample replicates were available, SAM and our methods showed similar performance. Finally, our proposed methods are competitive with traditional methods in classification tasks for microarrays. The results of simulation study and real data analysis have demonstrated that our proposed methods are effective for detecting significant genes and classification tasks, especially when the given data are noisy or have few sample replicates. By employing weighting schemes, we can obtain robust and reliable results for microarray data analysis.

Microfluidic-Based Robotic Sampling System for Radioactive Solutions

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

Jack D. Law; Julia L. Tripp; Tara E. Smith

A novel microfluidic based robotic sampling system has been developed for sampling and analysis of liquid solutions in nuclear processes. This system couples the use of a microfluidic sample chip with a robotic system designed to allow remote, automated sampling of process solutions in-cell and facilitates direct coupling of the microfluidic sample chip with analytical instrumentation. This system provides the capability for near real time analysis, reduces analytical waste, and minimizes the potential for personnel exposure associated with traditional sampling methods. A prototype sampling system was designed, built and tested. System testing demonstrated operability of the microfluidic based sample systemmore » and identified system modifications to optimize performance.« less

Real-Time Continuous Identification of Greenhouse Plant Pathogens Based on Recyclable Microfluidic Bioassay System.

PubMed

Qu, Xiangmeng; Li, Min; Zhang, Hongbo; Lin, Chenglie; Wang, Fei; Xiao, Mingshu; Zhou, Yi; Shi, Jiye; Aldalbahi, Ali; Pei, Hao; Chen, Hong; Li, Li

2017-09-20

The development of a real-time continuous analytical platform for the pathogen detection is of great scientific importance for achieving better disease control and prevention. In this work, we report a rapid and recyclable microfluidic bioassay system constructed from oligonucleotide arrays for selective and sensitive continuous identification of DNA targets of fungal pathogens. We employ the thermal denaturation method to effectively regenerate the oligonucleotide arrays for multiple sample detection, which could considerably reduce the screening effort and costs. The combination of thermal denaturation and laser-induced fluorescence detection technique enables real-time continuous identification of multiple samples (<10 min per sample). As a proof of concept, we have demonstrated that two DNA targets of fungal pathogens (Botrytis cinerea and Didymella bryoniae) can be sequentially analyzed using our rapid microfluidic bioassay system, which provides a new paradigm in the design of microfluidic bioassay system and will be valuable for chemical and biomedical analysis.

Rapidly differentiating grape seeds from different sources based on characteristic fingerprints using direct analysis in real time coupled with time-of-flight mass spectrometry combined with chemometrics.

PubMed

Song, Yuqiao; Liao, Jie; Dong, Junxing; Chen, Li

2015-09-01

The seeds of grapevine (Vitis vinifera) are a byproduct of wine production. To examine the potential value of grape seeds, grape seeds from seven sources were subjected to fingerprinting using direct analysis in real time coupled with time-of-flight mass spectrometry combined with chemometrics. Firstly, we listed all reported components (56 components) from grape seeds and calculated the precise m/z values of the deprotonated ions [M-H](-) . Secondly, the experimental conditions were systematically optimized based on the peak areas of total ion chromatograms of the samples. Thirdly, the seven grape seed samples were examined using the optimized method. Information about 20 grape seed components was utilized to represent characteristic fingerprints. Finally, hierarchical clustering analysis and principal component analysis were performed to analyze the data. Grape seeds from seven different sources were classified into two clusters; hierarchical clustering analysis and principal component analysis yielded similar results. The results of this study lay the foundation for appropriate utilization and exploitation of grape seed samples. Due to the absence of complicated sample preparation methods and chromatographic separation, the method developed in this study represents one of the simplest and least time-consuming methods for grape seed fingerprinting. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Standoff spectroscopic interrogation of samples irradiated by high energy lasers

NASA Astrophysics Data System (ADS)

Daigle, Jean-François; Pudo, Dominik; Théberge, Francis

2017-10-01

We report on a novel method that shows the potential to provide real-time, standoff forensic analysis of samples being irradiated by a high energy laser (HEL). The interaction of the HEL beam with matter produces specific optical signatures that can be detected from the location of the HEL system. A spectroscopic analysis of these signals can then provide useful information to the operator including the impact the laser has on the sample as well as providing data about the its structure and composition.

Petroleomics by Direct Analysis in Real Time-Mass Spectrometry.

PubMed

Romão, Wanderson; Tose, Lilian V; Vaz, Boniek G; Sama, Sara G; Lobinski, Ryszard; Giusti, Pierre; Carrier, Hervé; Bouyssiere, Brice

2016-01-01

The analysis of crude oil and its fractions by applying ambient ionization techniques remains underexplored in mass spectrometry (MS). Direct analysis in real time (DART) in the positive-ion mode was coupled to a linear quadrupole ion trap Orbitrap mass spectrometer (LTQ Orbitrap) to analyze crude oil, paraffin samples, and porphyrin standard compounds. The ionization parameters of DART-MS were optimized for crude oil analysis. DART-MS rendered the optimum conditions of the operation using paper as the substrate, T = 400°C, helium as the carrier gas, and a sample concentration ≥6 mg mL(-1). In the crude oils analysis, the DART(+)-Orbitrap mass spectra detected the typical N, NO, and O-containing compounds. In the paraffin samples, oxidized hydrocarbon species (Ox classes, where x = 1-4) with double-bond equivalent of 1-4 were detected, and their structures and connectivity were confirmed by collision-induced dissociation (CID) experiments. DART(+)-MS has identified the porphyrin standard compounds as [M + H](+) ions of m/z 615.2502 and 680.1763, where M = C44H30N4 and C44H28N4OV, respectively, based on the formula assignment and by phenyl losses observed on CID experiments.

"Soft"or "hard" ionisation? Investigation of metastable gas temperature effect on direct analysis in real-time analysis of Voriconazole.

PubMed

Lapthorn, Cris; Pullen, Frank

2009-01-01

The performance of the direct analysis in real-time (DART) technique was evaluated across a range of metastable gas temperatures for a pharmaceutical compound, Voriconazole, in order to investigate the effect of metastable gas temperature on molecular ion intensity and fragmentation. The DART source has been used to analyse a range of analytes and from a range of matrices including drugs in solid tablet form and preparations, active ingredients in ointment, naturally occurring plant alkaloids, flavours and fragrances, from thin layer chromatography (TLC) plates, melting point tubes and biological matrices including hair, urine and blood. The advantages of this technique include rapid analysis time (as little as 5 s), a reduction in sample preparation requirements, elimination of mobile phase requirement and analysis of samples not typically amenable to atmospheric pressure ionisation (API) techniques. This technology has therefore been proposed as an everyday tool for identification of components in crude organic reaction mixtures.

Feasibility of real-time geochemical analysis using LIBS (Laser-Induced Breakdown Spectroscopy) in oil wells

NASA Astrophysics Data System (ADS)

Shahin, Mohamed

2014-05-01

The oil and gas industry has attempted for many years to find new ways to analyze and determine the type of rocks drilled on a real time basis. Mud analysis logging is a direct method of detecting oil and gas in formations drilled, it depends on the "feel" of the bit to decide formation type, as well as, geochemical analysis which was introduced 30 years ago, starting with a pulsed-neutron generator (PNG) based wireline tool upon which LWD technology was based. In this paper, we are studying the feasibility of introducing a new technology for real-time geochemical analysis. Laser-induced breakdown spectroscopy (LIBS) is a type of atomic emission spectroscopy, It is a cutting-edge technology that is used for many applications such as determination of alloy composition, origin of manufacture (by monitoring trace components), and molecular analysis (unknown identification). LIBS can analyze any material regardless of its state (solid, liquid or gas), based upon that fact, we can analyze rocks, formation fluids' types and contacts between them. In cooperation with the National Institute of Laser Enhanced Science, Cairo University in Egypt, we've done tests on sandstone, limestone and coal samples acquired from different places using Nd: YAG Laser with in addition to other components that are explained in details through this paper to understand the ability of Laser to analyze rock samples and provide their elemental composition using LIBS technique. We've got promising results from the sample analysis via LIBS and discussed the possibility of deploying this technology in oilfields suggesting many applications and giving a base for achieving a quantitative elemental analysis method in view of its shortcomings and solutions.

Apparatus for the field determination of concentration of radioactive constituents in a medium

DOEpatents

Perkins, R.W.; Schilk, A.J.; Warner, R.A.; Wogman, N.A.

1995-08-15

The instant invention is an apparatus for determining the concentration of radioactive constituents in a test sample; such as surface soils, via rapid real-time analyses, and direct readout on location utilizing a probe made up of multiple layers of detection material used in combination with an analyzer and real-time readout unit. This is accomplished by comparing the signal received from the probe, which can discriminate between types of radiation and energies with stored patterns that are based upon experimental results. This comparison can be used in the calibration of a readout display that reads out in real-time the concentrations of constituents per given volume. For example, the concentration of constituents such as Cs-137, Sr-90, U-238 in the soil, and noble gas radionuclides such as Kr-85 in the atmosphere, can be measured in real-time, on location, without the need for laboratory analysis of samples. 14 figs.

A Dynamic Time Warping Approach to Real-Time Activity Recognition for Food Preparation

NASA Astrophysics Data System (ADS)

Pham, Cuong; Plötz, Thomas; Olivier, Patrick

We present a dynamic time warping based activity recognition system for the analysis of low-level food preparation activities. Accelerometers embedded into kitchen utensils provide continuous sensor data streams while people are using them for cooking. The recognition framework analyzes frames of contiguous sensor readings in real-time with low latency. It thereby adapts to the idiosyncrasies of utensil use by automatically maintaining a template database. We demonstrate the effectiveness of the classification approach by a number of real-world practical experiments on a publically available dataset. The adaptive system shows superior performance compared to a static recognizer. Furthermore, we demonstrate the generalization capabilities of the system by gradually reducing the amount of training samples. The system achieves excellent classification results even if only a small number of training samples is available, which is especially relevant for real-world scenarios.

Apparatus for the field determination of concentration of radioactive constituents in a medium

DOEpatents

Perkins, Richard W.; Schilk, Alan J.; Warner, Ray A.; Wogman, Ned A.

1995-01-01

The instant invention is an apparatus for determining the concentration of radioactive constituents in a test sample; such as surface soils, via rapid real-time analyses, and direct readout on location utilizing a probe made up of multiple layers of detection material used in combination with an analyzer and real-time readout unit. This is accomplished by comparing the signal received from the probe, which can discriminate between types of radiation and energies with stored patterns that are based upon experimental results. This comparison can be used in the calibration of a readout display that reads out in real-time the concentrations of constituents per given volume. For example, the concentration of constituents such as Cs-137, Sr-90, U-238 in the soil, and noble gas radionuclides such as Kr-85 in the atmosphere, can be measured in real-time, on location, without the need for laboratory analysis of samples.

Comparative Evaluation of Three Nucleic Acid-Based Assays for BK Virus Quantification

PubMed Central

Descamps, Veronique; Martin, Elodie; Morel, Virginie; François, Catherine; Helle, François; Duverlie, Gilles; Castelain, Sandrine

2015-01-01

With the growing importance of BK virus (BKV), effective and efficient screening for BKV replication in plasma and urine samples is very important for monitoring renal transplant and hematopoietic stem cell transplant recipients, who are at increased risk of BKV-associated diseases. However, recent assays proposed by many manufacturers have not been tested, and the available tests have not been standardized. The aim of the present study was to evaluate and compare the performances of three commercially available kits, R-gene, GeneProof, and RealStar, on plasma and urine specimens from patients infected with various genotypes and to determine the correlations with the results from a reference laboratory. A qualitatively excellent global agreement (96.8%) was obtained. RealStar PCR tended to give a higher sensitivity, especially for subtype Ib1 samples. Comparison of 30 plasma samples and 53 urine samples showed a good agreement between the three assays, with Spearman's Rho correlation coefficient values falling between 0.92 and 0.98 (P < 0.001). Moreover, a perfect correlation was obtained for comparison of the assay performances with the AcroMetrix BKV panel (P < 0.001 for all comparisons). According to Bland-Altman analysis, more than 95% (240/249 comparisons) of sample comparisons were situated in the range of the mean ± 2 standard deviations (SD). The greatest variability between assays was observed for 10.2% of subtype Ib2 samples, with differences of >1 log10 copies/ml. In conclusion, this study demonstrated the reliable and comparable performances of the R-gene, GeneProof, and RealStar real-time PCR systems for quantification of BKV in urine and plasma samples. All three real-time PCR assays are appropriate for screening of BKV replication in patients. PMID:26424842

SAVLOC, computer program for automatic control and analysis of X-ray fluorescence experiments

NASA Technical Reports Server (NTRS)

Leonard, R. F.

1977-01-01

A program for a PDP-15 computer is presented which provides for control and analysis of trace element determinations by using X-ray fluorescence. The program simultaneously handles data accumulation for one sample and analysis of data from previous samples. Data accumulation consists of sample changing, timing, and data storage. Analysis requires the locating of peaks in X-ray spectra, determination of intensities of peaks, identification of origins of peaks, and determination of a real density of the element responsible for each peak. The program may be run in either a manual (supervised) mode or an automatic (unsupervised) mode.

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

PubMed

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

2017-08-01

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

Estimation of real-time N load in surface water using dynamic data driven application system

Treesearch

Y. Ouyang; S.M. Luo; L.H. Cui; Q. Wang; J.E. Zhang

2011-01-01

Agricultural, industrial, and urban activities are the major sources for eutrophication of surface water ecosystems. Currently, determination of nutrients in surface water is primarily accomplished by manually collecting samples for laboratory analysis, which requires at least 24 h. In other words, little to no effort has been devoted to monitoring real-time variations...

Evaluation of an offline method for the analysis of atmospheric reactive gaseous mercury and particulate mercury

USGS Publications Warehouse

Rutter, A.P.; Hanford, K.L.; Zwers, J.T.; Perillo-Nicholas, A. L.; Schauer, J.J.; Olson, M.L.

2008-01-01

Reactive gaseous mercury (RGM) and particulate mercury (PHg) were collected in Milwaukee, WI, between April 2004 and May 2005, and in Riverside, CA, between July 25 and August 7, 2005 using sorbent and filter substrates. The substrates were analyzed for mercury by thermal desorption analysis (TDA) using a purpose-built instrument. Results from this offline-TDA method were compared with measurements using a real-time atmospheric mercury analyzer. RGM measurements made with the offline-TDA agreed well with a commercial real-time method. However, the offline TDA reported PHg concentrations 2.7 times higher than the real-time method, indicating evaporative losses might be occurring from the real-time instrument during sample collection. TDA combined with reactive mercury collection on filter and absorbent substrates was cheap, relatively easy to use, did not introduce biases due to a semicontinuous sample collection strategy, and had a dynamic range appropriate for use in rural and urban locations. The results of this study demonstrate that offline-TDA is a feasible method for collecting reactive mercury concentrations in a large network of filter-based samplers. Copyright 2008 Air & Waste Management Association.

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.

Rapid analysis of caffeine in various coffee samples employing direct analysis in real-time ionization-high-resolution mass spectrometry.

PubMed

Danhelova, Hana; Hradecky, Jaromir; Prinosilova, Sarka; Cajka, Tomas; Riddellova, Katerina; Vaclavik, Lukas; Hajslova, Jana

2012-07-01

The development and use of a fast method employing a direct analysis in real time (DART) ion source coupled to high-resolution time-of-flight mass spectrometry (TOFMS) for the quantitative analysis of caffeine in various coffee samples has been demonstrated in this study. A simple sample extraction procedure employing hot water was followed by direct, high-throughput (<1 min per run) examination of the extracts spread on a glass rod under optimized conditions of ambient mass spectrometry, without any prior chromatographic separation. For quantification of caffeine using DART-TOFMS, an external calibration was used. Isotopically labeled caffeine was used to compensate for the variations of the ion intensities of caffeine signal. Recoveries of the DART-TOFMS method were 97% for instant coffee at the spiking levels of 20 and 60 mg/g, respectively, while for roasted ground coffee, the obtained values were 106% and 107% at the spiking levels of 10 and 30 mg/g, respectively. The repeatability of the whole analytical procedure (expressed as relative standard deviation, RSD, %) was <5% for all tested spiking levels and matrices. Since the linearity range of the method was relatively narrow (two orders of magnitude), an optimization of sample dilution prior the DART-TOFMS measurement to avoid saturation of the detector was needed.

In-line real time air monitor

DOEpatents

Wise, M.B.; Thompson, C.V.

1998-07-14

An in-line gas monitor capable of accurate gas composition analysis in a continuous real time manner even under strong applied vacuum conditions operates by mixing an air sample with helium forming a sample gas in two complementary sample loops embedded in a manifold which includes two pairs of 3-way solenoid valves. The sample gas is then analyzed in an ion trap mass spectrometer on a continuous basis. Two valve drivers actuate the two pairs of 3-way valves in a reciprocating fashion, so that there is always flow through the in-line gas monitor via one or the other of the sample loops. The duty cycle for the two pairs of 3-way valves is varied by tuning the two valve drivers to a duty cycle typically between 0.2 to 0.7 seconds. 3 figs.

A real-time polymerase chain reaction-based protocol for low/medium-throughput Y-chromosome microdeletions analysis.

PubMed

Segat, Ludovica; Padovan, Lara; Doc, Darja; Petix, Vincenzo; Morgutti, Marcello; Crovella, Sergio; Ricci, Giuseppe

2012-12-01

We describe a real-time polymerase chain reaction (PCR) protocol based on the fluorescent molecule SYBR Green chemistry, for a low- to medium-throughput analysis of Y-chromosome microdeletions, optimized according to the European guidelines and aimed at making the protocol faster, avoiding post-PCR processing, and simplifying the results interpretation. We screened 156 men from the Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Institute for Maternal and Child Health IRCCS Burlo Garofolo (Trieste, Italy), 150 not presenting Y-chromosome microdeletion, and 6 with microdeletions in different azoospermic factor (AZF) regions. For each sample, the Zinc finger Y-chromosomal protein (ZFY), sex-determining region Y (SRY), sY84, sY86, sY127, sY134, sY254, and sY255 loci were analyzed by performing one reaction for each locus. AZF microdeletions were successfully detected in six individuals, confirming the results obtained with commercial kits. Our real-time PCR protocol proved to be a rapid, safe, and relatively cheap method that was suitable for a low- to medium-throughput diagnosis of Y-chromosome microdeletion, which allows an analysis of approximately 10 samples (with the addition of positive and negative controls) in a 96-well plate format, or approximately 46 samples in a 384-well plate for all markers simultaneously, in less than 2 h without the need of post-PCR manipulation.

Laboratory-based ROTEM(®) analysis: implementing pneumatic tube transport and real-time graphic transmission.

PubMed

Colucci, G; Giabbani, E; Barizzi, G; Urwyler, N; Alberio, L

2011-08-01

ROTEM(®) is considered a helpful point-of-care device to monitor blood coagulation. Centrally performed analysis is desirable but rapid transport of blood samples and real-time transmission of graphic results are an important prerequisite. The effect of sample transport through a pneumatic tube system on ROTEM(®) results is unknown. The aims of the present work were (i) to determine the influence of blood sample transport through a pneumatic tube system on ROTEM(®) parameters compared to manual transportation, and (ii) to verify whether graphic results can be transmitted on line via virtual network computing using local area network to the physician in charge of the patient. Single centre study with 30 normal volunteers. Two whole blood samples were transferred to the central haematology laboratory by either normal transport or pneumatic delivery. EXTEM, INTEM, FIBTEM and APTEM were analysed in parallel with two ROTEM(®) devices and compared. Connection between central laboratory, emergency and operating rooms was established using local area network. All collected ROTEM(®) parameters were within normal limits. No statistically significant differences between normal transport and pneumatic delivery were observed. Real-time transmission of the original ROTEM(®) curves using local area network is feasible and easy to establish. At our institution, transport of blood samples by pneumatic delivery does not influence ROTEM(®) parameters. Blood samples can be analysed centrally, and results transmitted live via virtual network computing to emergency or operating rooms. Prior to analyse blood samples centrally, the type of sample transport should be tested to exclude in vitro blood activation by local pneumatic transport system. © 2011 Blackwell Publishing Ltd.

Real-time oil-saturation monitoring in rock cores with low-field NMR.

PubMed

Mitchell, J; Howe, A M; Clarke, A

2015-07-01

Nuclear magnetic resonance (NMR) provides a powerful suite of tools for studying oil in reservoir core plugs at the laboratory scale. Low-field magnets are preferred for well-log calibration and to minimize magnetic-susceptibility-induced internal gradients in the porous medium. We demonstrate that careful data processing, combined with prior knowledge of the sample properties, enables real-time acquisition and interpretation of saturation state (relative amount of oil and water in the pores of a rock). Robust discrimination of oil and brine is achieved with diffusion weighting. We use this real-time analysis to monitor the forced displacement of oil from porous materials (sintered glass beads and sandstones) and to generate capillary desaturation curves. The real-time output enables in situ modification of the flood protocol and accurate control of the saturation state prior to the acquisition of standard NMR core analysis data, such as diffusion-relaxation correlations. Although applications to oil recovery and core analysis are demonstrated, the implementation highlights the general practicality of low-field NMR as an inline sensor for real-time industrial process control. Copyright © 2015 Elsevier Inc. All rights reserved.

Literature Reference for Entamoeba histolytica (Journal of Clinical Microbiology. 2005. 43(11): 5491–5497)

EPA Pesticide Factsheets

Procedures are described for analysis of clinical samples and may be adapted for assessment of solid, particulate, liquid and water samples. The method is a real-time PCR assay that targets the 18S rRNA gene sequence of Entamoeba histolytica.

Quantitation of mycotoxins using direct analysis in real time (DART)-mass spectrometry (MS)

USDA-ARS?s Scientific Manuscript database

Ambient ionization represents a new generation of mass spectrometry ion sources which is used for rapid ionization of small molecules under ambient conditions. The combination of ambient ionization and mass spectrometry allows analyzing multiple food samples with simple or no sample treatment, or in...

Analysis of selected antibiotics in surface freshwater and seawater using direct injection in liquid chromatography electrospray ionization tandem mass spectrometry.

PubMed

Bayen, Stéphane; Yi, Xinzhu; Segovia, Elvagris; Zhou, Zhi; Kelly, Barry C

2014-04-18

Emerging contaminants such as antibiotics have received recent attention as they have been detected in natural waters and health concerns over potential antibiotic resistance. With the purpose to investigate fast and high-throughput analysis, and eventually the continuous on-line analysis of emerging contaminants, this study presents results on the analysis of seven selected antibiotics (sulfadiazine, sulfamethazine, sulfamerazine, sulfamethoxazole, chloramphenicol, lincomycin, tylosin) in surface freshwater and seawater using direct injection of a small sample volume (20μL) in liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Notably, direct injection of seawater in the LC-ESI-MS/MS was made possible on account of the post-column switch on the system, which allows diversion of salt-containing solutions flushed out of the column to the waste. Mean recoveries based on the isotope dilution method average 95±14% and 96±28% amongst the compounds for spiked freshwater and seawater, respectively. Linearity across six spiking levels was assessed and the response was linear (r(2)>0.99) for all compounds. Direct injection concentrations were compared for real samples to those obtained with the conventional SPE-based analysis and both techniques concurs on the presence/absence and levels of the compounds in real samples. These results suggest direct injection is a reliable method to detect antibiotics in both freshwater and seawater. Method detection limits for the direct injection technique (37pg/L to 226ng/L in freshwater, and from 16pg/to 26ng/L in seawater) are sufficient for a number of environmental applications, for example the fast screening of water samples for ecological risk assessments. In the present study of real samples, this new method allowed for example the positive detection of some compounds (e.g. lincomycin) down to the sub ng/L range. The direct injection method appears to be relatively cheaper and faster, requires a smaller sample size, and is more robust to equipment cross-contamination as compared to the conventional SPE-based method. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantification of Campylobacter spp. in pig feces by direct real-time PCR with an internal control of extraction and amplification.

PubMed

Leblanc-Maridor, Mily; Garénaux, Amélie; Beaudeau, François; Chidaine, Bérangère; Seegers, Henri; Denis, Martine; Belloc, Catherine

2011-04-01

The rapid and direct quantification of Campylobacter spp. in complex substrates like feces or environmental samples is crucial to facilitate epidemiological studies on Campylobacter in pig production systems. We developed a real-time PCR assay for detecting and quantifying Campylobacter spp. directly in pig feces with the use of an internal control. Campylobacter spp. and Yersinia ruckeri primers-probes sets were designed and checked for specificity with diverse Campylobacter, related organisms, and other bacterial pathogens before being used in field samples. The quantification of Campylobacter spp. by the real-time PCR then was realized on 531 fecal samples obtained from experimentally and naturally infected pigs; the numeration of Campylobacter on Karmali plate was done in parallel. Yersinia ruckeri, used as bacterial internal control, was added to the samples before DNA extraction to control DNA-extraction and PCR-amplification. The sensitivity of the PCR assay was 10 genome copies. The established Campylobacter real-time PCR assay showed a 7-log-wide linear dynamic range of quantification (R²=0.99) with a detection limit of 200 Colony Forming Units of Campylobacter per gram of feces. A high correlation was found between the results obtained by real-time PCR and those by culture at both qualitative and quantitative levels. Moreover, DNA extraction followed by real-time PCR reduced the time needed for analysis to a few hours (within a working day). In conclusion, the real-time PCR developed in this study provides new tools for further epidemiological surveys to investigate the carriage and excretion of Campylobacter by pigs. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of a novel ultrasound-assisted headspace liquid-phase microextraction and its application to the analysis of chlorophenols in real aqueous samples.

PubMed

Xu, Hui; Liao, Ying; Yao, Jinrong

2007-10-05

A new sample pretreatment technique, ultrasound-assisted headspace liquid-phase microextraction was developed as mentioned in this paper. In the technique, the volatile analytes were headspace extracted into a small drop of solvent, which suspended on the bottom of a cone-shaped PCR tube instead of the needle tip of a microsyringe. More solvent could be suspended in the PCR tube than microsyringe due to the larger interfacial tension, thus the analysis sensitivity was significantly improved with the increase of the extractant volume. Moreover, ultrasound-assisted extraction and independent controlling temperature of the extractant and the sample were performed to enhance the extraction efficiency. Following the extraction, the solvent-loaded sample was analyzed by high-performance liquid chromatography. Chlorophenols (2-chlorophenol, 2,4-dichlorophenol and 2,6-dichlorophenol) were chosen as model analytes to investigate the feasibility of the method. The experimental conditions related to the extraction efficiency were systematically studied. Under the optimum experimental conditions, the detection limit (S/N=3), intra- and inter-day RSD were 6 ng mL(-1), 4.6%, 3.9% for 2-chlorophenol, 12 ng mL(-1), 2.4%, 8.8% for 2,4-dichlorophenol and 23 ng mL(-1), 3.3%, 5.3% for 2,6-dichlorophenol, respectively. The proposed method was successfully applied to determine chlorophenols in real aqueous samples. Good recoveries ranging from 84.6% to 100.7% were obtained. In addition, the extraction efficiency of our method and the conventional headspace liquid-phase microextraction were compared; the extraction efficiency of the former was about 21 times higher than that of the latter. The results demonstrated that the proposed method is a promising sample pretreatment approach, its advantages over the conventional headspace liquid-phase microextraction include simple setup, ease of operation, rapidness, sensitivity, precision and no cross-contamination. The method is very suitable for the analysis of trace volatile and semivolatile pollutants in real aqueous sample.

Continuous flow real-time PCR device using multi-channel fluorescence excitation and detection.

PubMed

Hatch, Andrew C; Ray, Tathagata; Lintecum, Kelly; Youngbull, Cody

2014-02-07

High throughput automation is greatly enhanced using techniques that employ conveyor belt strategies with un-interrupted streams of flow. We have developed a 'conveyor belt' analog for high throughput real-time quantitative Polymerase Chain Reaction (qPCR) using droplet emulsion technology. We developed a low power, portable device that employs LED and fiber optic fluorescence excitation in conjunction with a continuous flow thermal cycler to achieve multi-channel fluorescence detection for real-time fluorescence measurements. Continuously streaming fluid plugs or droplets pass through tubing wrapped around a two-temperature zone thermal block with each wrap of tubing fluorescently coupled to a 64-channel multi-anode PMT. This work demonstrates real-time qPCR of 0.1-10 μL droplets or fluid plugs over a range of 7 orders of magnitude concentration from 1 × 10(1) to 1 × 10(7). The real-time qPCR analysis allows dynamic range quantification as high as 1 × 10(7) copies per 10 μL reaction, with PCR efficiencies within the range of 90-110% based on serial dilution assays and a limit of detection of 10 copies per rxn. The combined functionality of continuous flow, low power thermal cycling, high throughput sample processing, and real-time qPCR improves the rates at which biological or environmental samples can be continuously sampled and analyzed.

Off-line real-time FTIR analysis of a process step in imipenem production

NASA Astrophysics Data System (ADS)

Boaz, Jhansi R.; Thomas, Scott M.; Meyerhoffer, Steven M.; Staskiewicz, Steven J.; Lynch, Joseph E.; Egan, Richard S.; Ellison, Dean K.

1992-08-01

We have developed an FT-IR method, using a Spectra-Tech Monit-IR 400 systems, to monitor off-line the completion of a reaction in real-time. The reaction is moisture-sensitive and analysis by more conventional methods (normal-phase HPLC) is difficult to reproduce. The FT-IR method is based on the shift of a diazo band when a conjugated beta-diketone is transformed into a silyl enol ether during the reaction. The reaction mixture is examined directly by IR and does not require sample workup. Data acquisition time is less than one minute. The method has been validated for specificity, precision and accuracy. The results obtained by the FT-IR method for known mixtures and in-process samples compare favorably with those from a normal-phase HPLC method.

Real-time subsecond voltammetric analysis of Pb in aqueous environmental samples.

PubMed

Yang, Yuanyuan; Pathirathna, Pavithra; Siriwardhane, Thushani; McElmurry, Shawn P; Hashemi, Parastoo

2013-08-06

Lead (Pb) pollution is an important environmental and public health concern. Rapid Pb transport during stormwater runoff significantly impairs surface water quality. The ability to characterize and model Pb transport during these events is critical to mitigating its impact on the environment. However, Pb analysis is limited by the lack of analytical methods that can afford rapid, sensitive measurements in situ. While electrochemical methods have previously shown promise for rapid Pb analysis, they are currently limited in two ways. First, because of Pb's limited solubility, test solutions that are representative of environmental systems are not typically employed in laboratory characterizations. Second, concerns about traditional Hg electrode toxicity, stability, and low temporal resolution have dampened opportunities for in situ analyses with traditional electrochemical methods. In this paper, we describe two novel methodological advances that bypass these limitations. Using geochemical models, we first create an environmentally relevant test solution that can be used for electrochemical method development and characterization. Second, we develop a fast-scan cyclic voltammetry (FSCV) method for Pb detection on Hg-free carbon fiber microelectrodes. We assess the method's sensitivity and stability, taking into account Pb speciation, and utilize it to characterize rapid Pb fluctuations in real environmental samples. We thus present a novel real-time electrochemical tool for Pb analysis in both model and authentic environmental solutions.

Characterization and application of droplet spray ionization for real-time reaction monitoring.

PubMed

Zhang, Hong; Li, Na; Li, Xiao-di; Jiang, Jie; Zhao, Dan-Dan; You, Hong

2016-08-01

The ionization source for real-time reaction monitoring has attracted tremendous interest in recent years. We have previously reported a reliable approach in which droplet spray ionization (DSI) was used for monitoring chemical reactions in real-time. Herein, we systematically investigated the characterization and application of DSI for real-time reaction monitoring. Analyte ions are generated by loading a sample solution onto a corner of a microscope cover glass positioned in front of the MS inlet and applying a high voltage to the sample. The tolerance to positioning, solvent effect, spray angle and spray time were investigated. Extension to real-time monitoring of macromolecule reactions was also demonstrated by the charge state change of cytochrome c in the presence of acetic acid. The corner could be positioned within an area of approximately 10 × 6 × 5 mm (x, y, z) in front of the MS inlet. The broad polarities of solvents from methanol to PhF were suitable for DSI. It featured monitoring real-time changes in reactions on the time scale of seconds to minutes. A real-time charge state change of cytochrome c was captured. DSI-MS features ease of use, durability of the spray platform and reusability of the ion source. Eliminating the need for a sample transport capillary, DSI opens a new avenue for the in situ analysis and real-time monitoring of short-lived key reaction intermediates even at subsecond dead times. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Alcohol affordability and alcohol demand: cross-country trends and panel data estimates, 1975 to 2008.

PubMed

Nelson, Jon P

2014-04-01

Relatively little is known about cross-country differences in alcohol affordability or factors that determine differences in affordability over time. This information is potentially important for alcohol policy, especially policies that focus on higher taxes or prices to reduce total alcohol consumption. This study estimates cross-country alcohol consumption relationships using economic models incorporating income and prices and alternative models based on alcohol affordability. The data and analysis are restricted to higher income countries. Data for alcohol consumption per capita (ages 15+) are analyzed for 2 samples: first, 17 countries in the Organisation for Economic Co-operation and Development for the period 1975 to 2000; second, 22 countries in the European Union for the period from 2000 to 2008. Panel data models are utilized, with country and time fixed-effects to control for confounding influences. In economic demand models, covariates are real per capita income and real alcohol price indices. In affordability models, income is divided by prices to yield an index of alcohol affordability. Analysis of data trends reveals that much of the increase in affordability is due to rising real incomes, and not falling real prices. Economic models of demand perform slightly better statistically, but differences are not substantial as income and affordability are highly correlated. For both samples, exogenous rates of growth of alcohol consumption are negative. Price and income elasticities, on average, are within the range of prior estimates. Affordability elasticities are between 0.21 and 0.25. Although alcohol affordability is a valid concept statistically, its use in policy discussions tends to hide underlying causes of changes in affordability. A better approach is a comparison and analysis of trends and cross-country differences in real incomes and real alcohol prices together with the affordability index. Country-level analysis of income and price elasticities also is required. Copyright © 2014 by the Research Society on Alcoholism.

Quantitative Digital Autoradiography for Environmental Swipe Sample Prioritization: System design, Characterization, and Initial Measurements

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

McDonald, Benjamin S.; Zalavadia, Mital A.; Miller, Brian W.

Environmental sampling and sample analyses by the International Atomic Energy Agency’s (IAEA) Network of Analytical Laboratories (NWAL) is a critical technical tool used to detect facility misuse under a Comprehensive Safeguards Agreement and to verify the absence of undeclared nuclear material activities under an Additional Protocol. Currently all environmental swipe samples (ESS) are screened using gamma spectrometry and x-ray fluorescence to estimate the amount of U and/or Pu in the ESS, to guide further analysis, and to assist in the shipment of ESS to the NWAL. Quantitative Digital Autoradiography for Environmental Samples (QDARES) is being developed to complement existing techniquesmore » through the use of a portable, real-time, high-spatial-resolution camera called the Ionizing-radiation Quantum Imaging Detector (iQID). The iQID constructs a spatial map of radionuclides within a sample or surface in real-time as charged particles (betas) and photons (gamma/x-rays) are detected and localized on an event-by-event basis. Knowledge of the location and nature of radioactive hot spots on the ESS could provide information for subsequent laboratory analysis. As a nondestructive technique, QDARES does not compromise the ESS chain of custody or subsequent laboratory analysis. In this paper we will present the system design and construction, characterization measurements with calibration sources, and initial measurements of ESS.« less

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

PubMed

Krämer, Nadine; Löfström, Charlotta; Vigre, Håkan; Hoorfar, Jeffrey; Bunge, Cornelia; Malorny, Burkhard

2011-03-01

Salmonella is a major zoonotic pathogen which causes outbreaks and sporadic cases of gastroenteritis in humans worldwide. The primary sources for Salmonella are food-producing animals such as pigs and poultry. For risk assessment and hazard analysis and critical control point (HACCP) concepts, it is essential to produce large amounts of quantitative data, which is currently not achievable with the standard cultural based methods for enumeration of Salmonella. This study presents the development of a novel strategy to enumerate low numbers of Salmonella in cork borer samples taken from pig carcasses as a first concept and proof of principle for a new sensitive and rapid quantification method based on combined enrichment and real-time PCR. The novelty of the approach is in the short pre-enrichment step, where for most bacteria, growth is in the log phase. The method consists of an 8h pre-enrichment of the cork borer sample diluted 1:10 in non-selective buffered peptone water, followed by DNA extraction, and Salmonella detection and quantification by real-time PCR. The limit of quantification was 1.4 colony forming units (CFU)/20 cm(2) (approximately 10 g) of artificially contaminated sample with 95% confidence interval of ± 0.7 log CFU/sample. The precision was similar to the standard reference most probable number (MPN) method. A screening of 200 potentially naturally contaminated cork borer samples obtained over seven weeks in a slaughterhouse resulted in 25 Salmonella-positive samples. The analysis of salmonellae within these samples showed that the PCR method had a higher sensitivity for samples with a low contamination level (<6.7 CFU/sample), where 15 of the samples negative with the MPN method was detected with the PCR method and 5 were found to be negative by both methods. For the samples with a higher contamination level (6.7-310 CFU/sample) a good agreement between the results obtained with the PCR and MPN methods was obtained. The quantitative real-time PCR method can easily be applied to other food and environmental matrices by adaptation of the pre-enrichment time and media. Copyright © 2010 Elsevier B.V. All rights reserved.

An optical fiber-based LSPR aptasensor for simple and rapid in-situ detection of ochratoxin A.

PubMed

Lee, Bobin; Park, Jin-Ho; Byun, Ju-Young; Kim, Joon Heon; Kim, Min-Gon

2018-04-15

Label-free biosensing methods that rely on the use of localized surface plasmon resonance (LSPR) have attracted great attention as a result of their simplicity, high sensitivity, and relatively low cost. However, in-situ analysis of real samples using these techniques has remained challenging because colloidal nanoparticles (NPs) can be unstable at certain levels of pH and salt concentration. Even in the case of a chip-type LSPR sensor that can resolve the instability problem by employing NPs immobilized on the substrate, loading of a sample to sensor chip with exact volume control can be difficult for unskilled users. Herein, we report an optical-fiber-based LSPR aptasensor that can avoid these problems and serve as a portable and simple system for sensitive detection of a small mycotoxin, ochratoxin A (OTA), in real samples. The optical fiber coated with aptamer-modified gold nanorods (GNRs) is simply dipped into a solution containing OTA and subjected to LSPR analysis. Quantitative analysis of OTA is performed by measuring the spectral red shift of the LSPR peak of GNRs. Under optimized conditions, the LSPR peak shift displays a linear response (R 2 = 0.9887) to OTA in the concentration range from 10pM to 100nM, with a limit of detection of 12.0pM (3S). The developed sensor shows a high selectivity for OTA over other mycotoxins such as zearalenone (ZEN) and ochratoxin B (OTB), and shows an accurate detection capability for OTA in real grape juice samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Method for Hot Real-Time Analysis of Pyrolysis Vapors at Pilot Scale

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

Pomeroy, Marc D

Pyrolysis oils contain more than 400 compounds, up to 60% of which do not re-volatilize for subsequent chemical analysis. Vapor chemical composition is also complicated as additional condensation reactions occur during quenching and collection of the product. Due to the complexity of the pyrolysis oil, and a desire to catalytically upgrade the vapor composition before condensation, online real-time analytical techniques such as Molecular Beam Mass Spectrometry (MBMS) are of great use. However, in order to properly sample hot pyrolysis vapors at the pilot scale, many challenges must be overcome.

Improving EEG-Based Motor Imagery Classification for Real-Time Applications Using the QSA Method.

PubMed

Batres-Mendoza, Patricia; Ibarra-Manzano, Mario A; Guerra-Hernandez, Erick I; Almanza-Ojeda, Dora L; Montoro-Sanjose, Carlos R; Romero-Troncoso, Rene J; Rostro-Gonzalez, Horacio

2017-01-01

We present an improvement to the quaternion-based signal analysis (QSA) technique to extract electroencephalography (EEG) signal features with a view to developing real-time applications, particularly in motor imagery (IM) cognitive processes. The proposed methodology (iQSA, improved QSA) extracts features such as the average, variance, homogeneity, and contrast of EEG signals related to motor imagery in a more efficient manner (i.e., by reducing the number of samples needed to classify the signal and improving the classification percentage) compared to the original QSA technique. Specifically, we can sample the signal in variable time periods (from 0.5 s to 3 s, in half-a-second intervals) to determine the relationship between the number of samples and their effectiveness in classifying signals. In addition, to strengthen the classification process a number of boosting-technique-based decision trees were implemented. The results show an 82.30% accuracy rate for 0.5 s samples and 73.16% for 3 s samples. This is a significant improvement compared to the original QSA technique that offered results from 33.31% to 40.82% without sampling window and from 33.44% to 41.07% with sampling window, respectively. We can thus conclude that iQSA is better suited to develop real-time applications.

Improving EEG-Based Motor Imagery Classification for Real-Time Applications Using the QSA Method

PubMed Central

Batres-Mendoza, Patricia; Guerra-Hernandez, Erick I.; Almanza-Ojeda, Dora L.; Montoro-Sanjose, Carlos R.

2017-01-01

We present an improvement to the quaternion-based signal analysis (QSA) technique to extract electroencephalography (EEG) signal features with a view to developing real-time applications, particularly in motor imagery (IM) cognitive processes. The proposed methodology (iQSA, improved QSA) extracts features such as the average, variance, homogeneity, and contrast of EEG signals related to motor imagery in a more efficient manner (i.e., by reducing the number of samples needed to classify the signal and improving the classification percentage) compared to the original QSA technique. Specifically, we can sample the signal in variable time periods (from 0.5 s to 3 s, in half-a-second intervals) to determine the relationship between the number of samples and their effectiveness in classifying signals. In addition, to strengthen the classification process a number of boosting-technique-based decision trees were implemented. The results show an 82.30% accuracy rate for 0.5 s samples and 73.16% for 3 s samples. This is a significant improvement compared to the original QSA technique that offered results from 33.31% to 40.82% without sampling window and from 33.44% to 41.07% with sampling window, respectively. We can thus conclude that iQSA is better suited to develop real-time applications. PMID:29348744

Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples.

PubMed

Mokhtari, W; Nsaibia, S; Gharbi, A; Aouni, M

2013-02-01

Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen. A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T(m) = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C(q)) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea. The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples. Copyright © 2012 Elsevier Ltd. All rights reserved.

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%.

Outbreak of hepatitis E virus infection in Darfur, Sudan: effectiveness of real-time reverse transcription-PCR analysis of dried blood spots.

PubMed

Mérens, Audrey; Guérin, Philippe Jean; Guthmann, Jean-Paul; Nicand, Elisabeth

2009-06-01

Biological samples collected in refugee camps during an outbreak of hepatitis E were used to compare the accuracy of hepatitis E virus RNA amplification by real-time reverse transcription-PCR (RT-PCR) for sera and dried blood spots (concordance of 90.6%). Biological profiles (RT-PCR and serology) of asymptomatic individuals were also analyzed.

Effects of Sample Selection Bias on the Accuracy of Population Structure and Ancestry Inference

PubMed Central

Shringarpure, Suyash; Xing, Eric P.

2014-01-01

Population stratification is an important task in genetic analyses. It provides information about the ancestry of individuals and can be an important confounder in genome-wide association studies. Public genotyping projects have made a large number of datasets available for study. However, practical constraints dictate that of a geographical/ethnic population, only a small number of individuals are genotyped. The resulting data are a sample from the entire population. If the distribution of sample sizes is not representative of the populations being sampled, the accuracy of population stratification analyses of the data could be affected. We attempt to understand the effect of biased sampling on the accuracy of population structure analysis and individual ancestry recovery. We examined two commonly used methods for analyses of such datasets, ADMIXTURE and EIGENSOFT, and found that the accuracy of recovery of population structure is affected to a large extent by the sample used for analysis and how representative it is of the underlying populations. Using simulated data and real genotype data from cattle, we show that sample selection bias can affect the results of population structure analyses. We develop a mathematical framework for sample selection bias in models for population structure and also proposed a correction for sample selection bias using auxiliary information about the sample. We demonstrate that such a correction is effective in practice using simulated and real data. PMID:24637351

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.

Trace analysis of energetic materials via direct analyte-probed nanoextraction coupled to direct analysis in real time mass spectrometry.

PubMed

Clemons, Kristina; Dake, Jeffrey; Sisco, Edward; Verbeck, Guido F

2013-09-10

Direct analysis in real time mass spectrometry (DART-MS) has proven to be a useful forensic tool for the trace analysis of energetic materials. While other techniques for detecting trace amounts of explosives involve extraction, derivatization, solvent exchange, or sample clean-up, DART-MS requires none of these. Typical DART-MS analyses directly from a solid sample or from a swab have been quite successful; however, these methods may not always be an optimal sampling technique in a forensic setting. For example, if the sample were only located in an area which included a latent fingerprint of interest, direct DART-MS analysis or the use of a swab would almost certainly destroy the print. To avoid ruining such potentially invaluable evidence, another method has been developed which will leave the fingerprint virtually untouched. Direct analyte-probed nanoextraction coupled to nanospray ionization-mass spectrometry (DAPNe-NSI-MS) has demonstrated excellent sensitivity and repeatability in forensic analyses of trace amounts of illicit drugs from various types of surfaces. This technique employs a nanomanipulator in conjunction with bright-field microscopy to extract single particles from a surface of interest and has provided a limit of detection of 300 attograms for caffeine. Combining DAPNe with DART-MS provides another level of flexibility in forensic analysis, and has proven to be a sufficient detection method for trinitrotoluene (TNT), RDX, and 1-methylaminoanthraquinone (MAAQ). Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

Real-time, continuous water-quality monitoring in Indiana and Kentucky

USGS Publications Warehouse

Shoda, Megan E.; Lathrop, Timothy R.; Risch, Martin R.

2015-01-01

Water-quality “super” gages (also known as “sentry” gages) provide real-time, continuous measurements of the physical and chemical characteristics of stream water at or near selected U.S. Geological Survey (USGS) streamgages in Indiana and Kentucky. A super gage includes streamflow and water-quality instrumentation and representative stream sample collection for laboratory analysis. USGS scientists can use statistical surrogate models to relate instrument values to analyzed chemical concentrations at a super gage. Real-time, continuous and laboratory-analyzed concentration and load data are publicly accessible on USGS Web pages.

Parker THM Analyzer

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

Hassan, Kazi; Siegal, Michael; Mowry, Curtis

This easy-to-operate, cost-effective, tabletop purge-and-trap gas chromatograph ensures safe drinking water and monitors disinfection by-product formation at water utilities in real-time without sample preparation or off-site analysis.

Ultrasonic Surface Measurements for the investigation of superficial alteration of natural stones

NASA Astrophysics Data System (ADS)

Meier, Thomas; Auras, Michael; Bilgili, Filiz; Christen, Sandra; Cristiano, Luigia; Krompholz, Rolf; Mosca, Ilaria; Rose, David

2013-04-01

Seismic waveform analysis is applicable also to the centimeter and decimeter scale for non-destructive testing of pavement, facades, plaster, sculptures, or load-bearing structures like pillars. Mostly transmission measurements are performed and travel-times of first arriving P-waves are considered that have limited resolution for the upper centimeters of an object. In contrast, surface measurements are well suited to quantify superficial alterations of material properties e.g. due to weathering. A number of surface measurements have been carried out in the laboratory as well as on real structures in order to study systematically the information content of ultrasonic waveforms and their variability under real conditions. As a preposition for ultrasonic waveform analysis, reproducible, broad-band measurements have to be carried out with a definite radiation pattern and an about 1 mm accuracy of the measurement geometry. We used special coupling devices for effective ultrasonic surface measurements in the laboratory as well as at real objects. Samples of concrete with varying composition and samples of natural stone - marble, tuff, and sandstone - were repeatedly weathered and tested by ultrasonic measurements. The resistance of the samples to weathering and the penetration depth of the weathering are analyzed. Furthermore, material specific calibration curves for changes in velocities of elastic waves due to weathering can be obtained by these tests. Tests on real structures have been carried out for marble (Schlossbrücke, Berlin) and sandstone (Porta Nigra, Trier). Altogether, these test measurements show clearly that despite of the internal inhomogeneity of many real objects, their surface roughness and topography especially ultrasonic Rayleigh waves are well suited to study material alterations in the upper centimeters. Dispersion of Rayleigh waves may be inverted for shear-wave velocity as a function of depth.

ELEMENTAL COMPOSITION OF FRESHLY NUCLEATED PARTICLES

EPA Science Inventory

The main objective of this work is to develop a method for real-time sampling and analysis of individual airborne nanoparticles in the 5 - 20 nm diameter range. The size range covered by this method is much smaller than existing single particle methods for chemical analysis. S...

A survey of the use of soy in processed Turkish meat products and detection of genetic modification.

PubMed

Ulca, Pelin; Balta, Handan; Senyuva, Hamide Z

2014-01-01

To screen for possible illegal use of soybeans in meat products, the performance characteristics of a commercial polymer chain reaction (PCR) kit for detection of soybean DNA in raw and cooked meat products were established. Minced chicken and beef products containing soybean at levels from 0.1% to 10.0% were analysed by real-time PCR to amplify the soybean lectin gene. The PCR method could reliably detect the addition of soybean at a level of 0.1%. A survey of 38 Turkish processed meat products found only six samples to be negative for the presence of soybean. In 32 (84%) positive samples, 13 (34%) contained levels of soy above 0.1%. Of soybean positive samples, further DNA analysis was conducted by real-time PCR to detect whether genetically modified (GM) soybean had been used. Of 32 meat samples containing soybean, two samples were positive for GM modification.

Raman spectroscopic analysis of real samples: Brazilian bauxite mineralogy

NASA Astrophysics Data System (ADS)

Faulstich, Fabiano Richard Leite; Castro, Harlem V.; de Oliveira, Luiz Fernando Cappa; Neumann, Reiner

2011-10-01

In this investigation, Raman spectroscopy with 1064 and 632.8 nm excitation was used to investigate real mineral samples of bauxite ore from mines of Northern Brazil, together with Raman mapping and X-rays diffraction. The obtained results show clearly that the use of microRaman spectroscopy is a powerful tool for the identification of all the minerals usually found in bauxites: gibbsite, kaolinite, goethite, hematite, anatase and quartz. Bulk samples can also be analysed, and FT-Raman is more adequate due to better signal-to-noise ratio and representativity, although not efficient for kaolinite. The identification of fingerprinting vibrations for all the minerals allows the acquisition of Raman-based chemical maps, potentially powerful tools for process mineralogy applied to bauxite ores.

Theoretical analysis of a dual-probe scanning tunneling microscope setup on graphene.

PubMed

Settnes, Mikkel; Power, Stephen R; Petersen, Dirch H; Jauho, Antti-Pekka

2014-03-07

Experimental advances allow for the inclusion of multiple probes to measure the transport properties of a sample surface. We develop a theory of dual-probe scanning tunneling microscopy using a Green's function formalism, and apply it to graphene. Sampling the local conduction properties at finite length scales yields real space conductance maps which show anisotropy for pristine graphene systems and quantum interference effects in the presence of isolated impurities. Spectral signatures in the Fourier transforms of real space conductance maps include characteristics that can be related to different scattering processes. We compute the conductance maps of graphene systems with different edge geometries or height fluctuations to determine the effects of nonideal graphene samples on dual-probe measurements.

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

PubMed

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

2006-01-01

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

Quarterly Update: July-September 1990

DTIC Science & Technology

1990-09-01

drafted a report that defines the process and the products of a feature oriented domain analysis ( FODA ) and provides a sample do- main analysis . This...receive ............... training to offer assessment services commercially. This section provides NASA adopted the rate monotonic scheduling analysis ...that the rate monotonic scheduling analysis approach will be the JulySeptember 1990 baseline methodology for its hard real-time operating system

Contribution of bulk mass spectrometry isotopic analysis to characterization of materials in the framework of CMX-4

DOE PAGES

Kuchkin, A.; Stebelkov, V.; Zhizhin, K.; ...

2018-01-30

Seven laboratories used the results of bulk uranium isotopic analysis by either inductively coupled plasma mass spectrometry (ICP-MS) or thermal ionization mass spectrometry (TIMS) for characterization of the samples in the Nuclear Forensic International Technical Working Group fourth international collaborative material exercise, CMX-4. Comparison of the measured isotopic compositions of uranium in three exercise samples is implemented for identifying any differences or similarities between the samples. The role of isotopic analyses in the context of a real nuclear forensic investigation is discussed. Several limitations in carrying out ICP-MS or TIMS analysis in CMX-4 are noted.

Contribution of bulk mass spectrometry isotopic analysis to characterization of materials in the framework of CMX-4

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

Kuchkin, A.; Stebelkov, V.; Zhizhin, K.

Seven laboratories used the results of bulk uranium isotopic analysis by either inductively coupled plasma mass spectrometry (ICP-MS) or thermal ionization mass spectrometry (TIMS) for characterization of the samples in the Nuclear Forensic International Technical Working Group fourth international collaborative material exercise, CMX-4. Comparison of the measured isotopic compositions of uranium in three exercise samples is implemented for identifying any differences or similarities between the samples. The role of isotopic analyses in the context of a real nuclear forensic investigation is discussed. Several limitations in carrying out ICP-MS or TIMS analysis in CMX-4 are noted.

Substrate-Coated Illumination Droplet Spray Ionization: Real-Time Monitoring of Photocatalytic Reactions

NASA Astrophysics Data System (ADS)

Zhang, Hong; Li, Na; Zhao, Dandan; Jiang, Jie; You, Hong

2017-09-01

Real-time monitoring of photocatalytic reactions facilitates the elucidation of the mechanisms of the reactions. However, suitable tools for real-time monitoring are lacking. Herein, a novel method based on droplet spray ionization named substrate-coated illumination droplet spray ionization (SCI-DSI) for direct analysis of photocatalytic reaction solution is reported. SCI-DSI addresses many of the analytical limitations of electrospray ionization (ESI) for analysis of photocatalytic-reaction intermediates, and has potential for both in situ analysis and real-time monitoring of photocatalytic reactions. In SCI-DSI-mass spectrometry (MS), a photocatalytic reaction occurs by loading sample solutions onto the substrate-coated cover slip and by applying UV light above the modified slip; one corner of this slip adjacent to the inlet of a mass spectrometer is the high-electric-field location for launching a charged-droplet spray. After both testing and optimizing the performance of SCI-DSI, the value of this method for in situ analysis and real-time monitoring of photocatalytic reactions was demonstrated by the removal of cyclophosphamide (CP) in TiO2/UV. Reaction times ranged from seconds to minutes, and the proposed reaction intermediates were captured and identified by tandem mass spectrometry. Moreover, the free hydroxyl radical (·OH) was identified as the main radicals for CP removal. These results show that SCI-DSI is suitable for in situ analysis and real-time monitoring of CP removal under TiO2-based photocatalytic reactions. SCI-DSI is also a potential tool for in situ analysis and real-time assessment of the roles of radicals during CP removal under TiO2-based photocatalytic reactions. Graphical Abstract[Figure not available: see fulltext.

Microwave plasma monitoring system for the elemental composition analysis of high temperature process streams

DOEpatents

Woskov, Paul P.; Cohn, Daniel R.; Titus, Charles H.; Surma, Jeffrey E.

1997-01-01

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, high temperature capability 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. The invention may be incorporated into a high temperature process device and implemented in situ for example, such as with a DC graphite electrode plasma arc furnace. The invention further provides a system for the elemental analysis of process streams by removing particulate and/or droplet samples therefrom and entraining such samples in the gas flow which passes through the plasma flame. Introduction of and entraining samples in the gas flow may be facilitated by a suction pump, regulating gas flow, gravity or combinations thereof.

Development of a multiplex real time PCR to detect thermophilic lactic acid bacteria in natural whey starters.

PubMed

Bottari, Benedetta; Agrimonti, Caterina; Gatti, Monica; Neviani, Erasmo; Marmiroli, Nelson

2013-01-01

A multiplex real time PCR (mRealT-PCR) useful to rapidly screen microbial composition of thermophilic starter cultures for hard cooked cheeses and to compare samples with potentially different technological properties was developed. Novel primers directed toward pheS gene were designed and optimized for multiple detection of Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus thermophilus and Lactobacillus fermentum. The assay was based on SYBR Green chemistry followed by melting curves analysis. The method was then evaluated for applications in the specific detection of the 4 lactic acid bacteria (LAB) in 29 different natural whey starters for Parmigiano Reggiano cheese production. The results obtained by mRealT-PCR were also compared with those obtained on the same samples by Fluorescence in Situ Hybridization (FISH) and Length-Heterogeneity PCR (LH-PCR). The mRealT-PCR developed in this study, was found to be effective for analyzing species present in the samples with an average sensitivity down to less than 600 copies of DNA and therefore sensitive enough to detect even minor LAB community members of thermophilic starter cultures. The assay was able to describe the microbial population of all the different natural whey starter samples analyzed, despite their natural variability. A higher number of whey starter samples with S. thermophilus and L. fermentum present in their microbial community were revealed, suggesting that these species could be more frequent in Parmigiano Reggiano natural whey starter samples than previously shown. The method was more effective than LH-PCR and FISH and, considering that these two techniques have to be used in combination to detect the less abundant species, the mRealT-PCR was also faster. Providing a single step sensitive detection of L. helveticus, L. delbrueckii, S. thermophilus and L. fermentum, the developed mRealT-PCR could be used for screening thermophilic starter cultures and to follow the presence of those species during ripening of derived dairy products. A major increase in understanding the starter culture contribution to cheese ecosystem could be harnessed to control cheese ripening and flavor formation. Copyright © 2012 Elsevier B.V. All rights reserved.

Real-Time PCR for the Detection and Quantification of Geodermatophilaceae from Stone Samples and Identification of New Members of the Genus Blastococcus†

PubMed Central

Salazar, Oscar; Valverde, Aranzazu; Genilloud, Olga

2006-01-01

Real-time PCR (RT-PCR) technology was used for the specific detection and quantification of members of the family Geodermatophilaceae in stone samples. Differences in the nucleotide sequences of the 16S rRNA gene region were used to design a pair of family-specific primers that were used to detect and quantify by RT-PCR DNA from members of this family in stone samples from different geographical origins in Spain. These primers were applied later to identify by PCR-specific amplification new members of the family Geodermatophilaceae isolated from the same stone samples. The diversity and taxonomic position of the wild-type strains identified from ribosomal sequence analysis suggest the presence of a new lineage within the genus Blastococcus. PMID:16391063

Salmonella detection in poultry samples. Comparison of two commercial real-time PCR systems with culture methods for the detection of Salmonella spp. in environmental and fecal samples of poultry.

PubMed

Sommer, D; Enderlein, D; Antakli, A; Schönenbrücher, H; Slaghuis, J; Redmann, T; Lierz, M

2012-01-01

The efficiency of two commercial PCR methods based on real-time technology, the foodproof® Salmonella detection system and the BAX® PCR Assay Salmonella system was compared to standardized culture methods (EN ISO 6579:2002 - Annex D) for the detection of Salmonella spp. in poultry samples. Four sample matrices (feed, dust, boot swabs, feces) obtained directly from poultry flocks, as well as artificially spiked samples of the same matrices, were used. All samples were tested for Salmonella spp. using culture methods first as the gold standard. In addition samples spiked with Salmonella Enteridis were tested to evaluate the sensitivity of both PCR methods. Furthermore all methods were evaluated in an annual ring-trial of the National Salmonella Reference Laboratory of Germany. Salmonella detection in the matrices feed, dust and boot swabs were comparable in both PCR systems whereas the results from feces differed markedly. The quality, especially the freshness, of the fecal samples had an influence on the sensitivity of the real-time PCR and the results of the culture methods. In fresh fecal samples an initial spiking level of 100cfu/25g Salmonella Enteritidis was detected. Two-days-dried fecal samples allowed the detection of 14cfu/25g. Both real- time PCR protocols appear to be suitable for the detection of Salmonella spp. in all four matrices. The foodproof® system detected eight samples more to be positive compared to the BAX® system, but had a potential false positive result in one case. In 7-days-dried samples none of the methods was able to detect Salmonella likely through letal cell damage. In general the advantage of PCR analyses over the culture method is the reduction of working time from 4-5 days to only 2 days. However, especially for the analysis of fecal samples official validation should be conducted according to the requirement of EN ISO6579:2002 - Annex D.

[Lice in Mali: frequency of infestation, genotyping, infection rate and case management].

PubMed

Sangaré, A K; Doumbo, S N; Koné, A K; Thera, M A; Dabo, A; Brouqui, P; Raoult, D; Doumbo, O K

2015-01-01

Because lice-transmitted infections are a real public health problem, epidemiological studies in different ecoclimatic zones of Africa are useful. This article aims to describe the frequency of lice infestation, their genotypes, and their infection by pathogens in the regions of Koulikoro and Mopti. A cross-sectional survey allowed us to collect lice from rural populations. Techniques of molecular biology (real-time PCR, standard PCR, and genotyping) were used for analysis of lice samples. Infestation rates were 57% (12/21) among subjects in Diankabou, in the Sahelian zone; 91% (39/43) in Doneguebougou, and 86% (59/69) in Zorocoro, in a savanna zone. The overall lice infestation rate in the samples in the three localities was 83% (110/133). Real-time PCR showed 3% (4/92) of Acinetobacter baumanii but no B. quintana in Diankabou. Phylogenetic analysis of the mitochondrial gene (Cytb) showed that head lice in Mali belong to genotype C. The high frequency of lice infestation in the study population indicates that it would be useful to conduct national epidemiological surveys to estimate the magnitude of this public health problem.

A Modified Content Analysis of Existing School Psychology Reports

ERIC Educational Resources Information Center

Mallin, Barry; Schellenberg, Miriam E.; Smith, Christiane

2012-01-01

Discrepancies between our university's training program's report-writing guidelines and common practice in Manitoba could not be resolved by reference to the literature. To inform the discussion, we collected a sample of local real world school psychology reports and undertook a modified content analysis to operationally define and measure…

The Lottery Is a Mathematics Powerball

ERIC Educational Resources Information Center

Lim, Vivian; Rubel, Laurie; Shookhoff, Lauren; Sullivan, Mathew; Williams, Sarah

2016-01-01

The lottery has rich potential for mathematical explorations. It serves as a real-world context to explore concepts of permutations, combinations, sample space, and probability in terms of making sense of the lottery games. The lottery offers additional possibilities in terms of scaling, data analysis, and spatial analysis. Finally, by readily…

Detection of Illicit Drugs on Surfaces Using Direct Analysis in Real Time (DART)/Time-of-Flight Mass Spectrometry

EPA Science Inventory

Methamphetamine (meth) residues from meth syntheses or habitual meth smoking pose human health hazards. State health departments require remediation of meth labs before properties are sold. NIOSH methods for meth analysis require wipe sampling, extraction, cleanup, solvent excha...

Clinical Application of Ambient Ionization Mass Spectrometry

PubMed Central

Li, Li-Hua; Hsieh, Hua-Yi; Hsu, Cheng-Chih

2017-01-01

Ambient ionization allows mass spectrometry analysis directly on the sample surface under atmospheric pressure with almost zero sample pretreatment. Since the development of desorption electrospray ionization (DESI) in 2004, many other ambient ionization techniques were developed. Due to their simplicity and low operation cost, rapid and on-site clinical mass spectrometry analysis becomes real. In this review, we will highlight some of the most widely used ambient ionization mass spectrometry approaches and their applications in clinical study. PMID:28337399

Improvement in the detection rate of diarrhoeagenic bacteria in human stool specimens by a rapid real-time PCR assay.

PubMed

Iijima, Yoshio; Asako, Nahoko T; Aihara, Masanori; Hayashi, Kozaburo

2004-07-01

A rapid laboratory system has been developed and evaluated that can simultaneously identify major diarrhoeagenic bacteria, including Salmonella enterica, Vibrio parahaemolyticus, Campylobacter jejuni and Shiga toxin-producing Escherichia coli, in stool specimens by real-time PCR. Specific identification was achieved by using selective TaqMan probes, detecting two targets in each pathogen. A positive result was scored only when both targets of a pathogen were amplified and the difference between threshold cycles for detection was less than five. Diagnosis of enteric bacterial infections using this highly sensitive method, including DNA extraction and real-time PCR, requires only 3 h. Forty stool specimens related to suspected food poisoning outbreaks were analysed: 16 (40%) of these samples were found to be positive for diarrhoeagenic bacteria using a conventional culture method; 28 (70%) were positive using the real-time PCR assay. Of the 12 PCR-positive but culture-negative cases, 11 patients had consumed pathogen-contaminated or high-risk food. Analysis of faecal samples from 105 outpatients who complained of diarrhoea and/or abdominal pain identified 19 (18%) patients as being positive for diarrhoeagenic bacteria using the culture method. An additional six (6%) patients were found to be positive by PCR analysis.

A semi-nested real-time PCR method to detect low chimerism percentage in small quantity of hematopoietic stem cell transplant DNA samples.

PubMed

Aloisio, Michelangelo; Bortot, Barbara; Gandin, Ilaria; Severini, Giovanni Maria; Athanasakis, Emmanouil

2017-02-01

Chimerism status evaluation of post-allogeneic hematopoietic stem cell transplantation samples is essential to predict post-transplant relapse. The most commonly used technique capable of detecting small increments of chimerism is quantitative real-time PCR. Although this method is already used in several laboratories, previously described protocols often lack sensitivity and the amount of the DNA required for each chimerism analysis is too high. In the present study, we compared a novel semi-nested allele-specific real-time PCR (sNAS-qPCR) protocol with our in-house standard allele-specific real-time PCR (gAS-qPCR) protocol. We selected two genetic markers and analyzed technical parameters (slope, y-intercept, R2, and standard deviation) useful to determine the performances of the two protocols. The sNAS-qPCR protocol showed better sensitivity and precision. Moreover, the sNAS-qPCR protocol requires, as input, only 10 ng of DNA, which is at least 10-fold less than the gAS-qPCR protocols described in the literature. Finally, the proposed sNAS-qPCR protocol could prove very useful for performing chimerism analysis with a small amount of DNA, as in the case of blood cell subsets.

[Selection of reference genes of Siraitia grosvenorii by real-time PCR].

PubMed

Tu, Dong-ping; Mo, Chang-ming; Ma, Xiao-jun; Zhao, Huan; Tang, Qi; Huang, Jie; Pan, Li-mei; Wei, Rong-chang

2015-01-01

Siraitia grosvenorii is a traditional Chinese medicine also as edible food. This study selected six candidate reference genes by real-time quantitative PCR, the expression stability of the candidate reference genes in the different samples was analyzed by using the software and methods of geNorm, NormFinder, BestKeeper, Delta CT method and RefFinder, reference genes for S. grosvenorii were selected for the first time. The results showed that 18SrRNA expressed most stable in all samples, was the best reference gene in the genetic analysis. The study has a guiding role for the analysis of gene expression using qRT-PCR methods, providing a suitable reference genes to ensure the results in the study on differential expressed gene in synthesis and biological pathways, also other genes of S. grosvenorii.

Comparative research of effectiveness of cellulose and fiberglass porous membrane carriers for bio sampling in veterinary and food industry monitoring

NASA Astrophysics Data System (ADS)

Gusev, Alexander; Vasyukova, Inna; Zakharova, Olga; Altabaeva, Yuliya; Saushkin, Nikolai; Samsonova, Jeanne; Kondakov, Sergey; Osipov, Alexander; Snegin, Eduard

2017-11-01

The aim of proposed research is to study the applicability of fiberglass porous membrane materials in a new strip format for dried blood storage in food industry monitoring. A comparative analysis of cellulosic and fiberglass porous membrane materials was carried out to obtain dried samples of serum or blood and the possibility of further species-specific analysis. Blood samples of Sus scrofa were used to study the comparative effectiveness of cellulose and fiberglass porous membrane carriers for long-term biomaterial storage allowing for further DNA detection by real-time polymerase chain reaction (PCR) method. Scanning electron microscopy of various membranes - native and with blood samples - indicate a fundamental difference in the form of dried samples. Membranes based on cellulosic materials sorb the components of the biological fluid on the surface of the fibers of their structure, partially penetrating the cellulose fibers, while in the case of glass fiber membranes the components of the biological fluid dry out as films in the pores of the membrane between the structural filaments. This fundamental difference in the retention mechanisms affects the rate of dissolution of the components of dry samples and contributes to an increase in the efficiency of the desorption process of the sample before subsequent analysis. Detecting of pig DNA in every analyzed sample under the performed Real-time PCR as well as good state of the biomaterial preservation on the glass fiber membranes was clearly demonstrated. Good biomaterials preservation has been revealed on the test cards for 4 days as well as for 1 hour.

Profiling and classification of French propolis by combined multivariate data analysis of planar chromatograms and scanning direct analysis in real time mass spectra.

PubMed

Chasset, Thibaut; Häbe, Tim T; Ristivojevic, Petar; Morlock, Gertrud E

2016-09-23

Quality control of propolis is challenging, as it is a complex natural mixture of compounds, and thus, very difficult to analyze and standardize. Shown on the example of 30 French propolis samples, a strategy for an improved quality control was demonstrated in which high-performance thin-layer chromatography (HPTLC) fingerprints were evaluated in combination with selected mass signals obtained by desorption-based scanning mass spectrometry (MS). The French propolis sample extracts were separated by a newly developed reversed phase (RP)-HPTLC method. The fingerprints obtained by two different detection modes, i.e. after (1) derivatization and fluorescence detection (FLD) at UV 366nm and (2) scanning direct analysis in real time (DART)-MS, were analyzed by multivariate data analysis. Thus, RP-HPTLC-FLD and RP-HPTLC-DART-MS fingerprints were explored and the best classification was obtained using both methods in combination with pattern recognition techniques, such as principal component analysis. All investigated French propolis samples were divided in two types and characteristic patterns were observed. Phenolic compounds such as caffeic acid, p-coumaric acid, chrysin, pinobanksin, pinobanksin-3-acetate, galangin, kaempferol, tectochrysin and pinocembrin were identified as characteristic marker compounds of French propolis samples. This study expanded the research on the European poplar type of propolis and confirmed the presence of two botanically different types of propolis, known as the blue and orange types. Copyright © 2016 Elsevier B.V. All rights reserved.

A TaqMan real-time PCR method based on alternative oxidase genes for detection of plant species in animal feed samples.

PubMed

Campos, Maria Doroteia; Valadas, Vera; Campos, Catarina; Morello, Laura; Braglia, Luca; Breviario, Diego; Cardoso, Hélia G

2018-01-01

Traceability of processed food and feed products has been gaining importance due to the impact that those products can have on human/animal health and to the associated economic and legal concerns, often related to adulterations and frauds as it can be the case for meat and milk. Despite mandatory traceability requirements for the analysis of feed composition, few reliable and accurate methods are presently available to enforce the legislative frame and allow the authentication of animal feeds. In this study, nine sensitive and species-specific real-time PCR TaqMan MGB assays are described for plant species detection in animal feed samples. The method is based on selective real-time qPCR (RT-qPCR) amplification of target genes belonging to the alternative oxidase (AOX) gene family. The plant species selected for detection in feed samples were wheat, maize, barley, soybean, rice and sunflower as common components of feeds, and cotton, flax and peanut as possible undesirable contaminants. The obtained results were compared with end-point PCR methodology. The applicability of the AOX TaqMan assays was evaluated through the screening of commercial feed samples, and by the analysis of plant mixtures with known composition. The RT-qPCR methodology allowed the detection of the most abundant species in feeds but also the identification of contaminant species present in lower amounts, down to 1% w/w. AOX-based methodology provides a suitable molecular marker approach to ascertain plant species composition of animal feed samples, thus supporting feed control and enforcement of the feed sector and animal production.

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

NASA Technical Reports Server (NTRS)

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

2014-01-01

The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a research platform capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR system, the Cepheid SmartCycler and will fly it in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid ramp times and the ability to detect up to four separate fluorescent channels at one time enabling multiplex assays that can be used for normalization and to study multiple genes of interest in each module. The team is currently working with Cepheid to enable the downlink of data from the ISS to the ground and provide uplink capabilities for programming, commanding, monitoring, and instrument maintenance. The project has adapted commercial technology to design a module that can lyse cells and extract RNA of sufficient quality and quantity for use in qRT-PCR reactions while using a housekeeping gene to normalize RNA concentration and integrity. The WetLab-2 system is capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on-orbit. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The system can be used to validate terrestrial analyses of samples returned from ISS by providing on-orbit gene expression benchmarking prior to sample return. The ability to get on orbit data will provide investigators with the opportunity to adjust experiment parameters for subsequent trials based on the real-time data analysis without need for sample return and re-flight. Researchers will also be able to sample multigenerational changes in organisms. Finally, the system can be used for analysis of air, surface, water, and clinical samples to monitor environmental contaminants and crew health. The verification flight of the instrument is scheduled to launch on SpaceX-7 in June 2015.

Rapid detection of highly pathogenic porcine reproductive and respiratory syndrome virus by a fluorescent probe-based isothermal recombinase polymerase amplification assay.

PubMed

Yang, Yang; Qin, Xiaodong; Sun, Yingjun; Chen, Ting; Zhang, Zhidong

2016-12-01

A novel fluorescent probe-based real-time reverse transcription recombinase polymerase amplification (real-time RT-RPA) assay was developed for rapid detection of highly pathogenic type 2 porcine reproductive and respiratory syndrome virus (HP-PRRSV). The sensitivity analysis showed that the detection limit of RPA was 70 copies of HP-PRRSV RNA/reaction. The real-time RT-RPA highly specific amplified HP-PRRSV with no cross-reaction with classic PRRSV, classic swine fever virus, pseudorabies virus, and foot-and-mouth disease virus. Assessment with 125 clinical samples showed that the developed real-time RT-RPA assay was well correlated with real-time RT-qPCR assays for detection of HP-PRRSV. These results suggest that the developed real-time RT-RPA assay is suitable for rapid detection of HP-PRRSV.

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...

Application of rapid onsite PCR (TaqMan) for Phytophthora ramorum under U.S. conditions

Treesearch

Kelvin Hughes; Jenny Tomlinson; Neil Boonham; Kelly Ivors; Matteo Garbelotto; Ian Barker

2006-01-01

Currently, diagnosis of Phytophthora ramorum involves sending samples to a laboratory for traditional isolation and morphological characterisation, and/or PCR analysis. This can take as long as 2 weeks from sampling to final diagnosis. However, the Plant Health Group, Central Science Laboratory, has produced on-site DNA extraction and real-time PCR (...

A noninvasive, direct real-time PCR method for sex determination in multiple avian species

USGS Publications Warehouse

Brubaker, Jessica L.; Karouna-Renier, Natalie K.; Chen, Yu; Jenko, Kathryn; Sprague, Daniel T.; Henry, Paula F.P.

2011-01-01

Polymerase chain reaction (PCR)-based methods to determine the sex of birds are well established and have seen few modifications since they were first introduced in the 1990s. Although these methods allowed for sex determination in species that were previously difficult to analyse, they were not conducive to high-throughput analysis because of the laboriousness of DNA extraction and gel electrophoresis. We developed a high-throughput real-time PCR-based method for analysis of sex in birds, which uses noninvasive sample collection and avoids DNA extraction and gel electrophoresis.

A novel real time PCR assay using melt curve analysis for ivory identification.

PubMed

Kitpipit, Thitika; Penchart, Kitichaya; Ouithavon, Kanita; Satasook, Chutamas; Linacre, Adrian; Thanakiatkrai, Phuvadol

2016-10-01

Demand for ivory and expansion of human settlements have resulted in a rapid decline in the number of elephants. Enforcement of local and international laws and regulations requires identification of the species from which any ivory, or ivory products, originated. Further geographical assignment of the dead elephant from which the ivory was taken can assist in forensic investigations. In this study, a real-time PCR assay using melt curve analysis was developed and fully validated for forensic use. The presence or absence of three Elephantidae-specific and elephant species-specific melting peaks was used to identify the elephant species. Using 141 blood and ivory samples from the three extant elephant species, the assay demonstrated very high reproducibility and accuracy. The limit of detection was as low as 0.031ng of input DNA for conventional amplification and 0.002ng for nested amplification. Both DNA concentrations are typically encountered in forensic casework, especially for degraded samples. No cross-reactivity was observed for non-target species. Evaluation of direct amplification and nested amplification demonstrated the assay's flexibility and capability of analyzing low-template DNA samples and aged samples. Additionally, blind trial testing showed the assay's suitability application in real casework. In conclusion, wildlife forensic laboratories could use this novel, quick, and low-cost assay to help combat the continuing poaching crises leading to the collapse of elephant numbers in the wild. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Design and development of a highly sensitive, field portable plasma source instrument for on-line liquid stream monitoring and real-time sample analysis

NASA Astrophysics Data System (ADS)

Duan, Yixiang; Su, Yongxuan; Jin, Zhe; Abeln, Stephen P.

2000-03-01

The development of a highly sensitive, field portable, low-powered instrument for on-site, real-time liquid waste stream monitoring is described in this article. A series of factors such as system sensitivity and portability, plasma source, sample introduction, desolvation system, power supply, and the instrument configuration, were carefully considered in the design of the portable instrument. A newly designed, miniature, modified microwave plasma source was selected as the emission source for spectroscopy measurement, and an integrated small spectrometer with a charge-coupled device detector was installed for signal processing and detection. An innovative beam collection system with optical fibers was designed and used for emission signal collection. Microwave plasma can be sustained with various gases at relatively low power, and it possesses high detection capabilities for both metal and nonmetal pollutants, making it desirable to use for on-site, real-time, liquid waste stream monitoring. An effective in situ sampling system was coupled with a high efficiency desolvation device for direct-sampling liquid samples into the plasma. A portable computer control system is used for data processing. The new, integrated instrument can be easily used for on-site, real-time monitoring in the field. The system possesses a series of advantages, including high sensitivity for metal and nonmetal elements; in situ sampling; compact structure; low cost; and ease of operation and handling. These advantages will significantly overcome the limitations of previous monitoring techniques and make great contributions to environmental restoration and monitoring.

Quantitative LIBS analysis of vanadium in samples of hexagonal mesoporous silica catalysts.

PubMed

Pouzar, Miloslav; Kratochvíl, Tomás; Capek, Libor; Smoláková, Lucie; Cernohorský, Tomás; Krejcová, Anna; Hromádko, Ludek

2011-02-15

The method for the analysis of vanadium in hexagonal mesoporous silica (V-HMS) catalysts using Laser Induced Breakdown Spectrometry (LIBS) was suggested. Commercially available LIBS spectrometer was calibrated with the aid of authentic V-HMS samples previously analyzed by ICP OES after microwave digestion. Deposition of the sample on the surface of adhesive tape was adopted as a sample preparation method. Strong matrix effect connected with the catalyst preparation technique (1st vanadium added in the process of HMS synthesis, 2nd already synthesised silica matrix was impregnated by vanadium) was observed. The concentration range of V in the set of nine calibration standards was 1.3-4.5% (w/w). Limit of detection was 0.13% (w/w) and it was calculated as a triple standard deviation from five replicated determinations of vanadium in the real sample with a very low vanadium concentration. Comparable results of LIBS and ED XRF were obtained if the same set of standards was used for calibration of both methods and vanadium was measured in the same type of real samples. LIBS calibration constructed using V-HMS-impregnated samples failed for measuring of V-HMS-synthesized samples. LIBS measurements seem to be strongly influenced with different chemical forms of vanadium in impregnated and synthesised samples. The combination of LIBS and ED XRF is able to provide new information about measured samples (in our case for example about procedure of catalyst preparation). Copyright © 2010 Elsevier B.V. All rights reserved.

Quantification of Endogenous Cholesterol in Human Serum on Paper Using Direct Analysis in Real Time Mass Spectrometry.

PubMed

Hsieh, Hua-Yi; Li, Li-Hua; Hsu, Ren-Yu; Kao, Wei-Fong; Huang, Ying-Chen; Hsu, Cheng-Chih

2017-06-06

Blood testing for endogenous small metabolites to determine physiological and biochemical states is routine for laboratory analysis. Here we demonstrate that by combining the commercial direct analysis in real time (DART) ion source with an ion trap mass spectrometer, native cholesterol in its free alcohol form is readily detected from a few hundred nanoliters of human serum loaded onto chromatography paper. Deuterium-labeled cholesterol was used as the internal standard to obtain the absolute quantity of the endogenous cholesterol. The amount of the cholesterol measured by this paper-loaded DART mass spectrometry (pDART-MS) is statistically comparable with that obtained by using commercially available fluorometric-enzymatic assay and liquid chromatography/mass spectrometry. Furthermore, sera from 21 participants at three different time points in an ultramarathon were collected to obtain their cholesterol levels. The test requires only very minimal sample preparation, and the concentrations of cholesterol in each sample were acquired within a minute.

Vector Doppler: spatial sampling analysis and presentation techniques for real-time systems

NASA Astrophysics Data System (ADS)

Capineri, Lorenzo; Scabia, Marco; Masotti, Leonardo F.

2001-05-01

The aim of the vector Doppler (VD) technique is the quantitative reconstruction of a velocity field independently of the ultrasonic probe axis to flow angle. In particular vector Doppler is interesting for studying vascular pathologies related to complex blood flow conditions. Clinical applications require a real-time operating mode and the capability to perform Doppler measurements over a defined volume. The combination of these two characteristics produces a real-time vector velocity map. In previous works the authors investigated the theory of pulsed wave (PW) vector Doppler and developed an experimental system capable of producing off-line 3D vector velocity maps. Afterwards, for producing dynamic velocity vector maps, we realized a new 2D vector Doppler system based on a modified commercial echograph. The measurement and presentation of a vector velocity field requires a correct spatial sampling that must satisfy the Shannon criterion. In this work we tackled this problem, establishing a relationship between sampling steps and scanning system characteristics. Another problem posed by the vector Doppler technique is the data representation in real-time that should be easy to interpret for the physician. With this in mine we attempted a multimedia solution that uses both interpolated images and sound to represent the information of the measured vector velocity map. These presentation techniques were experimented for real-time scanning on flow phantoms and preliminary measurements in vivo on a human carotid artery.

Sampling and analyte enrichment strategies for ambient mass spectrometry.

PubMed

Li, Xianjiang; Ma, Wen; Li, Hongmei; Ai, Wanpeng; Bai, Yu; Liu, Huwei

2018-01-01

Ambient mass spectrometry provides great convenience for fast screening, and has showed promising potential in analytical chemistry. However, its relatively low sensitivity seriously restricts its practical utility in trace compound analysis. In this review, we summarize the sampling and analyte enrichment strategies coupled with nine modes of representative ambient mass spectrometry (desorption electrospray ionization, paper vhspray ionization, wooden-tip spray ionization, probe electrospray ionization, coated blade spray ionization, direct analysis in real time, desorption corona beam ionization, dielectric barrier discharge ionization, and atmospheric-pressure solids analysis probe) that have dramatically increased the detection sensitivity. We believe that these advances will promote routine use of ambient mass spectrometry. Graphical abstract Scheme of sampling stretagies for ambient mass spectrometry.

Conducting On-orbit Gene Expression Analysis on ISS: WetLab-2

NASA Technical Reports Server (NTRS)

Parra, Macarena; Almeida, Eduardo; Boone, Travis; Jung, Jimmy; Lera, Matthew P.; Ricco, Antonio; Souza, Kenneth; Wu, Diana; Richey, C. Scott

2013-01-01

WetLab-2 will enable expanded genomic research on orbit by developing tools that support in situ sample collection, processing, and analysis on ISS. This capability will reduce the time-to-results for investigators and define new pathways for discovery on the ISS National Lab. The primary objective is to develop a research platform on ISS that will facilitate real-time quantitative gene expression analysis of biological samples collected on orbit. WetLab-2 will be capable of processing multiple sample types ranging from microbial cultures to animal tissues dissected on orbit. WetLab-2 will significantly expand the analytical capabilities onboard ISS and enhance science return from ISS.

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

Flow injection trace gas analysis method for on-site determination of organoarsenicals

DOEpatents

Aldstadt, III, Joseph H.

1997-01-01

A method for real-time determination of the concentration of Lewisite in the ambient atmosphere, the method includes separating and collecting a Lewisite sample from the atmosphere in a collection chamber, converting the collected Lewisite to an arsenite ion solution sample, pumping the arsenite ion containing sample to an electrochemical detector connected to the collection chamber, and electrochemically detecting the converted arsenite ions in the sample, whereby the concentration of arsenite ions detected is proportional to the concentration of Lewisite in the atmosphere.

Trace level and highly selective determination of urea in various real samples based upon voltammetric analysis of diacetylmonoxime-urea reaction product on the carbon nanotube/carbon paste electrode.

PubMed

Alizadeh, Taher; Ganjali, Mohammad Reza; Rafiei, Faride

2017-06-29

In this study an innovative method was introduced for selective and precise determination of urea in various real samples including urine, blood serum, soil and water. The method was based on the square wave voltammetry determination of an electroactive product, generated during diacetylmonoxime reaction with urea. A carbon paste electrode, modified with multi-walled carbon nanotubes (MWCNTs) was found to be an appropriate electrochemical transducer for recording of the electrochemical signal. It was found that the chemical reaction conditions influenced the analytical signal directly. The calibration graph of the method was linear in the range of 1 × 10 -7 - 1 × 10 -2  mol L -1 . The detection limit was calculated to be 52 nmol L -1 . Relative standard error of the method was also calculated to be 3.9% (n = 3). The developed determination procedure was applied for urea determination in various real samples including soil, urine, plasma and water samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Real-Time and Delayed Analysis of Tree and Shrub Cores as Indicators of Subsurface Volatile Organic Compound Contamination, Durham Meadows Superfund Site, Durham, Connecticut, August 29, 2006

USGS Publications Warehouse

Vroblesky, Don A.; Willey, Richard E.; Clifford, Scott; Murphy, James J.

2008-01-01

This study examined volatile organic compound concentrations in cores from trees and shrubs for use as indicators of vadose-zone contamination or potential vapor intrusion by volatile organic compounds into buildings at the Durham Meadows Superfund Site, Durham, Connecticut. The study used both (1) real-time tree- and shrub-core analysis, which involved field heating the core samples for 5 to 10 minutes prior to field analysis, and (2) delayed analysis, which involved allowing the gases in the cores to equilibrate with the headspace gas in the sample vials unheated for 1 to 2 days prior to analysis. General correspondence was found between the two approaches, indicating that preheating and field analysis of vegetation cores is a viable approach to real-time monitoring of subsurface volatile organic compounds. In most cases, volatile organic compounds in cores from trees and shrubs at the Merriam Manufacturing Company property showed a general correspondence to the distribution of volatile organic compounds detected in a soil-gas survey, despite the fact that most of the soil-gas survey data in close proximity to the relevant trees were collected about 3 years prior to the tree-core collection. Most of the trees cored at the Durham Meadows Superfund Site, outside of the Merriam Manufacturing Company property, contained no volatile organic compounds and were in areas where indoor air sampling and soil-gas sampling showed little or no volatile organic compound concentrations. An exception was tree DM11, which contained barely detectable concentrations of trichloroethene near a house where previous investigations found low concentrations of trichloroethene (0.13 to 1.2 parts per billion by volume) in indoor air and 7.7 micrograms per liter of trichloroethene in the ground water. The barely detectable concentration of trichloroethene in tree DM11 and the lack of volatile organic compound detection in nearby tree DM10 (adjacent to the well having 7.7 micrograms of trichloroethene) may be attributable to the relatively large depth to water (17.6 feet), the relatively low soil-vapor trichloroethene concentration, and the large amount of rainfall during and preceding the tree-coring event. The data indicate that real-time and delayed analyses of tree cores are viable approaches to examining subsurface volatile organic compound soil-gas or vadose-zone contamination at the Durham Meadows Superfund Site and other similar sites. Thus, the methods may have application for determining the potential for vapor intrusion into buildings.

Direct Analysis in Real Time (DART) of an Organothiophosphate at Ultrahigh Resolution by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry

PubMed Central

Prokai, Laszlo; Stevens, Stanley M.

2016-01-01

Direct analysis in real time (DART) is a recently developed ambient ionization technique for mass spectrometry to enable rapid and sensitive analyses with little or no sample preparation. After swab-based field sampling, the organothiophosphate malathion was analyzed using DART-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Mass resolution was documented to be over 800,000 in full-scan MS mode and over 1,000,000 for an MS/MS product ion produced by collision-induced dissociation of the protonated analyte. Mass measurement accuracy below 1 ppm was obtained for all DART-generated ions that belonged to the test compound in the mass spectra acquired using only external mass calibration. This high mass measurement accuracy, achievable at present only through FTMS, was required for unequivocal identification of the corresponding molecular formulae. PMID:26784186

Direct Analysis in Real Time (DART) of an Organothiophosphate at Ultrahigh Resolution by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry.

PubMed

Prokai, Laszlo; Stevens, Stanley M

2016-01-16

Direct analysis in real time (DART) is a recently developed ambient ionization technique for mass spectrometry to enable rapid and sensitive analyses with little or no sample preparation. After swab-based field sampling, the organothiophosphate malathion was analyzed using DART-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Mass resolution was documented to be over 800,000 in full-scan MS mode and over 1,000,000 for an MS/MS product ion produced by collision-induced dissociation of the protonated analyte. Mass measurement accuracy below 1 ppm was obtained for all DART-generated ions that belonged to the test compound in the mass spectra acquired using only external mass calibration. This high mass measurement accuracy, achievable at present only through FTMS, was required for unequivocal identification of the corresponding molecular formulae.

Magnetic stirrer induced dispersive ionic-liquid microextraction for the determination of vanadium in water and food samples prior to graphite furnace atomic absorption spectrometry.

PubMed

Naeemullah; Kazi, Tasneem Gul; Tuzen, Mustafa

2015-04-01

A new dispersive liquid-liquid microextraction, magnetic stirrer induced dispersive ionic-liquid microextraction (MS-IL-DLLME) was developed to quantify the trace level of vanadium in real water and food samples by graphite furnace atomic absorption spectrometry (GFAAS). In this extraction method magnetic stirrer was applied to obtained a dispersive medium of 1-butyl-3-methylimidazolium hexafluorophosphate [C4MIM][PF6] in aqueous solution of (real water samples and digested food samples) to increase phase transfer ratio, which significantly enhance the recovery of vanadium - 4-(2-pyridylazo) resorcinol (PAR) chelate. Variables having vital role on desired microextraction methods were optimised to obtain the maximum recovery of study analyte. Under the optimised experimental variables, enhancement factor (EF) and limit of detection (LOD) were achieved to be 125 and 18 ng L(-1), respectively. Validity and accuracy of the desired method was checked by analysis of certified reference materials (SLRS-4 Riverine water and NIST SRM 1515 Apple leaves). The relative standard deviation (RSD) for 10 replicate determinations at 0.5 μg L(-1) of vanadium level was found to be <5.0%. This method was successfully applied to real water and acid digested food samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

An in vitro comparative study of the adaptation and sealing ability of two carrier-based root canal obturators.

PubMed

Alkahtani, Ahmed; Al-Subait, Sara; Anil, Sukumaran

2013-01-01

The study was done to assess the sealing ability and adaptation of RealSeal 1, and to compare it with Thermafil. 65 single-rooted extracted teeth were selected and root canal treatment was performed. Root canals were obturated with RealSeal 1 or Thermafil. A double chamber bacterial leakage model using E. faecalis was developed to assess the sealing ability. Samples were monitored daily for 60 days. After the bacterial leakage test, samples were embedded in resin and sectioned horizontally at 2 and 4 mm from the apical foramen. Specimens were examined under scanning electron microscope and digitally photographed. AutoCAD software was used to measure the gap between the canal surface and obturation material. Results were statistically analyzed using nonparametric Kaplan-Meier survival analysis for the bacterial leakage and t-test to compare the means of gap in RealSeal 1 and Thermafil at 2 and 4 mm. There was no significant difference between the RealSeal 1 and Thermafil with respect to leakage over time. At 2 mm and 4 mm, RealSeal 1 had significantly more gaps than Thermafil. From the observations it can be concluded that RealSeal 1 and Thermafil have comparable performance in terms of adaptation and sealing ability.

An In Vitro Comparative Study of the Adaptation and Sealing Ability of Two Carrier-Based Root Canal Obturators

PubMed Central

Alkahtani, Ahmed; Al-Subait, Sara; Anil, Sukumaran

2013-01-01

The study was done to assess the sealing ability and adaptation of RealSeal 1, and to compare it with Thermafil. 65 single-rooted extracted teeth were selected and root canal treatment was performed. Root canals were obturated with RealSeal 1 or Thermafil. A double chamber bacterial leakage model using E. faecalis was developed to assess the sealing ability. Samples were monitored daily for 60 days. After the bacterial leakage test, samples were embedded in resin and sectioned horizontally at 2 and 4 mm from the apical foramen. Specimens were examined under scanning electron microscope and digitally photographed. AutoCAD software was used to measure the gap between the canal surface and obturation material. Results were statistically analyzed using nonparametric Kaplan-Meier survival analysis for the bacterial leakage and t-test to compare the means of gap in RealSeal 1 and Thermafil at 2 and 4 mm. There was no significant difference between the RealSeal 1 and Thermafil with respect to leakage over time. At 2 mm and 4 mm, RealSeal 1 had significantly more gaps than Thermafil. From the observations it can be concluded that RealSeal 1 and Thermafil have comparable performance in terms of adaptation and sealing ability. PMID:23710141

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.

Biochip for Real-Time Monitoring of Hepatitis B Virus (HBV) by Combined Loop-Mediated Isothermal Amplification and Solution-Phase Electrochemical Detection

NASA Astrophysics Data System (ADS)

Tien, Bui Quang; Ngoc, Nguyen Thy; Loc, Nguyen Thai; Thu, Vu Thi; Lam, Tran Dai

2017-06-01

Accurate in situ diagnostic tests play a key role in patient management and control of most infectious diseases. To achieve this, use of handheld biochips that implement sample handling, sample analysis, and result readout together is an ideal approach. We present herein a fluid-handling biochip for real-time electrochemical monitoring of nucleic acid amplification based on loop-mediated isothermal amplification and real-time electrochemical detection on a microfluidic platform. Intercalation between amplifying DNA and free redox probe in solution phase was used to monitor the number of DNA copies. The whole diagnostic process is completed within 70 min. Our platform offers a fast and easy tool for quantification of viral pathogens in shorter time and with limited risk of all potential forms of cross-contamination. Such diagnostic tools have potential to make a huge difference to the lives of millions of people worldwide.

Evaluation of an Improved U.S. Food and Drug Administration Method for the Detection of Cyclospora cayetanensis in Produce Using Real-Time PCR.

PubMed

Murphy, Helen R; Lee, Seulgi; da Silva, Alexandre J

2017-07-01

Cyclospora cayetanensis is a protozoan parasite that causes human diarrheal disease associated with the consumption of fresh produce or water contaminated with C. cayetanensis oocysts. In the United States, foodborne outbreaks of cyclosporiasis have been linked to various types of imported fresh produce, including cilantro and raspberries. An improved method was developed for identification of C. cayetanensis in produce at the U.S. Food and Drug Administration. The method relies on a 0.1% Alconox produce wash solution for efficient recovery of oocysts, a commercial kit for DNA template preparation, and an optimized TaqMan real-time PCR assay with an internal amplification control for molecular detection of the parasite. A single laboratory validation study was performed to assess the method's performance and compare the optimized TaqMan real-time PCR assay and a reference nested PCR assay by examining 128 samples. The samples consisted of 25 g of cilantro or 50 g of raspberries seeded with 0, 5, 10, or 200 C. cayetanensis oocysts. Detection rates for cilantro seeded with 5 and 10 oocysts were 50.0 and 87.5%, respectively, with the real-time PCR assay and 43.7 and 94.8%, respectively, with the nested PCR assay. Detection rates for raspberries seeded with 5 and 10 oocysts were 25.0 and 75.0%, respectively, with the real-time PCR assay and 18.8 and 68.8%, respectively, with the nested PCR assay. All unseeded samples were negative, and all samples seeded with 200 oocysts were positive. Detection rates using the two PCR methods were statistically similar, but the real-time PCR assay is less laborious and less prone to amplicon contamination and allows monitoring of amplification and analysis of results, making it more attractive to diagnostic testing laboratories. The improved sample preparation steps and the TaqMan real-time PCR assay provide a robust, streamlined, and rapid analytical procedure for surveillance, outbreak response, and regulatory testing of foods for detection of C. cayetanensis.

Seeded amplification of chronic wasting disease prions in nasal brushings and recto-anal mucosal associated lymphoid tissues from elk by real time quaking-induced conversion

USGS Publications Warehouse

Haley, Nicholas J.; Siepker, Chris; Hoon-Hanks , Laura L.; Mitchell, Gordon; Walter, W. David; Manca, Matteo; Monello, Ryan J.; Powers, Jenny G.; Wild, Margaret A.; Hoover, Edward A.; Caughey, Byron; Richt, Jürgen a.; Fenwick, B.W.

2016-01-01

Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of cervids, was first documented nearly 50 years ago in Colorado and Wyoming and has since been detected across North America and the Republic of Korea. The expansion of this disease makes the development of sensitive diagnostic assays and antemortem sampling techniques crucial for the mitigation of its spread; this is especially true in cases of relocation/reintroduction or prevalence studies of large or protected herds, where depopulation may be contraindicated. This study evaluated the sensitivity of the real-time quaking-induced conversion (RT-QuIC) assay of recto-anal mucosa-associated lymphoid tissue (RAMALT) biopsy specimens and nasal brushings collected antemortem. These findings were compared to results of immunohistochemistry (IHC) analysis of ante- and postmortem samples. RAMALT samples were collected from populations of farmed and free-ranging Rocky Mountain elk (Cervus elaphus nelsoni; n = 323), and nasal brush samples were collected from a subpopulation of these animals (n = 205). We hypothesized that the sensitivity of RT-QuIC would be comparable to that of IHC analysis of RAMALT and would correspond to that of IHC analysis of postmortem tissues. We found RAMALT sensitivity (77.3%) to be highly correlative between RT-QuIC and IHC analysis. Sensitivity was lower when testing nasal brushings (34%), though both RAMALT and nasal brush test sensitivities were dependent on both the PRNP genotype and disease progression determined by the obex score. These data suggest that RT-QuIC, like IHC analysis, is a relatively sensitive assay for detection of CWD prions in RAMALT biopsy specimens and, with further investigation, has potential for large-scale and rapid automated testing of antemortem samples for CWD.

FY*X real-time polymerase chain reaction with melting curve analysis associated with a complete one-step real-time FY genotyping.

PubMed

Ansart-Pirenne, H; Martin-Blanc, S; Le Pennec, P-Y; Rouger, P; Cartron, J-P; Tournamille, C

2007-02-01

The Duffy (FY) blood group system is controlled by four major alleles: FY*A and FY*B, the Caucasian common alleles, encoding Fy(a) and Fy(b) antigens; FY*X allele responsible for a poorly expressed Fy(b) antigen, and FY*Fy a silent predominant allele among Black population. Despite the recent development of a real-time fluorescent polymerase chain reaction (PCR) method for FY genotyping FY*X genotyping has not been described by this method. This study focused on the real-time FY*X genotyping development associated with a complete, one-step real-time FY genotyping, based on fluorescence resonance energy transfer (FRET) technology. Seventy-two blood samples from Fy(a+b-) Caucasian blood donors were studied by real-time PCR only. Forty-seven Caucasian and Black individual blood samples, referred to our laboratory, were studied by PCR-RFLP and real-time PCR. For each individual, the result of the genotype was compared to the known phenotype. The FY*X allele frequency calculated in an Fy(a+b-) Caucasian blood donors population was 0.014. With the Caucasian and Black patient samples we found a complete correlation between PCR-RFLP and the real-time PCR method whatever the alleles combination tested. When the known phenotype was not correlated to FY*X genotype, the presence of the Fy(b) antigen was always confirmed by adsorption-elution. The real-time technology method is rapid and accurate for FY genotyping. From now, we are able to detect the FY*X allele in all the alleles combinations studied. Regarding its significant frequency, the detection of the FY*X allele is useful for the correct typing of blood donors and recipients considering the therapeutic use of blood units and the preparation of test red blood cells for antibody screening.

Rapid analysis of Δ-9-tetrahydrocannabinol in hair using direct analysis in real time ambient ionization orbitrap mass spectrometry.

PubMed

Duvivier, Wilco F; van Beek, Teris A; Pennings, Ed J M; Nielen, Michel W F

2014-04-15

Forensic hair analysis methods are laborious, time-consuming and provide only a rough retrospective estimate of the time of drug intake. Recently, hair imaging methods using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) were reported, but these methods require the application of MALDI matrix and are performed under vacuum. Direct analysis of entire locks of hair without any sample pretreatment and with improved spatial resolution would thus address a need. Hair samples were attached to stainless steel mesh screens and scanned in the X-direction using direct analysis in real time (DART) ambient ionization orbitrap MS. The DART gas temperature and the accuracy of the probed hair zone were optimized using Δ-9-tetrahydrocannabinol (THC) as a model compound. Since external contamination is a major issue in forensic hair analysis, sub-samples were measured before and after dichloromethane decontamination. The relative intensity of the THC signal in spiked blank hair versus that of quinine as the internal standard showed good reproducibility (26% RSD) and linearity of the method (R(2)  = 0.991). With the DART hair scan THC could be detected in hair samples from different chronic cannabis users. The presence of THC was confirmed by quantitative liquid chromatography/tandem mass spectrometry. Zones with different THC content could be clearly distinguished, indicating that the method might be used for retrospective timeline assessments. Detection of THC in decontaminated drug user hair showed that the DART hair scan not only probes THC on the surface of hair, but penetrates deeply enough to measure incorporated THC. A new approach in forensic hair analysis has been developed by probing complete locks of hair using DART-MS. Longitudinal scanning enables detection of incorporated compounds and can be used as pre-screening for THC without sample preparation. The method could also be adjusted for the analysis of other drugs of abuse. Copyright © 2014 John Wiley & Sons, Ltd.

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.

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

PubMed

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

2014-09-07

To compare the performance of the Da-an real-time hepatitis B virus (HBV) DNA assay and Abbott RealTime HBV assay. HBV DNA standards as well as a total of 180 clinical serum samples from patients with chronic hepatitis B were measured using the Abbott and Da-an real-time polymerase chain reaction (PCR) assays. Correlation and Bland-Altman plot analysis was used to compare the performance of the Abbott and Da-an assays. The HBV DNA levels were logarithmically transformed for analysis. All statistical analyses were performed using SPSS for Windows version 18.0. The correlation between the two assays was analyzed by Pearson's correlation and linear regression. The Bland-Altman plots were used for the analysis of agreement between the two assays. A P value of < 0.05 was considered statistically significant. The HBV DNA values measured by the Abbott or Da-an assay were significantly correlated with the expected values of HBV DNA standards (r = 0.999, for Abbott; r = 0.987, for Da-an, P < 0.001). A Bland-Altman plot showed good agreement between these two assays in detecting HBV DNA standards. Among the 180 clinical serum samples, 126 were quantifiable by both assays. Fifty-two samples were detectable by the Abbott assay but below the detection limit of the Da-an assay. Moreover, HBV DNA levels measured by the Abbott assay were significantly higher than those of the Da-an assay (6.23 ± 1.76 log IU/mL vs 5.46 ± 1.55 log IU/mL, P < 0.001). A positive correlation was observed between HBV DNA concentrations determined by the two assays in 126 paired samples (r = 0.648, P < 0.001). One hundred and fifteen of 126 (91.3%) specimens tested with both assays were within mean difference ± 1.96 SD of HBV DNA levels. The Da-an assay presented lower sensitivity and a narrower linear range as compared to the Abbott assay, suggesting the need to be improved.

Development of recombinase polymerase amplification assays for the rapid detection of peste des petits ruminants virus.

PubMed

Zhang, Yongning; Wang, Jianchang; Zhang, Zhou; Mei, Lin; Wang, Jinfeng; Wu, Shaoqiang; Lin, Xiangmei

2018-04-01

Peste des petits ruminants (PPR) is a severe infectious disease of small ruminants caused by PPR virus (PPRV). Rapid and sensitive detection of PPRV is critical for controlling PPR. This report describes the development and evaluation of a conventional reverse transcription recombinase polymerase amplification (RT-RPA) assay and a real-time RT-RPA assay, targeting the PPRV N gene. Sensitivity analysis revealed that the conventional RT-RPA assay could detect 852 copies of standard PPRV RNA per reaction at 95% probability within 20 min at 41 °C, and the real-time RT-RPA assay could detect 103 copies of RNA molecules per reaction at 95% probability. Specificity analysis showed that both assays have no cross-reactivity with nucleic acid templates prepared from other selected viruses or common pathogens. Clinical evaluation using 162 ovine and hircine serum and nasal swab samples showed that the performance of both the real-time RT-RPA assay and the conventional RT-RPA assay were comparable to that of real-time RT-PCR. The overall agreements between real-time RT-PCR and real-time RT-RPA, and conventional RT-RPA were 99.4% (161/162) and 98.8% (160/162), respectively. The R 2 value of real-time RT-RPA and real-time RT-PCR was 0.900 by linear regression analysis. Our results suggest that both RT-RPA assays have a potential application in the rapid, sensitive and specific detection of PPRV. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of laser-induced breakdown spectroscopy sensor to assess groundwater quality impacts resulting from geologic carbon sequestration

NASA Astrophysics Data System (ADS)

Carson, Cantwell G.; Goueguel, Christian; Jain, Jinesh; McIntyre, Dustin

2015-05-01

The injection of CO2 into deep aquifers can potentially affect the quality of groundwater supplies were leakage to occur from the injection formation or fluids. Therefore, the detection of CO2 and/or entrained contaminants that migrate into shallow groundwater aquifers is important both to assess storage permanence and to evaluate impacts on water resources. Naturally occurring elements (i.e., Li, Sr) in conjunction with isotope ratios can be used to detect such leakage. We propose the use of laser induced breakdown spectroscopy (LIBS) as an analytical technique to detect a suite of elements in water samples. LIBS has real time monitoring capabilities and can be applied for elemental and isotopic analysis of solid, liquid, and gas samples. The flexibility of probe design and use of fiber optics make it a suitable technique for real time measurements in harsh conditions and in hard to reach places. The laboratory scale experiments to measure Li, K, Ca, and Sr composition of water samples indicate that the technique produces rapid and reliable data. Since CO2 leakage from saline aquifers may accompany a brine solution, we studied the effect of sodium salts on the accuracy of LIBS analysis. This work specifically also details the fabrication and application of a miniature ruggedized remotely operated diode pumped solid state passively Q-switched laser system for use as the plasma excitation source for a real time LIBS analysis. This work also proposes the optical distribution of many laser spark sources across a wide area for widespread leak detection and basin monitoring.

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.

Detection of Diarrhea Etiology Among U.S. Military Personnel During Exercise Balikatan 2014, Philippines, Using TaqMan Array Cards.

PubMed

Lertsethtakarn, Paphavee; Nakjarung, Kaewkanya; Silapong, Sasikorn; Neesanant, Pimmnapar; Sakpaisal, Pimmada; Bodhidatta, Ladaporn; Liu, Jie; Houpt, Eric; Velasco, John Mark; Macareo, Louis R; Swierczewski, Brett E; Mason, Carl J

2016-11-01

Military personnel are vulnerable to diarrhea. Diarrheal disease is common when deployed for operations or exercise in developing countries. Although diarrheal disease is transient, cumulative time lost and medical asset can have a significant impact on military operations. Currently, diagnostics of diarrheal etiology typically relies on a mixture of conventional bacteriology, enzyme-linked immunosorbent assay, and polymerase chain reaction (PCR)-based methods including real-time PCR. These methods, however, can be time and labor intensive, although the identification of diarrheal etiology needs to be informative and rapid for treatment and prevention. Real-time PCR has been increasingly used to identify pathogens. Real-time PCR panels of common diarrheal pathogens have been developed, but several diarrheal pathogens are not included in the panel. An expanded and customizable panel to detect diarrhea etiology has been developed employing TaqMan Array Card (TAC) technology. TAC performs 384 real-time PCR reactions simultaneously. As currently configured for diarrheal disease by the University of Virginia, a maximum of 8 samples can be tested simultaneously with approximately 48 target pathogens per sample including bacteria, fungi, helminths, protozoan parasites, and viruses. TAC diarrheal disease panels have been successfully applied to detect pathogens in acute diarrheal stool samples from young children in several international multicenter diarrhea studies. In this study, TAC was applied to stool samples collected under an approved human use protocol from military personnel with acute diarrhea participating in the annual joint military exercise, Balikatan, between the Republic of the Philippines and the United States in 2014. Several established pathogen-specific real-time PCR detection assays were also performed in parallel for comparative purposes. TAC was applied to 7 stool samples. Campylobacter spp. was the most common diarrheal disease pathogen detected. Results from TAC matched 5 out of 6 pathogen specific real-time PCR assays. TAC required a total of 5-6 hours to complete all the procedures from nucleic acid extraction and data analysis, whereas a minimum of 18 hours and 4 hours are required for conventional bacteriology and enzyme-linked immunosorbent assay, respectively, per pathogen. With TAC, pathogen load can be estimated from the amount of nucleic acid present for each pathogen, which can be analyzed further to better determine pathogen attribution and to compare pathogen load between case and control samples. Unfortunately, such correlative analysis was not possible because of the limited sample size available in this study. A larger sample size is needed for further evaluation of TAC on a specific population set, including military personnel. Regardless, TAC was found to be a useful and functional diagnostic platform that is less time-consuming, easy to use with high reproducibility, and costs less per sample compared to the current typically employed methods. The successful application of TAC in acute diarrhea stool samples from a US military population in the Philippines demonstrates its versatility as a potential candidate for a next-generation diagnostics platform. Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.

Development of a fast framing detector for electron microscopy

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

Johnson, Ian J.; Bustillo, Karen C.; Ciston, Jim

2016-10-01

A high frame rate detector system is described that enables fast real-time data analysis of scanning diffraction experiments in scanning transmission electron microscopy (STEM). This is an end-to-end development that encompasses the data producing detector, data transportation, and real-time processing of data. The detector will consist of a central pixel sensor that is surrounded by annular silicon diodes. Both components of the detector system will synchronously capture data at almost 100 kHz frame rate, which produces an approximately 400 Gb/s data stream. Low-level preprocessing will be implemented in firmware before the data is streamed from the National Center for Electronmore » Microscopy (NCEM) to the National Energy Research Scientific Computing Center (NERSC). Live data processing, before it lands on disk, will happen on the Cori supercomputer and aims to present scientists with prompt experimental feedback. This online analysis will provide rough information of the sample that can be utilized for sample alignment, sample monitoring and verification that the experiment is set up correctly. Only a compressed version of the relevant data is then selected for more in-depth processing.« less

Identification of the country of growth of Sophora flavescens using direct analysis in real time mass spectrometry (DART-MS).

PubMed

Fukuda, Eriko; Uesawa, Yoshihiro; Baba, Masaki; Suzuki, Ryuichiro; Fukuda, Tatsuo; Shirataki, Yoshiaki; Okada, Yoshihito

2014-11-01

In order to identify the country of growth of Sophora flavescens by chemical fingerprinting, extracts of plants grown in China and Japan were analyzed using direct analysis in real time mass spectrometry (DART)-MS. The peaks characteristic of each country of growth were statistically analyzed using a volcano plot to summarize the relationship between the p-values of a statistical test and the magnitude of the difference in the peak intensities of the samples in the groups. Peaks with ap value < 0.05 in the t-test and a ≥ 2 absolute difference were defined as characteristic. Peaks characteristic of Chinese S. flavescens were found at m/z 439 and 440. In contrast, peaks characteristic of Japanese S. flavescens were found at m/z 313, 423, 437 and 441. The intensity of the selected peaks was similar in Japanese samples, whereas the m/z 439 peak had a significantly higher intensity than the other peaks in Chinese samples. Therefore, differences in selected peak patterns may allow identification of the country of growth of S. flavescens.

Rapid quality assessment of Radix Aconiti Preparata using direct analysis in real time mass spectrometry.

PubMed

Zhu, Hongbin; Wang, Chunyan; Qi, Yao; Song, Fengrui; Liu, Zhiqiang; Liu, Shuying

2012-11-08

This study presents a novel and rapid method to identify chemical markers for the quality control of Radix Aconiti Preparata, a world widely used traditional herbal medicine. In the method, the samples with a fast extraction procedure were analyzed using direct analysis in real time mass spectrometry (DART MS) combined with multivariate data analysis. At present, the quality assessment approach of Radix Aconiti Preparata was based on the two processing methods recorded in Chinese Pharmacopoeia for the purpose of reducing the toxicity of Radix Aconiti and ensuring its clinical therapeutic efficacy. In order to ensure the safety and effectivity in clinical use, the processing degree of Radix Aconiti should be well controlled and assessed. In the paper, hierarchical cluster analysis and principal component analysis were performed to evaluate the DART MS data of Radix Aconiti Preparata samples in different processing times. The results showed that the well processed Radix Aconiti Preparata, unqualified processed and the raw Radix Aconiti could be clustered reasonably corresponding to their constituents. The loading plot shows that the main chemical markers having the most influence on the discrimination amongst the qualified and unqualified samples were mainly some monoester diterpenoid aconitines and diester diterpenoid aconitines, i.e. benzoylmesaconine, hypaconitine, mesaconitine, neoline, benzoylhypaconine, benzoylaconine, fuziline, aconitine and 10-OH-mesaconitine. The established DART MS approach in combination with multivariate data analysis provides a very flexible and reliable method for quality assessment of toxic herbal medicine. Copyright © 2012 Elsevier B.V. All rights reserved.

Theoretical analysis to interpret projected image data from in-situ 3-dimensional equiaxed nucleation and growth

NASA Astrophysics Data System (ADS)

Mooney, Robin P.; McFadden, Shaun

2017-12-01

In-situ observation of crystal growth in transparent media allows us to observe solidification phase change in real-time. These systems are analogous to opaque systems such as metals. The interpretation of transient 2-dimensional area projections from 3-dimensional phase change phenomena occurring in a bulky sample is problematic due to uncertainty of impingement and hidden nucleation events; in stereology this problem is known as over-projection. This manuscript describes and demonstrates a continuous model for nucleation and growth using the well-established Johnson-Mehl-Avrami-Kolmogorov model, and provides a method to relate 3-dimensional volumetric data (nucleation events, volume fraction) to observed data in a 2-dimensional projection (nucleation count, area fraction). A parametric analysis is performed; the projection phenomenon is shown to be significant in cases where nucleation is occurring continuously with a relatively large variance. In general, area fraction on a projection plane will overestimate the volume fraction within the sample and the nuclei count recorded on the projection plane will underestimate the number of real nucleation events. The statistical framework given in this manuscript provides a methodology to deal with the differences between the observed (projected) data and the real (volumetric) measures.

Zero valent Fe-reduced graphene oxide quantum dots as a novel magnetic dispersive solid phase microextraction sorbent for extraction of organophosphorus pesticides in real water and fruit juice samples prior to analysis by gas chromatography-mass spectrometry.

PubMed

Akbarzade, Samaneh; Chamsaz, Mahmoud; Rounaghi, Gholam Hossein; Ghorbani, Mahdi

2018-01-01

A selective and sensitive magnetic dispersive solid-phase microextraction (MDSPME) coupled with gas chromatography-mass spectrometry was developed for extraction and determination of organophosphorus pesticides (Sevin, Fenitrothion, Malathion, Parathion, and Diazinon) in fruit juice and real water samples. Zero valent Fe-reduced graphene oxide quantum dots (rGOQDs@ Fe) as a new and effective sorbent were prepared and applied for extraction of organophosphorus pesticides using MDSPME method. In order to study the performance of this new sorbent, the ability of rGOQDs@ Fe was compared with graphene oxide and magnetic graphene oxide nanocomposite by recovery experiments of the organophosphorus pesticides. Several affecting parameters in the microextraction procedure, including pH of donor phase, donor phase volume, stirring rate, extraction time, and desorption conditions such as the type and volume of solvents and desorption time were thoroughly investigated and optimized. Under the optimal conditions, the method showed a wide linear dynamic range with R-square between 0.9959 and 0.9991. The limit of detections, the intraday and interday relative standard deviations (n = 5) were less than 0.07 ngmL -1 , 4.7, and 8.6%, respectively. The method was successfully applied for extraction and determination of organophosphorus pesticides in real water samples (well, river and tap water) and fruit juice samples (apple and grape juice). The obtained relative recoveries were in the range of 82.9%-113.2% with RSD percentages of less than 5.8% for all the real samples.

A Class of Population Covariance Matrices in the Bootstrap Approach to Covariance Structure Analysis

ERIC Educational Resources Information Center

Yuan, Ke-Hai; Hayashi, Kentaro; Yanagihara, Hirokazu

2007-01-01

Model evaluation in covariance structure analysis is critical before the results can be trusted. Due to finite sample sizes and unknown distributions of real data, existing conclusions regarding a particular statistic may not be applicable in practice. The bootstrap procedure automatically takes care of the unknown distribution and, for a given…

Energy-filtered real- and k-space secondary and energy-loss electron imaging with Dual Emission Electron spectro-Microscope: Cs/Mo(110).

PubMed

Grzelakowski, Krzysztof P

2016-05-01

Since its introduction the importance of complementary k||-space (LEED) and real space (LEEM) information in the investigation of surface science phenomena has been widely demonstrated over the last five decades. In this paper we report the application of a novel kind of electron spectromicroscope Dual Emission Electron spectroMicroscope (DEEM) with two independent electron optical channels for reciprocal and real space quasi-simultaneous imaging in investigation of a Cs covered Mo(110) single crystal by using the 800eV electron beam from an "in-lens" electron gun system developed for the sample illumination. With the DEEM spectromicroscope it is possible to observe dynamic, irreversible processes at surfaces in the energy-filtered real space and in the corresponding energy-filtered kǁ-space quasi-simultaneously in two independent imaging columns. The novel concept of the high energy electron beam sample illumination in the cathode lens based microscopes allows chemically selective imaging and analysis under laboratory conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

Image analysis of representative food structures: application of the bootstrap method.

PubMed

Ramírez, Cristian; Germain, Juan C; Aguilera, José M

2009-08-01

Images (for example, photomicrographs) are routinely used as qualitative evidence of the microstructure of foods. In quantitative image analysis it is important to estimate the area (or volume) to be sampled, the field of view, and the resolution. The bootstrap method is proposed to estimate the size of the sampling area as a function of the coefficient of variation (CV(Bn)) and standard error (SE(Bn)) of the bootstrap taking sub-areas of different sizes. The bootstrap method was applied to simulated and real structures (apple tissue). For simulated structures, 10 computer-generated images were constructed containing 225 black circles (elements) and different coefficient of variation (CV(image)). For apple tissue, 8 images of apple tissue containing cellular cavities with different CV(image) were analyzed. Results confirmed that for simulated and real structures, increasing the size of the sampling area decreased the CV(Bn) and SE(Bn). Furthermore, there was a linear relationship between the CV(image) and CV(Bn) (.) For example, to obtain a CV(Bn) = 0.10 in an image with CV(image) = 0.60, a sampling area of 400 x 400 pixels (11% of whole image) was required, whereas if CV(image) = 1.46, a sampling area of 1000 x 100 pixels (69% of whole image) became necessary. This suggests that a large-size dispersion of element sizes in an image requires increasingly larger sampling areas or a larger number of images.

Effect of separate sampling on classification accuracy.

PubMed

Shahrokh Esfahani, Mohammad; Dougherty, Edward R

2014-01-15

Measurements are commonly taken from two phenotypes to build a classifier, where the number of data points from each class is predetermined, not random. In this 'separate sampling' scenario, the data cannot be used to estimate the class prior probabilities. Moreover, predetermined class sizes can severely degrade classifier performance, even for large samples. We employ simulations using both synthetic and real data to show the detrimental effect of separate sampling on a variety of classification rules. We establish propositions related to the effect on the expected classifier error owing to a sampling ratio different from the population class ratio. From these we derive a sample-based minimax sampling ratio and provide an algorithm for approximating it from the data. We also extend to arbitrary distributions the classical population-based Anderson linear discriminant analysis minimax sampling ratio derived from the discriminant form of the Bayes classifier. All the codes for synthetic data and real data examples are written in MATLAB. A function called mmratio, whose output is an approximation of the minimax sampling ratio of a given dataset, is also written in MATLAB. All the codes are available at: http://gsp.tamu.edu/Publications/supplementary/shahrokh13b.

Predictive modeling in Clostridium acetobutylicum fermentations employing Raman spectroscopy and multivariate data analysis for real-time culture monitoring

NASA Astrophysics Data System (ADS)

Zu, Theresah N. K.; Liu, Sanchao; Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Mackie, David M.; Sund, Christian J.

2016-05-01

The coupling of optical fibers with Raman instrumentation has proven to be effective for real-time monitoring of chemical reactions and fermentations when combined with multivariate statistical data analysis. Raman spectroscopy is relatively fast, with little interference from the water peak present in fermentation media. Medical research has explored this technique for analysis of mammalian cultures for potential diagnosis of some cancers. Other organisms studied via this route include Escherichia coli, Saccharomyces cerevisiae, and some Bacillus sp., though very little work has been performed on Clostridium acetobutylicum cultures. C. acetobutylicum is a gram-positive anaerobic bacterium, which is highly sought after due to its ability to use a broad spectrum of substrates and produce useful byproducts through the well-known Acetone-Butanol-Ethanol (ABE) fermentation. In this work, real-time Raman data was acquired from C. acetobutylicum cultures grown on glucose. Samples were collected concurrently for comparative off-line product analysis. Partial-least squares (PLS) models were built both for agitated cultures and for static cultures from both datasets. Media components and metabolites monitored include glucose, butyric acid, acetic acid, and butanol. Models were cross-validated with independent datasets. Experiments with agitation were more favorable for modeling with goodness of fit (QY) values of 0.99 and goodness of prediction (Q2Y) values of 0.98. Static experiments did not model as well as agitated experiments. Raman results showed the static experiments were chaotic, especially during and shortly after manual sampling.

European validation of Real-Time PCR method for detection of Salmonella spp. in pork meat.

PubMed

Delibato, Elisabetta; Rodriguez-Lazaro, David; Gianfranceschi, Monica; De Cesare, Alessandra; Comin, Damiano; Gattuso, Antonietta; Hernandez, Marta; Sonnessa, Michele; Pasquali, Frédérique; Sreter-Lancz, Zuzsanna; Saiz-Abajo, María-José; Pérez-De-Juan, Javier; Butrón, Javier; Prukner-Radovcic, Estella; Horvatek Tomic, Danijela; Johannessen, Gro S; Jakočiūnė, Džiuginta; Olsen, John E; Chemaly, Marianne; Le Gall, Francoise; González-García, Patricia; Lettini, Antonia Anna; Lukac, Maja; Quesne, Segolénè; Zampieron, Claudia; De Santis, Paola; Lovari, Sarah; Bertasi, Barbara; Pavoni, Enrico; Proroga, Yolande T R; Capuano, Federico; Manfreda, Gerardo; De Medici, Dario

2014-08-01

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

Number-Density Measurements of CO2 in Real Time with an Optical Frequency Comb for High Accuracy and Precision

NASA Astrophysics Data System (ADS)

Scholten, Sarah K.; Perrella, Christopher; Anstie, James D.; White, Richard T.; Al-Ashwal, Waddah; Hébert, Nicolas Bourbeau; Genest, Jérôme; Luiten, Andre N.

2018-05-01

Real-time and accurate measurements of gas properties are highly desirable for numerous real-world applications. Here, we use an optical-frequency comb to demonstrate absolute number-density and temperature measurements of a sample gas with state-of-the-art precision and accuracy. The technique is demonstrated by measuring the number density of 12C16O2 with an accuracy of better than 1% and a precision of 0.04% in a measurement and analysis cycle of less than 1 s. This technique is transferable to numerous molecular species, thus offering an avenue for near-universal gas concentration measurements.

Effect of the absolute statistic on gene-sampling gene-set analysis methods.

PubMed

Nam, Dougu

2017-06-01

Gene-set enrichment analysis and its modified versions have commonly been used for identifying altered functions or pathways in disease from microarray data. In particular, the simple gene-sampling gene-set analysis methods have been heavily used for datasets with only a few sample replicates. The biggest problem with this approach is the highly inflated false-positive rate. In this paper, the effect of absolute gene statistic on gene-sampling gene-set analysis methods is systematically investigated. Thus far, the absolute gene statistic has merely been regarded as a supplementary method for capturing the bidirectional changes in each gene set. Here, it is shown that incorporating the absolute gene statistic in gene-sampling gene-set analysis substantially reduces the false-positive rate and improves the overall discriminatory ability. Its effect was investigated by power, false-positive rate, and receiver operating curve for a number of simulated and real datasets. The performances of gene-set analysis methods in one-tailed (genome-wide association study) and two-tailed (gene expression data) tests were also compared and discussed.

PROBABILITY SAMPLING AND POPULATION INFERENCE IN MONITORING PROGRAMS

EPA Science Inventory

A fundamental difference between probability sampling and conventional statistics is that "sampling" deals with real, tangible populations, whereas "conventional statistics" usually deals with hypothetical populations that have no real-world realization. he focus here is on real ...

Sample preparation techniques for the determination of trace residues and contaminants in foods.

PubMed

Ridgway, Kathy; Lalljie, Sam P D; Smith, Roger M

2007-06-15

The determination of trace residues and contaminants in complex matrices, such as food, often requires extensive sample extraction and preparation prior to instrumental analysis. Sample preparation is often the bottleneck in analysis and there is a need to minimise the number of steps to reduce both time and sources of error. There is also a move towards more environmentally friendly techniques, which use less solvent and smaller sample sizes. Smaller sample size becomes important when dealing with real life problems, such as consumer complaints and alleged chemical contamination. Optimal sample preparation can reduce analysis time, sources of error, enhance sensitivity and enable unequivocal identification, confirmation and quantification. This review considers all aspects of sample preparation, covering general extraction techniques, such as Soxhlet and pressurised liquid extraction, microextraction techniques such as liquid phase microextraction (LPME) and more selective techniques, such as solid phase extraction (SPE), solid phase microextraction (SPME) and stir bar sorptive extraction (SBSE). The applicability of each technique in food analysis, particularly for the determination of trace organic contaminants in foods is discussed.

ANALYSIS OF ENTEROCOCCUS FAECALIS IN SAMPLES FROM TURKISH PATIENTS WITH PRIMARY ENDODONTIC INFECTIONS AND FAILED ENDODONTIC TREATMENT BY REAL-TIME PCR SYBR GREEN METHOD

PubMed Central

Ozbek, Selcuk M.; Ozbek, Ahmet; Erdogan, Aziz S.

2009-01-01

Objective: The aims of this study were to investigate the presence of Enterococcus faecalis in primary endodontic infections and failed endodontic treatments using real-time PCR and to determine the statistical importance of the presence of E. faecalis in a Turkish population with endodontic infections. Material and Methods: E. faecalis was investigated from 79 microbial samples collected from patients who were treated at the Endodontic Clinic of the Dental School of Atatürk University (Erzurum, Turkey). Microbial samples were taken from 43 patients (Group 1) with failed endodontic treatments and 36 patients (Group 2) with chronic apical periodontitis (primary endodontic infections). DNA was extracted from the samples by using a QIAamp® DNA mini-kit and analyzed with real-time PCR SYBR Green. Results: E. faecalis was detected in 41 out of 79 patients, suggesting that it exists in not less than 61% of all endodontic infections when the proportion test (z= -1.645,

Determination of biogenic amines in chocolate by ion chromatographic separation and pulsed integrated amperometric detection with implemented wave-form at Au disposable electrode.

PubMed

Pastore, Paolo; Favaro, Gabriella; Badocco, Denis; Tapparo, Andrea; Cavalli, Silvano; Saccani, Giovanna

2005-12-09

A rapid and selective cation exchange chromatographic method coupled to integrated pulsed amperometric detection (PAD) has been developed to quantify biogenic amines in chocolate. The method is based on gradient elution of aqueous methanesulfonic acid with post column addition of strong base to obtain suitable conditions for amperometric detection. A potential waveform able to keep long time performance of the Au disposable electrode was set up. Total analysis time is less than 20min. Concentration levels of dopamine, serotonin, tyramine, histamine and 2-phenylethylamine were measured, after extraction with perchloric acid from 2g samples previously defatted twice with petroleum ether. The method was used to determine the analytes in chocolate real matrices and their quantification was made with standard addition method. Only dopamine, histamine and serotonin were found in the analysed real samples. Repeatabilities of their signals, computed on their amounts in the real samples, were 5% for all of them. Repeatabilities of tyramine and phenethylamine were relative to standard additions to real samples (close to 1mg/l in the extract) and were 7 and 3%, respectively. Detection limits were computed with the 3s of the baseline noise combined with the calibration plot regression parameters. They were satisfactorily low for all amines: 3mg/kg for dopamine, 2mg/kg for tyramine, 1mg/kg for histamine, 2mg/kg for serotonin, 3mg/kg for 2-phenylethylamine.

[Application of transcription mediated amplification and real-time reverse transcription polymerase chain reaction in detection of human immunodeficiency virus RNA].

PubMed

Wu, Daxian; Tao, Shuhui; Liu, Shuiping; Zhou, Jiebin; Tan, Deming; Hou, Zhouhua

2017-07-28

To observe the sensitivity of transcription mediated amplification (TMA), and to compare its performance with real-time reverse transcription polymerase chain reaction (real-time RT-PCR) in detecting human immunodeficiency virus RNA (HIV RNA).
 Methods: TMA system was established with TaqMan probes, specific primers, moloney murine leukemia virus (MMLV) reverse transcriptase, T7 RNA polymerase, and reaction substrates. The sensitivity of TMA was evaluated by amplifying a group of 10-fold diluted HIV RNA standards which were transcribed in vitro. A total of 60 plasma of HIV infected patients were measured by TMA and Cobas Amplicor HIV-1 Monitor test to observe the positive rate. The correlation and concordance of the above two technologies were investigated by linear regression and Bland-Altman analysis.
 Results: TMA system was established successfully and HIV RNA transcribed standards at concentration of equal or more than 10 copies/mL could be detected by TMA technology. Among 60 samples of plasma from HIV infected patients, 46 were positively detected and 12 were negatively amplified by both TMA and Cobas reagents; 2 samples were positively tested by Cobas reagent but negatively tested by TMA system. The concordance rate of the two methods was 97.1% and the difference of positive detection rate between the two methods was not statistically significant (P>0.05). Linear regression was used for 46 samples which were positively detected by both TMA and Cobas reagents and showed an excellent correlation between the two reagents (r=0.997, P<0.001). Bland-Altma analysis revealed that the mean different value of HIV RNA levels for denary logarithm was 0.02. Forty-four samples were included in 95% of credibility interval of concordance.
 Conclusion: TMA system has the potential of high sensitivity. TMA and real-time RT-PCR keep an excellent correlation and consistency in detecting HIV RNA.

Synthetic geology - Exploring the "what if?" in geology

NASA Astrophysics Data System (ADS)

Klump, J. F.; Robertson, J.

2015-12-01

The spatial and temporal extent of geological phenomena makes experiments in geology difficult to conduct, if not entirely impossible and collection of data is laborious and expensive - so expensive that most of the time we cannot test a hypothesis. The aim, in many cases, is to gather enough data to build a predictive geological model. Even in a mine, where data are abundant, a model remains incomplete because the information at the level of a blasting block is two orders of magnitude larger than the sample from a drill core, and we have to take measurement errors into account. So, what confidence can we have in a model based on sparse data, uncertainties and measurement error? Synthetic geology does not attempt to model the real world in terms of geological processes with all their uncertainties, rather it offers an artificial geological data source with fully known properties. On the basis of this artificial geology, we can simulate geological sampling by established or future technologies to study the resulting dataset. Conducting these experiments in silico removes the constraints of testing in the field or in production, and provides us with a known ground-truth against which the steps in a data analysis and integration workflow can be validated.Real-time simulation of data sources can be used to investigate crucial questions such as the potential information gain from future sensing capabilities, or from new sampling strategies, or the combination of both, and it enables us to test many "what if?" questions, both in geology and in data engineering. What would we be able to see if we could obtain data at higher resolution? How would real-time data analysis change sampling strategies? Does our data infrastructure handle many new real-time data streams? What feature engineering can be deducted for machine learning approaches? By providing a 'data sandbox' able to scale to realistic geological scenarios we hope to start answering some of these questions.

DEVELOPMENT AND EVALUATION OF AN ENZYME-LINKED IMMUNOASSAY (ELISA) METHOD FOR THE MEASUREMENT OF 2,4-DICHLOROPHENOXYACETIC ACID IN HUMAN URINE

EPA Science Inventory

This paper describes the development of a 96-microwell high sample capacity ELISA method for measuring 2,4-D in urine; the analysis of 2,4-D in real-world urine samples by both ELISA and GC/MS methods; and compares the ELISA and GC/MS results in several key areas: accuracy, preci...

Calibration-free quantitative elemental analysis of meteor plasma using reference laser-induced breakdown spectroscopy of meteorite samples

NASA Astrophysics Data System (ADS)

Ferus, Martin; Koukal, Jakub; Lenža, Libor; Srba, Jiří; Kubelík, Petr; Laitl, Vojtěch; Zanozina, Ekaterina M.; Váňa, Pavel; Kaiserová, Tereza; Knížek, Antonín; Rimmer, Paul; Chatzitheodoridis, Elias; Civiš, Svatopluk

2018-03-01

Aims: We aim to analyse real-time Perseid and Leonid meteor spectra using a novel calibration-free (CF) method, which is usually applied in the laboratory for laser-induced breakdown spectroscopic (LIBS) chemical analysis. Methods: Reference laser ablation spectra of specimens of chondritic meteorites were measured in situ simultaneously with a high-resolution laboratory echelle spectrograph and a spectral camera for meteor observation. Laboratory data were subsequently evaluated via the CF method and compared with real meteor emission spectra. Additionally, spectral features related to airglow plasma were compared with the spectra of laser-induced breakdown and electric discharge in the air. Results: We show that this method can be applied in the evaluation of meteor spectral data observed in real time. Specifically, CF analysis can be used to determine the chemical composition of meteor plasma, which, in the case of the Perseid and Leonid meteors analysed in this study, corresponds to that of the C-group of chondrites.

Crystalline nucleation in undercooled liquids: a Bayesian data-analysis approach for a nonhomogeneous Poisson process.

PubMed

Filipponi, A; Di Cicco, A; Principi, E

2012-12-01

A Bayesian data-analysis approach to data sets of maximum undercooling temperatures recorded in repeated melting-cooling cycles of high-purity samples is proposed. The crystallization phenomenon is described in terms of a nonhomogeneous Poisson process driven by a temperature-dependent sample nucleation rate J(T). The method was extensively tested by computer simulations and applied to real data for undercooled liquid Ge. It proved to be particularly useful in the case of scarce data sets where the usage of binned data would degrade the available experimental information.

Real-Time Processing Library for Open-Source Hardware Biomedical Sensors

PubMed Central

Castro-García, Juan A.; Lebrato-Vázquez, Clara

2018-01-01

Applications involving data acquisition from sensors need samples at a preset frequency rate, the filtering out of noise and/or analysis of certain frequency components. We propose a novel software architecture based on open-software hardware platforms which allows programmers to create data streams from input channels and easily implement filters and frequency analysis objects. The performances of the different classes given in the size of memory allocated and execution time (number of clock cycles) were analyzed in the low-cost platform Arduino Genuino. In addition, 11 people took part in an experiment in which they had to implement several exercises and complete a usability test. Sampling rates under 250 Hz (typical for many biomedical applications) makes it feasible to implement filters, sliding windows and Fourier analysis, operating in real time. Participants rated software usability at 70.2 out of 100 and the ease of use when implementing several signal processing applications was rated at just over 4.4 out of 5. Participants showed their intention of using this software because it was percieved as useful and very easy to use. The performances of the library showed that it may be appropriate for implementing small biomedical real-time applications or for human movement monitoring, even in a simple open-source hardware device like Arduino Genuino. The general perception about this library is that it is easy to use and intuitive. PMID:29596394

Real-Time Processing Library for Open-Source Hardware Biomedical Sensors.

PubMed

Molina-Cantero, Alberto J; Castro-García, Juan A; Lebrato-Vázquez, Clara; Gómez-González, Isabel M; Merino-Monge, Manuel

2018-03-29

Applications involving data acquisition from sensors need samples at a preset frequency rate, the filtering out of noise and/or analysis of certain frequency components. We propose a novel software architecture based on open-software hardware platforms which allows programmers to create data streams from input channels and easily implement filters and frequency analysis objects. The performances of the different classes given in the size of memory allocated and execution time (number of clock cycles) were analyzed in the low-cost platform Arduino Genuino. In addition, 11 people took part in an experiment in which they had to implement several exercises and complete a usability test. Sampling rates under 250 Hz (typical for many biomedical applications) makes it feasible to implement filters, sliding windows and Fourier analysis, operating in real time. Participants rated software usability at 70.2 out of 100 and the ease of use when implementing several signal processing applications was rated at just over 4.4 out of 5. Participants showed their intention of using this software because it was percieved as useful and very easy to use. The performances of the library showed that it may be appropriate for implementing small biomedical real-time applications or for human movement monitoring, even in a simple open-source hardware device like Arduino Genuino. The general perception about this library is that it is easy to use and intuitive.

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

NASA Technical Reports Server (NTRS)

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

2013-01-01

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

Estimation and enhancement of real-time software reliability through mutation analysis

NASA Technical Reports Server (NTRS)

Geist, Robert; Offutt, A. J.; Harris, Frederick C., Jr.

1992-01-01

A simulation-based technique for obtaining numerical estimates of the reliability of N-version, real-time software is presented. An extended stochastic Petri net is employed to represent the synchronization structure of N versions of the software, where dependencies among versions are modeled through correlated sampling of module execution times. Test results utilizing specifications for NASA's planetary lander control software indicate that mutation-based testing could hold greater potential for enhancing reliability than the desirable but perhaps unachievable goal of independence among N versions.

Flow injection trace gas analysis method for on-site determination of organoarsenicals

DOEpatents

Aldstadt, J.H. III

1997-06-24

A method is described for real-time determination of the concentration of Lewisite in the ambient atmosphere, the method includes separating and collecting a Lewisite sample from the atmosphere in a collection chamber, converting the collected Lewisite to an arsenite ion solution sample, pumping the arsenite ion containing sample to an electrochemical detector connected to the collection chamber, and electrochemically detecting the converted arsenite ions in the sample, whereby the concentration of arsenite ions detected is proportional to the concentration of Lewisite in the atmosphere. 2 figs.

Qualitative analysis of tackifier resins in pressure sensitive adhesives using direct analysis in real time time-of-flight mass spectrometry.

PubMed

Mess, Aylin; Vietzke, Jens-Peter; Rapp, Claudius; Francke, Wittko

2011-10-01

Tackifier resins play an important role as additives in pressure sensitive adhesives (PSAs) to modulate their desired properties. With dependence on their origin and processing, tackifier resins can be multicomponent mixtures. Once they have been incorporated in a polymer matrix, conventional chemical analysis of tackifiers usually tends to be challenging because a suitable sample pretreatment and/or separation is necessary and all characteristic components have to be detected for an unequivocal identification of the resin additive. Nevertheless, a reliable analysis of tackifiers is essential for product quality and safety reasons. A promising approach for the examination of tackifier resins in PSAs is the novel direct analysis in real time mass spectrometry (DART-MS) technique, which enables screening analysis without time-consuming sample preparation. In the present work, four key classes of tackifier resins were studied (rosin, terpene phenolic, polyterpene, and hydrocarbon resins). Their corresponding complex mass spectra were interpreted and used as reference spectra for subsequent analyses. These data were used to analyze tackifier additives in synthetic rubber and acrylic adhesive matrixes. To prove the efficiency of the developed method, complete PSA products containing two or three different tackifiers were analyzed. The tackifier resins were successfully identified, while measurement time and interpretation took less than 10 mins per sample. Determination of resin additives in PSAs can be performed down to 0.1% (w/w, limit of detection) using the three most abundant signals for each tackifier. In summary, DART-MS is a rapid and efficient screening method for the analysis of various tackifiers in PSAs.

An investigation of the effects of relevant samples and a comparison of verification versus discovery based lab design

NASA Astrophysics Data System (ADS)

Rieben, James C., Jr.

This study focuses on the effects of relevance and lab design on student learning within the chemistry laboratory environment. A general chemistry conductivity of solutions experiment and an upper level organic chemistry cellulose regeneration experiment were employed. In the conductivity experiment, the two main variables studied were the effect of relevant (or "real world") samples on student learning and a verification-based lab design versus a discovery-based lab design. With the cellulose regeneration experiment, the effect of a discovery-based lab design vs. a verification-based lab design was the sole focus. Evaluation surveys consisting of six questions were used at three different times to assess student knowledge of experimental concepts. In the general chemistry laboratory portion of this study, four experimental variants were employed to investigate the effect of relevance and lab design on student learning. These variants consisted of a traditional (or verification) lab design, a traditional lab design using "real world" samples, a new lab design employing real world samples/situations using unknown samples, and the new lab design using real world samples/situations that were known to the student. Data used in this analysis were collected during the Fall 08, Winter 09, and Fall 09 terms. For the second part of this study a cellulose regeneration experiment was employed to investigate the effects of lab design. A demonstration creating regenerated cellulose "rayon" was modified and converted to an efficient and low-waste experiment. In the first variant students tested their products and verified a list of physical properties. In the second variant, students filled in a blank physical property chart with their own experimental results for the physical properties. Results from the conductivity experiment show significant student learning of the effects of concentration on conductivity and how to use conductivity to differentiate solution types with the use of real world samples. In the organic chemistry experiment, results suggest that the discovery-based design improved student retention of the chain length differentiation by physical properties relative to the verification-based design.

Clinical evaluation of β-tubulin real-time PCR for rapid diagnosis of dermatophytosis, a comparison with mycological methods.

PubMed

Motamedi, Marjan; Mirhendi, Hossein; Zomorodian, Kamiar; Khodadadi, Hossein; Kharazi, Mahboobeh; Ghasemi, Zeinab; Shidfar, Mohammad Reza; Makimura, Koichi

2017-10-01

Following our previous report on evaluation of the beta tubulin real-time PCR for detection of dermatophytosis, this study aimed to compare the real-time PCR assay with conventional methods for the clinical assessment of its diagnostic performance. Samples from a total of 853 patients with suspected dermatophyte lesions were subjected to direct examination (all samples), culture (499 samples) and real-time PCR (all samples). Fungal DNA was extracted directly from clinical samples using a conical steel bullet, followed by purification with a commercial kit and subjected to the Taq-Man probe-based real-time PCR. The study showed that among the 499 specimens for which all three methods were used, 156 (31.2%), 128 (25.6%) and 205 (41.0%) were found to be positive by direct microscopy, culture and real-time PCR respectively. Real-time PCR significantly increased the detection rate of dermatophytes compared with microscopy (288 vs 229) with 87% concordance between the two methods. The sensitivity, specificity, positive predictive value, and negative predictive value of the real-time PCR was 87.5%, 85%, 66.5% and 95.2% respectively. Although real-time PCR performed better on skin than on nail samples, it should not yet fully replace conventional diagnosis. © 2017 Blackwell Verlag GmbH.

Method for Hot Real-Time Sampling of Gasification Products

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

Pomeroy, Marc D

The Thermochemical Process Development Unit (TCPDU) at the National Renewable Energy Laboratory (NREL) is a highly instrumented half-ton/day pilot scale plant capable of demonstrating industrially relevant thermochemical technologies from lignocellulosic biomass conversion, including gasification. Gasification creates primarily Syngas (a mixture of Hydrogen and Carbon Monoxide) that can be utilized with synthesis catalysts to form transportation fuels and other valuable chemicals. Biomass derived gasification products are a very complex mixture of chemical components that typically contain Sulfur and Nitrogen species that can act as catalysis poisons for tar reforming and synthesis catalysts. Real-time hot online sampling techniques, such as Molecular Beammore » Mass Spectrometry (MBMS), and Gas Chromatographs with Sulfur and Nitrogen specific detectors can provide real-time analysis providing operational indicators for performance. Sampling typically requires coated sampling lines to minimize trace sulfur interactions with steel surfaces. Other materials used inline have also shown conversion of sulfur species into new components and must be minimized. Sample line Residence time within the sampling lines must also be kept to a minimum to reduce further reaction chemistries. Solids from ash and char contribute to plugging and must be filtered at temperature. Experience at NREL has shown several key factors to consider when designing and installing an analytical sampling system for biomass gasification products. They include minimizing sampling distance, effective filtering as close to source as possible, proper line sizing, proper line materials or coatings, even heating of all components, minimizing pressure drops, and additional filtering or traps after pressure drops.« less

Comprehensive two-dimensional liquid chromatography for polyphenol analysis in foodstuffs.

PubMed

Cacciola, Francesco; Farnetti, Sara; Dugo, Paola; Marriott, Philip John; Mondello, Luigi

2017-01-01

Polyphenols are a class of plant secondary metabolites that are recently drawing a special interest because of their broad spectrum of pharmacological effects. As they are characterized by an enormous structural variability, the identification of these molecules in food samples is a difficult task, and sometimes having only a limited number of commercially available reference materials is not of great help. One-dimensional liquid chromatography is the most widely applied analytical approach for their analysis. In particular, the hyphenation of liquid chromatography to mass spectrometry has come to play an influential role by allowing relatively fast tentative identification and accurate quantification of polyphenolic compounds at trace levels in vegetable media. However, when dealing with very complex real-world food samples, a single separation system often does not provide sufficient resolving power for attaining rewarding results. Comprehensive two-dimensional liquid chromatography is a technique of great analytical impact, since it offers much higher peak capacities than separations in a single dimension. In the present review, we describe applications in the field of comprehensive two-dimensional liquid chromatography for polyphenol analysis in real-world food samples. Comprehensive two-dimensional liquid chromatography applications to nonfood matrices fall outside the scope of the current report and will not be discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two-dimensional liquid chromatography consisting of twelve second-dimension columns for comprehensive analysis of intact proteins.

PubMed

Ren, Jiangtao; Beckner, Matthew A; Lynch, Kyle B; Chen, Huang; Zhu, Zaifang; Yang, Yu; Chen, Apeng; Qiao, Zhenzhen; Liu, Shaorong; Lu, Joann J

2018-05-15

A comprehensive two-dimensional liquid chromatography (LCxLC) system consisting of twelve columns in the second dimension was developed for comprehensive analysis of intact proteins in complex biological samples. The system consisted of an ion-exchange column in the first dimension and the twelve reverse-phase columns in the second dimension; all thirteen columns were monolithic and prepared inside 250 µm i.d. capillaries. These columns were assembled together through the use of three valves and an innovative configuration. The effluent from the first dimension was continuously fractionated and sequentially transferred into the twelve second-dimension columns, while the second-dimension separations were carried out in a series of batches (six columns per batch). This LCxLC system was tested first using standard proteins followed by real-world samples from E. coli. Baseline separation was observed for eleven standard proteins and hundreds of peaks were observed for the real-world sample analysis. Two-dimensional liquid chromatography, often considered as an effective tool for mapping proteins, is seen as laborious and time-consuming when configured offline. Our online LCxLC system with increased second-dimension columns promises to provide a solution to overcome these hindrances. Copyright © 2018 Elsevier B.V. All rights reserved.

How to test validity in orthodontic research: a mixed dentition analysis example.

PubMed

Donatelli, Richard E; Lee, Shin-Jae

2015-02-01

The data used to test the validity of a prediction method should be different from the data used to generate the prediction model. In this study, we explored whether an independent data set is mandatory for testing the validity of a new prediction method and how validity can be tested without independent new data. Several validation methods were compared in an example using the data from a mixed dentition analysis with a regression model. The validation errors of real mixed dentition analysis data and simulation data were analyzed for increasingly large data sets. The validation results of both the real and the simulation studies demonstrated that the leave-1-out cross-validation method had the smallest errors. The largest errors occurred in the traditional simple validation method. The differences between the validation methods diminished as the sample size increased. The leave-1-out cross-validation method seems to be an optimal validation method for improving the prediction accuracy in a data set with limited sample sizes. Copyright © 2015 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.

Capillary absorption spectrometer and process for isotopic analysis of small samples

DOEpatents

Alexander, M. Lizabeth; Kelly, James F.; Sams, Robert L.; Moran, James J.; Newburn, Matthew K.; Blake, Thomas A.

2016-03-29

A capillary absorption spectrometer and process are described that provide highly sensitive and accurate stable absorption measurements of analytes in a sample gas that may include isotopologues of carbon and oxygen obtained from gas and biological samples. It further provides isotopic images of microbial communities that allow tracking of nutrients at the single cell level. It further targets naturally occurring variations in carbon and oxygen isotopes that avoids need for expensive isotopically labeled mixtures which allows study of samples taken from the field without modification. The method also permits sampling in vivo permitting real-time ambient studies of microbial communities.

Capillary absorption spectrometer and process for isotopic analysis of small samples

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

Alexander, M. Lizabeth; Kelly, James F.; Sams, Robert L.

A capillary absorption spectrometer and process are described that provide highly sensitive and accurate stable absorption measurements of analytes in a sample gas that may include isotopologues of carbon and oxygen obtained from gas and biological samples. It further provides isotopic images of microbial communities that allow tracking of nutrients at the single cell level. It further targets naturally occurring variations in carbon and oxygen isotopes that avoids need for expensive isotopically labeled mixtures which allows study of samples taken from the field without modification. The process also permits sampling in vivo permitting real-time ambient studies of microbial communities.

Detection and genetic analysis of human sapoviruses in river water in Japan.

PubMed

Kitajima, Masaaki; Oka, Tomoichiro; Haramoto, Eiji; Katayama, Hiroyuki; Takeda, Naokazu; Katayama, Kazuhiko; Ohgaki, Shinichiro

2010-04-01

We investigated the prevalence of sapoviruses (SaVs) in the Tamagawa River in Japan from April 2003 to March 2004 and performed genetic analysis of the SaV genes identified in river water. A total of 60 river water samples were collected from five sites along the river, and 500 ml was concentrated using the cation-coated filter method. By use of a real-time reverse transcription (RT)-PCR assay, 12 (20%) of the 60 samples were positive for SaV. SaV sequences were obtained from 15 (25%) samples, and a total of 30 SaV strains were identified using six RT-PCR assays followed by cloning and sequence analysis. A newly developed nested RT-PCR assay utilizing a broadly reactive forward primer showed the highest detection efficiency and amplified more diverse SaV genomes in the samples. SaV sequences were frequently detected from November to March, whereas none were obtained in April, July, September, or October. No SaV sequences were detected in the upstream portion of the river, whereas the midstream portion showed high positive rates. Based on phylogenetic analysis, SaV strains identified in the river water samples were classified into nine genotypes, namely, GI/1, GI/2, GI/3, GI/5, GI/untyped, GII/1, GII/2, GII/3, and GV/1. To our knowledge, this is the first study describing seasonal and spatial distributions and genetic diversity of SaVs in river water. A combination of real-time RT-PCR assay and newly developed nested RT-PCR assay is useful for identifying and characterizing SaV strains in a water environment.

Purification of cardiac myocytes from human heart biopsies for gene expression analysis.

PubMed

Kosloski, L M; Bales, I K; Allen, K B; Walker, B L; Borkon, A M; Stuart, R S; Pak, A F; Wacker, M J

2009-09-01

The collection of gene expression data from human heart biopsies is important for understanding the cellular mechanisms of arrhythmias and diseases such as cardiac hypertrophy and heart failure. Many clinical and basic research laboratories conduct gene expression analysis using RNA from whole cardiac biopsies. This allows for the analysis of global changes in gene expression in areas of the heart, while eliminating the need for more complex and technically difficult single-cell isolation procedures (such as flow cytometry, laser capture microdissection, etc.) that require expensive equipment and specialized training. The abundance of fibroblasts and other cell types in whole biopsies, however, can complicate gene expression analysis and the interpretation of results. Therefore, we have designed a technique to quickly and easily purify cardiac myocytes from whole cardiac biopsies for RNA extraction. Human heart tissue samples were collected, and our purification method was compared with the standard nonpurification method. Cell imaging using acridine orange staining of the purified sample demonstrated that >98% of total RNA was contained within identifiable cardiac myocytes. Real-time RT-PCR was performed comparing nonpurified and purified samples for the expression of troponin T (myocyte marker), vimentin (fibroblast marker), and alpha-smooth muscle actin (smooth muscle marker). Troponin T expression was significantly increased, and vimentin and alpha-smooth muscle actin were significantly decreased in the purified sample (n = 8; P < 0.05). Extracted RNA was analyzed during each step of the purification, and no significant degradation occurred. These results demonstrate that this isolation method yields a more purified cardiac myocyte RNA sample suitable for downstream applications, such as real-time RT-PCR, and allows for more accurate gene expression changes in cardiac myocytes from heart biopsies.

Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall.

PubMed

Ooi, Delicia Shu Qin; Tan, Verena Ming Hui; Ong, Siong Gim; Chan, Yiong Huak; Heng, Chew Kiat; Lee, Yung Seng

2017-01-01

The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods. We found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples. Despite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed.

Real-time PCR detection and quantification of nine potential sources of fecal contamination by analysis of mitochondrial Cytochrome b targets

USGS Publications Warehouse

Schill, W.B.; Mathes, M.V.

2008-01-01

We designed and tested real-time PCR probe/primer sets to detect and quantify Cytochrome b sequences of mitochondrial DNA (mtDNA) from nine vertebrate species of pet (dog), farm (cow, chicken, sheep, horse, pig), wildlife (Canada goose, white-tailed deer), and human. Linear ranges of the assays were from 101 to 108 copies/??l. To formally test the performance of the assays, twenty blinded fecal suspension samples were analyzed by real-time PCR to identify the source of the feces. Sixteen of the twenty samples were correctly and unambiguously identified. Average sensitivity was calculated to be 0.850, while average specificity was found to be 0.994. One beef cow sample was not detected, but mtDNA from 11 other beef cattle of both sexes and varying physiological states was found in concentrations similar (3.45 ?? 107 copies/g) to thatfound in human feces (1.1 ?? 107 copies/g). Thus, environmental conditions and sample handling are probably important factors for successful detection of fecal mtDNA. When sewage samples were analyzed, only human mtDNA (7.2 ?? 104 copies/100 mL) was detected. With a detection threshold of 250 copies/reaction, an efficient concentration and purification method resulted in a final detection limit for human feces of 1.8 mg/100 mL water.

Diagnosis and identification of Leishmania spp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran.

PubMed

Khademvatan, S; Neisi, N; Maraghi, S; Saki, J

2011-12-01

The aim of present study was describing a real-time PCR assay for the diagnosis and direct identification of Leishmania species on Giemsa-stained slides in south-west of Iran. Altogether, 102 Giemsa-stained slides were collected from different part of south-west of Iran between 2008 and 2011. All the Giemsa-stained slides were examined under light microscope. After DNA extraction, real-time PCR amplification and detection were conducted with fluorescent SYBR Green I. For identification, PCR products were analysed with melting curve analysis. One hundred and two archived slides from suspected lesion examined by microscopy and real-time PCR. The sensitivity of the real-time PCR on Giemsa-stained slid was 98% (96/102). The melting curve analysis (T(m)) were 88·3±0·2°C for L. tropica (MHOM/IR/02/Mash10), 86·5±0·2°C for L. major (MHOM/IR/75/ER) and 89·4±0·3°C for L. infantum (MCAN/IR/97/LON 49), respectively. This study is first report in use of real-time PCR for diagnosis and identification of Leishmania spp. in Iran. Up to now, in Iran, the majority of identification of Leishmania species is restriction fragment length polymorphism (PCR-RFLP) of ITS1 and kinetoplast DNA. Our data showed that Giemsa-stained slides that were stored more than 3 years, can be use for Leishmania DNA extraction and amplification by real-time PCR. Compared to conventional PCR-based methods, the real-time PCR is extremely rapid with results and more samples can be processed at one time.

Multiplex real-time RT-PCR assay for bovine viral diarrhea virus type 1, type 2 and HoBi-like pestivirus.

PubMed

Mari, Viviana; Losurdo, Michele; Lucente, Maria Stella; Lorusso, Eleonora; Elia, Gabriella; Martella, Vito; Patruno, Giovanni; Buonavoglia, Domenico; Decaro, Nicola

2016-03-01

HoBi-like pestiviruses are emerging pestiviruses that infect cattle causing clinical forms overlapping to those induced by bovine viral diarrhea virus (BVDV) 1 and 2. As a consequence of their widespread distribution reported in recent years, molecular tools for rapid discrimination among pestiviruses infecting cattle are needed. The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. The assay was found to be sensitive, specific and repeatable, ensuring detection of as few as 10(0)-10(1) viral RNA copies. No cross-reactions between different pestiviral species were observed even in samples artificially contaminated with more than one pestivirus. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Development and validation of an UHPLC-MS/MS method for β2-agonists quantification in human urine and application to clinical samples.

PubMed

Bozzolino, Cristina; Leporati, Marta; Gani, Federica; Ferrero, Cinzia; Vincenti, Marco

2018-02-20

A fast analytical method for the simultaneous detection of 24 β 2 -agonists in human urine was developed and validated. The method covers the therapeutic drugs most commonly administered, but also potentially abused β 2 -agonists. The procedure is based on enzymatic deconjugation with β-glucuronidase followed by SPE clean up using mixed-phase cartridges with both ion-exchange and lipophilic properties. Instrumental analysis conducted by UHPLC-MS/MS allowed high peak resolution and rapid chromatographic separation, with reduced time and costs. The method was fully validated according ISO 17025:2005 principles. The following parameters were determined for each analyte: specificity, selectivity, linearity, limit of detection, limit of quantification, precision, accuracy, matrix effect, recovery and carry-over. The method was tested on real samples obtained from patients subjected to clinical treatment under chronic or acute therapy with either formoterol, indacaterol, salbutamol, or salmeterol. The drugs were administered using pressurized metered dose inhalers. All β 2 -agonists administered to the patients were detected in the real samples. The method proved adequate to accurately measure the concentration of these analytes in the real samples. The observed analytical data are discussed with reference to the administered dose and the duration of the therapy. Copyright © 2017 Elsevier B.V. All rights reserved.

Schistosoma real-time PCR as diagnostic tool for international travellers and migrants.

PubMed

Cnops, Lieselotte; Tannich, Egbert; Polman, Katja; Clerinx, Jan; Van Esbroeck, Marjan

2012-10-01

To evaluate the use of a genus-specific PCR that combines high sensitivity with the detection of different Schistosoma species for diagnosis in international travellers and migrants in comparison to standard microscopy. The genus-specific real-time PCR was developed to target the 28S ribosomal RNA gene of the major human Schistosoma species. It was validated for analytical specificity and reproducibility and demonstrated an analytical sensitivity of 0.2 eggs per gram of faeces. Its diagnostic performance was further evaluated on 152 faecal, 32 urine and 38 serum samples from patients presenting at the outpatient clinic of the Institute of Tropical Medicine in Antwerp (Belgium). We detected Schistosoma DNA in 76 faecal (50.0%) and five urine (15.6%) samples of which, respectively, nine and one were not detected by standard microscopy. Only two of the 38 serum samples of patients with confirmed schistosomiasis were positive with the presently developed PCR. Sequence analysis on positive faecal samples allowed identification of the Schistosoma species complex. The real-time PCR is highly sensitive and may offer added value in diagnosing imported schistosomiasis. The genus-specific PCR can detect all schistosome species that are infectious to humans and performs very well with faeces and urine, but not in serum. © 2012 Blackwell Publishing Ltd.

Surface Plasmon Resonance imaging-based sensing for anti-bovine immunoglobulins detection in human milk and serum.

PubMed

Scarano, S; Scuffi, C; Mascini, M; Minunni, M

2011-11-30

Only few papers deal with Surface Plasmon Resonance imaging (SPRi) direct detection on complex matrices, limiting the biosensor application to real analytical problems. In this work a SPRi biosensor for anti-bovine IgG detection in untreated human bodily fluids, i.e. diluted human serum and milk, was developed. Enhanced levels of cow's milk antibodies in children's serum are suspected for their possible correlation with Type 1 diabetes during childhood and their detection in real samples was up to now performed by classical immunoassays based on indirect detection. The biosensor was optimised in standard samples and then in untreated human milk for anti-bovine IgG direct detection. The key novelty of the work is the evaluation of matrix effect by applying to real samples an experimental and ex ante method previously developed for SPRi signal sampling in standard solutions, called "Data Analyzer"; it punctually visualises and analyses the behaviour of receptor spots of the array, to select only spot areas with the best specific vs. unspecific signal values. In this way, benefits provide by SPRi image analysis are exploited here to quantify and minimise drawbacks due to the matrix effect, allowing to by-pass every matrix pre-treatment except dilution. Copyright © 2011 Elsevier B.V. All rights reserved.

FIELD QUALITY CONTROL STRATEGIES ASSESSING SOLIDIFICATION/STABILIZATION

EPA Science Inventory

Existing regulatory mobility reduction (leaching) tests are not amenable to real time quality control because of the time required to perform sample extraction and chemical analysis. This is of conccern because the leaching test is the most important parameter used to relate trea...

Portable Solid Phase Micro-Extraction Coupled with Ion Mobility Spectrometry System for On-Site Analysis of Chemical Warfare Agents and Simulants in Water Samples

PubMed Central

Yang, Liu; Han, Qiang; Cao, Shuya; Yang, Jie; Yang, Junchao; Ding, Mingyu

2014-01-01

On-site analysis is an efficient approach to facilitate analysis at the location of the system under investigation as it can result in more accurate, more precise and quickly available analytical data. In our work, a novel self-made thermal desorption based interface was fabricated to couple solid-phase microextraction with ion mobility spectrometry for on-site water analysis. The portable interface can be connected with the front-end of an ion mobility spectrometer directly without other modifications. The analytical performance was evaluated via the extraction of chemical warfare agents and simulants in water samples. Several parameters including ionic strength and extraction time have been investigated in detail. The application of the developed method afforded satisfactory recoveries ranging from 72.9% to 114.4% when applied to the analysis of real water samples. PMID:25384006

Authentication of animal fats using direct analysis in real time (DART) ionization-mass spectrometry and chemometric tools.

PubMed

Vaclavik, Lukas; Hrbek, Vojtech; Cajka, Tomas; Rohlik, Bo-Anne; Pipek, Petr; Hajslova, Jana

2011-06-08

A combination of direct analysis in real time (DART) ionization coupled to time-of-flight mass spectrometry (TOFMS) and chemometrics was used for animal fat (lard and beef tallow) authentication. This novel instrumentation was employed for rapid profiling of triacylglycerols (TAGs) and polar compounds present in fat samples and their mixtures. Additionally, fat isolated from pork, beef, and pork/beef admixtures was analyzed. Mass spectral records were processed by principal component analysis (PCA) and stepwise linear discriminant analysis (LDA). DART-TOFMS profiles of TAGs were found to be more suitable for the purpose of discrimination among the examined fat types as compared to profiles of polar compounds. The LDA model developed using TAG data enabled not only reliable classification of samples representing neat fats but also detection of admixed lard and tallow at adulteration levels of 5 and 10% (w/w), respectively. The presented approach was also successfully applied to minced meat prepared from pork and beef with comparable fat content. Using the DART-TOFMS TAG profiles of fat isolated from meat mixtures, detection of 10% pork added to beef and vice versa was possible.

Cumulative Effective Hölder Exponent Based Indicator for Real-Time Fetal Heartbeat Analysis during Labour

NASA Astrophysics Data System (ADS)

Struzik, Zbigniew R.; van Wijngaarden, Willem J.

We introduce a special purpose cumulative indicator, capturing in real time the cumulative deviation from the reference level of the exponent h (local roughness, Hölder exponent) of the fetal heartbeat during labour. We verify that the indicator applied to the variability component of the heartbeat coincides with the fetal outcome as determined by blood samples. The variability component is obtained from running real time decomposition of fetal heartbeat into independent components using an adaptation of an oversampled Haar wavelet transform. The particular filters used and resolutions applied are motivated by obstetricial insight/practice. The methodology described has the potential for real-time monitoring of the fetus during labour and for the prediction of the fetal outcome, allerting the attending staff in the case of (threatening) hypoxia.

Single-protein nanomechanical mass spectrometry in real time

PubMed Central

Hanay, M.S.; Kelber, S.; Naik, A.K.; Chi, D.; Hentz, S.; Bullard, E.C.; Colinet, E.; Duraffourg, L.; Roukes, M.L.

2012-01-01

Nanoelectromechanical systems (NEMS) resonators can detect mass with exceptional sensitivity. Previously, mass spectra from several hundred adsorption events were assembled in NEMS-based mass spectrometry using statistical analysis. Here, we report the first realization of single-molecule NEMS-based mass spectrometry in real time. As each molecule in the sample adsorbs upon the NEMS resonator, its mass and the position-of-adsorption are determined by continuously tracking two driven vibrational modes of the device. We demonstrate the potential of multimode NEMS-based mass spectrometry by analyzing IgM antibody complexes in real-time. NEMS-MS is a unique and promising new form of mass spectrometry: it can resolve neutral species, provides resolving power that increases markedly for very large masses, and allows acquisition of spectra, molecule-by-molecule, in real-time. PMID:22922541

Identification by real-time PCR with SYBR Green of Leishmania spp. and Serratia marcescens in canine 'sterile' cutaneous nodular lesions.

PubMed

Cornegliani, Luisa; Corona, Antonio; Vercelli, Antonella; Roccabianca, Paola

2015-06-01

Noninfectious, non-neoplastic, nodular to diffuse, so-called 'sterile' granulomatous/pyogranulomatous skin lesions (SGPSLs) are infrequently identified in dogs and may represent a diagnostic challenge. Their correct identification is based on history, histopathology and absence of intralesional foreign bodies and micro-organisms. The aim of this study was to investigate the presence of Leishmania spp., Mycobacterium spp., Serratia marcescens and Nocardia spp. by real-time PCR in canine nodular skin lesions histologically diagnosed as putatively sterile. Formalin-fixed skin biopsies were collected from 40 dogs. All samples were associated with an SGPSL diagnosis characterized by multifocal, nodular to diffuse, periadnexal and perifollicular pyogranulomas/granulomas. Neither micro-organisms nor foreign bodies were detected with haematoxylin and eosin staining, under polarized light. Further analyses included periodic acid Schiff, Ziehl-Neelsen, Fite Faraco, Giemsa and Gram histochemical stains; anti-Bacillus Calmette-Guérin (BCG) and Leishmania spp. immunohistochemistry; and real-time PCR analysis for Leishmania spp., Mycobacterium spp., S. marcescens and Nocardia spp. Special stains and BCG/immunohistochemistry were negative in all samples. Real-time PCR was positive for Leishmania spp. in four of 40 biopsies and for S. marcescens in two of 40 samples. Real-time PCR for Mycobacterium spp. and Nocardia spp. was negative. No correlation between real-time PCR positivity and a specific histological pattern was identified. Leishmania spp. have been previously identified as possible agents of certain SGPSLs, while the involvement of S. marcescens has not been investigated previously. According to our findings, Serratia spp. should be included in the list of agents possibly associated with a subgroup of granulomatous/pyogranulomatous skin lesions in dogs. © 2015 ESVD and ACVD.

Comparative analysis of laparoscopic and ultrasound-guided biopsy methods for gene expression analysis in transgenic goats.

PubMed

Melo, C H; Sousa, F C; Batista, R I P T; Sanchez, D J D; Souza-Fabjan, J M G; Freitas, V J F; Melo, L M; Teixeira, D I A

2015-07-31

The present study aimed to compare laparoscopic (LP) and ultrasound-guided (US) biopsy methods to obtain either liver or splenic tissue samples for ectopic gene expression analysis in transgenic goats. Tissue samples were collected from human granulocyte colony stimulating factor (hG-CSF)-transgenic bucks and submitted to real-time PCR for the endogenous genes (Sp1, Baff, and Gapdh) and the transgene (hG-CSF). Both LP and US biopsy methods were successful in obtaining liver and splenic samples that could be analyzed by PCR (i.e., sufficient sample sizes and RNA yield were obtained). Although the number of attempts made to obtain the tissue samples was similar (P > 0.05), LP procedures took considerably longer than the US method (P = 0.03). Finally, transgene transcripts were not detected in spleen or liver samples. Thus, for the phenotypic characterization of a transgenic goat line, investigation of ectopic gene expression can be made successfully by LP or US biopsy, avoiding the traditional approach of euthanasia.

Comparison of the efficiency between two sampling plans for aflatoxins analysis in maize

PubMed Central

Mallmann, Adriano Olnei; Marchioro, Alexandro; Oliveira, Maurício Schneider; Rauber, Ricardo Hummes; Dilkin, Paulo; Mallmann, Carlos Augusto

2014-01-01

Variance and performance of two sampling plans for aflatoxins quantification in maize were evaluated. Eight lots of maize were sampled using two plans: manual, using sampling spear for kernels; and automatic, using a continuous flow to collect milled maize. Total variance and sampling, preparation, and analysis variance were determined and compared between plans through multifactor analysis of variance. Four theoretical distribution models were used to compare aflatoxins quantification distributions in eight maize lots. The acceptance and rejection probabilities for a lot under certain aflatoxin concentration were determined using variance and the information on the selected distribution model to build the operational characteristic curves (OC). Sampling and total variance were lower at the automatic plan. The OC curve from the automatic plan reduced both consumer and producer risks in comparison to the manual plan. The automatic plan is more efficient than the manual one because it expresses more accurately the real aflatoxin contamination in maize. PMID:24948911

Design of a portable electronic nose for real-fake detection of liquors

NASA Astrophysics Data System (ADS)

Qi, Pei-Feng; Zeng, Ming; Li, Zhi-Hua; Sun, Biao; Meng, Qing-Hao

2017-09-01

Portability is a major issue that influences the practical application of electronic noses (e-noses). For liquors detection, an e-nose must preprocess the liquid samples (e.g., using evaporation and thermal desorption), which makes the portable design even more difficult. To realize convenient and rapid detection of liquors, we designed a portable e-nose platform that consists of hardware and software systems. The hardware system contains an evaporation/sampling module, a reaction module, a control/data acquisition and analysis module, and a power module. The software system provides a user-friendly interface and can achieve automatic sampling and data processing. This e-nose platform has been applied to the real-fake recognition of Chinese liquors. Through parameter optimization of a one-class support vector machine classifier, the error rate of the negative samples is greatly reduced, and the overall recognition accuracy is improved. The results validated the feasibility of the designed portable e-nose platform.

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.

Molecular differentiation of Russian wild ginseng using mitochondrial nad7 intron 3 region.

PubMed

Li, Guisheng; Cui, Yan; Wang, Hongtao; Kwon, Woo-Saeng; Yang, Deok-Chun

2017-07-01

Cultivated ginseng is often introduced as a substitute and adulterant of Russian wild ginseng due to its lower cost or misidentification caused by similarity in appearance with wild ginseng. The aim of this study is to develop a simple and reliable method to differentiate Russian wild ginseng from cultivated ginseng. The mitochondrial NADH dehydrogenase subunit 7 ( nad 7) intron 3 regions of Russian wild ginseng and Chinese cultivated ginseng were analyzed. Based on the multiple sequence alignment result, a specific primer for Russian wild ginseng was designed by introducing additional mismatch and allele-specific polymerase chain reaction (PCR) was performed for identification of wild ginseng. Real-time allele-specific PCR with endpoint analysis was used for validation of the developed Russian wild ginseng single nucleotide polymorphism (SNP) marker. An SNP site specific to Russian wild ginseng was exploited by multiple alignments of mitochondrial nad 7 intron 3 regions of different ginseng samples. With the SNP-based specific primer, Russian wild ginseng was successfully discriminated from Chinese and Korean cultivated ginseng samples by allele-specific PCR. The reliability and specificity of the SNP marker was validated by checking 20 individuals of Russian wild ginseng samples with real-time allele-specific PCR assay. An effective DNA method for molecular discrimination of Russian wild ginseng from Chinese and Korean cultivated ginseng was developed. The established real-time allele-specific PCR was simple and reliable, and the present method should be a crucial complement of chemical analysis for authentication of Russian wild ginseng.

Detection of Leptospira interrogans DNA and antigen in fixed equine eyes affected with end-stage equine recurrent uveitis.

PubMed

Pearce, Jacqueline W; Galle, Laurence E; Kleiboeker, Steve B; Turk, James R; Schommer, Susan K; Dubielizig, Richard R; Mitchell, William J; Moore, Cecil P; Giuliano, Elizabeth A

2007-11-01

Equine recurrent uveitis (ERU) is the most frequent cause of blindness in horses worldwide. Leptospira has been implicated as an etiologic agent in some cases of ERU and has been detected in fresh ocular tissues of affected horses. The objective of this study was to determine the presence of Leptospira antigen and DNA in fixed equine ocular tissues affected with end-stage ERU. Sections of eyes from 30 horses were obtained. Controls included 1) 10 normal equine eyes and 2) 10 equine eyes with a nonrecurrent form of uveitis. The experimental group consisted of 10 eyes diagnosed with ERU based on clinical signs and histologic lesions. Sections were subjected to immunohistochemical staining with an array of rabbit anti-Leptospira polyclonal antibodies. DNA extractions were performed by using a commercial kit designed for fixed tissue. Real-time PCR analysis was completed on extracted DNA. The target sequence for PCR was designed from alignments of available Leptospira 16S rDNA partial sequences obtained from GenBank. Two of 10 test samples were positive for Leptospira antigen by immunohistochemical assay. Zero of 20 controls were positive for Leptospira antigen. All test samples and controls were negative for Leptospira DNA by real-time PCR analysis. Leptospira was detected at a lower frequency than that previously reported for fresh ERU-affected aqueous humor and vitreous samples. Leptospira is not frequently detectable in fixed ocular tissues of horses affected with ERU when using traditional immunohistochemical and real-time PCR techniques.

A pipeline VLSI design of fast singular value decomposition processor for real-time EEG system based on on-line recursive independent component analysis.

PubMed

Huang, Kuan-Ju; Shih, Wei-Yeh; Chang, Jui Chung; Feng, Chih Wei; Fang, Wai-Chi

2013-01-01

This paper presents a pipeline VLSI design of fast singular value decomposition (SVD) processor for real-time electroencephalography (EEG) system based on on-line recursive independent component analysis (ORICA). Since SVD is used frequently in computations of the real-time EEG system, a low-latency and high-accuracy SVD processor is essential. During the EEG system process, the proposed SVD processor aims to solve the diagonal, inverse and inverse square root matrices of the target matrices in real time. Generally, SVD requires a huge amount of computation in hardware implementation. Therefore, this work proposes a novel design concept for data flow updating to assist the pipeline VLSI implementation. The SVD processor can greatly improve the feasibility of real-time EEG system applications such as brain computer interfaces (BCIs). The proposed architecture is implemented using TSMC 90 nm CMOS technology. The sample rate of EEG raw data adopts 128 Hz. The core size of the SVD processor is 580×580 um(2), and the speed of operation frequency is 20MHz. It consumes 0.774mW of power during the 8-channel EEG system per execution time.

Microbial Monitoring of Common Opportunistic Pathogens by Comparing Multiple Real-time PCR Platforms for Potential Space Applications

NASA Technical Reports Server (NTRS)

Roman, Monserrate C.; Jones, Kathy U.; Oubre, Cherie M.; Castro, Victoria; Ott, Mark C.; Birmele, Michele; Venkateswaran, Kasthuri J.; Vaishampayan, Parag A.

2013-01-01

Current methods for microbial detection: a) Labor & time intensive cultivation-based approaches that can fail to detect or characterize all cells present. b) Requires collection of samples on orbit and transportation back to ground for analysis. Disadvantages to current detection methods: a) Unable to perform quick and reliable detection on orbit. b) Lengthy sampling intervals. c) No microbe identification.

Sampling in the light of Wigner distribution.

PubMed

Stern, Adrian; Javidi, Bahram

2004-03-01

We propose a new method for analysis of the sampling and reconstruction conditions of real and complex signals by use of the Wigner domain. It is shown that the Wigner domain may provide a better understanding of the sampling process than the traditional Fourier domain. For example, it explains how certain non-bandlimited complex functions can be sampled and perfectly reconstructed. On the basis of observations in the Wigner domain, we derive a generalization to the Nyquist sampling criterion. By using this criterion, we demonstrate simple preprocessing operations that can adapt a signal that does not fulfill the Nyquist sampling criterion. The preprocessing operations demonstrated can be easily implemented by optical means.

Evaluation of direct analysis in real time for the determination of highly polar pesticides in lettuce and celery using modified Quick Polar Pesticides Extraction method.

PubMed

Lara, Francisco J; Chan, Danny; Dickinson, Michael; Lloyd, Antony S; Adams, Stuart J

2017-05-05

Direct analysis in real time (DART) was evaluated for the determination of a number of highly polar pesticides using the Quick Polar Pesticides Extraction (QuPPe) method. DART was hyphenated to high resolution mass spectrometry (HRMS) in order to get the required selectivity that allows the determination of these compounds in complex samples such as lettuce and celery. Experimental parameters such as desorption temperature, scanning speed, and distances between the DART ion source and MS inlet were optimized. Two different mass analyzers (Orbitrap and QTOF) and two accessories for sample introduction (Dip-it ® tips and QuickStrip™ sample cards) were evaluated. An extra clean-up step using primary-secondary amine (PSA) was included in the QuPPe method to improve sensitivity. The main limitation found was in-source fragmentation, nevertheless QuPPe-DART-HRMS proved to be a fast and reliable tool with quantitative capabilities for at least seven compounds: amitrole, cyromazine, propamocarb, melamine, diethanolamine, triethanolamine and 1,2,4-triazole. The limits of detection ranged from 20 to 60μg/kg. Recoveries for fortified samples ranged from 71 to 115%, with relative standard deviations <18%. Copyright © 2017 Elsevier B.V. All rights reserved.

Toward decentralized analysis of mercury (II) in real samples. A critical review on nanotechnology-based methodologies.

PubMed

Botasini, Santiago; Heijo, Gonzalo; Méndez, Eduardo

2013-10-24

In recent years, it has increased the number of works focused on the development of novel nanoparticle-based sensors for mercury detection, mainly motivated by the need of low cost portable devices capable of giving fast and reliable analytical response, thus contributing to the analytical decentralization. Methodologies employing colorimetric, fluorometric, magnetic, and electrochemical output signals allowed reaching detection limits within the pM and nM ranges. Most of these developments proved their suitability in detecting and quantifying mercury (II) ions in synthetic solutions or spiked water samples. However, the state of art in these technologies is still behind the standard methods of mercury quantification, such as cold vapor atomic absorption spectrometry and inductively coupled plasma techniques, in terms of reliability and sensitivity. This is mainly because the response of nanoparticle-based sensors is highly affected by the sample matrix. The developed analytical nanosystems may fail in real samples because of the negative incidence of the ionic strength and the presence of exchangeable ligands. The aim of this review is to critically consider the recently published innovations in this area, and highlight the needs to include more realistic assays in future research in order to make these advances suitable for on-site analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Real-time analysis of self-assembled nucleobases by Venturi easy ambient sonic-spray ionization mass spectrometry.

PubMed

Na, Na; Shi, Ruixia; Long, Zi; Lu, Xin; Jiang, Fubin; Ouyang, Jin

2014-10-01

In this study, the real-time analysis of self-assembled nucleobases was employed by Venturi easy ambient sonic-spray ionization mass spectrometry (V-EASI-MS). With the analysis of three nucleobases including 6-methyluracil (6MU), uracil (U) and thymine (T) as examples, different orders of clusters centered with different metal ions were recorded in both positive and negative modes. Compared with the results obtained by traditional electrospray ionization mass spectrometry (ESI-MS) under the same condition, more clusters with high orders, such as [6MU7+Na](+), [6MU15+2NH4](2+), [6MU10+Na](+), [T7+Na](+), and [T15+2NH4](2+) were detected by V-EASI-MS, which demonstrated the soft ionization ability of V-EASI for studying the non-covalent interaction in a self-assembly process. Furthermore, with the injection of K(+) to the system by a syringe pumping, the real-time monitoring of the formation of nucleobases clusters was achieved by the direct extraction of samples from the system under the Venturi effect. Therefore, the effect of cations on the formation of clusters during self-assembly of nucleobases was demonstrated, which was in accordance with the reports. Free of high voltage, heating or radiation during the ionization, this technique is much soft and suitable for obtaining the real-time information of the self-assembly system, which also makes it quite convenient for extraction samples from the reaction system. This "easy and soft" ionization technique has provided a potential pathway for monitoring and controlling the self-assembly processes. Copyright © 2014 Elsevier B.V. All rights reserved.

NASA GRC and MSFC Space-Plasma Arc Testing Procedures

NASA Technical Reports Server (NTRS)

Ferguson, Dale C.; Vayner, Boris V.; Galofaro, Joel T.; Hillard, G. Barry; Vaughn, Jason; Schneider, Todd

2007-01-01

Tests of arcing and current collection in simulated space plasma conditions have been performed at the NASA Glenn Research Center (GRC) in Cleveland, Ohio, for over 30 years and at the Marshall Space Flight Center (MSFC) in Huntsville, Alabama, for almost as long. During this period, proper test conditions for accurate and meaningful space simulation have been worked out, comparisons with actual space performance in spaceflight tests and with real operational satellites have been made, and NASA has achieved our own internal standards for test protocols. It is the purpose of this paper to communicate the test conditions, test procedures, and types of analysis used at NASA GRC and MSFC to the space environmental testing community at large, to help with international space-plasma arcing-testing standardization. Discussed herein are neutral gas conditions, plasma densities and uniformity, vacuum chamber sizes, sample sizes and Debye lengths, biasing samples versus self-generated voltages, floating samples versus grounded samples, test electrical conditions, arc detection, preventing sustained discharges during testing, real samples versus idealized samples, validity of LEO tests for GEO samples, extracting arc threshold information from arc rate versus voltage tests, snapover, current collection, and glows at positive sample bias, Kapton pyrolysis, thresholds for trigger arcs, sustained arcs, dielectric breakdown and Paschen discharge, tether arcing and testing in very dense plasmas (i.e. thruster plumes), arc mitigation strategies, charging mitigation strategies, models, and analysis of test results. Finally, the necessity of testing will be emphasized, not to the exclusion of modeling, but as part of a complete strategy for determining when and if arcs will occur, and preventing them from occurring in space.

Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods.

PubMed

Camargo, Vinícius da Silva; Santana, Bruna Nicoleti; Ferrari, Elis Domingos; Nakamura, Alex Akira; Nagata, Walter Bertequini; Nardi, Ana Rita Moraes; Meireles, Marcelo Vasconcelos

2018-01-01

This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

Circulating tumor DNA profiling reveals clonal evolution and real-time disease progression in advanced hepatocellular carcinoma.

PubMed

Cai, Zhi-Xiong; Chen, Geng; Zeng, Yong-Yi; Dong, Xiu-Qing; Lin, Min-Jie; Huang, Xin-Hui; Zhang, Da; Liu, Xiao-Long; Liu, Jing-Feng

2017-09-01

Circulating tumor DNA (ctDNA) provides a potential non-invasive biomarker for cancer diagnosis and prognosis, but whether it could reflect tumor heterogeneity and monitor therapeutic responses in hepatocellular carcinoma (HCC) is unclear. Focusing on 574 cancer genes known to harbor actionable mutations, we identified the mutation repertoire of HCC tissues, and monitored the corresponding ctDNA features in blood samples to evaluate its clinical significance. Analysis of 3 HCC patients' mutation profiles revealed that ctDNA could overcome tumor heterogeneity and provide information of tumor burden and prognosis. Further analysis was conducted on the 4th HCC case with multiple lesion samples and sequential plasma samples. We identified 160 subclonal SNVs in tumor tissues as well as matched peritumor tissues with PBMC as control. 96.9% of this patient's tissue mutations could be also detected in plasma samples. These subclonal SNVs were grouped into 9 clusters according to their trends of cellular prevalence shift in tumor tissues. Two clusters constituted of tumor stem somatic mutations showed circulating levels relating with cancer progression. Analysis of tumor somatic mutations revealed that circulating level of such tumor stem somatic mutations could reflect tumor burden and even predict prognosis earlier than traditional strategies. Furthermore, HCK (p.V174M), identified as a recurrent/metastatic related mutation site, could promote migration and invasion of HCC cells. Taken together, study of mutation profiles in biopsy and plasma samples in HCC patients showed that ctDNA could overcome tumor heterogeneity and real-time track the therapeutic responses in the longitudinal monitoring. © 2017 UICC.

An evolution based biosensor receptor DNA sequence generation algorithm.

PubMed

Kim, Eungyeong; Lee, Malrey; Gatton, Thomas M; Lee, Jaewan; Zang, Yupeng

2010-01-01

A biosensor is composed of a bioreceptor, an associated recognition molecule, and a signal transducer that can selectively detect target substances for analysis. DNA based biosensors utilize receptor molecules that allow hybridization with the target analyte. However, most DNA biosensor research uses oligonucleotides as the target analytes and does not address the potential problems of real samples. The identification of recognition molecules suitable for real target analyte samples is an important step towards further development of DNA biosensors. This study examines the characteristics of DNA used as bioreceptors and proposes a hybrid evolution-based DNA sequence generating algorithm, based on DNA computing, to identify suitable DNA bioreceptor recognition molecules for stable hybridization with real target substances. The Traveling Salesman Problem (TSP) approach is applied in the proposed algorithm to evaluate the safety and fitness of the generated DNA sequences. This approach improves efficiency and stability for enhanced and variable-length DNA sequence generation and allows extension to generation of variable-length DNA sequences with diverse receptor recognition requirements.

Quantitative capillary electrophoresis and its application in analysis of alkaloids in tea, coffee, coca cola, and theophylline tablets.

PubMed

Li, Mengjia; Zhou, Junyi; Gu, Xue; Wang, Yan; Huang, Xiaojing; Yan, Chao

2009-01-01

A quantitative CE (qCE) system with high precision has been developed, in which a 4-port nano-valve was isolated from the electric field and served as sample injector. The accurate amount of sample was introduced into the CE system with high reproducibility. Based on this system, consecutive injections and separations were performed without voltage interruption. Reproducibilities in terms of RSD lower than 0.8% for retention time and 1.7% for peak area were achieved. The effectiveness of the system was demonstrated by the quantitative analysis of caffeine, theobromine, and theophylline in real samples, such as tea leaf, roasted coffee, coca cola, and theophylline tablets.

Development and optimization of a solid-phase microextraction gas chromatography-tandem mass spectrometry methodology to analyse ultraviolet filters in beach sand.

PubMed

Vila, Marlene; Llompart, Maria; Garcia-Jares, Carmen; Homem, Vera; Dagnac, Thierry

2018-06-06

A methodology based on solid-phase microextraction (SPME) followed by gas chromatography-tandem mass spectrometry (GC-MS/MS) has been developed for the simultaneous analysis of eleven multiclass ultraviolet (UV) filters in beach sand. To the best of our knowledge, this is the first time that this extraction technique is applied to the analysis of UV filters in sand samples, and in other kind of environmental solid samples. Main extraction parameters such as the fibre coating, the amount of sample, the addition of salt, the volume of water added to the sand, and the temperature were optimized. An experimental design approach was implemented in order to find out the most favourable conditions. The final conditions consisted of adding 1 mL of water to 1 g of sample followed by the headspace SPME for 20 min at 100 °C, using PDMS/DVB as fibre coating. The SPME-GC-MS/MS method was validated in terms of linearity, accuracy, limits of detection and quantification, and precision. Recovery studies were also performed at three concentration levels in real Atlantic and Mediterranean sand samples. The recoveries were generally above 85% and relative standard deviations below 11%. The limits of detection were in the pg g -1 level. The validated methodology was successfully applied to the analysis of real sand samples collected from Atlantic Ocean beaches in the Northwest coast of Spain and Portugal, Canary Islands (Spain), and from Mediterranean Sea beaches in Mallorca Island (Spain). The most frequently found UV filters were ethylhexyl salicylate (EHS), homosalate (HMS), 4-methylbenzylidene camphor (4MBC), 2-ethylhexyl methoxycinnamate (2EHMC) and octocrylene (OCR), with concentrations up to 670 ng g -1 . Copyright © 2018 Elsevier B.V. All rights reserved.

Real-time PCR and NASBA for rapid and sensitive detection of Vibrio cholerae in ballast water.

PubMed

Fykse, Else M; Nilsen, Trine; Nielsen, Agnete Dessen; Tryland, Ingun; Delacroix, Stephanie; Blatny, Janet M

2012-02-01

Transport of ballast water is one major factor in the transmission of aquatic organisms, including pathogenic bacteria. The IMO-guidelines of the Convention for the Control and Management of Ships' Ballast Water and Sediments, states that ships are to discharge <1 CFU per 100 ml ballast water of toxigenic Vibrio cholerae, emphasizing the need to establish test methods. To our knowledge, there are no methods sensitive and rapid enough available for cholera surveillance of ballast water. In this study real-time PCR and NASBA methods have been evaluated to specifically detect 1 CFU/100ml of V. cholerae in ballast water. Ballast water samples spiked with V. cholerae cells were filtered and enriched in alkaline peptone water before PCR or NASBA detection. The entire method, including sample preparation and analysis was performed within 7 h, and has the potential to be used for analysis of ballast water for inspection and enforcement control. Copyright © 2011 Elsevier Ltd. All rights reserved.

In-situ and real-time growth observation of high-quality protein crystals under quasi-microgravity on earth.

PubMed

Nakamura, Akira; Ohtsuka, Jun; Kashiwagi, Tatsuki; Numoto, Nobutaka; Hirota, Noriyuki; Ode, Takahiro; Okada, Hidehiko; Nagata, Koji; Kiyohara, Motosuke; Suzuki, Ei-Ichiro; Kita, Akiko; Wada, Hitoshi; Tanokura, Masaru

2016-02-26

Precise protein structure determination provides significant information on life science research, although high-quality crystals are not easily obtained. We developed a system for producing high-quality protein crystals with high throughput. Using this system, gravity-controlled crystallization are made possible by a magnetic microgravity environment. In addition, in-situ and real-time observation and time-lapse imaging of crystal growth are feasible for over 200 solution samples independently. In this paper, we also report results of crystallization experiments for two protein samples. Crystals grown in the system exhibited magnetic orientation and showed higher and more homogeneous quality compared with the control crystals. The structural analysis reveals that making use of the magnetic microgravity during the crystallization process helps us to build a well-refined protein structure model, which has no significant structural differences with a control structure. Therefore, the system contributes to improvement in efficiency of structural analysis for "difficult" proteins, such as membrane proteins and supermolecular complexes.

Smartphone-based assessment of blood alteration severity

NASA Astrophysics Data System (ADS)

Li, Xianglin; Xue, Jiaxin; Li, Wei; Li, Ting

2018-02-01

Blood quality and safety management is a critical issue for cold chain transportation of blood or blood-based biological reagent. The conventional methods of blood alteration severity assessment mainly rely on kit test or blood-gas analysis required opening the blood package to get samples, which cause possible blood pollution and are complicate, timeconsuming, and expensive. Here we proposed to develop a portable, real-time, safety, easy-operated and low cost method aimed at assessing blood alteration severity. Color images of the blood in transparent blood bags were collected with a smartphone and the alteration severity of the blood was assessed by the smartphone app offered analysis of RGB color values of the blood. The algorithm is based on a large number sample of RGB values of blood at different alteration degree. The blood quality results evaluated by the smartphone are in accordance with the actual data. This study indicates the potential of smart phone in real time, convenient, and reliable blood quality assessment.

Gas dispersion concentration of trace inorganic contaminants from fuel gas and analysis using head-column field-amplified sample stacking capillary electrophoresis.

PubMed

Yang, Jianmin; Li, Hai-Fang; Li, Meilan; Lin, Jin-Ming

2012-08-21

The presence of inorganic elements in fuel gas generally accelerates the corrosion and depletion of materials used in the fuel gas industry, and even leads to serious accidents. For identification of existing trace inorganic contaminants in fuel gas in a portable way, a highly efficient gas-liquid sampling collection system based on gas dispersion concentration is introduced in this work. Using the constructed dual path gas-liquid collection setup, inorganic cations and anions were simultaneously collected from real liquefied petroleum gas (LPG) and analyzed by capillary electrophoresis (CE) with indirect UV absorbance detection. The head-column field-amplified sample stacking technique was applied to improve the detection limits to 2-25 ng mL(-1). The developed collection and analytical methods have successfully determined existing inorganic contaminants in a real LPG sample in the range of 4.59-138.69 μg m(-3). The recoveries of cations and anions with spiked LPG samples were between 83.98 and 105.63%, and the relative standard deviations (RSDs) were less than 7.19%.

[Microinjection Monitoring System Design Applied to MRI Scanning].

PubMed

Xu, Yongfeng

2017-09-30

A microinjection monitoring system applied to the MRI scanning was introduced. The micro camera probe was used to stretch into the main magnet for real-time video injection monitoring of injection tube terminal. The programming based on LabVIEW was created to analysis and process the real-time video information. The feedback signal was used for intelligent controlling of the modified injection pump. The real-time monitoring system can make the best use of injection under the condition that the injection device was away from the sample which inside the magnetic room and unvisible. 9.4 T MRI scanning experiment showed that the system in ultra-high field can work stability and doesn't affect the MRI scans.

Payload and Components Real-Time Automated Test System (PACRATS), Data Acquisition of Leak Rate and Pressure Data Test Procedure

NASA Technical Reports Server (NTRS)

Rinehart, Maegan L.

2011-01-01

The purpose of this activity is to provide the Mechanical Components Test Facility (MCTF) with the capability to obtain electronic leak test and proof pressure data, Payload and Components Real-time Automated Test System (PACRATS) data acquisition software will be utilized to display real-time data. It will record leak rates and pressure/vacuum level(s) simultaneously. This added functionality will provide electronic leak test and pressure data at specified sampling frequencies. Electronically stored data will provide ES61 with increased data security, analysis, and accuracy. The tasks performed in this procedure are to verify PACRATS only, and are not intended to provide verifications for MCTF equipment.

Solid-phase extraction with the metal-organic framework MIL-101(Cr) combined with direct analysis in real time mass spectrometry for the fast analysis of triazine herbicides.

PubMed

Li, Xianjiang; Xing, Jiawei; Chang, Cuilan; Wang, Xin; Bai, Yu; Yan, Xiuping; Liu, Huwei

2014-06-01

MIL-101(Cr) is an excellent metal-organic framework with high surface area and nanoscale cavities, making it promising in solid-phase extraction. Herein, we used MIL-101(Cr) as a solid-phase extraction packing material combined with fast detection of direct analysis in real time mass spectrometry (DART-MS) for the analysis of triazine herbicides. After systematic optimization of the operation parameters, including the gas temperature of DART, the moving speed of the 1D platform, solvent for desorption, amount of MIL-101(Cr) extraction time, eluent volume and salt concentration, this method can realize the simultaneous detection of five kinds of triazine herbicides. The limits of detection were 0.1∼0.2 ng/mL and the linear ranges covered more than two orders of magnitude with the quantitation limits of 0.5∼1 ng/mL. Moreover, the developed method has been applied for the analysis of lake water samples and the recoveries for spiked analytes were in the range of 85∼110%. These results showed that solid-phase extraction with metal-organic frameworks is an efficient sample preparation approach for DART-MS analysis and could find more applications in environmental analysis. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Surface plasmon resonance biosensors for highly sensitive detection in real samples

NASA Astrophysics Data System (ADS)

Sepúlveda, B.; Carrascosa, L. G.; Regatos, D.; Otte, M. A.; Fariña, D.; Lechuga, L. M.

2009-08-01

In this work we summarize the main results obtained with the portable surface plasmon resonance (SPR) device developed in our group (commercialised by SENSIA, SL, Spain), highlighting its applicability for the real-time detection of extremely low concentrations of toxic pesticides in environmental water samples. In addition, we show applications in clinical diagnosis as, on the one hand, the real-time and label-free detection of DNA hybridization and single point mutations at the gene BRCA-1, related to the predisposition in women to develop an inherited breast cancer and, on the other hand, the analysis of protein biomarkers in biological samples (urine, serum) for early detection of diseases. Despite the large number of applications already proven, the SPR technology has two main drawbacks: (i) not enough sensitivity for some specific applications (where pM-fM or single-molecule detection are needed) (ii) low multiplexing capabilities. In order solve such drawbacks, we work in several alternative configurations as the Magneto-optical Surface Plasmon Resonance sensor (MOSPR) based on a combination of magnetooptical and ferromagnetic materials, to improve the SPR sensitivity, or the Localized Surface Plasmon Resonance (LSPR) based on nanostructures (nanoparticles, nanoholes,...), for higher multiplexing capabilities.

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

PubMed

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

2015-03-01

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

Design of point-of-care (POC) microfluidic medical diagnostic devices

NASA Astrophysics Data System (ADS)

Leary, James F.

2018-02-01

Design of inexpensive and portable hand-held microfluidic flow/image cytometry devices for initial medical diagnostics at the point of initial patient contact by emergency medical personnel in the field requires careful design in terms of power/weight requirements to allow for realistic portability as a hand-held, point-of-care medical diagnostics device. True portability also requires small micro-pumps for high-throughput capability. Weight/power requirements dictate use of super-bright LEDs and very small silicon photodiodes or nanophotonic sensors that can be powered by batteries. Signal-to-noise characteristics can be greatly improved by appropriately pulsing the LED excitation sources and sampling and subtracting noise in between excitation pulses. The requirements for basic computing, imaging, GPS and basic telecommunications can be simultaneously met by use of smartphone technologies, which become part of the overall device. Software for a user-interface system, limited real-time computing, real-time imaging, and offline data analysis can be accomplished through multi-platform software development systems that are well-suited to a variety of currently available cellphone technologies which already contain all of these capabilities. Microfluidic cytometry requires judicious use of small sample volumes and appropriate statistical sampling by microfluidic cytometry or imaging for adequate statistical significance to permit real-time (typically < 15 minutes) medical decisions for patients at the physician's office or real-time decision making in the field. One or two drops of blood obtained by pin-prick should be able to provide statistically meaningful results for use in making real-time medical decisions without the need for blood fractionation, which is not realistic in the field.

Structural aspects of digestion of medium chain triglycerides studied in real time using sSAXS and Cryo-TEM.

PubMed

Phan, Stephanie; Hawley, Adrian; Mulet, Xavier; Waddington, Lynne; Prestidge, Clive A; Boyd, Ben J

2013-12-01

The purpose of this study was to investigate the colloidal structures formed on digestion of medium chain triglyceride (MCT) with a specific objective of identifying and characterizing a previously reported vesicular phase, which has been linked to supersaturation and anomalous digestion kinetics, and to evaluate the influence of lipid mass and enzyme inhibition on self assembled structure. MCT was digested in vitro and nanostructure was monitored in real time using synchrotron small angle X-ray scattering (sSAXS), and morphology was studied using cryogenic transmission electron microscopy (cryo-TEM). Formation of the putative vesicular phase formed on digestion of MCT was confirmed and its structural attributes were determined. Vesicle formation was dependent on lipid mass and bile salt concentration. The use of enzyme inhibitor for offline analysis of lipolysis samples did influence structural aspects of the digestion medium when compared to real time evaluation. The formation of a vesicular phase was directly linked to the kinetics of lipid digestion. Vesicle formation is linked to lipid mass, or more specifically the ratio of lipid to bile salts present in the digestion mixture. Inhibition of lipase to halt digestion during sampling for offline analysis must be done with caution as structural aspects were shown to differ for the MCT digests with and without inhibitor present.

Molecular Analysis of Spinal Muscular Atrophy: A genotyping protocol based on TaqMan(®) real-time PCR.

PubMed

de Souza Godinho, Fernanda Marques; Bock, Hugo; Gheno, Tailise Conte; Saraiva-Pereira, Maria Luiza

2012-12-01

Spinal muscular atrophy (SMA) is an autosomal recessive inherited disorder caused by alterations in the survival motor neuron I (SMN1) gene. SMA patients are classified as type I-IV based on severity of symptoms and age of onset. About 95% of SMA cases are caused by the homozygous absence of SMN1 due to gene deletion or conversion into SMN2. PCR-based methods have been widely used in genetic testing for SMA. In this work, we introduce a new approach based on TaqMan(®)real-time PCR for research and diagnostic settings. DNA samples from 100 individuals with clinical signs and symptoms suggestive of SMA were analyzed. Mutant DNA samples as well as controls were confirmed by DNA sequencing. We detected 58 SMA cases (58.0%) by showing deletion of SMN1 exon 7. Considering clinical information available from 56 of them, the patient distribution was 26 (46.4%) SMA type I, 16 (28.6%) SMA type II and 14 (25.0%) SMA type III. Results generated by the new method was confirmed by PCR-RFLP and by DNA sequencing when required. In conclusion, a protocol based on real-time PCR was shown to be effective and specific for molecular analysis of SMA patients.

Description and interpretation of the bracts epidermis of Gramineae (Poaceae) with rotated image with maximum average power spectrum (RIMAPS) technique.

PubMed

Favret, Eduardo A; Fuentes, Néstor O; Molina, Ana M; Setten, Lorena M

2008-10-01

During the last few years, RIMAPS technique has been used to characterize the micro-relief of metallic surfaces and recently also applied to biological surfaces. RIMAPS is an image analysis technique which uses the rotation of an image and calculates its average power spectrum. Here, it is presented as a tool for describing the morphology of the trichodium net found in some grasses, which is developed on the epidermal cells of the lemma. Three different species of grasses (herbarium samples) are analyzed: Podagrostis aequivalvis (Trin.) Scribn. & Merr., Bromidium hygrometricum (Nees) Nees & Meyen and Bromidium ramboi (Parodi) Rúgolo. Simple schemes representing the real microstructure of the lemma are proposed and studied. RIMAPS spectra of both the schemes and the real microstructures are compared. These results allow inferring how similar the proposed geometrical schemes are to the real microstructures. Each geometrical pattern could be used as a reference for classifying other species. Finally, this kind of analysis is used to determine the morphology of the trichodium net of Agrostis breviculmis Hitchc. As the dried sample had shrunk and the microstructure was not clear, two kinds of morphology are proposed for the trichodium net of Agrostis L., one elliptical and the other rectilinear, the former being the most suitable.

Reconstruction of real-space linear matter power spectrum from multipoles of BOSS DR12 results

NASA Astrophysics Data System (ADS)

Lee, Seokcheon

2018-02-01

Recently, the power spectrum (PS) multipoles using the Baryon Oscillation Spectroscopic Survey (BOSS) Data Release 12 (DR12) sample are analyzed [1]. The based model for the analysis is the so-called TNS quasi-linear model and the analysis provides the multipoles up to the hexadecapole [2]. Thus, one might be able to recover the real-space linear matter PS by using the combinations of multipoles to investigate the cosmology [3]. We provide the analytic form of the ratio of quadrupole (hexadecapole) to monopole moments of the quasi-linear PS including the Fingers-of-God (FoG) effect to recover the real-space PS in the linear regime. One expects that observed values of the ratios of multipoles should be consistent with those of the linear theory at large scales. Thus, we compare the ratios of multipoles of the linear theory, including the FoG effect with the measured values. From these, we recover the linear matter power spectra in real-space. These recovered power spectra are consistent with the linear matter power spectra.

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.

Simplified Pan-species Real-time PCR-based Detection of Plasmodium Spp. in Blood Smear.

PubMed

Hassanpour, Gholamreza; Mirhendi, Hossein; Mohebali, Mehdi; Raeisi, Ahmad; Zeraati, Hojjat; Keshavarz, Hossein

2016-01-01

We aimed to quicken and simplify the detection of Plasmodium in blood samples by developing and testing a pan- Plasmodium real-time PCR for accurate screening of individuals suspected of malaria. A single primer/probe set for pan-species Plasmodium -specific real time PCR targeting a conserved region of the small subunit 18S ribosomal DNA was designed and evaluated for rapid diagnosis and screening of malaria infections using dried blood smears. FTA cards were used for rapid and simple DNA extraction. The primers and probes showed a positive response with the DNA extracted from bloods infected with P. falciparum and P. vivax but not with DNA extracted from various smears from uninfected blood samples. Seven positive cases positive by both microscopy and nested PCR were found among 280 blood samples taken from in South and Southeast Iran. Five samples were identified as positive for P. vivax and two as positive for P. falciparum . All positive samples were positive by real-time PCR. Furthermore, all 38-blood samples positive by microscopy were positive by real-time PCR. No microscopy-negative samples were positive by real-time PCR. By using a simple FTA card for DNA extraction and by application of the real-time PCR developed in this study, sensitivity similar to nested-PCR and microscopy was achieved. This format simplifies the detection of Plasmodium in large numbers of samples.

Assessing direct analysis in real-time-mass spectrometry (DART-MS) for the rapid identification of additives in food packaging.

PubMed

Ackerman, L K; Noonan, G O; Begley, T H

2009-12-01

The ambient ionization technique direct analysis in real time (DART) was characterized and evaluated for the screening of food packaging for the presence of packaging additives using a benchtop mass spectrometer (MS). Approximate optimum conditions were determined for 13 common food-packaging additives, including plasticizers, anti-oxidants, colorants, grease-proofers, and ultraviolet light stabilizers. Method sensitivity and linearity were evaluated using solutions and characterized polymer samples. Additionally, the response of a model additive (di-ethyl-hexyl-phthalate) was examined across a range of sample positions, DART, and MS conditions (temperature, voltage and helium flow). Under optimal conditions, molecular ion (M+H+) was the major ion for most additives. Additive responses were highly sensitive to sample and DART source orientation, as well as to DART flow rates, temperatures, and MS inlet voltages, respectively. DART-MS response was neither consistently linear nor quantitative in this setting, and sensitivity varied by additive. All additives studied were rapidly identified in multiple food-packaging materials by DART-MS/MS, suggesting this technique can be used to screen food packaging rapidly. However, method sensitivity and quantitation requires further study and improvement.

Rapid detection of HLA-B*51 by real-time polymerase chain reaction and high-resolution melting analysis.

PubMed

Imperiali, C; Alía-Ramos, P; Padró-Miquel, A

2015-08-01

HLA-B*51, a class I human leukocyte antigen (HLA) molecule, is the strongest known genetic risk factor for Behçet disease. However, there are only few articles reporting methods to determine the presence or absence of HLA-B51. For this reason, we designed and developed an easy, fast, and inexpensive real-time high-resolution melting (HRM) assay to detect HLA-B*51. We genotyped 61 samples by our HRM assay and by conventional polymerase chain reaction, and no discrepancies were found between results. Besides, a subgroup of 25 samples was also genotyped in a different laboratory, and another subgroup of 16 samples was obtained from the International Histocompatibility Working Group DNA Bank, and a full concordance of results was observed with those obtained by HRM. Regarding the identifying system evaluated, we obtained 100% of specificity, sensibility, and repeatability, and 0% of false positive and false negative rates. Therefore, this HRM analysis is easily applicable to the rapid detection of HLA-B*51, exhibits a high speed, and requires a very low budget. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

PubMed

Gianfranceschi, Monica Virginia; Rodriguez-Lazaro, David; Hernandez, Marta; González-García, Patricia; Comin, Damiano; Gattuso, Antonietta; Delibato, Elisabetta; Sonnessa, Michele; Pasquali, Frederique; Prencipe, Vincenza; Sreter-Lancz, Zuzsanna; Saiz-Abajo, María-José; Pérez-De-Juan, Javier; Butrón, Javier; Kozačinski, Lidija; Tomic, Danijela Horvatek; Zdolec, Nevijo; Johannessen, Gro S; Jakočiūnė, Džiuginta; Olsen, John Elmerdahl; De Santis, Paola; Lovari, Sarah; Bertasi, Barbara; Pavoni, Enrico; Paiusco, Antonella; De Cesare, Alessandra; Manfreda, Gerardo; De Medici, Dario

2014-08-01

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories. Copyright © 2014 Elsevier B.V. All rights reserved.

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

Long-term stability of morphine, codeine, and 6-acetylmorphine in real-life whole blood samples, stored at -20°C.

PubMed

Høiseth, Gudrun; Fjeld, Bente; Burns, Margrete Larsen; Strand, Dag Helge; Vindenes, Vigdis

2014-06-01

Stability of drugs during storage is important in forensic toxicology. For the analytes detected after intake of heroin (6-acetylmorphine (6-AM), morphine and codeine), long-time stability in real life whole blood samples are studied in only a small number of cases. Whole blood post mortem (n=37) and whole blood samples from living persons (n=22) containing morphine and codeine as well as 6-AM in blood or urine were selected. All cases represented intake of heroin. All samples contained fluoride and were initially analysed and stored in normal conditions (-20°C) for 4-9 years. All samples were then reanalysed using the same analytical methods and the results were compared. For samples from living persons, the median change in concentration was -3.7% for morphine and -5.3% for codeine. For post mortem samples, the median change in concentration was -12% for morphine and -11% for codeine. Both for samples from living persons and post mortem samples, the decrease in the concentrations from the original analysis to reanalysis were statistically significant for morphine and codeine. Regarding 6-AM, all living samples were negative at reanalysis. For post mortem samples, four cases still tested positive for 6-AM at reanalysis with a median change in the concentrations of -81%. There was no significant change in the morphine to codeine concentration ratios neither for living nor post mortem samples. This study showed that in real life whole blood samples, the concentrations of morphine and codeine are relatively stable during long-term storage at -20°C. 6-AM on the other hand, shows a considerable decrease in concentrations that is important to consider when interpreting results from reanalyses of forensic cases. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Real English Project Report.

ERIC Educational Resources Information Center

Cautin, Harvey; Regan, Edward

Requirements are discussed for an information retrieval language that enables users to employ natural language sentences in interaction with computer-stored files. Anticipated modes of operation of the system are outlined. These are: the search mode, the dictionary mode, the tables mode, and the statistical mode. Analysis of sample sentences…

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

PubMed

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

2015-12-01

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

Development and validation of a Pneumocystis jirovecii real-time polymerase chain reaction assay for diagnosis of Pneumocystis pneumonia

PubMed Central

Church, Deirdre L; Ambasta, Anshula; Wilmer, Amanda; Williscroft, Holly; Ritchie, Gordon; Pillai, Dylan R; Champagne, Sylvie; Gregson, Daniel G

2015-01-01

BACKGROUND: Pneumocystis jirovecii (PJ), a pathogenic fungus, causes severe interstitial Pneumocystis pneumonia (PCP) among immunocompromised patients. A laboratory-developed real-time polyermase chain reaction (PCR) assay was validated for PJ detection to improve diagnosis of PCP. METHODS: Forty stored bronchoalveolar lavage (BAL) samples (20 known PJ positive [PJ+] and 20 known PJ negative [PJ−]) were initially tested using the molecular assay. Ninety-two sequentially collected BAL samples were then analyzed using an immunofluorescence assay (IFA) and secondarily tested using the PJ real-time PCR assay. Discrepant results were resolved by retesting BAL samples using another real-time PCR assay with a different target. PJ real-time PCR assay performance was compared with the existing gold standard (ie, IFA) and a modified gold standard, in which a true positive was defined as a sample that tested positive in two of three methods in a patient suspected to have PCP. RESULTS: Ninety of 132 (68%) BAL fluid samples were collected from immunocompromised patients. Thirteen of 92 (14%) BALs collected were PJ+ when tested using IFA. A total of 40 BAL samples were PJ+ in the present study including: all IFA positive samples (n=13); all referred PJ+ BAL samples (n=20); and seven additional BAL samples that were IFA negative, but positive using the modified gold standard. Compared with IFA, the PJ real-time PCR had sensitivity, specificity, and positive and negative predictive values of 100%, 91%, 65% and 100%, respectively. Compared with the modified gold standard, PJ real-time PCR had a sensitivity, specificity, and positive and negative predictive values of 100%. CONCLUSION: PJ real-time PCR improved detection of PJ in immunocompromised patients. PMID:26600815

Rapid-viability PCR method for detection of live, virulent Bacillus anthracis in environmental samples.

PubMed

Létant, Sonia E; Murphy, Gloria A; Alfaro, Teneile M; Avila, Julie R; Kane, Staci R; Raber, Ellen; Bunt, Thomas M; Shah, Sanjiv R

2011-09-01

In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples.

Detection of bacteria and fungi in blood of patients with febrile neutropenia by real-time PCR with universal primers and probes.

PubMed

Teranishi, Hideto; Ohzono, Nanae; Inamura, Norikazu; Kato, Atsushi; Wakabayashi, Tokio; Akaike, Hiroto; Terada, Kihei; Ouchi, Kazunobu

2015-03-01

Febrile neutropenia is the main treatment-related cause of mortality in cancer patients. During June 2012 to April 2014, 97 blood culture samples were collected from patients receiving chemotherapy for hematological malignancy and cancer with febrile neutropenia episodes (FNEs). The samples were examined for the presence of bacteria and fungi using real-time PCR amplification and sequencing of 16S and 18S rRNA genes. Bacteria were identified in 20 of 97 samples (20.6%) by the real-time PCR assay and in 10 of 97 (10.3%) samples by blood culture. In 6 blood culture-positive samples, the real-time PCR assay detected the same type of bacteria. No fungi were detected by the real-time PCR assay or blood culture. During antibiotic therapy, all samples were negative by blood culture, but the real-time PCR assay yielded a positive result in 2 cases of 2 (100%). The bacterial DNA copy number was not well correlated with the serum C-reactive protein titer of patients with FNEs. We conclude that a real-time PCR assay could provide better detection of causative microbes' in a shorter time, and with a smaller blood sample than blood culture. Using a real-time PCR assay in combination with blood culture could improve microbiological documentation of FNEs. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Optimization of a direct analysis in real time/time-of-flight mass spectrometry method for rapid serum metabolomic fingerprinting.

PubMed

Zhou, Manshui; McDonald, John F; Fernández, Facundo M

2010-01-01

Metabolomic fingerprinting of bodily fluids can reveal the underlying causes of metabolic disorders associated with many diseases, and has thus been recognized as a potential tool for disease diagnosis and prognosis following therapy. Here we report a rapid approach in which direct analysis in real time (DART) coupled with time-of-flight (TOF) mass spectrometry (MS) and hybrid quadrupole TOF (Q-TOF) MS is used as a means for metabolomic fingerprinting of human serum. In this approach, serum samples are first treated to precipitate proteins, and the volatility of the remaining metabolites increased by derivatization, followed by DART MS analysis. Maximum DART MS performance was obtained by optimizing instrumental parameters such as ionizing gas temperature and flow rate for the analysis of identical aliquots of a healthy human serum samples. These variables were observed to have a significant effect on the overall mass range of the metabolites detected as well as the signal-to-noise ratios in DART mass spectra. Each DART run requires only 1.2 min, during which more than 1500 different spectral features are observed in a time-dependent fashion. A repeatability of 4.1% to 4.5% was obtained for the total ion signal using a manual sampling arm. With the appealing features of high-throughput, lack of memory effects, and simplicity, DART MS has shown potential to become an invaluable tool for metabolomic fingerprinting. 2010 American Society for Mass Spectrometry. Published by Elsevier Inc. All rights reserved.

Real life experimental determination of platinum group metals content in automotive catalytic converters

NASA Astrophysics Data System (ADS)

Yakoumis, I.; Moschovi, A. M.; Giannopoulou, I.; Panias, D.

2018-03-01

The real life experimental protocol for the preparation of spent automobile catalyst samples for elemental analysis is thoroughly described in the following study. Collection, sorting and dismantling, homogenization and sample preparation for X-Ray fluorescence spectroscopy and Atomic Adsorption Spectroscopy combined with Inductive coupled plasma mass spectrometry are discussed in detail for both ceramic and metallic spent catalysts. The concentrations of Platinum Group Metals (PGMs) in spent catalytic converters are presented based on typical consignments of recycled converters (more than 45,000 pieces) from the Greek Market. The conclusions clearly denoted commercial metallic catalytic foil contains higher PGMs loading than ceramic honeycombs. On the other hand, the total PGMs loading in spent ceramic catalytic converters has been found higher than the corresponding value for the metallic ones.

Analysis of munitions constituents in groundwater using a field-portable GC-MS.

PubMed

Bednar, A J; Russell, A L; Hayes, C A; Jones, W T; Tackett, P; Splichal, D E; Georgian, T; Parker, L V; Kirgan, R A; MacMillan, D K

2012-05-01

The use of munitions constituents (MCs) at military installations can produce soil and groundwater contamination that requires periodic monitoring even after training or manufacturing activities have ceased. Traditional groundwater monitoring methods require large volumes of aqueous samples (e.g., 2-4 L) to be shipped under chain of custody, to fixed laboratories for analysis. The samples must also be packed on ice and shielded from light to minimize degradation that may occur during transport and storage. The laboratory's turn-around time for sample analysis and reporting can be as long as 45 d. This process hinders the reporting of data to customers in a timely manner; yields data that are not necessarily representative of current site conditions owing to the lag time between sample collection and reporting; and incurs significant shipping costs for samples. The current work compares a field portable Gas Chromatograph-Mass Spectrometer (GC-MS) for analysis of MCs on-site with traditional laboratory-based analysis using High Performance Liquid Chromatography with UV absorption detection. The field method provides near real-time (within ~1 h of sampling) concentrations of MCs in groundwater samples. Mass spectrometry provides reliable confirmation of MCs and a means to identify unknown compounds that are potential false positives for methods with UV and other non-selective detectors. Published by Elsevier Ltd.

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

PubMed

Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Real-time image annotation by manifold-based biased Fisher discriminant analysis

NASA Astrophysics Data System (ADS)

Ji, Rongrong; Yao, Hongxun; Wang, Jicheng; Sun, Xiaoshuai; Liu, Xianming

2008-01-01

Automatic Linguistic Annotation is a promising solution to bridge the semantic gap in content-based image retrieval. However, two crucial issues are not well addressed in state-of-art annotation algorithms: 1. The Small Sample Size (3S) problem in keyword classifier/model learning; 2. Most of annotation algorithms can not extend to real-time online usage due to their low computational efficiencies. This paper presents a novel Manifold-based Biased Fisher Discriminant Analysis (MBFDA) algorithm to address these two issues by transductive semantic learning and keyword filtering. To address the 3S problem, Co-Training based Manifold learning is adopted for keyword model construction. To achieve real-time annotation, a Bias Fisher Discriminant Analysis (BFDA) based semantic feature reduction algorithm is presented for keyword confidence discrimination and semantic feature reduction. Different from all existing annotation methods, MBFDA views image annotation from a novel Eigen semantic feature (which corresponds to keywords) selection aspect. As demonstrated in experiments, our manifold-based biased Fisher discriminant analysis annotation algorithm outperforms classical and state-of-art annotation methods (1.K-NN Expansion; 2.One-to-All SVM; 3.PWC-SVM) in both computational time and annotation accuracy with a large margin.

Comparison among amoA Primers Suited for Quantification and Diversity Analyses of Ammonia-Oxidizing Bacteria in Soil

PubMed Central

Shimomura, Yumi; Morimoto, Sho; Hoshino, Yuko Takada; Uchida, Yoshitaka; Akiyama, Hiroko; Hayatsu, Masahito

2012-01-01

Ammonia monooxygenase subunit A gene (amoA) is frequently used as a functional gene marker for diversity analysis of ammonia-oxidizing bacteria (AOB). To select a suitable amoA primer for real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE), three reverse primers (degenerate primer amoA-2R; non-degenerate primers amoA-2R-GG and amoA-2IR) were examined. No significant differences were observed among the three primers in terms of quantitative values of amoA from environmental samples using real-time PCR. We found that PCR-DGGE analysis with the amoA-2IR primer gave the best results in this studied soil. These results indicate that amoA-2IR is a suitable primer for community analysis of AOB in the environment. PMID:22075625

DigOut: viewing differential expression genes as outliers.

PubMed

Yu, Hui; Tu, Kang; Xie, Lu; Li, Yuan-Yuan

2010-12-01

With regards to well-replicated two-conditional microarray datasets, the selection of differentially expressed (DE) genes is a well-studied computational topic, but for multi-conditional microarray datasets with limited or no replication, the same task is not properly addressed by previous studies. This paper adopts multivariate outlier analysis to analyze replication-lacking multi-conditional microarray datasets, finding that it performs significantly better than the widely used limit fold change (LFC) model in a simulated comparative experiment. Compared with the LFC model, the multivariate outlier analysis also demonstrates improved stability against sample variations in a series of manipulated real expression datasets. The reanalysis of a real non-replicated multi-conditional expression dataset series leads to satisfactory results. In conclusion, a multivariate outlier analysis algorithm, like DigOut, is particularly useful for selecting DE genes from non-replicated multi-conditional gene expression dataset.

Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.

PubMed

Shin, Saeam; Kim, Juwon; Kim, Yoonjung; Cho, Sun-Mi; Lee, Kyung-A

2017-10-26

EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples. A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples. All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%. We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.

Simplified Pan-species Real-time PCR-based Detection of Plasmodium Spp. in Blood Smear

PubMed Central

HASSANPOUR, Gholamreza; MIRHENDI, Hossein; MOHEBALI, Mehdi; RAEISI, Ahmad; ZERAATI, Hojjat; KESHAVARZ, Hossein

2016-01-01

Background: We aimed to quicken and simplify the detection of Plasmodium in blood samples by developing and testing a pan-Plasmodium real-time PCR for accurate screening of individuals suspected of malaria. Methods: A single primer/probe set for pan-species Plasmodium-specific real time PCR targeting a conserved region of the small subunit 18S ribosomal DNA was designed and evaluated for rapid diagnosis and screening of malaria infections using dried blood smears. FTA cards were used for rapid and simple DNA extraction. Results: The primers and probes showed a positive response with the DNA extracted from bloods infected with P. falciparum and P. vivax but not with DNA extracted from various smears from uninfected blood samples. Seven positive cases positive by both microscopy and nested PCR were found among 280 blood samples taken from in South and Southeast Iran. Five samples were identified as positive for P. vivax and two as positive for P. falciparum. All positive samples were positive by real-time PCR. Furthermore, all 38-blood samples positive by microscopy were positive by real-time PCR. No microscopy-negative samples were positive by real-time PCR. Conclusion: By using a simple FTA card for DNA extraction and by application of the real-time PCR developed in this study, sensitivity similar to nested-PCR and microscopy was achieved. This format simplifies the detection of Plasmodium in large numbers of samples. PMID:28127357

Comparison of Enterococcus qPCR analysis results from fresh and marine water samples on two real-time instruments - poster

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) will be recommending a quantitative polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for widespread implementation of this or ...

Simultaneous detection, typing and quantitation of oncogenic human papillomavirus by multiplex consensus real-time PCR.

PubMed

Jenkins, Andrew; Allum, Anne-Gry; Strand, Linda; Aakre, Randi Kersten

2013-02-01

A consensus multiplex real-time PCR test (PT13-RT) for the oncogenic human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 is described. The test targets the L1 gene. Analytical sensitivity is between 4 and 400 GU (genomic units) in the presence of 500 ng of human DNA, corresponding to 75,000 human cells. HPV types are grouped into multiplex groups of 3 or 4 resulting in the use of 4 wells per sample and permitting up to 24 samples per run (including controls) in a standard 96-well real-time PCR instrument. False negative results are avoided by (a) measuring sample DNA concentration to control that sufficient cellular material is present and (b) including HPV type 6 as a homologous internal control in order to detect PCR inhibition or competition from other (non-oncogenic) HPV types. Analysis time from refrigerator to report is 8 h, including 2.5 h hands-on time. Relative to the HC2 test, the sensitivity and specificity were respectively 98% and 83%, the lower specificity being attributable to the higher analytical sensitivity of PT13-RT. To assess type determination comparison was made with a reversed line-blot test. Type concordance was high (κ=0.79) with discrepancies occurring mostly in multiple-positive samples. Copyright © 2012 Elsevier B.V. All rights reserved.

Quantitative luminescence imaging system

DOEpatents

Erwin, D.N.; Kiel, J.L.; Batishko, C.R.; Stahl, K.A.

1990-08-14

The QLIS images and quantifies low-level chemiluminescent reactions in an electromagnetic field. It is capable of real time nonperturbing measurement and simultaneous recording of many biochemical and chemical reactions such as luminescent immunoassays or enzyme assays. The system comprises image transfer optics, a low-light level digitizing camera with image intensifying microchannel plates, an image process or, and a control computer. The image transfer optics may be a fiber image guide with a bend, or a microscope, to take the light outside of the RF field. Output of the camera is transformed into a localized rate of cumulative digitalized data or enhanced video display or hard-copy images. The system may be used as a luminescent microdosimetry device for radiofrequency or microwave radiation, as a thermal dosimeter, or in the dosimetry of ultra-sound (sonoluminescence) or ionizing radiation. It provides a near-real-time system capable of measuring the extremely low light levels from luminescent reactions in electromagnetic fields in the areas of chemiluminescence assays and thermal microdosimetry, and is capable of near-real-time imaging of the sample to allow spatial distribution analysis of the reaction. It can be used to instrument three distinctly different irradiation configurations, comprising (1) RF waveguide irradiation of a small Petri-dish-shaped sample cell, (2) RF irradiation of samples in a microscope for the microscopic imaging and measurement, and (3) RF irradiation of small to human body-sized samples in an anechoic chamber. 22 figs.

Quantitative luminescence imaging system

DOEpatents

Erwin, David N.; Kiel, Johnathan L.; Batishko, Charles R.; Stahl, Kurt A.

1990-01-01

The QLIS images and quantifies low-level chemiluminescent reactions in an electromagnetic field. It is capable of real time nonperturbing measurement and simultaneous recording of many biochemical and chemical reactions such as luminescent immunoassays or enzyme assays. The system comprises image transfer optics, a low-light level digitizing camera with image intensifying microchannel plates, an image process or, and a control computer. The image transfer optics may be a fiber image guide with a bend, or a microscope, to take the light outside of the RF field. Output of the camera is transformed into a localized rate of cumulative digitalized data or enhanced video display or hard-copy images. The system may be used as a luminescent microdosimetry device for radiofrequency or microwave radiation, as a thermal dosimeter, or in the dosimetry of ultra-sound (sonoluminescence) or ionizing radiation. It provides a near-real-time system capable of measuring the extremely low light levels from luminescent reactions in electromagnetic fields in the areas of chemiluminescence assays and thermal microdosimetry, and is capable of near-real-time imaging of the sample to allow spatial distribution analysis of the reaction. It can be used to instrument three distinctly different irradiation configurations, comprising (1) RF waveguide irradiation of a small Petri-dish-shaped sample cell, (2) RF irradiation of samples in a microscope for the microscopie imaging and measurement, and (3) RF irradiation of small to human body-sized samples in an anechoic chamber.

Utilization of biosensors and chemical sensors for space applications

NASA Technical Reports Server (NTRS)

Bonting, S. L.

1992-01-01

There will be a need for a wide array of chemical sensors for biomedical experimentation and for the monitoring of water and air recycling processes on Space Station Freedom. The infrequent logistics flights of the Space Shuttle will necessitate onboard analysis. The advantages of biosensors and chemical sensors over conventional analysis onboard spacecraft are manifold. They require less crew time, space, and power. Sample treatment is not needed. Real time or near-real time monitoring is possible, in some cases on a continuous basis. Sensor signals in digitized form can be transmitted to the ground. Types and requirements for chemical sensors to be used in biomedical experimentation and monitoring of water recycling during long-term space missions are discussed.

Continuous-flow water sampler for real-time isotopic water measurements

NASA Astrophysics Data System (ADS)

Carter, J.; Dennis, K.

2013-12-01

Measuring the stable isotopes of liquid water (δ18O and δD) is a tool familiar to many Earth scientists, but most current techniques require discrete sampling. For example, isotope ratio mass spectrometry requires the collection of aliquots of water that are then converted to CO2, CO or H2 for analysis. Similarly, laser-based techniques, such as Cavity Ring-Down Spectroscopy (CRDS) convert discrete samples (typically < 2μL) of liquid water to water vapor using a flash vaporization process. By requiring the use of discrete samples fine-scale spatial and temporal studies of changes in δ18O and δD are limited. Here we present a continuous-flow water sampler that will enable scientists to probe isotopic changes in real-time, with applications including, but not limited to, quantification of the 'amount effect' (Dansgaard, 1964) during an individual precipitation event or storm track, real-time mixing of water in river systems, and shipboard continuous water measurements (Munksgaard et al., 2012). Due to the inherent ability of CRDS to measure a continuous flow of water vapor it is an ideal candidate for interfacing with a continuous water sampling system. Here we present results from the first commercially available continuous-flow water sampler, developed by engineers at Picarro. This peripheral device is compatible with Picarro CRDS isotopic water analyzers, allowing real-time, continuous isotopic measurements of liquid water. The new device, which expands upon the design of Munskgaard et al. (2011), utilizes expanded polytetrafluoroethylene (ePTFE) membrane technology to continuously generate gas-phase water, while liquid water is pumped through the system. The water vapor subsequently travels to the CRDS analyzer where the isotopic ratios are measured and recorded. The generation of water vapor using membrane technology is sensitive to environmental conditions, which if not actively control, lead to sustainable experimental noise and drift. Consequently, our continuous-flow water sample employs active control for all pertinent parameters, significantly increasing its stability and usability. We will present data from controlled laboratory experiments demonstrating sample-to-sample precision and long-term stability. We will also show experimental data that highlights the instrumental sample-to-sample memory, which we have decreased significantly from previous implementations of this technology. Additionally, we will present field results from the Sacramento River, CA. Dansgaard, W. (1964) 'Stable isotopes in precipitation', Tellus, 16(4), p. 436-468. Munksgaard, N.C., Wurster, C.M., Bass, A., Zagorskis, I., and Bird, M.I. (2012) 'First continuous shipboard d18O and dD measurements in seawater by diffusion sampling--cavity ring-down spectrometry', Environmental Chemistry Letters, 10, p.301-307. Munksgaard, N.C., Wurster, C.M., and Bird, M.I., (2011), 'Continuous analysis of δ18O and δD values of water by diffusion sampling cavity ring-down spectrometry: a novel sampling device for unattended field monitoring of precipitation, ground and surface waters', Rapid Communications in Mass Spectrometry, 25, p. 3706-3712.

A Comprehensive Review of Low-Speed Rear Impact Volunteer Studies and a Comparison to Real-World Outcomes.

PubMed

Cormier, Joseph; Gwin, Lisa; Reinhart, Lars; Wood, Rawson; Bain, Charles

2018-02-27

This study combined all prior research involving human volunteers in low-speed rear-end impacts and performed a comparative analysis of real-world crashes using the National Automotive Sampling System - Crashworthiness Data System. The aim of this study was to assess the rates of neck pain between volunteer and real-world collisions as well as the likelihood of an injury beyond symptoms as a function of impact severity and occupant characteristics in real-world collisions. A total of 51 human volunteer studies were identified that produced a dataset of 1984 volunteer impacts along with a separate dataset of 515,601 weighted occupants in real-world rear impacts. Operating-characteristic curves were created to assess the utility of the volunteer dataset in making predictions regarding the overall population. Change in speed or delta-V was used to model the likelihood of reporting symptoms in both real-world and volunteer exposures and more severe injuries using real-world data. Logistic regression models were created for the volunteer data and survey techniques were used to analyze the weighted sampling scheme with the National Automotive Sampling System database. Symptom reporting rates were not different between males and females and were nearly identical between laboratory and real-world exposures. The minimal risk of injury predicted by real-world exposure is consistent with the statistical power of the large number of volunteer studies without any injury beyond the reporting of neck pain. This study shows that volunteer studies do not under-report symptoms and are sufficient in number to conclude that the risk of injury beyond neck strain under similar conditions is essentially zero. The real-world injury analyses demonstrate that rear impacts do not produce meaningful risks of cervical injury at impacts of similar and greater severity to those of the volunteer research. Future work concerning the mechanism of whiplash-related trauma should focus on impacts of severity greater than those in the current literature. 3This is an open access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0.

Mobile membrane introduction tandem mass spectrometry for on-the-fly measurements and adaptive sampling of VOCs around oil and gas projects in Alberta, Canada

NASA Astrophysics Data System (ADS)

Krogh, E.; Gill, C.; Bell, R.; Davey, N.; Martinsen, M.; Thompson, A.; Simpson, I. J.; Blake, D. R.

2012-12-01

The release of hydrocarbons into the environment can have significant environmental and economic consequences. The evolution of smaller, more portable mass spectrometers to the field can provide spatially and temporally resolved information for rapid detection, adaptive sampling and decision support. We have deployed a mobile platform membrane introduction mass spectrometer (MIMS) for the in-field simultaneous measurement of volatile and semi-volatile organic compounds. In this work, we report instrument and data handling advances that produce geographically referenced data in real-time and preliminary data where these improvements have been combined with high precision ultra-trace VOCs analysis to adaptively sample air plumes near oil and gas operations in Alberta, Canada. We have modified a commercially available ion-trap mass spectrometer (Griffin ICX 400) with an in-house temperature controlled capillary hollow fibre polydimethylsiloxane (PDMS) polymer membrane interface and in-line permeation tube flow cell for a continuously infused internal standard. The system is powered by 24 VDC for remote operations in a moving vehicle. Software modifications include the ability to run continuous, interlaced tandem mass spectrometry (MS/MS) experiments for multiple contaminants/internal standards. All data are time and location stamped with on-board GPS and meteorological data to facilitate spatial and temporal data mapping. Tandem MS/MS scans were employed to simultaneously monitor ten volatile and semi-volatile analytes, including benzene, toluene, ethylbenzene and xylene (BTEX), reduced sulfur compounds, halogenated organics and naphthalene. Quantification was achieved by calibrating against a continuously infused deuterated internal standard (toluene-d8). Time referenced MS/MS data were correlated with positional data and processed using Labview and Matlab to produce calibrated, geographical Google Earth data-visualizations that enable adaptive sampling protocols. This real-time approach has been employed in a moving vehicle to identify and track downwind plumes of fugitive VOC emissions near hydrocarbon upgrading and chemical processing facilities in Fort Saskatchewan, Alberta. This information was relayed to a trailing vehicle, which collected stationary grab samples in evacuated canisters for ultra trace analysis of over seventy VOC analytes. In addition, stationary time series data were collected and compared with grab samples co-located with our sampling line. Spatially and temporally resolved, time referenced MS/MS data for several air contaminants associated with oil and gas processing were processed in real time to produce geospatial data for visualization in Google Earth. This information was used to strategically locate grab samples for high precision, ultra trace analysis.

Evaluation of the Abbott Real Time HCV genotype II assay for Hepatitis C virus genotyping.

PubMed

Sariguzel, Fatma Mutlu; Berk, Elife; Gokahmetoglu, Selma; Ercal, Baris Derya; Celik, Ilhami

2015-01-01

The determination of HCV genotypes and subtypes is very important for the selection of antiviral therapy and epidemiological studies. The aim of this study was to evaluate the performance of Abbott Real Time HCV Genotype II assay in HCV genotyping of HCV infected patients in Kayseri, Turkey. One hundred patients with chronic hepatitis C admitted to our hospital were evaluated between June 2012 and December 2012, HCV RNA levels were determined by the COBAS® AmpliPrep/COBAS® TaqMan® 48 HCV test. HCV genotyping was investigated by the Abbott Real Time HCV Genotype II assay. With the exception of genotype 1, subtypes of HCV genotypes could not be determined by Abbott assay. Sequencing analysis was used as the reference method. Genotypes 1, 2, 3 and 4 were observed in 70, 4, 2 and 24 of the 100 patients, respectively, by two methods. The concordance between the two systems to determine HCV major genotypes was 100%. Of 70 patients with genotype 1, 66 showed infection with subtype 1b and 4 with subtype 1a by Abbott Real Time HCV Genotype II assay. Using sequence analysis, 61 showed infection with subtype 1b and 9 with subtype 1a. In determining of HCV genotype 1 subtypes, the difference between the two methods was not statistically significant (P>0.05). HCV genotype 4 and 3 samples were found to be subtype 4d and 3a, respectively, by sequence analysis. There were four patients with genotype 2. Sequence analysis revealed that two of these patients had type 2a and the other two had type 2b. The Abbott Real Time HCV Genotype II assay yielded results consistent with sequence analysis. However, further optimization of the Abbott Real Time HCV Genotype II assay for subtype identification of HCV is required.

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.

Carbon nanodots as a matrix for the analysis of low-molecular-weight molecules in both positive- and negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and quantification of glucose and uric acid in real samples.

PubMed

Chen, Suming; Zheng, Huzhi; Wang, Jianing; Hou, Jian; He, Qing; Liu, Huihui; Xiong, Caiqiao; Kong, Xianglei; Nie, Zongxiu

2013-07-16

Carbon nanodots were applied for the first time as a new matrix for the analysis of low-molecular-weight compounds by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in both positive- and negative-ion modes. A wide range of small molecules including amino acids, peptides, fatty acids, as well as β-agonists and neutral oligosaccharides were analyzed by MALDI MS with carbon nanodots as the matrix, and the lowest 0.2 fmol limits-of-detection were obtained for octadecanoic acid. Clear sodium and potassium adducts and deprotonated signals were produced in positive- and negative-ion modes. Furthermore, the glucose and uric acid in real samples were quantitatively determined by the internal standard method with the linear range of 0.5-9 mM and 0.1-1.8 mM (R(2) > 0.999), respectively. This work gives new insight into the application of carbon nanodots and provides a general approach for rapid analysis of low-molecular-weight compounds.

Reactive control and reasoning assistance for scientific laboratory instruments

NASA Technical Reports Server (NTRS)

Thompson, David E.; Levinson, Richard; Robinson, Peter

1993-01-01

Scientific laboratory instruments that are involved in chemical or physical sample identification frequently require substantial human preparation, attention, and interactive control during their operation. Successful real-time analysis of incoming data that supports such interactive control requires: (1) a clear recognition of variance of the data from expected results; and (2) rapid diagnosis of possible alternative hypotheses which might explain the variance. Such analysis then aids in decisions about modifying the experiment protocol, as well as being a goal itself. This paper reports on a collaborative project at the NASA Ames Research Center between artificial intelligence researchers and planetary microbial ecologists. Our team is currently engaged in developing software that autonomously controls science laboratory instruments and that provides data analysis of the real-time data in support of dynamic refinement of the experiment control. the first two instruments to which this technology has been applied are a differential thermal analyzer (DTA) and a gas chromatograph (GC). coupled together, they form a new geochemicstry and microbial analysis tool that is capable of rapid identification of the organiz and mineralogical constituents in soils. The thermal decomposition of the minerals and organics, and the attendance release of evolved gases, provides data about the structural and molecular chemistry of the soil samples.

Detection of Organophosphorus Pesticides with Colorimetry and Computer Image Analysis.

PubMed

Li, Yanjie; Hou, Changjun; Lei, Jincan; Deng, Bo; Huang, Jing; Yang, Mei

2016-01-01

Organophosphorus pesticides (OPs) represent a very important class of pesticides that are widely used in agriculture because of their relatively high-performance and moderate environmental persistence, hence the sensitive and specific detection of OPs is highly significant. Based on the inhibitory effect of acetylcholinesterase (AChE) induced by inhibitors, including OPs and carbamates, a colorimetric analysis was used for detection of OPs with computer image analysis of color density in CMYK (cyan, magenta, yellow and black) color space and non-linear modeling. The results showed that there was a gradually weakened trend of yellow intensity with the increase of the concentration of dichlorvos. The quantitative analysis of dichlorvos was achieved by Artificial Neural Network (ANN) modeling, and the results showed that the established model had a good predictive ability between training sets and predictive sets. Real cabbage samples containing dichlorvos were detected by colorimetry and gas chromatography (GC), respectively. The results showed that there was no significant difference between colorimetry and GC (P > 0.05). The experiments of accuracy, precision and repeatability revealed good performance for detection of OPs. AChE can also be inhibited by carbamates, and therefore this method has potential applications in real samples for OPs and carbamates because of high selectivity and sensitivity.

Versatile new ion source for the analysis of materials in open air under ambient conditions.

PubMed

Cody, Robert B; Laramée, James A; Durst, H Dupont

2005-04-15

A new ion source has been developed for rapid, noncontact analysis of materials at ambient pressure and at ground potential. The new source, termed DART (for "Direct Analysis in Real Time"), is based on the reactions of electronic or vibronic excited-state species with reagent molecules and polar or nonpolar analytes. DART has been installed on a high-resolution time-of-flight mass spectrometer (TOFMS) that provides improved selectivity and accurate elemental composition assignment through exact mass measurements. Although DART has been applied to the analysis of gases, liquids, and solids, a unique application is the direct detection of chemicals on surfaces without requiring sample preparation, such as wiping or solvent extraction. DART has demonstrated success in sampling hundreds of chemicals, including chemical agents and their signatures, pharmaceutics, metabolites, peptides and oligosaccharides, synthetic organics, organometallics, drugs of abuse, explosives, and toxic industrial chemicals. These species were detected on various surfaces, such as concrete, asphalt, human skin, currency, airline boarding passes, business cards, fruits, vegetables, spices, beverages, body fluids, horticultural leaves, cocktail glasses, and clothing. DART employs no radioactive components and is more versatile than devices using radioisotope-based ionization. Because its response is instantaneous, DART provides real-time information, a critical requirement for screening or high throughput.

[Identification of spill oil species based on low concentration synchronous fluorescence spectra and RBF neural network].

PubMed

Liu, Qian-qian; Wang, Chun-yan; Shi, Xiao-feng; Li, Wen-dong; Luan, Xiao-ning; Hou, Shi-lin; Zhang, Jin-liang; Zheng, Rong-er

2012-04-01

In this paper, a new method was developed to differentiate the spill oil samples. The synchronous fluorescence spectra in the lower nonlinear concentration range of 10(-2) - 10(-1) g x L(-1) were collected to get training data base. Radial basis function artificial neural network (RBF-ANN) was used to identify the samples sets, along with principal component analysis (PCA) as the feature extraction method. The recognition rate of the closely-related oil source samples is 92%. All the results demonstrated that the proposed method could identify the crude oil samples effectively by just one synchronous spectrum of the spill oil sample. The method was supposed to be very suitable to the real-time spill oil identification, and can also be easily applied to the oil logging and the analysis of other multi-PAHs or multi-fluorescent mixtures.

Initial operation of the NSTX-Upgrade real-time velocity diagnostic

DOE PAGES

Podestà, M.; Bell, R. E.

2016-11-03

A real-time velocity (RTV) diagnostic based on active charge-exchange recombination spectroscopy is now operational on the National Spherical Torus Experiment-Upgrade (NSTX-U) spherical torus (Menard et al 2012 Nucl. Fusion 52 083015). We designed the system in order to supply plasma velocity data in real time to the NSTX-U plasma control system, as required for the implementation of toroidal rotation control. Our measurements are available from four radii at a maximum sampling frequency of 5 kHz. Post-discharge analysis of RTV data provides additional information on ion temperature, toroidal velocity and density of carbon impurities. Furthermore, examples of physics studies enabled bymore » RTV measurements from initial operations of NSTX-U are discussed.« less

Bacterial microbiota of Kazakhstan cheese revealed by single molecule real time (SMRT) sequencing and its comparison with Belgian, Kalmykian and Italian artisanal cheeses.

PubMed

Li, Jing; Zheng, Yi; Xu, Haiyan; Xi, Xiaoxia; Hou, Qiangchuan; Feng, Shuzhen; Wuri, Laga; Bian, Yanfei; Yu, Zhongjie; Kwok, Lai-Yu; Sun, Zhihong; Sun, Tiansong

2017-01-09

In Kazakhstan, traditional artisanal cheeses have a long history and are widely consumed. The unique characteristics of local artisanal cheeses are almost completely preserved. However, their microbial communities have rarely been reported. The current study firstly generated the Single Molecule, Real-Time (SMRT) sequencing bacterial diversity profiles of 6 traditional artisanal cheese samples of Kazakhstan origin, followed by comparatively analyzed the microbiota composition between the current dataset and those from cheeses originated from Belgium, Russian Republic of Kalmykia (Kalmykia) and Italy. Across the Kazakhstan cheese samples, a total of 238 bacterial species belonging to 14 phyla and 140 genera were identified. Lactococcus lactis (28.93%), Lactobacillus helveticus (26.43%), Streptococcus thermophilus (12.18%) and Lactobacillus delbrueckii (12.15%) were the dominant bacterial species for these samples. To further evaluate the cheese bacterial diversity of Kazakhstan cheeses in comparison with those from other geographic origins, 16S rRNA datasets of 36 artisanal cheeses from Belgium, Russian Republic of Kalmykia (Kalmykia) and Italy were retrieved from public databases. The cheese bacterial microbiota communities were largely different across sample origins. By principal coordinate analysis (PCoA) and multivariate analysis of variance (MANOVA), the structure of the Kazakhstan artisanal cheese samples was found to be different from those of the other geographic origins. Furthermore, the redundancy analysis (RDA) identified 16 bacterial OTUs as the key variables responsible for such microbiota structural difference. Our results together suggest that the diversity of bacterial communities in different groups is stratified by geographic region. This study does not only provide novel information on the bacterial microbiota of traditional artisanal cheese of Kazakhstan at species level, but also interesting insights into the bacterial diversity of artisanal cheeses of various geographical origins.

Rapid, Real-time Methane Detection in Ground Water Using a New Gas-Water Equilibrator Design

NASA Astrophysics Data System (ADS)

Ruybal, C. J.; DiGiulio, D. C.; Wilkin, R. T.; Hargrove, K. D.; McCray, J. E.

2014-12-01

Recent increases in unconventional gas development have been accompanied by public concern for methane contamination in drinking water wells near production areas. Although not a regulated pollutant, methane may be a marker contaminant for others that are less mobile in groundwater and thus may be detected later, or at a location closer to the source. In addition, methane poses an explosion hazard if exsolved concentrations reach 5 - 15% volume in air. Methods for determining dissolved gases, such as methane, have evolved over 60 years. However, the response time of these methods is insufficient to monitor trends in methane concentration in real-time. To enable rapid, real-time monitoring of aqueous methane concentrations during ground water purging, a new gas-water equilibrator (GWE) was designed that increases gas-water mass exchange rates of methane for measurement. Monitoring of concentration trends allows a comparison of temporal trends between sampling events and comparison of baseline conditions with potential post-impact conditions. These trends may be a result of removal of stored casing water, pre-purge ambient borehole flow, formation physical and chemical heterogeneity, or flow outside of well casing due to inadequate seals. Real-time information in the field can help focus an investigation, aid in determining when to collect a sample, save money by limiting costs (e.g. analytical, sample transport and storage), and provide an immediate assessment of local methane concentrations. Four domestic water wells, one municipal water well, and one agricultural water well were sampled for traditional laboratory analysis and compared to the field GWE results. Aqueous concentrations measured on the GWE ranged from non-detect to 1,470 μg/L methane. Some trends in aqueous methane concentrations measured on the GWE were observed during purging. Applying a paired t-test comparing the new GWE method and traditional laboratory analysis yielded a p-value 0.383, suggesting no significant difference between the two methods for the current study. Additional field and laboratory experimentation are necessary to justify use beyond screening. However, early GWE use suggests promising results and applications.

Prevalence of mip virulence gene and PCR-base sequence typing of Legionella pneumophila from cooling water systems of two cities in Iran.

PubMed

Ahmadrajabi, Roya; Shakibaie, Mohammad Reza; Iranmanesh, Zahra; Mollaei, Hamid Reza; Sobhanipoor, Mohammad Hossein

2016-07-03

Legionella pneumophila is the primary respiratory pathogen and mostly transmitted to human through water cooling systems and cause mild to severe pneumonia with high mortality rate especially in elderly both in hospitals and community. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods. Here, we investigated the presence of L. pneumophila mip gene in water samples collected from different hospitals cooling towers, nursing homes and building/hotels water coolants from two geographical locations of Iran (Kerman and Bam cities) during summer season of 2015 by both nested and real-time PCR methods. Analysis of the 128 water samples for presence of the mip gene by nested-PCR revealed, 18 (23%) positive cases in Kerman and 7(14%) in Bam. However, when samples were tested by real-time PCR, we identified 4 more new cases of L. pneumophila in the hospitals as well as nursing homes water systems that were missed by nested-PCR. The highest rate of contamination was detected in water obtained from hospitals cooling towers in both the cities (p≤0.05). Dendrogram analysis and clonal relationship by PCR-base sequence typing (SBT) of the L. pneumophila genomic DNAs in Kerman water samples showed close clonal similarities among the isolates, in contrast, isolates identified from Bam city demonstrated two fingerprint patterns. The clones from hospital water samples were more related to the L. pneumophila serogroup- 1.

Rapid detection of G6PD mutations by multicolor melting curve analysis.

PubMed

Xia, Zhongmin; Chen, Ping; Tang, Ning; Yan, Tizhen; Zhou, Yuqiu; Xiao, Qizhi; Huang, Qiuying; Li, Qingge

2016-09-01

The MeltPro G6PD assay is the first commercial genetic test for glucose-6-phosphate dehydrogenase (G6PD) deficiency. This multicolor melting curve analysis-based real-time PCR assay is designed to genotype 16 G6PD mutations prevalent in the Chinese population. We comprehensively evaluated both the analytical and clinical performances of this assay. All 16 mutations were accurately genotyped, and the standard deviation of the measured Tm was <0.3°C. The limit of detection was 1.0ng/μL human genomic DNA. The assay could be run on four mainstream models of real-time PCR machines. The shortest running time (150min) was obtained with LightCycler 480 II. A clinical study using 763 samples collected from three hospitals indicated that, of 433 samples with reduced G6PD activity, the MeltPro assay identified 423 samples as mutant, yielding a clinical sensitivity of 97.7% (423/433). Of the 117 male samples with normal G6PD activity, the MeltPro assay confirmed that 116 samples were wild type, yielding a clinical specificity of 99.1% (116/117). Moreover, the MeltPro assay demonstrated 100% concordance with DNA sequencing for all targeted mutations. We concluded that the MeltPro G6PD assay is useful as a diagnostic or screening tool for G6PD deficiency in clinical settings. Copyright © 2016 Elsevier Inc. All rights reserved.

Prevalence of mip virulence gene and PCR-base sequence typing of Legionella pneumophila from cooling water systems of two cities in Iran

PubMed Central

Ahmadrajabi, Roya; Shakibaie, Mohammad Reza; Iranmanesh, Zahra; Mollaei, Hamid Reza; Sobhanipoor, Mohammad Hossein

2016-01-01

ABSTRACT Legionella pneumophila is the primary respiratory pathogen and mostly transmitted to human through water cooling systems and cause mild to severe pneumonia with high mortality rate especially in elderly both in hospitals and community. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods. Here, we investigated the presence of L. pneumophila mip gene in water samples collected from different hospitals cooling towers, nursing homes and building/hotels water coolants from two geographical locations of Iran (Kerman and Bam cities) during summer season of 2015 by both nested and real-time PCR methods. Analysis of the 128 water samples for presence of the mip gene by nested-PCR revealed, 18 (23%) positive cases in Kerman and 7(14%) in Bam. However, when samples were tested by real-time PCR, we identified 4 more new cases of L. pneumophila in the hospitals as well as nursing homes water systems that were missed by nested-PCR. The highest rate of contamination was detected in water obtained from hospitals cooling towers in both the cities (p≤0.05). Dendrogram analysis and clonal relationship by PCR-base sequence typing (SBT) of the L. pneumophila genomic DNAs in Kerman water samples showed close clonal similarities among the isolates, in contrast, isolates identified from Bam city demonstrated two fingerprint patterns. The clones from hospital water samples were more related to the L. pneumophila serogroup- 1. PMID:27028760

Microcomputer data acquisition and control.

PubMed

East, T D

1986-01-01

In medicine and biology there are many tasks that involve routine well defined procedures. These tasks are ideal candidates for computerized data acquisition and control. As the performance of microcomputers rapidly increases and cost continues to go down the temptation to automate the laboratory becomes great. To the novice computer user the choices of hardware and software are overwhelming and sadly most of the computer sales persons are not at all familiar with real-time applications. If you want to bill your patients you have hundreds of packaged systems to choose from; however, if you want to do real-time data acquisition the choices are very limited and confusing. The purpose of this chapter is to provide the novice computer user with the basics needed to set up a real-time data acquisition system with the common microcomputers. This chapter will cover the following issues necessary to establish a real time data acquisition and control system: Analysis of the research problem: Definition of the problem; Description of data and sampling requirements; Cost/benefit analysis. Choice of Microcomputer hardware and software: Choice of microprocessor and bus structure; Choice of operating system; Choice of layered software. Digital Data Acquisition: Parallel Data Transmission; Serial Data Transmission; Hardware and software available. Analog Data Acquisition: Description of amplitude and frequency characteristics of the input signals; Sampling theorem; Specification of the analog to digital converter; Hardware and software available; Interface to the microcomputer. Microcomputer Control: Analog output; Digital output; Closed-Loop Control. Microcomputer data acquisition and control in the 21st Century--What is in the future? High speed digital medical equipment networks; Medical decision making and artificial intelligence.

Real-Time Food Authentication Using a Miniature Mass Spectrometer.

PubMed

Gerbig, Stefanie; Neese, Stephan; Penner, Alexander; Spengler, Bernhard; Schulz, Sabine

2017-10-17

Food adulteration is a threat to public health and the economy. In order to determine food adulteration efficiently, rapid and easy-to-use on-site analytical methods are needed. In this study, a miniaturized mass spectrometer in combination with three ambient ionization methods was used for food authentication. The chemical fingerprints of three milk types, five fish species, and two coffee types were measured using electrospray ionization, desorption electrospray ionization, and low temperature plasma ionization. Minimum sample preparation was needed for the analysis of liquid and solid food samples. Mass spectrometric data was processed using the laboratory-built software MS food classifier, which allows for the definition of specific food profiles from reference data sets using multivariate statistical methods and the subsequent classification of unknown data. Applicability of the obtained mass spectrometric fingerprints for food authentication was evaluated using different data processing methods, leave-10%-out cross-validation, and real-time classification of new data. Classification accuracy of 100% was achieved for the differentiation of milk types and fish species, and a classification accuracy of 96.4% was achieved for coffee types in cross-validation experiments. Measurement of two milk mixtures yielded correct classification of >94%. For real-time classification, the accuracies were comparable. Functionality of the software program and its performance is described. Processing time for a reference data set and a newly acquired spectrum was found to be 12 s and 2 s, respectively. These proof-of-principle experiments show that the combination of a miniaturized mass spectrometer, ambient ionization, and statistical analysis is suitable for on-site real-time food authentication.

Rapid Analysis of Trace Drugs and Metabolites Using a Thermal Desorption DART-MS Configuration.

PubMed

Sisco, Edward; Forbes, Thomas P; Staymates, Matthew E; Gillen, Greg

2016-01-01

The need to analyze trace narcotic samples rapidly for screening or confirmatory purposes is of increasing interest to the forensic, homeland security, and criminal justice sectors. This work presents a novel method for the detection and quantification of trace drugs and metabolites off of a swipe material using a thermal desorption direct analysis in real time mass spectrometry (TD-DART-MS) configuration. A variation on traditional DART, this configuration allows for desorption of the sample into a confined tube, completely independent of the DART source, allowing for more efficient and thermally precise analysis of material present on a swipe. Over thirty trace samples of narcotics, metabolites, and cutting agents deposited onto swipes were rapidly differentiated using this methodology. The non-optimized method led to sensitivities ranging from single nanograms to hundreds of picograms. Direct comparison to traditional DART with a subset of the samples highlighted an improvement in sensitivity by a factor of twenty to thirty and an increase in reproducibility sample to sample from approximately 45 % RSD to less than 15 % RSD. Rapid extraction-less quantification was also possible.

Microbiological Quality and Food Safety of Plants Grown on ISS Project

NASA Technical Reports Server (NTRS)

Wheeler, Raymond M. (Compiler)

2014-01-01

The goal of this project is to select and advance methods to enable real-time sampling, microbiological analysis, and sanitation of crops grown on the International Space Station (ISS). These methods would validate the microbiological quality of crops grown for consumption to ensure safe and palatable fresh foods. This would be achieved through the development / advancement of microbiological sample collection, rapid pathogen detection and effective sanitation methods that are compatible with a microgravity environment.

Artificial neural networks applied to quantitative elemental analysis of organic material using PIXE

NASA Astrophysics Data System (ADS)

Correa, R.; Chesta, M. A.; Morales, J. R.; Dinator, M. I.; Requena, I.; Vila, I.

2006-08-01

An artificial neural network (ANN) has been trained with real-sample PIXE (particle X-ray induced emission) spectra of organic substances. Following the training stage ANN was applied to a subset of similar samples thus obtaining the elemental concentrations in muscle, liver and gills of Cyprinus carpio. Concentrations obtained with the ANN method are in full agreement with results from one standard analytical procedure, showing the high potentiality of ANN in PIXE quantitative analyses.

Development of magnetic graphene @hydrophilic polydopamine for the enrichment and analysis of phthalates in environmental water samples.

PubMed

Wang, Xianying; Song, Guoxin; Deng, Chunhui

2015-01-01

Magnetic graphene @hydrophilic polydopamine composites were successfully fabricated via a simple solvothermal reaction and self-polymerization of dopamine. Benefit from the excellent characteristics of strong magnetic responsivity, super-hydrophilicity and abundant π-electron system, the prepared material showed great potential as a magnetic solid phase extraction (MSPE) sorbent. In this work, six kinds of phthalates (PAEs) were selected as the target analytes to evaluate the extraction ability of the adsorbents combined with MSPE-GC-MS. And various extraction parameters were optimized by selecting the pH value of samples, the amount of sorbents, adsorption and desorption time, the type and volume of eluting solution. Meanwhile, the whole extraction process could be finished in 30 min. Under the optimized conditions, validations of the method were evaluated as well. And the results presented excellent linearity with a wide range of 50-20,000 μg/L (R(2)>0.9991). The detection of limits were in the range from 0.05-5 μg/L (S/N=3). Therefore, the novel magnetic graphene@polydopamine composites were successfully used as the sorbents for the enrichment and analysis of PAEs in real water samples. This proposed method provided a simple, efficient and sensitive approach for the determination of aromatic compounds in real environmental samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of forensic samples by using an infrared-based automatic DNA sequencer.

PubMed

Ricci, Ugo; Sani, Ilaria; Klintschar, Michael; Cerri, Nicoletta; De Ferrari, Francesco; Giovannucci Uzielli, Maria Luisa

2003-06-01

We have recently introduced a new protocol for analyzing all core loci of the Federal Bureau of Investigation's (FBI) Combined DNA Index System (CODIS) with an infrared (IR) automatic DNA sequencer (LI-COR 4200). The amplicons were labeled with forward oligonucleotide primers, covalently linked to a new infrared fluorescent molecule (IRDye 800). The alleles were displayed as familiar autoradiogram-like images with real-time detection. This protocol was employed for paternity testing, population studies, and identification of degraded forensic samples. We extensively analyzed some simulated forensic samples and mixed stains (blood, semen, saliva, bones, and fixed archival embedded tissues), comparing the results with donor samples. Sensitivity studies were also performed for the four multiplex systems. Our results show the efficiency, reliability, and accuracy of the IR system for the analysis of forensic samples. We also compared the efficiency of the multiplex protocol with ultraviolet (UV) technology. Paternity tests, undegraded DNA samples, and real forensic samples were analyzed with this approach based on IR technology and with UV-based automatic sequencers in combination with commercially-available kits. The comparability of the results with the widespread UV methods suggests that it is possible to exchange data between laboratories using the same core group of markers but different primer sets and detection methods.

First external quality assurance program for bloodstream Real-Time PCR monitoring of treatment response in clinical trials of Chagas disease.

PubMed

Ramírez, Juan C; Parrado, Rudy; Sulleiro, Elena; de la Barra, Anabelle; Rodríguez, Marcelo; Villarroel, Sandro; Irazu, Lucía; Alonso-Vega, Cristina; Alves, Fabiana; Curto, María A; García, Lineth; Ortiz, Lourdes; Torrico, Faustino; Gascón, Joaquim; Flevaud, Laurence; Molina, Israel; Ribeiro, Isabela; Schijman, Alejandro G

2017-01-01

Real-Time PCR (qPCR) testing is recommended as both a diagnostic and outcome measurement of etiological treatment in clinical practice and clinical trials of Chagas disease (CD), but no external quality assurance (EQA) program provides performance assessment of the assays in use. We implemented an EQA system to evaluate the performance of molecular biology laboratories involved in qPCR based follow-up in clinical trials of CD. An EQA program was devised for three clinical trials of CD: the E1224 (NCT01489228), a pro-drug of ravuconazole; the Sampling Study (NCT01678599), that used benznidazole, both conducted in Bolivia; and the CHAGASAZOL (NCT01162967), that tested posaconazole, conducted in Spain. Four proficiency testing panels containing negative controls and seronegative blood samples spiked with 1, 10 and 100 parasite equivalents (par. eq.)/mL of four Trypanosoma cruzi stocks, were sent from the Core Lab in Argentina to the participating laboratories located in Bolivia and Spain. Panels were analyzed simultaneously, blinded to sample allocation, at 4-month intervals. In addition, 302 random blood samples from both trials carried out in Bolivia were sent to Core Lab for retesting analysis. The analysis of proficiency testing panels gave 100% of accordance (within laboratory agreement) and concordance (between laboratory agreement) for all T. cruzi stocks at 100 par. eq./mL; whereas their values ranged from 71 to 100% and from 62 to 100% at 1 and 10 par. eq./mL, respectively, depending on the T. cruzi stock. The results obtained after twelve months of preparation confirmed the stability of blood samples in guanidine-EDTA buffer. No significant differences were found between qPCR results from Bolivian laboratory and Core Lab for retested clinical samples. This EQA program for qPCR analysis of CD patient samples may significantly contribute to ensuring the quality of laboratory data generated in clinical trials and molecular diagnostics laboratories of CD.

Sample introducing apparatus and sample modules for mass spectrometer

DOEpatents

Thompson, Cyril V.; Wise, Marcus B.

1993-01-01

An apparatus for introducing gaseous samples from a wide range of environmental matrices into a mass spectrometer for analysis of the samples is described. Several sample preparing modules including a real-time air monitoring module, a soil/liquid purge module, and a thermal desorption module are individually and rapidly attachable to the sample introducing apparatus for supplying gaseous samples to the mass spectrometer. The sample-introducing apparatus uses a capillary column for conveying the gaseous samples into the mass spectrometer and is provided with an open/split interface in communication with the capillary and a sample archiving port through which at least about 90 percent of the gaseous sample in a mixture with an inert gas that was introduced into the sample introducing apparatus is separated from a minor portion of the mixture entering the capillary discharged from the sample introducing apparatus.

Central Fetal Monitoring With and Without Computer Analysis: A Randomized Controlled Trial.

PubMed

Nunes, Inês; Ayres-de-Campos, Diogo; Ugwumadu, Austin; Amin, Pina; Banfield, Philip; Nicoll, Antony; Cunningham, Simon; Sousa, Paulo; Costa-Santos, Cristina; Bernardes, João

2017-01-01

To evaluate whether intrapartum fetal monitoring with computer analysis and real-time alerts decreases the rate of newborn metabolic acidosis or obstetric intervention when compared with visual analysis. A randomized clinical trial carried out in five hospitals in the United Kingdom evaluated women with singleton, vertex fetuses of 36 weeks of gestation or greater during labor. Continuous central fetal monitoring by computer analysis and online alerts (experimental arm) was compared with visual analysis (control arm). Fetal blood sampling and electrocardiographic ST waveform analysis were available in both arms. The primary outcome was incidence of newborn metabolic acidosis (pH less than 7.05 and base deficit greater than 12 mmol/L). Prespecified secondary outcomes included operative delivery, use of fetal blood sampling, low 5-minute Apgar score, neonatal intensive care unit admission, hypoxic-ischemic encephalopathy, and perinatal death. A sample size of 3,660 per group (N=7,320) was planned to be able to detect a reduction in the rate of metabolic acidosis from 2.8% to 1.8% (two-tailed α of 0.05 with 80% power). From August 2011 through July 2014, 32,306 women were assessed for eligibility and 7,730 were randomized: 3,961 to computer analysis and online alerts, and 3,769 to visual analysis. Baseline characteristics were similar in both groups. Metabolic acidosis occurred in 16 participants (0.40%) in the experimental arm and 22 participants (0.58%) in the control arm (relative risk 0.69 [0.36-1.31]). No statistically significant differences were found in the incidence of secondary outcomes. Compared with visual analysis, computer analysis of fetal monitoring signals with real-time alerts did not significantly reduce the rate of metabolic acidosis or obstetric intervention. A lower-than-expected rate of newborn metabolic acidosis was observed in both arms of the trial. ISRCTN Registry, http://www.isrctn.com, ISRCTN42314164.

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

PubMed Central

Junick, Jana

2012-01-01

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

Development of a selective and pH-independent method for the analysis of ultra trace amounts of nitrite in environmental water samples after dispersive magnetic solid phase extraction by spectrofluorimetry.

PubMed

Daneshvar Tarigh, Ghazale; Shemirani, Farzaneh

2014-10-01

This paper describes an innovative and rapidly dispersive magnetic solid phase extraction spectrofluorimetry (DMSPE-FL) method for the analysis of trace amounts of nitrite in some environmental water samples. The method includes derivatization of aqueous nitrite with 2, 3-diaminonaphthalene (DAN), analysis of highly fluorescent 2, 3-naphthotriazole (NAT) derivative using spectrofluorimetry after DSPME. The novelty of our method is based on forming NAT that was independent with the pH-responsive and was adsorbed on MMWCNT by hydrophobic attractions in both acidic and basic media. The extraction efficiency of the sorbent was investigated by extraction of nitrite. The optimum extraction conditions for NO2(-) were obtained as of extraction time, 1.5 min; 10mg sorbent from 160 mL of the sample solution, and elution with 1 mL of acetone/KOH. Under the optimal conditions, the calibration curves were obtained in the range of 0.1-80 µg L(-1) (R(2)=0.999) and LOD (S/N=3) was obtained in 34 ng L(-1). Relative standard deviations (RSD) were 0.6 % (five replicates at 5 μg L(-1)). In addition, the feasibility of the method was demonstrated with extraction and determination of nitrite from some real samples containing tap, mineral, sea, rain, snow and ground waters, with the recovery in standard addition to real matrix of 94-102 % and RSDs of 1.8-10.6%. Copyright © 2014 Elsevier B.V. All rights reserved.

Regression analysis and real-time water-quality monitoring to estimate constituent concentrations, loads, and yields in the Little Arkansas River, south-central Kansas, 1995-99

USGS Publications Warehouse

Christensen, Victoria G.; Jian, Xiaodong; Ziegler, Andrew C.

2000-01-01

Water from the Little Arkansas River is used as source water for artificial recharge to the Equus Beds aquifer, which provides water for the city of Wichita in south-central Kansas. To assess the quality of the source water, continuous in-stream water-quality monitors were installed at two U.S. Geological Survey stream-gaging stations to provide real-time measurement of specific conductance, pH, water temperature, dissolved oxygen, and turbidity in the Little Arkansas River. In addition, periodic water samples were collected manually and analyzed for selected constituents, including alkalinity, dissolved solids, total suspended solids, chloride, sulfate, atrazine, and fecal coliform bacteria. However, these periodic samples do not provide real-time data on which to base aquifer-recharge operational decisions to prevent degradation of the Equus Beds aquifer. Continuous and periodic monitoring enabled identification of seasonal trends in selected physical properties and chemical constituents and estimation of chemical mass transported in the Little Arkansas River. Identification of seasonal trends was especially important because high streamflows have a substantial effect on chemical loads and because concentration data from manually collected samples often were not available. Therefore, real-time water-quality monitoring of surrogates for the estimation of selected chemical constituents in streamflow can increase the accuracy of load and yield estimates and can decrease some manual data-collection activities. Regression equations, which were based on physical properties and analysis of water samples collected from 1995 through 1998 throughout 95 percent of the stream's flow duration, were developed to estimate alkalinity, dissolved solids, total suspended solids, chloride, sulfate, atrazine, and fecal coliform bacteria concentrations. Error was evaluated for the first year of data collection and each subsequent year, and a decrease in error was observed as the number of samples increased. Generally, 2 years of data (35 to 55 samples) collected throughout 90 to 95 percent of the stream's flow duration were sufficient to define the relation between a constituent and its surrogate(s). Relations and resulting equations were site specific. To test the regression equations developed from the first 3 years of data collection (1995-98), the equations were applied to the fourth year of data collection (1999) to calculate estimated constituent loads and the errors associated with these loads. Median relative percentage differences between measured constituent loads determined using the analysis of periodic, manual water samples and estimated constituent loads were less than 25 percent for alkalinity, dissolved solids, chloride, and sulfate. The percentage differences for total suspended solids, atrazine, and bacteria loads were more than 25 percent. Even for those constituents with large relative percentage differences between the measured and estimated loads, the estimation of constituent concentrations with regression analysis and real-time water-quality monitoring has numerous advantages over periodic manual sampling. The timely availability of bacteria and other constituent data may be important when considering recreation and the whole-body contact criteria established by the Kansas Department of Health and Environment for a specific water body. In addition, water suppliers would have timely information to use in adjusting water-treatment strategies; environmental changes could be assessed in time to prevent negative effects on fish or other aquatic life; and officials for the Equus Beds Ground-Water Recharge Demonstration project could use this information to prevent the possible degradation of the Equus Beds aquifer by choosing not to recharge when constituent concentrations in the source water are large. Constituent loads calculated from the regression equations may be useful for calculating total maximum daily loads (TMDL's), wh

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.

Comparative study on fast classification of brick samples by combination of principal component analysis and linear discriminant analysis using stand-off and table-top laser-induced breakdown spectroscopy

NASA Astrophysics Data System (ADS)

Vítková, Gabriela; Prokeš, Lubomír; Novotný, Karel; Pořízka, Pavel; Novotný, Jan; Všianský, Dalibor; Čelko, Ladislav; Kaiser, Jozef

2014-11-01

Focusing on historical aspect, during archeological excavation or restoration works of buildings or different structures built from bricks it is important to determine, preferably in-situ and in real-time, the locality of bricks origin. Fast classification of bricks on the base of Laser-Induced Breakdown Spectroscopy (LIBS) spectra is possible using multivariate statistical methods. Combination of principal component analysis (PCA) and linear discriminant analysis (LDA) was applied in this case. LIBS was used to classify altogether the 29 brick samples from 7 different localities. Realizing comparative study using two different LIBS setups - stand-off and table-top it is shown that stand-off LIBS has a big potential for archeological in-field measurements.

Hair analysis for cocaine: factors in laboratory contamination studies and their relevance to proficiency sample preparation and hair testing practices.

PubMed

Hill, Virginia; Cairns, Thomas; Schaffer, Michael

2008-03-21

Hair samples were contaminated by rubbing with cocaine (COC) followed by sweat application, multiple shampoo treatments and storage. The samples were then washed with isopropanol for 15min, followed by sequential aqueous washes totaling 3.5h. The amount of drug in the last wash was used to calculate a wash criterion to determine whether samples were positive due to use or contamination. Analyses of cocaine and metabolites were done by LC/MS/MS. These procedures were applied to samples produced by a U.S. government-sponsored cooperative study, in which this laboratory participated, and to samples in a parallel in-house study. All contaminated samples in both studies were correctly identified as contaminated by cutoff, benzoylecgonine (BE) presence, BE ratio, and/or the wash criterion. A method for determining hair porosity was applied to samples in both studies, and porosity characteristics of hair are discussed as they relate to experimental and real-world contamination of hair, preparation of proficiency survey samples, and analysis of unknown hair samples.

Searching for the best real-time RT-PCRs to detect Zika virus infections: the importance of comparing several protocols.

PubMed

de Moraes, F M; Espósito, D L A; Klein, T M; da Fonseca, B A L

2018-01-01

Clinical manifestations of Zika, dengue, and chikungunya virus infections are very similar, making it difficult to reach a diagnosis based only on clinical grounds. In addition, there is an intense cross-reactivity between antibodies directed to Zika virus and other flaviviruses, and an accurate Zika diagnosis is best achieved by real-time RT-PCR. However, some real-time RT-PCR show better performance than others. To reach the best possible Zika diagnosis, the analytic sensitivity of some probe-based real-time RT-PCR amplifying Zika virus RNA was evaluated in spiked and clinical samples. We evaluated primers and probes to detect Zika virus, which had been published before, and tested sensitivity using serum spiked and patient samples by real-time RT-PCR. When tested against spiked samples, the previously described primers showed different sensitivity, with very similar results when samples from patients (serum and urine) were analyzed. Real-time RT-PCR designed to amplify Zika virus NS1 showed the best analytical sensitivity for all samples.

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

An in-house assay for BK polyomavirus quantification using the Abbott m2000 RealTime system.

PubMed

Muldrew, Kenneth L; Lovett, Jennie L

2013-11-01

BK polyomavirus (BKPyV) quantification is useful for monitoring renal transplant patient response to therapy. The Abbott m2000 RealTime System employed by some clinical laboratories to perform US Food and Drug Administration-approved assays can also be used to develop in-house assays such as the one presented here. This study aimed to validate an in-house quantitative real-time PCR assay targeting the BKPyV major capsid VP1 gene for assessment of viral load using the Abbott m2000 RealTime System. BKPyV load was measured in 95 urine and plasma samples previously tested for BKPyV by one of three laboratories (46 BKPyV-positive samples consisting of 35 plasma and 11 urine samples; 49 samples negative for BKPyV consisting of 47 plasma and two urine samples). Two additional plasma specimens from the College of American Pathologists proficiency testing survey were also analysed. Precision studies were performed by diluting a high-viral-titre patient sample into BKPyV-negative pooled plasma to create high-positive (6.16 log10 copies ml(-1)) and low-positive (3.16 log10 copies ml(-1)) samples. For precision studies of inter-assay variability, a high-positive (7.0 log10 copies ml(-1)) and a low-positive (3.0 log10 copies ml(-1)) sample were measured in 20 separate runs. The assay's limit of quantification and limit of detection were 2.70 and 2.25 log10 copies ml(-1), respectively. The assay was linear from 2.70 to 9.26 log10 copies ml(-1). Of the 48 known positives, 43 were detected as positive, with three reported by the reference laboratory as values lower than the limit of detection. Two known positives at 3.27 and 3.80 log10 copies ml(-1) tested negative by the m2000 BKPyV assay. Of the 49 known negative samples, 48 were negative by the m2000 BKPyV load assay, with one sample confirmed positive by a reference laboratory. Qualitative analysis prior to discrepancy testing demonstrated a sensitivity of 89.58 % and a specificity of 97.96 %. Precision studies demonstrated inter-assay coefficients of variation of 0.63 % (high positive) and 4.38 % (low positive). Genotyping was performed on 22 patient samples, of which 21 (95.45 %) were type I and one (4.55 %) was type II. In conclusion, the m2000 BKPyV viral load assay sensitivity, specificity, linear range, precision and cost effectiveness make it an attractive methodology for clinical laboratories using the Abbott m2000 RealTime System.

Image Quality Assessment for Fake Biometric Detection: Application to Iris, Fingerprint, and Face Recognition.

PubMed

Galbally, Javier; Marcel, Sébastien; Fierrez, Julian

2014-02-01

To ensure the actual presence of a real legitimate trait in contrast to a fake self-manufactured synthetic or reconstructed sample is a significant problem in biometric authentication, which requires the development of new and efficient protection measures. In this paper, we present a novel software-based fake detection method that can be used in multiple biometric systems to detect different types of fraudulent access attempts. The objective of the proposed system is to enhance the security of biometric recognition frameworks, by adding liveness assessment in a fast, user-friendly, and non-intrusive manner, through the use of image quality assessment. The proposed approach presents a very low degree of complexity, which makes it suitable for real-time applications, using 25 general image quality features extracted from one image (i.e., the same acquired for authentication purposes) to distinguish between legitimate and impostor samples. The experimental results, obtained on publicly available data sets of fingerprint, iris, and 2D face, show that the proposed method is highly competitive compared with other state-of-the-art approaches and that the analysis of the general image quality of real biometric samples reveals highly valuable information that may be very efficiently used to discriminate them from fake traits.

Real-Time Polymerase Chain Reaction for Detection of Schistosoma DNA in Small-Volume Urine Samples Reflects Focal Distribution of Urogenital Schistosomiasis in Primary School Girls in KwaZulu Natal, South Africa

PubMed Central

Pillay, Pavitra; Taylor, Myra; Zulu, Siphosenkosi G.; Gundersen, Svein G.; Verweij, Jaco J.; Hoekstra, Pytsje; Brienen, Eric A. T.; Kleppa, Elisabeth; Kjetland, Eyrun F.; van Lieshout, Lisette

2014-01-01

Schistosoma haematobium eggs and Schistosoma DNA levels were measured in urine samples from 708 girls recruited from 18 randomly sampled primary schools in South Africa. Microscopic analysis of two 10-mL urine subsamples collected on three consecutive days confirmed high day-to-day variation; 103 (14.5%) girls had positive results at all six examinations, and at least one positive sample was seen in 225 (31.8%) girls. Schistosoma-specific DNA, which was measured in a 200-μL urine subsample by using real-time polymerase chain reaction, was detected in 180 (25.4%) cases, and levels of DNA corresponded significantly with average urine egg excretion. In concordance with microscopic results, polymerase chain reaction results were significantly associated with history of gynecologic symptoms and confirmed highly focal distribution of urogenital schistosomiasis. Parasite-specific DNA detection has a sensitivity comparable to single urine microscopy and could be used as a standardized high-throughput procedure to assess distribution of urogenital schistosomiasis in relatively large study populations by using small sample volumes. PMID:24470560

A new real-time PCR protocol for detection of avian haemosporidians.

PubMed

Bell, Jeffrey A; Weckstein, Jason D; Fecchio, Alan; Tkach, Vasyl V

2015-07-19

Birds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great genetic diversity and insufficient sampling in highly diverse regions. Studies aimed at surveying haemosporidian diversity involve collecting and screening samples from hundreds to thousands of individuals. Currently, screening relies on microscopy and/or single or nested standard PCR. Although effective, these methods are time and resource consuming, and in the case of microscopy require substantial expertise. Here we report a newly developed real-time PCR protocol designed to quickly and reliably detect all three genera of avian haemosporidians in a single biochemical reaction. Using available DNA sequences from avian haemosporidians we designed primers R330F and R480RL, which flank a 182 base pair fragment of mitochondrial conserved rDNA. These primers were initially tested using real-time PCR on samples from Malawi, Africa, previously screened for avian haemosporidians using traditional nested PCR. Our real time protocol was further tested on 94 samples from the Cerrado biome of Brazil, previously screened using a single PCR assay for haemosporidian parasites. These samples were also amplified using modified nested PCR protocols, allowing for comparisons between the three different screening methods (single PCR, nested PCR, real-time PCR). The real-time PCR protocol successfully identified all three genera of avian haemosporidians from both single and mixed infections previously detected from Malawi. There was no significant difference between the three different screening protocols used for the 94 samples from the Brazilian Cerrado (χ(2) = 0.3429, df = 2, P = 0.842). After proving effective, the real-time protocol was used to screen 2113 Brazilian samples, identifying 693 positive samples. Our real-time PCR assay proved as effective as two widely used molecular screening techniques, single PCR and nested PCR. However, the real-time protocol has the distinct advantage of detecting all three genera in a single reaction, which significantly increases efficiency by greatly decreasing screening time and cost. Our real-time PCR protocol is therefore a valuable tool in the quickly expanding field of avian haemosporidian research.

Introducing automation to the molecular diagnosis of Trypanosoma cruzi infection: A comparative study of sample treatments, DNA extraction methods and real-time PCR assays.

PubMed

Abras, Alba; Ballart, Cristina; Llovet, Teresa; Roig, Carme; Gutiérrez, Cristina; Tebar, Silvia; Berenguer, Pere; Pinazo, María-Jesús; Posada, Elizabeth; Gascón, Joaquim; Schijman, Alejandro G; Gállego, Montserrat; Muñoz, Carmen

2018-01-01

Polymerase chain reaction (PCR) has become a useful tool for the diagnosis of Trypanosoma cruzi infection. The development of automated DNA extraction methodologies and PCR systems is an important step toward the standardization of protocols in routine diagnosis. To date, there are only two commercially available Real-Time PCR assays for the routine laboratory detection of T. cruzi DNA in clinical samples: TCRUZIDNA.CE (Diagnostic Bioprobes Srl) and RealCycler CHAG (Progenie Molecular). Our aim was to evaluate the RealCycler CHAG assay taking into account the whole process. We assessed the usefulness of an automated DNA extraction system based on magnetic particles (EZ1 Virus Mini Kit v2.0, Qiagen) combined with a commercially available Real-Time PCR assay targeting satellite DNA (SatDNA) of T. cruzi (RealCycler CHAG), a methodology used for routine diagnosis in our hospital. It was compared with a well-known strategy combining a commercial DNA isolation kit based on silica columns (High Pure PCR Template Preparation Kit, Roche Diagnostics) with an in-house Real-Time PCR targeting SatDNA. The results of the two methodologies were in almost perfect agreement, indicating they can be used interchangeably. However, when variations in protocol factors were applied (sample treatment, extraction method and Real-Time PCR), the results were less convincing. A comprehensive fine-tuning of the whole procedure is the key to successful results. Guanidine EDTA-blood (GEB) samples are not suitable for DNA extraction based on magnetic particles due to inhibition, at least when samples are not processed immediately. This is the first study to evaluate the RealCycler CHAG assay taking into account the overall process, including three variables (sample treatment, extraction method and Real-Time PCR). Our findings may contribute to the harmonization of protocols between laboratories and to a wider application of Real-Time PCR in molecular diagnostic laboratories associated with health centers.

A New Method Using Single-Particle Mass Spectrometry Data to Distinguish Mineral Dust and Biological Aerosols

NASA Astrophysics Data System (ADS)

Al-Mashat, H.; Kristensen, L.; Sultana, C. M.; Prather, K. A.

2016-12-01

The ability to distinguish types of particles present within a cloud is important for determining accurate inputs to climate models. The chemical composition of particles within cloud liquid droplets and ice crystals can have a significant impact on the timing, location, and amount of precipitation that falls. Precipitation efficiency is increased by the presence of ice crystals in clouds, and both mineral dust and biological aerosols have been shown to be effective ice nucleating particles (INPs) in the atmosphere. A current challenge in aerosol science is distinguishing mineral dust and biological material in the analysis of real-time, ambient, single-particle mass spectral data. Single-particle mass spectrometers are capable of measuring the size-resolved chemical composition of individual atmospheric particles. However, there is no consistent analytical method for distinguishing dust and biological aerosols. Sampling and characterization of control samples (i.e. of known identity) of mineral dust and bacteria were performed by the Aerosol Time-of-Flight Mass Spectrometer (ATOFMS) as part of the Fifth Ice Nucleation (FIN01) Workshop at the Aerosol Interaction and Dynamics in the Atmosphere (AIDA) facility in Karlsruhe, Germany. Using data collected by the ATOFMS of control samples, a new metric has been developed to classify single particles as dust or biological independent of spectral cluster analysis. This method, involving the use of a ratio of mass spectral peak areas for organic nitrogen and silicates, is easily reproducible and does not rely on extensive knowledge of particle chemistry or the ionization characteristics of mass spectrometers. This represents a step toward rapidly distinguishing particle types responsible for ice nucleation activity during real-time sampling in clouds. The ability to distinguish types of particles present within a cloud is important for determining accurate inputs to climate models. The chemical composition of particles within cloud liquid droplets and ice crystals can have a significant impact on the timing, location, and amount of precipitation that falls. Precipitation efficiency is increased by the presence of ice crystals in clouds, and both mineral dust and biological aerosols have been shown to be effective ice nucleating particles (INPs) in the atmosphere. A current challenge in aerosol science is distinguishing mineral dust and biological material in the analysis of real-time, ambient, single-particle mass spectral data. Single-particle mass spectrometers are capable of measuring the size-resolved chemical composition of individual atmospheric particles. However, there is no consistent analytical method for distinguishing dust and biological aerosols. Sampling and characterization of control samples (i.e. of known identity) of mineral dust and bacteria were performed by the Aerosol Time-of-Flight Mass Spectrometer (ATOFMS) as part of the Fifth Ice Nucleation (FIN01) Workshop at the Aerosol Interaction and Dynamics in the Atmosphere (AIDA) facility in Karlsruhe, Germany. Using data collected by the ATOFMS of control samples, a new metric has been developed to classify single particles as dust or biological independent of spectral cluster analysis. This method, involving the use of a ratio of mass spectral peak areas for organic nitrogen and silicates, is easily reproducible and does not rely on extensive knowledge of particle chemistry or the ionization characteristics of mass spectrometers. This represents a step toward rapidly distinguishing particle types responsible for ice nucleation activity during real-time sampling in clouds.

A colorimetric indicator-displacement assay array for selective detection and identification of biological thiols.

PubMed

Qian, Sihua; Lin, Hengwei

2014-03-01

A simple, inexpensive yet highly selective colorimetric indicator-displacement assay array for the simultaneous detection and identification of three important biothiols at micromolar concentrations under physiological conditions and in real samples has been developed in this work. With use of an array composed of metal indicators and metal ions, clear differentiation among cysteine, homocysteine and glutathione was achieved. On the basis of the colour change of the array, quantification of each analyte was accomplished easily, and different biothiols were identified readily using standard chemometric approaches (hierarchical clustering analysis). Moreover, the colorimetric sensor array was not responsive to changes with 19 other natural amino acids, and it showed excellent reproducibility. Importantly, the sensor array developed was successfully applied to the determination and identification of the three biothiols in a real biological sample.

An exo probe-based recombinase polymerase amplification assay for the rapid detection of porcine parvovirus.

PubMed

Wang, Jian-Chang; Liu, Li-Bing; Han, Qing-An; Wang, Jin-Feng; Yuan, Wan-Zhe

2017-10-01

Recombinase polymerase amplification (RPA), an isothermal amplification technology, has been developed as an alternative to PCR in pathogen detection. A real-time RPA assay (rt-RPA) was developed to detect the porcine parvovirus (PPV) using primers and exo probe specific for the VP2 gene. The amplification was performed at 39°C for 20min. There was no cross-reaction with other pathogens tested. Using the recombinant plasmid pPPV-VP2 as template, the analytical sensitivity was 103 copies. The assay performance was evaluated by testing 115 field samples by rt-RPA and a real-time PCR assay. The diagnostic agreement between assays was 100%, and PPV DNA was detected in 94 samples. The R 2 value of rt-RPA and real-time PCR was 0.909 by linear regression analysis. The developed rt-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of PPV in diagnostic laboratories and at point-of-care, especially in remote and rural areas. Copyright © 2017 Elsevier B.V. All rights reserved.

An optimized magnetite microparticle-based phosphopeptide enrichment strategy for identifying multiple phosphorylation sites in an immunoprecipitated protein.

PubMed

Huang, Yi; Shi, Qihui; Tsung, Chia-Kuang; Gunawardena, Harsha P; Xie, Ling; Yu, Yanbao; Liang, Hongjun; Yang, Pengyuan; Stucky, Galen D; Chen, Xian

2011-01-01

To further improve the selectivity and throughput of phosphopeptide analysis for the samples from real-time cell lysates, here we demonstrate a highly efficient method for phosphopeptide enrichment via newly synthesized magnetite microparticles and the concurrent mass spectrometric analysis. The magnetite microparticles show excellent magnetic responsivity and redispersibility for a quick enrichment of those phosphopeptides in solution. The selectivity and sensitivity of magnetite microparticles in phosphopeptide enrichment are first evaluated by a known mixture containing both phosphorylated and nonphosphorylated proteins. Compared with the titanium dioxide-coated magnetic beads commercially available, our magnetite microparticles show a better specificity toward phosphopeptides. The selectively-enriched phosphopeptides from tryptic digests of β-casein can be detected down to 0.4 fmol μl⁻¹, whereas the recovery efficiency is approximately 90% for monophosphopeptides. This magnetite microparticle-based affinity technology with optimized enrichment conditions is then immediately applied to identify all possible phosphorylation sites on a signal protein isolated in real time from a stress-stimulated mammalian cell culture. A large fraction of peptides eluted from the magnetic particle enrichment step were identified and characterized as either single- or multiphosphorylated species by tandem mass spectrometry. With their high efficiency and utility for phosphopeptide enrichment, the magnetite microparticles hold great potential in the phosphoproteomic studies on real-time samples from cell lysates. Published by Elsevier Inc.

Detection of Yersinia Enterocolitica Species in Pig Tonsils and Raw Pork Meat by the Real-Time Pcr and Culture Methods.

PubMed

Stachelska, M A

2017-09-26

The aim of the present study was to establish a rapid and accurate real-time PCR method to detect pathogenic Yersinia enterocolitica in pork. Yersinia enterocolitica is considered to be a crucial zoonosis, which can provoke diseases both in humans and animals. The classical culture methods designated to detect Y. enterocolitica species in food matrices are often very time-consuming. The chromosomal locus _tag CH49_3099 gene, that appears in pathogenic Y. enterocolitica strains, was applied as DNA target for the 5' nuclease PCR protocol. The probe was labelled at the 5' end with the fluorescent reporter dye (FAM) and at the 3' end with the quencher dye (TAMRA). The real-time PCR cycling parameters included 41 cycles. A Ct value which reached a value higher than 40 constituted a negative result. The developed for the needs of this study qualitative real-time PCR method appeared to give very specific and reliable results. The detection rate of locus _tag CH49_3099 - positive Y. enterocolitica in 150 pig tonsils was 85 % and 32 % with PCR and culture methods, respectively. Both the Real-time PCR results and culture method results were obtained from material that was enriched during overnight incubation. The subject of the study were also raw pork meat samples. Among 80 samples examined, 7 ones were positive when real-time PCR was applied, and 6 ones were positive when classical culture method was applied. The application of molecular techniques based on the analysis of DNA sequences such as the Real-time PCR enables to detect this pathogenic bacteria very rapidly and with higher specificity, sensitivity and reliability in comparison to classical culture methods.

Development of real-time PCR and loop-mediated isothermal amplification (LAMP) assays for the differential detection of digital dermatitis associated treponemes.

PubMed

Anklam, Kelly; Kulow, Megan; Yamazaki, Wataru; Döpfer, Dörte

2017-01-01

Bovine digital dermatitis (DD) is a severe infectious cause of lameness in cattle worldwide, with important economic and welfare consequences. There are three treponeme phylogroups (T. pedis, T. phagedenis, and T. medium) that are implicated in playing an important causative role in DD. This study was conducted to develop real-time PCR and loop-mediated isothermal amplification (LAMP) assays for the detection and differentiation of the three treponeme phylogroups associated with DD. The real-time PCR treponeme phylogroup assays targeted the 16S-23S rDNA intergenic space (ITS) for T. pedis and T. phagedenis, and the flagellin gene (flaB2) for T. medium. The 3 treponeme phylogroup LAMP assays targeted the flagellin gene (flaB2) and the 16S rRNA was targeted for the Treponeme ssp. LAMP assay. The real-time PCR and LAMP assays correctly detected the target sequence of all control strains examined, and no cross-reactions were observed, representing 100% specificity. The limit of detection for each of the three treponeme phylogroup real-time PCR and LAMP assays was ≤ 70 fg/μl. The detection limit for the Treponema spp. LAMP assay ranged from 7-690 fg/μl depending on phylogroup. Treponemes were isolated from 40 DD lesion biopsies using an immunomagnetic separation culture method. The treponeme isolation samples were then subjected to the real-time PCR and LAMP assays for analysis. The treponeme phylogroup real-time PCR and LAMP assay results had 100% agreement, matching on all isolation samples. These results indicate that the developed assays are a sensitive and specific test for the detection and differentiation of the three main treponeme phylogroups implicated in DD.

Development of real-time PCR and loop-mediated isothermal amplification (LAMP) assays for the differential detection of digital dermatitis associated treponemes

PubMed Central

Kulow, Megan; Yamazaki, Wataru; Döpfer, Dörte

2017-01-01

Bovine digital dermatitis (DD) is a severe infectious cause of lameness in cattle worldwide, with important economic and welfare consequences. There are three treponeme phylogroups (T. pedis, T. phagedenis, and T. medium) that are implicated in playing an important causative role in DD. This study was conducted to develop real-time PCR and loop-mediated isothermal amplification (LAMP) assays for the detection and differentiation of the three treponeme phylogroups associated with DD. The real-time PCR treponeme phylogroup assays targeted the 16S-23S rDNA intergenic space (ITS) for T. pedis and T. phagedenis, and the flagellin gene (flaB2) for T. medium. The 3 treponeme phylogroup LAMP assays targeted the flagellin gene (flaB2) and the 16S rRNA was targeted for the Treponeme ssp. LAMP assay. The real-time PCR and LAMP assays correctly detected the target sequence of all control strains examined, and no cross-reactions were observed, representing 100% specificity. The limit of detection for each of the three treponeme phylogroup real-time PCR and LAMP assays was ≤ 70 fg/μl. The detection limit for the Treponema spp. LAMP assay ranged from 7–690 fg/μl depending on phylogroup. Treponemes were isolated from 40 DD lesion biopsies using an immunomagnetic separation culture method. The treponeme isolation samples were then subjected to the real-time PCR and LAMP assays for analysis. The treponeme phylogroup real-time PCR and LAMP assay results had 100% agreement, matching on all isolation samples. These results indicate that the developed assays are a sensitive and specific test for the detection and differentiation of the three main treponeme phylogroups implicated in DD. PMID:28542573

Rapid-Viability PCR Method for Detection of Live, Virulent Bacillus anthracis in Environmental Samples ▿

PubMed Central

Létant, Sonia E.; Murphy, Gloria A.; Alfaro, Teneile M.; Avila, Julie R.; Kane, Staci R.; Raber, Ellen; Bunt, Thomas M.; Shah, Sanjiv R.

2011-01-01

In the event of a biothreat agent release, hundreds of samples would need to be rapidly processed to characterize the extent of contamination and determine the efficacy of remediation activities. Current biological agent identification and viability determination methods are both labor- and time-intensive such that turnaround time for confirmed results is typically several days. In order to alleviate this issue, automated, high-throughput sample processing methods were developed in which real-time PCR analysis is conducted on samples before and after incubation. The method, referred to as rapid-viability (RV)-PCR, uses the change in cycle threshold after incubation to detect the presence of live organisms. In this article, we report a novel RV-PCR method for detection of live, virulent Bacillus anthracis, in which the incubation time was reduced from 14 h to 9 h, bringing the total turnaround time for results below 15 h. The method incorporates a magnetic bead-based DNA extraction and purification step prior to PCR analysis, as well as specific real-time PCR assays for the B. anthracis chromosome and pXO1 and pXO2 plasmids. A single laboratory verification of the optimized method applied to the detection of virulent B. anthracis in environmental samples was conducted and showed a detection level of 10 to 99 CFU/sample with both manual and automated RV-PCR methods in the presence of various challenges. Experiments exploring the relationship between the incubation time and the limit of detection suggest that the method could be further shortened by an additional 2 to 3 h for relatively clean samples. PMID:21764960

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

PubMed

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

2005-01-01

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

Comparison of sampling sites and laboratory diagnostic tests for S. equi subsp. equi in horses from confirmed strangles outbreaks.

PubMed

Lindahl, S; Båverud, V; Egenvall, A; Aspán, A; Pringle, J

2013-01-01

Strangles is a contagious equine-specific disease caused by Streptococcus equi subsp. equi. Unfortunately, detection of S. equi can fail in up to 40% of horses with strangles. Whereas recent molecular biologic methods and sampling techniques have improved recovery of S. equi optimal sampling methods and laboratory analyses remain ill-defined. To determine the yield of S. equi from horses with acute strangles in confirmed outbreaks by field-sampling methods subjected to culture and biochemical identification, and real-time PCR directly and after culture. Fifty-seven horses of varying breeds and ages from 8 strangles outbreaks. Prospective study. Culture with biochemical identification and real-time PCR directly, and from culture, were performed on nasal swabs, nasopharyngeal swabs, and nasopharyngeal lavages. Real-time PCR directly from samples identified the highest number of infected horses, with 45/57 nasal swabs, 41/57 nasopharyngeal swabs, and 48/57 nasopharyngeal lavages S. equi positive. Biochemical identification (highest positives 22/57) was inferior to real-time PCR for S. equi recovery regardless of sampling method. Real-time PCR of nasopharyngeal lavage directly and after culture yielded 52/57 positives whereas direct real-time PCR of nasopharyngeal lavage combined with either nasopharyngeal swabs or nasal swabs yielded 53/57 positives. Three horses were negative on all samples. Nasopharyngeal lavage analyzed by a combination of real-time PCR directly and after culture or, alternatively, real-time PCR directly on a nasopharyngeal lavage and a nasal/nasopharyngeal swab can identify S. equi in over 90% of acute strangles cases. Copyright © 2013 by the American College of Veterinary Internal Medicine.

Assessment of the Usefulness of Multiplex Real-Time PCR Tests in the Diagnostic and Therapeutic Process of Pneumonia in Hospitalized Children: A Single-Center Experience.

PubMed

Gowin, Ewelina; Bartkowska-Śniatkowska, Alicja; Jończyk-Potoczna, Katarzyna; Wysocka-Leszczyńska, Joanna; Bobkowski, Waldemar; Fichna, Piotr; Sobkowiak, Paulina; Mazur-Melewska, Katarzyna; Bręborowicz, Anna; Wysocki, Jacek; Januszkiewicz-Lewandowska, Danuta

2017-01-01

The aim of the study was assessment of the usefulness of multiplex real-time PCR tests in the diagnostic and therapeutic process in children hospitalized due to pneumonia and burdened with comorbidities. Methods . The study group included 97 children hospitalized due to pneumonia at the Karol Jonscher Teaching Hospital in Poznań, in whom multiplex real-time PCR tests (FTD respiratory pathogens 33; fast-track diagnostics) were used. Results . Positive test results of the test were achieved in 74 patients (76.3%). The average age in the group was 56 months. Viruses were detected in 61 samples (82% of all positive results); bacterial factors were found in 29 samples (39% of all positive results). The presence of comorbidities was established in 90 children (92.78%). On the basis of the obtained results, 5 groups of patients were established: viral etiology of infection, 34 patients; bacterial etiology, 7 patients; mixed etiology, 23 patients; pneumocystis, 9 patients; and no etiology diagnosed, 24 patients. Conclusions . Our analysis demonstrated that the participation of viruses in causing severe lung infections is significant in children with comorbidities. Multiplex real-time PCR tests proved to be more useful in establishing the etiology of pneumonia in hospitalized children than the traditional microbiological examinations.

Quantitative detection of Lactarius deliciosus extraradical soil mycelium by real-time PCR and its application in the study of fungal persistence and interspecific competition.

PubMed

Parladé, J; Hortal, S; Pera, J; Galipienso, L

2007-01-30

Real-Time PCR has been applied to quantify extraradical soil mycelium of the edible ectomycorrhizal fungus Lactarius deliciosus in an interspecific competition experiment under greenhouse conditions. Couples of Pinus pinea seedlings inoculated with either L. deliciosus, Rhizopogon roseolus, or non-inoculated (control) were transplanted into pots filled with two types of soil in all the possible combinations. Total DNA was extracted from soil samples at 3 and 6 months after transplantation to perform real-time PCR analysis. DNA extractions from soil mixed with known amounts of mycelium of L. deliciosus were used as standards. Six months after transplantation, the percentage of mycorrhizas of L. deliciosus and seedling growth were significantly affected by the soil type. Extraradical soil mycelium of L. deliciosus was positively correlated with the final percentage of mycorrhizas and significantly affected by the sampling time and soil depth. The competition effect of R. roseolus was not significant for any of the measured parameters, probably due to the sharp decrease of the mycorrhizal colonization by this fungus. We conclude that real-time PCR is a powerful technique for extraradical mycelium quantification in studies aimed at evaluating the persistence of introduced strains of L. deliciosus in field plantations.

Comparison of Enterococcus qPCR analysis results from fresh and marine water samples on two real-time instruments -

EPA Science Inventory

EPA is currently considering a quantitative polymerase chain reaction (qPCR) method, targeting Enterococcus spp., for beach monitoring. Improvements in the method’s cost-effectiveness may be realized by the use of newer instrumentation such as the Applied Biosystems StepOneTM a...

RadNet Map Interface for Near-Real-Time Radiation Monitoring Data

EPA Pesticide Factsheets

RadNet is a national network of monitoring stations that regularly collect air, precipitation, drinking water, and milk samples for analysis of radioactivity. The RadNet network, which has stations in each state, has been used to track environmental releases of radioactivity from nuclear weapons tests and nuclear accidents.

Methods for increasing cooperation rates for surveys of family forest owners

Treesearch

Brett J. Butler; Jaketon H. Hewes; Mary L. Tyrrell; Sarah M. Butler

2016-01-01

To maximize the representativeness of results from surveys, coverage, sampling, nonresponse, measurement, and analysis errors must be minimized. Although not a cure-all, one approach for mitigating nonresponse errors is to maximize cooperation rates. In this study, personalizing mailings, token financial incentives, and the use of real stamps were tested for their...

WetLab-2: Wet Lab RNA SmartCycler Providing PCR Capability on ISS

NASA Technical Reports Server (NTRS)

Parra, Macarena; Schonfeld, Julie

2015-01-01

The WetLab-2 system will provide sample preparation and qRT-PCR analysis on-board the ISS, a capability to enable using the ISS as a real laboratory. The system will be validated on SpX-7, and is planned for its first PI use on SpX-9.

The Real-World Effectiveness of Early Teaching Interventions for Children with Autism Spectrum Disorder

ERIC Educational Resources Information Center

Reed, Phil; Osborne, Lisa A.; Corness, Mark

2007-01-01

The effectiveness of 3 early teaching interventions (applied behavior analysis [ABA], special nursery placement, and portage) for children with autism spectrum disorder was studied in a community-based sample over 10 months. Measures of autism severity as well as intellectual, educational, and adaptive behavioral function were administered. In…

Turbo-Satori: a neurofeedback and brain-computer interface toolbox for real-time functional near-infrared spectroscopy.

PubMed

Lührs, Michael; Goebel, Rainer

2017-10-01

Turbo-Satori is a neurofeedback and brain-computer interface (BCI) toolbox for real-time functional near-infrared spectroscopy (fNIRS). It incorporates multiple pipelines from real-time preprocessing and analysis to neurofeedback and BCI applications. The toolbox is designed with a focus in usability, enabling a fast setup and execution of real-time experiments. Turbo-Satori uses an incremental recursive least-squares procedure for real-time general linear model calculation and support vector machine classifiers for advanced BCI applications. It communicates directly with common NIRx fNIRS hardware and was tested extensively ensuring that the calculations can be performed in real time without a significant change in calculation times for all sampling intervals during ongoing experiments of up to 6 h of recording. Enabling immediate access to advanced processing features also allows the use of this toolbox for students and nonexperts in the field of fNIRS data acquisition and processing. Flexible network interfaces allow third party stimulus applications to access the processed data and calculated statistics in real time so that this information can be easily incorporated in neurofeedback or BCI presentations.

Electricity-market price and nuclear power plant shutdown: Evidence from California

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

Woo, C. K.; Ho, T.; Zarnikau, J.

Japan's Fukushima nuclear disaster, triggered by the March 11, 2011 earthquake, has led to calls for shutting down existing nuclear plants. To maintain resource adequacy for a grid's reliable operation, one option is to expand conventional generation, whose marginal unit is typically fueled by natural-gas. Two timely and relevant questions thus arise for a deregulated wholesale electricity market: (1) what is the likely price increase due to a nuclear plant shutdown? and (2) what can be done to mitigate the price increase? To answer these questions, we perform a regression analysis of a large sample of hourly real-time electricity-market pricemore » data from the California Independent System Operator (CAISO) for the 33-month sample period of April 2010-December 2012. Our analysis indicates that the 2013 shutdown of the state's San Onofre plant raised the CAISO real-time hourly market prices by $6/MWH to $9/MWH, and that the price increases could have been offset by a combination of demand reduction, increasing solar generation, and increasing wind generation.« less

Complexity and multifractal behaviors of multiscale-continuum percolation financial system for Chinese stock markets

NASA Astrophysics Data System (ADS)

Zeng, Yayun; Wang, Jun; Xu, Kaixuan

2017-04-01

A new financial agent-based time series model is developed and investigated by multiscale-continuum percolation system, which can be viewed as an extended version of continuum percolation system. In this financial model, for different parameters of proportion and density, two Poisson point processes (where the radii of points represent the ability of receiving or transmitting information among investors) are applied to model a random stock price process, in an attempt to investigate the fluctuation dynamics of the financial market. To validate its effectiveness and rationality, we compare the statistical behaviors and the multifractal behaviors of the simulated data derived from the proposed model with those of the real stock markets. Further, the multiscale sample entropy analysis is employed to study the complexity of the returns, and the cross-sample entropy analysis is applied to measure the degree of asynchrony of return autocorrelation time series. The empirical results indicate that the proposed financial model can simulate and reproduce some significant characteristics of the real stock markets to a certain extent.

A new framework for comprehensive, robust, and efficient global sensitivity analysis: 2. Application

NASA Astrophysics Data System (ADS)

Razavi, Saman; Gupta, Hoshin V.

2016-01-01

Based on the theoretical framework for sensitivity analysis called "Variogram Analysis of Response Surfaces" (VARS), developed in the companion paper, we develop and implement a practical "star-based" sampling strategy (called STAR-VARS), for the application of VARS to real-world problems. We also develop a bootstrap approach to provide confidence level estimates for the VARS sensitivity metrics and to evaluate the reliability of inferred factor rankings. The effectiveness, efficiency, and robustness of STAR-VARS are demonstrated via two real-data hydrological case studies (a 5-parameter conceptual rainfall-runoff model and a 45-parameter land surface scheme hydrology model), and a comparison with the "derivative-based" Morris and "variance-based" Sobol approaches are provided. Our results show that STAR-VARS provides reliable and stable assessments of "global" sensitivity across the full range of scales in the factor space, while being 1-2 orders of magnitude more efficient than the Morris or Sobol approaches.

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

Gentry, T.; Schadt, C.; Zhou, J.

Microarray technology has the unparalleled potential tosimultaneously determine the dynamics and/or activities of most, if notall, of the microbial populations in complex environments such as soilsand sediments. Researchers have developed several types of arrays thatcharacterize the microbial populations in these samples based on theirphylogenetic relatedness or functional genomic content. Several recentstudies have used these microarrays to investigate ecological issues;however, most have only analyzed a limited number of samples withrelatively few experiments utilizing the full high-throughput potentialof microarray analysis. This is due in part to the unique analyticalchallenges that these samples present with regard to sensitivity,specificity, quantitation, and data analysis. Thismore » review discussesspecific applications of microarrays to microbial ecology research alongwith some of the latest studies addressing the difficulties encounteredduring analysis of complex microbial communities within environmentalsamples. With continued development, microarray technology may ultimatelyachieve its potential for comprehensive, high-throughput characterizationof microbial populations in near real-time.« less

The application of a novel optical SPM in biomedicine

NASA Astrophysics Data System (ADS)

Li, Yinli; Chen, Haibo; Wu, Shifa; Song, Linfeng; Zhang, Jian

2005-01-01

As an analysis tool, SPM has been broadly used in biomedicine in recent years, such as AFM and SNOM; they are effective instruments in detecting life nanostructures at atomic level. Atomic force and photon scanning tunneling microscope (AF/PSTM) is one of member of SPM, it can be used to obtain sample" optical and atomic fore images at once scanning, these images include the transmissivity image, reflection index image and topography image. This report mainly introduces the application of AF/PSTM in red blood membrane and the effect of different sample dealt with processes on the experiment result. The materials for preparing red cells membrane samples are anticoagulant blood, isotonic phosphatic buffer solution (PBS) and new two times distilled water. The images of AF/PSTM give real expression to the biology samples" fact despite of different sample dealt with processes, which prove that AF/PSTM suits to biology sample imaging. At the same time, the optical images and the topography image of AF/PSTM of the same sample are complementary with each other; this will make AF/PSTM a facile tool to analysis biologic samples" nanostructure. As another sample, this paper gives the application of AF/PSTM in immunoassay, the result shows that AF/PSTM is suit to analysis biologic sample, and it will become a new tool for biomedicine test.

Optimization of chemical and instrumental parameters in hydride generation laser-induced breakdown spectrometry for the determination of arsenic, antimony, lead and germanium in aqueous samples.

PubMed

Yeşiller, Semira Unal; Yalçın, Serife

2013-04-03

A laser induced breakdown spectrometry hyphenated with on-line continuous flow hydride generation sample introduction system, HG-LIBS, has been used for the determination of arsenic, antimony, lead and germanium in aqueous environments. Optimum chemical and instrumental parameters governing chemical hydride generation, laser plasma formation and detection were investigated for each element under argon and nitrogen atmosphere. Arsenic, antimony and germanium have presented strong enhancement in signal strength under argon atmosphere while lead has shown no sensitivity to ambient gas type. Detection limits of 1.1 mg L(-1), 1.0 mg L(-1), 1.3 mg L(-1) and 0.2 mg L(-1) were obtained for As, Sb, Pb and Ge, respectively. Up to 77 times enhancement in detection limit of Pb were obtained, compared to the result obtained from the direct analysis of liquids by LIBS. Applicability of the technique to real water samples was tested through spiking experiments and recoveries higher than 80% were obtained. Results demonstrate that, HG-LIBS approach is suitable for quantitative analysis of toxic elements and sufficiently fast for real time continuous monitoring in aqueous environments. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular Analysis of Oral Bacteria in Heart Valve of Patients With Cardiovascular Disease by Real-Time Polymerase Chain Reaction

PubMed Central

Oliveira, Francisco Artur Forte; Forte, Clarissa Pessoa Fernandes; Silva, Paulo Goberlânio de Barros; Lopes, Camile B.; Montenegro, Raquel Carvalho; dos Santos, Ândrea Kely Campos Ribeiro; Sobrinho, Carlos Roberto Martins Rodrigues; Mota, Mário Rogério Lima; Sousa, Fabrício Bitu; Alves, Ana Paula Negreiros Nunes

2015-01-01

Abstract Structural deficiencies and functional abnormalities of heart valves represent an important cause of cardiovascular morbidity and mortality, and a number of diseases, such as aortic stenosis, have been recently associated with infectious agents. This study aimed to analyze oral bacteria in dental plaque, saliva, and cardiac valves of patients with cardiovascular disease. Samples of supragingival plaque, subgingival plaque, saliva, and cardiac valve tissue were collected from 42 patients with heart valve disease. Molecular analysis of Streptococcus mutans, Prevotella intermedia, Porphyromonas gingivalis, and Treponema denticola was performed through real-time PCR. The micro-organism most frequently detected in heart valve samples was the S. mutans (89.3%), followed by P. intermedia (19.1%), P. gingivalis (4.2%), and T. denticola (2.1%). The mean decayed, missing, filled teeth (DMFT) was 26.4 ± 6.9 (mean ± SD), and according to the highest score of periodontal disease observed for each patient, periodontal pockets > 4 mm and dental calculus were detected in 43.4% and 34.7% of patients, respectively. In conclusion, oral bacteria, especially S. mutans, were found in the cardiac valve samples of patients with a high rate of caries and gingivitis/periodontitis. PMID:26632711

Study of the Wall Paintings of the Cenador Del Leon in the Real Alcazar of Seville

NASA Astrophysics Data System (ADS)

Robador, Maria Dolores; Mancera, Inmaculada; Perez-Maqueda, Rafael; Albardonedo, Antonio

2017-10-01

The paintings on the walls of the Cenador del Leon located in the gardens of the Real Alcazar in Seville next to the Pabellon de Carlos V in the Jardin Ingles area have been studied. The components of the wall paintings cross-sections, which were prepared using small samples taken from the walls of Cenador del Leon, were characterized using infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) with energy dispersive X-ray (EDX) analysis. The cross-sections of the collected samples indicated that the paint layer is well adhered to the preparation layer without any discontinuity, and only one carbonation layer exists at the top of the sequence of layers. These data suggest that the paint was applied on a fresco surface, and therefore, the adopted technique was fresco. Based on the different elements detected by EDX analysis of the cross-sections, the detected pigments included iron oxides accompanied by clay minerals (or earths) in the red pink, golden yellow and yellow colours, blue smelt for the blue colour and basic copper chloride (atacamite) for the green colour. In one sample, the particles were composed of Ba and S from barium sulphate and Ti and O from rutile titanium oxide due to a modern pigment.

Three-dimensional, automated, real-time video system for tracking limb motion in brain-machine interface studies.

PubMed

Peikon, Ian D; Fitzsimmons, Nathan A; Lebedev, Mikhail A; Nicolelis, Miguel A L

2009-06-15

Collection and analysis of limb kinematic data are essential components of the study of biological motion, including research into biomechanics, kinesiology, neurophysiology and brain-machine interfaces (BMIs). In particular, BMI research requires advanced, real-time systems capable of sampling limb kinematics with minimal contact to the subject's body. To answer this demand, we have developed an automated video tracking system for real-time tracking of multiple body parts in freely behaving primates. The system employs high-contrast markers painted on the animal's joints to continuously track the three-dimensional positions of their limbs during activity. Two-dimensional coordinates captured by each video camera are combined and converted to three-dimensional coordinates using a quadratic fitting algorithm. Real-time operation of the system is accomplished using direct memory access (DMA). The system tracks the markers at a rate of 52 frames per second (fps) in real-time and up to 100fps if video recordings are captured to be later analyzed off-line. The system has been tested in several BMI primate experiments, in which limb position was sampled simultaneously with chronic recordings of the extracellular activity of hundreds of cortical cells. During these recordings, multiple computational models were employed to extract a series of kinematic parameters from neuronal ensemble activity in real-time. The system operated reliably under these experimental conditions and was able to compensate for marker occlusions that occurred during natural movements. We propose that this system could also be extended to applications that include other classes of biological motion.

Real-scale comparison between simple and composite raw sewage sampling

NASA Astrophysics Data System (ADS)

Sergio Scalize, Paulo; Moraes Frazão, Juliana

2018-06-01

The present study performed a qualitative and quantitative characterization of the raw sewage collected at the entrance of the sewage treatment station of the city of Itumbiara, state of Goiás. Samples were collected every two hours over a period of seven consecutive days. Characterization of both point samples and composite samples was performed. The parameters analyzed were: temperature, pH, alkalinity, chemical oxygen demand, oil and grease, electric conductivity, total phosphorus, settleable solids, ammoniacal nitrogen, total suspended solids, volatile suspended solids, fixed suspended solids and turbidity. These results allowed us to verify that it is possible to perform the collection and analysis of a point sample, instead of a composite sample, as a way of monitoring the efficiency of a sewage treatment plant.

Comparison of culture, single and multiplex real-time PCR for detection of Sabin poliovirus shedding in recently vaccinated Indian children.

PubMed

Giri, Sidhartha; Rajan, Anand K; Kumar, Nirmal; Dhanapal, Pavithra; Venkatesan, Jayalakshmi; Iturriza-Gomara, Miren; Taniuchi, Mami; John, Jacob; Abraham, Asha Mary; Kang, Gagandeep

2017-08-01

Although, culture is considered the gold standard for poliovirus detection from stool samples, real-time PCR has emerged as a faster and more sensitive alternative. Detection of poliovirus from the stool of recently vaccinated children by culture, single and multiplex real-time PCR was compared. Of the 80 samples tested, 55 (68.75%) were positive by culture compared to 61 (76.25%) and 60 (75%) samples by the single and one step multiplex real-time PCR assays respectively. Real-time PCR (singleplex and multiplex) is more sensitive than culture for poliovirus detection in stool, although the difference was not statistically significant. © 2017 Wiley Periodicals, Inc.

Assessment of statistical uncertainty in the quantitative analysis of solid samples in motion using laser-induced breakdown spectroscopy

NASA Astrophysics Data System (ADS)

Cabalín, L. M.; González, A.; Ruiz, J.; Laserna, J. J.

2010-08-01

Statistical uncertainty in the quantitative analysis of solid samples in motion by laser-induced breakdown spectroscopy (LIBS) has been assessed. For this purpose, a LIBS demonstrator was designed and constructed in our laboratory. The LIBS system consisted of a laboratory-scale conveyor belt, a compact optical module and a Nd:YAG laser operating at 532 nm. The speed of the conveyor belt was variable and could be adjusted up to a maximum speed of 2 m s - 1 . Statistical uncertainty in the analytical measurements was estimated in terms of precision (reproducibility and repeatability) and accuracy. The results obtained by LIBS on shredded scrap samples under real conditions have demonstrated that the analytical precision and accuracy of LIBS is dependent on the sample geometry, position on the conveyor belt and surface cleanliness. Flat, relatively clean scrap samples exhibited acceptable reproducibility and repeatability; by contrast, samples with an irregular shape or a dirty surface exhibited a poor relative standard deviation.

Fire debris analysis for forensic fire investigation using laser induced breakdown spectroscopy (LIBS)

NASA Astrophysics Data System (ADS)

Choi, Soojin; Yoh, Jack J.

2017-08-01

The possibility verification of the first attempt to apply LIBS to arson investigation was performed. LIBS has capabilities for real time in-situ analysis and depth profiling. It can provide valuable information about the fire debris that are complementary to the classification of original sample components and combustion residues. In this study, fire debris was analyzed to determine the ignition source and existence of a fire accelerant using LIBS spectra and depth profiling analysis. Fire debris chemical composition and carbon layer thickness determines the possible ignition source while the carbon layer thickness of combusted samples represents the degree of sample carbonization. When a sample is combusted with fire accelerants, a thicker carbon layer is formed because the burning rate is increased. Therefore, depth profiling can confirm the existence of combustion accelerants, which is evidence of arson. Also investigation of fire debris by depth profiling is still possible when a fire is extinguished with water from fire hose. Such data analysis and in-situ detection of forensic signals via the LIBS may assist fire investigation at crime scenes.

Model-based frequency response characterization of a digital-image analysis system for epifluorescence microscopy

NASA Technical Reports Server (NTRS)

Hazra, Rajeeb; Viles, Charles L.; Park, Stephen K.; Reichenbach, Stephen E.; Sieracki, Michael E.

1992-01-01

Consideration is given to a model-based method for estimating the spatial frequency response of a digital-imaging system (e.g., a CCD camera) that is modeled as a linear, shift-invariant image acquisition subsystem that is cascaded with a linear, shift-variant sampling subsystem. The method characterizes the 2D frequency response of the image acquisition subsystem to beyond the Nyquist frequency by accounting explicitly for insufficient sampling and the sample-scene phase. Results for simulated systems and a real CCD-based epifluorescence microscopy system are presented to demonstrate the accuracy of the method.

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.

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

PubMed

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

2007-09-01

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

Optimization of the elution buffer and concentration method for detecting hepatitis E virus in swine liver using a nested reverse transcription-polymerase chain reaction and real-time reverse transcription-polymerase chain reaction.

PubMed

Son, Na Ry; Seo, Dong Joo; Lee, Min Hwa; Seo, Sheungwoo; Wang, Xiaoyu; Lee, Bog-Hieu; Lee, Jeong-Su; Joo, In-Sun; Hwang, In-Gyun; Choi, Changsun

2014-09-01

The aim of this study was to develop an optimal technique for detecting hepatitis E virus (HEV) in swine livers. Here, three elution buffers and two concentration methods were compared with respect to enhancing recovery of HEV from swine liver samples. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and nested RT-PCR were performed to detect HEV RNA. When phosphate-buffered saline (PBS, pH 7.4) was used to concentrate HEV in swine liver samples using ultrafiltration, real-time RT-PCR detected HEV in 6 of the 26 samples. When threonine buffer was used to concentrate HEV using polyethylene glycol (PEG) precipitation and ultrafiltration, real-time RT-PCR detected HEV in 1 and 3 of the 26 samples, respectively. When glycine buffer was used to concentrate HEV using ultrafiltration and PEG precipitation, real-time RT-PCR detected HEV in 1 and 3 samples of the 26 samples, respectively. When nested RT-PCR was used to detect HEV, all samples tested negative regardless of the type of elution buffer or concentration method used. Therefore, the combination of real-time RT-PCR and ultrafiltration with PBS buffer was the most sensitive and reliable method for detecting HEV in swine livers. Copyright © 2014 Elsevier B.V. All rights reserved.

Direct analysis in real time--high resolution mass spectrometry as a valuable tool for the pharmaceutical drug development.

PubMed

Srbek, Jan; Klejdus, Bořivoj; Douša, Michal; Břicháč, Jiří; Stasiak, Pawel; Reitmajer, Josef; Nováková, Lucie

2014-12-01

In this study, direct analysis in real time-mass spectrometry (DART-MS) was assessed for the analysis of various pharmaceutical formulations with intention to summarize possible applications for the routine pharmaceutical development. As DART is an ambient ionization technique, it allows direct analysis of pharmaceutical samples in solid or liquid form without complex sample preparation, which is often the most time-consuming part of the analytical method. This makes the technique suitable for many application fields, including pharmaceutical drug development. DART mass spectra of more than twenty selected tablets and other common pharmaceutical formulations, i.e. injection solutions, ointments and suppositories developed in the pharmaceutical industry during several recent years are presented. Moreover, as thin-layer chromatography (TLC) is still very popular for the monitoring of the reactions in the synthetic chemistry, several substances were analyzed directly from the TLC plates to demonstrate the simplicity of the technique. Pure substance solutions were spotted onto a TLC plate and then analyzed with DART without separation. This was the first DART-MS study of pharmaceutical dosage forms using DART-Orbitrap combination. The duration of sample analysis by the DART-MS technique lasted several seconds, allowing enough time to collect sufficient number of data points for compound identification. The experimental setup provided excellent mass accuracy and high resolution of the mass spectra which allowed unambiguous identification of the compounds of interest. Finally, DART mass spectrometry was also used for the monitoring of the selected impurity distribution in the atorvastatin tablets. These measurements demonstrated DART to be robust ionization technique, which provided easy-to-interpret mass spectra for the broad range of compounds. DART has high-throughput potential for various types of pharmaceutical analyses and therefore eliminates the time for sample cleanup and chromatographic separation. Copyright © 2014 Elsevier B.V. All rights reserved.

One Novel Multiple-Target Plasmid Reference Molecule Targeting Eight Genetically Modified Canola Events for Genetically Modified Canola Detection.

PubMed

Li, Zhuqing; Li, Xiang; Wang, Canhua; Song, Guiwen; Pi, Liqun; Zheng, Lan; Zhang, Dabing; Yang, Litao

2017-09-27

Multiple-target plasmid DNA reference materials have been generated and utilized as good substitutes of matrix-based reference materials in the analysis of genetically modified organisms (GMOs). Herein, we report the construction of one multiple-target plasmid reference molecule, pCAN, which harbors eight GM canola event-specific sequences (RF1, RF2, MS1, MS8, Topas 19/2, Oxy235, RT73, and T45) and a partial sequence of the canola endogenous reference gene PEP. The applicability of this plasmid reference material in qualitative and quantitative PCR assays of the eight GM canola events was evaluated, including the analysis of specificity, limit of detection (LOD), limit of quantification (LOQ), and performance of pCAN in the analysis of various canola samples, etc. The LODs are 15 copies for RF2, MS1, and RT73 assays using pCAN as the calibrator and 10 genome copies for the other events. The LOQ in each event-specific real-time PCR assay is 20 copies. In quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and PEP assays are between 91% and 97%, and the squared regression coefficients (R 2 ) are all higher than 0.99. The quantification bias values varied from 0.47% to 20.68% with relative standard deviation (RSD) from 1.06% to 24.61% in the quantification of simulated samples. Furthermore, 10 practical canola samples sampled from imported shipments in the port of Shanghai, China, were analyzed employing pCAN as the calibrator, and the results were comparable with those assays using commercial certified materials as the calibrator. Concluding from these results, we believe that this newly developed pCAN plasmid is one good candidate for being a plasmid DNA reference material in the detection and quantification of the eight GM canola events in routine analysis.

[Rapid identification of meningitis due to bacterial pathogens].

PubMed

Ubukata, Kimiko

2013-01-01

We constructed a new real-time PCR method to detect causative pathogens in cerebrospinal fluid (CSF) from patient due to bacterial meningitis. The eight pathogens targeted in the PCR are Streptococcus pneumoniae, Haemophilus influenzae, Streptococcus agalactiae, Staphylococcus aurues, Neisseria meningitides, Listeria monocytogenes, Esherichia coli, and Mycoplasma pneumoniae. The total time from DNA extraction from CSF to PCR analysis was 1.5 hour. The pathogens were detected in 72% of the CSF samples (n=115) by real-time PCR, but in only 48% by culture, although the microorganisms were completely concordant. The detection rate of pathogens with PCR was significantly better than that with cultures in patients with antibiotic administration.In conclusion, detection with real-time PCR is useful for rapidly identifying the causative pathogens of meningitis and for examining the clinical course of chemotherapy.

Separating twin images and locating the center of a microparticle in dense suspensions using correlations among reconstructed fields of two parallel holograms.

PubMed

Ling, Hangjian; Katz, Joseph

2014-09-20

This paper deals with two issues affecting the application of digital holographic microscopy (DHM) for measuring the spatial distribution of particles in a dense suspension, namely discriminating between real and virtual images and accurate detection of the particle center. Previous methods to separate real and virtual fields have involved applications of multiple phase-shifted holograms, combining reconstructed fields of multiple axially displaced holograms, and analysis of intensity distributions of weakly scattering objects. Here, we introduce a simple approach based on simultaneously recording two in-line holograms, whose planes are separated by a short distance from each other. This distance is chosen to be longer than the elongated trace of the particle. During reconstruction, the real images overlap, whereas the virtual images are displaced by twice the distance between hologram planes. Data analysis is based on correlating the spatial intensity distributions of the two reconstructed fields to measure displacement between traces. This method has been implemented for both synthetic particles and a dense suspension of 2 μm particles. The correlation analysis readily discriminates between real and virtual images of a sample containing more than 1300 particles. Consequently, we can now implement DHM for three-dimensional tracking of particles when the hologram plane is located inside the sample volume. Spatial correlations within the same reconstructed field are also used to improve the detection of the axial location of the particle center, extending previously introduced procedures to suspensions of microscopic particles. For each cross section within a particle trace, we sum the correlations among intensity distributions in all planes located symmetrically on both sides of the section. This cumulative correlation has a sharp peak at the particle center. Using both synthetic and recorded particle fields, we show that the uncertainty in localizing the axial location of the center is reduced to about one particle's diameter.

Multiplex Real-Time PCR for Detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) Genes from Selective Enrichments from Animals and Retail Meat

PubMed Central

Velasco, Valeria; Sherwood, Julie S.; Rojas-García, Pedro P.; Logue, Catherine M.

2014-01-01

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method. PMID:24849624

Multiplex real-time PCR for detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) genes from selective enrichments from animals and retail meat.

PubMed

Velasco, Valeria; Sherwood, Julie S; Rojas-García, Pedro P; Logue, Catherine M

2014-01-01

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68-0.88 (from substantial to almost perfect agreement) and 0.29-0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0-0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method.

Sample normalization methods in quantitative metabolomics.

PubMed

Wu, Yiman; Li, Liang

2016-01-22

To reveal metabolomic changes caused by a biological event in quantitative metabolomics, it is critical to use an analytical tool that can perform accurate and precise quantification to examine the true concentration differences of individual metabolites found in different samples. A number of steps are involved in metabolomic analysis including pre-analytical work (e.g., sample collection and storage), analytical work (e.g., sample analysis) and data analysis (e.g., feature extraction and quantification). Each one of them can influence the quantitative results significantly and thus should be performed with great care. Among them, the total sample amount or concentration of metabolites can be significantly different from one sample to another. Thus, it is critical to reduce or eliminate the effect of total sample amount variation on quantification of individual metabolites. In this review, we describe the importance of sample normalization in the analytical workflow with a focus on mass spectrometry (MS)-based platforms, discuss a number of methods recently reported in the literature and comment on their applicability in real world metabolomics applications. Sample normalization has been sometimes ignored in metabolomics, partially due to the lack of a convenient means of performing sample normalization. We show that several methods are now available and sample normalization should be performed in quantitative metabolomics where the analyzed samples have significant variations in total sample amounts. Copyright © 2015 Elsevier B.V. All rights reserved.

Sample introducing apparatus and sample modules for mass spectrometer

DOEpatents

Thompson, C.V.; Wise, M.B.

1993-12-21

An apparatus for introducing gaseous samples from a wide range of environmental matrices into a mass spectrometer for analysis of the samples is described. Several sample preparing modules including a real-time air monitoring module, a soil/liquid purge module, and a thermal desorption module are individually and rapidly attachable to the sample introducing apparatus for supplying gaseous samples to the mass spectrometer. The sample-introducing apparatus uses a capillary column for conveying the gaseous samples into the mass spectrometer and is provided with an open/split interface in communication with the capillary and a sample archiving port through which at least about 90 percent of the gaseous sample in a mixture with an inert gas that was introduced into the sample introducing apparatus is separated from a minor portion of the mixture entering the capillary discharged from the sample introducing apparatus. 5 figures.

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

PubMed

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

2018-02-15

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

Molecular identification and quantification of bacteria from endodontic infections using real-time polymerase chain reaction.

PubMed

Blome, B; Braun, A; Sobarzo, V; Jepsen, S

2008-10-01

It was the aim of the present study to evaluate root canal samples for the presence and numbers of specific species as well as for total bacterial load in teeth with chronic apical periodontitis using quantitative real-time polymerase chain reaction (PCR). Forty adult patients with one radiographically documented periapical lesion were included. Twenty teeth presented with primary infections and 20 with secondary infections, requiring retreatment. After removal of necrotic pulp tissue or root canal filling, a first bacterial sample was obtained. Following chemo-mechanical root canal preparation a second sample was taken and a third sample was obtained after 14 days of intracanal dressing with calcium hydroxide. Analysis by real-time PCR enabled the quantification of total bacterial counts and of nine selected species. Root canals with primary infections harbored significantly more bacteria (by total bacterial count) than teeth with secondary infections (P < 0.05). Mean total bacterial count in the retreatment group was 2.1 x 10(6) and was significantly reduced following root canal preparation (3.6 x 10(4)) and intracanal dressing (1.4 x 10(5)). Corresponding values for primary infections were: 4.6 x 10(7), 3.6 x 10(4), and 6.9 x 10(4). The numbers of the selected bacteria and their detection frequency were also significantly reduced. Root canals with primary infections contained a higher bacterial load. Chemo-mechanical root canal preparation reduced bacterial counts by at least 95%.

RASSF1A promoter methylation in high-grade serous ovarian cancer: A direct comparison study in primary tumors, adjacent morphologically tumor cell-free tissues and paired circulating tumor DNA.

PubMed

Giannopoulou, Lydia; Chebouti, Issam; Pavlakis, Kitty; Kasimir-Bauer, Sabine; Lianidou, Evi S

2017-03-28

The RASSF1A promoter is frequently methylated in high-grade serous ovarian cancer (HGSC). We examined RASSF1A promoter methylation in primary tumors, adjacent morphologically tumor cell-free tissues and corresponding circulating tumor DNA (ctDNA) samples of patients with HGSC, using a real-time methylation specific PCR (real-time MSP) and a methylation-sensitive high-resolution melting analysis (MS-HRMA) assay for the detection and semi-quantitative estimation of methylation, respectively. Two groups of primary HGSC tumor FFPE samples were recruited (Group A n=67 and Group B n=61), along with matched adjacent morphologically tumor cell-free tissues (n=58) and corresponding plasma samples (n=59) for group B. Using both assays, RASSF1A promoter was found highly methylated in primary tumors of both groups, and at lower percentages in the adjacent morphologically tumor cell-free tissues. Interestingly, RASSF1A promoter methylation was also observed in ctDNA by real-time MSP. Overall survival (OS) was significantly associated with RASSF1A promoter methylation in primary tumor samples using MS-HRMA (P=0.023). Our results clearly indicate that RASSF1A promoter is methylated in adjacent tissue surrounding the tumor in HGSC patients. We report for the first time that RASSF1A promoter methylation provides significant prognostic information in HGSC patients.

RASSF1A promoter methylation in high-grade serous ovarian cancer: A direct comparison study in primary tumors, adjacent morphologically tumor cell-free tissues and paired circulating tumor DNA

PubMed Central

Giannopoulou, Lydia; Chebouti, Issam; Pavlakis, Kitty; Kasimir-Bauer, Sabine; Lianidou, Evi S.

2017-01-01

The RASSF1A promoter is frequently methylated in high-grade serous ovarian cancer (HGSC). We examined RASSF1A promoter methylation in primary tumors, adjacent morphologically tumor cell-free tissues and corresponding circulating tumor DNA (ctDNA) samples of patients with HGSC, using a real-time methylation specific PCR (real-time MSP) and a methylation-sensitive high-resolution melting analysis (MS-HRMA) assay for the detection and semi-quantitative estimation of methylation, respectively. Two groups of primary HGSC tumor FFPE samples were recruited (Group A n=67 and Group B n=61), along with matched adjacent morphologically tumor cell-free tissues (n=58) and corresponding plasma samples (n=59) for group B. Using both assays, RASSF1A promoter was found highly methylated in primary tumors of both groups, and at lower percentages in the adjacent morphologically tumor cell-free tissues. Interestingly, RASSF1A promoter methylation was also observed in ctDNA by real-time MSP. Overall survival (OS) was significantly associated with RASSF1A promoter methylation in primary tumor samples using MS-HRMA (P=0.023). Our results clearly indicate that RASSF1A promoter is methylated in adjacent tissue surrounding the tumor in HGSC patients. We report for the first time that RASSF1A promoter methylation provides significant prognostic information in HGSC patients. PMID:28206954

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

PubMed Central

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

2018-01-01

Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura. In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in S. litura and ensure the data more accurate for the mechanism analysis of azadirachtin. PMID:29695976

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

PubMed

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

2018-01-01

Azadirachtin is an efficient and broad-spectrum botanical insecticide against more than 150 kinds of agricultural pests with the effects of mortality, antifeedant and growth regulation. Real-time quantitative polymerase chain reaction (RT-qPCR) could be one of the powerful tools to analyze the gene expression level and investigate the mechanism of azadirachtin at transcriptional level, however, the ideal reference genes are needed to normalize the expression profiling of target genes. In this present study, the fragments of eight candidate reference genes were cloned and identified from the pest Spodoptera litura . In addition, the expression stability of these genes in different samples from larvae of control and azadirachtin treatments were evaluated by the computational methods of NormFinder, BestKeeper, Delta CT, geNorm, and RefFinder. According to our results, two of the reference genes should be the optimal number for RT-qPCR analysis. Furthermore, the best reference genes for different samples were showed as followed: EF-1α and EF2 for cuticle, β-Tubulin and RPL7A for fat body, EF2 and Actin for midgut, EF2 and RPL13A for larva and RPL13A and RPL7A for all the samples. Our results established a reliable normalization for RT-qPCR experiments in S. litura and ensure the data more accurate for the mechanism analysis of azadirachtin.

[Analysis of phthalates in plastic food-packaging bags by thin layer chromatography].

PubMed

Chen, Hui; Wang, Yuan; Zhu, Ruohua

2006-01-01

The method for simultaneous determination of four phthalates, namely dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP) and di (2-ethylhexyl) phthalate (DEHP) in plastic food-packaging bags by thin layer chromatography (TLC) was developed. The plastic food-packaging bags were extracted with ethanol by ultrasonication, then the mixture was filtrated through membrane (0.45 microm). The mixture of ethyl acetate-anhydrous ether-isooctane (1 : 4 : 15, v/v) was used as developing agent on the TLC silica gel plate for development. The filtered liquid was spotted on the TLC plate dealt by acetone, and detected with scanning wavelength of 275 nm and reference wavelength of 340 nm. The qualitative analysis of the phthalates was performed using the R(f) values of the chromatogram. The quantitative analysis was performed with external standard method. Good linearities were obtained for DMP, DEP, DBP and DEHP. The detection limits were 2.1 ng for DMP, 2.4 ng for DEP, 3.4 ng for DBP and 4.0 ng for DEHP. The relative standard deviations (RSDs) of the four phthalates were 2.8% - 3.5%. The recoveries of the four phthalate standards in real sample were 78.58% - 111.04%. The method presented has the advantages of high precision, high sensitivity, small sample size, and simple pretreatment . The method was used to detect the four phthalates in the food-packaging bags. The contents in real samples were close to the results by gas chromatography.

The WAIS Melt Monitor: An automated ice core melting system for meltwater sample handling and the collection of high resolution microparticle size distribution data

NASA Astrophysics Data System (ADS)

Breton, D. J.; Koffman, B. G.; Kreutz, K. J.; Hamilton, G. S.

2010-12-01

Paleoclimate data are often extracted from ice cores by careful geochemical analysis of meltwater samples. The analysis of the microparticles found in ice cores can also yield unique clues about atmospheric dust loading and transport, dust provenance and past environmental conditions. Determination of microparticle concentration, size distribution and chemical makeup as a function of depth is especially difficult because the particle size measurement either consumes or contaminates the meltwater, preventing further geochemical analysis. Here we describe a microcontroller-based ice core melting system which allows the collection of separate microparticle and chemistry samples from the same depth intervals in the ice core, while logging and accurately depth-tagging real-time electrical conductivity and particle size distribution data. This system was designed specifically to support microparticle analysis of the WAIS Divide WDC06A deep ice core, but many of the subsystems are applicable to more general ice core melting operations. Major system components include: a rotary encoder to measure ice core melt displacement with 0.1 millimeter accuracy, a meltwater tracking system to assign core depths to conductivity, particle and sample vial data, an optical debubbler level control system to protect the Abakus laser particle counter from damage due to air bubbles, a Rabbit 3700 microcontroller which communicates with a host PC, collects encoder and optical sensor data and autonomously operates Gilson peristaltic pumps and fraction collectors to provide automatic sample handling, melt monitor control software operating on a standard PC allowing the user to control and view the status of the system, data logging software operating on the same PC to collect data from the melting, electrical conductivity and microparticle measurement systems. Because microparticle samples can easily be contaminated, we use optical air bubble sensors and high resolution ice core density profiles to guide the melting process. The combination of these data allow us to analyze melt head performance, minimize outer-to-inner fraction contamination and avoid melt head flooding. The WAIS Melt Monitor system allows the collection of real-time, sub-annual microparticle and electrical conductivity data while producing and storing enough sample for traditional Coulter-Counter particle measurements as well long term acid leaching of bioactive metals (e.g., Fe, Co, Cd, Cu, Zn) prior to chemical analysis.

Weighted analysis of paired microarray experiments.

PubMed

Kristiansson, Erik; Sjögren, Anders; Rudemo, Mats; Nerman, Olle

2005-01-01

In microarray experiments quality often varies, for example between samples and between arrays. The need for quality control is therefore strong. A statistical model and a corresponding analysis method is suggested for experiments with pairing, including designs with individuals observed before and after treatment and many experiments with two-colour spotted arrays. The model is of mixed type with some parameters estimated by an empirical Bayes method. Differences in quality are modelled by individual variances and correlations between repetitions. The method is applied to three real and several simulated datasets. Two of the real datasets are of Affymetrix type with patients profiled before and after treatment, and the third dataset is of two-colour spotted cDNA type. In all cases, the patients or arrays had different estimated variances, leading to distinctly unequal weights in the analysis. We suggest also plots which illustrate the variances and correlations that affect the weights computed by our analysis method. For simulated data the improvement relative to previously published methods without weighting is shown to be substantial.

Wireless, battery-operated data acquisition system for mobile spectrometry applications and (potentially) for the Internet of things

NASA Astrophysics Data System (ADS)

Fitzgerald, Ryan; Karanassios, Vassili

2017-05-01

There are many applications requiring chemical analysis in the field and analytical results in (near) real-time. For example, when accidental spills occur. In others, collecting samples in the field followed by analysis in a lab increases costs and introduces time-delays. In such cases, "bring part of the lab to the sample" would be ideal. Toward this ideal (and to further reduce size and weight), we developed a relatively inexpensive, battery-operated, wireless data acquisition hardware system around an Arduino nano micro-controller and a 16-bit ADC (Analog-to- Digital Converter) with a max sampling rate of 860 samples/s. The hardware communicates the acquired data using low-power Bluetooth. Software for data acquisition and data display was written in Python. Potential ways of making the hardware-software approach described here a part of the Internet-of-Things (IoT) are presented.

Advanced Non-Destructive Assessment Technology to Determine the Aging of Silicon Containing Materials for Generation IV Nuclear Reactors

NASA Astrophysics Data System (ADS)

Koenig, T. W.; Olson, D. L.; Mishra, B.; King, J. C.; Fletcher, J.; Gerstenberger, L.; Lawrence, S.; Martin, A.; Mejia, C.; Meyer, M. K.; Kennedy, R.; Hu, L.; Kohse, G.; Terry, J.

2011-06-01

To create an in-situ, real-time method of monitoring neutron damage within a nuclear reactor core, irradiated silicon carbide samples are examined to correlate measurable variations in the material properties with neutron fluence levels experienced by the silicon carbide (SiC) during the irradiation process. The reaction by which phosphorus doping via thermal neutrons occurs in the silicon carbide samples is known to increase electron carrier density. A number of techniques are used to probe the properties of the SiC, including ultrasonic and Hall coefficient measurements, as well as high frequency impedance analysis. Gamma spectroscopy is also used to examine residual radioactivity resulting from irradiation activation of elements in the samples. Hall coefficient measurements produce the expected trend of increasing carrier concentration with higher fluence levels, while high frequency impedance analysis shows an increase in sample impedance with increasing fluence.

Analytical Performances of Human Immunodeficiency Virus Type 1 RNA-Based Amplix® Real-Time PCR Platform for HIV-1 RNA Quantification

PubMed Central

Mboumba Bouassa, Ralph-Sydney; Jenabian, Mohammad-Ali; Wolyec, Serge Tonen; Robin, Leman; Matta, Mathieu; Longo, Jean de Dieu; Grésenguet, Gérard; Andreoletti, Laurent; Bélec, Laurent

2016-01-01

Objectives. We evaluated the performances of Amplix real-time PCR platform developed by Biosynex (Strasbourg, France), combining automated station extraction (Amplix station 16 Dx) and real-time PCR (Amplix NG), for quantifying plasma HIV-1 RNA by lyophilized HIV-1 RNA-based Amplix reagents targeting gag and LTR, using samples from HIV-1-infected adults from Central African Republic. Results. Amplix real-time PCR assay showed low limit of detection (28 copies/mL), across wide dynamic range (1.4–10 log copies/mL), 100% sensitivity and 99% specificity, high reproducibility, and accuracy with mean bias < 5%. The assay showed excellent correlations and concordance of 95.3% with the reference HIV-1 RNA load assay (Roche), with mean absolute bias of +0.097 log copies/mL by Bland-Altman analysis. The assay was able to detect and quantify the most prevalent HIV-1 subtype strains and the majority of non-B subtypes, CRFs of HIV-1 group M, and HIV-1 groups N and O circulating in Central Africa. The Amplix assay showed 100% sensitivity and 99.6% specificity to diagnose virological failure in clinical samples from antiretroviral drug-experienced patients. Conclusions. The HIV-1 RNA-based Amplix real-time PCR platform constitutes sensitive and reliable system for clinical monitoring of HIV-1 RNA load in HIV-1-infected children and adults, particularly adapted to intermediate laboratory facilities in sub-Saharan Africa. PMID:28050283

Simulated 'On-Line' Wear Metal Analysis of Lubricating Oils by X-Ray Fluorescence Spectroscopy

NASA Technical Reports Server (NTRS)

Kelliher, Warren C.; Partos, Richard D.; Nelson, Irina

1996-01-01

The objective of this project was to assess the sensitivity of X-ray Fluorescence Spectroscopy (XFS) for quantitative evaluation of metal particle content in engine oil suspensions and the feasibility of real-time, dynamic wear metal analysis. The study was focused on iron as the majority wear metal component. Variable parameters were: particle size, particle concentration and oil velocity. A commercial XFS spectrometer equipped with interchangeable static/dynamic (flow cell) sample chambers was used. XFS spectra were recorded for solutions of Fe-organometallic standard and for a series of DTE oil suspensions of high purity spherical iron particles of 2g, 4g, and 8g diameter, at concentrations from 5 ppm to 5,000 ppm. Real contaminated oil samples from Langley Air Force Base aircraft engines and NASA Langley Research Center wind tunnels were also analyzed. The experimental data conform the reliability of XFS as the analytical method of choice for this project. Intrinsic inadequacies of the instrument for precise analytic work at low metal concentrations were identified as being related to the particular x-ray beam definition, system geometry, and flow-cell materials selection. This work supports a proposal for the design, construction and testing of a conceptually new, miniature XFS spectrometer with superior performance, dedicated to on-line, real-time monitoring of lubricating oils in operating engines. Innovative design solutions include focalization of the incident x-ray beam, non-metal sample chamber, and miniaturization of the overall assembly. The instrument would contribute to prevention of catastrophic engine failures. A proposal for two-year funding has been presented to NASA Langley Research Center Internal Operation Group (IOG) Management, to continue the effort begun by this summer's project.

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

PubMed Central

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

2018-01-01

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

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.

Adjusting for multiple prognostic factors in the analysis of randomised trials

PubMed Central

2013-01-01

Background When multiple prognostic factors are adjusted for in the analysis of a randomised trial, it is unclear (1) whether it is necessary to account for each of the strata, formed by all combinations of the prognostic factors (stratified analysis), when randomisation has been balanced within each stratum (stratified randomisation), or whether adjusting for the main effects alone will suffice, and (2) the best method of adjustment in terms of type I error rate and power, irrespective of the randomisation method. Methods We used simulation to (1) determine if a stratified analysis is necessary after stratified randomisation, and (2) to compare different methods of adjustment in terms of power and type I error rate. We considered the following methods of analysis: adjusting for covariates in a regression model, adjusting for each stratum using either fixed or random effects, and Mantel-Haenszel or a stratified Cox model depending on outcome. Results Stratified analysis is required after stratified randomisation to maintain correct type I error rates when (a) there are strong interactions between prognostic factors, and (b) there are approximately equal number of patients in each stratum. However, simulations based on real trial data found that type I error rates were unaffected by the method of analysis (stratified vs unstratified), indicating these conditions were not met in real datasets. Comparison of different analysis methods found that with small sample sizes and a binary or time-to-event outcome, most analysis methods lead to either inflated type I error rates or a reduction in power; the lone exception was a stratified analysis using random effects for strata, which gave nominal type I error rates and adequate power. Conclusions It is unlikely that a stratified analysis is necessary after stratified randomisation except in extreme scenarios. Therefore, the method of analysis (accounting for the strata, or adjusting only for the covariates) will not generally need to depend on the method of randomisation used. Most methods of analysis work well with large sample sizes, however treating strata as random effects should be the analysis method of choice with binary or time-to-event outcomes and a small sample size. PMID:23898993

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

PubMed

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

2014-01-01

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

Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

PubMed Central

Gandelman, Olga A.; Church, Vicki L.; Moore, Cathy A.; Kiddle, Guy; Carne, Christopher A.; Parmar, Surendra; Jalal, Hamid; Tisi, Laurence C.; Murray, James A. H.

2010-01-01

Background The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. Principal Findings Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. Conclusions The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings. PMID:21152399

A sheeppox outbreak in Morocco: isolation and identification of virus responsible for the new clinical form of disease.

PubMed

Zro, Khalil; Zakham, Fathiah; Melloul, Marouane; El Fahime, Elmostafa; Ennaji, Moulay Mustapha

2014-01-27

Sheeppoxvirus (SPPV) is a member of the Capripoxvirus genus of the Poxviridae family, which causes significant economic losses in Morocco. The resurgence of the sheeppox disease during 2010 was characterized by an emergence of a classical nodular form for the first time in Morocco. However, little is known about the virus strain responsible for nodular form. In this study, thirty three sheep, from the eastern region of Morocco, clinically infected were examined and dead animals were autopsied.A rapid diagnostic assay for SPPV using different type of clinical samples would be useful for outbreak management. The aim of this work was to isolate the virus strain responsible for nodular form and we identified and compared by phylogenetic analysis the field strain with Moroccan vaccine strain targeting the thymidine kinase (TK) gene and the chemokine analogue receptor of interleukin (IL8) gene. Further, it was important to investigate and validate a real-time PCR using different clinical and post-mortem samples to manage epidemic sheeppox disease. The nodular form of sheeppox disease observed in Morocco was clinically characterized by fever, depression, lacrimation, diarrhea in lambs and nodule. At necropsy, the most affected organ was the lung. The etiological strain was successfully isolated from lung nodule in a dead lamb and was identified by using real-time PCR that has been tested and validated on different types of clinical and post mortem samples from naturally infected animals. Sequence and phylogenetic analysis of TK and IL8 gene showed that there was a very close relationship between field and vaccine strain. They were clustered within other SPPV strains. In the current study, we show for the first time the nodular form of sheeppox in Morocco. We demonstrate a robust real-time PCR-based diagnostic assay to detect the sheeppox virus in multiple sample that can be implemented to efficiently manage the disease outbreak. Our study also offers the prospect for future molecular studies to understand the clinical forms.

Arthropod Surveillance Programs: Basic Components, Strategies, and Analysis.

PubMed

Cohnstaedt, Lee W; Rochon, Kateryn; Duehl, Adrian J; Anderson, John F; Barrera, Roberto; Su, Nan-Yao; Gerry, Alec C; Obenauer, Peter J; Campbell, James F; Lysyk, Tim J; Allan, Sandra A

2012-03-01

Effective entomological surveillance planning stresses a careful consideration of methodology, trapping technologies, and analysis techniques. Herein, the basic principles and technological components of arthropod surveillance plans are described, as promoted in the symposium "Advancements in arthropod monitoring technology, techniques, and analysis" presented at the 58th annual meeting of the Entomological Society of America in San Diego, CA. Interdisciplinary examples of arthropod monitoring for urban, medical, and veterinary applications are reviewed. Arthropod surveillance consists of the three components: 1) sampling method, 2) trap technology, and 3) analysis technique. A sampling method consists of selecting the best device or collection technique for a specific location and sampling at the proper spatial distribution, optimal duration, and frequency to achieve the surveillance objective. Optimized sampling methods are discussed for several mosquito species (Diptera: Culicidae) and ticks (Acari: Ixodidae). The advantages and limitations of novel terrestrial and aerial insect traps, artificial pheromones and kairomones are presented for the capture of red flour beetle (Coleoptera: Tenebrionidae), small hive beetle (Coleoptera: Nitidulidae), bed bugs (Hemiptera: Cimicidae), and Culicoides (Diptera: Ceratopogonidae) respectively. After sampling, extrapolating real world population numbers from trap capture data are possible with the appropriate analysis techniques. Examples of this extrapolation and action thresholds are given for termites (Isoptera: Rhinotermitidae) and red flour beetles.

Detection of Listeria monocytogenes in ready-to-eat food by Step One real-time polymerase chain reaction.

PubMed

Pochop, Jaroslav; Kačániová, Miroslava; Hleba, Lukáš; Lopasovský, L'ubomír; Bobková, Alica; Zeleňáková, Lucia; Stričík, Michal

2012-01-01

The aim of this study was to follow contamination of ready-to-eat food with Listeria monocytogenes by using the Step One real time polymerase chain reaction (PCR). We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and MicroSEQ® Listeria monocytogenes Detection Kit for the real-time PCR performance. In 30 samples of ready-to-eat milk and meat products without incubation we detected strains of Listeria monocytogenes in five samples (swabs). Internal positive control (IPC) was positive in all samples. Our results indicated that the real-time PCR assay developed in this study could sensitively detect Listeria monocytogenes in ready-to-eat food without incubation.

Noninvasive analysis of volatile biomarkers in human emanations for health and early disease diagnosis.

PubMed

Kataoka, Hiroyuki; Saito, Keita; Kato, Hisato; Masuda, Kazufumi

2013-06-01

Early disease diagnosis is crucial for human healthcare and successful therapy. Since any changes in homeostatic balance can alter human emanations, the components of breath exhalations and skin emissions may be diagnostic biomarkers for various diseases and metabolic disorders. Since hundreds of endogenous and exogenous volatile organic compounds (VOCs) are released from the human body, analysis of these VOCs may be a noninvasive, painless, and easy diagnostic tool. Sampling and preconcentration by sorbent tubes/traps and solid-phase microextraction, in combination with GC or GC-MS, are usually used to analyze VOCs. In addition, GC-MS-olfactometry is useful for simultaneous analysis of odorants and odor quality. Direct MS techniques are also useful for the online real-time detection of VOCs. This review focuses on recent developments in sampling and analysis of volatile biomarkers in human odors and/or emanations, and discusses future use of VOC analysis.

Screening for trace explosives by AccuTOF™-DART®: an in-depth validation study.

PubMed

Sisco, Edward; Dake, Jeffrey; Bridge, Candice

2013-10-10

Ambient ionization mass spectrometry is finding increasing utility as a rapid analysis technique in a number of fields. In forensic science specifically, analysis of many types of samples, including drugs, explosives, inks, bank dye, and lotions, has been shown to be possible using these techniques [1]. This paper focuses on one type of ambient ionization mass spectrometry, Direct Analysis in Real Time Mass Spectrometry (DART-MS or DART), and its viability as a screening tool for trace explosives analysis. In order to assess viability, a validation study was completed which focused on the analysis of trace amounts of nitro and peroxide based explosives. Topics which were studied, and are discussed, include method optimization, reproducibility, sensitivity, development of a search library, discrimination of mixtures, and blind sampling. Advantages and disadvantages of this technique over other similar screening techniques are also discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Feasibility of corona discharge ion mobility spectrometry for direct analysis of samples extracted by dispersive liquid-liquid microextraction.

PubMed

Jafari, Mohammad T; Riahi, Farhad

2014-05-23

The capability of corona discharge ionization ion mobility spectrometry (CD-IMS) for direct analysis of the samples extracted by dispersive liquid-liquid microextraction (DLLME) was investigated and evaluated, for the first time. To that end, an appropriate new injection port was designed and constructed, resulting in possibility of direct injection of the known sample volume, without tedious sample preparation steps (e.g. derivatization, solvent evaporation, and re-solving in another solvent…). Malathion as a test compound was extracted from different matrices by a rapid and convenient DLLME method. The positive ion mobility spectra of the extracted malathion were obtained after direct injection of carbon tetrachloride or methanol solutions. The analyte responses were compared and the statistical results revealed the feasibility of direct analysis of the extracted samples in carbon tetrachloride, resulting in a convenient methodology. The coupled method of DLLME-CD-IMS was exhaustively validated in terms of sensitivity, dynamic range, recovery, and enrichment factor. Finally, various real samples of apple, river and underground water were analyzed, all verifying the feasibility and success of the proposed method for the easy extraction of the analyte using DLLME separation before the direct analysis by CD-IMS. Copyright © 2014 Elsevier B.V. All rights reserved.

Performance of Panfungal- and Specific-PCR-Based Procedures for Etiological Diagnosis of Invasive Fungal Diseases on Tissue Biopsy Specimens with Proven Infection: a 7-Year Retrospective Analysis from a Reference Laboratory

PubMed Central

Bernal-Martinez, L.; Castelli, M. V.; Rodriguez-Tudela, J. L.; Cuenca-Estrella, M.

2014-01-01

A retrospective analysis of real-time PCR (RT-PCR) results for 151 biopsy samples obtained from 132 patients with proven invasive fungal diseases was performed. PCR-based techniques proved to be fast and sensitive and enabled definitive diagnosis in all cases studied, with detection of a total of 28 fungal species. PMID:24574295

Revealing retroperitoneal liposarcoma morphology using optical coherence tomography

NASA Astrophysics Data System (ADS)

Carbajal, Esteban F.; Baranov, Stepan A.; Manne, Venu G. R.; Young, Eric D.; Lazar, Alexander J.; Lev, Dina C.; Pollock, Raphael E.; Larin, Kirill V.

2011-02-01

A new approach to distinguish normal fat, well-differentiated (WD), and dedifferentiated liposarcoma (LS) tumors is demonstrated, based on the use of optical coherence tomography (OCT). OCT images show the same structures seen with conventional histological methods. Our visual grading analysis is supported by numerical analysis of observed structures for normal fat and WDLS samples. Further development could apply the real-time and high resolution advantages of OCT for use in liposarcoma diagnosis and clinical procedures.

Advanced functional materials in solid phase extraction for ICP-MS determination of trace elements and their species - A review.

PubMed

He, Man; Huang, Lijin; Zhao, Bingshan; Chen, Beibei; Hu, Bin

2017-06-22

For the determination of trace elements and their species in various real samples by inductively coupled plasma mass spectrometry (ICP-MS), solid phase extraction (SPE) is a commonly used sample pretreatment technique to remove complex matrix, pre-concentrate target analytes and make the samples suitable for subsequent sample introduction and measurements. The sensitivity, selectivity/anti-interference ability, sample throughput and application potential of the methodology of SPE-ICP-MS are greatly dependent on SPE adsorbents. This article presents a general overview of the use of advanced functional materials (AFMs) in SPE for ICP-MS determination of trace elements and their species in the past decade. Herein the AFMs refer to the materials featuring with high adsorption capacity, good selectivity, fast adsorption/desorption dynamics and satisfying special requirements in real sample analysis, including nanometer-sized materials, porous materials, ion imprinting polymers, restricted access materials and magnetic materials. Carbon/silica/metal/metal oxide nanometer-sized adsorbents with high surface area and plenty of adsorption sites exhibit high adsorption capacity, and porous adsorbents would provide more adsorption sites and faster adsorption dynamics. The selectivity of the materials for target elements/species can be improved by using physical/chemical modification, ion imprinting and restricted accessed technique. Magnetic adsorbents in conventional batch operation offer unique magnetic response and high surface area-volume ratio which provide a very easy phase separation, greater extraction capacity and efficiency over conventional adsorbents, and chip-based magnetic SPE provides a versatile platform for special requirement (e.g. cell analysis). The performance of these adsorbents for the determination of trace elements and their species in different matrices by ICP-MS is discussed in detail, along with perspectives and possible challenges in the future development. Copyright © 2017 Elsevier B.V. All rights reserved.

Moving your laboratories to the field – Advantages and limitations of the use of field portable instruments in environmental sample analysis

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

Gałuszka, Agnieszka, E-mail: Agnieszka.Galuszka@ujk.edu.pl; Migaszewski, Zdzisław M.; Namieśnik, Jacek

The recent rapid progress in technology of field portable instruments has increased their applications in environmental sample analysis. These instruments offer a possibility of cost-effective, non-destructive, real-time, direct, on-site measurements of a wide range of both inorganic and organic analytes in gaseous, liquid and solid samples. Some of them do not require the use of reagents and do not produce any analytical waste. All these features contribute to the greenness of field portable techniques. Several stationary analytical instruments have their portable versions. The most popular ones include: gas chromatographs with different detectors (mass spectrometer (MS), flame ionization detector, photoionization detector),more » ultraviolet–visible and near-infrared spectrophotometers, X-ray fluorescence spectrometers, ion mobility spectrometers, electronic noses and electronic tongues. The use of portable instruments in environmental sample analysis gives a possibility of on-site screening and a subsequent selection of samples for routine laboratory analyses. They are also very useful in situations that require an emergency response and for process monitoring applications. However, quantification of results is still problematic in many cases. The other disadvantages include: higher detection limits and lower sensitivity than these obtained in laboratory conditions, a strong influence of environmental factors on the instrument performance and a high possibility of sample contamination in the field. This paper reviews recent applications of field portable instruments in environmental sample analysis and discusses their analytical capabilities. - Highlights: • Field portable instruments are widely used in environmental sample analysis. • Field portable instruments are indispensable for analysis in emergency response. • Miniaturization of field portable instruments reduces resource consumption. • In situ analysis is in agreement with green analytical chemistry principles. • Performance requirements in field analysis stimulate technological progress.« less

Development of a Water-Quality Lab That Enhances Learning & Connects Students to the Land

ERIC Educational Resources Information Center

Enos-Berlage, Jodi

2012-01-01

A 3-week laboratory module was developed for an undergraduate microbiology course that would connect student learning to a real-life challenge, specifically a local water-quality project. The laboratory series included multiple field trips, sampling of soil and water, and subsequent analysis for bacteria and nitrate. Laboratory results confirmed…

Categories and Underlying Processes, or Representative Behavior Samples and S-R Analysis: Opposing Strategies.

ERIC Educational Resources Information Center

Staats, Arthur W.

Psychological researchers should deal with the concrete stimulus-response principles of learning on which behavior is based, and study behaviors that are representative of real life behaviors. The present research strategy has come from two faulty ideas: first, a concern with underlying, inferred mental processes, rather than with actual tasks or…

An Inexpensive Autosampler to Maximize Throughput for an Ion Source that Samples Surfaces in Open Air

EPA Science Inventory

An autosampler was built to pull cotton swab heads mounted into a 3-foot long, square Al rod in ambient air through the He ionizing beam of a Direct Analysis in Real Time (DART) ion source interfaced to an orthogonal acceleration, time-of-flight mass spectrometer. The cost of th...

Environmental Indicators of Metal Pollution and Emission: An Experiment for the Instrumental Analysis Laboratory

ERIC Educational Resources Information Center

Bowden, John A.; Nocito, Brian A.; Lowers, Russell H.; Guillette, Louis J., Jr.; Williams, Kathryn R.; Young, Vaneica Y.

2012-01-01

This experiment enlightens students on the use of environmental indicators and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and demonstrates the ability of these monitoring tools to measure metal deposition in environmental samples (both as a result of lab-simulated and real events). In this two-part study, the initial…

Study of pipe thickness loss using a neutron radiography method

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

Mohamed, Abdul Aziz; Wahab, Aliff Amiru Bin; Yazid, Hafizal B.

2014-02-12

The purpose of this preliminary work is to study for thickness changes in objects using neutron radiography. In doing the project, the technique for the radiography was studied. The experiment was done at NUR-2 facility at TRIGA research reactor in Malaysian Nuclear Agency, Malaysia. Test samples of varying materials were used in this project. The samples were radiographed using direct technique. Radiographic images were recorded using Nitrocellulose film. The films obtained were digitized to processed and analyzed. Digital processing is done on the images using software Isee!. The images were processed to produce better image for analysis. The thickness changesmore » in the image were measured to be compared with real thickness of the objects. From the data collected, percentages difference between measured and real thickness are below than 2%. This is considerably very low variation from original values. Therefore, verifying the neutron radiography technique used in this project.« less

Assessment of bacterial pathogens in fresh rainwater and airborne particulate matter using Real-Time PCR

NASA Astrophysics Data System (ADS)

Kaushik, Rajni; Balasubramanian, Rajasekhar

2012-01-01

Bacterial pathogens in airborne particulate matter (PM) and in rainwater (RW) were detected using a robust and sensitive Real-Time PCR method. Both RW and PM were collected simultaneously in the tropical atmosphere of Singapore, which were then subjected to analysis for the presence of selected bacterial pathogens and potential pathogen of health concern ( Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Aeromonas hydrophila). These pathogens were found to be prevalent in both PM and RW samples with E. coli being the most prevalent potential pathogen in both types of samples. The temporal distribution of these pathogens in PM and RW was found to be similar to each other. Using the proposed microbiological technique, the atmospheric deposition (dry and wet deposition) of bacterial pathogens to lakes and reservoirs can be studied in view of growing concerns about the outbreak of waterborne diseases.

Towards real-time metabolic profiling of a biopsy specimen during a surgical operation by 1H high resolution magic angle spinning nuclear magnetic resonance: a case report.

PubMed

Piotto, Martial; Moussallieh, François-Marie; Neuville, Agnès; Bellocq, Jean-Pierre; Elbayed, Karim; Namer, Izzie Jacques

2012-01-18

Providing information on cancerous tissue samples during a surgical operation can help surgeons delineate the limits of a tumoral invasion more reliably. Here, we describe the use of metabolic profiling of a colon biopsy specimen by high resolution magic angle spinning nuclear magnetic resonance spectroscopy to evaluate tumoral invasion during a simulated surgical operation. Biopsy specimens (n = 9) originating from the excised right colon of a 66-year-old Caucasian women with an adenocarcinoma were automatically analyzed using a previously built statistical model. Metabolic profiling results were in full agreement with those of a histopathological analysis. The time-response of the technique is sufficiently fast for it to be used effectively during a real operation (17 min/sample). Metabolic profiling has the potential to become a method to rapidly characterize cancerous biopsies in the operation theater.

High-rate real-time GPS network at Parkfield: Utility for detecting fault slip and seismic displacements

USGS Publications Warehouse

Langbein, J.; Bock, Y.

2004-01-01

A network of 13 continuous GPS stations near Parkfield, California has been converted from 30 second to 1 second sampling with positions of the stations estimated in real-time relative to a master station. Most stations are near the trace of the San Andreas fault, which exhibits creep. The noise spectra of the instantaneous 1 Hz positions show flicker noise at high frequencies and change to frequency independence at low frequencies; the change in character occurs between 6 to 8 hours. Our analysis indicates that 1-second sampled GPS can estimate horizontal displacements of order 6 mm at the 99% confidence level from a few seconds to a few hours. High frequency GPS can augment existing measurements in capturing large creep events and postseismic slip that would exceed the range of existing creepmeters, and can detect large seismic displacements. Copyright 2004 by the American Geophysical Union.

Peptide Modified ZnO Nanoparticles as Gas Sensors Array for Volatile Organic Compounds (VOCs)

PubMed Central

Mascini, Marcello; Gaggiotti, Sara; Della Pelle, Flavio; Di Natale, Corrado; Qakala, Sinazo; Iwuoha, Emmanuel; Pittia, Paola; Compagnone, Dario

2018-01-01

In this work a peptide based gas sensor array based of ZnO nanoparticles (ZnONPs) has been realized. Four different pentapeptides molecularly modeled for alcohols and esters having cysteine as a common spacer have been immobilized onto ZnONPs. ZnONPs have been morphologically and spectroscopically characterized. Modified nanoparticles have been then deposited onto quartz crystal microbalances (QCMs) and used as gas sensors with nitrogen as carrier gas. Analysis of the pure compounds modeled demonstrated a nice fitting of modeling with real data. The peptide based ZnONPs had very low sensitivity to water, compared to previously studied AuNPs peptide based gas sensors allowing the use of the array on samples with high water content. Real samples of fruit juices have been assayed; stability of the signal, good repeatability, and discrimination ability of the array was achieved. PMID:29713626

Peptide Modified ZnO Nanoparticles as Gas Sensors Array for Volatile Organic Compounds (VOCs).

PubMed

Mascini, Marcello; Gaggiotti, Sara; Della Pelle, Flavio; Di Natale, Corrado; Qakala, Sinazo; Iwuoha, Emmanuel; Pittia, Paola; Compagnone, Dario

2018-01-01

In this work a peptide based gas sensor array based of ZnO nanoparticles (ZnONPs) has been realized. Four different pentapeptides molecularly modeled for alcohols and esters having cysteine as a common spacer have been immobilized onto ZnONPs. ZnONPs have been morphologically and spectroscopically characterized. Modified nanoparticles have been then deposited onto quartz crystal microbalances (QCMs) and used as gas sensors with nitrogen as carrier gas. Analysis of the pure compounds modeled demonstrated a nice fitting of modeling with real data. The peptide based ZnONPs had very low sensitivity to water, compared to previously studied AuNPs peptide based gas sensors allowing the use of the array on samples with high water content. Real samples of fruit juices have been assayed; stability of the signal, good repeatability, and discrimination ability of the array was achieved.

Peptide modified ZnO nanoparticles as gas sensors array for volatile organic compounds (VOCs)

NASA Astrophysics Data System (ADS)

Mascini, Marcello; Gaggiotti, Sara; Della Pelle, Flavio; Di Natale, Corrado; Qakala, Sinazo; Iwuoha, Emmanuel; Pittia, Paola; Compagnone, Dario

2018-04-01

In this work a peptide based gas sensor array based of ZnO nanoparticles (ZnONPs) has been realized. Four different pentapeptides molecularly modelled for alcohols and esters having cysteine as a common spacer have been immobilized onto ZnONPs. ZnONPs have been morphologically and spectroscopically characterized. Modified nanoparticles have been then deposited onto quartz crystal microbalances (QCMs) and used as gas sensors with nitrogen as carrier gas. Analysis of the pure compounds modelled demonstrated a nice fitting of modelling with real data. The peptide based ZnONPs had very low sensitivity to water, compared to previously studied AuNPs peptide based gas sensors allowing the use of the array on samples with high water content. Real samples of fruit juices have been assayed; stability of the signal, good repeatability and discrimination ability of the array was achieved.

Capillary isoelectric focusing of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification.

PubMed

Horká, Marie; Karásek, Pavel; Salplachta, Jiří; Růžička, Filip; Vykydalová, Marie; Kubesová, Anna; Dráb, Vladimír; Roth, Michal; Slais, Karel

2013-07-25

In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cow's milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2×10(6) mL(-1). Finally, cow's milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample. Copyright © 2013 Elsevier B.V. All rights reserved.

Thermal stability of inorganic and organic compounds in atmospheric particulate matter

NASA Astrophysics Data System (ADS)

Perrino, Cinzia; Marconi, Elisabetta; Tofful, Luca; Farao, Carmela; Materazzi, Stefano; Canepari, Silvia

2012-07-01

The thermal behaviour of atmospheric particulate matter (PM) has been investigated by using different analytical approaches to explore the added value offered by these technique in environmental studies. The thermogravimetric analysis (TGA), carried out on both certified material and real PM samples, has shown that several mass losses can be detected starting from 80 °C up to above 500 °C, when pyrolysis occur. Thermo-optical analysis of PM and ion chromatographic analysis of the residual have shown that the mass losses in the temperature range 80-180 °C are not justified by the release of either organic or inorganic compounds; it can be thus attributed to the release of weakly and strongly bound water. Release of water has also been evidenced in the temperature range 225-275 °C. The release of ammonium chloride and nitrate has been detected only above 80 °C. This indicates that the release of nitric acid, hydrochloric acid and ammonia, which is observed downstream of the filters during the sampling of atmospheric PM at ambient temperature, cannot be reproduced off-line, after the end of the sampling. We successfully explored one of the possible explanations, that is the desorption of HNO3, HCl and NH3 adsorbed on collected particles. NH4NO3 and NH4Cl, which can be thermally released by the filter, exhibit a different thermal behaviour from NaNO3 and NaCl, which are thermally stable up to 370 °C. This different behaviour can be used to discriminate between natural and secondary sources of atmospheric inorganic salts, as the interconversion that is observed when heating mixtures of pure salts resulted to be not relevant when heating real PM samples.

Antemortem detection of chronic wasting disease prions in nasal brush collections and rectal biopsies from white-tailed deer by real time quaking-induced conversion

USGS Publications Warehouse

Haley, Nicholas J.; Siepker, Chris; Walter, W. David; Thomsen, Bruce V.; Greenlee, Justin J.; Lehmkuhl, Aaron D.; Richt, Jürgen a.

2016-01-01

Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of cervids, was first documented nearly 50 years ago in Colorado and Wyoming and has since spread to cervids in 23 states, two Canadian provinces, and the Republic of Korea. The expansion of this disease makes the development of sensitive diagnostic assays and antemortem sampling techniques crucial for the mitigation of its spread; this is especially true in cases of relocation/reintroduction of farmed or free-ranging deer and elk or surveillance studies of private or protected herds, where depopulation is contraindicated. This study sought to evaluate the sensitivity of the real-time quaking-induced conversion (RT-QuIC) assay by using recto-anal mucosa-associated lymphoid tissue (RAMALT) biopsy specimens and nasal brush samples collected antemortem from farmed white-tailed deer (n = 409). Antemortem findings were then compared to results from ante- and postmortem samples (RAMALT, brainstem, and medial retropharyngeal lymph nodes) evaluated by using the current gold standard in vitro assay, immunohistochemistry (IHC) analysis. We hypothesized that the sensitivity of RT-QuIC would be comparable to IHC analysis in antemortem tissues and would correlate with both the genotype and the stage of clinical disease. Our results showed that RAMALT testing by RT-QuIC assay had the highest sensitivity (69.8%) compared to that of postmortem testing, with a specificity of >93.9%. These data suggest that RT-QuIC, like IHC analysis, is an effective assay for detection of PrPCWD in rectal biopsy specimens and other antemortem samples and, with further research to identify more sensitive tissues, bodily fluids, or experimental conditions, has potential for large-scale and rapid automated testing for CWD diagnosis.

A molecular approach for the rapid, selective and sensitive detection of Exophiala jeanselmei in environmental samples: development and performance assessment of a real-time PCR assay.

PubMed

Libert, X; Chasseur, C; Packeu, A; Bureau, F; Roosens, N H; De Keersmaecker, S J C

2016-02-01

Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized into the air and subsequently cause health problems, its monitoring is recommended. Currently, this monitoring is based on culture and microscopic identification which are complex, sometimes ambiguous and time-demanding, i.e., up to 21 days. Therefore, molecular, culture-independent methods could be more advantageous for the monitoring of E. jeanselmei. In this study, we developed a SYBR®green real-time PCR assay based on the internal transcribed spacer 2 from the 18S ribosomal DNA complex for the specific detection of E. jeanselmei. The selectivity (100 %), PCR efficiency (95.5 %), dynamic range and repeatability of this qPCR assay were subsequently evaluated. The limit of detection for this qPCR assay was determined to be 1 copy of genomic DNA of E. jeanselmei. Finally, water samples collected from cooling reservoirs were analyzed using this qPCR assay to deliver a proof of concept for the molecular detection of E. jeanselmei in environmental samples. The results obtained by molecular analysis were compared with those of classical methods (i.e., culture and microscopic identification) used in routine analysis and were 100 % matching. This comparison demonstrated that this SYBR®green qPCR assay can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by E. jeanselmei, while eliminating the need for culturing and thereby considerably decreasing the required analysis time to 2 days.

Spectral refractive index assessment of turbid samples by combining spatial frequency near-infrared spectroscopy with Kramers-Kronig analysis

NASA Astrophysics Data System (ADS)

Meitav, Omri; Shaul, Oren; Abookasis, David

2018-03-01

A practical algorithm for estimating the wavelength-dependent refractive index (RI) of a turbid sample in the spatial frequency domain with the aid of Kramers-Kronig (KK) relations is presented. In it, phase-shifted sinusoidal patterns (structured illumination) are serially projected at a high spatial frequency onto the sample surface (mouse scalp) at different near-infrared wavelengths while a camera mounted normally to the sample surface captures the reflected diffuse light. In the offline analysis pipeline, recorded images at each wavelength are converted to spatial absorption maps by logarithmic function, and once the absorption coefficient information is obtained, the imaginary part (k) of the complex RI (CRI), based on Maxell's equations, can be calculated. Using the data represented by k, the real part of the CRI (n) is then resolved by KK analysis. The wavelength dependence of n ( λ ) is then fitted separately using four standard dispersion models: Cornu, Cauchy, Conrady, and Sellmeier. In addition, three-dimensional surface-profile distribution of n is provided based on phase profilometry principles and a phase-unwrapping-based phase-derivative-variance algorithm. Experimental results demonstrate the capability of the proposed idea for sample's determination of a biological sample's RI value.

Analytical performance of benchtop total reflection X-ray fluorescence instrumentation for multielemental analysis of wine samples

NASA Astrophysics Data System (ADS)

Dalipi, Rogerta; Marguí, Eva; Borgese, Laura; Bilo, Fabjola; Depero, Laura E.

2016-06-01

Recent technological improvements have led to a widespread adoption of benchtop total reflection X-ray fluorescence systems (TXRF) for analysis of liquid samples. However, benchtop TXRF systems usually present limited sensitivity compared with high-scale instrumentation which can restrict its application in some fields. The aim of the present work was to evaluate and compare the analytical capabilities of two TXRF systems, equipped with low power Mo and W target X-ray tubes, for multielemental analysis of wine samples. Using the Mo-TXRF system, the detection limits for most elements were one order of magnitude lower than those attained using the W-TXRF system. For the detection of high Z elements like Cd and Ag, however, W-TXRF remains a very good option due to the possibility of K-Lines detection. Accuracy and precision of the obtained results have been evaluated analyzing spiked real wine samples and comparing the TXRF results with those obtained by inductively coupled plasma emission spectroscopy (ICP-OES). In general, good agreement was obtained between ICP-OES and TXRF results for the analysis of both red and white wine samples except for light elements (i.e., K) which TXRF concentrations were underestimated. However, a further achievement of analytical quality of TXRF results can be achieved if wine analysis is performed after dilution of the sample with de-ionized water.

Direct analysis in real time--a critical review on DART-MS.

PubMed

Gross, Jürgen H

2014-01-01

Direct analysis in real time mass spectrometry (DART-MS) has become an established technique for rapid mass spectral analysis of a large variety of samples. DART-MS is capable of analyzing the sample at atmospheric pressure, essentially in the open laboratory environment. DART-MS can be applied to compounds that have been deposited or adsorbed on to surfaces or that are being desorbed therefrom into the atmosphere. This makes DART-MS suitable and well-known for analysis of ingredients of plant materials, pesticide monitoring on vegetables, forensic and safety applications such as screening for traces of explosives, warfare agents, or illicit drugs on luggage, clothes, or bank notes, etc. DART can also be used for analysis of either solid or liquid bulk materials, as may be required in quality control, or to quickly investigate the identity of a compound from chemical synthesis. Even living organisms can be subjected to DART-MS. Driven by different needs in analytical practice, the combination of the DART ionization source and interface can be configured in multiple geometries and with various accessories to adapt the setup as required. Analysis by DART-MS relies on some sort of gas-phase ionization mechanism. In DART, initial generation of the ionizing species is by use of a corona discharge in a pure helium atmosphere which delivers excited helium atoms that, upon their release into the atmosphere, will initiate a cascade of gas-phase reactions. In the end, this results in reagent ions created from atmospheric water or (solvent) vapor in the vicinity of the surface subject to analysis where they effect a chemical ionization process. DART ionization processes may generate positive or negative ions, predominantly even-electron species, but odd-electron species do also occur. The prevailing process of analyte ion formation from a given sample is highly dependent on analyte properties.

Sample entropy predicts lifesaving interventions in trauma patients with normal vital signs.

PubMed

Naraghi, L; Mejaddam, A Y; Birkhan, O A; Chang, Y; Cropano, C M; Mesar, T; Larentzakis, A; Peev, M; Sideris, A C; Van der Wilden, G M; Imam, A M; Hwabejire, J O; Velmahos, G C; Fagenholz, P J; Yeh, D; de Moya, M A; King, D R

2015-08-01

Heart rate complexity, commonly described as a "new vital sign," has shown promise in predicting injury severity, but its use in clinical practice is not yet widely adopted. We previously demonstrated the ability of this noninvasive technology to predict lifesaving interventions (LSIs) in trauma patients. This study was conducted to prospectively evaluate the utility of real-time, automated, noninvasive, instantaneous sample entropy (SampEn) analysis to predict the need for an LSI in a trauma alert population presenting with normal vital signs. Prospective enrollment of patients who met criteria for trauma team activation and presented with normal vital signs was conducted at a level I trauma center. High-fidelity electrocardiogram recording was used to calculate SampEn and SD of the normal-to-normal R-R interval (SDNN) continuously in real time for 2 hours with a portable, handheld device. Patients who received an LSI were compared to patients without any intervention (non-LSI). Multivariable analysis was performed to control for differences between the groups. Treating clinicians were blinded to results. Of 129 patients enrolled, 38 (29%) received 136 LSIs within 24 hours of hospital arrival. Initial systolic blood pressure was similar in both groups. Lifesaving intervention patients had a lower Glasgow Coma Scale. The mean SampEn on presentation was 0.7 (0.4-1.2) in the LSI group compared to 1.5 (1.1-2.0) in the non-LSI group (P < .0001). The area under the curve with initial SampEn alone was 0.73 (95% confidence interval [CI], 0.64-0.81) and increased to 0.93 (95% CI, 0.89-0.98) after adding sedation to the model. Sample entropy of less than 0.8 yields sensitivity, specificity, negative predictive value, and positive predictive value of 58%, 86%, 82%, and 65%, respectively, with an overall accuracy of 76% for predicting an LSI. SD of the normal-to-normal R-R interval had no predictive value. In trauma patients with normal presenting vital signs, decreased SampEn is an independent predictor of the need for LSI. Real-time SampEn analysis may be a useful adjunct to standard vital signs monitoring. Adoption of real-time, instantaneous SampEn monitoring for trauma patients, especially in resource-constrained environments, should be considered. Copyright © 2015 Elsevier Inc. All rights reserved.

Authentication of virgin olive oil by a novel curve resolution approach combined with visible spectroscopy.

PubMed

Ferreiro-González, Marta; Barbero, Gerardo F; Álvarez, José A; Ruiz, Antonio; Palma, Miguel; Ayuso, Jesús

2017-04-01

Adulteration of olive oil is not only a major economic fraud but can also have major health implications for consumers. In this study, a combination of visible spectroscopy with a novel multivariate curve resolution method (CR), principal component analysis (PCA) and linear discriminant analysis (LDA) is proposed for the authentication of virgin olive oil (VOO) samples. VOOs are well-known products with the typical properties of a two-component system due to the two main groups of compounds that contribute to the visible spectra (chlorophylls and carotenoids). Application of the proposed CR method to VOO samples provided the two pure-component spectra for the aforementioned families of compounds. A correlation study of the real spectra and the resolved component spectra was carried out for different types of oil samples (n=118). LDA using the correlation coefficients as variables to discriminate samples allowed the authentication of 95% of virgin olive oil samples. Copyright © 2016 Elsevier Ltd. All rights reserved.

The real relationship inventory: development and psychometric investigation of the client form.

PubMed

Kelley, Frances A; Gelso, Charles J; Fuertes, Jairo N; Marmarosh, Cheri; Lanier, Stacey Holmes

2010-12-01

The development and validation of a client version of the Real Relationship Inventory (RRI-C) is reported. Using a sample of clients (n = 94) who were currently in psychotherapy, a 24-item measure was developed consisting of two subscales (Realism and Genuineness) and a total score. This 24-item version and other measures used for validation were completed by 93 additional clients. Results of the present study offer initial support for the validity and reliability of the RRI-C. The RRI-C correlated significantly in theoretically expected ways with measures of the client-rated working alliance and therapists' congruence, clients' observing ego, and client ratings of client and therapist real relationship on an earlier measure of the real relationship (Eugster & Wampold, 1996). A nonsignificant relation was found between the RRI-C and a measure of social desirability, providing support for discriminant validity. A confirmatory factor analysis supported the two theorized factors of the RRI-C. The authors discuss the importance of measuring clients' perceptions of the real relationship. (PsycINFO Database Record (c) 2010 APA, all rights reserved).

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.

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.

Evaluation of non-extracted genital swabs for real-time HSV PCR.

PubMed

Miari, Victoria F; Wall, Gavin R; Clark, Duncan A

2015-01-01

Nucleic acid extraction of clinical samples is accepted as a key requirement in molecular diagnostics. At Barts Health NHS Trust, swabs taken from patients with clinical suspicion of HSV infection were routinely extracted on the Qiagen MDx BioRobot prior to testing with a real-time triplex PCR for HSV1, HSV2, and VZV. The aim of this study was to adapt an existing HSV1/HSV2/VZV real-time PCR by replacing VZV with phocine herpesvirus 1 (PhHV) as an internal control (IC) and evaluate whether this adapted assay required the nucleic acid extraction step for predominantly genital swabs. First 313 non-extracted and extracted swabs were tested in parallel with the existing triplex HSV1/HSV2/VZV real-time PCR. The second stage involved testing 176 non-extracted swabs using a triplex real-time PCR for HSV1, HSV2, and PhHV and comparing the results with the samples extracted and tested by the original triplex assay. The results correlated well when the existing assay was used, with only three non-extracted samples that would have been reported as negative compared to the extracted sample result (Cq s 33, 39, 35-two samples HSV1, one sample HSV2). In the evaluation using the adapted assay containing the IC, two of 176 samples were discordant, where a HSV negative non-extracted sample result would have been reported differently to the extracted sample result (Cq s 32, 33-both HSV1). This study demonstrated that it is feasible to test non-extracted swabs for HSV in a real-time PCR that includes an IC. J. Med. Virol. 87: 125-129, 2015. © 2014 Wiley Periodicals, Inc. © 2014 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

Sampling properties of directed networks

NASA Astrophysics Data System (ADS)

Son, S.-W.; Christensen, C.; Bizhani, G.; Foster, D. V.; Grassberger, P.; Paczuski, M.

2012-10-01

For many real-world networks only a small “sampled” version of the original network may be investigated; those results are then used to draw conclusions about the actual system. Variants of breadth-first search (BFS) sampling, which are based on epidemic processes, are widely used. Although it is well established that BFS sampling fails, in most cases, to capture the IN component(s) of directed networks, a description of the effects of BFS sampling on other topological properties is all but absent from the literature. To systematically study the effects of sampling biases on directed networks, we compare BFS sampling to random sampling on complete large-scale directed networks. We present new results and a thorough analysis of the topological properties of seven complete directed networks (prior to sampling), including three versions of Wikipedia, three different sources of sampled World Wide Web data, and an Internet-based social network. We detail the differences that sampling method and coverage can make to the structural properties of sampled versions of these seven networks. Most notably, we find that sampling method and coverage affect both the bow-tie structure and the number and structure of strongly connected components in sampled networks. In addition, at a low sampling coverage (i.e., less than 40%), the values of average degree, variance of out-degree, degree autocorrelation, and link reciprocity are overestimated by 30% or more in BFS-sampled networks and only attain values within 10% of the corresponding values in the complete networks when sampling coverage is in excess of 65%. These results may cause us to rethink what we know about the structure, function, and evolution of real-world directed networks.

Multiplex Real-Time PCR Method for Simultaneous Identification and Toxigenic Type Characterization of Clostridium difficile From Stool Samples

PubMed Central

Alam, Mohammad J.; Tisdel, Naradah L.; Shah, Dhara N.; Yapar, Mehmet; Lasco, Todd M.; Garey, Kevin W.

2015-01-01

Background The aim of this study was to develop and validate a multiplex real-time PCR assay for simultaneous identification and toxigenic type characterization of Clostridium difficile. Methods The multiplex real-time PCR assay targeted and simultaneously detected triose phosphate isomerase (tpi) and binary toxin (cdtA) genes, and toxin A (tcdA) and B (tcdB) genes in the first and sec tubes, respectively. The results of multiplex real-time PCR were compared to those of the BD GeneOhm Cdiff assay, targeting the tcdB gene alone. The toxigenic culture was used as the reference, where toxin genes were detected by multiplex real-time PCR. Results A total of 351 stool samples from consecutive patients were included in the study. Fifty-five stool samples (15.6%) were determined to be positive for the presence of C. difficile by using multiplex real-time PCR. Of these, 48 (87.2%) were toxigenic (46 tcdA and tcdB-positive, two positive for only tcdB) and 11 (22.9%) were cdtA-positive. The sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) of the multiplex real-time PCR compared with the toxigenic culture were 95.6%, 98.6%, 91.6%, and 99.3%, respectively. The analytical sensitivity of the multiplex real-time PCR assay was determined to be 103colonyforming unit (CFU)/g spiked stool sample and 0.0625 pg genomic DNA from culture. Analytical specificity determined by using 15 enteric and non-clostridial reference strains was 100%. Conclusions The multiplex real-time PCR assay accurately detected C. difficile isolates from diarrheal stool samples and characterized its toxin genes in a single PCR run. PMID:25932438

Validation of two commercial real-time RT-PCR kits for rapid and specific diagnosis of classical swine fever virus.

PubMed

Le Dimna, M; Vrancken, R; Koenen, F; Bougeard, S; Mesplède, A; Hutet, E; Kuntz-Simon, G; Le Potier, M F

2008-01-01

Two real-time RT-PCR kits, developed by LSI (TaqVet CSF) and ADIAGENE (Adiavet CSF), obtained an agreement to be commercialised in France, subject to conditions, defined by the French Classical Swine Fever (CSF) National Reference Laboratory. The producers were asked to introduce an internal control to check the RNA extraction efficacy. The different criteria assessed were sensitivity, "pestivirus specificity", reproducibility and ease of handling, using 189 different samples. These samples were either CSFV inactivated strains or blood/serum/organs collected from CSFV experimentally infected pigs or naturally infected wild boars. The reproducibility of the assays was confirmed by the analysis of a batch-to-batch panel control that was used for inter-laboratory tests involving nine laboratories. The two kits were also tested for the use in mass diagnostics and the results proved the kits to be suited using pools of blood, serum and tonsils. Moreover, a field evaluation, carried out on spleen samples collected from the CSF surveillance of wild boars in an area known to be infected and from domestic pigs at a slaughterhouse, confirmed the high sensitivity and specificity of the two kits. This step-by-step evaluation procedure confirmed that the two commercial CSF real-time RT-PCR kits have a higher predictive value than the current diagnostic standard, Virus Isolation.

Environmental Measurement-While-Drilling system for real-time field screening of contaminants

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

Lockwood, G.J.; Normann, R.A.; Bishop, L.B.

Sampling during environmental drilling is essential to fully characterize the spatial distribution and migration of near surface contaminants. However, the analysis of these samples is not only expensive, but can take weeks or months when sent to an off-site laboratory. In contrast, measurement-while-drilling (MWD) screening capability could save money and valuable time by quickly distinguishing between contaminated and uncontaminated areas. Real-time measurements provided by a MVM system would enable on-the-spot decisions to be made regarding sampling strategies, enhance worker safety, and provide the added flexibility of being able to ``steer`` the drill bit in or out hazardous zones. During measurement-while-drilling,more » down-hole sensors are located behind the drill bit and linked by a rapid data transmission system to a computer at the surface. As drilling proceeds, data are collected on the nature and extent of the subsurface contamination in real-time. The down-hole sensor is a Geiger-Mueller tube (GMT) gamma radiation detector. In addition to the GMT signal, the MWD system monitors these required down-hole voltages and two temperatures associated with the detector assembly. The Gamma Ray Detection System (GRDS) and electronics package are discussed in as well as the results of the field test. Finally, our conclusions and discussion of future work are presented.« less

Recent Updates in the Endoscopic Diagnosis of Barrett's Oesophagus.

PubMed

Sharma, Neel; Ho, Khek Yu

2016-10-01

Barrett's oesophagus (BO) is a premalignant condition associated with the development of oesophageal adenocarcinoma (OAC). Despite the low risk of progression per annum, OAC is associated with significant morbidity and mortality, with an estimated 5-year survival of 10%. Furthermore, the incidence of OAC continues to rise globally. Therefore, it is imperative to detect the premalignant phase of BO and follow up such patients accordingly. The mainstay diagnosis of BO is endoscopy and biopsy sampling. However, limitations with white light endoscopy (WLE) and undertaking biopsies have shifted the current focus towards real-time image analysis. Utilization of additional tools such as chromoendoscopy, narrow-band imaging (NBI), confocal laser endomicroscopy (CLE), and optical coherence tomography (OCT) are proving beneficial. Furthermore, it is also becoming more apparent that often these tools are utilized by experts in the field. Therefore, for the non-expert, training in these systems is key. Currently as yet, the methodologies used for training optimization require further inquiry. (1) Real-time imaging can serve to minimize excess biopsies. (2) Tools such as chromoendoscopy, NBI, CLE, and OCT can help to compliment WLE. WLE is associated with limited sensitivity. Biopsy sampling is cost-ineffective and associated with sampling error. Hence, from a practical perspective, endoscopists should aim to utilize additional tools to help in real-time image interpretation and minimize an overreliance on histology.

Recent Updates in the Endoscopic Diagnosis of Barrett's Oesophagus

PubMed Central

Sharma, Neel; Ho, Khek Yu

2016-01-01

Background Barrett's oesophagus (BO) is a premalignant condition associated with the development of oesophageal adenocarcinoma (OAC). Despite the low risk of progression per annum, OAC is associated with significant morbidity and mortality, with an estimated 5-year survival of 10%. Furthermore, the incidence of OAC continues to rise globally. Therefore, it is imperative to detect the premalignant phase of BO and follow up such patients accordingly. Summary The mainstay diagnosis of BO is endoscopy and biopsy sampling. However, limitations with white light endoscopy (WLE) and undertaking biopsies have shifted the current focus towards real-time image analysis. Utilization of additional tools such as chromoendoscopy, narrow-band imaging (NBI), confocal laser endomicroscopy (CLE), and optical coherence tomography (OCT) are proving beneficial. Furthermore, it is also becoming more apparent that often these tools are utilized by experts in the field. Therefore, for the non-expert, training in these systems is key. Currently as yet, the methodologies used for training optimization require further inquiry. Key Message (1) Real-time imaging can serve to minimize excess biopsies. (2) Tools such as chromoendoscopy, NBI, CLE, and OCT can help to compliment WLE. Practical Implications WLE is associated with limited sensitivity. Biopsy sampling is cost-ineffective and associated with sampling error. Hence, from a practical perspective, endoscopists should aim to utilize additional tools to help in real-time image interpretation and minimize an overreliance on histology. PMID:27904863

Functionalization of magnetic hollow porous oval shape NiFe2O4 as a highly selective sorbent for the simultaneous determination of five heavy metals in real samples.

PubMed

Liu, Mingyang; Yang, Lijun; Zhang, Lei

2016-12-01

In this study, a functionalized magnetic hollow porous oval-shape NiFe 2 O 4 (MHPO-NiFe 2 O 4 ) was designed by a facile synthesis procedure, and employed as magnetic solid phase extraction (MSPE) material to extract several heavy metal ions. As-prepared MHPO-NiFe 2 O 4 exhibited superior adsorption capacities of 20.17, 16.64, 16.82, 9.69 and 16.58mgg -1 , for Cu(II), Cd(II), Cr(III), Co(II) and Zn(II), and was then used to detect these heavy metals elements in real samples by combining with inductively coupled plasma optical emission spectroscopy (ICP-OES). The possible mechanism of the enrichment of heavy metals ions on MHPO-NiFe 2 O 4 was proposed, which involved the dominant adsorption and desorption. The detection limits were as low as 0.015, 0.13, 0.062, 0.035 and 0.46μgL -1 for Cu(II), Cd(II), Cr(III), Co(II) and Zn(II), respectively. A good repeatability was obtained with the relative standard deviation (RSD) of 3.87%. Moreover, the method was successfully utilized for the analysis of five heavy metals in real samples (cabbage, lettuce, apple, seawater), with satisfactory recoveries in the range of 92-108%. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

Busman, Mark; Liu, Jihong; Zhong, Hongjian; Bobell, John R; Maragos, Chris M

2014-01-01

Direct analysis in real time (DART) ionisation 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 parameter settings were optimised to obtain sensitive and accurate determination of aflatoxin AFB1. 84:16 acetonitrile water extracts of corn were analysed by DART-MS. The lowest calibration level (LCL) for aflatoxin AFB1 was 4 μg kg⁻¹. Quantitative analysis was performed with the use of matrix-matched standards employing the ¹³C-labelled internal standard for AFB1. DART-MS of spiked corn extracts gave linear response in the range 4-1000 μg kg⁻¹. Good recoveries (94-110%) and repeatabilities (RSD = 0.7-6.9%) were obtained at spiking levels of 20 and 100 μg kg⁻¹ with the use of an isotope dilution technique. Trueness of data obtained for AFB1 in maize by DART-MS was demonstrated by analysis of corn certified reference materials.

Real-Time Binding Kinetic Analyses of the Interaction of the Dietary Stain Orange II with Dentin Matrix.

PubMed

Alraies, Amr; Cole, David K; S Rees, Jeremy; Glasse, Carl; Young, Nigel; Waddington, Rachel J; Sloan, Alastair J

2018-06-09

Dietary stains can be adsorbed into the dentin of teeth. Using Orange II as a model dietary stain, this study investigated the strength of its interaction with the mineral and protein components of dentin matrix and how hydrogen peroxide (H 2 O 2 ) treatment influences this interaction. Dentin slices were prepared from human teeth and were either deproteinized (5.6% sodium hypochlorite, 12 days), demineralised (0.5 M EDTA, 3 days) or left as intact control samples. Samples were stained with Orange II for 1-168 h, during which staining intensity was quantified by image analysis. Similarly, uptake of stain by deproteinized / demineralized samples treated with 10 or 30% H 2 O 2 was investigated. Using surface plasmon resonance technology, real-time binding kinetics were determined assessing the interaction of orange II with the dentin matrix protein constituents, collagen type I, biglycan, decorin, dentin sialoprotein and osteopontin. Deproteinization of dentin matrix reduced the uptake of the orange II compared to the intact control. Conversely, demineralization of dentin samples increased the uptake of the dye. Treatment of samples for 48 h with H 2 O 2 reduced subsequent uptake of the orange II. Real-time kinetic analysis indicated moderate strength of binding for Orange II with collagen type I, weak binding with decorin and biglycan and negligible binding with dentine sialoprotein and osteopontin. These results indicate a predominant role for collagen type I, which accounts for 90% of the organic protein matrix of teeth, for attracting dietary stains. Binding analyses indicate that the interaction is highly dissociable, and further binding is reduced following H 2 O 2 treatment. This study provides new information regarding adsorption of dietary stains into tooth dentin, suggesting that they are attracted and moderately bound to the collagen type I matrix. This study also contributes valuable information for discussion for considering the effect of H 2 O 2 on bleaching teeth and its influence on subsequent uptake of dietary stains following whitening treatments. Copyright © 2018. Published by Elsevier Ltd.

Statistical Analysis of Large Scale Structure by the Discrete Wavelet Transform

NASA Astrophysics Data System (ADS)

Pando, Jesus

1997-10-01

The discrete wavelet transform (DWT) is developed as a general statistical tool for the study of large scale structures (LSS) in astrophysics. The DWT is used in all aspects of structure identification including cluster analysis, spectrum and two-point correlation studies, scale-scale correlation analysis and to measure deviations from Gaussian behavior. The techniques developed are demonstrated on 'academic' signals, on simulated models of the Lymanα (Lyα) forests, and on observational data of the Lyα forests. This technique can detect clustering in the Ly-α clouds where traditional techniques such as the two-point correlation function have failed. The position and strength of these clusters in both real and simulated data is determined and it is shown that clusters exist on scales as large as at least 20 h-1 Mpc at significance levels of 2-4 σ. Furthermore, it is found that the strength distribution of the clusters can be used to distinguish between real data and simulated samples even where other traditional methods have failed to detect differences. Second, a method for measuring the power spectrum of a density field using the DWT is developed. All common features determined by the usual Fourier power spectrum can be calculated by the DWT. These features, such as the index of a power law or typical scales, can be detected even when the samples are geometrically complex, the samples are incomplete, or the mean density on larger scales is not known (the infrared uncertainty). Using this method the spectra of Ly-α forests in both simulated and real samples is calculated. Third, a method for measuring hierarchical clustering is introduced. Because hierarchical evolution is characterized by a set of rules of how larger dark matter halos are formed by the merging of smaller halos, scale-scale correlations of the density field should be one of the most sensitive quantities in determining the merging history. We show that these correlations can be completely determined by the correlations between discrete wavelet coefficients on adjacent scales and at nearly the same spatial position, Cj,j+12/cdot2. Scale-scale correlations on two samples of the QSO Ly-α forests absorption spectra are computed. Lastly, higher order statistics are developed to detect deviations from Gaussian behavior. These higher order statistics are necessary to fully characterize the Ly-α forests because the usual 2nd order statistics, such as the two-point correlation function or power spectrum, give inconclusive results. It is shown how this technique takes advantage of the locality of the DWT to circumvent the central limit theorem. A non-Gaussian spectrum is defined and this spectrum reveals not only the magnitude, but the scales of non-Gaussianity. When applied to simulated and observational samples of the Ly-α clouds, it is found that different popular models of structure formation have different spectra while two, independent observational data sets, have the same spectra. Moreover, the non-Gaussian spectra of real data sets are significantly different from the spectra of various possible random samples. (Abstract shortened by UMI.)

Real rock-microfluidic flow cell: A test bed for real-time in situ analysis of flow, transport, and reaction in a subsurface reactive transport environment.

PubMed

Singh, Rajveer; Sivaguru, Mayandi; Fried, Glenn A; Fouke, Bruce W; Sanford, Robert A; Carrera, Martin; Werth, Charles J

2017-09-01

Physical, chemical, and biological interactions between groundwater and sedimentary rock directly control the fundamental subsurface properties such as porosity, permeability, and flow. This is true for a variety of subsurface scenarios, ranging from shallow groundwater aquifers to deeply buried hydrocarbon reservoirs. Microfluidic flow cells are now commonly being used to study these processes at the pore scale in simplified pore structures meant to mimic subsurface reservoirs. However, these micromodels are typically fabricated from glass, silicon, or polydimethylsiloxane (PDMS), and are therefore incapable of replicating the geochemical reactivity and complex three-dimensional pore networks present in subsurface lithologies. To address these limitations, we developed a new microfluidic experimental test bed, herein called the Real Rock-Microfluidic Flow Cell (RR-MFC). A porous 500μm-thick real rock sample of the Clair Group sandstone from a subsurface hydrocarbon reservoir of the North Sea was prepared and mounted inside a PDMS microfluidic channel, creating a dynamic flow-through experimental platform for real-time tracking of subsurface reactive transport. Transmitted and reflected microscopy, cathodoluminescence microscopy, Raman spectroscopy, and confocal laser microscopy techniques were used to (1) determine the mineralogy, geochemistry, and pore networks within the sandstone inserted in the RR-MFC, (2) analyze non-reactive tracer breakthrough in two- and (depth-limited) three-dimensions, and (3) characterize multiphase flow. The RR-MFC is the first microfluidic experimental platform that allows direct visualization of flow and transport in the pore space of a real subsurface reservoir rock sample, and holds potential to advance our understandings of reactive transport and other subsurface processes relevant to pollutant transport and cleanup in groundwater, as well as energy recovery. Copyright © 2017 Elsevier B.V. All rights reserved.

Generation of realistic scene using illuminant estimation and mixed chromatic adaptation

NASA Astrophysics Data System (ADS)

Kim, Jae-Chul; Hong, Sang-Gi; Kim, Dong-Ho; Park, Jong-Hyun

2003-12-01

The algorithm of combining a real image with a virtual model was proposed to increase the reality of synthesized images. Currently, synthesizing a real image with a virtual model facilitated the surface reflection model and various geometric techniques. In the current methods, the characteristics of various illuminants in the real image are not sufficiently considered. In addition, despite the chromatic adaptation plays a vital role for accommodating different illuminants in the two media viewing conditions, it is not taken into account in the existing methods. Thus, it is hardly to get high-quality synthesized images. In this paper, we proposed the two-phase image synthesis algorithm. First, the surface reflectance of the maximum high-light region (MHR) was estimated using the three eigenvectors obtained from the principal component analysis (PCA) applied to the surface reflectances of 1269 Munsell samples. The combined spectral value, i.e., the product of surface reflectance and the spectral power distributions (SPDs) of an illuminant, of MHR was then estimated using the three eigenvectors obtained from PCA applied to the products of surface reflectances of Munsell 1269 samples and the SPDs of four CIE Standard Illuminants (A, C, D50, D65). By dividing the average combined spectral values of MHR by the average surface reflectances of MHR, we could estimate the illuminant of a real image. Second, the mixed chromatic adaptation (S-LMS) using an estimated and an external illuminants was applied to the virtual-model image. For evaluating the proposed algorithm, experiments with synthetic and real scenes were performed. It was shown that the proposed method was effective in synthesizing the real and the virtual scenes under various illuminants.

Sensitivity and specificity of real-time reverse transcription polymerase chain reaction, histopathology, and immunohistochemical labeling for the detection of Rift Valley fever virus in naturally infected cattle and sheep.

PubMed

Odendaal, Lieza; Fosgate, Geoffrey T; Romito, Marco; Coetzer, Jacobus A W; Clift, Sarah J

2014-01-01

Real-time reverse transcription polymerase chain reaction (real-time RT-PCR), histopathology, and immunohistochemical labeling (IHC) were performed on liver specimens from 380 naturally infected cattle and sheep necropsied during the 2010 Rift Valley fever (RVF) epidemic in South Africa. Sensitivity (Se) and specificity (Sp) of real-time RT-PCR, histopathology, and IHC were estimated in a latent-class model using a Bayesian framework. The Se and Sp of real-time RT-PCR were estimated as 97.4% (95% confidence interval [CI] = 95.2-98.8%) and 71.7% (95% CI = 65-77.9%) respectively. The Se and Sp of histopathology were estimated as 94.6% (95% CI = 91-97.2%) and 92.3% (95% CI = 87.6-95.8%), respectively. The Se and Sp of IHC were estimated as 97.6% (95% CI = 93.9-99.8%) and 99.4% (95% CI = 96.9-100%), respectively. Decreased Sp of real-time RT-PCR was ascribed to cross-contamination of samples. Stratified analysis of the data suggested variations in test accuracy with fetuses and severely autolyzed specimens. The Sp of histopathology in fetuses (83%) was 9.3% lower than the sample population (92.3%). The Se of IHC decreased from 97.6% to 81.5% in the presence of severe autolysis. The diagnostic Se and Sp of histopathology was higher than expected, confirming the value of routine postmortem examinations and histopathology of liver specimens. Aborted fetuses, however, should be screened using a variety of tests in areas endemic for RVF, and results from severely autolyzed specimens should be interpreted with caution. The most feasible testing option for countries lacking suitably equipped laboratories seems to be routine histology in combination with IHC.

Sequential injection analysis with chemiluminescence detection for rapid monitoring of commercial Calendula officinalis extractions.

PubMed

Hughes, Rachel R; Scown, David; Lenehan, Claire E

2015-01-01

Plant extracts containing high levels of antioxidants are desirable due to their reported health benefits. Most techniques capable of determining the antioxidant activity of plant extracts are unsuitable for rapid at-line analysis as they require extensive sample preparation and/or long analysis times. Therefore, analytical techniques capable of real-time or pseudo real-time at-line monitoring of plant extractions, and determination of extraction endpoints, would be useful to manufacturers of antioxidant-rich plant extracts. To develop a reliable method for the rapid at-line extraction monitoring of antioxidants in plant extracts. Calendula officinalis extracts were prepared from dried flowers and analysed for antioxidant activity using sequential injection analysis (SIA) with chemiluminescence (CL) detection. The intensity of CL emission from the reaction of acidic potassium permanganate with antioxidants within the extract was used as the analytical signal. The SIA-CL method was applied to monitor the extraction of C. officinalis over the course of a batch extraction to determine the extraction endpoint. Results were compared with those from ultra high performance liquid chromatography (UHPLC). Pseudo real-time, at-line monitoring showed the level of antioxidants in a batch extract of Calendula officinalis plateaued after 100 min of extraction. These results correlated well with those of an offline UHPLC study. SIA-CL was found to be a suitable method for pseudo real-time monitoring of plant extractions and determination of extraction endpoints with respect to antioxidant concentrations. The method was applied at-line in the manufacturing industry. Copyright © 2015 John Wiley & Sons, Ltd.

Detection of α-thalassemia-1 Southeast Asian and Thai Type Deletions and β-thalassemia 3.5-kb Deletion by Single-tube Multiplex Real-time PCR with SYBR Green1 and High-resolution Melting Analysis

PubMed Central

Wiengkum, Thanatcha; Srithep, Sarinee; Chainoi, Isarapong; Singboottra, Panthong; Wongwiwatthananukit, Sanchai

2011-01-01

Background Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. Methods Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of α-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and β-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with α-thalassemia-1 SEA type deletion, 2 with α-thalassemia-1 Thai type deletion, and 2 with heterozygous β-thalassemia 3.5-kb gene deletion. Results HRM analysis indicated that the amplified fragments from α-thalassemia-1 SEA type deletion, α-thalassemia-1 Thai type deletion, β-thalassemia 3.5-kb gene deletion, and the wild-type β-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89℃, 85.66℃, 77.24℃, and 74.92℃, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. Conclusions Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of α- and β-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate. PMID:21779184

A field-deployable mobile molecular diagnostic system for malaria at the point of need.

PubMed

Choi, Gihoon; Song, Daniel; Shrestha, Sony; Miao, Jun; Cui, Liwang; Guan, Weihua

2016-11-01

In response to the urgent need of a field-deployable and highly sensitive malaria diagnosis, we developed a standalone, "sample-in-answer-out" molecular diagnostic system (AnyMDx) to enable quantitative molecular analysis of blood-borne malaria in low resource areas. The system consists of a durable battery-powered analyzer and a disposable microfluidic compact disc loaded with reagents ready for use. A low power thermal module and a novel fluorescence-sensing module are integrated into the analyzer for real-time monitoring of loop-mediated isothermal nucleic acid amplification (LAMP) of target parasite DNA. With 10 μL of raw blood sample, the AnyMDx system automates the nucleic acid sample preparation and subsequent LAMP and real-time detection. Under laboratory conditions with whole-blood samples spiked with cultured Plasmodium falciparum, we achieved a detection limit of ∼0.6 parasite per μL, much lower than those for the conventional microscopy and rapid diagnostic tests (∼50-100 parasites per μL). The turnaround time from sample to answer is less than 40 minutes. The AnyMDx is user-friendly requiring minimal technological training. The analyzer and the disposable reagent compact discs are cost-effective, making AnyMDx a potential tool for malaria molecular diagnosis under field settings for malaria elimination.

Applications of DART-MS for food quality and safety assurance in food supply chain.

PubMed

Guo, Tianyang; Yong, Wei; Jin, Yong; Zhang, Liya; Liu, Jiahui; Wang, Sai; Chen, Qilong; Dong, Yiyang; Su, Haijia; Tan, Tianwei

2017-03-01

Direct analysis in real time (DART) represents a new generation of ion source which is used for rapid ionization of small molecules under ambient conditions. The combination of DART and various mass spectrometers allows analyzing multiple food samples with simple or no sample treatment, or in conjunction with prevailing protocolized sample preparation methods. Abundant applications by DART-MS have been reviewed in this paper. The DART-MS strategy applied to food supply chain (FSC), including production, processing, and storage and transportation, provides a comprehensive solution to various food components, contaminants, authenticity, and traceability. Additionally, typical applications available in food analysis by other ambient ionization mass spectrometers were summarized, and fundamentals mainly including mechanisms, devices, and parameters were discussed as well. © 2015 Wiley Periodicals, Inc. Mass Spec Rev. 36:161-187, 2017. © 2015 Wiley Periodicals, Inc.

Rapid sample classification using an open port sampling interface coupled with liquid introduction atmospheric pressure ionization mass spectrometry

DOE PAGES

Van Berkel, Gary J.; Kertesz, Vilmos

2016-11-15

An “Open Access”-like mass spectrometric platform to fully utilize the simplicity of the manual open port sampling interface for rapid characterization of unprocessed samples by liquid introduction atmospheric pressure ionization mass spectrometry has been lacking. The in-house developed integrated software with a simple, small and relatively low-cost mass spectrometry system introduced here fills this void. Software was developed to operate the mass spectrometer, to collect and process mass spectrometric data files, to build a database and to classify samples using such a database. These tasks were accomplished via the vendorprovided software libraries. Sample classification based on spectral comparison utilized themore » spectral contrast angle method. As a result, using the developed software platform near real-time sample classification is exemplified using a series of commercially available blue ink rollerball pens and vegetable oils. In the case of the inks, full scan positive and negative ion ESI mass spectra were both used for database generation and sample classification. For the vegetable oils, full scan positive ion mode APCI mass spectra were recorded. The overall accuracy of the employed spectral contrast angle statistical model was 95.3% and 98% in case of the inks and oils, respectively, using leave-one-out cross-validation. In conclusion, this work illustrates that an open port sampling interface/mass spectrometer combination, with appropriate instrument control and data processing software, is a viable direct liquid extraction sampling and analysis system suitable for the non-expert user and near real-time sample classification via database matching.« less

Rapid sample classification using an open port sampling interface coupled with liquid introduction atmospheric pressure ionization mass spectrometry

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

Van Berkel, Gary J.; Kertesz, Vilmos

An “Open Access”-like mass spectrometric platform to fully utilize the simplicity of the manual open port sampling interface for rapid characterization of unprocessed samples by liquid introduction atmospheric pressure ionization mass spectrometry has been lacking. The in-house developed integrated software with a simple, small and relatively low-cost mass spectrometry system introduced here fills this void. Software was developed to operate the mass spectrometer, to collect and process mass spectrometric data files, to build a database and to classify samples using such a database. These tasks were accomplished via the vendorprovided software libraries. Sample classification based on spectral comparison utilized themore » spectral contrast angle method. As a result, using the developed software platform near real-time sample classification is exemplified using a series of commercially available blue ink rollerball pens and vegetable oils. In the case of the inks, full scan positive and negative ion ESI mass spectra were both used for database generation and sample classification. For the vegetable oils, full scan positive ion mode APCI mass spectra were recorded. The overall accuracy of the employed spectral contrast angle statistical model was 95.3% and 98% in case of the inks and oils, respectively, using leave-one-out cross-validation. In conclusion, this work illustrates that an open port sampling interface/mass spectrometer combination, with appropriate instrument control and data processing software, is a viable direct liquid extraction sampling and analysis system suitable for the non-expert user and near real-time sample classification via database matching.« less

Real time quantitative colourimetric test for methamphetamine detection using digital and mobile phone technology.

PubMed

Choodum, Aree; Parabun, Kaewalee; Klawach, Nantikan; Daeid, Niamh Nic; Kanatharana, Proespichaya; Wongniramaikul, Worawit

2014-02-01

The Simon presumptive color test was used in combination with the built-in digital camera on a mobile phone to detect methamphetamine. The real-time Red-Green-Blue (RGB) basic color data was obtained using an application installed on the mobile phone and the relationship profile between RGB intensity, including other calculated values, and the colourimetric product was investigated. A wide linear range (0.1-2.5mg mL(-1)) and a low detection limit (0.0110±0.0001-0.044±0.002mg mL(-1)) were achieved. The method also required a small sample size (20μL). The results obtained from the analysis of illicit methamphetamine tablets were comparable to values obtained from gas chromatograph-flame ionization detector (GC-FID) analysis. Method validation indicated good intra- and inter-day precision (2.27-4.49%RSD and 2.65-5.62%RSD, respectively). The results suggest that this is a powerful real-time mobile method with the potential to be applied in field tests. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Real-Time PCR for the Detection of Precise Transgene Copy Number in Wheat.

PubMed

Giancaspro, Angelica; Gadaleta, Agata; Blanco, Antonio

2017-01-01

Despite the unceasing advances in genetic transformation techniques, the success of common delivery methods still lies on the behavior of the integrated transgenes in the host genome. Stability and expression of the introduced genes are influenced by several factors such as chromosomal location, transgene copy number and interaction with the host genotype. Such factors are traditionally characterized by Southern blot analysis, which can be time-consuming, laborious, and often unable to detect the exact copy number of rearranged transgenes. Recent research in crop field suggests real-time PCR as an effective and reliable tool for the precise quantification and characterization of transgene loci. This technique overcomes most problems linked to phenotypic segregation analysis and can analyze hundreds of samples in a day, making it an efficient method for estimating a gene copy number integrated in a transgenic line. This protocol describes the use of real-time PCR for the detection of transgene copy number in durum wheat transgenic lines by means of two different chemistries (SYBR ® Green I dye and TaqMan ® probes).

Biofilm bacterial communities in urban drinking water distribution systems transporting waters with different purification strategies.

PubMed

Wu, Huiting; Zhang, Jingxu; Mi, Zilong; Xie, Shuguang; Chen, Chao; Zhang, Xiaojian

2015-02-01

Biofilm formation in drinking water distribution systems (DWDS) has many adverse consequences. Knowledge of microbial community structure of DWDS biofilm can aid in the design of an effective control strategy. However, biofilm bacterial community in real DWDS and the impact of drinking water purification strategy remain unclear. The present study investigated the composition and diversity of biofilm bacterial community in real DWDSs transporting waters with different purification strategies (conventional treatment and integrated treatment). High-throughput Illumina MiSeq sequencing analysis illustrated a large shift in the diversity and structure of biofilm bacterial community in real DWDS. Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Nitrospirae, and Cyanobacteria were the major components of biofilm bacterial community. Proteobacteria (mainly Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria) predominated in each DWDS biofilm, but the compositions of the dominant proteobacterial classes and genera and their proportions varied among biofilm samples. Drinking water purification strategy could shape DWDS biofilm bacterial community. Moreover, Pearson's correlation analysis indicated that Actinobacteria was positively correlated with the levels of total alkalinity and dissolved organic carbon in tap water, while Firmicutes had a significant positive correlation with nitrite nitrogen.

A nonlinear dynamic age-structured model of e-commerce in spain: Stability analysis of the equilibrium by delay and stochastic perturbations

NASA Astrophysics Data System (ADS)

Burgos, C.; Cortés, J.-C.; Shaikhet, L.; Villanueva, R.-J.

2018-11-01

First, we propose a deterministic age-structured epidemiological model to study the diffusion of e-commerce in Spain. Afterwards, we determine the parameters (death, birth and growth rates) of the underlying demographic model as well as the parameters (transmission of the use of e-commerce rates) of the proposed epidemiological model that best fit real data retrieved from the Spanish National Statistical Institute. Motivated by the two following facts: first the dynamics of acquiring the use of a new technology as e-commerce is mainly driven by the feedback after interacting with our peers (family, friends, mates, mass media, etc.), hence having a certain delay, and second the inherent uncertainty of sampled real data and the social complexity of the phenomena under analysis, we introduce aftereffect and stochastic perturbations in the initial deterministic model. This leads to a delayed stochastic model for e-commerce. We then investigate sufficient conditions in order to guarantee the stability in probability of the equilibrium point of the dynamic e-commerce delayed stochastic model. Our theoretical findings are numerically illustrated using real data.

An enhanced cluster analysis program with bootstrap significance testing for ecological community analysis

USGS Publications Warehouse

McKenna, J.E.

2003-01-01

The biosphere is filled with complex living patterns and important questions about biodiversity and community and ecosystem ecology are concerned with structure and function of multispecies systems that are responsible for those patterns. Cluster analysis identifies discrete groups within multivariate data and is an effective method of coping with these complexities, but often suffers from subjective identification of groups. The bootstrap testing method greatly improves objective significance determination for cluster analysis. The BOOTCLUS program makes cluster analysis that reliably identifies real patterns within a data set more accessible and easier to use than previously available programs. A variety of analysis options and rapid re-analysis provide a means to quickly evaluate several aspects of a data set. Interpretation is influenced by sampling design and a priori designation of samples into replicate groups, and ultimately relies on the researcher's knowledge of the organisms and their environment. However, the BOOTCLUS program provides reliable, objectively determined groupings of multivariate data.

Quantitative microbiological study of human carious dentine by culture and real-time PCR: association of anaerobes with histopathological changes in chronic pulpitis.

PubMed

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

2002-05-01

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

Avian-specific real-time PCR assay for authenticity control in farm animal feeds and pet foods.

PubMed

Pegels, Nicolette; González, Isabel; García, Teresa; Martín, Rosario

2014-01-01

A highly sensitive TaqMan real-time PCR assay targeting the mitochondrial 12S rRNA gene was developed for detection of an avian-specific DNA fragment (68bp) in farm animal and pet feeds. The specificity of the assay was verified against a wide representation of animal and plant species. Applicability assessment of the avian real-time PCR was conducted through representative analysis of two types of compound feeds: industrial farm animal feeds (n=60) subjected to extreme temperatures, and commercial dog and cat feeds (n=210). Results obtained demonstrated the suitability of the real-time PCR assay to detect the presence of low percentages of highly processed avian material in the feed samples analysed. Although quantification results were well reproducible under the experimental conditions tested, an accurate estimation of the target content in feeds is impossible in practice. Nevertheless, the method may be useful as an alternative tool for traceability purposes within the framework of feed control. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evaluation of Novel Broad-Range Real-Time PCR Assay for Rapid Detection of Human Pathogenic Fungi in Various Clinical Specimens▿

PubMed Central

Vollmer, Tanja; Störmer, Melanie; Kleesiek, Knut; Dreier, Jens

2008-01-01

In the present study, a novel broad-range real-time PCR was developed for the rapid detection of human pathogenic fungi. The assay targets a part of the 28S large-subunit ribosomal RNA (rDNA) gene. We investigated its application for the most important human pathogenic fungal genera, including Aspergillus, Candida, Cryptococcus, Mucor, Penicillium, Pichia, Microsporum, Trichophyton, and Scopulariopsis. Species were identified in PCR-positive reactions by direct DNA sequencing. A noncompetitive internal control was applied to prevent false-negative results due to PCR inhibition. The minimum detection limit for the PCR was determined to be one 28S rDNA copy per PCR, and the 95% detection limit was calculated to 15 copies per PCR. To assess the clinical applicability of the PCR method, intensive-care patients with artificial respiration and patients with infective endocarditis were investigated. For this purpose, 76 tracheal secretion samples and 70 heart valve tissues were analyzed in parallel by real-time PCR and cultivation. No discrepancies in results were observed between PCR analysis and cultivation methods. Furthermore, the application of the PCR method was investigated for other clinical specimens, including cervical swabs, nail and horny skin scrapings, and serum, blood, and urine samples. The combination of a broad-range real-time PCR and direct sequencing facilitates rapid screening for fungal infection in various clinical specimens. PMID:18385440

Evaluation of novel broad-range real-time PCR assay for rapid detection of human pathogenic fungi in various clinical specimens.

PubMed

Vollmer, Tanja; Störmer, Melanie; Kleesiek, Knut; Dreier, Jens

2008-06-01

In the present study, a novel broad-range real-time PCR was developed for the rapid detection of human pathogenic fungi. The assay targets a part of the 28S large-subunit ribosomal RNA (rDNA) gene. We investigated its application for the most important human pathogenic fungal genera, including Aspergillus, Candida, Cryptococcus, Mucor, Penicillium, Pichia, Microsporum, Trichophyton, and Scopulariopsis. Species were identified in PCR-positive reactions by direct DNA sequencing. A noncompetitive internal control was applied to prevent false-negative results due to PCR inhibition. The minimum detection limit for the PCR was determined to be one 28S rDNA copy per PCR, and the 95% detection limit was calculated to 15 copies per PCR. To assess the clinical applicability of the PCR method, intensive-care patients with artificial respiration and patients with infective endocarditis were investigated. For this purpose, 76 tracheal secretion samples and 70 heart valve tissues were analyzed in parallel by real-time PCR and cultivation. No discrepancies in results were observed between PCR analysis and cultivation methods. Furthermore, the application of the PCR method was investigated for other clinical specimens, including cervical swabs, nail and horny skin scrapings, and serum, blood, and urine samples. The combination of a broad-range real-time PCR and direct sequencing facilitates rapid screening for fungal infection in various clinical specimens.

Diversity in the 18S SSU rRNA V4 hyper-variable region of Theileria spp. in Cape buffalo (Syncerus caffer) and cattle from southern Africa.

PubMed

Mans, Ben J; Pienaar, Ronel; Latif, Abdalla A; Potgieter, Fred T

2011-05-01

Sequence variation within the 18S SSU rRNA V4 hyper-variable region can affect the accuracy of real-time hybridization probe-based diagnostics for the detection of Theileria spp. infections. This is relevant for assays that use non-specific primers, such as the real-time hybridization assay for T. parva (Sibeko et al. 2008). To assess the effect of sequence variation on this test, the Theileria 18S gene from 62 buffalo and 49 cattle samples was cloned and ∼1000 clones sequenced. Twenty-six genotypes were detected which included known and novel genotypes for the T. buffeli, T. mutans, T. taurotragi and T. velifera clades. A novel genotype related to T. sp. (sable) was also detected in 1 bovine sample. Theileria genotypic diversity was higher in buffalo compared to cattle. Polymorphism within the T. parva hyper-variable region was confirmed by aberrant real-time melting peaks and supported by sequencing of the S5 ribosomal gene. Analysis of the S5 gene suggests that this gene can be a marker for species differentiation. T. parva, T. sp. (buffalo) and T. sp. (bougasvlei) remain the only genotypes amplified by the primer set of the hybridization assay. Therefore, the 18S sequence diversity observed does not seem to affect the current real-time hybridization assay for T. parva.

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.

Analysing neutron scattering data using McStas virtual experiments

NASA Astrophysics Data System (ADS)

Udby, L.; Willendrup, P. K.; Knudsen, E.; Niedermayer, Ch.; Filges, U.; Christensen, N. B.; Farhi, E.; Wells, B. O.; Lefmann, K.

2011-04-01

With the intention of developing a new data analysis method using virtual experiments we have built a detailed virtual model of the cold triple-axis spectrometer RITA-II at PSI, Switzerland, using the McStas neutron ray-tracing package. The parameters characterising the virtual instrument were carefully tuned against real experiments. In the present paper we show that virtual experiments reproduce experimentally observed linewidths within 1-3% for a variety of samples. Furthermore we show that the detailed knowledge of the instrumental resolution found from virtual experiments, including sample mosaicity, can be used for quantitative estimates of linewidth broadening resulting from, e.g., finite domain sizes in single-crystal samples.

Nonuniform sampling theorems for random signals in the linear canonical transform domain

NASA Astrophysics Data System (ADS)

Shuiqing, Xu; Congmei, Jiang; Yi, Chai; Youqiang, Hu; Lei, Huang

2018-06-01

Nonuniform sampling can be encountered in various practical processes because of random events or poor timebase. The analysis and applications of the nonuniform sampling for deterministic signals related to the linear canonical transform (LCT) have been well considered and researched, but up to now no papers have been published regarding the various nonuniform sampling theorems for random signals related to the LCT. The aim of this article is to explore the nonuniform sampling and reconstruction of random signals associated with the LCT. First, some special nonuniform sampling models are briefly introduced. Second, based on these models, some reconstruction theorems for random signals from various nonuniform samples associated with the LCT have been derived. Finally, the simulation results are made to prove the accuracy of the sampling theorems. In addition, the latent real practices of the nonuniform sampling for random signals have been also discussed.

A Nonlinear Framework of Delayed Particle Smoothing Method for Vehicle Localization under Non-Gaussian Environment.

PubMed

Xiao, Zhu; Havyarimana, Vincent; Li, Tong; Wang, Dong

2016-05-13

In this paper, a novel nonlinear framework of smoothing method, non-Gaussian delayed particle smoother (nGDPS), is proposed, which enables vehicle state estimation (VSE) with high accuracy taking into account the non-Gaussianity of the measurement and process noises. Within the proposed method, the multivariate Student's t-distribution is adopted in order to compute the probability distribution function (PDF) related to the process and measurement noises, which are assumed to be non-Gaussian distributed. A computation approach based on Ensemble Kalman Filter (EnKF) is designed to cope with the mean and the covariance matrix of the proposal non-Gaussian distribution. A delayed Gibbs sampling algorithm, which incorporates smoothing of the sampled trajectories over a fixed-delay, is proposed to deal with the sample degeneracy of particles. The performance is investigated based on the real-world data, which is collected by low-cost on-board vehicle sensors. The comparison study based on the real-world experiments and the statistical analysis demonstrates that the proposed nGDPS has significant improvement on the vehicle state accuracy and outperforms the existing filtering and smoothing methods.

Identification of Microbial Profile of Koji Using Single Molecule, Real-Time Sequencing Technology.

PubMed

Hui, Wenyan; Hou, Qiangchuan; Cao, Chenxia; Xu, Haiyan; Zhen, Yi; Kwok, Lai-Yu; Sun, Tiansong; Zhang, Heping; Zhang, Wenyi

2017-05-01

Koji is a kind of Japanese traditional fermented starter that has been used for centuries. Many fermented foods are made from koji, such as sake, miso, and soy sauce. This study used the single molecule real-time sequencing technology (SMRT) to investigate the bacterial and fungal microbiota of 3 Japanese koji samples. After SMRT analysis, a total of 39121 high-quality sequences were generated, including 14354 bacterial and 24767 fungal sequence reads. The high-quality gene sequences were assigned to 5 bacterial and 2 fungal plyla, dominated by Proteobacteria and Ascomycota, respectively. At the genus level, Ochrobactrum and Wickerhamomyces were the most abundant bacterial and fungal genera, respectively. The predominant bacterial and fungal species were Ochrobactrum lupini and Wickerhamomyces anomalus, respectively. Our study profiled the microbiota composition of 3 Japanese koji samples to the species level precision. The results may be useful for further development of traditional fermented products, especially optimization of koji preparation. Meanwhile, this study has demonstrated that SMRT is a robust tool for analyzing the microbial composition in food samples. © 2017 Institute of Food Technologists®.

Fast cholesterol detection using flow injection microfluidic device with functionalized carbon nanotubes based electrochemical sensor.

PubMed

Wisitsoraat, A; Sritongkham, P; Karuwan, C; Phokharatkul, D; Maturos, T; Tuantranont, A

2010-12-15

This work reports a new cholesterol detection scheme using functionalized carbon nanotube (CNT) electrode in a polydimethylsiloxane/glass based flow injection microfluidic chip. CNTs working, silver reference and platinum counter electrode layers were fabricated on the chip by sputtering and low temperature chemical vapor deposition methods. Cholesterol oxidase prepared in polyvinyl alcohol solution was immobilized on CNTs by in-channel flow technique. Cholesterol analysis based on flow injection chronoamperometric measurement was performed in 150-μm-wide and 150-μm-deep microchannels. Fast and sensitive real-time detection was achieved with high throughput of more than 60 samples per hour and small sample volume of 15 μl. The cholesterol sensor had a linear detection range between 50 and 400 mg/dl. In addition, low cross-sensitivities toward glucose, ascorbic acid, acetaminophen and uric acid were confirmed. The proposed system is promising for clinical diagnostics of cholesterol with high speed real-time detection capability, very low sample consumption, high sensitivity, low interference and good stability. Copyright © 2010 Elsevier B.V. All rights reserved.

NASA GRC and MSFC Space-Plasma Arc Testing Procedures

NASA Technical Reports Server (NTRS)

Ferguson, Dale C.; Vayner, Boris V.; Galofaro, Joel T,; Hillard, G. Barry; Vaughn, Jason; Schneider, Todd

2005-01-01

Tests of arcing and current collection in simulated space plasma conditions have been performed at the NASA Glenn Research Center (GRC) in Cleveland, Ohio, for over 30 years and at the Marshall Space Flight Center (MSFC) in Huntsville, Alabama, for almost as long. During this period, proper test conditions for accurate and meaningful space simulation have been worked out, comparisons with actual space performance in spaceflight tests and with real operational satellites have been made, and NASA has achieved our own internal standards for test protocols. It is the purpose of this paper to communicate the test conditions, test procedures, and types of analysis used at NASA GRC and MSFC to the space environmental testing community at large, to help with international space-plasma arcing-testing standardization. To be discussed are: 1.Neutral pressures, neutral gases, and vacuum chamber sizes. 2. Electron and ion densities, plasma uniformity, sample sizes, and Debuy lengths. 3. Biasing samples versus self-generated voltages. Floating samples versus grounded. 4. Power supplies and current limits. Isolation of samples from power supplies during arcs. 5. Arc circuits. Capacitance during biased arc-threshold tests. Capacitance during sustained arcing and damage tests. Arc detection. Prevention sustained discharges during testing. 6. Real array or structure samples versus idealized samples. 7. Validity of LEO tests for GEO samples. 8. Extracting arc threshold information from arc rate versus voltage tests. 9. Snapover and current collection at positive sample bias. Glows at positive bias. Kapon (R) pyrolisis. 10. Trigger arc thresholds. Sustained arc thresholds. Paschen discharge during sustained arcing. 11. Testing for Paschen discharge threshold. Testing for dielectric breakdown thresholds. Testing for tether arcing. 12. Testing in very dense plasmas (ie thruster plumes). 13. Arc mitigation strategies. Charging mitigation strategies. Models. 14. Analysis of test results. Finally, the necessity of testing will be emphasized, not to the exclusion of modeling, but as part of a complete strategy for determining when and if arcs will occur, and preventing them from occurring in space.

NASA GRC and MSFC Space-Plasma Arc Testing Procedures

NASA Technical Reports Server (NTRS)

Ferguson, Dale C.a; Vayner, Boris V.; Galofaro, Joel T.; Hillard, G. Barry; Vaughn, Jason; Schneider, Todd

2005-01-01

Tests of arcing and current collection in simulated space plasma conditions have been performed at the NASA Glenn Research Center (GRC) in Cleveland, Ohio, for over 30 years and at the Marshall Space flight Center (MSFC) for almost as long. During this period, proper test conditions for accurate and meaningful space simulation have been worked out, comparisons with actual space performance in spaceflight tests and with real operational satellites have been made, and NASA has achieved our own internal standards for test protocols. It is the purpose of this paper to communicate the test conditions, test procedures, and types of analysis used at NASA GRC and MSFC to the space environmental testing community at large, to help with international space-plasma arcing testing standardization. To be discussed are: 1. Neutral pressures, neutral gases, and vacuum chamber sizes. 2. Electron and ion densities, plasma uniformity, sample sizes, and Debye lengths. 3. Biasing samples versus self-generated voltages. Floating samples versus grounded. 4. Power supplies and current limits. Isolation of samples from power supplies during arcs. Arc circuits. Capacitance during biased arc-threshold tests. Capacitance during sustained arcing and damage tests. Arc detection. Preventing sustained discharges during testing. 5. Real array or structure samples versus idealized samples. 6. Validity of LEO tests for GEO samples. 7. Extracting arc threshold information from arc rate versus voltage tests. 8 . Snapover and current collection at positive sample bias. Glows at positive bias. Kapton pyrolization. 9. Trigger arc thresholds. Sustained arc thresholds. Paschen discharge during sustained arcing. 10. Testing for Paschen discharge thresholds. Testing for dielectric breakdown thresholds. Testing for tether arcing. 11. Testing in very dense plasmas (ie thruster plumes). 12. Arc mitigation strategies. Charging mitigation strategies. Models. 13. Analysis of test results. Finally, the necessity of testing will be emphasized, not to the exclusion of modeling, but as part of a complete strategy for determining when and if arcs will occur, and preventing them from occurring in space.

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.

A real negative selection algorithm with evolutionary preference for anomaly detection

NASA Astrophysics Data System (ADS)

Yang, Tao; Chen, Wen; Li, Tao

2017-04-01

Traditional real negative selection algorithms (RNSAs) adopt the estimated coverage (c0) as the algorithm termination threshold, and generate detectors randomly. With increasing dimensions, the data samples could reside in the low-dimensional subspace, so that the traditional detectors cannot effectively distinguish these samples. Furthermore, in high-dimensional feature space, c0 cannot exactly reflect the detectors set coverage rate for the nonself space, and it could lead the algorithm to be terminated unexpectedly when the number of detectors is insufficient. These shortcomings make the traditional RNSAs to perform poorly in high-dimensional feature space. Based upon "evolutionary preference" theory in immunology, this paper presents a real negative selection algorithm with evolutionary preference (RNSAP). RNSAP utilizes the "unknown nonself space", "low-dimensional target subspace" and "known nonself feature" as the evolutionary preference to guide the generation of detectors, thus ensuring the detectors can cover the nonself space more effectively. Besides, RNSAP uses redundancy to replace c0 as the termination threshold, in this way RNSAP can generate adequate detectors under a proper convergence rate. The theoretical analysis and experimental result demonstrate that, compared to the classical RNSA (V-detector), RNSAP can achieve a higher detection rate, but with less detectors and computing cost.

[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.

Indirect estimation of signal-dependent noise with nonadaptive heterogeneous samples.

PubMed

Azzari, Lucio; Foi, Alessandro

2014-08-01

We consider the estimation of signal-dependent noise from a single image. Unlike conventional algorithms that build a scatterplot of local mean-variance pairs from either small or adaptively selected homogeneous data samples, our proposed approach relies on arbitrarily large patches of heterogeneous data extracted at random from the image. We demonstrate the feasibility of our approach through an extensive theoretical analysis based on mixture of Gaussian distributions. A prototype algorithm is also developed in order to validate the approach on simulated data as well as on real camera raw images.