Epidemiologic Evaluation of Measurement Data in the Presence of Detection Limits

PubMed Central

Lubin, Jay H.; Colt, Joanne S.; Camann, David; Davis, Scott; Cerhan, James R.; Severson, Richard K.; Bernstein, Leslie; Hartge, Patricia

2004-01-01

Quantitative measurements of environmental factors greatly improve the quality of epidemiologic studies but can pose challenges because of the presence of upper or lower detection limits or interfering compounds, which do not allow for precise measured values. We consider the regression of an environmental measurement (dependent variable) on several covariates (independent variables). Various strategies are commonly employed to impute values for interval-measured data, including assignment of one-half the detection limit to nondetected values or of “fill-in” values randomly selected from an appropriate distribution. On the basis of a limited simulation study, we found that the former approach can be biased unless the percentage of measurements below detection limits is small (5–10%). The fill-in approach generally produces unbiased parameter estimates but may produce biased variance estimates and thereby distort inference when 30% or more of the data are below detection limits. Truncated data methods (e.g., Tobit regression) and multiple imputation offer two unbiased approaches for analyzing measurement data with detection limits. If interest resides solely on regression parameters, then Tobit regression can be used. If individualized values for measurements below detection limits are needed for additional analysis, such as relative risk regression or graphical display, then multiple imputation produces unbiased estimates and nominal confidence intervals unless the proportion of missing data is extreme. We illustrate various approaches using measurements of pesticide residues in carpet dust in control subjects from a case–control study of non-Hodgkin lymphoma. PMID:15579415

A probe-based quantitative PCR assay for detecting Tetracapsuloides bryosalmonae in fish tissue and environmental DNA water samples

USGS Publications Warehouse

Hutchins, Patrick; Sepulveda, Adam; Martin, Renee; Hopper, Lacey

2017-01-01

A probe-based quantitative real-time PCR assay was developed to detect Tetracapsuloides bryosalmonae, which causes proliferative kidney disease in salmonid fish, in kidney tissue and environmental DNA (eDNA) water samples. The limits of detection and quantification were 7 and 100 DNA copies for calibration standards and T. bryosalmonae was reliably detected down to 100 copies in tissue and eDNA samples. The assay presented here is a highly sensitive and quantitative tool for detecting T. bryosalmonae with potential applications for tissue diagnostics and environmental detection.

Supramolecular assembly affording a ratiometric two-photon fluorescent nanoprobe for quantitative detection and bioimaging.

PubMed

Wang, Peng; Zhang, Cheng; Liu, Hong-Wen; Xiong, Mengyi; Yin, Sheng-Yan; Yang, Yue; Hu, Xiao-Xiao; Yin, Xia; Zhang, Xiao-Bing; Tan, Weihong

2017-12-01

Fluorescence quantitative analyses for vital biomolecules are in great demand in biomedical science owing to their unique detection advantages with rapid, sensitive, non-damaging and specific identification. However, available fluorescence strategies for quantitative detection are usually hard to design and achieve. Inspired by supramolecular chemistry, a two-photon-excited fluorescent supramolecular nanoplatform ( TPSNP ) was designed for quantitative analysis with three parts: host molecules (β-CD polymers), a guest fluorophore of sensing probes (Np-Ad) and a guest internal reference (NpRh-Ad). In this strategy, the TPSNP possesses the merits of (i) improved water-solubility and biocompatibility; (ii) increased tissue penetration depth for bioimaging by two-photon excitation; (iii) quantitative and tunable assembly of functional guest molecules to obtain optimized detection conditions; (iv) a common approach to avoid the limitation of complicated design by adjustment of sensing probes; and (v) accurate quantitative analysis by virtue of reference molecules. As a proof-of-concept, we utilized the two-photon fluorescent probe NHS-Ad-based TPSNP-1 to realize accurate quantitative analysis of hydrogen sulfide (H 2 S), with high sensitivity and good selectivity in live cells, deep tissues and ex vivo -dissected organs, suggesting that the TPSNP is an ideal quantitative indicator for clinical samples. What's more, TPSNP will pave the way for designing and preparing advanced supramolecular sensors for biosensing and biomedicine.

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

PubMed

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

2016-07-01

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

Accounting for Limited Detection Efficiency and Localization Precision in Cluster Analysis in Single Molecule Localization Microscopy

PubMed Central

Shivanandan, Arun; Unnikrishnan, Jayakrishnan; Radenovic, Aleksandra

2015-01-01

Single Molecule Localization Microscopy techniques like PhotoActivated Localization Microscopy, with their sub-diffraction limit spatial resolution, have been popularly used to characterize the spatial organization of membrane proteins, by means of quantitative cluster analysis. However, such quantitative studies remain challenged by the techniques’ inherent sources of errors such as a limited detection efficiency of less than 60%, due to incomplete photo-conversion, and a limited localization precision in the range of 10 – 30nm, varying across the detected molecules, mainly depending on the number of photons collected from each. We provide analytical methods to estimate the effect of these errors in cluster analysis and to correct for them. These methods, based on the Ripley’s L(r) – r or Pair Correlation Function popularly used by the community, can facilitate potentially breakthrough results in quantitative biology by providing a more accurate and precise quantification of protein spatial organization. PMID:25794150

Hallmarks in prostate cancer imaging with Ga68-PSMA-11-PET/CT with reference to detection limits and quantitative properties.

PubMed

Sanchez-Crespo, Alejandro; Jussing, Emma; Björklund, Ann-Charlotte; Pokrovskaja Tamm, Katja

2018-04-04

Gallium-68-labeled prostate-specific antigen positron emission tomography/computed tomography imaging (Ga68-PSMA-11-PET/CT) has emerged as a potential gold standard for prostate cancer (PCa) diagnosis. However, the imaging limitations of this technique at the early state of PCa recurrence/metastatic spread are still not well characterized. The aim of this study was to determine the quantitative properties and the fundamental imaging limits of Ga68-PSMA-11-PET/CT in localizing small PCa cell deposits. The human PCa LNCaP cells (PSMA expressing) were grown and collected as single cell suspension or as 3D-spheroids at different cell numbers and incubated with Ga68-PSMA-11. Thereafter, human HCT116 cells (PSMA negative) were added to a total cell number of 2 × 10 5 cells per tube. The tubes were then pelleted and the supernatant aspirated. A whole-body PET/CT scanner with a clinical routine protocol was used for imaging the pellets inside of a cylindrical water phantom with increasing amounts of background activity. The actual activity bound to the cells was also measured in an automatic gamma counter. Imaging detection limits and activity recovery coefficients as a function of LNCaP cell number were obtained. The effect of Ga68-PSMA-11 mass concentration on cell binding was also investigated in samples of LnCaP cells incubated with increasing concentrations of radioligand. A total of 1 × 10 4 LNCaP cells mixed in a pellet of 2 × 10 5 cells were required to reach a 50% detection probability with Ga68-PSMA-11-PET/CT without background. With a background level of 1 kBq/ml, between 4 × 10 5 and 1 × 10 6 cells are required. The radioligand equilibrium dissociation constant was 27.05 nM, indicating high binding affinity. Hence, the specific activity of the radioligand has a profound effect on image quantification. Ga68-PSMA-11-PET detects a small number of LNCaP cells even when they are mixed in a population of non-PSMA expressing cells and in the

Single-Plex Quantitative Assays for the Detection and Quantification of Most Pneumococcal Serotypes

PubMed Central

Chochua, Sopio; Satzke, Catherine; Dunne, Eileen M.; Mulholland, Kim; Klugman, Keith P.

2015-01-01

Streptococcus pneumoniae globally kills more children than any other infectious disease every year. A prerequisite for pneumococcal disease and transmission is colonization of the nasopharynx. While the introduction of pneumococcal conjugate vaccines has reduced the burden of pneumococcal disease, understanding the impact of vaccination on nasopharyngeal colonization has been hampered by the lack of sensitive quantitative methods for the detection of >90 known S. pneumoniae serotypes. In this work, we developed 27 new quantitative (q)PCR reactions and optimized 26 for a total of 53 qPCR reactions targeting pneumococcal serotypes or serogroups, including all vaccine types. Reactions proved to be target-specific with a limit of detection of 2 genome equivalents per reaction. Given the number of probes required for these assays and their unknown shelf-life, the stability of cryopreserved reagents was evaluated. Our studies demonstrate that two-year cryopreserved probes had similar limit of detection as freshly-diluted probes. Moreover, efficiency and limit of detection of 1-month cryopreserved, ready-to-use, qPCR reaction mixtures were similar to those of freshly prepared mixtures. Using these reactions, our proof-of-concept studies utilizing nasopharyngeal samples (N=30) collected from young children detected samples containing ≥2 serotypes/serogroups. Samples colonized by multiple serotypes/serogroups always had a serotype that contributes at least 50% of the pneumococcal load. In addition, a molecular approach called S6-q(PCR)2 was developed and proven to individually detect and quantify epidemiologically-important serogroup 6 strains including 6A, 6B, 6C and 6D. This technology will be useful for epidemiological studies, diagnostic platforms and to study the pneumobiome. PMID:25798884

Targeted Analyte Detection by Standard Addition Improves Detection Limits in MALDI Mass Spectrometry

PubMed Central

Eshghi, Shadi Toghi; Li, Xingde; Zhang, Hui

2014-01-01

Matrix-assisted laser desorption/ionization has proven an effective tool for fast and accurate determination of many molecules. However, the detector sensitivity and chemical noise compromise the detection of many invaluable low-abundance molecules from biological and clinical samples. To challenge this limitation, we developed a targeted analyte detection (TAD) technique. In TAD, the target analyte is selectively elevated by spiking a known amount of that analyte into the sample, thereby raising its concentration above the noise level, where we take advantage of the improved sensitivity to detect the presence of the endogenous analyte in the sample. We assessed TAD on three peptides in simple and complex background solutions with various exogenous analyte concentrations in two MALDI matrices. TAD successfully improved the limit of detection (LOD) of target analytes when the target peptides were added to the sample in a concentration close to optimum concentration. The optimum exogenous concentration was estimated through a quantitative method to be approximately equal to the original LOD for each target. Also, we showed that TAD could achieve LOD improvements on an average of 3-fold in a simple and 2-fold in a complex sample. TAD provides a straightforward assay to improve the LOD of generic target analytes without the need for costly hardware modifications. PMID:22877355

Apricot DNA as an indicator for persipan: detection and quantitation in marzipan using ligation-dependent probe amplification.

PubMed

Luber, Florian; Demmel, Anja; Hosken, Anne; Busch, Ulrich; Engel, Karl-Heinz

2012-06-13

The confectionery ingredient marzipan is exclusively prepared from almond kernels and sugar. The potential use of apricot kernels, so-called persipan, is an important issue for the quality assessment of marzipan. Therefore, a ligation-dependent probe amplification (LPA) assay was developed that enables a specific and sensitive detection of apricot DNA, as an indicator for the presence of persipan. The limit of detection was determined to be 0.1% persipan in marzipan. The suitability of the method was confirmed by the analysis of 20 commercially available food samples. The integration of a Prunus -specific probe in the LPA assay as a reference allowed for the relative quantitation of persipan in marzipan. The limit of quantitation was determined to be 0.5% persipan in marzipan. The analysis of two self-prepared mixtures of marzipan and persipan demonstrated the applicability of the quantitation method at concentration levels of practical relevance for quality control.

Culture-free, highly sensitive, quantitative detection of bacteria from minimally processed samples using fluorescence imaging by smartphone.

PubMed

Shrivastava, Sajal; Lee, Won-Il; Lee, Nae-Eung

2018-06-30

A critical unmet need in the diagnosis of bacterial infections, which remain a major cause of human morbidity and mortality, is the detection of scarce bacterial pathogens in a variety of samples in a rapid and quantitative manner. Herein, we demonstrate smartphone-based detection of Staphylococcus aureus in a culture-free, rapid, quantitative manner from minimally processed liquid samples using aptamer-functionalized fluorescent magnetic nanoparticles. The tagged S. aureus cells were magnetically captured in a detection cassette, and then fluorescence was imaged using a smartphone camera with a light-emitting diode as the excitation source. Our results showed quantitative detection capability with a minimum detectable concentration as low as 10 cfu/ml by counting individual bacteria cells, efficiently capturing S. aureus cells directly from a peanut milk sample within 10 min. When the selectivity of detection was investigated using samples spiked with other pathogenic bacteria, no significant non-specific detection occurred. Furthermore, strains of S. aureus from various origins showed comparable results, ensuring that the approach can be widely adopted. Therefore, the quantitative fluorescence imaging platform on a smartphone could allow on-site detection of bacteria, providing great potential assistance during major infectious disease outbreaks in remote and resource-limited settings. Copyright © 2018 Elsevier B.V. All rights reserved.

Optimization of Evans blue quantitation in limited rat tissue samples

PubMed Central

Wang, Hwai-Lee; Lai, Ted Weita

2014-01-01

Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting. PMID:25300427

Optimization of Evans blue quantitation in limited rat tissue samples

NASA Astrophysics Data System (ADS)

Wang, Hwai-Lee; Lai, Ted Weita

2014-10-01

Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader. First, ethanol ensured a consistent optic path length in each well and substantially enhanced the sensitivity of EBD fluorescence spectroscopy. Second, trichloroacetic acid (TCA) removed false-positive EBD measurements as a result of biological solutes and partially extracted EBD into the supernatant. Moreover, a 1:2 volume ratio of 50% TCA ([TCA final] = 33.3%) optimally extracted EBD from the rat plasma protein-EBD complex in vitro and in vivo, and 1:2 and 1:3 weight-volume ratios of 50% TCA optimally extracted extravasated EBD from the rat brain and liver, respectively, in vivo. This procedure is particularly useful in the detection of EBD extravasation into the BBB-intact brain, but it can also be applied to detect dye extravasation into tissues where vascular permeability is less limiting.

Comparative analysis of techniques for detection of quiescent Botrytis cinerea in grapes by quantitative PCR

USDA-ARS?s Scientific Manuscript database

Quantitative PCR (qPCR) can be used to detect and monitor pathogen colonization, but early attempts to apply the technology to quiescent Botrytis cinerea infections of grape berries identified some specific limitations. In this study, four DNA extraction methods, two tissue-grinding methods, two gra...

Comparison of detection limits in environmental analysis--is it possible? An approach on quality assurance in the lower working range by verification.

PubMed

Geiss, S; Einax, J W

2001-07-01

Detection limit, reporting limit and limit of quantitation are analytical parameters which describe the power of analytical methods. These parameters are used for internal quality assurance and externally for competing, especially in the case of trace analysis in environmental compartments. The wide variety of possibilities for computing or obtaining these measures in literature and in legislative rules makes any comparison difficult. Additionally, a host of terms have been used within the analytical community to describe detection and quantitation capabilities. Without trying to create an order for the variety of terms, this paper is aimed at providing a practical proposal for answering the main questions for the analysts concerning quality measures above. These main questions and related parameters were explained and graphically demonstrated. Estimation and verification of these parameters are the two steps to get real measures. A rule for a practical verification is given in a table, where the analyst can read out what to measure, what to estimate and which criteria have to be fulfilled. In this manner verified parameters detection limit, reporting limit and limit of quantitation now are comparable and the analyst himself is responsible to the unambiguity and reliability of these measures.

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

NASA Astrophysics Data System (ADS)

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

2015-11-01

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

Quantitative multiplex detection of pathogen biomarkers

DOEpatents

Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I.; Martinez, Jennifer; Grace, Wynne K.

2016-02-09

The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

Quantitative multiplex detection of pathogen biomarkers

DOEpatents

Mukundan, Harshini; Xie, Hongzhi; Swanson, Basil I; Martinez, Jennifer; Grace, Wynne K

2014-10-14

The present invention addresses the simultaneous detection and quantitative measurement of multiple biomolecules, e.g., pathogen biomarkers through either a sandwich assay approach or a lipid insertion approach. The invention can further employ a multichannel, structure with multi-sensor elements per channel.

Facile and quantitative electrochemical detection of yeast cell apoptosis

NASA Astrophysics Data System (ADS)

Yue, Qiulin; Xiong, Shiquan; Cai, Dongqing; Wu, Zhengyan; Zhang, Xin

2014-03-01

An electrochemical method based on square wave anodic stripping voltammetry (SWASV) was developed to detect the apoptosis of yeast cells conveniently and quantitatively through the high affinity between Cu2+ and phosphatidylserine (PS) translocated from the inner to the outer plasma membrane of the apoptotic cells. The combination of negatively charged PS and Cu2+ could decrease the electrochemical response of Cu2+ on the electrode. The results showed that the apoptotic rates of cells could be detected quantitatively through the variations of peak currents of Cu2+ by SWASV, and agreed well with those obtained through traditional flow cytometry detection. This work thus may provide a novel, simple, immediate and accurate detection method for cell apoptosis.

Reproducibility and quantitation of amplicon sequencing-based detection

PubMed Central

Zhou, Jizhong; Wu, Liyou; Deng, Ye; Zhi, Xiaoyang; Jiang, Yi-Huei; Tu, Qichao; Xie, Jianping; Van Nostrand, Joy D; He, Zhili; Yang, Yunfeng

2011-01-01

To determine the reproducibility and quantitation of the amplicon sequencing-based detection approach for analyzing microbial community structure, a total of 24 microbial communities from a long-term global change experimental site were examined. Genomic DNA obtained from each community was used to amplify 16S rRNA genes with two or three barcode tags as technical replicates in the presence of a small quantity (0.1% wt/wt) of genomic DNA from Shewanella oneidensis MR-1 as the control. The technical reproducibility of the amplicon sequencing-based detection approach is quite low, with an average operational taxonomic unit (OTU) overlap of 17.2%±2.3% between two technical replicates, and 8.2%±2.3% among three technical replicates, which is most likely due to problems associated with random sampling processes. Such variations in technical replicates could have substantial effects on estimating β-diversity but less on α-diversity. A high variation was also observed in the control across different samples (for example, 66.7-fold for the forward primer), suggesting that the amplicon sequencing-based detection approach could not be quantitative. In addition, various strategies were examined to improve the comparability of amplicon sequencing data, such as increasing biological replicates, and removing singleton sequences and less-representative OTUs across biological replicates. Finally, as expected, various statistical analyses with preprocessed experimental data revealed clear differences in the composition and structure of microbial communities between warming and non-warming, or between clipping and non-clipping. Taken together, these results suggest that amplicon sequencing-based detection is useful in analyzing microbial community structure even though it is not reproducible and quantitative. However, great caution should be taken in experimental design and data interpretation when the amplicon sequencing-based detection approach is used for quantitative

Quantitative Detection of Cracks in Steel Using Eddy Current Pulsed Thermography.

PubMed

Shi, Zhanqun; Xu, Xiaoyu; Ma, Jiaojiao; Zhen, Dong; Zhang, Hao

2018-04-02

Small cracks are common defects in steel and often lead to catastrophic accidents in industrial applications. Various nondestructive testing methods have been investigated for crack detection; however, most current methods focus on qualitative crack identification and image processing. In this study, eddy current pulsed thermography (ECPT) was applied for quantitative crack detection based on derivative analysis of temperature variation. The effects of the incentive parameters on the temperature variation were analyzed in the simulation study. The crack profile and position are identified in the thermal image based on the Canny edge detection algorithm. Then, one or more trajectories are determined through the crack profile in order to determine the crack boundary through its temperature distribution. The slope curve along the trajectory is obtained. Finally, quantitative analysis of the crack sizes was performed by analyzing the features of the slope curves. The experimental verification showed that the crack sizes could be quantitatively detected with errors of less than 1%. Therefore, the proposed ECPT method was demonstrated to be a feasible and effective nondestructive approach for quantitative crack detection.

Design and synthesis of target-responsive aptamer-cross-linked hydrogel for visual quantitative detection of ochratoxin A.

PubMed

Liu, Rudi; Huang, Yishun; Ma, Yanli; Jia, Shasha; Gao, Mingxuan; Li, Jiuxing; Zhang, Huimin; Xu, Dunming; Wu, Min; Chen, Yan; Zhu, Zhi; Yang, Chaoyong

2015-04-01

A target-responsive aptamer-cross-linked hydrogel was designed and synthesized for portable and visual quantitative detection of the toxin Ochratoxin A (OTA), which occurs in food and beverages. The hydrogel network forms by hybridization between one designed DNA strand containing the OTA aptamer and two complementary DNA strands grafting on linear polyacrylamide chains. Upon the introduction of OTA, the aptamer binds with OTA, leading to the dissociation of the hydrogel, followed by release of the preloaded gold nanoparticles (AuNPs), which can be observed by the naked eye. To enable sensitive visual and quantitative detection, we encapsulated Au@Pt core-shell nanoparticles (Au@PtNPs) in the hydrogel to generate quantitative readout in a volumetric bar-chart chip (V-Chip). In the V-Chip, Au@PtNPs catalyzes the oxidation of H2O2 to generate O2, which induces movement of an ink bar to a concentration-dependent distance for visual quantitative readout. Furthermore, to improve the detection limit in complex real samples, we introduced an immunoaffinity column (IAC) of OTA to enrich OTA from beer. After the enrichment, as low as 1.27 nM (0.51 ppb) OTA can be detected by the V-Chip, which satisfies the test requirement (2.0 ppb) by the European Commission. The integration of a target-responsive hydrogel with portable enrichment by IAC, as well as signal amplification and quantitative readout by a simple microfluidic device, offers a new method for portable detection of food safety hazard toxin OTA.

Quantitative detection method for Roundup Ready soybean in food using duplex real-time PCR MGB chemistry.

PubMed

Samson, Maria Cristina; Gullì, Mariolina; Marmiroli, Nelson

2010-07-01

Methodologies that enable the detection of genetically modified organisms (GMOs) (authorized and non-authorized) in food and feed strongly influence the potential for adequate updating and implementation of legislation together with labeling requirements. Quantitative polymerase chain reaction (qPCR) systems were designed to boost the sensitivity and specificity on the identification of GMOs in highly degraded DNA samples; however, such testing will become economically difficult to cope with due to increasing numbers of approved genetically modified (GM) lines. Multiplexing approaches are therefore in development to provide cost-efficient solution. Construct-specific primers and probe were developed for quantitative analysis of Roundup Ready soybean (RRS) event glyphosate-tolerant soybean (GTS) 40-3-2. The lectin gene (Le1) was used as a reference gene, and its specificity was verified. RRS- and Le1-specific quantitative real-time PCR (qRTPCR) were optimized in a duplex platform that has been validated with respect to limit of detection (LOD) and limit of quantification (LOQ), as well as accuracy. The analysis of model processed food samples showed that the degradation of DNA has no adverse or little effects on the performance of quantification assay. In this study, a duplex qRTPCR using TaqMan minor groove binder-non-fluorescent quencher (MGB-NFQ) chemistry was developed for specific detection and quantification of RRS event GTS 40-3-2 that can be used for practical monitoring in processed food products.

Quantitative analysis of sitagliptin using the (19)F-NMR method: a universal technique for fluorinated compound detection.

PubMed

Zhang, Fen-Fen; Jiang, Meng-Hong; Sun, Lin-Lin; Zheng, Feng; Dong, Lei; Shah, Vishva; Shen, Wen-Bin; Ding, Ya

2015-01-07

To expand the application scope of nuclear magnetic resonance (NMR) technology in quantitative analysis of pharmaceutical ingredients, (19)F nuclear magnetic resonance ((19)F-NMR) spectroscopy has been employed as a simple, rapid, and reproducible approach for the detection of a fluorine-containing model drug, sitagliptin phosphate monohydrate (STG). ciprofloxacin (Cipro) has been used as the internal standard (IS). Influential factors, including the relaxation delay time (d1) and pulse angle, impacting the accuracy and precision of spectral data are systematically optimized. Method validation has been carried out in terms of precision and intermediate precision, linearity, limit of detection (LOD) and limit of quantification (LOQ), robustness, and stability. To validate the reliability and feasibility of the (19)F-NMR technology in quantitative analysis of pharmaceutical analytes, the assay result has been compared with that of (1)H-NMR. The statistical F-test and student t-test at 95% confidence level indicate that there is no significant difference between these two methods. Due to the advantages of (19)F-NMR, such as higher resolution and suitability for biological samples, it can be used as a universal technology for the quantitative analysis of other fluorine-containing pharmaceuticals and analytes.

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

PubMed

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

2015-12-14

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

Quantitative multiplex detection of biomarkers on a waveguide-based biosensor using quantum dots

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

Xie, Hongzhi; Mukundan, Harshini; Martinez, Jennifer S

2009-01-01

The quantitative, simultaneous detection of multiple biomarkers with high sensitivity and specificity is critical for biomedical diagnostics, drug discovery and biomarker characterization [Wilson 2006, Tok 2006, Straub 2005, Joos 2002, Jani 2000]. Detection systems relying on optical signal transduction are, in general, advantageous because they are fast, portable, inexpensive, sensitive, and have the potential for multiplex detection of analytes of interest. However, conventional immunoassays for the detection of biomarkers, such as the Enzyme Linked Immunosorbant Assays (ELISAs) are semi-quantitative, time consuming and insensitive. ELISA assays are also limited by high non-specific binding, especially when used with complex biological samples suchmore » as serum and urine (REF). Organic fluorophores that are commonly used in such applications lack photostability and possess a narrow Stoke's shift that makes simultaneous detection of multiple fluorophores with a single excitation source difficult, thereby restricting their use in multiplex assays. The above limitations with traditional assay platforms have resulted in the increased use of nanotechnology-based tools and techniques in the fields of medical imaging [ref], targeted drug delivery [Caruthers 2007, Liu 2007], and sensing [ref]. One such area of increasing interest is the use of semiconductor quantum dots (QDs) for biomedical research and diagnostics [Gao and Cui 2004, Voura 2004, Michalet 2005, Chan 2002, Jaiswal 2004, Gao 2005, Medintz 2005, So 2006 2006, Wu 2003]. Compared to organic dyes, QDs provide several advantages for use in immunoassay platforms, including broad absorption bands with high extinction coefficients, narrow and symmetric emission bands with high quantum yields, high photostablility, and a large Stokes shift [Michalet 2005, Gu 2002]. These features prompted the use of QDs as probes in biodetection [Michalet 2005, Medintz 2005]. For example, Jaiswal et al. reported long term

Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

PubMed Central

2012-01-01

Background The accurate quantification of antigens at low concentrations over a wide dynamic range is needed for identifying biomarkers associated with disease and detecting protein interactions in high-throughput microarrays used in proteomics. Here we report the development of an ultrasensitive quantitative assay format called immunoliposome polymerase chain reaction (ILPCR) that fulfills these requirements. This method uses a liposome, with reporter DNA encapsulated inside and biotin-labeled polyethylene glycol (PEG) phospholipid conjugates incorporated into the outer surface of the liposome, as a detection reagent. The antigenic target is immobilized in the well of a microplate by a capture antibody and the liposome detection reagent is then coupled to a biotin-labeled second antibody through a NeutrAvidin bridge. The liposome is ruptured to release the reporter DNA, which serves as a surrogate to quantify the protein target using real-time PCR. Results A liposome detection reagent was prepared, which consisted of a population of liposomes ~120 nm in diameter with each liposome possessing ~800 accessible biotin receptors and ~220 encapsulated reporters. This liposome detection reagent was used in an assay to quantify the concentration of carcinoembryonic antigen (CEA) in human serum. This ILPCR assay exhibited a linear dose–response curve from 10-10 M to 10-16 M CEA. Within this range the assay coefficient of variance was <6 % for repeatability and <2 % for reproducibility. The assay detection limit was 13 fg/mL, which is 1,500-times more sensitive than current clinical assays for CEA. An ILPCR assay to quantify HIV-1 p24 core protein in buffer was also developed. Conclusions The ILPCR assay has several advantages over other immuno-PCR methods. The reporter DNA and biotin-labeled PEG phospholipids spontaneously incorporate into the liposomes as they form, simplifying preparation of the detection reagent. Encapsulation of the reporter inside the

Limits of detection and decision. Part 4

NASA Astrophysics Data System (ADS)

Voigtman, E.

2008-02-01

Probability density functions (PDFs) have been derived for a number of commonly used limit of detection definitions, including several variants of the Relative Standard Deviation of the Background-Background Equivalent Concentration (RSDB-BEC) method, for a simple linear chemical measurement system (CMS) having homoscedastic, Gaussian measurement noise and using ordinary least squares (OLS) processing. All of these detection limit definitions serve as both decision and detection limits, thereby implicitly resulting in 50% rates of Type 2 errors. It has been demonstrated that these are closely related to Currie decision limits, if the coverage factor, k, is properly defined, and that all of the PDFs are scaled reciprocals of noncentral t variates. All of the detection limits have well-defined upper and lower limits, thereby resulting in finite moments and confidence limits, and the problem of estimating the noncentrality parameter has been addressed. As in Parts 1-3, extensive Monte Carlo simulations were performed and all the simulation results were found to be in excellent agreement with the derived theoretical expressions. Specific recommendations for harmonization of detection limit methodology have also been made.

Targeted analyte detection by standard addition improves detection limits in matrix-assisted laser desorption/ionization mass spectrometry.

PubMed

Toghi Eshghi, Shadi; Li, Xingde; Zhang, Hui

2012-09-18

Matrix-assisted laser desorption/ionization (MALDI) has proven an effective tool for fast and accurate determination of many molecules. However, the detector sensitivity and chemical noise compromise the detection of many invaluable low-abundance molecules from biological and clinical samples. To challenge this limitation, we developed a targeted analyte detection (TAD) technique. In TAD, the target analyte is selectively elevated by spiking a known amount of that analyte into the sample, thereby raising its concentration above the noise level, where we take advantage of the improved sensitivity to detect the presence of the endogenous analyte in the sample. We assessed TAD on three peptides in simple and complex background solutions with various exogenous analyte concentrations in two MALDI matrices. TAD successfully improved the limit of detection (LOD) of target analytes when the target peptides were added to the sample in a concentration close to optimum concentration. The optimum exogenous concentration was estimated through a quantitative method to be approximately equal to the original LOD for each target. Also, we showed that TAD could achieve LOD improvements on an average of 3-fold in a simple and 2-fold in a complex sample. TAD provides a straightforward assay to improve the LOD of generic target analytes without the need for costly hardware modifications.

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

USDA-ARS?s Scientific Manuscript database

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

Use of a capillary electrophoresis instrument with laser-induced fluorescence detection for DNA quantitation. Comparison of YO-PRO-1 and PicoGreen assays.

PubMed

Guillo, Christelle; Ferrance, Jerome P; Landers, James P

2006-04-28

Highly selective and sensitive assays are required for detection and quantitation of the small masses of DNA typically encountered in clinical and forensic settings. High detection sensitivity is achieved using fluorescent labeling dyes and detection techniques such as spectrofluorometers, microplate readers and cytometers. This work describes the use of a laser-induced fluorescence (LIF) detector in conjunction with a commercial capillary electrophoresis instrument for DNA quantitation. PicoGreen and YO-PRO-1, two fluorescent DNA labeling dyes, were used to assess the potential of the system for routine DNA analysis. Linearity, reproducibility, sensitivity, limits of detection and quantitation, and sample stability were examined for the two assays. The LIF detector response was found to be linear (R2 > 0.999) and reproducible (RSD < 9%) in both cases. The PicoGreen assay displayed lower limits of detection and quantitation (20 pg and 60 pg, respectively) than the YO-PRO-1 assay (60 pg and 260 pg, respectively). Although a small variation in fluorescence was observed for the DNA/dye complexes over time, quantitation was not significantly affected and the solutions were found to be relatively stable for 80 min. The advantages of the technique include a 4- to 40-fold reduction in the volume of sample required compared to traditional assays, a 2- to 20-fold reduction in the volume of reagents consumed, fast and automated analysis, and low cost (no specific instrumentation required).

Quantitative and multiplexed detection for blood typing based on quantum dot-magnetic bead assay.

PubMed

Xu, Ting; Zhang, Qiang; Fan, Ya-Han; Li, Ru-Qing; Lu, Hua; Zhao, Shu-Ming; Jiang, Tian-Lun

2017-01-01

Accurate and reliable blood grouping is essential for safe blood transfusion. However, conventional methods are qualitative and use only single-antigen detection. We overcame these limitations by developing a simple, quantitative, and multiplexed detection method for blood grouping using quantum dots (QDs) and magnetic beads. In the QD fluorescence assay (QFA), blood group A and B antigens were quantified using QD labeling and magnetic beads, and the blood groups were identified according to the R value (the value was calculated with the fluorescence intensity from dual QD labeling) of A and B antigens. The optimized performance of QFA was established by blood typing 791 clinical samples. Quantitative and multiplexed detection for blood group antigens can be completed within 35 min with more than 10 5 red blood cells. When conditions are optimized, the assay performance is satisfactory for weak samples. The coefficients of variation between and within days were less than 10% and the reproducibility was good. The ABO blood groups of 791 clinical samples were identified by QFA, and the accuracy obtained was 100% compared with the tube test. Receiver-operating characteristic curves revealed that the QFA has high sensitivity and specificity toward clinical samples, and the cutoff points of the R value of A and B antigens were 1.483 and 1.576, respectively. In this study, we reported a novel quantitative and multiplexed method for the identification of ABO blood groups and presented an effective alternative for quantitative blood typing. This method can be used as an effective tool to improve blood typing and further guarantee clinical transfusion safety.

Quantitative PCR detection of Batrachochytrium dendrobatidis DNA from sediments and water

USGS Publications Warehouse

Kirshtein, Julie D.; Anderson, Chauncey W.; Wood, J.S.; Longcore, Joyce E.; Voytek, Mary A.

2007-01-01

The fungal pathogen Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a disease implicated in amphibian declines on 5 continents. Polymerase chain reaction (PCR) primer sets exist with which amphibians can be tested for this disease, and advances in sampling techniques allow non-invasive testing of animals. We developed filtering and PCR based quantitative methods by modifying existing PCR assays to detect Bd DNA in water and sediments, without the need for testing amphibians; we tested the methods at 4 field sites. The SYBR based assay using Boyle primers (SYBR/Boyle assay) and the Taqman based assay using Wood primers performed similarly with samples generated in the laboratory (Bd spiked filters), but the SYBR/Boyle assay detected Bd DNA in more field samples. We detected Bd DNA in water from 3 of 4 sites tested, including one pond historically negative for chytridiomycosis. Zoospore equivalents in sampled water ranged from 19 to 454 l-1 (nominal detection limit is 10 DNA copies, or about 0.06 zoospore). We did not detect DNA of Bd from sediments collected at any sites. Our filtering and amplification methods provide a new tool to investigate critical aspects of Bd in the environment. ?? Inter-Research 2007.

Quantitative confirmation of diffusion-limited oxidation theories

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

Gillen, K.T.; Clough, R.L.

1990-01-01

Diffusion-limited (heterogeneous) oxidation effects are often important for studies of polymer degradation. Such effects are common in polymers subjected to ionizing radiation at relatively high dose rate. To better understand the underlying oxidation processes and to aid in the planning of accelerated aging studies, it would be desirable to be able to monitor and quantitatively understand these effects. In this paper, we briefly review a theoretical diffusion approach which derives model profiles for oxygen surrounded sheets of material by combining oxygen permeation rates with kinetically based oxygen consumption expressions. The theory leads to a simple governing expression involving the oxygenmore » consumption and permeation rates together with two model parameters {alpha} and {beta}. To test the theory, gamma-initiated oxidation of a sheet of commercially formulated EPDM rubber was performed under conditions which led to diffusion-limited oxidation. Profile shapes from the theoretical treatments are shown to accurately fit experimentally derived oxidation profiles. In addition, direct measurements on the same EPDM material of the oxygen consumption and permeation rates, together with values of {alpha} and {beta} derived from the fitting procedure, allow us to quantitatively confirm for the first time the governing theoretical relationship. 17 refs., 3 figs.« less

Rapid Detection of Ceratocystis platani Inoculum by Quantitative Real-Time PCR Assay

PubMed Central

Ghelardini, Luisa; Belbahri, Lassaâd; Quartier, Marion; Santini, Alberto

2013-01-01

Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread. The goal of this study was to design a rapid, real-time quantitative PCR assay to detect a C. platani airborne inoculum. Airborne inoculum traps (AITs) were placed in an urban setting in the city of Florence, Italy, where the disease was present. Primers and TaqMan minor groove binder (MGB) probes were designed to target cerato-platanin (CP) and internal transcribed spacer 2 (ITS2) genes. The detection limits of the assay were 0.05 pg/μl and 2 fg/μl of fungal DNA for CP and ITS, respectively. Pathogen detection directly from AITs demonstrated specificity and high sensitivity for C. platani, detecting DNA concentrations as low as 1.2 × 10−2 to 1.4 × 10−2 pg/μl, corresponding to ∼10 conidia per ml. Airborne inoculum traps were able to detect the C. platani inoculum within 200 m of the closest symptomatic infected plane tree. The combination of airborne trapping and real-time quantitative PCR assay provides a rapid and sensitive method for the specific detection of a C. platani inoculum. This technique may be used to identify the period of highest risk of pathogen spread in a site, thus helping disease management. PMID:23811499

Nanomolar colorimetric quantitative detection of Fe3 + and PPi with high selectivity

NASA Astrophysics Data System (ADS)

Li, Zhanxian; Li, Haixia; Shi, Caixia; Yu, Mingming; Wei, Liuhe; Ni, Zhonghai

2016-04-01

A novel rhodamine and 8-hydroxyquinoline-based derivative was synthesized, which is shown to act as a colorimetric chemosensor for Fe3 + in aqueous solution with high selectivity over various environmentally and biologically relevant metal ions and anions with a distinct color change from colorless to pink in very fast response time (< 1 min). Fe3 + can be detected quantitatively in the concentration range from 6.7 to 16 μM and the detection limit (LOD) on UV-vis response of the sensor can be as low as 15 nM. The 'in situ' prepared Fe3 + complex (1 ṡ Fe) showed high selectivity toward PPi against many common anions, and sensitivity (the LOD can be as low as 71 nM). In addition, both the chemosensor and the 'in situ' prepared Fe3 + complex are reusable for the detection of Fe3 + and PPi respectively.

12 CFR 223.41 - What covered transactions are exempt from the quantitative limits and collateral requirements?

Code of Federal Regulations, 2013 CFR

2013-01-01

... quantitative limits and collateral requirements? 223.41 Section 223.41 Banks and Banking FEDERAL RESERVE SYSTEM... covered transactions are exempt from the quantitative limits and collateral requirements? The following transactions are not subject to the quantitative limits of §§ 223.11 and 223.12 or the collateral requirements...

12 CFR 223.41 - What covered transactions are exempt from the quantitative limits and collateral requirements?

Code of Federal Regulations, 2014 CFR

2014-01-01

... quantitative limits and collateral requirements? 223.41 Section 223.41 Banks and Banking FEDERAL RESERVE SYSTEM... covered transactions are exempt from the quantitative limits and collateral requirements? The following transactions are not subject to the quantitative limits of §§ 223.11 and 223.12 or the collateral requirements...

12 CFR 223.41 - What covered transactions are exempt from the quantitative limits and collateral requirements?

Code of Federal Regulations, 2010 CFR

2010-01-01

... quantitative limits and collateral requirements? 223.41 Section 223.41 Banks and Banking FEDERAL RESERVE SYSTEM... are exempt from the quantitative limits and collateral requirements? The following transactions are not subject to the quantitative limits of §§ 223.11 and 223.12 or the collateral requirements of § 223...

12 CFR 223.41 - What covered transactions are exempt from the quantitative limits and collateral requirements?

Code of Federal Regulations, 2012 CFR

2012-01-01

... quantitative limits and collateral requirements? 223.41 Section 223.41 Banks and Banking FEDERAL RESERVE SYSTEM... are exempt from the quantitative limits and collateral requirements? The following transactions are not subject to the quantitative limits of §§ 223.11 and 223.12 or the collateral requirements of § 223...

12 CFR 223.41 - What covered transactions are exempt from the quantitative limits and collateral requirements?

Code of Federal Regulations, 2011 CFR

2011-01-01

... quantitative limits and collateral requirements? 223.41 Section 223.41 Banks and Banking FEDERAL RESERVE SYSTEM... are exempt from the quantitative limits and collateral requirements? The following transactions are not subject to the quantitative limits of §§ 223.11 and 223.12 or the collateral requirements of § 223...

Distance-based microfluidic quantitative detection methods for point-of-care testing.

PubMed

Tian, Tian; Li, Jiuxing; Song, Yanling; Zhou, Leiji; Zhu, Zhi; Yang, Chaoyong James

2016-04-07

Equipment-free devices with quantitative readout are of great significance to point-of-care testing (POCT), which provides real-time readout to users and is especially important in low-resource settings. Among various equipment-free approaches, distance-based visual quantitative detection methods rely on reading the visual signal length for corresponding target concentrations, thus eliminating the need for sophisticated instruments. The distance-based methods are low-cost, user-friendly and can be integrated into portable analytical devices. Moreover, such methods enable quantitative detection of various targets by the naked eye. In this review, we first introduce the concept and history of distance-based visual quantitative detection methods. Then, we summarize the main methods for translation of molecular signals to distance-based readout and discuss different microfluidic platforms (glass, PDMS, paper and thread) in terms of applications in biomedical diagnostics, food safety monitoring, and environmental analysis. Finally, the potential and future perspectives are discussed.

Comparison of salivary collection and processing methods for quantitative HHV-8 detection.

PubMed

Speicher, D J; Johnson, N W

2014-10-01

Saliva is a proved diagnostic fluid for the qualitative detection of infectious agents, but the accuracy of viral load determinations is unknown. Stabilising fluids impede nucleic acid degradation, compared with collection onto ice and then freezing, and we have shown that the DNA Genotek P-021 prototype kit (P-021) can produce high-quality DNA after 14 months of storage at room temperature. Here we evaluate the quantitative capability of 10 collection/processing methods. Unstimulated whole mouth fluid was spiked with a mixture of HHV-8 cloned constructs, 10-fold serial dilutions were produced, and samples were extracted and then examined with quantitative PCR (qPCR). Calibration curves were compared by linear regression and qPCR dynamics. All methods extracted with commercial spin columns produced linear calibration curves with large dynamic range and gave accurate viral loads. Ethanol precipitation of the P-021 does not produce a linear standard curve, and virus is lost in the cell pellet. DNA extractions from the P-021 using commercial spin columns produced linear standard curves with wide dynamic range and excellent limit of detection. When extracted with spin columns, the P-021 enables accurate viral loads down to 23 copies μl(-1) DNA. The quantitative and long-term storage capability of this system makes it ideal for study of salivary DNA viruses in resource-poor settings. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The Limitations of Quantitative Social Science for Informing Public Policy

ERIC Educational Resources Information Center

Jerrim, John; de Vries, Robert

2017-01-01

Quantitative social science (QSS) has the potential to make an important contribution to public policy. However it also has a number of limitations. The aim of this paper is to explain these limitations to a non-specialist audience and to identify a number of ways in which QSS research could be improved to better inform public policy.

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

PubMed

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

2016-12-01

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

A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples.

PubMed

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

2016-03-18

Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems.

A Novel Pretreatment-Free Duplex Chamber Digital PCR Detection System for the Absolute Quantitation of GMO Samples

PubMed Central

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

2016-01-01

Digital polymerase chain reaction (PCR) has developed rapidly since it was first reported in the 1990s. However, pretreatments are often required during preparation for digital PCR, which can increase operation error. The single-plex amplification of both the target and reference genes may cause uncertainties due to the different reaction volumes and the matrix effect. In the current study, a quantitative detection system based on the pretreatment-free duplex chamber digital PCR was developed. The dynamic range, limit of quantitation (LOQ), sensitivity and specificity were evaluated taking the GA21 event as the experimental object. Moreover, to determine the factors that may influence the stability of the duplex system, we evaluated whether the pretreatments, the primary and secondary structures of the probes and the SNP effect influence the detection. The results showed that the LOQ was 0.5% and the sensitivity was 0.1%. We also found that genome digestion and single nucleotide polymorphism (SNP) sites affect the detection results, whereas the unspecific hybridization within different probes had little side effect. This indicated that the detection system was suited for both chamber-based and droplet-based digital PCR. In conclusion, we have provided a simple and flexible way of achieving absolute quantitation for genetically modified organism (GMO) genome samples using commercial digital PCR detection systems. PMID:26999129

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

12 CFR 223.42 - What covered transactions are exempt from the quantitative limits, collateral requirements, and...

Code of Federal Regulations, 2014 CFR

2014-01-01

... quantitative limits, collateral requirements, and low-quality asset prohibition? 223.42 Section 223.42 Banks... Provisions of Section 23A § 223.42 What covered transactions are exempt from the quantitative limits... quantitative limits of §§ 223.11 and 223.12, the collateral requirements of § 223.14, or the prohibition on the...

12 CFR 223.42 - What covered transactions are exempt from the quantitative limits, collateral requirements, and...

Code of Federal Regulations, 2013 CFR

2013-01-01

... quantitative limits, collateral requirements, and low-quality asset prohibition? 223.42 Section 223.42 Banks... Provisions of Section 23A § 223.42 What covered transactions are exempt from the quantitative limits... quantitative limits of §§ 223.11 and 223.12, the collateral requirements of § 223.14, or the prohibition on the...

Development and application of a real-time PCR assay for the detection and quantitation of lymphocystis disease virus.

PubMed

Ciulli, Sara; Pinheiro, Ana Cristina de Aguiar Saldana; Volpe, Enrico; Moscato, Michele; Jung, Tae Sung; Galeotti, Marco; Stellino, Sabrina; Farneti, Riccardo; Prosperi, Santino

2015-03-01

Lymphocystis disease virus (LCDV) is responsible for a chronic self-limiting disease that affects more than 125 teleosts. Viral isolation of LCDV is difficult, time-consuming and often ineffective; the development of a rapid and specific tool to detect and quantify LCDV is desirable for both diagnosis and pathogenic studies. In this study, a quantitative real-time PCR (qPCR) assay was developed using a Sybr-Green-based assay targeting a highly conserved region of the MCP gene. Primers were designed on a multiple alignment that included all known LCDV genotypes. The viral DNA segment was cloned within a plasmid to generate a standard curve. The limit of detection was as low as 2.6DNA copies/μl of plasmid and the qPCR was able to detect viral DNA from cell culture lysates and tissues at levels ten-times lower than conventional PCR. Both gilthead seabream and olive flounder LCDV has been amplified, and an in silico assay showed that LCDV of all genotypes can be amplified. LCDV was detected in target and non-target tissues of both diseased and asymptomatic fish. The LCDV qPCR assay developed in this study is highly sensitive, specific, reproducible and versatile for the detection and quantitation of Lymphocystivirus, and may also be used for asymptomatic carrier detection or pathogenesis studies of different LCDV strains. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitative fucK gene polymerase chain reaction on sputum and nasopharyngeal secretions to detect Haemophilus influenzae pneumonia.

PubMed

Abdeldaim, Guma M K; Strålin, Kristoffer; Olcén, Per; Blomberg, Jonas; Mölling, Paula; Herrmann, Björn

2013-06-01

A quantitative polymerase chain reaction (PCR) for the fucK gene was developed for specific detection of Haemophilus influenzae. The method was tested on sputum and nasopharyngeal aspirate (NPA) from 78 patients with community-acquired pneumonia (CAP). With a reference standard of sputum culture and/or serology against the patient's own nasopharyngeal isolate, H. influenzae etiology was detected in 20 patients. Compared with the reference standard, fucK PCR (using the detection limit 10(5) DNA copies/mL) on sputum and NPA showed a sensitivity of 95.0% (19/20) in both cases, and specificities of 87.9% (51/58) and 89.5% (52/58), respectively. In a receiver operating characteristic curve analysis, sputum fucK PCR was found to be significantly superior to sputum P6 PCR for detection of H. influenzae CAP. NPA fucK PCR was positive in 3 of 54 adult controls without respiratory symptoms. In conclusion, quantitative fucK real-time PCR provides a sensitive and specific identification of H. influenzae in respiratory secretions. Copyright © 2013 Elsevier Inc. All rights reserved.

12 CFR 223.42 - What covered transactions are exempt from the quantitative limits, collateral requirements, and...

Code of Federal Regulations, 2012 CFR

2012-01-01

... quantitative limits, collateral requirements, and low-quality asset prohibition? 223.42 Section 223.42 Banks... 23A § 223.42 What covered transactions are exempt from the quantitative limits, collateral requirements, and low-quality asset prohibition? The following transactions are not subject to the quantitative...

12 CFR 223.42 - What covered transactions are exempt from the quantitative limits, collateral requirements, and...

Code of Federal Regulations, 2011 CFR

2011-01-01

... quantitative limits, collateral requirements, and low-quality asset prohibition? 223.42 Section 223.42 Banks... 23A § 223.42 What covered transactions are exempt from the quantitative limits, collateral requirements, and low-quality asset prohibition? The following transactions are not subject to the quantitative...

12 CFR 223.42 - What covered transactions are exempt from the quantitative limits, collateral requirements, and...

Code of Federal Regulations, 2010 CFR

2010-01-01

... quantitative limits, collateral requirements, and low-quality asset prohibition? 223.42 Section 223.42 Banks... 23A § 223.42 What covered transactions are exempt from the quantitative limits, collateral requirements, and low-quality asset prohibition? The following transactions are not subject to the quantitative...

Fundamental limits of measurement in telecommunications: Experimental and modeling studies in a test optical network on proposal for the reform of telecommunication quantitations

NASA Astrophysics Data System (ADS)

Egan, James; McMillan, Normal; Denieffe, David

2011-08-01

Proposals for a review of the limits of measurement for telecommunications are made. The measures are based on adapting work from the area of chemical metrology for the field of telecommunications. Currie has introduced recommendations for defining the limits of measurement in chemical metrology and has identified three key fundamental limits of measurement. These are the critical level, the detection limit and the determination limit. Measurements on an optical system are used to illustrate the utility of these measures and discussion is given into the advantages of using these fundamental quantitations over existing methods.

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

PubMed Central

Joyner, Jessica; Wanless, David; Sinigalliano, Christopher D.

2014-01-01

Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml−1 and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml−1. This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health. PMID:24375136

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

PubMed

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

2014-03-01

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

Quantitative risk stratification in Markov chains with limiting conditional distributions.

PubMed

Chan, David C; Pollett, Philip K; Weinstein, Milton C

2009-01-01

Many clinical decisions require patient risk stratification. The authors introduce the concept of limiting conditional distributions, which describe the equilibrium proportion of surviving patients occupying each disease state in a Markov chain with death. Such distributions can quantitatively describe risk stratification. The authors first establish conditions for the existence of a positive limiting conditional distribution in a general Markov chain and describe a framework for risk stratification using the limiting conditional distribution. They then apply their framework to a clinical example of a treatment indicated for high-risk patients, first to infer the risk of patients selected for treatment in clinical trials and then to predict the outcomes of expanding treatment to other populations of risk. For the general chain, a positive limiting conditional distribution exists only if patients in the earliest state have the lowest combined risk of progression or death. The authors show that in their general framework, outcomes and population risk are interchangeable. For the clinical example, they estimate that previous clinical trials have selected the upper quintile of patient risk for this treatment, but they also show that expanded treatment would weakly dominate this degree of targeted treatment, and universal treatment may be cost-effective. Limiting conditional distributions exist in most Markov models of progressive diseases and are well suited to represent risk stratification quantitatively. This framework can characterize patient risk in clinical trials and predict outcomes for other populations of risk.

Event-specific qualitative and quantitative detection of five genetically modified rice events using a single standard reference molecule.

PubMed

Kim, Jae-Hwan; Park, Saet-Byul; Roh, Hyo-Jeong; Shin, Min-Ki; Moon, Gui-Im; Hong, Jin-Hwan; Kim, Hae-Yeong

2017-07-01

One novel standard reference plasmid, namely pUC-RICE5, was constructed as a positive control and calibrator for event-specific qualitative and quantitative detection of genetically modified (GM) rice (Bt63, Kemingdao1, Kefeng6, Kefeng8, and LLRice62). pUC-RICE5 contained fragments of a rice-specific endogenous reference gene (sucrose phosphate synthase) as well as the five GM rice events. An existing qualitative PCR assay approach was modified using pUC-RICE5 to create a quantitative method with limits of detection correlating to approximately 1-10 copies of rice haploid genomes. In this quantitative PCR assay, the square regression coefficients ranged from 0.993 to 1.000. The standard deviation and relative standard deviation values for repeatability ranged from 0.02 to 0.22 and 0.10% to 0.67%, respectively. The Ministry of Food and Drug Safety (Korea) validated the method and the results suggest it could be used routinely to identify five GM rice events. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantitative analysis of ginger components in commercial products using liquid chromatography with electrochemical array detection

PubMed Central

Shao, Xi; Lv, Lishuang; Parks, Tiffany; Wu, Hou; Ho, Chi-Tang; Sang, Shengmin

2010-01-01

For the first time, a sensitive reversed-phase HPLC electrochemical array method has been developed for the quantitative analysis of eight major ginger components ([6]-, [8]-, and [10]-gingerol, [6]-, [8]-, and [10]-shogaol, [6]-paradol, and [1]-dehydrogingerdione) in eleven ginger-containing commercial products. This method was valid with unrivaled sensitivity as low as 7.3 – 20.2 pg of limit of detection and a range of 14.5 to 40.4 pg of limit of quantification. Using this method, we quantified the levels of eight ginger components in eleven different commercial products. Our results found that both levels and ratios among the eight compounds vary greatly in commercial products. PMID:21090746

Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5'-transgene integration sequence.

PubMed

Li, P; Jia, J W; Jiang, L X; Zhu, H; Bai, L; Wang, J B; Tang, X M; Pan, A H

2012-04-27

To ensure the implementation of genetically modified organism (GMO)-labeling regulations, an event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic carnation variety Moonlite. The 5'-transgene integration sequence was isolated by thermal asymmetric interlaced PCR. Based upon the 5'-transgene integration sequence, the event-specific primers and TaqMan probe were designed to amplify the fragments, which spanned the exogenous DNA and carnation genomic DNA. Qualitative and quantitative PCR assays were developed employing the designed primers and probe. The detection limit of the qualitative PCR assay was 0.05% for Moonlite in 100 ng total carnation genomic DNA, corresponding to about 79 copies of the carnation haploid genome; the limit of detection and quantification of the quantitative PCR assay were estimated to be 38 and 190 copies of haploid carnation genomic DNA, respectively. Carnation samples with different contents of genetically modified components were quantified and the bias between the observed and true values of three samples were lower than the acceptance criterion (<25%) of the GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite.

An event-specific method for the detection and quantification of ML01, a genetically modified Saccharomyces cerevisiae wine strain, using quantitative PCR.

PubMed

Vaudano, Enrico; Costantini, Antonella; Garcia-Moruno, Emilia

2016-10-03

The availability of genetically modified (GM) yeasts for winemaking and, in particular, transgenic strains based on the integration of genetic constructs deriving from other organisms into the genome of Saccharomyces cerevisiae, has been a reality for several years. Despite this, their use is only authorized in a few countries and limited to two strains: ML01, able to convert malic acid into lactic acid during alcoholic fermentation, and ECMo01 suitable for reducing the risk of carbamate production. In this work we propose a quali-quantitative culture-independent method for the detection of GM yeast ML01 in commercial preparations of ADY (Active Dry Yeast) consisting of efficient extraction of DNA and qPCR (quantitative PCR) analysis based on event-specific assay targeting MLC (malolactic cassette), and a taxon-specific S. cerevisiae assay detecting the MRP2 gene. The ADY DNA extraction methodology has been shown to provide good purity DNA suitable for subsequent qPCR. The MLC and MRP2 qPCR assay showed characteristics of specificity, dynamic range, limit of quantification (LOQ) limit of detection (LOD), precision and trueness, which were fully compliant with international reference guidelines. The method has been shown to reliably detect 0.005% (mass/mass) of GM ML01 S. cerevisiae in commercial preparations of ADY. Copyright © 2016 Elsevier B.V. All rights reserved.

A paper/polymer hybrid microfluidic microplate for rapid quantitative detection of multiple disease biomarkers.

PubMed

Sanjay, Sharma T; Dou, Maowei; Sun, Jianjun; Li, XiuJun

2016-07-26

Enzyme linked immunosorbent assay (ELISA) is one of the most widely used laboratory disease diagnosis methods. However, performing ELISA in low-resource settings is limited by long incubation time, large volumes of precious reagents, and well-equipped laboratories. Herein, we developed a simple, miniaturized paper/PMMA (poly(methyl methacrylate)) hybrid microfluidic microplate for low-cost, high throughput, and point-of-care (POC) infectious disease diagnosis. The novel use of porous paper in flow-through microwells facilitates rapid antibody/antigen immobilization and efficient washing, avoiding complicated surface modifications. The top reagent delivery channels can simply transfer reagents to multiple microwells thus avoiding repeated manual pipetting and costly robots. Results of colorimetric ELISA can be observed within an hour by the naked eye. Quantitative analysis was achieved by calculating the brightness of images scanned by an office scanner. Immunoglobulin G (IgG) and Hepatitis B surface Antigen (HBsAg) were quantitatively analyzed with good reliability in human serum samples. Without using any specialized equipment, the limits of detection of 1.6 ng/mL for IgG and 1.3 ng/mL for HBsAg were achieved, which were comparable to commercial ELISA kits using specialized equipment. We envisage that this simple POC hybrid microplate can have broad applications in various bioassays, especially in resource-limited settings.

A paper/polymer hybrid microfluidic microplate for rapid quantitative detection of multiple disease biomarkers

PubMed Central

Sanjay, Sharma T.; Dou, Maowei; Sun, Jianjun; Li, XiuJun

2016-01-01

Enzyme linked immunosorbent assay (ELISA) is one of the most widely used laboratory disease diagnosis methods. However, performing ELISA in low-resource settings is limited by long incubation time, large volumes of precious reagents, and well-equipped laboratories. Herein, we developed a simple, miniaturized paper/PMMA (poly(methyl methacrylate)) hybrid microfluidic microplate for low-cost, high throughput, and point-of-care (POC) infectious disease diagnosis. The novel use of porous paper in flow-through microwells facilitates rapid antibody/antigen immobilization and efficient washing, avoiding complicated surface modifications. The top reagent delivery channels can simply transfer reagents to multiple microwells thus avoiding repeated manual pipetting and costly robots. Results of colorimetric ELISA can be observed within an hour by the naked eye. Quantitative analysis was achieved by calculating the brightness of images scanned by an office scanner. Immunoglobulin G (IgG) and Hepatitis B surface Antigen (HBsAg) were quantitatively analyzed with good reliability in human serum samples. Without using any specialized equipment, the limits of detection of 1.6 ng/mL for IgG and 1.3 ng/mL for HBsAg were achieved, which were comparable to commercial ELISA kits using specialized equipment. We envisage that this simple POC hybrid microplate can have broad applications in various bioassays, especially in resource-limited settings. PMID:27456979

A paper/polymer hybrid microfluidic microplate for rapid quantitative detection of multiple disease biomarkers

NASA Astrophysics Data System (ADS)

Sanjay, Sharma T.; Dou, Maowei; Sun, Jianjun; Li, Xiujun

2016-07-01

Enzyme linked immunosorbent assay (ELISA) is one of the most widely used laboratory disease diagnosis methods. However, performing ELISA in low-resource settings is limited by long incubation time, large volumes of precious reagents, and well-equipped laboratories. Herein, we developed a simple, miniaturized paper/PMMA (poly(methyl methacrylate)) hybrid microfluidic microplate for low-cost, high throughput, and point-of-care (POC) infectious disease diagnosis. The novel use of porous paper in flow-through microwells facilitates rapid antibody/antigen immobilization and efficient washing, avoiding complicated surface modifications. The top reagent delivery channels can simply transfer reagents to multiple microwells thus avoiding repeated manual pipetting and costly robots. Results of colorimetric ELISA can be observed within an hour by the naked eye. Quantitative analysis was achieved by calculating the brightness of images scanned by an office scanner. Immunoglobulin G (IgG) and Hepatitis B surface Antigen (HBsAg) were quantitatively analyzed with good reliability in human serum samples. Without using any specialized equipment, the limits of detection of 1.6 ng/mL for IgG and 1.3 ng/mL for HBsAg were achieved, which were comparable to commercial ELISA kits using specialized equipment. We envisage that this simple POC hybrid microplate can have broad applications in various bioassays, especially in resource-limited settings.

Gold Nanoparticle Labeling Based ICP-MS Detection/Measurement of Bacteria, and Their Quantitative Photothermal Destruction

PubMed Central

Lin, Yunfeng

2015-01-01

Bacteria such as Salmonella and E. coli present a great challenge in public health care in today’s society. Protection of public safety against bacterial contamination and rapid diagnosis of infection require simple and fast assays for the detection and elimination of bacterial pathogens. After utilizing Salmonella DT104 as an example bacterial strain for our investigation, we report a rapid and sensitive assay for the qualitative and quantitative detection of bacteria by using antibody affinity binding, popcorn shaped gold nanoparticle (GNPOPs) labeling, surfance enchanced Raman spectroscopy (SERS), and inductively coupled plasma mass spectrometry (ICP-MS) detection. For qualitative analysis, our assay can detect Salmonella within 10 min by Raman spectroscopy; for quantitative analysis, our assay has the ability to measure as few as 100 Salmonella DT104 in a 1 mL sample (100 CFU/mL) within 40 min. Based on the quantitative detection, we investigated the quantitative destruction of Salmonella DT104, and the assay’s photothermal efficiency in order to reduce the amount of GNPOPs in the assay to ultimately to eliminate any potential side effects/toxicity to the surrounding cells in vivo. Results suggest that our assay may serve as a promising candidate for qualitative and quantitative detection and elimination of a variety of bacterial pathogens. PMID:26417447

Smartphone-Based Dual-Modality Imaging System for Quantitative Detection of Color or Fluorescent Lateral Flow Immunochromatographic Strips

NASA Astrophysics Data System (ADS)

Hou, Yafei; Wang, Kan; Xiao, Kun; Qin, Weijian; Lu, Wenting; Tao, Wei; Cui, Daxiang

2017-04-01

Nowadays, lateral flow immunochromatographic assays are increasingly popular as a diagnostic tool for point-of-care (POC) test based on their simplicity, specificity, and sensitivity. Hence, quantitative detection and pluralistic popular application are urgently needed in medical examination. In this study, a smartphone-based dual-modality imaging system was developed for quantitative detection of color or fluorescent lateral flow test strips, which can be operated anywhere at any time. In this system, the white and ultra-violet (UV) light of optical device was designed, which was tunable with different strips, and the Sobel operator algorithm was used in the software, which could enhance the identification ability to recognize the test area from the background boundary information. Moreover, this technology based on extraction of the components from RGB format (red, green, and blue) of color strips or only red format of the fluorescent strips can obviously improve the high-signal intensity and sensitivity. Fifty samples were used to evaluate the accuracy of this system, and the ideal detection limit was calculated separately from detection of human chorionic gonadotropin (HCG) and carcinoembryonic antigen (CEA). The results indicated that smartphone-controlled dual-modality imaging system could provide various POC diagnoses, which becomes a potential technology for developing the next-generation of portable system in the near future.

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

PubMed

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

2017-02-01

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

Development of a quantitative fluorescence single primer isothermal amplification-based method for the detection of Salmonella.

PubMed

Wang, Jianchang; Li, Rui; Hu, Lianxia; Sun, Xiaoxia; Wang, Jinfeng; Li, Jing

2016-02-16

Food-borne disease caused by Salmonella has long been, and continues to be, an important global public health problem, necessitating rapid and accurate detection of Salmonella in food. Real time PCR is the most recently developed approach for Salmonella detection. Single primer isothermal amplification (SPIA), a novel gene amplification technique, has emerged as an attractive microbiological testing method. SPIA is performed under a constant temperature, eliminating the need for an expensive thermo-cycler. In addition, SPIA reactions can be accomplished in 30 min, faster than real time PCR that usually takes over 2h. We developed a quantitative fluorescence SPIA-based method for the detection of Salmonella. Using Salmonella Typhimurium genomic DNA as template and a primer targeting Salmonella invA gene, we showed the detection limit of SPIA was 2.0 × 10(1)fg DNA. Its successful amplification of different serotypic Salmonella genomic DNA but not non-Salmonella bacterial DNA demonstrated the specificity of SPIA. Furthermore, this method was validated with artificially contaminated beef. In conclusion, we showed high sensitivity and specificity of SPIA in the detection of Salmonella, comparable to real time PCR. In addition, SPIA is faster and more cost-effective (non-use of expensive cyclers), making it a potential alternative for field detection of Salmonella in resource-limited settings that are commonly encountered in developing countries. Copyright © 2015 Elsevier B.V. All rights reserved.

A practical approach to determination of laboratory GC-MS limits of detection.

PubMed

Underwood, P J; Kananen, G E; Armitage, E K

1997-01-01

Determination of limit of detection (LOD) values in a forensic laboratory serves a fundamental forensic requirement for assay performance. In addition to demonstrating assay capability, LOD values can also be used to fulfill certification requirements of a high-volume forensic drug laboratory. The LOD was defined as the lowest concentration of drug that the laboratory can detect in a specimen with forensic certainty at a minimum of 85% of the time. Overall batch acceptance criteria included acceptable quantitation of control materials (within 20% of target), acceptable chromatography (symmetry, peak integration, peak shape, peak, and baseline resolution), retention time within +/-1% of the extracted standard, and mass ion ratios within +/-20% of the extracted standard mass ion ratios. Individual specimen acceptance criteria were the same as the batch acceptance criteria excluding the quantitation requirement. Data were collected from all instruments on different runs. A minimum of ten data points was required for each certified instrument, and a minimum of 85% of data points was acceptable. Quantitation within +/-20% of the LOD concentration was not required, but acceptable mass ratios were required. Data points with poor chromatography (internal standard failed mass ratios; interference of the baseline, for example, shoulders; asymmetry; and baseline resolution) was omitted from the acceptable rate calculation. Data points with good chromatography with failed mass ion ratios were included in the acceptable rate calculation. With these criteria, we established the following LODs: 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid, 2 ng/mL; benzoylecgonine, 5 ng/mL; phencyclidine, 2.5 ng/mL; amphetamine, 150 ng/mL; methamphetamine, 100 ng/mL; codeine, 500 ng/mL; and morphine, 1000 ng/mL.

Statistical behavior of ten million experimental detection limits

NASA Astrophysics Data System (ADS)

Voigtman, Edward; Abraham, Kevin T.

2011-02-01

Using a lab-constructed laser-excited fluorimeter, together with bootstrapping methodology, the authors have generated many millions of experimental linear calibration curves for the detection of rhodamine 6G tetrafluoroborate in ethanol solutions. The detection limits computed from them are in excellent agreement with both previously published theory and with comprehensive Monte Carlo computer simulations. Currie decision levels and Currie detection limits, each in the theoretical, chemical content domain, were found to be simply scaled reciprocals of the non-centrality parameter of the non-central t distribution that characterizes univariate linear calibration curves that have homoscedastic, additive Gaussian white noise. Accurate and precise estimates of the theoretical, content domain Currie detection limit for the experimental system, with 5% (each) probabilities of false positives and false negatives, are presented.

Comparison of different approaches to quantitative adenovirus detection in stool specimens of hematopoietic stem cell transplant recipients.

PubMed

Kosulin, K; Dworzak, S; Lawitschka, A; Matthes-Leodolter, S; Lion, T

2016-12-01

Adenoviruses almost invariably proliferate in the gastrointestinal tract prior to dissemination, and critical threshold concentrations in stool correlate with the risk of viremia. Monitoring of adenovirus loads in stool may therefore be important for timely initiation of treatment in order to prevent invasive infection. Comparison of a manual DNA extraction kit in combination with a validated in-house PCR assay with automated extraction on the NucliSENS-EasyMAG device coupled with the Adenovirus R-gene kit (bioMérieux) for quantitative adenovirus analysis in stool samples. Stool specimens spiked with adenovirus concentrations in a range from 10E2-10E11 copies/g and 32 adenovirus-positive clinical stool specimens from pediatric stem cell transplant recipients were tested along with appropriate negative controls. Quantitative analysis of viral load in adenovirus-positive stool specimens revealed a median difference of 0.5 logs (range 0.1-2.2) between the detection systems tested and a difference of 0.3 logs (range 0.0-1.7) when the comparison was restricted to the PCR assays only. Spiking experiments showed a detection limit of 10 2 -10 3 adenovirus copies/g stool revealing a somewhat higher sensitivity offered by the automated extraction. The dynamic range of accurate quantitative analysis by both systems investigated was between 10 3 and 10 8 virus copies/g. The differences in quantitative analysis of adenovirus copy numbers between the systems tested were primarily attributable to the DNA extraction method used, while the qPCR assays revealed a high level of concordance. Both systems showed adequate performance for detection and monitoring of adenoviral load in stool specimens. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative detection of 4-hydroxyequilenin-DNA adducts in mammalian cells using an immunoassay with a novel monoclonal antibody.

PubMed

Okahashi, Yumiko; Iwamoto, Takaaki; Suzuki, Naomi; Shibutani, Shinya; Sugiura, Shigeki; Itoh, Shinji; Nishiwaki, Tomohisa; Ueno, Satoshi; Mori, Toshio

2010-07-01

Estrogen-DNA adducts are potential biomarkers for assessing the risk and development of estrogen-associated cancers. 4-Hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4-OHEQ), the metabolites of equine estrogens present in common hormone replacement therapy (HRT) formulations, are capable of producing bulky 4-OHEN-DNA adducts. Although the formation of 4-OHEN-DNA adducts has been reported, their quantitative detection in mammalian cells has not been done. To quantify such DNA adducts, we generated a novel monoclonal antibody (4OHEN-1) specific for 4-OHEN-DNA adducts. The primary epitope recognized is one type of stereoisomers of 4-OHEN-dA adducts and of 4-OHEN-dC adducts in DNA. An immunoassay with 4OHEN-1 revealed a linear dose-response between known amounts of 4-OHEN-DNA adducts and the antibody binding to those adducts, with a detection limit of approximately five adducts/10(8) bases in 1 microg DNA sample. In human breast cancer cells, the quantitative immunoassay revealed that 4-OHEN produces five times more 4-OHEN-DNA adducts than does 4-OHEQ. Moreover, in a mouse model for HRT, oral administration of Premarin increased the levels of 4-OHEN-DNA adducts in various tissues, including the uterus and ovaries, in a time-dependent manner. Thus, we succeeded in establishing a novel immunoassay for quantitative detection of 4-OHEN-DNA adducts in mammalian cells.

Quantitative analysis of trace levels of surface contamination by X-ray photoelectron spectroscopy Part I: statistical uncertainty near the detection limit.

PubMed

Hill, Shannon B; Faradzhev, Nadir S; Powell, Cedric J

2017-12-01

We discuss the problem of quantifying common sources of statistical uncertainties for analyses of trace levels of surface contamination using X-ray photoelectron spectroscopy. We examine the propagation of error for peak-area measurements using common forms of linear and polynomial background subtraction including the correlation of points used to determine both background and peak areas. This correlation has been neglected in previous analyses, but we show that it contributes significantly to the peak-area uncertainty near the detection limit. We introduce the concept of relative background subtraction variance (RBSV) which quantifies the uncertainty introduced by the method of background determination relative to the uncertainty of the background area itself. The uncertainties of the peak area and atomic concentration and of the detection limit are expressed using the RBSV, which separates the contributions from the acquisition parameters, the background-determination method, and the properties of the measured spectrum. These results are then combined to find acquisition strategies that minimize the total measurement time needed to achieve a desired detection limit or atomic-percentage uncertainty for a particular trace element. Minimization of data-acquisition time is important for samples that are sensitive to x-ray dose and also for laboratories that need to optimize throughput.

Temporal lobe epilepsy: quantitative MR volumetry in detection of hippocampal atrophy.

PubMed

Farid, Nikdokht; Girard, Holly M; Kemmotsu, Nobuko; Smith, Michael E; Magda, Sebastian W; Lim, Wei Y; Lee, Roland R; McDonald, Carrie R

2012-08-01

To determine the ability of fully automated volumetric magnetic resonance (MR) imaging to depict hippocampal atrophy (HA) and to help correctly lateralize the seizure focus in patients with temporal lobe epilepsy (TLE). This study was conducted with institutional review board approval and in compliance with HIPAA regulations. Volumetric MR imaging data were analyzed for 34 patients with TLE and 116 control subjects. Structural volumes were calculated by using U.S. Food and Drug Administration-cleared software for automated quantitative MR imaging analysis (NeuroQuant). Results of quantitative MR imaging were compared with visual detection of atrophy, and, when available, with histologic specimens. Receiver operating characteristic analyses were performed to determine the optimal sensitivity and specificity of quantitative MR imaging for detecting HA and asymmetry. A linear classifier with cross validation was used to estimate the ability of quantitative MR imaging to help lateralize the seizure focus. Quantitative MR imaging-derived hippocampal asymmetries discriminated patients with TLE from control subjects with high sensitivity (86.7%-89.5%) and specificity (92.2%-94.1%). When a linear classifier was used to discriminate left versus right TLE, hippocampal asymmetry achieved 94% classification accuracy. Volumetric asymmetries of other subcortical structures did not improve classification. Compared with invasive video electroencephalographic recordings, lateralization accuracy was 88% with quantitative MR imaging and 85% with visual inspection of volumetric MR imaging studies but only 76% with visual inspection of clinical MR imaging studies. Quantitative MR imaging can depict the presence and laterality of HA in TLE with accuracy rates that may exceed those achieved with visual inspection of clinical MR imaging studies. Thus, quantitative MR imaging may enhance standard visual analysis, providing a useful and viable means for translating volumetric analysis into

Automatic rectum limit detection by anatomical markers correlation.

PubMed

Namías, R; D'Amato, J P; del Fresno, M; Vénere, M

2014-06-01

Several diseases take place at the end of the digestive system. Many of them can be diagnosed by means of different medical imaging modalities together with computer aided detection (CAD) systems. These CAD systems mainly focus on the complete segmentation of the digestive tube. However, the detection of limits between different sections could provide important information to these systems. In this paper we present an automatic method for detecting the rectum and sigmoid colon limit using a novel global curvature analysis over the centerline of the segmented digestive tube in different imaging modalities. The results are compared with the gold standard rectum upper limit through a validation scheme comprising two different anatomical markers: the third sacral vertebra and the average rectum length. Experimental results in both magnetic resonance imaging (MRI) and computed tomography colonography (CTC) acquisitions show the efficacy of the proposed strategy in automatic detection of rectum limits. The method is intended for application to the rectum segmentation in MRI for geometrical modeling and as contextual information source in virtual colonoscopies and CAD systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

A quantitative TaqMan PCR assay for the detection of Ureaplasma diversum.

PubMed

Marques, Lucas M; Amorim, Aline T; Martins, Hellen Braga; Rezende, Izadora Souza; Barbosa, Maysa Santos; Lobão, Tassia Neves; Campos, Guilherme B; Timenetsky, Jorge

2013-12-27

Ureaplasma diversum in veterinary studies is an undesirable microbe, which may cause infection in bulls and may result in seminal vesiculitis, balanopostitis, and alterations in spermatozoids, whereas in cows, it may cause placentitis, fetal alveolitis, abortion, and birth of weak calves. U. diversum is released through organic secretions, especially semen, preputial and vaginal mucus, conjunctival secretion, and milk. The aim of the present study was to develop a TaqMan probe, highly sensitive and specific quantitative PCR (qPCR) assay for the detection and quantification of U. diversum from genital swabs of bovines. Primers and probes specific to U. diversum 16S rRNA gene were designed. The specificity, detection limit, intra- and inter-assay variability of qPCR to detect this ureaplasma was compared with the results of the conventional PCR assay (cPCR). Swabs of vaginal mucus from 169 cows were tested. The qPCR assay detected as few as 10 copies of U. diversum and was 100-fold more sensitive than the cPCR. No cross-reactivity with other Mollicutes or eubacteria was observed. U. diversum was detected in 79 swabs (46.42%) by qPCR, while using cPCR it was detected in 42 (25%) samples. The difference in cPCR and qPCR ureaplasma detection between healthy and sick animals was not statistically significant. But the U. diversum load in samples from animals with genital disorders was higher than in healthy animals. The qPCR assay developed herein is highly sensitive and specific for the detection and quantification of U. diversum in vaginal bovine samples. Copyright © 2013. Published by Elsevier B.V.

Accounting for imperfect detection in ecology: a quantitative review.

PubMed

Kellner, Kenneth F; Swihart, Robert K

2014-01-01

Detection in studies of species abundance and distribution is often imperfect. Assuming perfect detection introduces bias into estimation that can weaken inference upon which understanding and policy are based. Despite availability of numerous methods designed to address this assumption, many refereed papers in ecology fail to account for non-detection error. We conducted a quantitative literature review of 537 ecological articles to measure the degree to which studies of different taxa, at various scales, and over time have accounted for imperfect detection. Overall, just 23% of articles accounted for imperfect detection. The probability that an article incorporated imperfect detection increased with time and varied among taxa studied; studies of vertebrates were more likely to incorporate imperfect detection. Among articles that reported detection probability, 70% contained per-survey estimates of detection that were less than 0.5. For articles in which constancy of detection was tested, 86% reported significant variation. We hope that our findings prompt more ecologists to consider carefully the detection process when designing studies and analyzing results, especially for sub-disciplines where incorporation of imperfect detection in study design and analysis so far has been lacking.

Rapid and Quantitative Detection of Vibrio parahemolyticus by the Mixed-Dye-Based Loop-Mediated Isothermal Amplification Assay on a Self-Priming Compartmentalization Microfluidic Chip.

PubMed

Pang, Bo; Ding, Xiong; Wang, Guoping; Zhao, Chao; Xu, Yanan; Fu, Kaiyue; Sun, Jingjing; Song, Xiuling; Wu, Wenshuai; Liu, Yushen; Song, Qi; Hu, Jiumei; Li, Juan; Mu, Ying

2017-12-27

Vibrio parahemolyticus (VP) mostly isolated from aquatic products is one of the major causes of bacterial food-poisoning events worldwide, which could be reduced using a promising on-site detection method. Herein, a rapid and quantitative method for VP detection was developed by applying a mixed-dye-loaded loop-mediated isothermal amplification (LAMP) assay on a self-priming compartmentalization (SPC) microfluidic chip, termed on-chip mixed-dye-based LAMP (CMD-LAMP). In comparison to conventional approaches, CMD-LAMP was advantageous on the limit of detection, which reached down to 1 × 10 3 CFU/mL in food-contaminated samples without the pre-enrichment of bacteria. Additionally, as a result of the use of a mixed dye and SPC chip, the quantitative result could be easily acquired, avoiding the requirement of sophisticated instruments and tedious operation. Also, CMD-LAMP was rapid and cost-effective. Conclusively, CMD-LAMP has great potential in realizing the on-site quantitative analysis of VP for food safety.

Limits of linearity and detection for some drugs of abuse.

PubMed

Needleman, S B; Romberg, R W

1990-01-01

The limits of linearity (LOL) and detection (LOD) are important factors in establishing the reliability of an analytical procedure for accurately assaying drug concentrations in urine specimens. Multiple analyses of analyte over an extended range of concentrations provide a measure of the ability of the analytical procedure to correctly identify known quantities of drug in a biofluid matrix. Each of the seven drugs of abuse gives linear analytical responses from concentrations at or near their LOD to concentrations several-fold higher than those generally encountered in the drug screening laboratory. The upper LOL exceeds the Department of Navy (DON) cutoff values by factors of approximately 2 to 160. The LOD varies from 0.4 to 5.0% of the DON cutoff value for each drug. The limit of quantitation (LOQ) is calculated as the LOD + 7 SD. The range for LOL is greater for drugs analyzed with deuterated internal standards compared with those using conventional internal standards. For THC acid, cocaine, PCP, and morphine, LOLs are 8 to 160-fold greater than the defined cutoff concentrations. For the other drugs, the LOL's are only 2 to 4-fold greater than the defined cutoff concentrations.

Universal and specific quantitative detection of botulinum neurotoxin genes

PubMed Central

2010-01-01

Background Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA)-accepted assay to detect and type botulinum neurotoxins (BoNTs) is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR) technology to determine the specific serotype of the neurotoxin. Results We assayed purified C. botulinum DNA and crude toxin preparations, as well as food and stool from healthy individuals spiked with purified BoNT DNA, and one stool sample from a case of infant botulism for the presence of the NTNH gene, which is part of the BoNT gene cluster, and for the presence of serotype-specific BoNT genes. The PCR surpassed the mouse bioassay both in specificity and sensitivity, detecting positive signals in BoNT preparations containing well below the 1 LD50 required for detection via the mouse bioassay. These results were type-specific and we were reliably able to quantify as few as 10 genomic copies. Conclusions While other studies have reported

Detection of sex chromosome aneuploidies using quantitative fluorescent PCR in the Hungarian population.

PubMed

Nagy, Balint; Nagy, Richard Gyula; Lazar, Levente; Schonleber, Julianna; Papp, Csaba; Rigo, Janos

2015-05-20

Aneuploidies are the most frequent chromosomal abnormalities at birth. Autosomal aneuploidies cause serious malformations like trisomy 21, trisomy 18 and trisomy 13. However sex chromosome aneuploidies are causing less severe syndromes. For the detection of these aneuploidies, the "gold standard" method is the cytogenetic analysis of fetal cells, karyograms show all numerical and structural abnormalities, but it takes 2-4 weeks to get the reports. Molecular biological methods were developed to overcome the long culture time, thus, FISH and quantitative fluorescent PCR were introduced. In this work we show our experience with a commercial kit for the detection of sex chromosome aneuploidies. We analyzed 20.173 amniotic fluid samples for the period of 2006-2013 in our department. A conventional cytogenetic analysis was performed on the samples. We checked the reliability of quantitative fluorescent PCR and DNA fragment analysis on those samples where sex chromosomal aneuploidy was diagnosed. From the 20.173 amniotic fluid samples we found 50 samples with sex chromosome aneuploidy. There were 19 samples showing 46, XO, 17 samples with 46, XXY, 9 samples with 47, XXX and 5 samples with 47, XYY karyotypes. The applied quantitative fluorescent PCR and DNA fragment analyses method are suitable to detect all abnormal sex chromosome aneuploidies. Quantitative fluorescent PCR is a fast and reliable method for detection of sex chromosome aneuploidies. Copyright © 2015. Published by Elsevier B.V.

A double-label time-resolved fluorescent strip for rapidly quantitative detection of carbofuran residues in agro-products.

PubMed

Zhang, Qi; Qu, Qiaoyu; Chen, Shanshan; Liu, Xiaowei; Li, Peiwu

2017-09-15

A rapid and quantitative time-resolved fluorescent immunochromatographic assay (TRFICA) for detecting carbofuran residues in agro-products was reported in this paper. This assay was developed based on double-label immunoprobes, one of which was a carbofuran-specific antibody coupled with europium microbeads for the test (T) line signal while the other was mouse IgG coupled with europium microbeads for the control (C) line signal. Quantitative relationships between carbofuran concentrations and T/C ratios were established to determine the analyte concentration. To increase assay accuracy, four standard curves were established for the agro-products (green bean, cabbage, apple, and pear). The limits of detection (LODs) ranged from 0.04 to 0.76mgL -1 . The spiked recoveries of carbofuran in the agro-products were in the range of 81-103%, which was in good agreement with a standard HPLC method. Therefore, we provided a new and reliable method for determination of N-methylcarbamate pesticide carbofuran residues in agro-products including vegetables and fruits. Copyright © 2017. Published by Elsevier Ltd.

Nitromethane K-9 Detection Limit

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

Strobel, R; Kury, J

2003-08-29

The Bureau of Alcohol, Tobacco and Firearms (ATF) trains canine/handler teams to detect explosives for government and other agencies worldwide. After completing the training program the teams are tested on an array containing explosives and numerous other samples designed to distract a canine. Passing this test results in a team's certification. These teams can be considered as ''detection instruments'' freshly calibrated just before leaving the ''factory''. Using these teams to examine special experimental arrays immediately following certification can lead to a better understanding of a canine's detection capabilities. Forty-one of these ''detection instruments'' were used in four test series withmore » arrays containing dilute nitromethane-in-water solutions. (The canines had been trained on the amount of nitromethane vapor in equilibrium with the undiluted liquid explosive.) By diluting liquid nitromethane with water, the amount of explosive vapor can be reduced many orders of magnitude to test the lower limit of the canine's nitromethane vapor detection response. The results are presented in this paper.« less

Influence analysis in quantitative trait loci detection.

PubMed

Dou, Xiaoling; Kuriki, Satoshi; Maeno, Akiteru; Takada, Toyoyuki; Shiroishi, Toshihiko

2014-07-01

This paper presents systematic methods for the detection of influential individuals that affect the log odds (LOD) score curve. We derive general formulas of influence functions for profile likelihoods and introduce them into two standard quantitative trait locus detection methods-the interval mapping method and single marker analysis. Besides influence analysis on specific LOD scores, we also develop influence analysis methods on the shape of the LOD score curves. A simulation-based method is proposed to assess the significance of the influence of the individuals. These methods are shown useful in the influence analysis of a real dataset of an experimental population from an F2 mouse cross. By receiver operating characteristic analysis, we confirm that the proposed methods show better performance than existing diagnostics. © 2014 The Author. Biometrical Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development and evaluation of a quantitative PCR assay for detection of Hepatozoon sp.

PubMed

Criado-Fornelio, A; Buling, A; Cunha-Filho, N A; Ruas, J L; Farias, N A R; Rey-Valeiron, C; Pingret, J L; Etievant, M; Barba-Carretero, J C

2007-12-25

With the aim to improve current molecular diagnostic techniques of Hepatozoon sp. in carnivore mammals, we developed a quantitative PCR (qPCR) assay with SYBR Green I((R)). The method, consisting of amplification of a 235bp fragment of the 18S rRNA gene, is able to detect at least 0.1fg of parasite DNA. Reproducible quantitative results were obtained over a range of 0.1ng-0.1fg of Hepatozoon sp. DNA. To assess the performance of the qPCR assay, DNA samples from dogs (140) and cats (50) were tested with either standard PCR or qPCR. Positive samples were always confirmed by partial sequencing of the 18S rRNA gene. Quantitative PCR was 15.8% more sensitive than standard PCR to detect H. canis in dogs. In cats, no infections were detected by standard PCR, compared to two positives by qPCR (which were infected by H. canis as shown by sequencing).

ARE WE THERE YET? TIME TO DETECTION OF NANOHERTZ GRAVITATIONAL WAVES BASED ON PULSAR-TIMING ARRAY LIMITS

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

Taylor, S. R.; Vallisneri, M.; Ellis, J. A.

2016-03-01

Decade-long timing observations of arrays of millisecond pulsars have placed highly constraining upper limits on the amplitude of the nanohertz gravitational-wave stochastic signal from the mergers of supermassive black hole binaries (∼10{sup −15} strain at f = 1 yr{sup −1}). These limits suggest that binary merger rates have been overestimated, or that environmental influences from nuclear gas or stars accelerate orbital decay, reducing the gravitational-wave signal at the lowest, most sensitive frequencies. This prompts the question whether nanohertz gravitational waves (GWs) are likely to be detected in the near future. In this Letter, we answer this question quantitatively using simple statistical estimates,more » deriving the range of true signal amplitudes that are compatible with current upper limits, and computing expected detection probabilities as a function of observation time. We conclude that small arrays consisting of the pulsars with the least timing noise, which yield the tightest upper limits, have discouraging prospects of making a detection in the next two decades. By contrast, we find large arrays are crucial to detection because the quadrupolar spatial correlations induced by GWs can be well sampled by many pulsar pairs. Indeed, timing programs that monitor a large and expanding set of pulsars have an ∼80% probability of detecting GWs within the next 10 years, under assumptions on merger rates and environmental influences ranging from optimistic to conservative. Even in the extreme case where 90% of binaries stall before merger and environmental coupling effects diminish low-frequency gravitational-wave power, detection is delayed by at most a few years.« less

A differential mobility spectrometry/mass spectrometry platform for the rapid detection and quantitation of DNA adduct dG-ABP.

PubMed

Kafle, Amol; Klaene, Joshua; Hall, Adam B; Glick, James; Coy, Stephen L; Vouros, Paul

2013-07-15

There is continued interest in exploring new analytical technologies for the detection and quantitation of DNA adducts, biomarkers which provide direct evidence of exposure and genetic damage in cells. With the goal of reducing clean-up steps and improving sample throughput, a Differential Mobility Spectrometry/Mass Spectrometry (DMS/MS) platform has been introduced for adduct analysis. A DMS/MS platform has been utilized for the analysis of dG-ABP, the deoxyguanosine adduct of the bladder carcinogen 4-aminobiphenyl (4-ABP). After optimization of the DMS parameters, each sample was analyzed in just 30 s following a simple protein precipitation step of the digested DNA. A detection limit of one modification in 10^6 nucleosides has been achieved using only 2 µg of DNA. A brief comparison (quantitative and qualitative) with liquid chromatography/mass spectrometry is also presented highlighting the advantages of using the DMS/MS method as a high-throughput platform. The data presented demonstrate the successful application of a DMS/MS/MS platform for the rapid quantitation of DNA adducts using, as a model analyte, the deoxyguanosine adduct of the bladder carcinogen 4-aminobiphenyl. Copyright © 2013 John Wiley & Sons, Ltd.

Rapid Trace Detection and Isomer Quantitation of Pesticide Residues via Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.

PubMed

Wu, Xinzhou; Li, Weifeng; Guo, Pengran; Zhang, Zhixiang; Xu, Hanhong

2018-04-18

Matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) has been applied for rapid, sensitive, undisputed, and quantitative detection of pesticide residues on fresh leaves with little sample pretreatment. Various pesticides (insecticides, bactericides, herbicides, and acaricides) are detected directly in the complex matrix with excellent limits of detection down to 4 μg/L. FTICR-MS could unambiguously identify pesticides with tiny mass differences (∼0.017 75 Da), thereby avoiding false-positive results. Remarkably, pesticide isomers can be totally discriminated by use of diagnostic fragments, and quantitative analysis of pesticide isomers is demonstrated. The present results expand the horizons of the MALDI-FTICR-MS platform in the reliable determination of pesticides, with integrated advantages of ultrahigh mass resolution and accuracy. This method provides growing evidence for the resultant detrimental effects of pesticides, expediting the identification and evaluation of innovative pesticides.

Temporal Lobe Epilepsy: Quantitative MR Volumetry in Detection of Hippocampal Atrophy

PubMed Central

Farid, Nikdokht; Girard, Holly M.; Kemmotsu, Nobuko; Smith, Michael E.; Magda, Sebastian W.; Lim, Wei Y.; Lee, Roland R.

2012-01-01

Purpose: To determine the ability of fully automated volumetric magnetic resonance (MR) imaging to depict hippocampal atrophy (HA) and to help correctly lateralize the seizure focus in patients with temporal lobe epilepsy (TLE). Materials and Methods: This study was conducted with institutional review board approval and in compliance with HIPAA regulations. Volumetric MR imaging data were analyzed for 34 patients with TLE and 116 control subjects. Structural volumes were calculated by using U.S. Food and Drug Administration–cleared software for automated quantitative MR imaging analysis (NeuroQuant). Results of quantitative MR imaging were compared with visual detection of atrophy, and, when available, with histologic specimens. Receiver operating characteristic analyses were performed to determine the optimal sensitivity and specificity of quantitative MR imaging for detecting HA and asymmetry. A linear classifier with cross validation was used to estimate the ability of quantitative MR imaging to help lateralize the seizure focus. Results: Quantitative MR imaging–derived hippocampal asymmetries discriminated patients with TLE from control subjects with high sensitivity (86.7%–89.5%) and specificity (92.2%–94.1%). When a linear classifier was used to discriminate left versus right TLE, hippocampal asymmetry achieved 94% classification accuracy. Volumetric asymmetries of other subcortical structures did not improve classification. Compared with invasive video electroencephalographic recordings, lateralization accuracy was 88% with quantitative MR imaging and 85% with visual inspection of volumetric MR imaging studies but only 76% with visual inspection of clinical MR imaging studies. Conclusion: Quantitative MR imaging can depict the presence and laterality of HA in TLE with accuracy rates that may exceed those achieved with visual inspection of clinical MR imaging studies. Thus, quantitative MR imaging may enhance standard visual analysis, providing a

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

PubMed

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

2005-11-30

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

Search times and probability of detection in time-limited search

NASA Astrophysics Data System (ADS)

Wilson, David; Devitt, Nicole; Maurer, Tana

2005-05-01

When modeling the search and target acquisition process, probability of detection as a function of time is important to war games and physical entity simulations. Recent US Army RDECOM CERDEC Night Vision and Electronics Sensor Directorate modeling of search and detection has focused on time-limited search. Developing the relationship between detection probability and time of search as a differential equation is explored. One of the parameters in the current formula for probability of detection in time-limited search corresponds to the mean time to detect in time-unlimited search. However, the mean time to detect in time-limited search is shorter than the mean time to detect in time-unlimited search and the relationship between them is a mathematical relationship between these two mean times. This simple relationship is derived.

Quantitative detection of type A staphylococcal enterotoxin by Laurell electroimmunodiffusion.

PubMed

Gasper, E; Heimsch, R C; Anderson, A W

1973-03-01

The detection of staphylococcal enterotoxin A by the quantitative technique of electroimmunodiffusion is described. High dilutions of type-specific rabbit antiserum were used in 1% agarose gels, 1 mm thick, and prepared in 0.05-mug barbital buffer, pH 8.6. Volumes of 10 muliters containing 1.5 to 10 ng of toxin were electrophoresed out of 4-mm diameter wells at 5 mA/cm width of gel. The precipitin cones formed were made visible by first immersing the agarose gels in 0.2 M NaCl and then overlaying the surface with the purified globulin fraction of sheep serum against rabbit globulin, followed by soaking of the gels in 1% aqueous cadmium acetate and staining with 0.1% thiazine red in 1% glacial acetic acid. Fully extended cones, 4 to 23 mm in length depending on toxin concentration and antiserum dilution, were developed in 2 to 5 h of electrophoresis, and visualization was achieved within 2 to 3 h. Because the method is qualitative, quantitative, simple, rapid, and sensitive, it offers a practical tool for the detection of small amounts of bacterial toxins in contaminated foods. The method should also qualify as a sensitive detection device in biochemical procedures which attempt to trace, detect, and identify biological substances in nanogram quantities, provided these substances are antigenic and capable of forming a precipitate with their specific antibodies.

Validated semiquantitative/quantitative screening of 51 drugs in whole blood as silylated derivatives by gas chromatography-selected ion monitoring mass spectrometry and gas chromatography electron capture detection.

PubMed

Gunnar, Teemu; Mykkänen, Sirpa; Ariniemi, Kari; Lillsunde, Pirjo

2004-07-05

A comprehensively validated procedure is presented for simultaneous semiquantitative/quantitative screening of 51 drugs of abuse or drugs potentially hazardous for traffic safety in serum, plasma or whole blood. Benzodiazepines (12), cannabinoids (3), opioids (8), cocaine, antidepressants (13), antipsychotics (5) and antiepileptics (2) as well as zolpidem, zaleplon, zopiclone, meprobamate, carisoprodol, tizanidine and orphenadrine and internal standard flurazepam, were isolated by high-yield liquid-liquid extraction (LLE). The dried extracts were derivatized by two-step silylation and analyzed by the combination of two different gas chromatographic (GC) separations with both electron capture detection (ECD) and mass spectrometry (MS) operating in a selected ion-monitoring (SIM) mode. Quantitative or semiquantitative results were obtained for each substance based on four-point calibration. In the validation tests, accuracy, reproducibility, linearity, limit of detection (LOD) and limit of quantitation (LOQ), selectivity, as well as extraction efficiency and stability of standard stock solutions were tested, and derivatization was optimized in detail. Intra- and inter-day precisions were within 2.5-21.8 and 6.0-22.5%, and square of correlation coefficients of linearity ranged from 0.9896 to 0.9999. The limit of quantitation (LOQ) varied from 2 to 2000 ng/ml due to a variety of the relevant concentrations of the analyzed substances in blood. The method is feasible for highly sensitive, reliable and possibly routinely performed clinical and forensic toxicological analyses.

Development of a quantitative PCR for detection of Lactobacillus plantarum starters during wine malolactic fermentation.

PubMed

Cho, Gyu-Sung; Krauss, Sabrina; Huch, Melanie; Du Toit, Maret; Franz, Charles M A P

2011-12-01

A quantitative, real-time PCR method was developed to enumerate Lactobacillus plantarum IWBT B 188 during the malolactic fermentation (MLF) in Grauburgunder wine. The qRT-PCR was strain-specific, as it was based on primers targeting a plasmid DNA sequence, or it was L. plantarum-specific, as it targeted a chromosomally located plantaricin gene sequence. Two 50 l wine fermentations were prepared. One was inoculated with 15 g/hl Saccharomyces cerevisiae, followed by L. plantarum IWBT B 188 at 3.6 × 10(6) CFU/ml, whereas the other was not inoculated (control). Viable cell counts were performed for up to 25 days on MRS agar, and the same cells were enumerated by qRT-PCR with both the plasmid or chromosomally encoded gene primers. The L. plantarum strain survived under the harsh conditions in the wine fermentation at levels above 10(5)/ml for approx. 10 days, after which cell numbers decreased to levels of 10(3) CFU/ml at day 25, and to below the detection limit after day 25. In the control, no lactic acid bacteria could be detected throughout the fermentation, with the exception of two sampling points where ca. 1 × 10(2) CFU/ml was detected. The minimum detection level for quantitative PCR in this study was 1 × 10(2) to 1 × 10(3) CFU/ml. The qRT-PCR results determined generally overestimated the plate count results by about 1 log unit, probably as a result of the presence of DNA from dead cells. Overall, qRT-PCR appeared to be well suited for specifically enumerating Lactobacillus plantarum starter cultures in the MLF in wine.

Quantitative surface-enhanced resonance Raman scattering of phthalocyanine-labelled oligonucleotides

PubMed Central

Macaskill, A.; Chernonosov, A. A.; Koval, V. V.; Lukyanets, E. A.; Fedorova, O. S.; Smith, W. E.; Faulds, K.; Graham, D.

2007-01-01

The evaluation of phthalocyanine labels for the surface-enhanced resonance Raman scattering (SERRS) detection of oligonucleotides is reported. Three phthalocyanine-labelled oligonucleotides were assessed, each containing a different metal centre. Detection limits for each labelled oligonucleotide were determined using two excitation frequencies where possible. Limits of detection as low as 2.8 × 10−11 mol. dm−3 were obtained which are comparable to standard fluorescently labelled probes used in previous SERRS studies. The identification of two phthalocyanine-labelled oligonucleotides without separation was also demonstrated indicating their suitability for multiplexing. This study extends the range of labels suitable for quantitative surface-enhanced resonance Raman scattering with silver nanoparticles and offers more flexibility and choice when considering SERRS for quantitative DNA detection. PMID:17289751

Development of strain-specific PCR primers for quantitative detection of Bacillus mesentericus strain TO-A in human feces.

PubMed

Sato, Naoki; Seo, Genichiro; Benno, Yoshimi

2014-01-01

Strain-specific polymerase chain reaction (PCR) primers for detection of Bacillus mesentericus strain TO-A (BM TO-A) were developed. The randomly amplified polymorphic DNA (RAPD) technique was used to produce potential strain-specific markers. A 991-bp RAPD marker found to be strain-specific was sequenced, and two primer pairs specific to BM TO-A were constructed based on this sequence. In addition, we explored a more specific DNA region using inverse PCR, and designed a strain-specific primer set for use in real-time quantitative PCR (qPCR). These primer pairs were tested against 25 Bacillus subtilis strains and were found to be strain-specific. After examination of the detection limit and linearity of detection of BM TO-A in feces, the qPCR method and strain-specific primers were used to quantify BM TO-A in the feces of healthy volunteers who had ingested 3×10(8) colony forming unit (CFU) of BM TO-A per day in tablets. During the administration period, BM TO-A was detected in the feces of all 24 subjects, and the average number of BM TO-A detected using the culture method and qPCR was about 10(4.8) and 10(5.8) cells per gram of feces, respectively. Using the qPCR method, BM TO-A was detected in the feces of half of the subjects 3 d after withdrawal, and was detected in the feces of only one subject 1 week after withdrawal. These results suggest that the qPCR method using BM TO-A strain-specific primers is useful for the quantitative detection of this strain in feces.

Detection and quantitation of Kaposi's sarcoma-associated herpesvirus (KSHV) by a single competitive-quantitative polymerase chain reaction.

PubMed

Curreli, Francesca; Robles, Monica A; Friedman-Kien, Alvin E; Flore, Ornella

2003-02-01

Kaposi's sarcoma-associated herpesvirus is a novel herpesvirus linked to AIDS-related neoplasms. Currently it is difficult to evaluate the number of virions in viral preparation or in samples obtained from patients with Kaposi's sarcoma (KS), since no protocol for determining the plaque forming units of KSHV exists. We constructed a fragment of a different size than the target viral DNA to carry out a competitive-quantitative PCR. Both fragment and viral DNA were added to a single PCR reaction to compete for the same set of primers. By knowing the amount of the competitor added to the reaction, we could determine the number of viral DNA molecules. We used this assay successfully to detect and quantify KSHV genomes from KS skin biopsies and pleural effusion lymphoma, and from different viral preparations. To date, this is the most convenient and economic method that allows an accurate and fast viral detection/quantitation with a single PCR.

Quantitative PCR and Digital PCR for Detection of Ascaris lumbricoides Eggs in Reclaimed Water

PubMed Central

Santísima-Trinidad, Ana Belén; Bornay-Llinares, Fernando Jorge; Martín González, Marcos; Pascual Valero, José Antonio; Ros Muñoz, Margarita

2017-01-01

The reuse of reclaimed water from wastewater depuration is a widespread and necessary practice in many areas around the world and must be accompanied by adequate and continuous quality control. Ascaris lumbricoides is one of the soil-transmitted helminths (STH) with risk for humans due to its high infectivity and an important determinant of transmission is the inadequacy of water supplies and sanitation. The World Health Organization (WHO) recommends a limit equal to or lower than one parasitic helminth egg per liter, to reuse reclaimed water for unrestricted irrigation. We present two new protocols of DNA extraction from large volumes of reclaimed water. Quantitative PCR (qPCR) and digital PCR (dPCR) were able to detect low amounts of A. lumbricoides eggs. By using the first extraction protocol, which processes 500 mL of reclaimed water, qPCR can detect DNA concentrations as low as one A. lumbricoides egg equivalent, while dPCR can detect DNA concentrations as low as five A. lumbricoides egg equivalents. By using the second protocol, which processes 10 L of reclaimed water, qPCR was able to detect DNA concentrations equivalent to 20 A. lumbricoides eggs. This fact indicated the importance of developing new methodologies to detect helminth eggs with higher sensitivity and precision avoiding possible human infection risks. PMID:28377928

Measurement issues associated with quantitative molecular biology analysis of complex food matrices for the detection of food fraud.

PubMed

Burns, Malcolm; Wiseman, Gordon; Knight, Angus; Bramley, Peter; Foster, Lucy; Rollinson, Sophie; Damant, Andrew; Primrose, Sandy

2016-01-07

Following a report on a significant amount of horse DNA being detected in a beef burger product on sale to the public at a UK supermarket in early 2013, the Elliott report was published in 2014 and contained a list of recommendations for helping ensure food integrity. One of the recommendations included improving laboratory testing capacity and capability to ensure a harmonised approach for testing for food authenticity. Molecular biologists have developed exquisitely sensitive methods based on the polymerase chain reaction (PCR) or mass spectrometry for detecting the presence of particular nucleic acid or peptide/protein sequences. These methods have been shown to be specific and sensitive in terms of lower limits of applicability, but they are largely qualitative in nature. Historically, the conversion of these qualitative techniques into reliable quantitative methods has been beset with problems even when used on relatively simple sample matrices. When the methods are applied to complex sample matrices, as found in many foods, the problems are magnified resulting in a high measurement uncertainty associated with the result which may mean that the assay is not fit for purpose. However, recent advances in the technology and the understanding of molecular biology approaches have further given rise to the re-assessment of these methods for their quantitative potential. This review focuses on important issues for consideration when validating a molecular biology assay and the various factors that can impact on the measurement uncertainty of a result associated with molecular biology approaches used in detection of food fraud, with a particular focus on quantitative PCR-based and proteomics assays.

Detection of Prostate Cancer: Quantitative Multiparametric MR Imaging Models Developed Using Registered Correlative Histopathology.

PubMed

Metzger, Gregory J; Kalavagunta, Chaitanya; Spilseth, Benjamin; Bolan, Patrick J; Li, Xiufeng; Hutter, Diane; Nam, Jung W; Johnson, Andrew D; Henriksen, Jonathan C; Moench, Laura; Konety, Badrinath; Warlick, Christopher A; Schmechel, Stephen C; Koopmeiners, Joseph S

2016-06-01

Purpose To develop multiparametric magnetic resonance (MR) imaging models to generate a quantitative, user-independent, voxel-wise composite biomarker score (CBS) for detection of prostate cancer by using coregistered correlative histopathologic results, and to compare performance of CBS-based detection with that of single quantitative MR imaging parameters. Materials and Methods Institutional review board approval and informed consent were obtained. Patients with a diagnosis of prostate cancer underwent multiparametric MR imaging before surgery for treatment. All MR imaging voxels in the prostate were classified as cancer or noncancer on the basis of coregistered histopathologic data. Predictive models were developed by using more than one quantitative MR imaging parameter to generate CBS maps. Model development and evaluation of quantitative MR imaging parameters and CBS were performed separately for the peripheral zone and the whole gland. Model accuracy was evaluated by using the area under the receiver operating characteristic curve (AUC), and confidence intervals were calculated with the bootstrap procedure. The improvement in classification accuracy was evaluated by comparing the AUC for the multiparametric model and the single best-performing quantitative MR imaging parameter at the individual level and in aggregate. Results Quantitative T2, apparent diffusion coefficient (ADC), volume transfer constant (K(trans)), reflux rate constant (kep), and area under the gadolinium concentration curve at 90 seconds (AUGC90) were significantly different between cancer and noncancer voxels (P < .001), with ADC showing the best accuracy (peripheral zone AUC, 0.82; whole gland AUC, 0.74). Four-parameter models demonstrated the best performance in both the peripheral zone (AUC, 0.85; P = .010 vs ADC alone) and whole gland (AUC, 0.77; P = .043 vs ADC alone). Individual-level analysis showed statistically significant improvement in AUC in 82% (23 of 28) and 71% (24 of 34

A competitive enzyme immunoassay for the quantitative detection of cocaine from banknotes and latent fingermarks.

PubMed

van der Heide, Susan; Garcia Calavia, Paula; Hardwick, Sheila; Hudson, Simon; Wolff, Kim; Russell, David A

2015-05-01

A sensitive and versatile competitive enzyme immunoassay (cEIA) has been developed for the quantitative detection of cocaine in complex forensic samples. Polyclonal anti-cocaine antibody was purified from serum and deposited onto microtiter plates. The concentration of the cocaine antibody adsorbed onto the plates, and the dilution of the cocaine-HRP hapten were both studied to achieve an optimised immunoassay. The method was successfully used to quantify cocaine in extracts taken from both paper currency and latent fingermarks. The limit of detection (LOD) of 0.162ngmL(-1) achieved with the assay compares favourably to that of conventional chromatography-mass spectroscopy techniques, with an appropriate sensitivity for the quantification of cocaine at the low concentrations present in some forensic samples. The cEIA was directly compared to LC-MS for the analysis of ten UK banknote samples. The results obtained from both techniques were statistically similar, suggesting that the immunoassay was unaffected by cross-reactivity with potentially interfering compounds. The cEIA was used also for the detection of cocaine in extracts from latent fingermarks. The results obtained were compared to the cocaine concentrations detected in oral fluid sampled from the same individual. Using the cEIA, we have shown, for the first time, that endogeneously excreted cocaine can be detected and quantified from a single latent fingermark. Additionally, it has been shown that the presence of cocaine, at similar concentrations, in more than one latent fingermark from the same individual can be linked with those concentrations found in oral fluid. These results show that detection of drugs in latent fingermarks could directly indicate whether an individual has consumed the drug. The specificity and feasibility of measuring low concentrations of cocaine in complex forensic samples demonstrate the effectiveness and robustness of the assay. The immunoassay presents a simple and cost

SU-D-210-03: Limited-View Multi-Source Quantitative Photoacoustic Tomography

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

Feng, J; Gao, H

2015-06-15

Purpose: This work is to investigate a novel limited-view multi-source acquisition scheme for the direct and simultaneous reconstruction of optical coefficients in quantitative photoacoustic tomography (QPAT), which has potentially improved signal-to-noise ratio and reduced data acquisition time. Methods: Conventional QPAT is often considered in two steps: first to reconstruct the initial acoustic pressure from the full-view ultrasonic data after each optical illumination, and then to quantitatively reconstruct optical coefficients (e.g., absorption and scattering coefficients) from the initial acoustic pressure, using multi-source or multi-wavelength scheme.Based on a novel limited-view multi-source scheme here, We have to consider the direct reconstruction of opticalmore » coefficients from the ultrasonic data, since the initial acoustic pressure can no longer be reconstructed as an intermediate variable due to the incomplete acoustic data in the proposed limited-view scheme. In this work, based on a coupled photo-acoustic forward model combining diffusion approximation and wave equation, we develop a limited-memory Quasi-Newton method (LBFGS) for image reconstruction that utilizes the adjoint forward problem for fast computation of gradients. Furthermore, the tensor framelet sparsity is utilized to improve the image reconstruction which is solved by Alternative Direction Method of Multipliers (ADMM). Results: The simulation was performed on a modified Shepp-Logan phantom to validate the feasibility of the proposed limited-view scheme and its corresponding image reconstruction algorithms. Conclusion: A limited-view multi-source QPAT scheme is proposed, i.e., the partial-view acoustic data acquisition accompanying each optical illumination, and then the simultaneous rotations of both optical sources and ultrasonic detectors for next optical illumination. Moreover, LBFGS and ADMM algorithms are developed for the direct reconstruction of optical coefficients from

Development and in-house validation of the event-specific qualitative and quantitative PCR detection methods for genetically modified cotton MON15985.

PubMed

Jiang, Lingxi; Yang, Litao; Rao, Jun; Guo, Jinchao; Wang, Shu; Liu, Jia; Lee, Seonghun; Zhang, Dabing

2010-02-01

To implement genetically modified organism (GMO) labeling regulations, an event-specific analysis method based on the junction sequence between exogenous integration and host genomic DNA has become the preferential approach for GMO identification and quantification. In this study, specific primers and TaqMan probes based on the revealed 5'-end junction sequence of GM cotton MON15985 were designed, and qualitative and quantitative polymerase chain reaction (PCR) assays were established employing the designed primers and probes. In the qualitative PCR assay, the limit of detection (LOD) was 0.5 g kg(-1) in 100 ng total cotton genomic DNA, corresponding to about 17 copies of haploid cotton genomic DNA, and the LOD and limit of quantification (LOQ) for quantitative PCR assay were 10 and 17 copies of haploid cotton genomic DNA, respectively. Furthermore, the developed quantitative PCR assays were validated in-house by five different researchers. Also, five practical samples with known GM contents were quantified using the developed PCR assay in in-house validation, and the bias between the true and quantification values ranged from 2.06% to 12.59%. This study shows that the developed qualitative and quantitative PCR methods are applicable for the identification and quantification of GM cotton MON15985 and its derivates.

Quantitation of free polyethylene glycol in PEGylated protein conjugate by size exclusion HPLC with refractive index (RI) detection.

PubMed

Li, Ning; Ziegemeier, Daisy; Bass, Laura; Wang, Wei

2008-12-15

In this study, size exclusion high performance liquid chromatography was evaluated for its application in separation and quantitation of free polyethylene glycol (PEG) and its PEGylated-protein-conjugate (PEG-conjugate). Although the large mass of the free PEG (2-fold greater than the protein) made separation difficult, chromatographic conditions were identified enabling resolution and quantitation of the free PEG, PEG-conjugate and non-PEGylated protein with Shodex Protein KW803 and KW804 columns in series and refractive index detection. The optimum resolution of 1.7 and 2.0 was achieved for the free PEG and PEG-conjugate as well as the free PEG and non-PEGylated protein using 20mM HEPES buffer at pH 6.5. Under this condition, the plot of log(10)MW of all the pertinent analytes against retention time showed a linear relationship with a correlation coefficient of 1. Limited assay performance evaluation demonstrated that the method was linear in the concentration range of 10 to 250 microg/mL of free PEG with correlation coefficients of > or = 0.99. When free PEG in this concentration range was spiked into PEG-conjugate samples at 1mg/mL, the recovery was in the range of 78%-120%. Detection and quantitation limits were determined to be, respectively, 10 and 25 microg/mL for free PEG. The R.S.D. for intra- and inter-day precision was 0.09% or less for retention time measurements and 2.9% or less for area count measurements. Robustness testing was performed by deliberately deviating +/-0.2 pH units away from the desired pH as well as by increasing the flow rate. These deviations resulted in no significant impact on area percent distribution of all species. However, separation was found to be sensitive to high ionic strength and buffer species.

Quantitative PCR: an appropriate tool to detect viable but not culturable Brettanomyces bruxellensis in wine.

PubMed

Willenburg, Elize; Divol, Benoit

2012-11-15

Quantitative PCR as a tool has been used to detect Brettanomyces bruxellensis directly from wine samples. Accurate and timely detection of this yeast is important to prevent unwanted spoilage of wines and beverages. The aim of this study was to distinguish differences between DNA and mRNA as template for the detection of this yeast. The study was also used to determine if it is possible to accurately detect cells in the viable but not culturable (VBNC) state of B. bruxellensis by qPCR. Several methods including traditional plating, epifluorescence counts and qPCR were used to amplify DNA and mRNA. It was observed that mRNA was a better template for the detection in terms of standard curve analysis and qPCR efficiencies. Various primers previously published were tested for their specificity, qPCR efficiency and accuracy of enumeration. A single primer set was selected which amplified a region of the actin-encoding gene. The detection limit for this assay was 10cellsmL(-1). B. bruxellensis could also be quantified in naturally contaminated wines with this assay. The mRNA gave a better indication of the viability of the cells which compared favourably to fluorescent microscopy and traditional cell counts. The ability of the assay to accurately estimate the number of cells in the VBNC state was also demonstrated. Copyright © 2012 Elsevier B.V. All rights reserved.

USE OF METHOD DETECTION LIMITS IN ENVIRONMENTAL MEASUREMENTS

EPA Science Inventory

Environmental measurements often produce values below the method detection limit (MDL). Because low or zero values may be used in determining compliance with regulatory limits, in determining emission factors (typical concentrations emitted by a given type of source), or in model...

Quantitative Ultrasound Assessment of Duchenne Muscular Dystrophy Using Edge Detection Analysis.

PubMed

Koppaka, Sisir; Shklyar, Irina; Rutkove, Seward B; Darras, Basil T; Anthony, Brian W; Zaidman, Craig M; Wu, Jim S

2016-09-01

The purpose of this study was to investigate the ability of quantitative ultrasound (US) using edge detection analysis to assess patients with Duchenne muscular dystrophy (DMD). After Institutional Review Board approval, US examinations with fixed technical parameters were performed unilaterally in 6 muscles (biceps, deltoid, wrist flexors, quadriceps, medial gastrocnemius, and tibialis anterior) in 19 boys with DMD and 21 age-matched control participants. The muscles of interest were outlined by a tracing tool, and the upper third of the muscle was used for analysis. Edge detection values for each muscle were quantified by the Canny edge detection algorithm and then normalized to the number of edge pixels in the muscle region. The edge detection values were extracted at multiple sensitivity thresholds (0.01-0.99) to determine the optimal threshold for distinguishing DMD from normal. Area under the receiver operating curve values were generated for each muscle and averaged across the 6 muscles. The average age in the DMD group was 8.8 years (range, 3.0-14.3 years), and the average age in the control group was 8.7 years (range, 3.4-13.5 years). For edge detection, a Canny threshold of 0.05 provided the best discrimination between DMD and normal (area under the curve, 0.96; 95% confidence interval, 0.84-1.00). According to a Mann-Whitney test, edge detection values were significantly different between DMD and controls (P < .0001). Quantitative US imaging using edge detection can distinguish patients with DMD from healthy controls at low Canny thresholds, at which discrimination of small structures is best. Edge detection by itself or in combination with other tests can potentially serve as a useful biomarker of disease progression and effectiveness of therapy in muscle disorders.

Quantitative polymerase chain reaction detection of circulating DNA in serum for early diagnosis of mucormycosis in immunocompromised patients.

PubMed

Millon, Laurence; Larosa, Fabrice; Lepiller, Quentin; Legrand, Faezeh; Rocchi, Steffi; Daguindau, Etienne; Scherer, Emeline; Bellanger, Anne-Pauline; Leroy, Joel; Grenouillet, Frederic

2013-05-01

The aim of our study was to assess the detection of circulating DNA from the most common species of Mucorales for early diagnosis of mucormycosis in at-risk patients. We retrospectively evaluated a combination of 3 quantitative polymerase chain reaction (qPCR) assays using hydrolysis probes targeting Mucor/Rhizopus, Lichtheimia (formerly Absidia), and Rhizomucor for circulating Mucorales detection. Serial serum samples from 10 patients diagnosed with proven mucormycosis (2-9 samples per patient) were analyzed. No cross-reactivity was detected in the 3 qPCR assays using 19 reference strains of opportunistic fungi, and the limit of detection ranged from 3.7 to 15 femtograms/10 µL, depending on the species. DNA from Mucorales was detected in the serum of 9 of 10 patients between 68 and 3 days before mucormycosis diagnosis was confirmed by histopathological examination and/or positive culture. All the qPCR results were concordant with culture and/or PCR-based identification of the causing agents in tissue (Lichtheimia species, Rhizomucor species, and Mucor/Rhizopus species in 4, 3, and 2 patients, respectively). Quantitative PCR was negative in only 1 patient with proven disseminated mucormycosis caused by Lichtheimia species. Our study suggests that using specific qPCR targeting several species of Mucorales according to local ecology to screen at-risk patients could be useful in a clinical setting. The cost and efficacy of this strategy should be evaluated. However, given the human and economic cost of mucormycosis and the need for rapid diagnosis to initiate prompt directed antifungal therapy, this strategy could be highly attractive.

PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method.

PubMed

Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R

2008-05-15

A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target-nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2,2'-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.

Colloidal silver nanoparticles prepared by UV-light induced citrate reduction technique for the quantitative detection of uric acid

NASA Astrophysics Data System (ADS)

Maity, Anupam; Panda, Sovan Kumar

2018-04-01

Reddish-yellow color colloid consisting of silver nanoparticles (Ag NPs) has been synthesized by reducing aqueous AgNO3 solution by photo-induced citrate reduction technique under UV light. As prepared colloid exhibits single and intense plasmonic absorption peak in the violet region of the visible spectra with the peak centered at 405 nm. The NPs are fine and spherical with diameter ranging from 5 to 10 nm. These colloidal NPs have been used for the quantitative detection of uric acid by UV-VIS spectroscopy. A linear red shifting of the characteristics Plasmonic absorption peak of Ag NPs is observed with uric acid concentration. Uric acid can be detected by UV-VIS spectroscopy down to 5 nM limit using the prepared colloid.

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

PubMed Central

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

2006-01-01

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

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

PubMed

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

2006-09-01

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

Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.

PubMed

Xiang, Dongshan; Zhai, Kun; Xiang, Wenjun; Wang, Lianzhi

2014-11-01

A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

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

2009-12-09

Accurate laboratory diagnosis of malaria species in returning travelers is paramount in the treatment of this potentially fatal infectious disease. A total of 466 blood specimens from returning travelers to Africa, Asia, and South/Central America with suspected malaria infection were collected between 2007 and 2009 at the reference public health laboratory. These specimens were assessed by reference microscopy, multipex real-time quantitative polymerase chain reaction (QPCR), and two rapid diagnostic immuno-chromatographic tests (ICT) in a blinded manner. Key clinical laboratory parameters such as limit of detection (LOD) analysis on clinical specimens by parasite stage, inter-reader variability of ICTs, staffing implications, quality assurance and cost analysis were evaluated. QPCR is the most analytically sensitive method (sensitivity 99.41%), followed by CARESTART (sensitivity 88.24%), and BINAXNOW (sensitivity 86.47%) for the diagnosis of malaria in returning travelers when compared to reference microscopy. However, microscopy was unable to specifically identify Plasmodia spp. in 18 out of 170 positive samples by QPCR. Moreover, the 17 samples that were negative by microscopy and positive by QPCR were also positive by ICTs. Quality assurance was achieved for QPCR by exchanging a blinded proficiency panel with another reference laboratory. The Kappa value of inter-reader variability among three readers for BINAXNOW and CARESTART was calculated to be 0.872 and 0.898 respectively. Serial dilution studies demonstrated that the QPCR cycle threshold correlates linearly with parasitemia (R(2) = 0.9746) in a clinically relevant dynamic range and retains a LOD of 11 rDNA copies/microl for P. falciparum, which was several log lower than reference microscopy and ICTs. LOD for QPCR is affected not only by parasitemia but the parasite stage distribution of each clinical specimen. QPCR was approximately 6-fold more costly than reference microscopy. These data suggest that

Principles of quantitation of viral loads using nucleic acid sequence-based amplification in combination with homogeneous detection using molecular beacons.

PubMed

Weusten, Jos J A M; Carpay, Wim M; Oosterlaken, Tom A M; van Zuijlen, Martien C A; van de Wiel, Paul A

2002-03-15

For quantitative NASBA-based viral load assays using homogeneous detection with molecular beacons, such as the NucliSens EasyQ HIV-1 assay, a quantitation algorithm is required. During the amplification process there is a constant growth in the concentration of amplicons to which the beacon can bind while generating a fluorescence signal. The overall fluorescence curve contains kinetic information on both amplicon formation and beacon binding, but only the former is relevant for quantitation. In the current paper, mathematical modeling of the relevant processes is used to develop an equation describing the fluorescence curve as a function of the amplification time and the relevant kinetic parameters. This equation allows reconstruction of RNA formation, which is characterized by an exponential increase in concentrations as long as the primer concentrations are not rate limiting and by linear growth over time after the primer pool is depleted. During the linear growth phase, the actual quantitation is based on assessing the amplicon formation rate from the viral RNA relative to that from a fixed amount of calibrator RNA. The quantitation procedure has been successfully applied in the NucliSens EasyQ HIV-1 assay.

Detection and quantitation of HPV in genital and oral tissues and fluids by real time PCR

PubMed Central

2010-01-01

Background Human papillomaviruses (HPVs) remain a serious world health problem due to their association with anogenital/oral cancers and warts. While over 100 HPV types have been identified, a subset is associated with malignancy. HPV16 and 18 are the most prevalent oncogenic types, while HPV6 and 11 are most commonly responsible for anogenital warts. While other quantitative PCR (qPCR) assays detect oncogenic HPV, there is no single tube assay distinguishing the most frequent oncogenic types and the most common types found in warts. Results A Sybr Green-based qPCR assay was developed utilizing degenerate primers to the highly conserved HPV E1 theoretically detecting any HPV type. A single tube multiplex qPCR assay was also developed using type-specific primer pairs and TaqMan probes that allowed for detection and quantitation of HPV6,11,16,18. Each HPV type was detected over a range from 2 × 101 to 2 × 106copies/reaction providing a reliable method of quantitating type-specific HPV in 140 anogenital/cutaneous/oral benign and malignant specimens. 35 oncogenic and low risk alpha genus HPV types were detected. Concordance was detected in previously typed specimens. Comparisons to the gold standard detected an overall sensitivity of 89% (95% CI: 77% - 96%) and specificity of 90% (95%CI: 52% - 98%). Conclusion There was good agreement between the ability of the qPCR assays described here to identify HPV types in malignancies previously typed using standard methods. These novel qPCR assays will allow rapid detection and quantitation of HPVs to assess their role in viral pathogenesis. PMID:20723234

Augmenting Amyloid PET Interpretations With Quantitative Information Improves Consistency of Early Amyloid Detection.

PubMed

Harn, Nicholas R; Hunt, Suzanne L; Hill, Jacqueline; Vidoni, Eric; Perry, Mark; Burns, Jeffrey M

2017-08-01

Establishing reliable methods for interpreting elevated cerebral amyloid-β plaque on PET scans is increasingly important for radiologists, as availability of PET imaging in clinical practice increases. We examined a 3-step method to detect plaque in cognitively normal older adults, focusing on the additive value of quantitative information during the PET scan interpretation process. Fifty-five F-florbetapir PET scans were evaluated by 3 experienced raters. Scans were first visually interpreted as having "elevated" or "nonelevated" plaque burden ("Visual Read"). Images were then processed using a standardized quantitative analysis software (MIMneuro) to generate whole brain and region of interest SUV ratios. This "Quantitative Read" was considered elevated if at least 2 of 6 regions of interest had an SUV ratio of more than 1.1. The final interpretation combined both visual and quantitative data together ("VisQ Read"). Cohen kappa values were assessed as a measure of interpretation agreement. Plaque was elevated in 25.5% to 29.1% of the 165 total Visual Reads. Interrater agreement was strong (kappa = 0.73-0.82) and consistent with reported values. Quantitative Reads were elevated in 45.5% of participants. Final VisQ Reads changed from initial Visual Reads in 16 interpretations (9.7%), with most changing from "nonelevated" Visual Reads to "elevated." These changed interpretations demonstrated lower plaque quantification than those initially read as "elevated" that remained unchanged. Interrater variability improved for VisQ Reads with the addition of quantitative information (kappa = 0.88-0.96). Inclusion of quantitative information increases consistency of PET scan interpretations for early detection of cerebral amyloid-β plaque accumulation.

Quantitative and simultaneous detection of four foodborne bacterial pathogens with a multi-channel SPR sensor.

PubMed

Taylor, Allen D; Ladd, Jon; Yu, Qiuming; Chen, Shengfu; Homola, Jirí; Jiang, Shaoyi

2006-12-15

We report the quantitative and simultaneous detection of four species of bacteria, Escherichia coli O157:H7, Salmonella choleraesuis serotype typhimurium, Listeria monocytogenes, and Campylobacter jejuni, using an eight-channel surface plasmon resonance (SPR) sensor based on wavelength division multiplexing. Detection curves showing SPR response versus analyte concentration were established for each species of bacteria in buffer at pH 7.4, apple juice at native pH 3.7, and apple juice at an adjusted pH of 7.4, as well as for a mixture containing all four species of bacteria in buffer. Control experiments were performed to show the non-fouling characteristics of the sensor surface as well as the specificity of the amplification antibodies used in this study. The limit of detection (LOD) for each of the four species of bacteria in the tested matrices ranges from 3.4 x 10(3) to 1.2 x 10(5) cfu/ml. Detection curves in buffer of an individual species of bacteria in a mixture of all four species of bacteria correlated well with detection curves of the individual species of bacteria alone. SPR responses were higher for bacteria in apple juice at pH 7.4 than in apple juice at pH 3.7. This difference in sensor response could be partly attributed to the pH dependence of antibody-antigen binding.

Stochastic fluctuations and the detectability limit of network communities.

PubMed

Floretta, Lucio; Liechti, Jonas; Flammini, Alessandro; De Los Rios, Paolo

2013-12-01

We have analyzed the detectability limits of network communities in the framework of the popular Girvan and Newman benchmark. By carefully taking into account the inevitable stochastic fluctuations that affect the construction of each and every instance of the benchmark, we come to the conclusion that the native, putative partition of the network is completely lost even before the in-degree/out-degree ratio becomes equal to that of a structureless Erdös-Rényi network. We develop a simple iterative scheme, analytically well described by an infinite branching process, to provide an estimate of the true detectability limit. Using various algorithms based on modularity optimization, we show that all of them behave (semiquantitatively) in the same way, with the same functional form of the detectability threshold as a function of the network parameters. Because the same behavior has also been found by further modularity-optimization methods and for methods based on different heuristics implementations, we conclude that indeed a correct definition of the detectability limit must take into account the stochastic fluctuations of the network construction.

A web-based quantitative signal detection system on adverse drug reaction in China.

PubMed

Li, Chanjuan; Xia, Jielai; Deng, Jianxiong; Chen, Wenge; Wang, Suzhen; Jiang, Jing; Chen, Guanquan

2009-07-01

To establish a web-based quantitative signal detection system for adverse drug reactions (ADRs) based on spontaneous reporting to the Guangdong province drug-monitoring database in China. Using Microsoft Visual Basic and Active Server Pages programming languages and SQL Server 2000, a web-based system with three software modules was programmed to perform data preparation and association detection, and to generate reports. Information component (IC), the internationally recognized measure of disproportionality for quantitative signal detection, was integrated into the system, and its capacity for signal detection was tested with ADR reports collected from 1 January 2002 to 30 June 2007 in Guangdong. A total of 2,496 associations including known signals were mined from the test database. Signals (e.g., cefradine-induced hematuria) were found early by using the IC analysis. In addition, 291 drug-ADR associations were alerted for the first time in the second quarter of 2007. The system can be used for the detection of significant associations from the Guangdong drug-monitoring database and could be an extremely useful adjunct to the expert assessment of very large numbers of spontaneously reported ADRs for the first time in China.

Quantitation and detection of vanadium in biologic and pollution materials

NASA Technical Reports Server (NTRS)

Gordon, W. A.

1974-01-01

A review is presented of special considerations and methodology for determining vanadium in biological and air pollution materials. In addition to descriptions of specific analysis procedures, general sections are included on quantitation of analysis procedures, sample preparation, blanks, and methods of detection of vanadium. Most of the information presented is applicable to the determination of other trace elements in addition to vanadium.

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients.

PubMed

Zhai, Juping; Ding, Mengyuan; Yang, Tianjie; Zuo, Bin; Weng, Zhen; Zhao, Yunxiao; He, Jun; Wu, Qingyu; Ruan, Changgeng; He, Yang

2017-10-23

Platelet autoantibody detection is critical for immune thrombocytopenia (ITP) diagnosis and prognosis. Therefore, we aimed to establish a quantitative flow cytometric immunobead assay (FCIA) for ITP platelet autoantibodies evaluation. Capture microbeads coupled with anti-GPIX, -GPIb, -GPIIb, -GPIIIa and P-selectin antibodies were used to bind the platelet-bound autoantibodies complex generated from plasma samples of 250 ITP patients, 163 non-ITP patients and 243 healthy controls, a fluorescein isothiocyanate (FITC)-conjugated secondary antibody was the detector reagent and mean fluorescence intensity (MFI) signals were recorded by flow cytometry. Intra- and inter-assay variations of the quantitative FCIA assay were assessed. Comparisons of the specificity, sensitivity and accuracy between quantitative and qualitative FCIA or monoclonal antibody immobilization of platelet antigen (MAIPA) assay were performed. Finally, treatment process was monitored by our quantitative FCIA in 8 newly diagnosed ITPs. The coefficient of variations (CV) of the quantitative FCIA assay were respectively 9.4, 3.8, 5.4, 5.1 and 5.8% for anti-GPIX, -GPIb, -GPIIIa, -GPIIb and -P-selectin autoantibodies. Elevated levels of autoantibodies against platelet glycoproteins GPIX, GPIb, GPIIIa, GPIIb and P-selectin were detected by our quantitative FCIA in ITP patients compared to non-ITP patients or healthy controls. The sensitivity, specificity and accuracy of our quantitative assay were respectively 73.13, 81.98 and 78.65% when combining all 5 autoantibodies, while the sensitivity, specificity and accuracy of MAIPA assay were respectively 41.46, 90.41 and 72.81%. A quantitative FCIA assay was established. Reduced levels of platelet autoantibodies could be confirmed by our quantitative FCIA in ITP patients after corticosteroid treatment. Our quantitative assay is not only good for ITP diagnosis but also for ITP treatment monitoring.

A miniaturized optoelectronic system for rapid quantitative label-free detection of harmful species in food

NASA Astrophysics Data System (ADS)

Raptis, Ioannis; Misiakos, Konstantinos; Makarona, Eleni; Salapatas, Alexandros; Petrou, Panagiota; Kakabakos, Sotirios; Botsialas, Athanasios; Jobst, Gerhard; Haasnoot, Willem; Fernandez-Alba, Amadeo; Lees, Michelle; Valamontes, Evangelos

2016-03-01

Optical biosensors have emerged in the past decade as the most promising candidates for portable, highly-sensitive bioanalytical systems that can be employed for in-situ measurements. In this work, a miniaturized optoelectronic system for rapid, quantitative, label-free detection of harmful species in food is presented. The proposed system has four distinctive features that can render to a powerful tool for the next generation of Point-of-Need applications, namely it accommodates the light sources and ten interferometric biosensors on a single silicon chip of a less-than-40mm2 footprint, each sensor can be individually functionalized for a specific target analyte, the encapsulation can be performed at the wafer-scale, and finally it exploits a new operation principle, Broad-band Mach-Zehnder Interferometry to ameliorate its analytical capabilities. Multi-analyte evaluation schemes for the simultaneous detection of harmful contaminants, such as mycotoxins, allergens and pesticides, proved that the proposed system is capable of detecting within short time these substances at concentrations below the limits imposed by regulatory authorities, rendering it to a novel tool for the near-future food safety applications.

Detection of human herpesvirus 8 by quantitative polymerase chain reaction: development and standardisation of methods.

PubMed

Speicher, David J; Johnson, Newell W

2012-09-11

Human herpesvirus 8 (HHV-8), the aetiological agent of Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL) is rare in Australia, but endemic in Sub-Saharan Africa, parts of South-east Asia and Oceania. While the treatment of external KS lesions can be monitored by clinical observation, the internal lesions of KS, MCD and PEL require extensive and expensive internal imaging, or autopsy. In patients with MCD and PEL, if HHV-8 viraemia is not reduced quickly, ~50% die within 24 months. HHV-8 qPCR is a valuable tool for monitoring HHV-8 viraemia, but is not available in many parts of the world, including those with high prevalence of KS and HHV-8. A new molecular facility with stringent three-phase workflow was established, adhering to NPAAC and CLSI guidelines. Three fully validated quantitative assays were developed: two for detection and quantification of HHV-8; one for GAPDH, necessary for normalisation of viral loads in tissue and peripheral blood. The HHV-8 ORF73 and ORF26 qPCR assays were 100% specific. All qPCR assays, displayed a broad dynamic range (102 to 1010 copies/μL TE Buffer) with a limit of detection of 4.85x103, 5.61x102, and 2.59x102 copies/μL TE Buffer and a limit of quantification of 4.85x103, 3.01x102, and 1.38x102 copies/μL TE Buffer for HHV-8 ORF73, HHV-8 ORF26, and GAPDH respectively.The assays were tested on a panel of 35 KS biopsies from Queensland. All were HHV-8 qPCR positive with average viral load of 2.96x105 HHV-8 copies/μL DNA extract (range: 4.37x103 to 1.47x106 copies/μL DNA extract): When normalised these equate to an average viral load of 2.44x104 HHV-8 copies/103 cells (range: 2.20x102 to 7.38x105 HHV-8 copies/103 cells). These are the first fully optimised, validated and MIQE compliant HHV-8 qPCR assays established in Australia. They worked well for qualitative detection of HHV-8 in archival tissue, and are well-suited for quantitative detection in whole blood. They are now

Detection, monitoring, and quantitative analysis of wildfires with the BIRD satellite

NASA Astrophysics Data System (ADS)

Oertel, Dieter A.; Briess, Klaus; Lorenz, Eckehard; Skrbek, Wolfgang; Zhukov, Boris

2004-02-01

Increasing concern about environment and interest to avoid losses led to growing demands on space borne fire detection, monitoring and quantitative parameter estimation of wildfires. The global change research community intends to quantify the amount of gaseous and particulate matter emitted from vegetation fires, peat fires and coal seam fires. The DLR Institute of Space Sensor Technology and Planetary Exploration (Berlin-Adlershof) developed a small satellite called BIRD (Bi-spectral Infrared Detection) which carries a sensor package specially designed for fire detection. BIRD was launched as a piggy-back satellite on October 22, 2001 with ISRO"s Polar Satellite Launch Vehicle (PSLV). It is circling the Earth on a polar and sun-synchronous orbit at an altitude of 572 km and it is providing unique data for detailed analysis of high temperature events on Earth surface. The BIRD sensor package is dedicated for high resolution and reliable fire recognition. Active fire analysis is possible in the sub-pixel domain. The leading channel for fire detection and monitoring is the MIR channel at 3.8 μm. The rejection of false alarms is based on procedures using MIR/NIR (Middle Infra Red/Near Infra Red) and MIR/TIR (Middle Infra Red/Thermal Infra Red) radiance ratio thresholds. Unique results of BIRD wildfire detection and analysis over fire prone regions in Australia and Asia will be presented. BIRD successfully demonstrates innovative fire recognition technology for small satellites which permit to retrieve quantitative characteristics of active burning wildfires, such as the equivalent fire temperature, fire area, radiative energy release, fire front length and fire front strength.

Quantitative detection of the colloidal gold immunochromatographic strip in HSV color space

NASA Astrophysics Data System (ADS)

Wu, Yuanshu; Gao, Yueming; Du, Min

2014-09-01

In this paper, a fast, reliable and accurate quantitative detection method for the colloidal gold immunochromatographic strip(GICA) is presented. An image acquisition device which is mainly composed of annular LED source, zoom ratio lens, and 10bit CMOS image sensors with 54.5dB SNR is designed for the detection. Firstly, the test line is extracted from the strip window through using the H component peak points of the HSV space as the clustering centers via the Fuzzy C-Means(FCM) clustering method. Then, a two dimensional eigenvalue composed with the hue(H) and saturation(S) of HSV space was proposed to improve the accuracy of the quantitative detection. At last, the experiment of human chorionic gonadotropin(HCG) with the concentration range 0-500mIU/mL is carried out. The results show that the linear correlation coefficient between this method and optical density(OD) values measured by the fiber optical sensor reach 96.74%. Meanwhile, the linearity of fitting curve constructed with concentration was greater than 95.00%.

Detection limit used for early warning in public health surveillance.

PubMed

Kobari, Tsuyoshi; Iwaki, Kazuo; Nagashima, Tomomi; Ishii, Fumiyoshi; Hayashi, Yuzuru; Yajima, Takehiko

2009-06-01

A theory of detection limit, developed in analytical chemistry, is applied to public health surveillance to detect an outbreak of national emergencies such as natural disaster and bioterrorism. In this investigation, the influenza epidemic around the Tokyo area from 2003 to 2006 is taken as a model of normal and large-scale epidemics. The detection limit of the normal epidemic is used as a threshold with a specified level of significance to identify a sign of the abnormal epidemic among the daily variation in anti-influenza drug sales at community pharmacies. While auto-correlation of data is often an obstacle to an unbiased estimator of standard deviation involved in the detection limit, the analytical theory (FUMI) can successfully treat the auto-correlation of the drug sales in the same way as the auto-correlation appearing as 1/f noise in many analytical instruments.

Semi-quantitative visual detection of loop mediated isothermal amplification (LAMP)-generated DNA by distance-based measurement on a paper device.

PubMed

Hongwarittorrn, Irin; Chaichanawongsaroj, Nuntaree; Laiwattanapaisal, Wanida

2017-12-01

A distance-based paper analytical device (dPAD) for loop mediated isothermal amplification (LAMP) detection based on distance measurement was proposed. This approach relied on visual detection by the length of colour developed on the dPAD with reference to semi-quantitative determination of the initial amount of genomic DNA. In this communication, E. coli DNA was chosen as a template DNA for LAMP reaction. In accordance with the principle, the dPAD was immobilized by polyethylenimine (PEI), which is a strong cationic polymer, in the hydrophilic channel of the paper device. Hydroxynaphthol blue (HNB), a colourimetric indicator for monitoring the change of magnesium ion concentration in the LAMP reaction, was used to react with the immobilized PEI. The positive charges of PEI react with the negative charges of free HNB in the LAMP reaction, producing a blue colour deposit on the paper device. Consequently, the apparently visual distance appeared within 5min and length of distance correlated to the amount of DNA in the sample. The distance-based PAD for the visual detection of the LAMP reaction could quantify the initial concentration of genomic DNA as low as 4.14 × 10 3 copiesµL -1 . This distance-based visual semi-quantitative platform is suitable for choice of LAMP detection method, particular in resource-limited settings because of the advantages of low cost, simple fabrication and operation, disposability and portable detection of the dPAD device. Copyright © 2017 Elsevier B.V. All rights reserved.

Sandwiched gold/PNIPAm/gold microstructures for smart plasmonics application: towards the high detection limit and Raman quantitative measurements.

PubMed

Elashnikov, R; Mares, D; Podzimek, T; Švorčík, V; Lyutakov, O

2017-08-07

A smart plasmonic sensor, comprising a layer of a stimuli-responsive polymer sandwiched between two gold layers, is reported. As a stimuli-responsive material, a monolayer of poly(N-isopropylacrylamide) (PNIPAm) crosslinked globules is used. A quasi-periodic structure of the top gold layer facilitates efficient excitation and serves as a support for plasmon excitation and propagation. The intermediate layer of PNIPAm efficiently entraps targeted molecules from solutions. The sensor structure was optimized for efficient light focusing in the "active" PNIPAm layer. The optimization was based on the time-resolved finite-element simulations, which take into account the thickness of gold layers, size of PNIPAm globules and Raman excitation wavelength (780 nm). The prepared structures were characterized using SEM, AFM, UV-Vis refractometry and goniometry. Additional AFM scans were performed in water at two temperatures corresponding to the collapsed and swollen PNIPAm states. The Raman measurements demonstrate a high detection limit and perfect reproducibility of the Raman scattering signal for the prepared sensor. In addition, the use of created SERS structures for the detection of relevant molecules in the medical, biological and safety fields was demonstrated.

Quantitative photoacoustic elasticity and viscosity imaging for cirrhosis detection

NASA Astrophysics Data System (ADS)

Wang, Qian; Shi, Yujiao; Yang, Fen; Yang, Sihua

2018-05-01

Elasticity and viscosity assessments are essential for understanding and characterizing the physiological and pathological states of tissue. In this work, by establishing a photoacoustic (PA) shear wave model, an approach for quantitative PA elasticity imaging based on measurement of the rise time of the thermoelastic displacement was developed. Thus, using an existing PA viscoelasticity imaging method that features a phase delay measurement, quantitative PA elasticity imaging and viscosity imaging can be obtained in a simultaneous manner. The method was tested and validated by imaging viscoelastic agar phantoms prepared at different agar concentrations, and the imaging data were in good agreement with rheometry results. Ex vivo experiments on liver pathological models demonstrated the capability for cirrhosis detection, and the results were consistent with the corresponding histological results. This method expands the scope of conventional PA imaging and has potential to become an important alternative imaging modality.

PCR-free quantitative detection of genetically modified organism from raw materials – A novel electrochemiluminescence-based bio-barcode method

PubMed Central

Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R.

2018-01-01

Bio-barcode assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio-barcode assay requires lengthy experimental procedures including the preparation and release of barcode DNA probes from the target-nanoparticle complex, and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio-barcode assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2’2’-bipyridyl) ruthenium (TBR)-labele barcode DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products. PMID:18386909

Statistical Limits to Super Resolution

NASA Astrophysics Data System (ADS)

Lucy, L. B.

1992-08-01

The limits imposed by photon statistics on the degree to which Rayleigh's resolution limit for diffraction-limited images can be surpassed by applying image restoration techniques are investigated. An approximate statistical theory is given for the number of detected photons required in the image of an unresolved pair of equal point sources in order that its information content allows in principle resolution by restoration. This theory is confirmed by numerical restoration experiments on synthetic images, and quantitative limits are presented for restoration of diffraction-limited images formed by slit and circular apertures.

The limit of detection for explosives in spectroscopic differential reflectometry

NASA Astrophysics Data System (ADS)

Dubroca, Thierry; Vishwanathan, Karthik; Hummel, Rolf E.

2011-05-01

In the wake of recent terrorist attacks, such as the 2008 Mumbai hotel explosion or the December 25th 2009 "underwear bomber", our group has developed a technique (US patent #7368292) to apply differential reflection spectroscopy to detect traces of explosives. Briefly, light (200-500 nm) is shone on a surface such as a piece of luggage at an airport. Upon reflection, the light is collected with a spectrometer combined with a CCD camera. A computer processes the data and produces in turn a differential reflection spectrum involving two adjacent areas of the surface. This differential technique is highly sensitive and provides spectroscopic data of explosives. As an example, 2,4,6, trinitrotoluene (TNT) displays strong and distinct features in differential reflectograms near 420 nm. Similar, but distinctly different features are observed for other explosives. One of the most important criteria for explosive detection techniques is the limit of detection. This limit is defined as the amount of explosive material necessary to produce a signal to noise ratio of three. We present here, a method to evaluate the limit of detection of our technique. Finally, we present our sample preparation method and experimental set-up specifically developed to measure the limit of detection for our technology. This results in a limit ranging from 100 nano-grams to 50 micro-grams depending on the method and the set-up parameters used, such as the detector-sample distance.

Development of a comprehensive analytical platform for the detection and quantitation of food fraud using a biomarker approach. The oregano adulteration case study.

PubMed

Wielogorska, Ewa; Chevallier, Olivier; Black, Connor; Galvin-King, Pamela; Delêtre, Marc; Kelleher, Colin T; Haughey, Simon A; Elliott, Christopher T

2018-01-15

Due to increasing number of food fraud incidents, there is an inherent need for the development and implementation of analytical platforms enabling detection and quantitation of adulteration. In this study a set of unique biomarkers of commonly found oregano adulterants became the targets in the development of a LC-MS/MS method which underwent a rigorous in-house validation. The method presented very high selectivity and specificity, excellent linearity (R 2 >0.988) low decision limits and detection capabilities (<2%), acceptable accuracy (intra-assay 92-113%, inter-assay 69-138%) and precision (CV<20%). The method was compared with an established FTIR screening assay and revealed a good correlation of quali- and quantitative results (R 2 >0.81). An assessment of 54 suspected adulterated oregano samples revealed that almost 90% of them contained at least one bulking agent, with a median level of adulteration of 50%. Such innovative methodologies need to be established as routine testing procedures to detect and ultimately deter food fraud. Copyright © 2017 Elsevier Ltd. All rights reserved.

QUANTITATIVE DETECTION OF ENVIRONMENTALLY IMPORTANT DYES USING DIODE LASER/FIBER-OPTIC RAMAN

EPA Science Inventory

A compact diode laser/fiber-optic Raman spectrometer is used for quantitative detection of environmentally important dyes. This system is based on diode laser excitation at 782 mm, fiber optic probe technology, an imaging spectrometer, and state-of-the-art scientific CCD camera. ...

Quantitation of acrylamide in foods by high-resolution mass spectrometry.

PubMed

Troise, Antonio Dario; Fiore, Alberto; Fogliano, Vincenzo

2014-01-08

Acrylamide detection still represents one of the hottest topics in food chemistry. Solid phase cleanup coupled to liquid chromatography separation and tandem mass spectrometry detection along with GC-MS detection are nowadays the gold standard procedure for acrylamide quantitation thanks to high reproducibility, good recovery, and low relative standard deviation. High-resolution mass spectrometry (HRMS) is particularly suitable for the detection of low molecular weight amides, and it can provide some analytical advantages over other MS techniques. In this paper a liquid chromatography (LC) method for acrylamide determination using HRMS detection was developed and compared to LC coupled to tandem mass spectrometry. The procedure applied a simplified extraction, no cleanup steps, and a 4 min chromatography. It proved to be solid and robust with an acrylamide mass accuracy of 0.7 ppm, a limit of detection of 2.65 ppb, and a limit of quantitation of 5 ppb. The method was tested on four acrylamide-containing foods: cookies, French fries, ground coffee, and brewed coffee. Results were perfectly in line with those obtained by LC-MS/MS.

Quantitative Detection of Trace Malachite Green in Aquiculture Water Samples by Extractive Electrospray Ionization Mass Spectrometry

PubMed Central

Fang, Xiaowei; Yang, Shuiping; Chingin, Konstantin; Zhu, Liang; Zhang, Xinglei; Zhou, Zhiquan; Zhao, Zhanfeng

2016-01-01

Exposure to malachite green (MG) may pose great health risks to humans; thus, it is of prime importance to develop fast and robust methods to quantitatively screen the presence of malachite green in water. Herein the application of extractive electrospray ionization mass spectrometry (EESI-MS) has been extended to the trace detection of MG within lake water and aquiculture water, due to the intensive use of MG as a biocide in fisheries. This method has the advantage of obviating offline liquid-liquid extraction or tedious matrix separation prior to the measurement of malachite green in native aqueous medium. The experimental results indicate that the extrapolated detection limit for MG was ~3.8 μg·L−1 (S/N = 3) in lake water samples and ~0.5 μg·L−1 in ultrapure water under optimized experimental conditions. The signal intensity of MG showed good linearity over the concentration range of 10–1000 μg·L−1. Measurement of practical water samples fortified with MG at 0.01, 0.1 and 1.0 mg·L−1 gave a good validation of the established calibration curve. The average recoveries and relative standard deviation (RSD) of malachite green in lake water and Carassius carassius fish farm effluent water were 115% (6.64% RSD), 85.4% (9.17% RSD) and 96.0% (7.44% RSD), respectively. Overall, the established EESI-MS/MS method has been demonstrated suitable for sensitive and rapid (<2 min per sample) quantitative detection of malachite green in various aqueous media, indicating its potential for online real-time monitoring of real life samples. PMID:27529262

Quantitative Detection of Trace Malachite Green in Aquiculture Water Samples by Extractive Electrospray Ionization Mass Spectrometry.

PubMed

Fang, Xiaowei; Yang, Shuiping; Chingin, Konstantin; Zhu, Liang; Zhang, Xinglei; Zhou, Zhiquan; Zhao, Zhanfeng

2016-08-11

Exposure to malachite green (MG) may pose great health risks to humans; thus, it is of prime importance to develop fast and robust methods to quantitatively screen the presence of malachite green in water. Herein the application of extractive electrospray ionization mass spectrometry (EESI-MS) has been extended to the trace detection of MG within lake water and aquiculture water, due to the intensive use of MG as a biocide in fisheries. This method has the advantage of obviating offline liquid-liquid extraction or tedious matrix separation prior to the measurement of malachite green in native aqueous medium. The experimental results indicate that the extrapolated detection limit for MG was ~3.8 μg·L(-1) (S/N = 3) in lake water samples and ~0.5 μg·L(-1) in ultrapure water under optimized experimental conditions. The signal intensity of MG showed good linearity over the concentration range of 10-1000 μg·L(-1). Measurement of practical water samples fortified with MG at 0.01, 0.1 and 1.0 mg·L(-1) gave a good validation of the established calibration curve. The average recoveries and relative standard deviation (RSD) of malachite green in lake water and Carassius carassius fish farm effluent water were 115% (6.64% RSD), 85.4% (9.17% RSD) and 96.0% (7.44% RSD), respectively. Overall, the established EESI-MS/MS method has been demonstrated suitable for sensitive and rapid (<2 min per sample) quantitative detection of malachite green in various aqueous media, indicating its potential for online real-time monitoring of real life samples.

A Colorimetric Sensor for Qualitative Discrimination and Quantitative Detection of Volatile Amines

PubMed Central

Tang, Zhonglin; Yang, Jianhua; Yu, Junyun; Cui, Bo

2010-01-01

We have developed a novel colorimetric sensor based on a digital camera and white LED illumination. Colorimetric sensor arrays (CSAs) were made from a set of six chemically responsive dyes impregnated on an inert substrate plate by solution casting. Six common amine aqueous solutions, including dimethylamine, triethylamine, diisopropylamine, aniline, cyclohexylamine, and pyridine vaporized at 25 °C and six health-related trimethylamine (TMA) concentrations including 170 ppm, 51 ppm, 8 ppm, 2 ppm, 125 ppb and 50 ppb were analyzed by the sensor to test its ability for the qualitative discrimination and quantitative detection of volatile amines. We extracted the feature vectors of the CSA's response to the analytes from a fusional color space, which was obtained by conducting a joint search algorithm of sequential forward selection and sequential backward selection (SFS&SBS) based on the linear discriminant criteria (LDC) in a mixed color space composed of six common color spaces. The principle component analysis (PCA) followed by the hierarchical cluser analysis (HCA) were utilized to discriminate 12 analytes. Results showed that the colorimetric sensor grouped the six amine vapors and five TMA concentrations correctly, while TMA concentrations of 125 ppb and 50 ppb were indiscriminable from each other. The limitation of detection (LOD) of the sensor for TMA was found to be lower than 50 ppb. The CSAs were reusable for TMA concentrations below 8 ppm. PMID:22163560

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

PubMed

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

2010-03-01

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

Detection of colloidal silver chloride near solubility limit

NASA Astrophysics Data System (ADS)

Putri, K. Y.; Adawiah, R.

2018-03-01

Detection of nanoparticles in solution has been made possible by several means; one of them is laser-induced breakdown detection (LIBD). LIBD is able to distinguish colloids of various sizes and concentrations. This technique has been used in several solubility studies. In this study, the formation of colloids in a mixed system of silver nitrate and sodium chloride was observed by acoustic LIBD. Silver chloride has low solubility limit, therefore LIBD measurement is appropriate. Silver and chloride solutions with equal concentrations, set at below and above the solubility of silver chloride as the expected solid product, were mixed and the resulting colloids were observed. The result of LIBD measurement showed that larger particles were present as more silver and chloride introduced. However, once the concentrations exceeded the solubility limit of silver chloride, the detected particle size seemed to be decreasing, hence suggested the occurrence of coprecipitation process. This phenomenon indicated that the ability of LIBD to detect even small changes in colloid amounts might be a useful tool in study on formation and stability of colloids, i.e. to confirm whether nanoparticles synthesis has been successfully performed and whether the system is stable or not.

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

PubMed

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

2008-03-01

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

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

PubMed

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

2011-05-01

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

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

PubMed

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

1999-12-01

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

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

PubMed

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

2016-11-01

The TaqMan® real-time PCR assay using the mitochondrial D-loop region was developed for the quantitative detection of pork in processed meat products. The newly designed primers and probe specifically amplified pork without any cross-reactivity with non-target animal species. The limit of detection of the real-time PCR assay was 0.1pg of heat-treated pork meat and 0.1% (w/w) pork meat in beef and chicken meat mixtures. The quantitative real-time PCR assay was applied to analyze the pork meat content in 22 commercial processed meat products including jerkies, press hams, sausages, hamburger patties and steaks, grilled short rib patties, and nuggets. The developed real-time PCR method was able to detect pork meat in various types of processed meat products that declared the use of pork meat on their label. All processed meat products that declared no use of pork meat showed a negative result in the assay. The method developed in this study showed sensitivity and specificity in the quantification of pork meat in commercial processed meat products. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantitative detection of melamine based on terahertz time-domain spectroscopy

NASA Astrophysics Data System (ADS)

Zhao, Xiaojing; Wang, Cuicui; Liu, Shangjian; Zuo, Jian; Zhou, Zihan; Zhang, Cunlin

2018-01-01

Melamine is an organic base and a trimer of cyanamide, with a 1, 3, 5-triazine skeleton. It is usually used for the production of plastics, glue and flame retardants. Melamine combines with acid and related compounds to form melamine cyanurate and related crystal structures, which have been implicated as contaminants or biomarkers in protein adulterations by lawbreakers, especially in milk powder. This paper is focused on developing an available method for quantitative detection of melamine in the fields of security inspection and nondestructive testing based on THz-TDS. Terahertz (THz) technology has promising applications for the detection and identification of materials because it exhibits the properties of spectroscopy, good penetration and safety. Terahertz time-domain spectroscopy (THz-TDS) is a key technique that is applied to spectroscopic measurement of materials based on ultrafast femtosecond laser. In this study, the melamine and its mixture with polyethylene powder in different consistence are measured using the transmission THz-TDS. And we obtained the refractive index spectra and the absorption spectrum of different concentrations of melamine on 0.2-2.8THz. In the refractive index spectra, it is obvious to see that decline trend with the decrease of concentration; and in the absorption spectrum, two peaks of melamine at 1.98THz and 2.28THz can be obtained. Based on the experimental result, the absorption coefficient and the consistence of the melamine in the mixture are determined. Finally, methods for quantitative detection of materials in the fields of nondestructive testing and quality control based on THz-TDS have been studied.

Development and validation of a quantitative PCR for rapid and specific detection of California sea lion adenovirus 1 and prevalence in wild and managed populations.

PubMed

Cortés-Hinojosa, Galaxia; Gulland, Frances M D; Goldstein, Tracey; Venn-Watson, Stephanie; Rivera, Rebecca; Archer, Linda L; Waltzek, Thomas B; Gray, Gregory C; Wellehan, James F X

2017-03-01

California sea lion adenovirus 1 (CSLAdV-1) has been associated with hepatitis and enteritis in several wild and captive populations of diverse pinniped species. Currently available tests have been limited to pan-adenoviral polymerase chain reaction (PCR) followed by sequencing. We present the development of a quantitative probe-hybridization PCR (qPCR) assay for rapid, sensitive, and specific detection of this virus in California sea lions ( Zalophus californianus) and other pinnipeds. This assay did not amplify other mammalian adenoviruses and is able to detect consistently down to 10 viral copies per well. Compared with the gold standard conventional pan-adenovirus PCR/sequencing assay, diagnostic sensitivity and specificity of 100% and 88.2% were found, respectively. The lower diagnostic specificity of this qPCR assay may be the result of the lower limit of detection of this assay compared with the gold standard rather than the result of detection of true false-positives.

Dose limited reliability of quantitative annular dark field scanning transmission electron microscopy for nano-particle atom-counting.

PubMed

De Backer, A; Martinez, G T; MacArthur, K E; Jones, L; Béché, A; Nellist, P D; Van Aert, S

2015-04-01

Quantitative annular dark field scanning transmission electron microscopy (ADF STEM) has become a powerful technique to characterise nano-particles on an atomic scale. Because of their limited size and beam sensitivity, the atomic structure of such particles may become extremely challenging to determine. Therefore keeping the incoming electron dose to a minimum is important. However, this may reduce the reliability of quantitative ADF STEM which will here be demonstrated for nano-particle atom-counting. Based on experimental ADF STEM images of a real industrial catalyst, we discuss the limits for counting the number of atoms in a projected atomic column with single atom sensitivity. We diagnose these limits by combining a thorough statistical method and detailed image simulations. Copyright © 2014 Elsevier B.V. All rights reserved.

Suppression of plasma virus load below the detection limit of a human immunodeficiency virus kit is associated with longer virologic response than suppression below the limit of quantitation.

PubMed

Raboud, J M; Rae, S; Hogg, R S; Yip, B; Sherlock, C H; Harrigan, P R; O'Shaughnessy, M V; Montaner, J S

1999-10-01

Suppression of human immunodeficiency virus type 1 plasma virus load (PVL) to <20 copies/mL is associated with a longer virologic response after initiation of antiretroviral therapy. The relationship between duration of virologic response and PVL nadir according to a less sensitive assay was explored. When compared with subjects with a PVL nadir >500 copies/mL, the relative risks of PVL rising above 1000 copies/mL for participants in the INCAS trial and the British Columbia Drug Treatment Program with a PVL nadir below the limit of detection (LOD) were 0.04 (95% confidence interval [CI], 0.02-0.09) and 0.06 (95% CI, 0.03-0.12), respectively. The corresponding relative risks for persons with a detectable but not quantifiable PVL nadir were 0.25 (95% CI, 0.13-0.50) and 0.54 (95% CI, 0.25-1.19). The relative risks of virologic failure associated with a PVL nadir detectable but not quantifiable and a PVL nadir below the LOD were statistically different (P<.0001) in both data sets.

Quantitative analysis of PEG-functionalized colloidal gold nanoparticles using charged aerosol detection.

PubMed

Smith, Mackensie C; Crist, Rachael M; Clogston, Jeffrey D; McNeil, Scott E

2015-05-01

Surface characteristics of a nanoparticle, such as functionalization with polyethylene glycol (PEG), are critical to understand and achieve optimal biocompatibility. Routine physicochemical characterization such as UV-vis spectroscopy (for gold nanoparticles), dynamic light scattering, and zeta potential are commonly used to assess the presence of PEG. However, these techniques are merely qualitative and are not sensitive enough to distinguish differences in PEG quantity, density, or presentation. As an alternative, two methods are described here which allow for quantitative measurement of PEG on PEGylated gold nanoparticles. The first, a displacement method, utilizes dithiothreitol to displace PEG from the gold surface. The dithiothreitol-coated gold nanoparticles are separated from the mixture via centrifugation, and the excess dithiothreitol and dissociated PEG are separated through reversed-phase high-performance liquid chromatography (RP-HPLC). The second, a dissolution method, utilizes potassium cyanide to dissolve the gold nanoparticles and liberate PEG. Excess CN(-), Au(CN)2 (-), and free PEG are separated using RP-HPLC. In both techniques, the free PEG can be quantified against a standard curve using charged aerosol detection. The displacement and dissolution methods are validated here using 2-, 5-, 10-, and 20-kDa PEGylated 30-nm colloidal gold nanoparticles. Further value in these techniques is demonstrated not only by quantitating the total PEG fraction but also by being able to be adapted to quantitate the free unbound PEG and the bound PEG fractions. This is an important distinction, as differences in the bound and unbound PEG fractions can affect biocompatibility, which would not be detected in techniques that only quantitate the total PEG fraction.

A self-powered, one-step chip for rapid, quantitative and multiplexed detection of proteins from pinpricks of whole blood.

PubMed

Wang, Jun; Ahmad, Habib; Ma, Chao; Shi, Qihui; Vermesh, Ophir; Vermesh, Udi; Heath, James

2010-11-21

We describe an automated, self-powered chip based on lateral flow immunoassay for rapid, quantitative, and multiplex protein detection from pinpricks of whole blood. The device incorporates on-chip purification of blood plasma by employing inertial forces to focus blood cells away from the assay surface, where plasma proteins are captured and detected on antibody "barcode" arrays. Power is supplied from the capillary action of a piece of adsorbent paper, and sequentially drives, over a 40 minute period, the four steps required to capture serum proteins and then develop a multiplex immunoassay. An 11 protein panel is assayed from whole blood, with high sensitivity and high reproducibility. This inexpensive, self-contained, and easy to operate chip provides a useful platform for point-of-care diagnoses, particularly in resource-limited settings.

A self-powered, one-step chip for rapid, quantitative and multiplexed detection of proteins from pinpricks of whole blood†

PubMed Central

Wang, Jun; Ahmad, Habib; Ma, Chao; Shi, Qihui; Vermesh, Ophir; Vermesh, Udi; Heath, James

2012-01-01

We describe an automated, self-powered chip based on lateral flow immunoassay for rapid, quantitative, and multiplex protein detection from pinpricks of whole blood. The device incorporates on-chip purification of blood plasma by employing inertial forces to focus blood cells away from the assay surface, where plasma proteins are captured and detected on antibody “barcode” arrays. Power is supplied from the capillary action of a piece of adsorbent paper, and sequentially drives, over a 40 minute period, the four steps required to capture serum proteins and then develop a multiplex immunoassay. An 11 protein panel is assayed from whole blood, with high sensitivity and high reproducibility. This inexpensive, self-contained, and easy to operate chip provides a useful platform for point-of-care diagnoses, particularly in resource-limited settings. PMID:20924527

Detection of Microcystin-Producing Cyanobacteria in Missisquoi Bay, Quebec, Canada, Using Quantitative PCR▿

PubMed Central

Fortin, Nathalie; Aranda-Rodriguez, Rocio; Jing, Hongmei; Pick, Frances; Bird, David; Greer, Charles W.

2010-01-01

Toxic cyanobacterial blooms, as well as their increasing global occurrence, pose a serious threat to public health, domestic animals, and livestock. In Missisquoi Bay, Lake Champlain, public health advisories have been issued from 2001 to 2009, and local microcystin concentrations found in the lake water regularly exceeded the Canadian drinking water guideline of 1.5 μg liter−1. A quantitative PCR (Q-PCR) approach was developed for the detection of blooms formed by microcystin-producing cyanobacteria. Primers were designed for the β-ketoacyl synthase (mcyDKS) and the first dehydratase domain (mcyDDH) of the mcyD gene, involved in microcystin synthesis. The Q-PCR method was used to track the toxigenic cyanobacteria in Missisquoi Bay during the summers of 2006 and 2007. Two toxic bloom events were detected in 2006: more than 6.5 × 104 copies of the mcyDKS gene ml−1 were detected in August, and an average of 4.0 × 104 copies ml−1 were detected in September, when microcystin concentrations were more than 4 μg liter−1 and approximately 2 μg liter−1, respectively. Gene copy numbers and total microcystin concentrations (determined by enzyme-linked immunosorbent assay [ELISA]) were highly correlated in the littoral (r = 0.93, P < 0.001) and the pelagic station (r = 0.87, P < 0.001) in 2006. In contrast to the situation in 2006, a cyanobacterial bloom occurred only in late summer-early fall of 2007, reaching only 3 × 102 mcyDKS copies ml−1, while the microcystin concentration was barely detectable. The Q-PCR method allowed the detection of microcystin-producing cyanobacteria when toxins and toxigenic cyanobacterial abundance were still below the limit of detection by high-pressure liquid chromatography (HPLC) and microscopy. Toxin gene copy numbers grew exponentially at a steady rate over a period of 7 weeks. Onshore winds selected for cells with a higher cell quota of microcystin. This technique could be an effective approach for the routine monitoring

Censoring approach to the detection limits in X-ray fluorescence analysis

NASA Astrophysics Data System (ADS)

Pajek, M.; Kubala-Kukuś, A.

2004-10-01

We demonstrate that the effect of detection limits in the X-ray fluorescence analysis (XRF), which limits the determination of very low concentrations of trace elements and results in appearance of the so-called "nondetects", can be accounted for using the statistical concept of censoring. More precisely, the results of such measurements can be viewed as the left random censored data, which can further be analyzed using the Kaplan-Meier method correcting the data for the presence of nondetects. Using this approach, the results of measured, detection limit censored concentrations can be interpreted in a nonparametric manner including the correction for the nondetects, i.e. the measurements in which the concentrations were found to be below the actual detection limits. Moreover, using the Monte Carlo simulation technique we show that by using the Kaplan-Meier approach the corrected mean concentrations for a population of the samples can be estimated within a few percent uncertainties with respect of the simulated, uncensored data. This practically means that the final uncertainties of estimated mean values are limited in fact by the number of studied samples and not by the correction procedure itself. The discussed random-left censoring approach was applied to analyze the XRF detection-limit-censored concentration measurements of trace elements in biomedical samples.

Using multiple PCR and CE with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism.

PubMed

Guo, Longhua; Qiu, Bin; Chi, Yuwu; Chen, Guonan

2008-09-01

In this paper, an ultrasensitive CE-CL detection system coupled with a novel double-on-column coaxial flow detection interface was developed for the detection of PCR products. A reliable procedure based on this system had been demonstrated for qualitative and quantitative analysis of genetically modified organism-the detection of Roundup Ready Soy (RRS) samples was presented as an example. The promoter, terminator, function and two reference genes of RRS were amplified with multiplex PCR simultaneously. After that, the multiplex PCR products were labeled with acridinium ester at the 5'-terminal through an amino modification and then analyzed by the proposed CE-CL system. Reproducibility of analysis times and peak heights for the CE-CL analysis were determined to be better than 0.91 and 3.07% (RSD, n=15), respectively, for three consecutive days. It was shown that this method could accurately and qualitatively detect RRS standards and the simulative samples. The evaluation in terms of quantitative analysis of RRS provided by this new method was confirmed by comparing our assay results with those of the standard real-time quantitative PCR (RT-QPCR) using SYBR Green I dyes. The results showed a good coherence between the two methods. This approach demonstrated the possibility for accurate qualitative and quantitative detection of GM plants in a single run.

Highly sensitive and selective lateral flow immunoassay based on magnetic nanoparticles for quantitative detection of carcinoembryonic antigen.

PubMed

Liu, Fangming; Zhang, Honglian; Wu, Zhenhua; Dong, Haidao; Zhou, Lin; Yang, Dawei; Ge, Yuqing; Jia, Chunping; Liu, Huiying; Jin, Qinghui; Zhao, Jianlong; Zhang, Qiqing; Mao, Hongju

2016-12-01

Carcinoembryonic antigen (CEA) is an important biomarker in cancer diagnosis. Here, we present an efficient, selective lateral-flow immunoassay (LFIA) based on magnetic nanoparticles (MNPs) for in situ sensitive and accurate point-of-care detection of CEA. Signal amplification mechanism involved linking of detection MNPs with signal MNPs through biotin-modified single-stranded DNA (ssDNA) and streptavidin. To verify the effectiveness of this modified LFIA system, the sensitivity and specificity were evaluated. Sensitivity evaluation showed a broad detection range of 0.25-1000ng/ml for CEA protein by the modified LFIA, and the limit of detection (LOD) of the modified LFIA was 0.25ng/ml, thus producing significant increase in detection threshold compared with the traditional LFIA. The modified LFIA could selectively recognize CEA in presence of several interfering proteins. In addition, this newly developed assay was applied for quantitative detection of CEA in human serum specimens collected from 10 randomly selected patients. The modified LFIA system detected minimum 0.27ng/ml of CEA concentration in serum samples. The results were consistent with the clinical data obtained using commercial electrochemiluminescence immunoassay (ECLIA) (p<0.01). In conclusion, the MNPs based LFIA system not only demonstrated enhanced signal to noise ratio, it also detected CEA with higher sensitivity and selectivity, and thus has great potential to be commercially applied as a sensitive tumor marker filtration system. Copyright © 2016 Elsevier B.V. All rights reserved.

True detection limits in an experimental linearly heteroscedastic system.. Part 2

NASA Astrophysics Data System (ADS)

Voigtman, Edward; Abraham, Kevin T.

2011-11-01

Despite much different processing of the experimental fluorescence detection data presented in Part 1, essentially the same estimates were obtained for the true theoretical Currie decision levels ( YC and XC) and true Currie detection limits ( YD and XD). The obtained experimental values, for 5% probability of false positives and 5% probability of false negatives, were YC = 56.0 mV, YD = 125. mV, XC = 0.132 μg/mL and XD = 0.293 μg/mL. For 5% probability of false positives and 1% probability of false negatives, the obtained detection limits were YD = 158 . mV and XD = 0.371 μg/mL. Furthermore, by using bootstrapping methodology on the experimental data for the standards and the analytical blank, it was possible to validate previously published experimental domain expressions for the decision levels ( yC and xC) and detection limits ( yD and xD). This was demonstrated by testing the generated decision levels and detection limits for their performance in regard to false positives and false negatives. In every case, the obtained numbers of false negatives and false positives were as specified a priori.

Calculation of the detection limit in radiation measurements with systematic uncertainties

NASA Astrophysics Data System (ADS)

Kirkpatrick, J. M.; Russ, W.; Venkataraman, R.; Young, B. M.

2015-06-01

The detection limit (LD) or Minimum Detectable Activity (MDA) is an a priori evaluation of assay sensitivity intended to quantify the suitability of an instrument or measurement arrangement for the needs of a given application. Traditional approaches as pioneered by Currie rely on Gaussian approximations to yield simple, closed-form solutions, and neglect the effects of systematic uncertainties in the instrument calibration. These approximations are applicable over a wide range of applications, but are of limited use in low-count applications, when high confidence values are required, or when systematic uncertainties are significant. One proposed modification to the Currie formulation attempts account for systematic uncertainties within a Gaussian framework. We have previously shown that this approach results in an approximation formula that works best only for small values of the relative systematic uncertainty, for which the modification of Currie's method is the least necessary, and that it significantly overestimates the detection limit or gives infinite or otherwise non-physical results for larger systematic uncertainties where such a correction would be the most useful. We have developed an alternative approach for calculating detection limits based on realistic statistical modeling of the counting distributions which accurately represents statistical and systematic uncertainties. Instead of a closed form solution, numerical and iterative methods are used to evaluate the result. Accurate detection limits can be obtained by this method for the general case.

Collection of quantitative chemical release field data.

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

Demirgian, J.; Macha, S.; Loyola Univ.

1999-01-01

Detection and quantitation of chemicals in the environment requires Fourier-transform infrared (FTIR) instruments that are properly calibrated and tested. This calibration and testing requires field testing using matrices that are representative of actual instrument use conditions. Three methods commonly used for developing calibration files and training sets in the field are a closed optical cell or chamber, a large-scale chemical release, and a small-scale chemical release. There is no best method. The advantages and limitations of each method should be considered in evaluating field results. Proper calibration characterizes the sensitivity of an instrument, its ability to detect a component inmore » different matrices, and the quantitative accuracy and precision of the results.« less

sFIDA automation yields sub-femtomolar limit of detection for Aβ aggregates in body fluids.

PubMed

Herrmann, Yvonne; Kulawik, Andreas; Kühbach, Katja; Hülsemann, Maren; Peters, Luriano; Bujnicki, Tuyen; Kravchenko, Kateryna; Linnartz, Christina; Willbold, Johannes; Zafiu, Christian; Bannach, Oliver; Willbold, Dieter

2017-03-01

Alzheimer's disease (AD) is a neurodegenerative disorder with yet non-existent therapeutic and limited diagnostic options. Reliable biomarker-based AD diagnostics are of utmost importance for the development and application of therapeutic substances. We have previously introduced a platform technology designated 'sFIDA' for the quantitation of amyloid β peptide (Aβ) aggregates as AD biomarker. In this study we implemented the sFIDA assay on an automated platform to enhance robustness and performance of the assay. In sFIDA (surface-based fluorescence intensity distribution analysis) Aβ species are immobilized by a capture antibody to a glass surface. Aβ aggregates are then multiply loaded with fluorescent antibodies and quantitated by high resolution fluorescence microscopy. As a model system for Aβ aggregates, we used Aβ-conjugated silica nanoparticles (Aβ-SiNaPs) diluted in PBS buffer and cerebrospinal fluid, respectively. Automation of the assay was realized on a liquid handling system in combination with a microplate washer. The automation of the sFIDA assay results in improved intra-assay precision, linearity and sensitivity in comparison to the manual application, and achieved a limit of detection in the sub-femtomolar range. Automation improves the precision and sensitivity of the sFIDA assay, which is a prerequisite for high-throughput measurements and future application of the technology in routine AD diagnostics. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Validation of quantitative and qualitative methods for detecting allergenic ingredients in processed foods in Japan.

PubMed

Sakai, Shinobu; Adachi, Reiko; Akiyama, Hiroshi; Teshima, Reiko

2013-06-19

A labeling system for food allergenic ingredients was established in Japan in April 2002. To monitor the labeling, the Japanese government announced official methods for detecting allergens in processed foods in November 2002. The official methods consist of quantitative screening tests using enzyme-linked immunosorbent assays (ELISAs) and qualitative confirmation tests using Western blotting or polymerase chain reactions (PCR). In addition, the Japanese government designated 10 μg protein/g food (the corresponding allergenic ingredient soluble protein weight/food weight), determined by ELISA, as the labeling threshold. To standardize the official methods, the criteria for the validation protocol were described in the official guidelines. This paper, which was presented at the Advances in Food Allergen Detection Symposium, ACS National Meeting and Expo, San Diego, CA, Spring 2012, describes the validation protocol outlined in the official Japanese guidelines, the results of interlaboratory studies for the quantitative detection method (ELISA for crustacean proteins) and the qualitative detection method (PCR for shrimp and crab DNAs), and the reliability of the detection methods.

Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses.

PubMed

Patel, Pranav; Landt, Olfert; Kaiser, Marco; Faye, Oumar; Koppe, Tanja; Lass, Ulrich; Sall, Amadou A; Niedrig, Matthias

2013-02-14

The genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses. A Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively. Two degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10-100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays. The assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis.

Quantitative PCR for HTLV-1 provirus in adult T-cell leukemia/lymphoma using paraffin tumor sections.

PubMed

Kato, Junki; Masaki, Ayako; Fujii, Keiichiro; Takino, Hisashi; Murase, Takayuki; Yonekura, Kentaro; Utsunomiya, Atae; Ishida, Takashi; Iida, Shinsuke; Inagaki, Hiroshi

2016-11-01

Detection of HTLV-1 provirus using paraffin tumor sections may assist the diagnosis of adult T-cell leukemia/lymphoma (ATLL). For the detection, non-quantitative PCR assay has been reported, but its usefulness and limitations remain unclear. To our knowledge, quantitative PCR assay using paraffin tumor sections has not been reported. Using paraffin sections from ATLLs and non-ATLL T-cell lymphomas, we first performed non-quantitative PCR for HTLV-1 provirus. Next, we determined tumor ratios and carried out quantitative PCR to obtain provirus copy numbers. The results were analyzed with a simple regression model and a novel criterion, cut-off using 95 % rejection limits. Our quantitative PCR assay showed an excellent association between tumor ratios and the copy numbers (r = 0.89, P < 0.0001). The 95 % rejection limits provided a statistical basis for the range for the determination of HTLV-1 involvement. Its application suggested that results of non-quantitative PCR assay should be interpreted very carefully and that our quantitative PCR assay is useful to estimate the status of HTLV-1 involvement in the tumor cases. In conclusion, our quantitative PCR assay using paraffin tumor sections may be useful for the screening of ATLL cases, especially in HTLV-1 non-endemic areas where easy access to serological testing for HTLV-1 infection is limited. © 2016 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.

Quantitative measures detect sensory and motor impairments in multiple sclerosis.

PubMed

Newsome, Scott D; Wang, Joseph I; Kang, Jonathan Y; Calabresi, Peter A; Zackowski, Kathleen M

2011-06-15

Sensory and motor dysfunction in multiple sclerosis (MS) is often assessed with rating scales which rely heavily on clinical judgment. Quantitative devices may be more precise than rating scales. To quantify lower extremity sensorimotor measures in individuals with MS, evaluate the extent to which they can detect functional systems impairments, and determine their relationship to global disability measures. We tested 145 MS subjects and 58 controls. Vibration thresholds were quantified using a Vibratron-II device. Strength was quantified by a hand-held dynamometer. We also recorded Expanded Disability Status Scale (EDSS) and Timed 25-Foot Walk (T25FW). t-tests and Wilcoxon-rank sum were used to compare group data. Spearman correlations were used to assess relationships between each measure. We also used a step-wise linear regression model to determine how much the quantitative measures explain the variance in the respective functional systems scores (FSS). EDSS scores ranged from 0-7.5, mean disease duration was 10.4 ± 9.6 years, and 66% were female. In relapsing-remitting MS, but not progressive MS, poorer vibration sensation correlated with a worse EDSS score, whereas progressive groups' ankle/hip strength changed significantly with EDSS progression. Interestingly, not only did sensorimotor measures significantly correlate with global disability measures (i.e., EDSS), but they had improved sensitivity, as they detected impairments in up to 32% of MS subjects with normal sensory and pyramidal FSS. Sensory and motor deficits in MS can be quantified using clinically accessible tools and distinguish differences among MS subtypes. We show that quantitative sensorimotor measures are more sensitive than FSS from the EDSS. These tools have the potential to be used as clinical outcome measures in practice and for future MS clinical trials of neurorehabilitative and neuroreparative interventions. Copyright © 2011 Elsevier B.V. All rights reserved.

Quantitative measures detect sensory and motor impairments in multiple sclerosis

PubMed Central

Newsome, Scott D.; Wang, Joseph I.; Kang, Jonathan Y.; Calabresi, Peter A.; Zackowski, Kathleen M.

2011-01-01

Background Sensory and motor dysfunction in multiple sclerosis (MS) is often assessed with rating scales which rely heavily on clinical judgment. Quantitative devices may be more precise than rating scales. Objective To quantify lower extremity sensorimotor measures in individuals with MS, evaluate the extent to which they can detect functional systems impairments, and determine their relationship to global disability measures. Methods We tested 145 MS subjects and 58 controls. Vibration thresholds were quantified using a Vibratron-II device. Strength was quantified by a hand-held dynamometer. We also recorded Expanded Disability Status Scale (EDSS) and timed 25-foot walk (T25FW). T-tests and Wilcoxon-rank sum were used to compare group data. Spearman correlations were used to assess relationships between each measure. We also used a step-wise linear regression model to determine how much the quantitative measures explain the variance in the respective functional systems scores (FSS). Results EDSS scores ranged from 0-7.5, mean disease duration was 10.4±9.6 years, and 66% were female. In RRMS, but not progressive MS, poorer vibration sensation correlated with a worse EDSS score, whereas progressive groups’ ankle/hip strength changed significantly with EDSS progression. Interestingly, not only did sensorimotor measures significantly correlate with global disability measures (EDSS), but they had improved sensitivity, as they detected impairments in up to 32% of MS subjects with normal sensory FSS. Conclusions Sensory and motor deficits can be quantified using clinically accessible tools and distinguish differences among MS subtypes. We show that quantitative sensorimotor measures are more sensitive than FSS from the EDSS. These tools have the potential to be used as clinical outcome measures in practice and for future MS clinical trials of neurorehabilitative and neuroreparative interventions. PMID:21458828

Differential plating medium for quantitative detection of histamine-producing bacteria.

PubMed Central

Niven, C F; Jeffrey, M B; Corlett, D A

1981-01-01

A histidine-containing agar medium has been devised for quantitative detection of histamine-producing bacteria that are alleged to be associated with scombroid fish poisoning outbreaks. The responsible bacteria produce a marked pH change in the agar, with attendant color change of pH indicator adjacent to the colonies, thus facilitating their recognition. Proteus morganii and Klebsiella pneumoniae were the two most common histidine-decarboxylating species isolated from scombroid fish and mahi mahi. PMID:7013698

Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution

EPA Science Inventory

Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described cow feces-spec...

Estimating the resolution limit of the map equation in community detection

NASA Astrophysics Data System (ADS)

Kawamoto, Tatsuro; Rosvall, Martin

2015-01-01

A community detection algorithm is considered to have a resolution limit if the scale of the smallest modules that can be resolved depends on the size of the analyzed subnetwork. The resolution limit is known to prevent some community detection algorithms from accurately identifying the modular structure of a network. In fact, any global objective function for measuring the quality of a two-level assignment of nodes into modules must have some sort of resolution limit or an external resolution parameter. However, it is yet unknown how the resolution limit affects the so-called map equation, which is known to be an efficient objective function for community detection. We derive an analytical estimate and conclude that the resolution limit of the map equation is set by the total number of links between modules instead of the total number of links in the full network as for modularity. This mechanism makes the resolution limit much less restrictive for the map equation than for modularity; in practice, it is orders of magnitudes smaller. Furthermore, we argue that the effect of the resolution limit often results from shoehorning multilevel modular structures into two-level descriptions. As we show, the hierarchical map equation effectively eliminates the resolution limit for networks with nested multilevel modular structures.

Organic Cyanide Decorated SERS Active Nanopipettes for Quantitative Detection of Hemeproteins and Fe3+ in Single Cells.

PubMed

Hanif, Sumaira; Liu, Hailing; Chen, Ming; Muhammad, Pir; Zhou, Yue; Cao, Jiao; Ahmed, Saud Asif; Xu, Jingjuan; Xia, Xinghua; Chen, Hongyuan; Wang, Kang

2017-02-21

It is challenging to develop a robust nanoprobe for real-time operational and accurate detection of heavy metals in single cells. Fe-CN coordination chemistry has been well studied to determine the structural characteristics of hemeproteins by different techniques. However, the frequently used cyanide ligands are inorganic molecules that release cyanide anion under particular conditions and cause cyanide poisoning. In the present study, organic cyanide (4-mercaptobenzonitrile, MBN) was utilized for the first time in developing a facile nanoprobe based on surface-enhanced Raman scattering (SERS) for quantitative detection of hemeproteins (oxy-Hb) and trivalent iron (Fe 3+ ) ions. The nanoprobe prepared by coating the glass capillary tip (100 nm) with a thin gold film, which enables highly localized study in living cell system. The cyanide stretching vibration in MBN was highly sensitive and selective to Fe 3+ and oxy-Hb with excellent binding affinity (K d 0.4 pM and 0.1 nM, respectively). The high sensitivity of the nanoprobe to analyte (Fe 3+ ) was attributed to the two adsorption conformations (-SH and -CN) of MBN to the gold surface. Therefore, MBN showed an exceptional dual-peak (2126 and 2225 cm -1 ) behavior. Furthermore, the special Raman peaks of cyanide in 2100-2300 cm -1 (silent region of SERS spectra) are distinguishable from other biomolecules characteristic peaks. The selective detection of Fe 3+ in both free and protein-bound states in aqueous solution is achieved with 0.1 pM and 0.08 μM levels of detection limits, respectively. Furthermore, practical applicability of fabricated nanoprobe was validated by detection of free Fe 3+ in pretreated living HeLa cells by direct insertion of a SERS active nanoprobe. Regarding the appropriate precision, good reproducibility (relative standard deviation, RSD 7.2-7.6%), and recyclability (retain good Raman intensity even after three renewing cycles) of the method, the developed sensing strategy on a nanopipette

Shot noise limited detection of OH using the technique of laser induced fluorescence

NASA Technical Reports Server (NTRS)

Bakalyar, D. M.; Davis, L. I., Jr.; Guo, C.; James, J. V.; Kakos, S.; Morris, P. T.; Wang, C. C.

1984-01-01

Nearly shot-noise limited detection of OH using the technique of laser-induced fluorescence is reported. A LIDAR configuration is used to excite fluorescence in a large volume and a narrow-bandwidth interference filter provides spectral discrimination. This arrangement alleviates the effect of ozone interference and facilitates image processing at relatively close distances. The detection limit is determined mainly by the shot-noise of the solar background. Ground-based measurements in Dearborn indicate a detection limit of better than 1 x 10 to the 6th power OH/cubic cm over a forty-minute acquisition period. Under favorable conditions, a comparable detection limit was also observed for airborne measurements.

Shot noise limited detection of OH using the technique of laser-induced fluorescence

NASA Technical Reports Server (NTRS)

Bakalyar, D. M.; Davis, L. I., Jr.; Guo, C.; James, J. V.; Wang, C. C.; Kakos, S.; Morris, P. T.

1984-01-01

Nearly shot-noise limited detection of OH using the technique of laser-induced fluorescence is reported. A LIDAR configuration is used to excite fluoresence in a large volume and a narrow-bandwidth interference filter provides spectral discrimination. This arrangement alleviates the effect of ozone interference and facilitates image processing at relatively close distances. The detection limit is determined mainly by the short-noise of the solar background. Ground-based measurements in Dearborn indicate a detection limit of better than 1 x 10 to the 6th power OH/cubic cm over a forty-minute acquisition period. Under favorable conditions, a comparable detection limit was also observed for airborne measurements.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye

PubMed Central

Ahberg, Christian D.; Manz, Andreas; Neuzil, Pavel

2015-01-01

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio. PMID:26088868

Novel ionic liquid matrices for qualitative and quantitative detection of carbohydrates by matrix assisted laser desorption/ionization mass spectrometry.

PubMed

Zhao, Xiaoyong; Shen, Shanshan; Wu, Datong; Cai, Pengfei; Pan, Yuanjiang

2017-09-08

Analysis of carbohydrates based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is still challenging and researchers have been devoting themselves to efficient matrices discovery. In the present study, the design, synthesis, qualitative and quantitative performance of non-derivative ionic liquid matrices (ILMs) were reported. DHB/N-methylaniline (N-MA) and DHB/N-ethylaniline (N-EA), performing best for carbohydrate detection, have been screened out. The limit of detection for oligosaccharide provided by DHB/N-MA and DHB/N-EA were as low as 10 fmol. DHB/N-MA and DHB/N-EA showed significantly higher ion generation efficiency than DHB. The comparison of capacity to probe polysaccharide between these two ILMs and DHB also revealed their powerful potential. Their outstanding performance were probably due to lower proton affinities and stronger UV absorption at λ = 355 nm. What is more, taking DHB/N-MA as an example, quantitative analysis of fructo-oligosaccharide mixtures extracted and identified from rice noodles has been accomplished sensitively using an internal standard method. Overall, DHB/N-MA and DHB/N-EA exhibited excellent performance and might be significant sources as the carbohydrate matrices. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of urine for Leishmania infantum DNA detection by real-time quantitative PCR.

PubMed

Pessoa-E-Silva, Rômulo; Mendonça Trajano-Silva, Lays Adrianne; Lopes da Silva, Maria Almerice; da Cunha Gonçalves-de-Albuquerque, Suênia; de Goes, Tayná Correia; Silva de Morais, Rayana Carla; Lopes de Melo, Fábio; de Paiva-Cavalcanti, Milena

2016-12-01

The availability of some sorts of biological samples which require noninvasive collection methods has led to an even greater interest in applying molecular biology on visceral leishmaniasis (VL) diagnosis, since these samples increase the safety and comfort of both patients and health professionals. In this context, this work aimed to evaluate the suitability of the urine as a specimen for Leishmania infantum kinetoplast DNA detection by real-time quantitative PCR (qPCR). Subsequent to the reproducibility analysis, the detection limit of the qPCR assay was set at 5fg (~0.025 parasites) per μL of urine. From the comparative analysis performed with a set of diagnostic criteria (serological and molecular reference tests), concordance value of 96.08% was obtained (VL-suspected and HIV/AIDS patients, n=51) (P>0.05). Kappa coefficient (95% CI) indicated a good agreement between the test and the set of diagnostic criteria (k=0.778±0.151). The detection of Leishmania DNA in urine by qPCR was possible in untreated individuals, and in those with or without suggestive renal impairment. Fast depletion of the parasite's DNA in urine after treatment (from one dose of meglumine antimoniate) was suggested by negative qPCR results, thus indicating it as a potential alternative specimen to follow up the efficacy of therapeutic approaches. Even when evaluated in a clinically heterogeneous set of patients, the urine showed good prospect as sample for VL diagnosis by qPCR, also indicating a good negative predictive value for untreated suspected patients. Copyright © 2016 Elsevier B.V. All rights reserved.

Limits of detection and decision. Part 3

NASA Astrophysics Data System (ADS)

Voigtman, E.

2008-02-01

It has been shown that the MARLAP (Multi-Agency Radiological Laboratory Analytical Protocols) for estimating the Currie detection limit, which is based on 'critical values of the non-centrality parameter of the non-central t distribution', is intrinsically biased, even if no calibration curve or regression is used. This completed the refutation of the method, begun in Part 2. With the field cleared of obstructions, the true theory underlying Currie's limits of decision, detection and quantification, as they apply in a simple linear chemical measurement system (CMS) having heteroscedastic, Gaussian measurement noise and using weighted least squares (WLS) processing, was then derived. Extensive Monte Carlo simulations were performed, on 900 million independent calibration curves, for linear, "hockey stick" and quadratic noise precision models (NPMs). With errorless NPM parameters, all the simulation results were found to be in excellent agreement with the derived theoretical expressions. Even with as much as 30% noise on all of the relevant NPM parameters, the worst absolute errors in rates of false positives and false negatives, was only 0.3%.

Of Detection Limits and Effective Mitigation: The Use of Infrared Cameras for Methane Leak Detection

NASA Astrophysics Data System (ADS)

Ravikumar, A. P.; Wang, J.; McGuire, M.; Bell, C.; Brandt, A. R.

2017-12-01

Mitigating methane emissions, a short-lived and potent greenhouse gas, is critical to limiting global temperature rise to two degree Celsius as outlined in the Paris Agreement. A major source of anthropogenic methane emissions in the United States is the oil and gas sector. To this effect, state and federal governments have recommended the use of optical gas imaging systems in periodic leak detection and repair (LDAR) surveys to detect for fugitive emissions or leaks. The most commonly used optical gas imaging systems (OGI) are infrared cameras. In this work, we systematically evaluate the limits of infrared (IR) camera based OGI system for use in methane leak detection programs. We analyze the effect of various parameters that influence the minimum detectable leak rates of infrared cameras. Blind leak detection tests were carried out at the Department of Energy's MONITOR natural gas test-facility in Fort Collins, CO. Leak sources included natural gas wellheads, separators, and tanks. With an EPA mandated 60 g/hr leak detection threshold for IR cameras, we test leak rates ranging from 4 g/hr to over 350 g/hr at imaging distances between 5 ft and 70 ft from the leak source. We perform these experiments over the course of a week, encompassing a wide range of wind and weather conditions. Using repeated measurements at a given leak rate and imaging distance, we generate detection probability curves as a function of leak-size for various imaging distances, and measurement conditions. In addition, we estimate the median detection threshold - leak-size at which the probability of detection is 50% - under various scenarios to reduce uncertainty in mitigation effectiveness. Preliminary analysis shows that the median detection threshold varies from 3 g/hr at an imaging distance of 5 ft to over 150 g/hr at 50 ft (ambient temperature: 80 F, winds < 4 m/s). Results from this study can be directly used to improve OGI based LDAR protocols and reduce uncertainty in estimated

Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

PubMed

Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

2017-04-01

Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

Detection limits of antimicrobials in ewe milk by delvotest photometric measurements.

PubMed

Althaus, R L; Torres, A; Montero, A; Balasch, S; Molina, M P

2003-02-01

The Delvotest method detection limits per manufacturer's instructions at a fixed reading time of 3 h for 24 antimicrobial agents were determined in ewe milk by photometric measurement. For each drug, eight concentrations were tested on 20 ewe milk samples from individual ewes. Detection limits, determined by means of logistic regression models, were (microg/kg): 3, amoxycillin; 2, ampicillin; 18, cloxacillin; 1, penicillin "G"; 34, cefadroxil; 430, cephalosporin "C"; 40, cephalexin; 20, cefoperazone; 33, Ceftiofur; 18, cefuroxime; 6100, streptomycin; 1200, gentamycin; 2600, neomycin; 830, erythromycin; 100, tylosin; 180, doxycycline; 320, oxytetracycline; 590, tetracycline; 88, sulfadiazine; 44, sulfamethoxazole; 140, sulfametoxypyridazine; 48, sulfaquinoxaline; 12,000, chloramphenicol; and 290, trimethoprim. Whereas the beta-lactam antibiotics, sulphonamides, and tylosin were detected by Delvotest method at levels equal to those of maximum residue limits, its sensitivity needs to be enhanced to detect aminoglycosides, tetracyclines, streptomycin, chloramphenicol, and trimethoprim residues in ewe milk or to develop an integrated residue detection system for ewe milk with different sensitive microorganisms for each group of antiinfectious agents.

Rapid Determination of Lymphogranuloma Venereum Serovars of Chlamydia trachomatis by Quantitative High-Resolution Melt Analysis (HRMA)

PubMed Central

Stevens, Matthew P.; Garland, Suzanne M.; Zaia, Angelo M.; Tabrizi, Sepehr N.

2012-01-01

A quantitative high-resolution melt analysis assay was developed to differentiate lymphogranuloma venereum-causing serovars of Chlamydia trachomatis (L1 to L3) from other C. trachomatis serovars (D to K). The detection limit of this assay is approximately 10 copies per reaction, comparable to the limits of other quantitative-PCR-based methods. PMID:22933594

Detecting Genetic Interactions for Quantitative Traits Using m-Spacing Entropy Measure

PubMed Central

Yee, Jaeyong; Kwon, Min-Seok; Park, Taesung; Park, Mira

2015-01-01

A number of statistical methods for detecting gene-gene interactions have been developed in genetic association studies with binary traits. However, many phenotype measures are intrinsically quantitative and categorizing continuous traits may not always be straightforward and meaningful. Association of gene-gene interactions with an observed distribution of such phenotypes needs to be investigated directly without categorization. Information gain based on entropy measure has previously been successful in identifying genetic associations with binary traits. We extend the usefulness of this information gain by proposing a nonparametric evaluation method of conditional entropy of a quantitative phenotype associated with a given genotype. Hence, the information gain can be obtained for any phenotype distribution. Because any functional form, such as Gaussian, is not assumed for the entire distribution of a trait or a given genotype, this method is expected to be robust enough to be applied to any phenotypic association data. Here, we show its use to successfully identify the main effect, as well as the genetic interactions, associated with a quantitative trait. PMID:26339620

Endotoxin Detection in Pharmaceuticals and Medical Devices with Kinetic-QCL, a Kinetic-Quantitative Chromogenic Limulus Amebocyte Lysate Assay.

PubMed

Berzofsky, Ronald N.

1995-01-01

The observation that endotoxin caused gelation in extracts of Limulus amebocytes has been expanded to the development of an in vitro kinetic, quantitative chromogenic LAL assay (Kinetic-QCL) for the detection of endotoxin in aqueous fluids. Within the last 15 years, the use of Limulus amebocyte lysate to detect and control the presence of pyrogenic substances in pharmaceuticals and medical devices has gained wide international acceptance. Both the United States and European Pharmacopoeias contain descriptions of and requirements for the LAL Bacterial Endotoxin Test. Both pharmacopoeias have begun to remove the rabbit pyrogen test requirement in a majority of drug monographs and have substituted endotoxin limits to be determined by LAL. The use of LAL has proved invaluable in controlling the level of endotoxin in finished product. The endotoxin contribution of raw materials and packaging material can be monitored as well. In-process testing at critical production steps can identify additional sources of endotoxin contamination, and depyrogenation processes can be validated by quantitating the degradation of endotoxin challenges. The speed, reproducibility, sensitivity, and economics of the Kinetic-QCL assay, in conjunction with the ppropriate equipment and software, over both the in vivo rabbit pyrogen test and the more traditional LAL gel-clot assay allow a more in-depth approach to the control of endotoxin in pharmaceuticals and medical devices.

Quantitative SERS detection of low-concentration aromatic polychlorinated biphenyl-77 and 2,4,6-trinitrotoluene.

PubMed

Bao, Zhi Yong; Liu, Xin; Chen, Y; Wu, Yucheng; Chan, Helen L W; Dai, Jiyan; Lei, Dang Yuan

2014-09-15

This paper reports a simple label-free high-sensitive method for detecting low-concentration persistent organic pollutants and explosive materials. The proposed method combines surface-enhanced Raman spectroscopy (SERS) and magnetomotive enrichment of the target molecules on the surface of Ag nanoparticles (NPs). This structure can be achieved through self-assembling integration of Ag NPs with ferromagnetic Fe3O4 microspheres, forming a hybrid SERS nanoprobe with both optical and magnetic properties. Moreover, the magnetic response of ferromagnetic Fe3O4 microspheres can be used to dynamically modulate the optical property of Ag NPs through controlling their geometric arrangement on the substrate by applying an external magnetic field. It is also demonstrated from the full-wave numerical simulation results that the maximum electromagnetic field enhancement can be greatly increased by shortening the distance of neighboring Ag NPs and therefore resulting in an improved SERS detecting limit. More importantly, by using the prepared substrate, the SERS signals from organic pollution substances, i.e. aromatic polychlorinated biphenyl-77 and 2,4,6-trinitrotoluene, were quantitatively analyzed. Copyright © 2014 Elsevier B.V. All rights reserved.

True detection limits in an experimental linearly heteroscedastic system. Part 1

NASA Astrophysics Data System (ADS)

Voigtman, Edward; Abraham, Kevin T.

2011-11-01

Using a lab-constructed laser-excited filter fluorimeter deliberately designed to exhibit linearly heteroscedastic, additive Gaussian noise, it has been shown that accurate estimates may be made of the true theoretical Currie decision levels ( YC and XC) and true Currie detection limits ( YD and XD) for the detection of rhodamine 6 G tetrafluoroborate in ethanol. The obtained experimental values, for 5% probability of false positives and 5% probability of false negatives, were YC = 56.1 mV, YD = 125. mV, XC = 0.132 μg /mL and XD = 0.294 μg /mL. For 5% probability of false positives and 1% probability of false negatives, the obtained detection limits were YD = 158. mV and XD = 0.372 μg /mL. These decision levels and corresponding detection limits were shown to pass the ultimate test: they resulted in observed probabilities of false positives and false negatives that were statistically equivalent to the a priori specified values.

Repeatability Assessment by ISO 11843-7 in Quantitative HPLC for Herbal Medicines.

PubMed

Chen, Liangmian; Kotani, Akira; Hakamata, Hideki; Tsutsumi, Risa; Hayashi, Yuzuru; Wang, Zhimin; Kusu, Fumiyo

2015-01-01

We have proposed an assessment methods to estimate the measurement relative standard deviation (RSD) of chromatographic peaks in quantitative HPLC for herbal medicines by the methodology of ISO 11843 Part 7 (ISO 11843-7:2012), which provides detection limits stochastically. In quantitative HPLC with UV detection (HPLC-UV) of Scutellaria Radix for the determination of baicalin, the measurement RSD of baicalin by ISO 11843-7:2012 stochastically was within a 95% confidence interval of the statistically obtained RSD by repetitive measurements (n = 6). Thus, our findings show that it is applicable for estimating of the repeatability of HPLC-UV for determining baicalin without repeated measurements. In addition, the allowable limit of the "System repeatability" in "Liquid Chromatography" regulated in a pharmacopoeia can be obtained by the present assessment method. Moreover, the present assessment method was also successfully applied to estimate the measurement RSDs of quantitative three-channel liquid chromatography with electrochemical detection (LC-3ECD) of Chrysanthemi Flos for determining caffeoylquinic acids and flavonoids. By the present repeatability assessment method, reliable measurement RSD was obtained stochastically, and the experimental time was remarkably reduced.

Effects of detector dead-time on quantitative analyses involving boron and multi-hit detection events in atom probe tomography.

PubMed

Meisenkothen, Frederick; Steel, Eric B; Prosa, Ty J; Henry, Karen T; Prakash Kolli, R

2015-12-01

In atom probe tomography (APT), some elements tend to field evaporate preferentially in multi-hit detection events. Boron (B) is one such element. It is thought that a large fraction of the B signal may be lost during data acquisition and is not reported in the mass spectrum or in the 3-D APT reconstruction. Understanding the relationship between the field evaporation behavior of B and the limitations for detecting multi-hit events can provide insight into the signal loss mechanism for B and may suggest ways to improve B detection accuracy. The present work reports data for nominally pure B and for B-implanted silicon (Si) (NIST-SRM2137) at dose levels two-orders of magnitude lower than previously studied by Da Costa, et al. in 2012. Boron concentration profiles collected from SRM2137 specimens qualitatively confirmed a signal loss mechanism is at work in laser pulsed atom probe measurements of B in Si. Ion correlation analysis was used to graphically demonstrate that the detector dead-time results in few same isotope, same charge-state (SISCS) ion pairs being properly recorded in the multi-hit data, explaining why B is consistently under-represented in quantitative analyses. Given the important role of detector dead-time as a signal loss mechanism, the results from three different methods of estimating the detector dead-time are presented. The findings of this study apply to all quantitative analyses that involve multi-hit data, but the dead-time will have the greatest effect on the elements that have a significant quantity of ions detected in multi-hit events. Published by Elsevier B.V.

Improving membrane based multiplex immunoassays for semi-quantitative detection of multiple cytokines in a single sample

PubMed Central

2014-01-01

Background Inflammatory mediators can serve as biomarkers for the monitoring of the disease progression or prognosis in many conditions. In the present study we introduce an adaptation of a membrane-based technique in which the level of up to 40 cytokines and chemokines can be determined in both human and rodent blood in a semi-quantitative way. The planar assay was modified using the LI-COR (R) detection system (fluorescence based) rather than chemiluminescence and semi-quantitative outcomes were achieved by normalizing the outcomes using the automated exposure settings of the Odyssey readout device. The results were compared to the gold standard assay, namely ELISA. Results The improved planar assay allowed the detection of a considerably higher number of analytes (n = 30 and n = 5 for fluorescent and chemiluminescent detection, respectively). The improved planar method showed high sensitivity up to 17 pg/ml and a linear correlation of the normalized fluorescence intensity with the results from the ELISA (r = 0.91). Conclusions The results show that the membrane-based technique is a semi-quantitative assay that correlates satisfactorily to the gold standard when enhanced by the use of fluorescence and subsequent semi-quantitative analysis. This promising technique can be used to investigate inflammatory profiles in multiple conditions, particularly in studies with constraints in sample sizes and/or budget. PMID:25022797

Quantitative relationship between the octanol/water partition coefficient and the diffusion limitation of the exchange between adipose and blood.

PubMed

Levitt, David G

2010-01-07

The goal of physiologically based pharmacokinetics (PBPK) is to predict drug kinetics from an understanding of the organ/blood exchange. The standard approach is to assume that the organ is "flow limited" which means that the venous blood leaving the organ equilibrates with the well-stirred tissue compartment. Although this assumption is valid for most solutes, it has been shown to be incorrect for several very highly fat soluble compounds which appear to be "diffusion limited". This paper describes the physical basis of this adipose diffusion limitation and its quantitative dependence on the blood/water (Kbld-wat) and octanol/water (Kow) partition coefficient. Experimental measurements of the time dependent rat blood and adipose concentration following either intravenous or oral input were used to estimate the "apparent" adipose perfusion rate (FA) assuming that the tissue is flow limited. It is shown that the ratio of FA to the anatomic perfusion rate (F) provides a measure of the diffusion limitation. A quantitative relationship between this diffusion limitation and Kbld-wat and Kow is derived. This analysis was applied to previously published data, including the Oberg et. al. measurements of the rat plasma and adipose tissue concentration following an oral dose of a mixture of 13 different polychlorinated biphenyls. Solutes become diffusion limited at values of log Kow greater than about 5.6, with the adipose-blood exchange rate reduced by a factor of about 30 for a solute with a log Kow of 7.36. Quantitatively, a plot of FA/F versus Kow is well described assuming an adipose permeability-surface area product (PS) of 750/min. This PS corresponds to a 0.14 micron aqueous layer separating the well-stirred blood from the adipose lipid. This is approximately equal to the thickness of the rat adipose capillary endothelium. These results can be used to quantitate the adipose-blood diffusion limitation as a function of Kow. This is especially important for the highly

Theoretical detection limit of PIXE analysis using 20 MeV proton beams

NASA Astrophysics Data System (ADS)

Ishii, Keizo; Hitomi, Keitaro

2018-02-01

Particle-induced X-ray emission (PIXE) analysis is usually performed using proton beams with energies in the range 2∼3 MeV because at these energies, the detection limit is low. The detection limit of PIXE analysis depends on the X-ray production cross-section, the continuous background of the PIXE spectrum and the experimental parameters such as the beam currents and the solid angle and detector efficiency of X-ray detector. Though the continuous background increases as the projectile energy increases, the cross-section of the X-ray increases as well. Therefore, the detection limit of high energy proton PIXE is not expected to increase significantly. We calculated the cross sections of continuous X-rays produced in several bremsstrahlung processes and estimated the detection limit of a 20 MeV proton PIXE analysis by modelling the Compton tail of the γ-rays produced in the nuclear reactions, and the escape effect on the secondary electron bremsstrahlung. We found that the Compton tail does not affect the detection limit when a thin X-ray detector is used, but the secondary electron bremsstrahlung escape effect does have an impact. We also confirmed that the detection limit of the PIXE analysis, when used with 4 μm polyethylene backing film and an integrated beam current of 1 μC, is 0.4∼2.0 ppm for proton energies in the range 10∼30 MeV and elements with Z = 16-90. This result demonstrates the usefulness of several 10 MeV cyclotrons for performing PIXE analysis. Cyclotrons with these properties are currently installed in positron emission tomography (PET) centers.

Detection of Legionella by quantitative-polymerase chain reaction (qPCR) for monitoring and risk assessment.

PubMed

Krøjgaard, Louise H; Krogfelt, Karen A; Albrechtsen, Hans-Jørgen; Uldum, Søren A

2011-11-21

Culture and quantitative polymerase chain reaction (qPCR) assays for the detection of Legionella were compared on samples from a residential area before and after two interventions. A total of 84 samples were collected from shower hoses and taps as first flush samples and at constant temperature. Samples were grouped according to the origin of the sample, a) circulation water b) water from empty apartments c) water from shower hoses. The aims were to investigate the usefulness of qPCR compared to culture for monitoring remedial actions for elimination of Legionella bacteria and as a tool for risk assessment. In water collected from the apartments Legionella spp were detected by qPCR in the concentration range from LOQ to 9.6*105GU/L while L. pneumophila were detected in a range from LOQ to 6.8*105 GU/L. By culturing, the legionellae were detected in the range from below detection limit (> 10 CFU/L) to 1.6*106 CFU/L. In circulating water and in first flush water from shower hoses, culture and qPCR showed the same tendencies. The overall correlation between the bacteria number detected by culture and the two developed qPCR assays (L. spp and L. pneumophila) was relatively poor (r2 = 0.31 for culture and Legionella spp. assay, r2 = 0.20 for culture and L. pneumophila assay). Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc is dubious. However, the rapid detection by qPCR of high concentrations of Legionella - especially Legionella pneumophila - is valuable as an indicator of risk, although it may be false positive compared to culture results. On the other hand, the detection of a low number of bacteria by qPCR is a strong indication for the absence of risk.

A globotetraosylceramide (Gb₄) receptor-based ELISA for quantitative detection of Shiga toxin 2e.

PubMed

Togashi, Katsuhiro; Sasaki, Shiho; Sato, Wataru

2015-08-01

Currently, no simple assays are available for routine quantitative detection of Escherichia coli-produced Shiga toxin 2e (Stx2e) that causes porcine edema disease. Here, we present a novel quantitative detection method for Stx2e based on the measurement of Stx2e binding to the specific globotetraosylceramide (Gb4) receptor by ELISA (Gb4-ELISA). No cross-reactivity was found with the other Shiga toxins Stx1 and Stx2, indicating high specificity. When the recombinant Stx2e B subunit (Stx2eB) was used, the absorbance measured by Gb4-ELISA increased linearly with Stx2eB concentration in the range of 20-2,500 ng/ml. The Gb4-ELISA method can be easily performed, suggesting that it would be a useful diagnostic tool for porcine edema disease.

Censoring: a new approach for detection limits in total-reflection X-ray fluorescence

NASA Astrophysics Data System (ADS)

Pajek, M.; Kubala-Kukuś, A.; Braziewicz, J.

2004-08-01

It is shown that the detection limits in the total-reflection X-ray fluorescence (TXRF), which restrict quantification of very low concentrations of trace elements in the samples, can be accounted for using the statistical concept of censoring. We demonstrate that the incomplete TXRF measurements containing the so-called "nondetects", i.e. the non-measured concentrations falling below the detection limits and represented by the estimated detection limit values, can be viewed as the left random-censored data, which can be further analyzed using the Kaplan-Meier (KM) method correcting for nondetects. Within this approach, which uses the Kaplan-Meier product-limit estimator to obtain the cumulative distribution function corrected for the nondetects, the mean value and median of the detection limit censored concentrations can be estimated in a non-parametric way. The Monte Carlo simulations performed show that the Kaplan-Meier approach yields highly accurate estimates for the mean and median concentrations, being within a few percent with respect to the simulated, uncensored data. This means that the uncertainties of KM estimated mean value and median are limited in fact only by the number of studied samples and not by the applied correction procedure for nondetects itself. On the other hand, it is observed that, in case when the concentration of a given element is not measured in all the samples, simple approaches to estimate a mean concentration value from the data yield erroneous, systematically biased results. The discussed random-left censoring approach was applied to analyze the TXRF detection-limit-censored concentration measurements of trace elements in biomedical samples. We emphasize that the Kaplan-Meier approach allows one to estimate the mean concentrations being substantially below the mean level of detection limits. Consequently, this approach gives a new access to lower the effective detection limits for TXRF method, which is of prime interest for

Developing the Quantitative Histopathology Image Ontology (QHIO): A case study using the hot spot detection problem.

PubMed

Gurcan, Metin N; Tomaszewski, John; Overton, James A; Doyle, Scott; Ruttenberg, Alan; Smith, Barry

2017-02-01

Interoperability across data sets is a key challenge for quantitative histopathological imaging. There is a need for an ontology that can support effective merging of pathological image data with associated clinical and demographic data. To foster organized, cross-disciplinary, information-driven collaborations in the pathological imaging field, we propose to develop an ontology to represent imaging data and methods used in pathological imaging and analysis, and call it Quantitative Histopathological Imaging Ontology - QHIO. We apply QHIO to breast cancer hot-spot detection with the goal of enhancing reliability of detection by promoting the sharing of data between image analysts. Copyright © 2016 Elsevier Inc. All rights reserved.

A sensitive one-step method for quantitative detection of α-amylase in serum and urine using a personal glucose meter.

PubMed

Wang, Qing; Wang, Hui; Yang, Xiaohai; Wang, Kemin; Liu, Rongjuan; Li, Qing; Ou, Jinqing

2015-02-21

Assays of α-amylase (AMS) activity in serum and urine constitute the important indicator for the diagnosis of acute pancreatitis, mumps, renal disease and abdominal disorders. Since these diseases confer a heavy financial burden on the health care system, AMS detection in point-of-care is fundamental. Here, a one-step assay for direct determination of the AMS activity was developed using a portable personal glucose meter (PGM). In this assay, maltopentaose was used as a substrate for sensitive detection of AMS with the assistance of α-glucosidase. In the presence of AMS, maltopentaose can be readily hydrolyzed to form maltotriose and maltose quickly. With the enzymatic hydrolysis of α-glucosidase, maltotriose and maltose can be turned into glucose rapidly, which can be quantitatively measured using a portable PGM. This assay did not require any cumbersome and time consuming operations, such as surface modification, synthesis of invertase conjugate, washing and centrifugation. Detection of AMS can be achieved using only a one-step mixture, and the limit of detection was 20 U L(-1) which was lower than the clinical cutoff for AMS. More importantly, this sensitive and selective assay was also used for the detection of AMS in human serum/urine samples. The results showed that the recovery of AMS from human serum/urine samples was 91-107%. The rapid and easy-to-operate assay may have potential application in the fields of point-of-care (POC) clinical diagnosis, particularly in rural and remote areas where lab equipment may be limited.

Rapid detection and quantification of Ebola Zaire virus by one-step real-time quantitative reverse transcription-polymerase chain reaction.

PubMed

Ro, Young-Tae; Ticer, Anysha; Carrion, Ricardo; Patterson, Jean L

2017-04-01

Given that Ebola virus causes severe hemorrhagic fever in humans with mortality rates as high as 90%, rapid and accurate detection of this virus is essential both for controlling infection and preventing further transmission. Here, a one-step qRT-PCR assay for rapid and quantitative detection of an Ebola Zaire strain using GP, VP24 or VP40 genes as a target is introduced. Routine assay conditions for hydrolysis probe detection were established from the manufacturer's protocol used in the assays. The analytical specificity and sensitivity of each assay was evaluated using in vitro synthesized viral RNA transcripts. The assays were highly specific for the RNA transcripts, no cross-reactivity being observed among them. The limits of detection of the assays ranged from 10 2 to 10 3 copies per reaction. The assays were also evaluated using viral RNAs extracted from cell culture-propagated viruses (Ebola Zaire, Sudan and Reston strains), confirming that they are gene- and strain-specific. The RT-PCR assays detected viral RNAs in blood samples from virus-infected animal, suggesting that they can be also a useful method for identifying Ebola virus in clinical samples. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Sniper detection using infrared camera: technical possibilities and limitations

NASA Astrophysics Data System (ADS)

Kastek, M.; Dulski, R.; Trzaskawka, P.; Bieszczad, G.

2010-04-01

The paper discusses technical possibilities to build an effective system for sniper detection using infrared cameras. Descriptions of phenomena which make it possible to detect sniper activities in infrared spectra as well as analysis of physical limitations were performed. Cooled and uncooled detectors were considered. Three phases of sniper activities were taken into consideration: before, during and after the shot. On the basis of experimental data the parameters defining the target were determined which are essential in assessing the capability of infrared camera to detect sniper activity. A sniper body and muzzle flash were analyzed as targets. The simulation of detection ranges was done for the assumed scenario of sniper detection task. The infrared sniper detection system was discussed, capable of fulfilling the requirements. The discussion of the results of analysis and simulations was finally presented.

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.

A new molecular diagnostic tool for quantitatively detecting and genotyping “Candidatus Liberibacter species”

USDA-ARS?s Scientific Manuscript database

A new molecular diagnostic method was developed for quantitative detection of “Candidatus Liberibacter” species associated with citrus Huanglongbing (“Ca. Liberibacter asiaticus”, “Ca. Liberibacter africanus” and “Ca. Liberibacter americanus”) and potato zebra chip disorder (“Ca. Liberibacter solana...

Diazonium Salt-Based Surface-Enhanced Raman Spectroscopy Nanosensor: Detection and Quantitation of Aromatic Hydrocarbons in Water Samples

PubMed Central

Tijunelyte, Inga; Betelu, Stéphanie; Moreau, Jonathan; Ignatiadis, Ioannis; Berho, Catherine; Lidgi-Guigui, Nathalie; Guénin, Erwann; David, Catalina; Vergnole, Sébastien; Rinnert, Emmanuel; Lamy de la Chapelle, Marc

2017-01-01

Here, we present a surface-enhanced Raman spectroscopy (SERS) nanosensor for environmental pollutants detection. This study was conducted on three polycyclic aromatic hydrocarbons (PAHs): benzo[a]pyrene (BaP), fluoranthene (FL), and naphthalene (NAP). SERS substrates were chemically functionalized using 4-dodecyl benzenediazonium-tetrafluoroborate and SERS analyses were conducted to detect the pollutants alone and in mixtures. Compounds were first measured in water-methanol (9:1 volume ratio) samples. Investigation on solutions containing concentrations ranging from 10−6 g L−1 to 10−3 g L−1 provided data to plot calibration curves and to determine the performance of the sensor. The calculated limit of detection (LOD) was 0.026 mg L−1 (10−7 mol L−1) for BaP, 0.064 mg L−1 (3.2 × 10−7 mol L−1) for FL, and 3.94 mg L−1 (3.1 × 10−5 mol L−1) for NAP, respectively. The correlation between the calculated LOD values and the octanol-water partition coefficient (Kow) of the investigated PAHs suggests that the developed nanosensor is particularly suitable for detecting highly non-polar PAH compounds. Measurements conducted on a mixture of the three analytes (i) demonstrated the ability of the developed technology to detect and identify the three analytes in the mixture; (ii) provided the exact quantitation of pollutants in a mixture. Moreover, we optimized the surface regeneration step for the nanosensor. PMID:28538680

Diazonium Salt-Based Surface-Enhanced Raman Spectroscopy Nanosensor: Detection and Quantitation of Aromatic Hydrocarbons in Water Samples.

PubMed

Tijunelyte, Inga; Betelu, Stéphanie; Moreau, Jonathan; Ignatiadis, Ioannis; Berho, Catherine; Lidgi-Guigui, Nathalie; Guénin, Erwann; David, Catalina; Vergnole, Sébastien; Rinnert, Emmanuel; Lamy de la Chapelle, Marc

2017-05-24

Here, we present a surface-enhanced Raman spectroscopy (SERS) nanosensor for environmental pollutants detection. This study was conducted on three polycyclic aromatic hydrocarbons (PAHs): benzo[a]pyrene (BaP), fluoranthene (FL), and naphthalene (NAP). SERS substrates were chemically functionalized using 4-dodecyl benzenediazonium-tetrafluoroborate and SERS analyses were conducted to detect the pollutants alone and in mixtures. Compounds were first measured in water-methanol (9:1 volume ratio) samples. Investigation on solutions containing concentrations ranging from 10 -6 g L -1 to 10 -3 g L -1 provided data to plot calibration curves and to determine the performance of the sensor. The calculated limit of detection (LOD) was 0.026 mg L -1 (10 -7 mol L -1 ) for BaP, 0.064 mg L -1 (3.2 × 10 -7 mol L -1 ) for FL, and 3.94 mg L -1 (3.1 × 10 -5 mol L -1 ) for NAP, respectively. The correlation between the calculated LOD values and the octanol-water partition coefficient (K ow ) of the investigated PAHs suggests that the developed nanosensor is particularly suitable for detecting highly non-polar PAH compounds. Measurements conducted on a mixture of the three analytes (i) demonstrated the ability of the developed technology to detect and identify the three analytes in the mixture; (ii) provided the exact quantitation of pollutants in a mixture. Moreover, we optimized the surface regeneration step for the nanosensor.

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

NASA Astrophysics Data System (ADS)

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

2008-04-01

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

Simultaneous detection and quantitation of organic impurities in methamphetamine by ultra-high-performance liquid chromatography-tandem mass spectrometry, a complementary technique for methamphetamine profiling.

PubMed

Li, Li; Brown, Jaclyn L; Toske, Steven G

2018-04-06

The analysis of organic impurities plays an important role in the impurity profiling of methamphetamine, which in turn provides valuable information about methamphetamine manufacturing, in particular its synthetic route, chemicals, and precursors used. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) is ideally suited for this purpose due to its excellent sensitivity, selectivity, and wide linear range in multiple reaction monitoring (MRM) mode. In this study, a dilute-and-shoot UHPLC-MS/MS method was developed for the simultaneous identification and quantitation of 23 organic manufacturing impurities in illicit methamphetamine. The developed method was validated in terms of stability, limit of detection (LOD), lower limit of quantification (LLOQ), accuracy, and precision. More than 100 illicitly prepared methamphetamine samples were analyzed. Due to its ability to detect ephedrine/pseudoephedrine and its high sensitivity for critical target markers (eg, chloro-pseudoephedrine, N-cyclohexylamphetamine, and compounds B and P), more impurities and precursor/pre-precursors were identified and quantified versus the current procedure by gas chromatography-mass spectrometry (GC-MS). Consequently, more samples could be classified by their synthetic routes. However, the UHPLC-MS/MS method has difficulty in detecting neutral and untargeted emerging manufacturing impurities and can therefore only serve as a complement to the current method. Despite this deficiency, the quantitative information acquired by the presented UHPLC-MS/MS methodology increased the sample discrimination power, thereby enhancing the capacity of methamphetamine profiling program (MPP) to conduct sample-sample comparisons. Published 2018. This article is a U.S. Government work and is in the public domain in the USA.

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

PubMed

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

2017-08-01

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

Detectability limit and uncertainty considerations for laser induced fluorescence spectroscopy in flames

NASA Technical Reports Server (NTRS)

Daily, J. W.

1978-01-01

Laser induced fluorescence spectroscopy of flames is discussed, and derived uncertainty relations are used to calculate detectability limits due to statistical errors. Interferences due to Rayleigh scattering from molecules as well as Mie scattering and incandescence from particles have been examined for their effect on detectability limits. Fluorescence trapping is studied, and some methods for reducing the effect are considered. Fluorescence trapping places an upper limit on the number density of the fluorescing species that can be measured without signal loss.

Detection limit of a VCO based detection chain dedicated to particles recognition and tracking

NASA Astrophysics Data System (ADS)

Coulié, K.; Rahajandraibe, W.; Aziza, H.; Micolau, G.; Vauché, R.

2018-01-01

A particle detection chain based on CMOS-SOI VCO circuit is presented. The solution is used for the recognition and the tracking of a given particle at circuit level. TCAD simulation of the detector has been performed on a 3×3 matrix of diodes based detector for particles recognition and tracking. The current response of the detector has been used for a case study in order to determine the ability of the chain to recognize an alpha particle crossing a 3×3 detection cell. The detection limit of the proposed solution is investigated and discussed in this paper.

On the detection of early osteoarthritis by quantitative microscopic imaging

NASA Astrophysics Data System (ADS)

Mittelstaedt, Daniel John

Articular cartilage is a thin layer of connective tissue that protects the ends of bones in diarthroidal joints. Cartilage distributes mechanical forces during daily movement throughout its unique depth-dependent structure. The extracellular matrix (ECM) of cartilage primarily contains water, collagen, and glycosaminoglycan (GAG). The collagen fibers are intertwined with negatively charged GAG and surround the cells (i.e. chondrocytes) in cartilage. Degradation to the ECM reduces the load bearing properties of cartilage which can be initiated by injury (e.g. anterior cruciate ligament (ACL) rupture) or disease (e.g. osteoarthritis (OA)). Magnetic resonance imaging (MRI) and x-ray computed tomography (CT) are noninvasive imaging techniques that are increasingly being used in the clinical detection of cartilage degradation. The aim of the first project in this dissertation was to quantify and compare the depth-dependent GAG concentration from healthy and biochemically degraded humeral ex vivo articular cartilage using quantitative contrast enhanced micro-computed tomography (qCECT) at high resolution. The second project in this dissertation was aimed to measure the topographical and depth-dependent GAG concentration using qCECT and delayed gadolinium enhanced magnetic resonance imaging of cartilage (dGEMRIC) from the medial tibia cartilage three weeks after unilateral ACL transection which is an animal model of OA (i.e. modified Pond-Nuki model). These GAG measurements were correlated with a biochemical method, inductively couple plasma optical emission spectrometry, to compare the degradation on the medial tibia between the OA and contralateral cartilage. The third project in this dissertation used the same cartilage specimens as in project two to investigate the change in T2 due to OA and the effect on T2 from a contrast agent. Furthermore, the change in T2 relaxation was investigated from static unconfined compression with correlations by biomechanical

Quantitative analysis of fragrance in selectable one dimensional or two dimensional gas chromatography-mass spectrometry with simultaneous detection of multiple detectors in single injection.

PubMed

Tan, Hui Peng; Wan, Tow Shi; Min, Christina Liew Shu; Osborne, Murray; Ng, Khim Hui

2014-03-14

A selectable one-dimensional ((1)D) or two-dimensional ((2)D) gas chromatography-mass spectrometry (GC-MS) system coupled with flame ionization detector (FID) and olfactory detection port (ODP) was employed in this study to analyze perfume oil and fragrance in shower gel. A split/splitless (SSL) injector and a programmable temperature vaporization (PTV) injector are connected via a 2-way splitter of capillary flow technology (CFT) in this selectable (1)D/(2)D GC-MS/FID/ODP system to facilitate liquid sample injections and thermal desorption (TD) for stir bar sorptive extraction (SBSE) technique, respectively. The dual-linked injectors set-up enable the use of two different injector ports (one at a time) in single sequence run without having to relocate the (1)D capillary column from one inlet to another. Target analytes were separated in (1)D GC-MS/FID/ODP and followed by further separation of co-elution mixture from (1)D in (2)D GC-MS/FID/ODP in single injection without any instrumental reconfiguration. A (1)D/(2)D quantitative analysis method was developed and validated for its repeatability - tR; calculated linear retention indices (LRI); response ratio in both MS and FID signal, limit of detection (LOD), limit of quantitation (LOQ), as well as linearity over a concentration range. The method was successfully applied in quantitative analysis of perfume solution at different concentration level (RSD≤0.01%, n=5) and shower gel spiked with perfume at different dosages (RSD≤0.04%, n=5) with good recovery (96-103% for SSL injection; 94-107% for stir bar sorptive extraction-thermal desorption (SBSE-TD). Copyright © 2014 Elsevier B.V. All rights reserved.

Quantitative food web analysis supports the energy-limitation hypothesis in cave stream ecosystems.

PubMed

Venarsky, Michael P; Huntsman, Brock M; Huryn, Alexander D; Benstead, Jonathan P; Kuhajda, Bernard R

2014-11-01

Energy limitation has long been the primary assumption underlying conceptual models of evolutionary and ecological processes in cave ecosystems. However, the prediction that cave communities are actually energy-limited in the sense that constituent populations are consuming all or most of their resource supply is untested. We assessed the energy-limitation hypothesis in three cave streams in northeastern Alabama (USA) by combining measurements of animal production, demand, and resource supplies (detritus, primarily decomposing wood particles). Comparisons of animal consumption and detritus supply rates in each cave showed that all, or nearly all, available detritus was required to support macroinvertebrate production. Furthermore, only a small amount of macroinvertebrate prey production remained to support other predatory taxa (i.e., cave fish and salamanders) after accounting for crayfish consumption. Placing the energy demands of a cave community within the context of resource supply rates provided quantitative support for the energy-limitation hypothesis, confirming the mechanism (limited energy surpluses) that likely influences the evolutionary processes and population dynamics that shape cave communities. Detritus-based surface ecosystems often have large detrital surpluses. Thus, cave ecosystems, which show minimal surpluses, occupy the extreme oligotrophic end of the spectrum of detritus-based food webs.

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

PubMed

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

2005-07-01

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

A quantitative and direct PCR assay for the subspecies-specific detection of Clavibacter michiganensis subsp. michiganensis based on a ferredoxin reductase gene.

PubMed

Cho, Min Seok; Lee, Jang Ha; Her, Nam Han; Kim, Changkug; Seol, Young-Joo; Hahn, Jang Ho; Baeg, Ji Hyoun; Kim, Hong Gi; Park, Dong Suk

2012-06-01

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis is the causal agent of canker disease in tomato. Because it is very important to control newly introduced inoculum sources from commercial materials, the specific detection of this pathogen in seeds and seedlings is essential for effective disease control. In this study, a novel and efficient assay for the detection and quantitation of C. michiganensis subsp. michiganensis in symptomless tomato and red pepper seeds was developed. A pair of polymerase chain reaction (PCR) primers (Cmm141F/R) was designed to amplify a specific 141 bp fragment on the basis of a ferredoxin reductase gene of C. michiganensis subsp. michiganensis NCPPB 382. The specificity of the primer set was evaluated using purified DNA from 16 isolates of five C. michiganensis subspecies, one other Clavibacter species, and 17 other reference bacteria. The primer set amplified a single band of expected size from the genomic DNA obtained from the C. michiganensis subsp. michiganensis strains but not from the other C. michiganensis subspecies or from other Clavibacter species. The detection limit was a single cloned copy of the ferredoxin reductase gene of C. michiganensis subsp. michiganensis. In conclusion, this quantitative direct PCR assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of seeds and seedlings with a low level or latent infection of C. michiganensis subsp. michiganensis.

Assessment of and standardization for quantitative nondestructive test

NASA Technical Reports Server (NTRS)

Neuschaefer, R. W.; Beal, J. B.

1972-01-01

Present capabilities and limitations of nondestructive testing (NDT) as applied to aerospace structures during design, development, production, and operational phases are assessed. It will help determine what useful structural quantitative and qualitative data may be provided from raw materials to vehicle refurbishment. This assessment considers metal alloys systems and bonded composites presently applied in active NASA programs or strong contenders for future use. Quantitative and qualitative data has been summarized from recent literature, and in-house information, and presented along with a description of those structures or standards where the information was obtained. Examples, in tabular form, of NDT technique capabilities and limitations have been provided. NDT techniques discussed and assessed were radiography, ultrasonics, penetrants, thermal, acoustic, and electromagnetic. Quantitative data is sparse; therefore, obtaining statistically reliable flaw detection data must be strongly emphasized. The new requirements for reusable space vehicles have resulted in highly efficient design concepts operating in severe environments. This increases the need for quantitative NDT evaluation of selected structural components, the end item structure, and during refurbishment operations.

Detection and quantitation of chromosomal mosaicism in human blastocysts using copy number variation sequencing.

PubMed

Ruttanajit, Tida; Chanchamroen, Sujin; Cram, David S; Sawakwongpra, Kritchakorn; Suksalak, Wanwisa; Leng, Xue; Fan, Junmei; Wang, Li; Yao, Yuanqing; Quangkananurug, Wiwat

2016-02-01

Currently, our understanding of the nature and reproductive potential of blastocysts associated with trophectoderm (TE) lineage chromosomal mosaicism is limited. The objective of this study was to first validate copy number variation sequencing (CNV-Seq) for measuring the level of mosaicism and second, examine the nature and level of mosaicism in TE biopsies of patient's blastocysts. TE biopy samples were analysed by array comparative genomic hybridization (CGH) and CNV-Seq to discriminate between euploid, aneuploid and mosaic blastocysts. Using artificial models of TE mosaicism for five different chromosomes, CNV-Seq accurately and reproducibly quantitated mosaicism at levels of 50% and 20%. In a comparative 24-chromosome study of 49 blastocysts by array CGH and CNV-Seq, 43 blastocysts (87.8%) had a concordant diagnosis and 6 blastocysts (12.2%) were discordant. The discordance was attributed to low to medium levels of chromosomal mosaicism (30-70%) not detected by array CGH. In an expanded study of 399 blastocysts using CNV-Seq as the sole diagnostic method, the proportion of diploid-aneuploid mosaics (34, 8.5%) was significantly higher than aneuploid mosaics (18, 4.5%) (p < 0.02). Mosaicism is a significant chromosomal abnormality associated with the TE lineage of human blastocysts that can be reliably and accurately detected by CNV-Seq. © 2015 John Wiley & Sons, Ltd.

Confocal laser-induced fluorescence detector for narrow capillary system with yoctomole limit of detection.

PubMed

Weaver, Mitchell T; Lynch, Kyle B; Zhu, Zaifang; Chen, Huang; Lu, Joann J; Pu, Qiaosheng; Liu, Shaorong

2017-04-01

Laser-induced fluorescence (LIF) detectors for low-micrometer and sub-micrometer capillary on-column detection are not commercially available. In this paper, we describe in details how to construct a confocal LIF detector to address this issue. We characterize the detector by determining its limit of detection (LOD), linear dynamic range (LDR) and background signal drift; a very low LOD (~70 fluorescein molecules or 12 yoctomole fluorescein), a wide LDR (greater than 3 orders of magnitude) and a small background signal drift (~1.2-fold of the root mean square noise) are obtained. For detecting analytes inside a low-micrometer and sub-micrometer capillary, proper alignment is essential. We present a simple protocol to align the capillary with the optical system and use the position-lock capability of a translation stage to fix the capillary in position during the experiment. To demonstrate the feasibility of using this detector for narrow capillary systems, we build a 2-μm-i.d. capillary flow injection analysis (FIA) system using the newly developed LIF prototype as a detector and obtain an FIA LOD of 14 zeptomole fluorescein. We also separate a DNA ladder sample by bare narrow capillary - hydrodynamic chromatography and use the LIF prototype to monitor the resolved DNA fragments. We obtain not only well-resolved peaks but also the quantitative information of all DNA fragments. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative relationship between the octanol/water partition coefficient and the diffusion limitation of the exchange between adipose and blood

PubMed Central

2010-01-01

Background The goal of physiologically based pharmacokinetics (PBPK) is to predict drug kinetics from an understanding of the organ/blood exchange. The standard approach is to assume that the organ is "flow limited" which means that the venous blood leaving the organ equilibrates with the well-stirred tissue compartment. Although this assumption is valid for most solutes, it has been shown to be incorrect for several very highly fat soluble compounds which appear to be "diffusion limited". This paper describes the physical basis of this adipose diffusion limitation and its quantitative dependence on the blood/water (Kbld-wat) and octanol/water (Kow) partition coefficient. Methods Experimental measurements of the time dependent rat blood and adipose concentration following either intravenous or oral input were used to estimate the "apparent" adipose perfusion rate (FA) assuming that the tissue is flow limited. It is shown that the ratio of FA to the anatomic perfusion rate (F) provides a measure of the diffusion limitation. A quantitative relationship between this diffusion limitation and Kbld-wat and Kow is derived. This analysis was applied to previously published data, including the Oberg et. al. measurements of the rat plasma and adipose tissue concentration following an oral dose of a mixture of 13 different polychlorinated biphenyls. Results Solutes become diffusion limited at values of log Kow greater than about 5.6, with the adipose-blood exchange rate reduced by a factor of about 30 for a solute with a log Kow of 7.36. Quantitatively, a plot of FA/F versus Kow is well described assuming an adipose permeability-surface area product (PS) of 750/min. This PS corresponds to a 0.14 micron aqueous layer separating the well-stirred blood from the adipose lipid. This is approximately equal to the thickness of the rat adipose capillary endothelium. Conclusions These results can be used to quantitate the adipose-blood diffusion limitation as a function of Kow. This

Automated detection and quantitation of bacterial RNA by using electrical microarrays.

PubMed

Elsholz, B; Wörl, R; Blohm, L; Albers, J; Feucht, H; Grunwald, T; Jürgen, B; Schweder, T; Hintsche, Rainer

2006-07-15

Low-density electrical 16S rRNA specific oligonucleotide microarrays and an automated analysis system have been developed for the identification and quantitation of pathogens. The pathogens are Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis, which are typically involved in urinary tract infections. Interdigitated gold array electrodes (IDA-electrodes), which have structures in the nanometer range, have been used for very sensitive analysis. Thiol-modified oligonucleotides are immobilized on the gold IDA as capture probes. They mediate the specific recognition of the target 16S rRNA by hybridization. Additionally three unlabeled oligonucleotides are hybridized in close proximity to the capturing site. They are supporting molecules, because they improve the RNA hybridization at the capturing site. A biotin labeled detector oligonucleotide is also allowed to hybridize to the captured RNA sequence. The biotin labels enable the binding of avidin alkaline phophatase conjugates. The phosphatase liberates the electrochemical mediator p-aminophenol from its electrically inactive phosphate derivative. The electrical signals were generated by amperometric redox cycling and detected by a unique multipotentiostat. The read out signals of the microarray are position specific current and change over time in proportion to the analyte concentration. If two additional biotins are introduced into the affinity binding complex via the supporting oligonucleotides, the sensitivity of the assays increase more than 60%. The limit of detection of Escherichia coli total RNA has been determined to be 0.5 ng/microL. The control of fluidics for variable assay formats as well as the multichannel electrical read out and data handling have all been fully automated. The fast and easy procedure does not require any amplification of the targeted nucleic acids by PCR.

A highly selective and fast-response fluorescent probe based on Cd-MOF for the visual detection of Al3+ ion and quantitative detection of Fe3+ ion

NASA Astrophysics Data System (ADS)

Lv, Rui; Chen, Zhihengyu; Fu, Xin; Yang, Boyi; Li, Hui; Su, Jian; Gu, Wen; Liu, Xin

2018-03-01

A new luminescent Cd(II)-based metal-organic framework, [Cd(PAM)(4-bpdb)1.5]·DMF (Cd-MOF, PAM = 4,4‧-methylenebis(3-hydroxy-2-naphthalene-carboxylic acid) and 4-bpdb = 1,4-bis(4-pyridyl)-2,3-diaza-1,3-butadiene) was successfully synthesized by solvothermal synthesis method. The Cd-MOF reveals excellent luminescence property which can selectively detect Al3+ and Fe3+ ions among other interfering metal ions. The detection limit is 0.56 μM for Al3+ ion in aqueous solutions, and it is obvious lower than the maximum standard of Al3+ ion in drinking water of 7.41 μM which is defined by the WHO. More importantly, the Cd-MOF shows an obvious luminescent color change from yellow to blue under the UV lamp irradiation at 365 nm with the dropping of Al3+ ion, which can make it apply to the visual detection. And, the detection based on the test paper was explored for the first time. In addition, the Cd-MOF can also be used for quantitative detecting Fe3+ ion, and the LOD for Fe3+ ion can be as low as 0.3 μM which is lower than most reported MOFs. It is worth noting that Fe3+ and Al3+ ions can not interfere with each other. These properties make it become an excellent luminescence sensor for the detection of Al3+ and Fe3+ ions.

On the Determination of Uncertainty and Limit of Detection in Label-Free Biosensors.

PubMed

Lavín, Álvaro; Vicente, Jesús de; Holgado, Miguel; Laguna, María F; Casquel, Rafael; Santamaría, Beatriz; Maigler, María Victoria; Hernández, Ana L; Ramírez, Yolanda

2018-06-26

A significant amount of noteworthy articles reviewing different label-free biosensors are being published in the last years. Most of the times, the comparison among the different biosensors is limited by the procedure used of calculating the limit of detection and the measurement uncertainty. This article clarifies and establishes a simple procedure to determine the calibration function and the uncertainty of the concentration measured at any point of the measuring interval of a generic label-free biosensor. The value of the limit of detection arises naturally from this model as the limit at which uncertainty tends when the concentration tends to zero. The need to provide additional information, such as the measurement interval and its linearity, among others, on the analytical systems and biosensor in addition to the detection limit is pointed out. Finally, the model is applied to curves that are typically obtained in immunoassays and a discussion is made on the application validity of the model and its limitations.

Towards quantitative magnetic particle imaging: A comparison with magnetic particle spectroscopy

NASA Astrophysics Data System (ADS)

Paysen, Hendrik; Wells, James; Kosch, Olaf; Steinhoff, Uwe; Trahms, Lutz; Schaeffter, Tobias; Wiekhorst, Frank

2018-05-01

Magnetic Particle Imaging (MPI) is a quantitative imaging modality with promising features for several biomedical applications. Here, we study quantitatively the raw data obtained during MPI measurements. We present a method for the calibration of the MPI scanner output using measurements from a magnetic particle spectrometer (MPS) to yield data in units of magnetic moments. The calibration technique is validated in a simplified MPI mode with a 1D excitation field. Using the calibrated results from MPS and MPI, we determine and compare the detection limits for each system. The detection limits were found to be 5.10-12 Am2 for MPS and 3.6.10-10 Am2 for MPI. Finally, the quantitative information contained in a standard MPI measurement with a 3D excitation is analyzed and compared to the previous results, showing a decrease in signal amplitudes of the odd harmonics related to the case of 1D excitation. We propose physical explanations for all acquired results; and discuss the possible benefits for the improvement of MPI technology.

Development of a Quantitative Competitive PCR Assay for Detection and Quantification of Escherichia coli O157:H7 Cells

PubMed Central

Li, Wenli; Drake, Mary Anne

2001-01-01

A quantitative competitive PCR (QC-PCR) assay was developed to detect and quantify Escherichia coli O157:H7 cells. From 103 to 108 CFU of E. coli O157:H7 cells/ml was quantified in broth or skim milk, and cell densities predicted by QC-PCR were highly related to viable cell counts (r2 = 0.99 and 0.93, respectively). QC-PCR has potential for quantitative detection of pathogenic bacteria in foods. PMID:11425755

Prediction of the limit of detection of an optical resonant reflection biosensor.

PubMed

Hong, Jongcheol; Kim, Kyung-Hyun; Shin, Jae-Heon; Huh, Chul; Sung, Gun Yong

2007-07-09

A prediction of the limit of detection of an optical resonant reflection biosensor is presented. An optical resonant reflection biosensor using a guided-mode resonance filter is one of the most promising label-free optical immunosensors due to a sharp reflectance peak and a high sensitivity to the changes of optical path length. We have simulated this type of biosensor using rigorous coupled wave theory to calculate the limit of detection of the thickness of the target protein layer. Theoretically, our biosensor has an estimated ability to detect thickness change approximately the size of typical antigen proteins. We have also investigated the effects of the absorption and divergence of the incident light on the detection ability of the biosensor.

A Quantitative Polymerase Chain Reaction Assay for the Detection and Quantification of Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus 3).

PubMed

Glenney, Gavin W; Barbash, Patricia A; Coll, John A

2016-03-01

Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R(2) = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR. Received April 20, 2015; accepted November 10, 2015.

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

NASA Astrophysics Data System (ADS)

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

2008-10-01

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

Nonparametric rank regression for analyzing water quality concentration data with multiple detection limits.

PubMed

Fu, Liya; Wang, You-Gan

2011-02-15

Environmental data usually include measurements, such as water quality data, which fall below detection limits, because of limitations of the instruments or of certain analytical methods used. The fact that some responses are not detected needs to be properly taken into account in statistical analysis of such data. However, it is well-known that it is challenging to analyze a data set with detection limits, and we often have to rely on the traditional parametric methods or simple imputation methods. Distributional assumptions can lead to biased inference and justification of distributions is often not possible when the data are correlated and there is a large proportion of data below detection limits. The extent of bias is usually unknown. To draw valid conclusions and hence provide useful advice for environmental management authorities, it is essential to develop and apply an appropriate statistical methodology. This paper proposes rank-based procedures for analyzing non-normally distributed data collected at different sites over a period of time in the presence of multiple detection limits. To take account of temporal correlations within each site, we propose an optimal linear combination of estimating functions and apply the induced smoothing method to reduce the computational burden. Finally, we apply the proposed method to the water quality data collected at Susquehanna River Basin in United States of America, which clearly demonstrates the advantages of the rank regression models.

Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

PubMed

Smith, Cindy J; Osborn, A Mark

2009-01-01

Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

LIMITATIONS ON THE USES OF MULTIMEDIA EXPOSURE MEASUREMENTS FOR MULTIPATHWAY EXPOSURE ASSESSMENT - PART I: HANDLING OBSERVATIONS BELOW DETECTION LIMITS

EPA Science Inventory

Multimedia data from two probability-based exposure studies were investigated in terms of how censoring of non-detects affected estimation of population parameters and associations. Appropriate methods for handling censored below-detection-limit (BDL) values in this context were...

Quantitative Detection of Prostatic-Specific Antigens by Using Scanning Electron Microscopy for the Analysis of Protein Chips.

PubMed

Lee, Jisu; Jung, Moon Youn; Park, Hyung Ju

2017-04-01

We reported that quantitative detection of prostatic-specific antigen (PSA), which is the biomarker of prostate cancer, could be carried out by calculating the number density and the area ratio of gold nanoparticle probes on the surface of silicon oxide chips. When chips selectively activated with PSA were immersed in the gold nanoparticles conjugated with prostatic specific antigens-poly clonal antibodies (PSA-pAb), it was possible to observe changes in the number density and the area ratio of gold nanoparticles on the surface of the chips according to the concentration of PSA with scanning electron microscopy (SEM) images. As PSA concentration increased, the number density and the area ratio of gold nanoparticle probes on the surfaces of the chips increased accordingly. Conversely, with lower concentration, the number density and the area ratio of gold nanoparticle probes on the surfaces decreased at a certain ratio. We observed the correlations between PSA concentration and number density, area ratio of gold nanoparticle probes through the analysis of SEM images. In addition, it was confirmed that the sizes of the gold nanoparticles affected the detection limit of the number density and the area ratio of gold nanoparticle probes on the surface.

High performance liquid chromatographic assay for the quantitation of total glutathione in plasma

NASA Technical Reports Server (NTRS)

Abukhalaf, Imad K.; Silvestrov, Natalia A.; Menter, Julian M.; von Deutsch, Daniel A.; Bayorh, Mohamed A.; Socci, Robin R.; Ganafa, Agaba A.

2002-01-01

A simple and widely used homocysteine HPLC procedure was applied for the HPLC identification and quantitation of glutathione in plasma. The method, which utilizes SBDF as a derivatizing agent utilizes only 50 microl of sample volume. Linear quantitative response curve was generated for glutathione over a concentration range of 0.3125-62.50 micromol/l. Linear regression analysis of the standard curve exhibited correlation coefficient of 0.999. Limit of detection (LOD) and limit of quantitation (LOQ) values were 5.0 and 15 pmol, respectively. Glutathione recovery using this method was nearly complete (above 96%). Intra-assay and inter-assay precision studies reflected a high level of reliability and reproducibility of the method. The applicability of the method for the quantitation of glutathione was demonstrated successfully using human and rat plasma samples.

On-site semi-quantitative analysis for ammonium nitrate detection using digital image colourimetry.

PubMed

Choodum, Aree; Boonsamran, Pichapat; NicDaeid, Niamh; Wongniramaikul, Worawit

2015-12-01

Digital image colourimetry was successfully applied in the semi-quantitative analysis of ammonium nitrate using Griess's test with zinc reduction. A custom-built detection box was developed to enable reproducible lighting of samples, and was used with the built-in webcams of a netbook and an ultrabook for on-site detection. The webcams were used for colour imaging of chemical reaction products in the samples, while the netbook was used for on-site colour analysis. The analytical performance was compared to a commercial external webcam and a digital single-lens reflex (DSLR) camera. The relationship between Red-Green-Blue intensities and ammonium nitrate concentration was investigated. The green channel intensity (IG) was the most sensitive for the pink-violet products from ammonium nitrate that revealed a spectrometric absorption peak at 546 nm. A wide linear range (5 to 250 mgL⁻¹) with a high sensitivity was obtained with the built-in webcam of the ultrabook. A considerably lower detection limit (1.34 ± 0.05mgL⁻¹) was also obtained using the ultrabook, in comparison with the netbook (2.6 ± 0.2 mgL⁻¹), the external web cam (3.4 ± 0.1 mgL⁻¹) and the DSLR (8.0 ± 0.5 mgL⁻¹). The best inter-day precision (over 3 days) was obtained with the external webcam (0.40 to 1.34%RSD), while the netbook and the ultrabook had 0.52 to 3.62% and 1.25 to 4.99% RSDs, respectively. The relative errors were +3.6, +5.6 and -7.1%, on analysing standard ammonium nitrate solutions of known concentration using IG, for the ultrabook, the external webcam, and the netbook, respectively, while the DSLR gave -4.4% relative error. However, the IG of the pink-violet reaction product suffers from interference by soil, so that blank subtraction (|IG-IGblank| or |AG-AGblank|) is recommended for soil sample analysis. This method also gave very good accuracies of -0.11 to -5.61% for spiked soil samples and the results presented for five seized samples showed good correlations between

Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

PubMed

Youmans, Bonnie P; Ajami, Nadim J; Jiang, Zhi-Dong; Petrosino, Joseph F; DuPont, Herbert L; Highlander, Sarah K

2014-01-01

Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

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

EPA Science Inventory

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

Development and validation of a reverse transcription quantitative polymerase chain reaction for tilapia lake virus detection in clinical samples and experimentally challenged fish.

PubMed

Tattiyapong, P; Sirikanchana, K; Surachetpong, W

2018-02-01

Tilapia lake virus (TiLV) is an emerging pathogen associated with high mortalities of wild and farm-raised tilapia in different countries. In this study, a SYBR green-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay targeting segment three of the virus was developed to detect and quantify TiLV in clinical samples and experimentally challenged fish. All 30 field samples with clinical signs and history consistent with TiLV infection were positive for TiLV as detected by the developed RT-qPCR method. The RT-qPCR technique provided 100 and 10,000 times more sensitive for virus detection than those offered by the RT-PCR and virus isolation in cell culture methods, respectively. The detection limit of the RT-qPCR method was as low as two viral copies/μl. Moreover, the RT-qPCR technique could be applied for TiLV detection in various fish tissues including gills, liver, brain, heart, anterior kidney and spleen. Significantly, this study delivered an accurate and reliable method for rapid detection of TiLV viruses that facilitates active surveillance programme and disease containment. © 2017 John Wiley & Sons Ltd.

Quantitative Detection of Small Molecule/DNA Complexes Employing a Force-Based and Label-Free DNA-Microarray

PubMed Central

Ho, Dominik; Dose, Christian; Albrecht, Christian H.; Severin, Philip; Falter, Katja; Dervan, Peter B.; Gaub, Hermann E.

2009-01-01

Force-based ligand detection is a promising method to characterize molecular complexes label-free at physiological conditions. Because conventional implementations of this technique, e.g., based on atomic force microscopy or optical traps, are low-throughput and require extremely sensitive and sophisticated equipment, this approach has to date found only limited application. We present a low-cost, chip-based assay, which combines high-throughput force-based detection of dsDNA·ligand interactions with the ease of fluorescence detection. Within the comparative unbinding force assay, many duplicates of a target DNA duplex are probed against a defined reference DNA duplex each. The fractions of broken target and reference DNA duplexes are determined via fluorescence. With this assay, we investigated the DNA binding behavior of artificial pyrrole-imidazole polyamides. These small compounds can be programmed to target specific dsDNA sequences and distinguish between D- and L-DNA. We found that titration with polyamides specific for a binding motif, which is present in the target DNA duplex and not in the reference DNA duplex, reliably resulted in a shift toward larger fractions of broken reference bonds. From the concentration dependence nanomolar to picomolar dissociation constants of dsDNA·ligand complexes were determined, agreeing well with prior quantitative DNAase footprinting experiments. This finding corroborates that the forced unbinding of dsDNA in presence of a ligand is a nonequilibrium process that produces a snapshot of the equilibrium distribution between dsDNA and dsDNA·ligand complexes. PMID:19486688

Comparison of culture-based, vital stain and PMA-qPCR methods for the quantitative detection of viable hookworm ova.

PubMed

Gyawali, P; Sidhu, J P S; Ahmed, W; Jagals, P; Toze, S

2017-06-01

Accurate quantitative measurement of viable hookworm ova from environmental samples is the key to controlling hookworm re-infections in the endemic regions. In this study, the accuracy of three quantitative detection methods [culture-based, vital stain and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR)] was evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by the culture-based method were significantly (P < 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stain methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over the vital stain method in scenarios where ova speciation is needed.

Quantitative SERS Detection of Dopamine in Cerebrospinal Fluid by Dual-Recognition-Induced Hot Spot Generation.

PubMed

Zhang, Kun; Liu, Yu; Wang, Yuning; Zhang, Ren; Liu, Jiangang; Wei, Jia; Qian, Hufei; Qian, Kun; Chen, Ruoping; Liu, Baohong

2018-05-09

Reliable profiling of the extracellular dopamine (DA) concentration in the central nervous system is essential for a deep understanding of its biological and pathological functions. However, quantitative determination of this neurotransmitter remains a challenge because of the extremely low concentration of DA in the cerebrospinal fluid (CSF) of patients. Herein, on the basis of the specific recognition of boronate toward diol and N-hydroxysuccinimide ester toward the amine group, a simple and highly sensitive strategy was presented for DA detection by using surface-enhanced Raman scattering (SERS) spectroscopy as a signal readout. This was realized by first immobilizing 3,3'-dithiodipropionic acid di( N-hydroxysuccinimide ester) on gold thin film surfaces to capture DA, followed by introducing 3-mercaptophenylboronic acid (3-MPBA)-functionalized silver nanoparticles to generate numerous plasmonic "hot spots" with the nanoparticle-on-mirror geometry. Such a dual-recognition mechanism not only avoids complicated bioelement-based manipulations but also efficiently decreases the background signal. With the direct use of the recognition probe 3-MPBA as a Raman reporter, the "signal-on" SERS method was employed to quantify the concentration of DA from 1 pM to 1 μM with a detection limit of 0.3 pM. Moreover, our dual-recognition-directed SERS assay exhibited a high resistance to cerebral interference and was successfully applied to monitoring of DA in CSF samples of patients.

Natural history of optical coherence tomography-detected non-flow-limiting edge dissections following drug-eluting stent implantation.

PubMed

Radu, Maria D; Räber, Lorenz; Heo, Jungho; Gogas, Bill D; Jørgensen, Erik; Kelbæk, Henning; Muramatsu, Takashi; Farooq, Vasim; Helqvist, Steffen; Garcia-Garcia, Hector M; Windecker, Stephan; Saunamäki, Kari; Serruys, Patrick W

2014-01-22

Angiographic evidence of edge dissections has been associated with a risk of early stent thrombosis. Optical coherence tomography (OCT) is a high-resolution technology detecting a greater number of edge dissections--particularly non-flow-limiting--compared to angiography. Their natural history and clinical implications remain unclear. The objectives of the present study were to assess the morphology, healing response, and clinical outcomes of OCT-detected edge dissections using serial OCT imaging at baseline and at one year following drug-eluting stent (DES) implantation. Edge dissections were defined as disruptions of the luminal surface in the 5 mm segments proximal and distal to the stent, and categorised as flaps, cavities, double-lumen dissections or fissures. Qualitative and quantitative OCT analyses were performed every 0.5 mm at baseline and one year, and clinical outcomes were assessed. Sixty-three lesions (57 patients) were studied with OCT at baseline and one-year follow-up. Twenty-two non-flow-limiting edge dissections in 21 lesions (20 patients) were identified by OCT; only two (9%) were angiographically visible. Flaps were found in 96% of cases. The median longitudinal dissection length was 2.9 mm (interquartile range [IQR] 1.6-4.2 mm), whereas the circumferential and axial extensions amounted to 1.2 mm (IQR: 0.9-1.7 mm) and 0.6 mm (IQR: 0.4-0.7 mm), respectively. Dissections extended into the media and adventitia in seven (33%) and four (20%) cases, respectively. Eighteen (82%) OCT-detected edge dissections were also evaluated with intravascular ultrasound which identified nine (50%) of these OCT-detected dissections. No stent thrombosis or target lesion revascularisation occurred up to one year. At follow-up, 20 (90%) edge dissections were completely healed on OCT. The two cases exhibiting persistent dissection had the longest flaps (2.81 mm and 2.42 mm) at baseline. OCT-detected edge dissections which are angiographically silent in the majority of

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

PubMed Central

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

2010-01-01

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

A Direct, Competitive Enzyme-Linked Immunosorbent Assay (ELISA) as a Quantitative Technique for Small Molecules

ERIC Educational Resources Information Center

Powers, Jennifer L.; Rippe, Karen Duda; Imarhia, Kelly; Swift, Aileen; Scholten, Melanie; Islam, Naina

2012-01-01

ELISA (enzyme-linked immunosorbent assay) is a widely used technique with applications in disease diagnosis, detection of contaminated foods, and screening for drugs of abuse or environmental contaminants. However, published protocols with a focus on quantitative detection of small molecules designed for teaching laboratories are limited. A…

A Targeted LC-MS/MS Method for the Simultaneous Detection and Quantitation of Egg, Milk, and Peanut Allergens in Sugar Cookies.

PubMed

Boo, Chelsea C; Parker, Christine H; Jackson, Lauren S

2018-01-01

Food allergy is a growing public health concern, with many individuals reporting allergies to multiple food sources. Compliance with food labeling regulations and prevention of inadvertent cross-contact in manufacturing requires the use of reliable methods for the detection and quantitation of allergens in processed foods. In this work, a novel liquid chromatography-tandem mass spectrometry multiple-reaction monitoring method for multiallergen detection and quantitation of egg, milk, and peanut was developed and evaluated in an allergen-incurred baked sugar cookie matrix. A systematic evaluation of method parameters, including sample extraction, concentration, and digestion, were optimized for candidate allergen peptide markers. The optimized method enabled the reliable detection and quantitation of egg, milk, and peanut allergens in sugar cookies, with allergen concentrations as low as 5 ppm allergen-incurred ingredient.

Qualitative and quantitative analysis of the diuretic component ergone in Polyporus umbellatus by HPLC with fluorescence detection and HPLC-APCI-MS/MS.

PubMed

Zhao, Ying-Yong; Zhao, Ye; Zhang, Yong-Min; Lin, Rui-Chao; Sun, Wen-Ji

2009-06-01

Polyporus umbellatus is a widely used anti-aldosteronic diuretic in Traditional Chinese medicine (TCM). A new, sensitive and selective high-performance liquid chromatography-fluorescence detector (HPLC-FLD) and high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS/MS) method for quantitative and qualitative determination of ergosta-4,6,8(14),22-tetraen-3-one(ergone), which is the main diuretic component, was provided for quality control of P. umbellatus crude drug. The ergone in the ethanolic extract of P. umbellatus was unambiguously characterized by HPLC-APCI, and further confirmed by comparing with a standard compound. The trace ergone was detected by the sensitive and selective HPLC-FLD. Linearity (r2 > 0.9998) and recoveries of low, medium and high concentration (100.5%, 100.2% and 100.4%) were consistent with the experimental criteria. The limit of detection (LOD) of ergone was around 0.2 microg/mL. Our results indicated that the content of ergone in P. umbellatus varied significantly from habitat to habitat with contents ranging from 2.13 +/- 0.02 to 59.17 +/- 0.05 microg/g. Comparison among HPLC-FLD and HPLC-UV or HPLC-APCI-MS/MS demonstrated that the HPLC-FLD and HPLC-APCI-MS/MS methods gave similar quantitative results for the selected herb samples, the HPLC-UV methods gave lower quantitative results than HPLC-FLD and HPLC-APCI-MS/MS methods. The established new HPLC-FLD method has the advantages of being rapid, simple, selective and sensitive, and could be used for the routine analysis of P. umbellatus crude drug.

[Research on rapid and quantitative detection method for organophosphorus pesticide residue].

PubMed

Sun, Yuan-Xin; Chen, Bing-Tai; Yi, Sen; Sun, Ming

2014-05-01

The methods of physical-chemical inspection is adopted in the traditional pesticide residue detection, which require a lot of pretreatment processes, are time-consuming and complicated. In the present study, the authors take chlorpyrifos applied widely in the present agricultural field as the research object and propose a rapid and quantitative detection method for organophosphorus pesticide residues. At first, according to the chemical characteristics of chlorpyrifos and comprehensive chromogenic effect of several colorimetric reagents and secondary pollution, the pretreatment of the scheme of chromogenic reaction of chlorpyrifos with resorcin in a weak alkaline environment was determined. Secondly, by analyzing Uv-Vis spectrum data of chlorpyrifos samples whose content were between 0. 5 and 400 mg kg-1, it was confirmed that the characteristic information after the color reaction mainly was concentrated among 360 approximately 400 nm. Thirdly, the full spectrum forecasting model was established based on the partial least squares, whose correlation coefficient of calibration was 0. 999 6, correlation coefficient of prediction reached 0. 995 6, standard deviation of calibration (RMSEC) was 2. 814 7 mg kg-1, and standard deviation of verification (RMSEP) was 8. 012 4 mg kg-1. Fourthly, the wavelengths whose center wavelength is 400 nm was extracted as characteristic region to build a forecasting model, whose correlation coefficient of calibration was 0. 999 6, correlation coefficient of prediction reached 0. 999 3, standard deviation of calibration (RMSEC) was 2. 566 7 mg kg-1 , standard deviation of verification (RMSEP) was 4. 886 6 mg kg-1, respectively. At last, by analyzing the near infrared spectrum data of chlorpyrifos samples with contents between 0. 5 and 16 mg kg-1, the authors found that although the characteristics of the chromogenic functional group are not obvious, the change of absorption peaks of resorcin itself in the neighborhood of 5 200 cm

A Complementary Isothermal Amplification Method to the U.S. EPA Quantitative Polymerase Chain Reaction Approach for the Detection of Enterococci in Environmental Waters

PubMed Central

2017-01-01

We report a novel molecular assay, based on helicase-dependent amplification (HDA), for the detection of enterococci as markers for fecal pollution in water. This isothermal assay targets the same Enterococcus 23S rRNA gene region as the existing quantitative polymerase chain reaction (qPCR) assays of U.S. Environmental Protection Agency Methods 1611 and 1609 but can be entirely performed on a simple heating block. The developed Enterococcus HDA assay successfully discriminated 15 enterococcal from 15 non-enterococcal reference strains and reliably detected 48 environmental isolates of enterococci. The limit of detection was 25 target copies per reaction, only 3 times higher than that of qPCR. The applicability of the assay was tested on 30 environmental water sample DNA extracts, simulating a gradient of fecal pollution. Despite the isothermal nature of the reaction, the HDA results were consistent with those of the qPCR reference. Given this performance, we conclude that the developed Enterococcus HDA assay has great potential as a qualitative molecular screening method for resource-limited settings when combined with compatible up- and downstream processes. This amplification strategy can pave the way for developing a new generation of rapid, low-cost, and field-deployable molecular diagnostic tools for water quality monitoring. PMID:28541661

Development of an enzyme-linked immunosorbent assay for the detection of dicamba.

PubMed

Clegg, B S; Stephenson, G R; Hall, J C

2001-05-01

A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed to quantitate the herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) in water. The CI-ELISA has a detection limit of 2.3 microg L(-1) and a linear working range of 10--10000 microg L(-1) with an IC(50) value of 195 microg L(-1). The dicamba polyclonal antisera did not cross-react with a number of other herbicides tested but did cross-react with a dicamba metabolite, 5-hydroxydicamba, and structurally related chlorobenzoic acids. The assay was used to estimate quantitatively dicamba concentrations in water samples. Water samples were analyzed directly, and no sample preparation was required. To improve detection limits, a C(18) (reversed phase) column concentration step was devised prior to analysis, and the detection limits were increased by at least by 10-fold. After the sample preconcentration, the detection limit, IC(50), and linear working range were 0.23, 19.5, and 5-200 microg L(-1), respectively. The CI-ELISA estimations in water correlated well with those from gas chromatography-mass spectrometry (GC-MS) analysis (r(2) = 0.9991). This assay contributes to reducing laboratory costs associated with the conventional GC-MS residue analysis techniques for the quantitation of dicamba in water.

Photoelectrochemical detection of benzaldehyde in foodstuffs

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

LaCourse, W.R.; Krull, I.S.

Photoelectrochemical detection (PED) coupled with high performance liquid chromatography was used to quantitatively determine benzaldehyde in extracts, beverages, and foodstuffs. Photoelectrochemical detection is responsive to alkyl and aryl ketones and aldehydes and offers the advantages of 2-3 orders of magnitude linearity, 5-1-ng limits of detection, and a high degree of selectivity without chemical derivatization. This is the first application of the PED to sample analysis.

Characterization and Comparison of Galactomannan Enzyme Immunoassay and Quantitative Real-Time PCR Assay for Detection of Aspergillus fumigatus in Bronchoalveolar Lavage Fluid from Experimental Invasive Pulmonary Aspergillosis

PubMed Central

Francesconi, Andrea; Kasai, Miki; Petraitiene, Ruta; Petraitis, Vidmantas; Kelaher, Amy M.; Schaufele, Robert; Hope, William W.; Shea, Yvonne R.; Bacher, John; Walsh, Thomas J.

2006-01-01

Bronchoalveolar lavage (BAL) is widely used for evaluation of patients with suspected invasive pulmonary aspergillosis (IPA). However, the diagnostic yield of BAL for detection of IPA by culture and direct examination is limited. Earlier diagnosis may be facilitated by assays that can detect Aspergillus galactomannan antigen or DNA in BAL fluid. We therefore characterized and compared the diagnostic yields of a galactomannan enzyme immunoassay (GM EIA), quantitative real-time PCR (qPCR), and quantitative cultures in experiments using BAL fluid from neutropenic rabbits with experimentally induced IPA defined as microbiologically and histologically evident invasion. The qPCR assay targeted the rRNA gene complex of Aspergillus fumigatus. The GM EIA and qPCR assay were characterized by receiver operator curve analysis. With an optimal cutoff of 0.75, the GM EIA had a sensitivity and specificity of 100% in untreated controls. A decline in sensitivity (92%) was observed when antifungal therapy (AFT) was administered. The optimal cutoff for qPCR was a crossover of 36 cycles, with sensitivity and specificity of 80% and 100%, respectively. The sensitivity of qPCR also decreased with AFT to 50%. Quantitative culture of BAL had a sensitivity of 46% and a specificity of 100%. The sensitivity of quantitative culture decreased with AFT to 16%. The GM EIA and qPCR assay had greater sensitivity than culture in detection of A. fumigatus in BAL fluid in experimentally induced IPA (P ± 0.04). Use of the GM EIA and qPCR assay in conjunction with culture-based diagnostic methods applied to BAL fluid could facilitate accurate diagnosis and more-timely initiation of specific therapy. PMID:16825367

Cellular chromosome DNA interferes with fluorescence quantitative real-time PCR detection of HBV DNA in culture medium.

PubMed

Pan, Xiao-Ben; Wei, Lai; Han, Jin-Chao; Gao, Yan

2008-01-01

Fluorescence quantitative real-time PCR (FQ-PCR) is a recently developed technique increasingly used for clinical diagnosis by detection of hepatitis B virus (HBV) DNA in serum. FQ-PCR is also used in scientific research for detection of HBV DNA in cell culture. Understanding potential FQ-PCR interference factors can improve the accuracy of HBV DNA quantification in cell culture medium. HBV positive serum was diluted with culture medium to produce three test groups with HBV DNA levels of 5 x 10(7) copies/ml (high), 5 x 10(5) copies/ml (medium), and 5 x 10(3) copies/ml (low). Chromosome DNA was extracted from HepG2 cells and then added to high, medium, and low group samples at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. The samples were quantified by FQ-PCR and data were evaluated using statistical software. No marked changes were seen in the quantitative curves for high level HBV DNA samples when the samples were supplemented with 0-100 microg/ml of chromosome DNA. Interference was observed in medium level samples when 50 and 100 microg/ml of chromosome DNA was added. Interference was also observed in low level HBV DNA samples when the concentration of added chromosome DNA was greater than 25 microg/ml. The interference was eliminated when samples were digested by DNase I prior to PCR detection. In Conclusions, the presence of cellular chromosome DNA can interfere with the detection of HBV DNA by FQ-PCR. Removal of cellular chromosome DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection. (c) 2007 Wiley-Liss, Inc.

Quantitative detection of settled coal dust over green canopy

NASA Astrophysics Data System (ADS)

Brook, Anna; Sahar, Nir

2017-04-01

The main task of environmental and geoscience applications are efficient and accurate quantitative classification of earth surfaces and spatial phenomena. In the past decade, there has been a significant interest in employing spectral unmixing in order to retrieve accurate quantitative information latent in in situ data. Recently, the ground-truth and laboratory measured spectral signatures promoted by advanced algorithms are proposed as a new path toward solving the unmixing problem in semi-supervised fashion. This study presents a practical implementation of field spectroscopy as a quantitative tool to detect settled coal dust over green canopy in free/open environment. Coal dust is a fine powdered form of coal, which is created by the crushing, grinding, and pulverizing of coal. Since the inelastic nature of coal, coal dust can be created during transportation, or by mechanically handling coal. Coal dust, categorized at silt-clay particle size, of particular concern due to heavy metals (lead, mercury, nickel, tin, cadmium, mercury, antimony, arsenic, isotopes of thorium and strontium) which are toxic also at low concentrations. This hazard exposes risk on both environment and public health. It has been identified by medical scientist around the world as causing a range of diseases and health problems, mainly heart and respiratory diseases like asthma and lung cancer. It is due to the fact that the fine invisible coal dust particles (less than 2.5 microns) long lodge in the lungs and are not naturally expelled, so long-term exposure increases the risk of health problems. Numerus studies reported that data to conduct study of geographic distribution of the very fine coal dust (smaller than PM 2.5) and related health impacts from coal exports, is not being collected. Sediment dust load in an indoor environment can be spectrally assessed using reflectance spectroscopy (Chudnovsky and Ben-Dor, 2009). Small amounts of particulate pollution that may carry a signature

Effect of saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus in oral fluid by quantitative reverse transcriptase real-time PCR.

PubMed

Decorte, Inge; Van der Stede, Yves; Nauwynck, Hans; De Regge, Nick; Cay, Ann Brigitte

2013-08-01

This study evaluated the effect of extraction-amplification methods, storage temperature and saliva stabilisers on detection of porcine reproductive and respiratory syndrome virus (PRRSV) RNA by quantitative reverse transcriptase real-time PCR (qRT-PCR) in porcine oral fluid. The diagnostic performance of different extraction-amplification methods was examined using a dilution series of oral fluid spiked with PRRSV. To determine RNA stability, porcine oral fluid, with or without commercially available saliva stabilisers, was spiked with PRRSV, stored at 4°C or room temperature and tested for the presence of PRRSV RNA by qRT-PCR. PRRSV RNA could be detected in oral fluid using all extraction-amplification combinations, but the limit of detection varied amongst different combinations. Storage temperature and saliva stabilisers had an effect on the stability of PRRSV RNA, which could only be detected for 7 days when PRRSV spiked oral fluid was kept at 4°C or stabilised at room temperature with a commercial mRNA stabiliser. Copyright © 2013 Elsevier Ltd. All rights reserved.

Bioelectrocatalytic application of titania nanotube array for molecule detection.

PubMed

Xie, Yibing; Zhou, Limin; Huang, Haitao

2007-06-15

A bioelectrocatalysis system based on titania nanotube electrode has been developed for the quantitative detection application. Highly ordered titania nanotube array with inner diameter of 60 nm and total length of 540 nm was formed by anodizing titanium foils. The functionalization modification was achieved by embedding glucose oxidases inside tubule channels and electropolymerizing pyrrole for interfacial immobilization. Morphology and microstructure characterization, electrochemical properties and bioelectrocatalytic reactivities of this composite were fully investigated. The direct detection of hydrogen peroxide by electrocatalytic reduction reaction was fulfilled on pure titania nanotube array with a detection limit up to 2.0 x 10(-4)mM. A biosensor based on the glucose oxidase-titania/titanium electrode was constructed for amperometric detection and quantitative determination of glucose in a phosphate buffer solution (pH 6.8) under a potentiostatic condition (-0.4V versus SCE). The resulting glucose biosensor showed an excellent performance with a response time below 5.6s and a detection limit of 2.0 x 10(-3)mM. The corresponding detection sensitivity was 45.5 microA mM(-1)cm(-2). A good operational reliability was also achieved with relative standard deviations below 3.0%. This novel biosensor exhibited quite high response sensitivity and low detection limit for potential applications.

An adaptive confidence limit for periodic non-steady conditions fault detection

NASA Astrophysics Data System (ADS)

Wang, Tianzhen; Wu, Hao; Ni, Mengqi; Zhang, Milu; Dong, Jingjing; Benbouzid, Mohamed El Hachemi; Hu, Xiong

2016-05-01

System monitoring has become a major concern in batch process due to the fact that failure rate in non-steady conditions is much higher than in steady ones. A series of approaches based on PCA have already solved problems such as data dimensionality reduction, multivariable decorrelation, and processing non-changing signal. However, if the data follows non-Gaussian distribution or the variables contain some signal changes, the above approaches are not applicable. To deal with these concerns and to enhance performance in multiperiod data processing, this paper proposes a fault detection method using adaptive confidence limit (ACL) in periodic non-steady conditions. The proposed ACL method achieves four main enhancements: Longitudinal-Standardization could convert non-Gaussian sampling data to Gaussian ones; the multiperiod PCA algorithm could reduce dimensionality, remove correlation, and improve the monitoring accuracy; the adaptive confidence limit could detect faults under non-steady conditions; the fault sections determination procedure could select the appropriate parameter of the adaptive confidence limit. The achieved result analysis clearly shows that the proposed ACL method is superior to other fault detection approaches under periodic non-steady conditions.

An electrochemical immunosensor for quantitative detection of ficolin-3

NASA Astrophysics Data System (ADS)

San, Lili; Zeng, Dongdong; Song, Shiping; Zuo, Xiaolei; Zhang, Huan; Wang, Chenguang; Wu, Jiarui; Mi, Xianqiang

2016-06-01

Diabetes mellitus (DM) is one of the most common metabolic disorders in the world, of which more than 90% is type-2 diabetes mellitus (T2DM). There is a rather urgent need for reliable, sensitive and quick detection techniques in clinical application of T2DM. Ficolin-3 is a potential biomarker of T2DM, because serum ficolin-3 levels are associated with insulin resistance and predict the incidence of T2DM. Herein, a sandwich-type electrochemical immunosensor was developed for the detection of ficolin-3 in human serum. Cyclic voltammetry and the amperometric current versus time were used to characterize the performance of the immunosensor. Under optimal conditions, the detection limitation of ficolin-3 was 100 ng ml-1 and the linear dynamic range was between 2 and 50 μg ml-1. The method has ideal accuracy, excellent stability and selectivity and has wide application prospects in clinical research.

Gold nanoparticle-based low limit of detection Love wave biosensor for carcinoembryonic antigens.

PubMed

Li, Shuangming; Wan, Ying; Su, Yan; Fan, Chunhai; Bhethanabotla, Venkat R

2017-09-15

In this work, a Love wave biosensing platform is described for detecting cancer-related biomarker carcinoembryonic antigen (CEA). An ST 90°-X quartz Love wave device with a layer of SiO 2 waveguide was combined with gold nanoparticles (Au NPs) to amplify the mass loading effect of the acoustic wave sensor to achieve a limit of detection of 37pg/mL. The strategy involves modifying the Au NPs with anti-CEA antibody conjugates to form nanoprobes in a sandwich immunoassay. The unamplified detection limit of the Love wave biosensor is 9.4ng/mL. This 2-3 order of magnitude reduction in the limit of detection brings the SAW platform into the range useful for clinical diagnosis. Measurement electronics and microfluidics are easily constructed for acoustic wave biosensors, such as the Love wave device described here, allowing for robust platforms for point of care applications for cancer biomarkers in general. Copyright © 2017 Elsevier B.V. All rights reserved.

Strategies and limitations for fluorescence detection of XAFS at high flux beamlines

DOE PAGES

Heald, Steve M.

2015-02-17

The issue of detecting the XAFS signal from dilute samples is discussed in detail with the aim of making best use of high flux beamlines that provide up to 10 13 photons -1. Various detection methods are compared, including filters with slits, solid state detectors, crystal analyzers and combinations of these. These comparisons rely on simulations that use experimentally determined parameters. It is found that inelastic scattering places a fundamental limit on detection, and that it is important to take proper account of the polarization dependence of the signals. The combination of a filter–slit system with a solid state detectormore » is a promising approach. With an optimized system good performance can be obtained even if the total count rate is limited to 10 7 Hz. Detection schemes with better energy resolution can help at the largest dilutions if their collection efficiency and count rate limits can be improved.« less

Strategies and limitations for fluorescence detection of XAFS at high flux beamlines

PubMed Central

Heald, Steve M.

2015-01-01

The issue of detecting the XAFS signal from dilute samples is discussed in detail with the aim of making best use of high flux beamlines that provide up to 1013 photons s−1. Various detection methods are compared, including filters with slits, solid state detectors, crystal analyzers and combinations of these. These comparisons rely on simulations that use experimentally determined parameters. It is found that inelastic scattering places a fundamental limit on detection, and that it is important to take proper account of the polarization dependence of the signals. The combination of a filter–slit system with a solid state detector is a promising approach. With an optimized system good performance can be obtained even if the total count rate is limited to 107 Hz. Detection schemes with better energy resolution can help at the largest dilutions if their collection efficiency and count rate limits can be improved. PMID:25723945

Limit of detection of 15{sub N} by gas-chromatography atomic emission detection: Optimization using an experimental design

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

Deruaz, D.; Bannier, A.; Pionchon, C.

1995-08-01

This paper deals with the optimal conditions for the detection of {sup 15}N determined using a four-factor experimental design from [2{sup 13}C,-1,3 {sup 15}N] caffeine measured with an atomic emission detector (AED) coupled to gas chromatography (GC). Owing to the capability of a photodiodes array, AED can simultaneously detect several elements using their specific emission lines within a wavelength range of 50 nm. So, the emissions of {sup 15}N and {sup 14}N are simultaneously detected at 420.17 nm and 421.46 nm respectively. Four independent experimental factors were tested (1) helium flow rate (plasma gas); (2) methane pressure (reactant gas); (3)more » oxygen pressure; (4) hydrogen pressure. It has been shown that these four gases had a significant influence on the analytical response of {sup 15}N. The linearity of the detection was determined using {sup 15}N amounts ranging from 1.52 pg to 19 ng under the optimal conditions obtained from the experimental design. The limit of detection was studied using different methods. The limits of detection of {sup 15}N was 1.9 pg/s according to the IUPAC method (International-Union of Pure and Applied Chemistry). The method proposed by Quimby and Sullivan gave a value of 2.3 pg/s and that of Oppenheimer gave a limit of 29 pg/s. For each determination, and internal standard: 1-isobutyl-3.7 dimethylxanthine was used. The results clearly demonstrate that GC AED is sensitive and selective enough to detect and measure {sup 15}N-labelled molecules after gas chromatographic separation.« less

Method for improving the limit of detection in a data signal

DOEpatents

Synovec, Robert E.; Yueng, Edward S.

1989-10-17

A method for improving the limit of detection for a data set in which experimental noise is uncorrelated along a given abscissa and an analytical signal is correlated to the abscissa, the steps comprising collecting the data set, converting the data set into a data signal including an analytical portion and the experimental noise portion, designating and adjusting a baseline of the data signal to center the experimental noise numerically about a zero reference, and integrating the data signal preserving the corresponding information for each point of the data signal. The steps of the method produce an enhanced integrated data signal which improves the limit of detection of the data signal.

Quantitative Phase Fraction Detection in Organic Photovoltaic Materials through EELS Imaging

DOE PAGES

Dyck, Ondrej; Hu, Sheng; Das, Sanjib; ...

2015-11-24

Organic photovoltaic materials have recently seen intense interest from the research community. Improvements in device performance are occurring at an impressive rate; however, visualization of the active layer phase separation still remains a challenge. Our paper outlines the application of two electron energy-loss spectroscopic (EELS) imaging techniques that can complement and enhance current phase detection techniques. Specifically, the bulk plasmon peak position, often used to produce contrast between phases in energy filtered transmission electron microscopy (EFTEM), is quantitatively mapped across a sample cross section. One complementary spectrum image capturing the carbon and sulfur core loss edges is compared with themore » plasmon peak map and found to agree quite well, indicating that carbon and sulfur density differences between the two phases also allows phase discrimination. Additionally, an analytical technique for determining absolute atomic areal density is used to produce an absolute carbon and sulfur areal density map. We also show how these maps may be re-interpreted as a phase ratio map, giving quantitative information about the purity of the phases within the junction.« less

Study on the Simultaneously Quantitative Detection for β-Lactoglobulin and Lactoferrin of Cow Milk by Using Protein Chip Technique.

PubMed

Yin, Ji Yong; Huo, Jun Sheng; Ma, Xin Xin; Sun, Jing; Huang, Jian

2017-12-01

To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin (β-L) and Lactoferrin (Lf) at one time. Protein chip printer was used to print both anti-β-L antibodies and anti-Lf antibodies on each block of protein chip. And then an improved sandwich detection method was applied while the other two detecting antibodies for the two antigens were added in the block after they were mixed. The detection conditions of the quantitative detection for simultaneous measurement of β-L and Lf with protein chip were optimized and evaluated. Based on these detected conditions, two standard curves of the two proteins were simultaneously established on one protein chip. Finally, the new detection method was evaluated by using the analysis of precision and accuracy. By comparison experiment, mouse monoclonal antibodies of the two antigens were chosen as the printing probe. The concentrations of β-L and Lf probes were 0.5 mg/mL and 0.5 mg/mL, respectively, while the titers of detection antibodies both of β-L and Lf were 1:2,000. Intra- and inter-assay variability was between 4.88% and 38.33% for all tests. The regression coefficients of protein chip comparing with ELISA for β-L and Lf were better than 0.734, and both of the two regression coefficients were statistically significant (r = 0.734, t = 2.644, P = 0.038; and r = 0.774, t = 2.998, P = 0.024). A protein chip method of simultaneously quantitative detection for β-L and Lf has been established and this method is worthy in further application. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Rapid quantitative detection of glucose content in glucose injection by reaction headspace gas chromatography.

PubMed

Xie, Wei-Qi; Gong, Yi-Xian; Yu, Kong-Xian

2017-10-20

This work investigates an automated technique for rapid detecting the glucose content in glucose injection by reaction headspace gas chromatography (HS-GC). This method is based on the oxidation reaction of glucose in glucose injection with potassium dichromate. The carbon dioxide (CO 2 ) formed from the oxidation reaction can be quantitatively detected by GC. The results show that the relative standard deviation (RSD) of the present method was within 2.91%, and the measured glucose contents in glucose injection closely match those quantified by the reference method (relative differences <6.45%). The new HS-GC technique is rapid, practical and can be used to the batch detection of the glucose content in glucose injection related applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluating detection limits of next-generation sequencing for the surveillance and monitoring of international marine pests.

PubMed

Pochon, Xavier; Bott, Nathan J; Smith, Kirsty F; Wood, Susanna A

2013-01-01

Most surveillance programmes for marine invasive species (MIS) require considerable taxonomic expertise, are laborious, and are unable to identify species at larval or juvenile stages. Therefore, marine pests may go undetected at the initial stages of incursions when population densities are low. In this study, we evaluated the ability of the benchtop GS Junior™ 454 pyrosequencing system to detect the presence of MIS in complex sample matrices. An initial in-silico evaluation of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear small subunit ribosomal DNA (SSU) genes, found that multiple primer sets (targeting a ca. 400 base pair region) would be required to obtain species level identification within the COI gene. In contrast a single universal primer set was designed to target the V1-V3 region of SSU, allowing simultaneous PCR amplification of a wide taxonomic range of MIS. To evaluate the limits of detection of this method, artificial contrived communities (10 species from 5 taxonomic groups) were created using varying concentrations of known DNA samples and PCR products. Environmental samples (water and sediment) spiked with one or five 160 hr old Asterias amurensis larvae were also examined. Pyrosequencing was able to recover DNA/PCR products of individual species present at greater than 0.64% abundance from all tested contrived communities. Additionally, single A. amurensis larvae were detected from both water and sediment samples despite the co-occurrence of a large array of environmental eukaryotes, indicating an equivalent sensitivity to quantitative PCR. NGS technology has tremendous potential for the early detection of marine invasive species worldwide.

Theoretical limitations of quantification for noncompetitive sandwich immunoassays.

PubMed

Woolley, Christine F; Hayes, Mark A; Mahanti, Prasun; Douglass Gilman, S; Taylor, Tom

2015-11-01

Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations.

Determination of Minor and Trace Metals in Aluminum and Aluminum Alloys by ICP-AES; Evaluation of the Uncertainty and Limit of Quantitation from Interlaboratory Testing.

PubMed

Uemoto, Michihisa; Makino, Masanori; Ota, Yuji; Sakaguchi, Hiromi; Shimizu, Yukari; Sato, Kazuhiro

2018-01-01

Minor and trace metals in aluminum and aluminum alloys have been determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) as an interlaboratory testing toward standardization. The trueness of the measured data was successfully investigated to improve the analytical protocols, using certified reference materials of aluminum. Their precision could also be evaluated, feasible to estimate the uncertainties separately. The accuracy (trueness and precision) of the data were finally in good agreement with the certified values and assigned uncertainties. Repeated measurements of aluminum solutions with different concentrations of the analytes revealed the relative standard deviations of the measurements with concentrations, thus enabling their limits of quantitation. They differed separately and also showed slightly higher values with an aluminum matrix than those without one. In addition, the upper limit of the detectable concentration of silicon with simple acid digestion was estimated to be 0.03 % in the mass fraction.

METHODS OF DEALING WITH VALUES BELOW THE LIMIT OF DETECTION USING SAS

EPA Science Inventory

Due to limitations of chemical analysis procedures, small concentrations cannot be precisely measured. These concentrations are said to be below the limit of detection (LOD). In statistical analyses, these values are often censored and substituted with a constant value, such ...

Detection of Head and Neck Cancer in Surgical Specimens Using Quantitative Hyperspectral Imaging.

PubMed

Lu, Guolan; Little, James V; Wang, Xu; Zhang, Hongzheng; Patel, Mihir R; Griffith, Christopher C; El-Deiry, Mark W; Chen, Amy Y; Fei, Baowei

2017-09-15

Purpose: This study intends to investigate the feasibility of using hyperspectral imaging (HSI) to detect and delineate cancers in fresh, surgical specimens of patients with head and neck cancers. Experimental Design: A clinical study was conducted in order to collect and image fresh, surgical specimens from patients ( N = 36) with head and neck cancers undergoing surgical resection. A set of machine-learning tools were developed to quantify hyperspectral images of the resected tissue in order to detect and delineate cancerous regions which were validated by histopathologic diagnosis. More than two million reflectance spectral signatures were obtained by HSI and analyzed using machine-learning methods. The detection results of HSI were compared with autofluorescence imaging and fluorescence imaging of two vital-dyes of the same specimens. Results: Quantitative HSI differentiated cancerous tissue from normal tissue in ex vivo surgical specimens with a sensitivity and specificity of 91% and 91%, respectively, and which was more accurate than autofluorescence imaging ( P < 0.05) or fluorescence imaging of 2-NBDG ( P < 0.05) and proflavine ( P < 0.05). The proposed quantification tools also generated cancer probability maps with the tumor border demarcated and which could provide real-time guidance for surgeons regarding optimal tumor resection. Conclusions: This study highlights the feasibility of using quantitative HSI as a diagnostic tool to delineate the cancer boundaries in surgical specimens, and which could be translated into the clinic application with the hope of improving clinical outcomes in the future. Clin Cancer Res; 23(18); 5426-36. ©2017 AACR . ©2017 American Association for Cancer Research.

Quantitative phase-filtered wavelength-modulated differential photoacoustic radar tumor hypoxia imaging toward early cancer detection.

PubMed

Dovlo, Edem; Lashkari, Bahman; Soo Sean Choi, Sung; Mandelis, Andreas; Shi, Wei; Liu, Fei-Fei

2017-09-01

Overcoming the limitations of conventional linear spectroscopy used in multispectral photoacoustic imaging, wherein a linear relationship is assumed between the absorbed optical energy and the absorption spectra of the chromophore at a specific location, is crucial for obtaining accurate spatially-resolved quantitative functional information by exploiting known chromophore-specific spectral characteristics. This study introduces a non-invasive phase-filtered differential photoacoustic technique, wavelength-modulated differential photoacoustic radar (WM-DPAR) imaging that addresses this issue by eliminating the effect of the unknown wavelength-dependent fluence. It employs two laser wavelengths modulated out-of-phase to significantly suppress background absorption while amplifying the difference between the two photoacoustic signals. This facilitates pre-malignant tumor identification and hypoxia monitoring, as minute changes in total hemoglobin concentration and hemoglobin oxygenation are detectable. The system can be tuned for specific applications such as cancer screening and SO 2 quantification by regulating the amplitude ratio and phase shift of the signal. The WM-DPAR imaging of a head and neck carcinoma tumor grown in the thigh of a nude rat demonstrates the functional PA imaging of small animals in vivo. The PA appearance of the tumor in relation to tumor vascularity is investigated by immunohistochemistry. Phase-filtered WM-DPAR imaging is also illustrated, maximizing quantitative SO 2 imaging fidelity of tissues. Oxygenation levels within a tumor grown in the thigh of a nude rat using the two-wavelength phase-filtered differential PAR method. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhanced Detection of Surface-Associated Bacteria in Indoor Environments by Quantitative PCR

PubMed Central

Buttner, Mark P.; Cruz-Perez, Patricia; Stetzenbach, Linda D.

2001-01-01

Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitate Bacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling

Optimal Hotspots of Dynamic Surfaced-Enhanced Raman Spectroscopy for Drugs Quantitative Detection.

PubMed

Yan, Xiunan; Li, Pan; Zhou, Binbin; Tang, Xianghu; Li, Xiaoyun; Weng, Shizhuang; Yang, Liangbao; Liu, Jinhuai

2017-05-02

Surface-enhanced Raman spectroscopy (SERS) as a powerful qualitative analysis method has been widely applied in many fields. However, SERS for quantitative analysis still suffers from several challenges partially because of the absence of stable and credible analytical strategy. Here, we demonstrate that the optimal hotspots created from dynamic surfaced-enhanced Raman spectroscopy (D-SERS) can be used for quantitative SERS measurements. In situ small-angle X-ray scattering was carried out to in situ real-time monitor the formation of the optimal hotspots, where the optimal hotspots with the most efficient hotspots were generated during the monodisperse Au-sol evaporating process. Importantly, the natural evaporation of Au-sol avoids the nanoparticles instability of salt-induced, and formation of ordered three-dimensional hotspots allows SERS detection with excellent reproducibility. Considering SERS signal variability in the D-SERS process, 4-mercaptopyridine (4-mpy) acted as internal standard to validly correct and improve stability as well as reduce fluctuation of signals. The strongest SERS spectra at the optimal hotspots of D-SERS have been extracted to statistics analysis. By using the SERS signal of 4-mpy as a stable internal calibration standard, the relative SERS intensity of target molecules demonstrated a linear response versus the negative logarithm of concentrations at the point of strongest SERS signals, which illustrates the great potential for quantitative analysis. The public drugs 3,4-methylenedioxymethamphetamine and α-methyltryptamine hydrochloride obtained precise analysis with internal standard D-SERS strategy. As a consequence, one has reason to believe our approach is promising to challenge quantitative problems in conventional SERS analysis.

Automated Detection of P. falciparum Using Machine Learning Algorithms with Quantitative Phase Images of Unstained Cells.

PubMed

Park, Han Sang; Rinehart, Matthew T; Walzer, Katelyn A; Chi, Jen-Tsan Ashley; Wax, Adam

2016-01-01

Malaria detection through microscopic examination of stained blood smears is a diagnostic challenge that heavily relies on the expertise of trained microscopists. This paper presents an automated analysis method for detection and staging of red blood cells infected by the malaria parasite Plasmodium falciparum at trophozoite or schizont stage. Unlike previous efforts in this area, this study uses quantitative phase images of unstained cells. Erythrocytes are automatically segmented using thresholds of optical phase and refocused to enable quantitative comparison of phase images. Refocused images are analyzed to extract 23 morphological descriptors based on the phase information. While all individual descriptors are highly statistically different between infected and uninfected cells, each descriptor does not enable separation of populations at a level satisfactory for clinical utility. To improve the diagnostic capacity, we applied various machine learning techniques, including linear discriminant classification (LDC), logistic regression (LR), and k-nearest neighbor classification (NNC), to formulate algorithms that combine all of the calculated physical parameters to distinguish cells more effectively. Results show that LDC provides the highest accuracy of up to 99.7% in detecting schizont stage infected cells compared to uninfected RBCs. NNC showed slightly better accuracy (99.5%) than either LDC (99.0%) or LR (99.1%) for discriminating late trophozoites from uninfected RBCs. However, for early trophozoites, LDC produced the best accuracy of 98%. Discrimination of infection stage was less accurate, producing high specificity (99.8%) but only 45.0%-66.8% sensitivity with early trophozoites most often mistaken for late trophozoite or schizont stage and late trophozoite and schizont stage most often confused for each other. Overall, this methodology points to a significant clinical potential of using quantitative phase imaging to detect and stage malaria infection

Automated Detection of P. falciparum Using Machine Learning Algorithms with Quantitative Phase Images of Unstained Cells

PubMed Central

Park, Han Sang; Rinehart, Matthew T.; Walzer, Katelyn A.; Chi, Jen-Tsan Ashley; Wax, Adam

2016-01-01

Malaria detection through microscopic examination of stained blood smears is a diagnostic challenge that heavily relies on the expertise of trained microscopists. This paper presents an automated analysis method for detection and staging of red blood cells infected by the malaria parasite Plasmodium falciparum at trophozoite or schizont stage. Unlike previous efforts in this area, this study uses quantitative phase images of unstained cells. Erythrocytes are automatically segmented using thresholds of optical phase and refocused to enable quantitative comparison of phase images. Refocused images are analyzed to extract 23 morphological descriptors based on the phase information. While all individual descriptors are highly statistically different between infected and uninfected cells, each descriptor does not enable separation of populations at a level satisfactory for clinical utility. To improve the diagnostic capacity, we applied various machine learning techniques, including linear discriminant classification (LDC), logistic regression (LR), and k-nearest neighbor classification (NNC), to formulate algorithms that combine all of the calculated physical parameters to distinguish cells more effectively. Results show that LDC provides the highest accuracy of up to 99.7% in detecting schizont stage infected cells compared to uninfected RBCs. NNC showed slightly better accuracy (99.5%) than either LDC (99.0%) or LR (99.1%) for discriminating late trophozoites from uninfected RBCs. However, for early trophozoites, LDC produced the best accuracy of 98%. Discrimination of infection stage was less accurate, producing high specificity (99.8%) but only 45.0%-66.8% sensitivity with early trophozoites most often mistaken for late trophozoite or schizont stage and late trophozoite and schizont stage most often confused for each other. Overall, this methodology points to a significant clinical potential of using quantitative phase imaging to detect and stage malaria infection

Method for improving the limit of detection in a data signal

DOEpatents

Synovec, R.E.; Yueng, E.S.

1989-10-17

Disclosed is a method for improving the limit of detection for a data set in which experimental noise is uncorrelated along a given abscissa and an analytical signal is correlated to the abscissa, the steps comprising collecting the data set, converting the data set into a data signal including an analytical portion and the experimental noise portion, designating and adjusting a baseline of the data signal to center the experimental noise numerically about a zero reference, and integrating the data signal preserving the corresponding information for each point of the data signal. The steps of the method produce an enhanced integrated data signal which improves the limit of detection of the data signal. 8 figs.

Signal detection evidence for limited capacity in visual search

PubMed Central

Fencsik, David E.; Flusberg, Stephen J.; Horowitz, Todd S.; Wolfe, Jeremy M.

2014-01-01

The nature of capacity limits (if any) in visual search has been a topic of controversy for decades. In 30 years of work, researchers have attempted to distinguish between two broad classes of visual search models. Attention-limited models have proposed two stages of perceptual processing: an unlimited-capacity preattentive stage, and a limited-capacity selective attention stage. Conversely, noise-limited models have proposed a single, unlimited-capacity perceptual processing stage, with decision processes influenced only by stochastic noise. Here, we use signal detection methods to test a strong prediction of attention-limited models. In standard attention-limited models, performance of some searches (feature searches) should only be limited by a preattentive stage. Other search tasks (e.g., spatial configuration search for a “2” among “5”s) should be additionally limited by an attentional bottleneck. We equated average accuracies for a feature and a spatial configuration search over set sizes of 1–8 for briefly presented stimuli. The strong prediction of attention-limited models is that, given overall equivalence in performance, accuracy should be better on the spatial configuration search than on the feature search for set size 1, and worse for set size 8. We confirm this crossover interaction and show that it is problematic for at least one class of one-stage decision models. PMID:21901574

Apparatus and method for quantitatively evaluating total fissile and total fertile nuclide content in samples

DOEpatents

Caldwell, John T.; Kunz, Walter E.; Cates, Michael R.; Franks, Larry A.

1985-01-01

Simultaneous photon and neutron interrogation of samples for the quantitative determination of total fissile nuclide and total fertile nuclide material present is made possible by the use of an electron accelerator. Prompt and delayed neutrons produced from resulting induced fissions are counted using a single detection system and allow the resolution of the contributions from each interrogating flux leading in turn to the quantitative determination sought. Detection limits for .sup.239 Pu are estimated to be about 3 mg using prompt fission neutrons and about 6 mg using delayed neutrons.

Improved PCR assay for the specific detection and quantitation of Escherichia coli serotype O157 in water.

PubMed

Cho, Min Seok; Joh, Kiseong; Ahn, Tae-Young; Park, Dong Suk

2014-09-01

Escherichia coli serotype O157 is still a major global healthcare problem. However, only limited information is now available on the molecular and serological detection of pathogenic bacteria. Therefore, the development of appropriate strategies for their rapid identification and monitoring is still needed. In general, the sequence analysis based on stx, slt, eae, hlyA, rfb, and fliCh7 genes is widely employed for the identification of E. coli serotype O157; but there have been critical defects in the diagnosis and identification of E. coli serotype O157, in that they are also present in other E. coli serogroups. In this study, NCBI-BLAST searches using the nucleotide sequences of the putative regulatory protein gene from E. coli O157:H7 str. Sakai found sequence difference at the serotype level. The specific primers from the putative regulatory protein gene were designed and investigated for their sensitivity and specificity for detecting the pathogen in environment water samples. The specificity of the primer set was evaluated using genomic DNA from 8 isolates of E. coli serotype O157 and 32 other reference strains. In addition, the sensitivity and specificity of this assay were confirmed by successful identification of E. coli serotype O157 in environmental water samples. In conclusion, this study showed that the newly developed quantitative serotype-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk E. coli serotype O157.

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

EPA Science Inventory

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

The detection of Yersinia enterocolitica in surface water by quantitative PCR amplification of the ail and yadA genes.

PubMed

Cheyne, Bo M; Van Dyke, Michele I; Anderson, William B; Huck, Peter M

2010-09-01

Yersinia enterocolitica has been detected in surface water, and drinking untreated water is a risk factor for infection. PCR-based methods have been used to detect Y. enterocolitica in various sample types, but quantitative studies have not been conducted in water. In this study, quantitative PCR (qPCR)-based methods targeting the Yersinia virulence genes ail and yadA were used to survey the Grand River watershed in southern Ontario, Canada. Initial testing of reference strains showed that ail and yadA PCR assays were specific for pathogenic biotypes of Y. enterocolitica; however the genes were also detected in one clinical Yersinia intermedia isolate. A survey of surface water from the Grand River watershed showed that both genes were detected at five sampling locations, with the ail and yadA genes detected in 38 and 21% of samples, respectively. Both genes were detected more frequently at colder water temperatures. A screening of Yersinia strains isolated from the watershed showed that the ail gene was detected in three Y. enterocolitica 1A/O:5 isolates. Results of this study show that Yersinia virulence genes were commonly detected in a watershed used as a source of drinking water, and that the occurrence of these genes was seasonal.

Re-evaluation of groundwater monitoring data for glyphosate and bentazone by taking detection limits into account.

PubMed

Hansen, Claus Toni; Ritz, Christian; Gerhard, Daniel; Jensen, Jens Erik; Streibig, Jens Carl

2015-12-01

Current regulatory assessment of pesticide contamination of Danish groundwater is exclusively based on samples with pesticide concentrations above detection limit. Here we demonstrate that a realistic quantification of pesticide contamination requires the inclusion of "non-detect" samples i.e. samples with concentrations below the detection limit, as left-censored observations. The median calculated pesticide concentrations are shown to be reduced 10(4) to 10(5) fold for two representative herbicides (glyphosate and bentazone) relative to the median concentrations based upon observations above detection limits alone. Copyright © 2015 Elsevier B.V. All rights reserved.

Vascular Corrosion Casting: Review of Advantages and Limitations in the Application of Some Simple Quantitative Methods.

PubMed

Hossler, Fred E.; Douglas, John E.

2001-05-01

Vascular corrosion casting has been used for about 40 years to produce replicas of normal and abnormal vasculature and microvasculature of various tissues and organs that could be viewed at the ultrastructural level. In combination with scanning electron microscopy (SEM), the primary application of corrosion casting has been to describe the morphology and anatomical distribution of blood vessels in these tissues. However, such replicas should also contain quantitative information about that vasculature. This report summarizes some simple quantitative applications of vascular corrosion casting. Casts were prepared by infusing Mercox resin or diluted Mercox resin into the vasculature. Surrounding tissues were removed with KOH, hot water, and formic acid, and the resulting dried casts were observed with routine SEM. The orientation, size, and frequency of vascular endothelial cells were determined from endothelial nuclear imprints on various cast surfaces. Vascular volumes of heart, lung, and avian salt gland were calculated using tissue and resin densities, and weights. Changes in vascular volume and functional capillary density in an experimentally induced emphysema model were estimated from confocal images of casts. Clearly, corrosion casts lend themselves to quantitative analysis. However, because blood vessels differ in their compliances, in their responses to the toxicity of casting resins, and in their response to varying conditions of corrosion casting procedures, it is prudent to use care in interpreting this quantitative data. Some of the applications and limitations of quantitative methodology with corrosion casts are reviewed here.

New Performance Metrics for Quantitative Polymerase Chain Reaction-Based Microbial Source Tracking Methods

EPA Science Inventory

Binary sensitivity and specificity metrics are not adequate to describe the performance of quantitative microbial source tracking methods because the estimates depend on the amount of material tested and limit of detection. We introduce a new framework to compare the performance ...

Laboratory Information Bulletin: Quantitation of Aflatoxin M1 in Bovine Milk by Liquid Chromatography with Fluorescence Detection.

PubMed

Vega, Victor A; Young, Michelle; Todd, Sarah

2016-01-01

An extraction for aflatoxin M1 from bovine milk samples is described. The samples were extracted by adding 10 mL acetonitrile to 10 g of sample. The extract was salted out with sodium chloride and magnesium sulfate to separate the water and acetonitrile. The organic layer was dried down and reconstituted in water before being subjected to an immunoaffinity column for cleanup. Once the analyte was isolated, quantitation was obtained by LC with fluorescence detection. LC/fluorescence parameters were optimized with an Agilent Poroshell 120 C18 LC column resulting in a 4 min run time. To test the procedure's robustness, three different kinds of matrixes were fortified at three different levels each. Whole milk, reduced fat milk, and skim milk samples were fortified at approximately 0.25, 0.5, and 1.0 μg/kg. Recoveries from all samples ranged from 70 to 100%. Confirmation was accomplished by injecting the samples in an ion trap mass spectrometer. The method presented here entails an extraction step followed by an immunoaffinity column clean-up that leads to fast analysis time and consistent recoveries with an uncertainty measurement of 10.5% and method detection limit of less than 0.011 μg/kg.

METHODS OF DEALING WITH VALUES BELOW THE LIMIT OF DETECTION USING SAS

EPA Science Inventory

Due to limitations of chemical analysis procedures, small values cannot be precisely measured. These values are said to be below the limit of detection (LOD). In statistical analyses, these values are often censored and substituted with a constant value, such as half the LOD,...

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

PubMed

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

2018-05-02

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

The fragmented testis method: development and its advantages of a new quantitative evaluation technique for detection of testis-ova in male fish.

PubMed

Lin, Bin-Le; Hagino, Satoshi; Kagoshima, Michio; Iwamatsu, Takashi

2009-02-01

A new quantitative evaluation technique, termed the fragmented testis method, has been developed for the detection of testis-ova in genotypic male fish using the medaka (Oryzias latipes). The routine traditional histological method for detection of testis-ova in male fish exposed to estrogens or suspected endocrine-disrupting chemicals has several disadvantages, including possible oversight of testis-ova due to limited sampling of selected tissue sections. The method we have developed here allows for the accurate determination of the developmental stages and the number and the size of testis-ova in a whole testis. Each testis was removed from the fish specimen, fixed with 10% buffered formalin solution, and then divided into small fragments on a glass slide with a dissecting needle or scalpel and aciform forceps in glycerin solution containing a small amount of methylene blue or toluidine blue. If present, all developing testis-ova of various sizes in fragmented testicular tissues were clearly stained and were observable under a dissecting microscope. Testis-ova occurred in controls were ascertained, while spermatozoa were also distinguishable using this method. This proved to be a convenient and cost-effective method for quantitatively evaluating testis-ova appearance in fish, and it may help to clarify the mechanism of testis-ova formation and the biological significance of testis-ova in future studies of endocrine disruption.

Global limits and interference patterns in dark matter direct detection

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

Catena, Riccardo; Gondolo, Paolo

2015-08-13

We compare the general effective theory of one-body dark matter nucleon interactions to current direct detection experiments in a global multidimensional statistical analysis. We derive exclusion limits on the 28 isoscalar and isovector coupling constants of the theory, and show that current data place interesting constraints on dark matter-nucleon interaction operators usually neglected in this context. We characterize the interference patterns that can arise in dark matter direct detection from pairs of dark matter-nucleon interaction operators, or from isoscalar and isovector components of the same operator. We find that commonly neglected destructive interference effects weaken standard direct detection exclusion limitsmore » by up to one order of magnitude in the coupling constants.« less

Global limits and interference patterns in dark matter direct detection

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

Catena, Riccardo; Gondolo, Paolo, E-mail: riccardo.catena@theorie.physik.uni-goettingen.de, E-mail: paolo.gondolo@utah.edu

2015-08-01

We compare the general effective theory of one-body dark matter nucleon interactions to current direct detection experiments in a global multidimensional statistical analysis. We derive exclusion limits on the 28 isoscalar and isovector coupling constants of the theory, and show that current data place interesting constraints on dark matter-nucleon interaction operators usually neglected in this context. We characterize the interference patterns that can arise in dark matter direct detection from pairs of dark matter-nucleon interaction operators, or from isoscalar and isovector components of the same operator. We find that commonly neglected destructive interference effects weaken standard direct detection exclusion limitsmore » by up to one order of magnitude in the coupling constants.« less

Optical detection of glyphosate in water

NASA Astrophysics Data System (ADS)

de Góes, R. E.; Possetti, G. R. C.; Muller, M.; Fabris, J. L.

2017-04-01

This work shows preliminary results of the detection of Glyphosate in water by using optical fiber spectroscopy. A colloid with citrate-caped silver nanoparticles was employed as substrate for the measurements. A cross analysis between optical absorption and inelastic scattering evidenced a controlled aggregation of the sample constituents, leading to the possibility of quantitative detection of the analyte. The estimate limit of detection for Glyphosate in water for the proposed sensing scheme was about 1.7 mg/L.

Residual eDNA detection sensitivity assessed by quantitative real-time PCR in a river ecosystem.

PubMed

Balasingham, Katherine D; Walter, Ryan P; Heath, Daniel D

2017-05-01

Several studies have demonstrated that environmental DNA (eDNA) can be used to detect the presence of aquatic species, days to weeks after the target species has been removed. However, most studies used eDNA analysis in lentic systems (ponds or lakes), or in controlled laboratory experiments. While eDNA degrades rapidly in all aquatic systems, it also undergoes dilution effects and physical destruction in flowing systems, complicating detection in rivers. However, some eDNA (i.e. residual eDNA) can be retained in aquatic systems, even those subject to high flow regimes. Our goal was to determine residual eDNA detection sensitivity using quantitative real-time polymerase chain reaction (qRT-PCR), in a flowing, uncontrolled river after the eDNA source was removed from the system; we repeated the experiment over 2 years. Residual eDNA had the strongest signal strength at the original source site and was detectable there up to 11.5 h after eDNA source removal. Residual eDNA signal strength decreased as sampling distance downstream from the eDNA source site increased, and was no longer detectable at the source site 48 h after the eDNA source water was exhausted in both experiments. This experiment shows that residual eDNA sampled in surface water can be mapped quantitatively using qRT-PCR, which allows a more accurate spatial identification of the target species location in lotic systems, and relative residual eDNA signal strength may allow the determination of the timing of the presence of target species. © 2016 John Wiley & Sons Ltd.

Sensitive and quantitative detection of botulinum neurotoxin in neurons derived from mouse embryonic stem cells.

PubMed

Pellett, Sabine; Du, Zhong-wei; Pier, Christina L; Tepp, William H; Zhang, Su-chun; Johnson, Eric A

2011-01-07

Botulinum neurotoxins (BoNTs), the most poisonous protein toxins known, represent a serious bioterrorism threat but are also used as a unique and important bio-pharmaceutical to treat an increasing myriad of neurological disorders. The only currently accepted detection method by the United States Food and Drug Administration for biological activity of BoNTs and for potency determination of pharmaceutical preparations is the mouse bioassay (MBA). Recent advances have indicated that cell-based assays using primary neuronal cells can provide an equally sensitive and robust detection platform as the MBA to reliably and quantitatively detect biologically active BoNTs. This study reports for the first time a BoNT detection assay using mouse embryonic stem cells to produce a neuronal cell culture. The data presented indicate that this assay can reliably detect BoNT/A with a similar sensitivity as the MBA. Published by Elsevier Inc.

The Applicability of TaqMan-Based Quantitative Real-Time PCR Assays for Detecting and Enumerating Cryptosporidium spp. Oocysts in the Environment

PubMed Central

Staggs, Sarah E.; Beckman, Erin M.; Keely, Scott P.; Mackwan, Reena; Ware, Michael W.; Moyer, Alan P.; Ferretti, James A.; Sayed, Abu; Xiao, Lihua; Villegas, Eric N.

2013-01-01

Quantitative real-time polymerase chain reaction (qPCR) assays to detect Cryptosporidium oocysts in clinical samples are increasingly being used to diagnose human cryptosporidiosis, but a parallel approach for detecting and identifying Cryptosporidium oocyst contamination in surface water sources has yet to be established for current drinking water quality monitoring practices. It has been proposed that Cryptosporidium qPCR-based assays could be used as viable alternatives to current microscopic-based detection methods to quantify levels of oocysts in drinking water sources; however, data on specificity, analytical sensitivity, and the ability to accurately quantify low levels of oocysts are limited. The purpose of this study was to provide a comprehensive evaluation of TaqMan-based qPCR assays, which were developed for either clinical or environmental investigations, for detecting Cryptosporidium oocyst contamination in water. Ten different qPCR assays, six previously published and four developed in this study were analyzed for specificity and analytical sensitivity. Specificity varied between all ten assays, and in one particular assay, which targeted the Cryptosporidium 18S rRNA gene, successfully detected all Cryptosporidium spp. tested, but also cross-amplified T. gondii, fungi, algae, and dinoflagellates. When evaluating the analytical sensitivity of these qPCR assays, results showed that eight of the assays could reliably detect ten flow-sorted oocysts in reagent water or environmental matrix. This study revealed that while a qPCR-based detection assay can be useful for detecting and differentiating different Cryptosporidium species in environmental samples, it cannot accurately measure low levels of oocysts that are typically found in drinking water sources. PMID:23805235

Wheat-specific gene, ribosomal protein l21, used as the endogenous reference gene for qualitative and real-time quantitative polymerase chain reaction detection of transgenes.

PubMed

Liu, Yi-Ke; Li, He-Ping; Huang, Tao; Cheng, Wei; Gao, Chun-Sheng; Zuo, Dong-Yun; Zhao, Zheng-Xi; Liao, Yu-Cai

2014-10-29

Wheat-specific ribosomal protein L21 (RPL21) is an endogenous reference gene suitable for genetically modified (GM) wheat identification. This taxon-specific RPL21 sequence displayed high homogeneity in different wheat varieties. Southern blots revealed 1 or 3 copies, and sequence analyses showed one amplicon in common wheat. Combined analyses with sequences from common wheat (AABBDD) and three diploid ancestral species, Triticum urartu (AA), Aegilops speltoides (BB), and Aegilops tauschii (DD), demonstrated the presence of this amplicon in the AA genome. Using conventional qualitative polymerase chain reaction (PCR), the limit of detection was 2 copies of wheat haploid genome per reaction. In the quantitative real-time PCR assay, limits of detection and quantification were about 2 and 8 haploid genome copies, respectively, the latter of which is 2.5-4-fold lower than other reported wheat endogenous reference genes. Construct-specific PCR assays were developed using RPL21 as an endogenous reference gene, and as little as 0.5% of GM wheat contents containing Arabidopsis NPR1 were properly quantified.

A volumetric meter chip for point-of-care quantitative detection of bovine catalase for food safety control.

PubMed

Cui, Xingye; Hu, Jie; Choi, Jane Ru; Huang, Yalin; Wang, Xuemin; Lu, Tian Jian; Xu, Feng

2016-09-07

A volumetric meter chip was developed for quantitative point-of-care (POC) analysis of bovine catalase, a bioindicator of bovine mastitis, in milk samples. The meter chip displays multiplexed quantitative results by presenting the distance of ink bar advancement that is detectable by the naked eye. The meter chip comprises a poly(methyl methacrylate) (PMMA) layer, a double-sided adhesive (DSA) layer and a glass slide layer fabricated by the laser-etching method, which is typically simple, rapid (∼3 min per chip), and cost effective (∼$0.2 per chip). Specially designed "U shape" reaction cells are covered by an adhesive tape that serves as an on-off switch, enabling the simple operation of the assay. As a proof of concept, we employed the developed meter chip for the quantification of bovine catalase in raw milk samples to detect catalase concentrations as low as 20 μg/mL. The meter chip has great potential to detect various target analytes for a wide range of POC applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitative Proteomics via High Resolution MS Quantification: Capabilities and Limitations

PubMed Central

Higgs, Richard E.; Butler, Jon P.; Han, Bomie; Knierman, Michael D.

2013-01-01

Recent improvements in the mass accuracy and resolution of mass spectrometers have led to renewed interest in label-free quantification using data from the primary mass spectrum (MS1) acquired from data-dependent proteomics experiments. The capacity for higher specificity quantification of peptides from samples enriched for proteins of biological interest offers distinct advantages for hypothesis generating experiments relative to immunoassay detection methods or prespecified peptide ions measured by multiple reaction monitoring (MRM) approaches. Here we describe an evaluation of different methods to post-process peptide level quantification information to support protein level inference. We characterize the methods by examining their ability to recover a known dilution of a standard protein in background matrices of varying complexity. Additionally, the MS1 quantification results are compared to a standard, targeted, MRM approach on the same samples under equivalent instrument conditions. We show the existence of multiple peptides with MS1 quantification sensitivity similar to the best MRM peptides for each of the background matrices studied. Based on these results we provide recommendations on preferred approaches to leveraging quantitative measurements of multiple peptides to improve protein level inference. PMID:23710359

Limits to behavioral evolution: the quantitative genetics of a complex trait under directional selection.

PubMed

Careau, Vincent; Wolak, Matthew E; Carter, Patrick A; Garland, Theodore

2013-11-01

Replicated selection experiments provide a powerful way to study how "multiple adaptive solutions" may lead to differences in the quantitative-genetic architecture of selected traits and whether this may translate into differences in the timing at which evolutionary limits are reached. We analyze data from 31 generations (n=17,988) of selection on voluntary wheel running in house mice. The rate of initial response, timing of selection limit, and height of the plateau varied significantly between sexes and among the four selected lines. Analyses of litter size and realized selection differentials seem to rule out counterposing natural selection as a cause of the selection limits. Animal-model analyses showed that although the additive genetic variance was significantly lower in selected than control lines, both before and after the limits, the decrease was not sufficient to explain the limits. Moreover, directional selection promoted a negative covariance between additive and maternal genetic variance over the first 10 generations. These results stress the importance of replication in selection studies of higher-level traits and highlight the fact that long-term predictions of response to selection are not necessarily expected to be linear because of the variable effects of selection on additive genetic variance and maternal effects. © 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.

Evaluating Detection Limits of Next-Generation Sequencing for the Surveillance and Monitoring of International Marine Pests

PubMed Central

Pochon, Xavier; Bott, Nathan J.; Smith, Kirsty F.; Wood, Susanna A.

2013-01-01

Most surveillance programmes for marine invasive species (MIS) require considerable taxonomic expertise, are laborious, and are unable to identify species at larval or juvenile stages. Therefore, marine pests may go undetected at the initial stages of incursions when population densities are low. In this study, we evaluated the ability of the benchtop GS Junior™ 454 pyrosequencing system to detect the presence of MIS in complex sample matrices. An initial in-silico evaluation of the mitochondrial cytochrome c oxidase subunit I (COI) and the nuclear small subunit ribosomal DNA (SSU) genes, found that multiple primer sets (targeting a ca. 400 base pair region) would be required to obtain species level identification within the COI gene. In contrast a single universal primer set was designed to target the V1–V3 region of SSU, allowing simultaneous PCR amplification of a wide taxonomic range of MIS. To evaluate the limits of detection of this method, artificial contrived communities (10 species from 5 taxonomic groups) were created using varying concentrations of known DNA samples and PCR products. Environmental samples (water and sediment) spiked with one or five 160 hr old Asterias amurensis larvae were also examined. Pyrosequencing was able to recover DNA/PCR products of individual species present at greater than 0.64% abundance from all tested contrived communities. Additionally, single A. amurensis larvae were detected from both water and sediment samples despite the co-occurrence of a large array of environmental eukaryotes, indicating an equivalent sensitivity to quantitative PCR. NGS technology has tremendous potential for the early detection of marine invasive species worldwide. PMID:24023913

Specific and quantitative detection of human polyomaviruses BKV, JCV, and SV40 by real time PCR.

PubMed

McNees, Adrienne L; White, Zoe S; Zanwar, Preeti; Vilchez, Regis A; Butel, Janet S

2005-09-01

The polyomaviruses that infect humans, BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40), typically establish subclinical persistent infections. However, reactivation of these viruses in immunocompromised hosts is associated with renal nephropathy and hemorrhagic cystitis (HC) caused by BKV and with progressive multifocal leukoencephalopathy (PML) caused by JCV. Additionally, SV40 is associated with several types of human cancers including primary brain and bone cancers, mesotheliomas, and non-Hodgkin's lymphoma. Advancements in detection of these viruses may contribute to improved diagnosis and treatment of affected patients. To develop sensitive and specific real time quantitative polymerase chain reaction (RQ-PCR) assays for the detection of T-antigen DNA sequences of the human polyomaviruses BKV, JCV, and SV40 using the ABI Prism 7000 Sequence Detection System. Assays for absolute quantification of the viral T-ag sequences were designed and the sensitivity and specificity were evaluated. A quantitative assay to measure the single copy human RNAse P gene was also developed and evaluated in order to normalize viral gene copy numbers to cell numbers. Quantification of the target genes is sensitive and specific over a 7 log dynamic range. Ten copies each of the viral and cellular genes are reproducibly and accurately detected. The sensitivity of detection of the RQ-PCR assays is increased 10- to 100-fold compared to conventional PCR and agarose gel protocols. The primers and probes used to detect the viral genes are specific for each virus and there is no cross reactivity within the dynamic range of the standard dilutions. The sensitivity of detection for these assays is not reduced in human cellular extracts; however, different DNA extraction protocols may affect quantification. These assays provide a technique for rapid and specific quantification of polyomavirus genomes per cell in human samples.

Quantitation of N-ethyl-3,4-methylenedioxyamphetamine and its major metabolites in human plasma by high-performance liquid chromatography and fluorescence detection.

PubMed

Brunnenberg, M; Lindenblatt, H; Gouzoulis-Mayfrank, E; Kovar, K A

1998-11-20

A HPLC method has been developed for the analogue of Ecstasy MDE and its major metabolites N-ethyl-4-hydroxy-3-methoxyamphetamine (HME) and 3,4-methylenedioxyamphetamine (MDA) in human plasma. In the course of our investigations we found that the methylenedioxyamphetamines and HME exhibit fluorescence at 322 nm. Therefore the detection could be carried out with a fluorescence (FL) detector. Solid-phase extraction was used for sample preparation and yielded high recovery rates greater than 95%. The limit of quantitation for MDE and its metabolites in the extracts was between 1.5 and 8.9 ng/ml and the method standard deviations were less than 5%. This sensitive, rapid and reliable analytical method has been used successfully in the quantitation of the substances in plasma samples obtained from 14 volunteers in two clinical studies after p.o. administration of 100 to 140 mg MDE*HCI. The maximum plasma concentrations were 235-465 ng/ml (MDE), 67-673 ng/ml (HME) and 7-33 ng/ml (MDA), respectively. Pharmacokinetic parameters have been investigated using the plasma concentration curves.

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

EPA Science Inventory

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

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA

PubMed Central

Zozaya-Valdés, Enrique; Porter, Jessica L.; Coventry, John; Fyfe, Janet A. M.; Carter, Glen P.; Gonçalves da Silva, Anders; Schultz, Mark B.; Seemann, Torsten; Johnson, Paul D. R.; Stewardson, Andrew J.; Bastian, Ivan; Roberts, Sally A.; Howden, Benjamin P.; Williamson, Deborah A.

2017-01-01

ABSTRACT Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium-M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera. Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera. We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico-predicted specificity for M. chimaera. Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera. PMID:28381604

Immuno-analysis of microparticles: probing at the limits of detection

PubMed Central

Latham, Sharissa L.; Tiberti, Natalia; Gokoolparsadh, Naveena; Holdaway, Karen; Olivier Couraud, Pierre; Grau, Georges E. R.; Combes, Valery

2015-01-01

Microparticle (MP) research is clouded by debate regarding the accuracy and validity of flow cytometry (FCM) as an analytical methodology, as it is influenced by many variables including the pre-analytical conditions, instruments physical capabilities and detection parameters. This study utilises a simplistic in vitro system for generating MP, and through comparative analysis with immuno-electron microscopy (Immuno-EM) assesses the strengths and limitations of probe selection and high-sensitivity FCM. Of the markers examined, MP were most specifically labelled with phosphatidylserine ligands, annexin V and lactadherin, although only ~60% MP are PS positive. Whilst these two ligands detect comparable absolute MP numbers, they interact with the same population in distinct manners; annexin V binding is enhanced on TNF induced MP. CD105 and CD54 expression were, as expected, consistent and enhanced following TNF activation respectively. Their labelling however accounted for as few as 30–40% of MP. The greatest discrepancies between FCM and I-EM were observed in the population solely labelled for the surface antigen. These findings demonstrate that despite significant improvements in resolution, high-sensitivity FCM remains limited in detecting small-size MP expressing low antigen levels. This study highlights factors to consider when selecting endothelial MP probes, as well as interpreting and representing data. PMID:26553743

[Influence of Spectral Pre-Processing on PLS Quantitative Model of Detecting Cu in Navel Orange by LIBS].

PubMed

Li, Wen-bing; Yao, Lin-tao; Liu, Mu-hua; Huang, Lin; Yao, Ming-yin; Chen, Tian-bing; He, Xiu-wen; Yang, Ping; Hu, Hui-qin; Nie, Jiang-hui

2015-05-01

Cu in navel orange was detected rapidly by laser-induced breakdown spectroscopy (LIBS) combined with partial least squares (PLS) for quantitative analysis, then the effect on the detection accuracy of the model with different spectral data ptetreatment methods was explored. Spectral data for the 52 Gannan navel orange samples were pretreated by different data smoothing, mean centralized and standard normal variable transform. Then 319~338 nm wavelength section containing characteristic spectral lines of Cu was selected to build PLS models, the main evaluation indexes of models such as regression coefficient (r), root mean square error of cross validation (RMSECV) and the root mean square error of prediction (RMSEP) were compared and analyzed. Three indicators of PLS model after 13 points smoothing and processing of the mean center were found reaching 0. 992 8, 3. 43 and 3. 4 respectively, the average relative error of prediction model is only 5. 55%, and in one word, the quality of calibration and prediction of this model are the best results. The results show that selecting the appropriate data pre-processing method, the prediction accuracy of PLS quantitative model of fruits and vegetables detected by LIBS can be improved effectively, providing a new method for fast and accurate detection of fruits and vegetables by LIBS.

Air Monitoring for Hazardous Gas Detection

NASA Technical Reports Server (NTRS)

Arkin, C. Richard; Griffin, Timothy P.; Adams, Frederick W.; Naylor, Guy; Haskell, William; Floyd, David; Curley, Charles; Follistein, Duke W.

2004-01-01

The Hazardous Gas Detection Lab (HGDL) at Kennedy Space Center is involved in the design and development of instrumentation that can detect and quantify various hazardous gases. Traditionally these systems are designed for leak detection of the cryogenic gases used for the propulsion of the Shuttle and other vehicles. Mass spectrometers are the basis of these systems, which provide excellent quantitation, sensitivity, selectivity, response times and detection limits. A Table lists common gases monitored for aerospace applications. The first five gases, hydrogen, helium, nitrogen, oxygen, and argon are historically the focus of the HGDL.

Improved Quantitation of Gluten in Wheat Starch for Celiac Disease Patients by Gel-Permeation High-Performance Liquid Chromatography with Fluorescence Detection (GP-HPLC-FLD).

PubMed

Scherf, Katharina Anne; Wieser, Herbert; Koehler, Peter

2016-10-12

Purified wheat starch (WSt) is commonly used in gluten-free products for celiac disease (CD) patients. It is mostly well-tolerated, but doubts about its safety for CD patients persist. One reason may be that most ELISA kits primarily recognize the alcohol-soluble gliadin fraction of gluten, but insufficiently target the alcohol-insoluble glutenin fraction. To address this problem, a new sensitive method based on the sequential extraction of gliadins, glutenins, and gluten from WSt followed by gel-permeation high-performance liquid chromatography with fluorescence detection (GP-HPLC-FLD) was developed. It revealed that considerable amounts of glutenins were present in most WSt. The gluten contents quantitated by GP-HPLC-FLD as sum of gliadins and glutenins were higher than those by R5 ELISA (gluten as gliadin content multiplied by a factor of 2) in 19 out of 26 WSt. Despite its limited selectivity, GP-HPLC-FLD may be applied as confirmatory method to ELISA to quantitate gluten in WSt.

Quantitative Glycomics Strategies*

PubMed Central

Mechref, Yehia; Hu, Yunli; Desantos-Garcia, Janie L.; Hussein, Ahmed; Tang, Haixu

2013-01-01

The correlations between protein glycosylation and many biological processes and diseases are increasing the demand for quantitative glycomics strategies enabling sensitive monitoring of changes in the abundance and structure of glycans. This is currently attained through multiple strategies employing several analytical techniques such as capillary electrophoresis, liquid chromatography, and mass spectrometry. The detection and quantification of glycans often involve labeling with ionic and/or hydrophobic reagents. This step is needed in order to enhance detection in spectroscopic and mass spectrometric measurements. Recently, labeling with stable isotopic reagents has also been presented as a very viable strategy enabling relative quantitation. The different strategies available for reliable and sensitive quantitative glycomics are herein described and discussed. PMID:23325767

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

PubMed

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

2014-01-01

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

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

PubMed Central

Field, Christopher R.; Lubrano, Adam; Woytowitz, Morgan; Giordano, Braden C.; Rose-Pehrsson, Susan L.

2014-01-01

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples. PMID:25145416

Quantitative detection of trace explosive vapors by programmed temperature desorption gas chromatography-electron capture detector.

PubMed

Field, Christopher R; Lubrano, Adam; Woytowitz, Morgan; Giordano, Braden C; Rose-Pehrsson, Susan L

2014-07-25

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

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

PubMed Central

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

2007-01-01

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

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

PubMed

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

2012-03-01

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

Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR.

PubMed

Kim, Jeong-Soon; Wang, Nian

2009-03-06

Citrus Huanglongbing (HLB) is one of the most devastating diseases on citrus and is associated with Candidatus Liberibacter spp.. The pathogens are phloem limited and have not been cultured in vitro. The current management strategy of HLB is to remove infected citrus trees and reduce psyllid populations with insecticides to prevent the spreading. This strategy requires sensitive and reliable diagnostic methods for early detection. We investigated the copy numbers of the 16S rDNA and 16S rRNA of the HLB pathogen and the implication of improving the diagnosis of HLB for early detection using Quantitative PCR. We compared the detection of HLB with different Quantitative PCR based methods with primers/probe targeting either 16S rDNA, beta-operon DNA, 16S rRNA, or beta-operon RNA. The 16S rDNA copy number of Ca. Liberibacter asiaticus was estimated to be three times of that of the beta-operon region, thus allowing detection of lower titer of Ca. L. asiaticus. Quantitative reverse transcriptional PCR (QRT-PCR) indicated that the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples. Dilution analysis also indicates that QRT-PCR targeting 16S rRNA is 10 time more sensitive than QPCR targeting 16S rDNA. Thus QRT-PCR was able to increase the sensitivity of detection by targeting 16S rRNA. Our result indicates that Candidatus Liberibacter asiaticus contains three copies of 16S rDNA. The copy number of 16S rRNA of Ca. L. asiaticus in planta averaged about 7.8 times of 16S rDNA for the same set of samples tested in this study. Detection sensitivity of HLB could be improved through the following approaches: using 16S rDNA based primers/probe in the QPCR assays; and using QRT-PCR assays targeting 16S rRNA.

Development of a reverse transcription quantitative polymerase chain reaction-based assay for broad coverage detection of African and Asian Zika virus lineages.

PubMed

Yang, Yang; Wong, Gary; Ye, Baoguo; Li, Shihua; Li, Shanqin; Zheng, Haixia; Wang, Qiang; Liang, Mifang; Gao, George F; Liu, Lei; Liu, Yingxia; Bi, Yuhai

2017-06-01

The Zika virus (ZIKV) is an arbovirus that has spread rapidly worldwide within recent times. There is accumulating evidence that associates ZIKV infections with Guillain-Barré Syndrome (GBS) and microcephaly in humans. The ZIKV is genetically diverse and can be separated into Asian and African lineages. A rapid, sensitive, and specific assay is needed for the detection of ZIKV across various pandemic regions. So far, the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains. To this end, we have developed a one-step quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based on conserved sequences in the ZIKV envelope (E) gene. The detection limit of the assay was determined to be five RNA transcript copies and 2.94 × 10 -3 50% tissue culture infectious doses (TCID 50 ) of live ZIKV per reaction. The assay was highly specific and able to detect five different ZIKV strains covering the Asian and African lineages without nonspecific amplification, when tested against other flaviviruses. The assay was also successful in testing for ZIKV in clinical samples. Our assay represents an improvement over the current methods available for the detection ZIKV and would be valuable as a diagnostic tool in various pandemic regions.

Automatic detection and quantitative analysis of cells in the mouse primary motor cortex

NASA Astrophysics Data System (ADS)

Meng, Yunlong; He, Yong; Wu, Jingpeng; Chen, Shangbin; Li, Anan; Gong, Hui

2014-09-01

Neuronal cells play very important role on metabolism regulation and mechanism control, so cell number is a fundamental determinant of brain function. Combined suitable cell-labeling approaches with recently proposed three-dimensional optical imaging techniques, whole mouse brain coronal sections can be acquired with 1-μm voxel resolution. We have developed a completely automatic pipeline to perform cell centroids detection, and provided three-dimensional quantitative information of cells in the primary motor cortex of C57BL/6 mouse. It involves four principal steps: i) preprocessing; ii) image binarization; iii) cell centroids extraction and contour segmentation; iv) laminar density estimation. Investigations on the presented method reveal promising detection accuracy in terms of recall and precision, with average recall rate 92.1% and average precision rate 86.2%. We also analyze laminar density distribution of cells from pial surface to corpus callosum from the output vectorizations of detected cell centroids in mouse primary motor cortex, and find significant cellular density distribution variations in different layers. This automatic cell centroids detection approach will be beneficial for fast cell-counting and accurate density estimation, as time-consuming and error-prone manual identification is avoided.

Detection limit for rate fluctuations in inhomogeneous Poisson processes

NASA Astrophysics Data System (ADS)

Shintani, Toshiaki; Shinomoto, Shigeru

2012-04-01

Estimations of an underlying rate from data points are inevitably disturbed by the irregular occurrence of events. Proper estimation methods are designed to avoid overfitting by discounting the irregular occurrence of data, and to determine a constant rate from irregular data derived from a constant probability distribution. However, it can occur that rapid or small fluctuations in the underlying density are undetectable when the data are sparse. For an estimation method, the maximum degree of undetectable rate fluctuations is uniquely determined as a phase transition, when considering an infinitely long series of events drawn from a fluctuating density. In this study, we analytically examine an optimized histogram and a Bayesian rate estimator with respect to their detectability of rate fluctuation, and determine whether their detectable-undetectable phase transition points are given by an identical formula defining a degree of fluctuation in an underlying rate. In addition, we numerically examine the variational Bayes hidden Markov model in its detectability of rate fluctuation, and determine whether the numerically obtained transition point is comparable to those of the other two methods. Such consistency among these three principled methods suggests the presence of a theoretical limit for detecting rate fluctuations.

Detection limit for rate fluctuations in inhomogeneous Poisson processes.

PubMed

Shintani, Toshiaki; Shinomoto, Shigeru

2012-04-01

Estimations of an underlying rate from data points are inevitably disturbed by the irregular occurrence of events. Proper estimation methods are designed to avoid overfitting by discounting the irregular occurrence of data, and to determine a constant rate from irregular data derived from a constant probability distribution. However, it can occur that rapid or small fluctuations in the underlying density are undetectable when the data are sparse. For an estimation method, the maximum degree of undetectable rate fluctuations is uniquely determined as a phase transition, when considering an infinitely long series of events drawn from a fluctuating density. In this study, we analytically examine an optimized histogram and a Bayesian rate estimator with respect to their detectability of rate fluctuation, and determine whether their detectable-undetectable phase transition points are given by an identical formula defining a degree of fluctuation in an underlying rate. In addition, we numerically examine the variational Bayes hidden Markov model in its detectability of rate fluctuation, and determine whether the numerically obtained transition point is comparable to those of the other two methods. Such consistency among these three principled methods suggests the presence of a theoretical limit for detecting rate fluctuations.

A versatile quantitation platform based on platinum nanoparticles incorporated volumetric bar-chart chip for highly sensitive assays.

PubMed

Wang, Yuzhen; Zhu, Guixian; Qi, Wenjin; Li, Ying; Song, Yujun

2016-11-15

Platinum nanoparticles incorporated volumetric bar-chart chip (PtNPs-V-Chip) is able to be used for point-of-care tests by providing quantitative and visualized readout without any assistance from instruments, data processing, or graphic plotting. To improve the sensitivity of PtNPs-V-Chip, hybridization chain reaction was employed in this quantitation platform for highly sensitive assays that can detect as low as 16 pM Ebola Virus DNA, 0.01ng/mL carcinoembryonic antigen (CEA), and the 10 HER2-expressing cancer cells. Based on this amplified strategy, a 100-fold decrease of detection limit was achieved for DNA by improving the number of platinum nanoparticle catalyst for the captured analyte. This quantitation platform can also distinguish single base mismatch of DNA hybridization and observe the concentration threshold of CEA. The new strategy lays the foundation for this quantitation platform to be applied in forensic analysis, biothreat detection, clinical diagnostics and drug screening. Copyright © 2016 Elsevier B.V. All rights reserved.

Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay.

PubMed

Hendricks, D A; Stowe, B J; Hoo, B S; Kolberg, J; Irvine, B D; Neuwald, P D; Urdea, M S; Perrillo, R P

1995-11-01

The aim of this study was to establish the performance characteristics of a nonradioisotopic branched DNA (bDNA) signal amplification assay for quantitation of hepatitis B virus (HBV) DNA in human serum. Quantitation was determined from a standard curve and expressed as HBV DNA equivalents/mL (Eq/mL; 285,000 Eq = 1 pg of double stranded HBV DNA). The bDNA assay exhibited a nearly four log dynamic range of quantitation and an analytical detection limit of approximately 100,000 Eq/mL. To ensure a specificity of 99.7%, the quantitation limit was set at 700,000 Eq/mL. The interassay percent coefficient of variance for quantification values ranged from 10% to 15% when performed by novice users with different sets of reagents. Using the bDNA assay, HBV DNA was detected in 94% to 100% of hepatitis B e antigen-positive specimens and 27% to 31% of hepatitis B e antigen-negative specimens from chronic HBV-infected patients. The bDNA assay may be useful as a prognostic and therapy monitoring tool for the management of HBV-infected patients undergoing antiviral treatment.

Quantitative Surface Chirality Detection with Sum Frequency Generation Vibrational Spectroscopy: Twin Polarization Angle Approach

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

Wei, Feng; Xu, Yanyan; Guo, Yuan

2009-12-27

Here we report a novel twin polarization angle (TPA) approach in the quantitative chirality detection with the surface sum-frequency generation vibrational spectroscopy (SFG-VS). Generally, the achiral contribution dominates the surface SFG-VS signal, and the pure chiral signal is usually two or three orders of magnitude smaller. Therefore, it has been difficult to make quantitative detection and analysis of the chiral contributions to the surface SFG- VS signal. In the TPA method, by varying together the polarization angles of the incoming visible light and the sum frequency signal at fixed s or p polarization of the incoming infrared beam, the polarizationmore » dependent SFG signal can give not only direct signature of the chiral contribution in the total SFG-VS signal, but also the accurate measurement of the chiral and achiral components in the surface SFG signal. The general description of the TPA method is presented and the experiment test of the TPA approach is also presented for the SFG-VS from the S- and R-limonene chiral liquid surfaces. The most accurate degree of chiral excess values thus obtained for the 2878 cm⁻¹ spectral peak of the S- and R-limonene liquid surfaces are (23.7±0.4)% and ({25.4±1.3)%, respectively.« less

Quantitative subpixel spectral detection of targets in multispectral images. [terrestrial and planetary surfaces

NASA Technical Reports Server (NTRS)

Sabol, Donald E., Jr.; Adams, John B.; Smith, Milton O.

1992-01-01

The conditions that affect the spectral detection of target materials at the subpixel scale are examined. Two levels of spectral mixture analysis for determining threshold detection limits of target materials in a spectral mixture are presented, the cases where the target is detected as: (1) a component of a spectral mixture (continuum threshold analysis) and (2) residuals (residual threshold analysis). The results of these two analyses are compared under various measurement conditions. The examples illustrate the general approach that can be used for evaluating the spectral detectability of terrestrial and planetary targets at the subpixel scale.

Surface-Enhanced Raman Scattering Active Plasmonic Nanoparticles with Ultrasmall Interior Nanogap for Multiplex Quantitative Detection and Cancer Cell Imaging.

PubMed

Li, Jiuxing; Zhu, Zhi; Zhu, Bingqing; Ma, Yanli; Lin, Bingqian; Liu, Rudi; Song, Yanling; Lin, Hui; Tu, Song; Yang, Chaoyong

2016-08-02

Due to its large enhancement effect, nanostructure-based surface-enhanced Raman scattering (SERS) technology had been widely applied for bioanalysis and cell imaging. However, most SERS nanostructures suffer from poor signal reproducibility, which hinders the application of SERS nanostructures in quantitative detection. We report an etching-assisted approach to synthesize SERS-active plasmonic nanoparticles with 1 nm interior nanogap for multiplex quantitative detection and cancer cell imaging. Raman dyes and methoxy poly(ethylene glycol) thiol (mPEG-SH) were attached to gold nanoparticles (AuNPs) to prepare gold cores. Next, Ag atoms were deposited on gold cores in the presence of Pluronic F127 to form a Ag shell. HAuCl4 was used to etch the Ag shell and form an interior nanogap in Au@AgAuNPs, leading to increased Raman intensity of dyes. SERS intensity distribution of Au@AgAuNPs was found to be more uniform than that of aggregated AuNPs. Finally, Au@AgAuNPs were used for multiplex quantitative detection and cancer cell imaging. With the advantages of simple and rapid preparation of Au@AgAuNPs with highly uniform, stable, and reproducible Raman intensity, the method reported here will widen the applications of SERS-active nanoparticles in diagnostics and imaging.

Ultrafast scene detection and recognition with limited visual information

PubMed Central

Hagmann, Carl Erick; Potter, Mary C.

2016-01-01

Humans can detect target color pictures of scenes depicting concepts like picnic or harbor in sequences of six or twelve pictures presented as briefly as 13 ms, even when the target is named after the sequence (Potter, Wyble, Hagmann, & McCourt, 2014). Such rapid detection suggests that feedforward processing alone enabled detection without recurrent cortical feedback. There is debate about whether coarse, global, low spatial frequencies (LSFs) provide predictive information to high cortical levels through the rapid magnocellular (M) projection of the visual path, enabling top-down prediction of possible object identities. To test the “Fast M” hypothesis, we compared detection of a named target across five stimulus conditions: unaltered color, blurred color, grayscale, thresholded monochrome, and LSF pictures. The pictures were presented for 13–80 ms in six-picture rapid serial visual presentation (RSVP) sequences. Blurred, monochrome, and LSF pictures were detected less accurately than normal color or grayscale pictures. When the target was named before the sequence, all picture types except LSF resulted in above-chance detection at all durations. Crucially, when the name was given only after the sequence, performance dropped and the monochrome and LSF pictures (but not the blurred pictures) were at or near chance. Thus, without advance information, monochrome and LSF pictures were rarely understood. The results offer only limited support for the Fast M hypothesis, suggesting instead that feedforward processing is able to activate conceptual representations without complementary reentrant processing. PMID:28255263

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

EPA Science Inventory

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

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

PubMed Central

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

2006-01-01

Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage. PMID:17088381

ICP-MS as a novel detection system for quantitative element-tagged immunoassay of hidden peanut allergens in foods.

PubMed

Careri, Maria; Elviri, Lisa; Mangia, Alessandro; Mucchino, Claudio

2007-03-01

A novel ICP-MS-based ELISA immunoassay via element-tagged determination was devised for quantitative analysis of hidden allergens in food. The method was able to detect low amounts of peanuts (down to approximately 2 mg peanuts kg(-1) cereal-based matrix) by using a europium-tagged antibody. Selectivity was proved by the lack of detectable cross-reaction with a number of protein-rich raw materials.

Quantitative Studies on the Propagation and Extinction of Near-Limit Premixed Flames Under Normal and Microgravity

NASA Technical Reports Server (NTRS)

Dong, Y.; Spedding, G. R.; Egolfopoulos, F. N.; Miller, F. J.

2003-01-01

The main objective of this research is to introduce accurate fluid mechanics measurements diagnostics in the 2.2-s drop tower for the determination of the detailed flow-field at the states of extinction. These results are important as they can then be compared with confidence with detailed numerical simulations so that important insight is provided into near-limit phenomena that are controlled by not well-understood kinetics and thermal radiation processes. Past qualitative studies did enhance our general understanding on the subject. However, quantitative studies are essential for the validation of existing models that subsequently be used to describe near-limit phenomena that can initiate catastrophic events in micro- and/or reduced gravity environments.

Comparison of propidium monoazide-quantitative PCR and reverse transcription quantitative PCR for viability detection of fresh Cryptosporidium oocysts following disinfection and after long-term storage in water samples

EPA Science Inventory

Purified oocysts of Cryptosporidium parvum were used to evaluate applicability of two quantitative PCR (qPCR) viability detection methods in raw surface water and disinfection treated water. Propidium monoazide-qPCR targeting hsp70 gene was compared to reverse transcription (RT)-...

Failure Detecting Method of Fault Current Limiter System with Rectifier

NASA Astrophysics Data System (ADS)

Tokuda, Noriaki; Matsubara, Yoshio; Asano, Masakuni; Ohkuma, Takeshi; Sato, Yoshibumi; Takahashi, Yoshihisa

A fault current limiter (FCL) is extensively needed to suppress fault current, particularly required for trunk power systems connecting high-voltage transmission lines, such as 500kV class power system which constitutes the nucleus of the electric power system. We proposed a new type FCL system (rectifier type FCL), consisting of solid-state diodes, DC reactor and bypass AC reactor, and demonstrated the excellent performances of this FCL by developing the small 6.6kV and 66kV model. It is important to detect the failure of power devices used in the rectifier under the normal operating condition, for keeping the excellent reliability of the power system. In this paper, we have proposed a new failure detecting method of power devices most suitable for the rectifier type FCL. This failure detecting system is simple and compact. We have adapted the proposed system to the 66kV prototype single-phase model and successfully demonstrated to detect the failure of power devices.

Dual color fluorescence quantitative detection of specific single-stranded DNA with molecular beacons and nucleic acid dye SYBR Green I.

PubMed

Xiang, Dong-Shan; Zhou, Guo-Hua; Luo, Ming; Ji, Xing-Hu; He, Zhi-Ke

2012-08-21

We have developed a dual color fluorescence quantitative detection method for specific single-stranded DNA with molecular beacons (MBs) and nucleic acid dye SYBR Green I by synchronous scanning fluorescence spectrometry. It is demonstrated by a reverse-transcription oligonucleotide sequence (target DNA, 33 bases) of RNA fragment of human immunodeficiency virus (HIV) as a model system. In the absence of target DNA, the MBs are in the stem-closed state, the fluorescence of 5-carboxy-X-rhodamine (ROX) is quenched by black hole quencher-2 (BHQ-2), and the interaction between SYBR Green I and the MBs is very weak. At this time the fluorescence signals of ROX and SYBR Green I are all very weak. In the presence of target DNA, MBs hybridize with target DNA and form a double-strand structure, the fluorophore ROX is separated from the quencher BHQ-2, and the fluorescence of ROX recovers. At the same time, SYBR Green I binds to hybridized dsDNA, whose fluorescence intensity is significantly enhanced. Thus, dual color fluorescence quantitative detection for the target DNA can be realized by synchronous scanning fluorescence spectrometry. In this strategy, the fluorescence signal of SYBR Green I is far larger than that of ROX, so the quantitative analysis of target DNA with the fluorescence intensity of SYBR Green I can significantly improve the detection sensitivity. In addition, the false-positive signals of MBs do not affect the fluorescence signals of nucleic acid dye SYBR Green I. Thereby, in the analysis of complex samples, quantitative analysis of target DNA with SYBR Green I can avoid the false-positive signals of MBs and improve the detection accuracy.

Apparatus and method for quantitatively evaluating total fissile and total fertile nuclide content in samples. [Patent application

DOEpatents

Caldwell, J.T.; Kunz, W.E.; Cates, M.R.; Franks, L.A.

1982-07-07

Simultaneous photon and neutron interrogation of samples for the quantitative determination of total fissile nuclide and total fertile nuclide material present is made possible by the use of an electron accelerator. Prompt and delayed neutrons produced from resulting induced fission are counted using a single detection system and allow the resolution of the contributions from each interrogating flux leading in turn to the quantitative determination sought. Detection limits for /sup 239/Pu are estimated to be about 3 mg using prompt fission neutrons and about 6 mg using delayed neutrons.

Novel approach to quantitative polymerase chain reaction using real-time detection: application to the detection of gene amplification in breast cancer.

PubMed

Bièche, I; Olivi, M; Champème, M H; Vidaud, D; Lidereau, R; Vidaud, M

1998-11-23

Gene amplification is a common event in the progression of human cancers, and amplified oncogenes have been shown to have diagnostic, prognostic and therapeutic relevance. A kinetic quantitative polymerase-chain-reaction (PCR) method, based on fluorescent TaqMan methodology and a new instrument (ABI Prism 7700 Sequence Detection System) capable of measuring fluorescence in real-time, was used to quantify gene amplification in tumor DNA. Reactions are characterized by the point during cycling when PCR amplification is still in the exponential phase, rather than the amount of PCR product accumulated after a fixed number of cycles. None of the reaction components is limited during the exponential phase, meaning that values are highly reproducible in reactions starting with the same copy number. This greatly improves the precision of DNA quantification. Moreover, real-time PCR does not require post-PCR sample handling, thereby preventing potential PCR-product carry-over contamination; it possesses a wide dynamic range of quantification and results in much faster and higher sample throughput. The real-time PCR method, was used to develop and validate a simple and rapid assay for the detection and quantification of the 3 most frequently amplified genes (myc, ccndl and erbB2) in breast tumors. Extra copies of myc, ccndl and erbB2 were observed in 10, 23 and 15%, respectively, of 108 breast-tumor DNA; the largest observed numbers of gene copies were 4.6, 18.6 and 15.1, respectively. These results correlated well with those of Southern blotting. The use of this new semi-automated technique will make molecular analysis of human cancers simpler and more reliable, and should find broad applications in clinical and research settings.

The detection limits of antimicrobial agents in cow's milk by a simple Yoghurt Culture Test.

PubMed

Mohsenzadeh, M; Bahrainipour, A

2008-09-15

The aim of this study was to study performance of Yoghurt Culture Test (YCT) in the detection of antimicrobial residues in milk. For this purpose, the sensitivity of YCT for 15 antibiotics were determined. For each drug, 8 concentrations were tested. The detection limits of YCT at 2.5 h and 4 h incubation were determined (microg kg(-1)): 15 and 37.5, penicillin G; 4 and 5, ampicillin; 5 and 7.5, amoxycillin; 100 and 200, cephalexin; 80 and 100, cefazoline; 100 and 200, oxytetracycline; 500 and 100, chlortetracycline; 100 and 200, tetracycline; 150 and 200, doxycycline; 200 and 400, sulphadimidine; 500 and 1000, gentamycin; 1000 and 1500, spectinomycin; 400 and 500, erythromycin; 50 and 100, tylosin; 5000 and 10000, chloramphenicol. The YCT detection limits at 2.5 h incubation for ampicillin, cephalexin, tetracycline, oxytetracycline and tylosin are similar to those obtained as Maximum Residue Limit (MRL) according to Regulation 2377/90 EEC as set out by the European Union. In addition the detection limits of YCT for some antibiotics were lower than some of microbial inhibitor test.

A simple and highly sensitive colorimetric detection method for gaseous formaldehyde.

PubMed

Feng, Liang; Musto, Christopher J; Suslick, Kenneth S

2010-03-31

A colorimetric detection method using amine-functionalized polymer films doped with a pH indicator has been developed for the rapid, sensitive, and quantitative detection of gaseous formaldehyde at concentrations well below the immediately dangerous to life or health (IDLH) limit. In 1 min, visible color changes are easily observed, even down to the permissible exposure limit (PEL) at 750 ppb. The limit of detection is below 50 ppb (7% of the PEL) after 10 min of exposure. This sensor is essentially unaffected by changes in humidity or temperature (4 to 50 degrees C) and is not sensitive to common interferents.

Quantitative prediction of perceptual decisions during near-threshold fear detection

NASA Astrophysics Data System (ADS)

Pessoa, Luiz; Padmala, Srikanth

2005-04-01

A fundamental goal of cognitive neuroscience is to explain how mental decisions originate from basic neural mechanisms. The goal of the present study was to investigate the neural correlates of perceptual decisions in the context of emotional perception. To probe this question, we investigated how fluctuations in functional MRI (fMRI) signals were correlated with behavioral choice during a near-threshold fear detection task. fMRI signals predicted behavioral choice independently of stimulus properties and task accuracy in a network of brain regions linked to emotional processing: posterior cingulate cortex, medial prefrontal cortex, right inferior frontal gyrus, and left insula. We quantified the link between fMRI signals and behavioral choice in a whole-brain analysis by determining choice probabilities by means of signal-detection theory methods. Our results demonstrate that voxel-wise fMRI signals can reliably predict behavioral choice in a quantitative fashion (choice probabilities ranged from 0.63 to 0.78) at levels comparable to neuronal data. We suggest that the conscious decision that a fearful face has been seen is represented across a network of interconnected brain regions that prepare the organism to appropriately handle emotionally challenging stimuli and that regulate the associated emotional response. decision making | emotion | functional MRI

Modified Linnik microscopic interferometry for quantitative depth evaluation of diffraction-limited microgroove

NASA Astrophysics Data System (ADS)

Ye, Shiwei; Takahashi, Satoru; Michihata, Masaki; Takamasu, Kiyoshi

2018-05-01

The quality control of microgrooves is extremely crucial to ensure the performance and stability of microstructures and improve their fabrication efficiency. This paper introduces a novel optical inspection method and a modified Linnik microscopic interferometer measurement system to detect the depth of microgrooves with a width less than the diffraction limit. Using this optical method, the depth of diffraction-limited microgrooves can be related to the near-field optical phase difference, which cannot be practically observed but can be computed from practical far-field observations. Thus, a modified Linnik microscopic interferometer system based on three identical objective lenses and an optical path reversibility principle were developed. In addition, experiments for standard grating microgrooves on the silicon surface were carried out to demonstrate the feasibility and repeatability of the proposed method and developed measurement system.

Attentional Capacity Limits Gap Detection during Concurrent Sound Segregation.

PubMed

Leung, Ada W S; Jolicoeur, Pierre; Alain, Claude

2015-11-01

Detecting a brief silent interval (i.e., a gap) is more difficult when listeners perceive two concurrent sounds rather than one in a sound containing a mistuned harmonic in otherwise in-tune harmonics. This impairment in gap detection may reflect the interaction of low-level encoding or the division of attention between two sound objects, both of which could interfere with signal detection. To distinguish between these two alternatives, we compared ERPs during active and passive listening with complex harmonic tones that could include a gap, a mistuned harmonic, both features, or neither. During active listening, participants indicated whether they heard a gap irrespective of mistuning. During passive listening, participants watched a subtitled muted movie of their choice while the same sounds were presented. Gap detection was impaired when the complex sounds included a mistuned harmonic that popped out as a separate object. The ERP analysis revealed an early gap-related activity that was little affected by mistuning during the active or passive listening condition. However, during active listening, there was a marked decrease in the late positive wave that was thought to index attention and response-related processes. These results suggest that the limitation in detecting the gap is related to attentional processing, possibly divided attention induced by the concurrent sound objects, rather than deficits in preattentional sensory encoding.

Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters

EPA Science Inventory

Modern techniques for tracking fecal pollution in environmental waters require investing in DNA-based methods to determine the presence of specific fecal sources. To help water quality managers decide whether to employ routine polymerase chain reaction (PCR) or quantitative PC...

A Quantitative PCR Protocol for Detection of Oxyspirura petrowi in Northern Bobwhites (Colinus virginianus).

PubMed

Kistler, Whitney M; Parlos, Julie A; Peper, Steven T; Dunham, Nicholas R; Kendall, Ronald J

2016-01-01

Oxyspirura petrowi is a parasitic nematode that infects wild birds. This parasite has a broad host range, but has recently been reported in high prevalences from native Galliformes species in the United States. In order to better understand the impact O. petrowi has on wild bird populations, we developed a quantitative PCR protocol to detect infections in wild northern bobwhites (Colinus virginianus). We used paired fecal and cloacal swab samples from wild caught and experimentally infected northern bobwhites and matching fecal float data from experimentally infected birds to validate our assay. Overall we detected more positive birds from fecal samples than the paired cloacal swabs and there was strong agreement between the qPCR results from fecal samples and from fecal flotation (84%; κ = 0.69 [0.53-0.84 95% CI]). We also detected O. petrowi DNA in ten replicates of samples spiked with one O. petrowi egg. This qPCR assay is an effective assay to detect O. petrowi infections in wild birds. Our results suggest that fecal samples are the most appropriate sample for detecting infections; although, cloacal swabs can be useful for determining if O. petrowi is circulating in a population.

A Quantitative PCR Protocol for Detection of Oxyspirura petrowi in Northern Bobwhites (Colinus virginianus)

PubMed Central

Kistler, Whitney M.; Parlos, Julie A.; Peper, Steven T.; Dunham, Nicholas R.; Kendall, Ronald J.

2016-01-01

Oxyspirura petrowi is a parasitic nematode that infects wild birds. This parasite has a broad host range, but has recently been reported in high prevalences from native Galliformes species in the United States. In order to better understand the impact O. petrowi has on wild bird populations, we developed a quantitative PCR protocol to detect infections in wild northern bobwhites (Colinus virginianus). We used paired fecal and cloacal swab samples from wild caught and experimentally infected northern bobwhites and matching fecal float data from experimentally infected birds to validate our assay. Overall we detected more positive birds from fecal samples than the paired cloacal swabs and there was strong agreement between the qPCR results from fecal samples and from fecal flotation (84%; κ = 0.69 [0.53–0.84 95% CI]). We also detected O. petrowi DNA in ten replicates of samples spiked with one O. petrowi egg. This qPCR assay is an effective assay to detect O. petrowi infections in wild birds. Our results suggest that fecal samples are the most appropriate sample for detecting infections; although, cloacal swabs can be useful for determining if O. petrowi is circulating in a population. PMID:27893772

Detection and differentiation of early acute and following age stages of myocardial infarction with quantitative post-mortem cardiac 1.5T MR.

PubMed

Schwendener, Nicole; Jackowski, Christian; Persson, Anders; Warntjes, Marcel J; Schuster, Frederick; Riva, Fabiano; Zech, Wolf-Dieter

2017-01-01

Recently, quantitative MR sequences have started being used in post-mortem imaging. The goal of the present study was to evaluate if early acute and following age stages of myocardial infarction can be detected and discerned by quantitative 1.5T post-mortem cardiac magnetic resonance (PMCMR) based on quantitative T1, T2 and PD values. In 80 deceased individuals (25 female, 55 male), a cardiac MR quantification sequence was performed prior to cardiac dissection at autopsy in a prospective study. Focal myocardial signal alterations detected in synthetically generated MR images were MR quantified for their T1, T2 and PD values. The locations of signal alteration measurements in PMCMR were targeted at autopsy heart dissection and cardiac tissue specimens were taken for histologic examinations. Quantified signal alterations in PMCMR were correlated to their according histologic age stage of myocardial infarction. In PMCMR seventy-three focal myocardial signal alterations were detected in 49 of 80 investigated hearts. These signal alterations were diagnosed histologically as early acute (n=39), acute (n=14), subacute (n=10) and chronic (n=10) age stages of myocardial infarction. Statistical analysis revealed that based on their quantitative T1, T2 and PD values, a significant difference between all defined age groups of myocardial infarction can be determined. It can be concluded that quantitative 1.5T PMCMR quantification based on quantitative T1, T2 and PD values is feasible for characterization and differentiation of early acute and following age stages of myocardial infarction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Comparison of nested PCR and qPCR for the detection and quantitation of BoHV6 DNA.

PubMed

Kubiś, Piotr; Materniak, Magdalena; Kuźmak, Jacek

2013-12-01

Nested PCR and qPCR (quantitative PCR) tests based on glycoprotein B (gB) gene were designed for detecting Bovine herpesvirus 6 (BoHV6) in bovine whole blood samples and wild ruminant blood clots (deer and roe-deer). This virus, commonly known as BLHV (bovine lymphotropic herpesvirus) belongs to the Herpesviridae family, subfamily Gammaherpesvirinae and Macavirus genus. DNA isolated from 92 dairy cow blood samples and 69 wild ruminant clots were examined for the presence of BoHV6 using nested PCR and qPCR tests. Viral DNA was detected by using nested PCR in 59 out of 92 bovine blood samples (64.1%), and by qPCR in 68 out of 92 bovine blood samples (73.9%), but none out of 69 DNA samples isolated from wild ruminant blood clots, was positive in both assays. The specificity of nested PCR and qPCR was confirmed by using BoHV1, BoHV4, BoHV6, BFV, BIV, and BLV DNA. The sensitivity of nested PCR and qPCR was determined using a serially 10-fold diluted vector pCR2.1HgB (2 × 10(0)-2 × 10(6)copies/reaction). In this testing, qPCR was more sensitive than the nested PCR, detecting two copies of BoHV6 whilst the limit of detection for nested PCR was 20 copies. In all qPCR assays, the coefficients of determination (R(2)) ranged between 0.990 and 0.999, and the calculated amplification efficiencies (Eff%) within the range of 89.7-106.9. The intra- and inter-assay CV (coefficient of variation) values did not exceed 4%. Copyright © 2013 Elsevier B.V. All rights reserved.

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

PubMed

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

2012-06-01

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

Quantitative detection of RASSF1A DNA promoter methylation in tumors and serum of patients with serous epithelial ovarian cancer.

PubMed

Bondurant, Amy E; Huang, Zhiqing; Whitaker, Regina S; Simel, Lauren R; Berchuck, Andrew; Murphy, Susan K

2011-12-01

Detection of cell free tumor-specific DNA methylation has been proposed as a potentially useful noninvasive mechanism to detect malignancies, including ovarian cancer, and to monitor response to treatment. However, there are few easily implemented quantitative approaches available for DNA methylation analysis. Our objectives were to develop an absolute quantitative method for detection of DNA methylation using RASSF1A, a known target of promoter methylation in ovarian cancer, and test the ability to detect RASSF1A methylation in tumors and serum specimens of women with ovarian cancer. Bisulfite modified DNAs were subjected to real time PCR using nondiscriminatory PCR primers and a probe with sequence containing a single CpG site, theoretically able to capture the methylation status of that CpG for every allele within a given specimen. Input DNA was normalized to ACTB levels detected simultaneously by assay multiplexing. Methylation levels were established by comparison to results obtained from universally methylated DNA. The assay was able to detect one methylated RASSF1A allele in 100,000 unmethylated alleles. RASSF1A was methylated in 54 of 106 (51%) invasive serous ovarian cancers analyzed and methylation status was concordant in 20/20 matched preoperative serum-tumor pairs. Serial serum specimens taken over the course of treatment for 8 of 9 patients showed fluctuations in RASSF1A methylation concomitant with disease status. This novel assay provides a real-time PCR-based method for absolute quantitation of DNA methylation. Our results support feasibility of monitoring RASSF1A methylation from serum samples taken over the course of treatment from women with ovarian cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

Multifunctional sample preparation kit and on-chip quantitative nucleic acid sequence-based amplification tests for microbial detection.

PubMed

Zhao, Xinyan; Dong, Tao

2012-10-16

This study reports a quantitative nucleic acid sequence-based amplification (Q-NASBA) microfluidic platform composed of a membrane-based sampling module, a sample preparation cassette, and a 24-channel Q-NASBA chip for environmental investigations on aquatic microorganisms. This low-cost and highly efficient sampling module, having seamless connection with the subsequent steps of sample preparation and quantitative detection, is designed for the collection of microbial communities from aquatic environments. Eight kinds of commercial membrane filters are relevantly analyzed using Saccharomyces cerevisiae, Escherichia coli, and Staphylococcus aureus as model microorganisms. After the microorganisms are concentrated on the membrane filters, the retentate can be easily conserved in a transport medium (TM) buffer and sent to a remote laboratory. A Q-NASBA-oriented sample preparation cassette is originally designed to extract DNA/RNA molecules directly from the captured cells on the membranes. Sequentially, the extract is analyzed within Q-NASBA chips that are compatible with common microplate readers in laboratories. Particularly, a novel analytical algorithmic method is developed for simple but robust on-chip Q-NASBA assays. The reported multifunctional microfluidic system could detect a few microorganisms quantitatively and simultaneously. Further research should be conducted to simplify and standardize ecological investigations on aquatic environments.

Combining markers with and without the limit of detection

PubMed Central

Dong, Ting; Liu, Catherine Chunling; Petricoin, Emanuel F.; Tang, Liansheng Larry

2014-01-01

In this paper, we consider the combination of markers with and without the limit of detection (LOD). LOD is often encountered when measuring proteomic markers. Because of the limited detecting ability of an equipment or instrument, it is difficult to measure markers at a relatively low level. Suppose that after some monotonic transformation, the marker values approximately follow multivariate normal distributions. We propose to estimate distribution parameters while taking the LOD into account, and then combine markers using the results from the linear discriminant analysis. Our simulation results show that the ROC curve parameter estimates generated from the proposed method are much closer to the truth than simply using the linear discriminant analysis to combine markers without considering the LOD. In addition, we propose a procedure to select and combine a subset of markers when many candidate markers are available. The procedure based on the correlation among markers is different from a common understanding that a subset of the most accurate markers should be selected for the combination. The simulation studies show that the accuracy of a combined marker can be largely impacted by the correlation of marker measurements. Our methods are applied to a protein pathway dataset to combine proteomic biomarkers to distinguish cancer patients from non-cancer patients. PMID:24132938

Detection and quantitation of trace phenolphthalein (in pharmaceutical preparations and in forensic exhibits) by liquid chromatography-tandem mass spectrometry, a sensitive and accurate method.

PubMed

Sharma, Kakali; Sharma, Shiba P; Lahiri, Sujit C

2013-01-01

Phenolphthalein, an acid-base indicator and laxative, is important as a constituent of widely used weight-reducing multicomponent food formulations. Phenolphthalein is an useful reagent in forensic science for the identification of blood stains of suspected victims and for apprehending erring officials accepting bribes in graft or trap cases. The pink-colored alkaline hand washes originating from the phenolphthalein-smeared notes can easily be determined spectrophotometrically. But in many cases, colored solution turns colorless with time, which renders the genuineness of bribe cases doubtful to the judiciary. No method is known till now for the detection and identification of phenolphthalein in colorless forensic exhibits with positive proof. Liquid chromatography-tandem mass spectrometry had been found to be most sensitive, accurate method capable of detection and quantitation of trace phenolphthalein in commercial formulations and colorless forensic exhibits with positive proof. The detection limit of phenolphthalein was found to be 1.66 pg/L or ng/mL, and the calibration curve shows good linearity (r(2) = 0.9974). © 2012 American Academy of Forensic Sciences.

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus

USGS Publications Warehouse

Jia, Peng; Purcell, Maureen; Pan, Guang; Wang, Jinjin; Kan, Shifu; Liu, Yin; Zheng, Xiaocong; SHi, Xiujie; He, Junqiang; Yu, Li; Hua, Qunyi; Lu, Tikang; Lan, Wensheng; Winton, James; Jin, Ningyi; Liu, Hong

2017-01-01

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4–100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

A quantitative x-ray detection system for gold nanoparticle tumour biomarkers.

PubMed

Ricketts, K; Castoldi, A; Guazzoni, C; Ozkan, C; Christodoulou, C; Gibson, A P; Royle, G J

2012-09-07

X-ray fluorescence techniques have proven beneficial for identifying and quantifying trace elements in biological tissues. A novel approach is being developed that employs x-ray fluorescence with an aim to locate heavy nanoparticles, such as gold, which are embedded into tissues. Such nanoparticles can be functionalized to act as markers for tumour characteristics to map the disease state, with the future aim of imaging them to inform cancer therapy regimes. The uptake of functionalized nanoparticles by cancer cells will also enable detection of small clusters of infiltrating cancer cells which are currently missed by commonly used imaging modalities. The novel system, consisting of an energy-resolving silicon drift detector with high spectral resolution, shows potential in both quantification of and sensitivity to nanoparticle concentrations typically found in tumours. A series of synchrotron measurements are presented; a linear relationship between fluorescence intensity and gold nanoparticle (GNP) concentration was found down to 0.005 mgAu ml(-1), the detection limit of the system. Successful use of a bench-top source, suitable for possible future clinical use, is also demonstrated, and found not to degrade the detection limit or accuracy of the GNP concentration measurement. The achieved system sensitivity suggests possible future clinical usefulness in measuring tumour uptake in vivo, particularly in shallow tumour sites and small animals, in ex vivo tissue and in 3D in vitro research samples.

Detection limits for nanoparticles in solution with classical turbidity spectra

NASA Astrophysics Data System (ADS)

Le Blevennec, G.

2013-09-01

Detection of nanoparticles in solution is required to manage safety and environmental problems. Spectral transmission turbidity method has now been known for a long time. It is derived from the Mie Theory and can be applied to any number of spheres, randomly distributed and separated by large distance compared to wavelength. Here, we describe a method for determination of size, distribution and concentration of nanoparticles in solution using UV-Vis transmission measurements. The method combines Mie and Beer Lambert computation integrated in a best fit approximation. In a first step, a validation of the approach is completed on silver nanoparticles solution. Verification of results is realized with Transmission Electronic Microscopy measurements for size distribution and an Inductively Coupled Plasma Mass Spectrometry for concentration. In view of the good agreement obtained, a second step of work focuses on how to manage the concentration to be the most accurate on the size distribution. Those efficient conditions are determined by simple computation. As we are dealing with nanoparticles, one of the key points is to know what the size limits reachable are with that kind of approach based on classical electromagnetism. In taking into account the transmission spectrometer accuracy limit we determine for several types of materials, metals, dielectrics, semiconductors the particle size limit detectable by such a turbidity method. These surprising results are situated at the quantum physics frontier.

Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

PubMed Central

Worbs, Sylvia; Fiebig, Uwe; Zeleny, Reinhard; Schimmel, Heinz; Rummel, Andreas; Luginbühl, Werner; Dorner, Brigitte G.

2015-01-01

In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay. PMID:26703724

Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

PubMed

Yegnasubramanian, Srinivasan; Lin, Xiaohui; Haffner, Michael C; DeMarzo, Angelo M; Nelson, William G

2006-02-09

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina.

PubMed

Buling, A; Criado-Fornelio, A; Asenzo, G; Benitez, D; Barba-Carretero, J C; Florin-Christensen, M

2007-06-20

The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome b gene, this method shows both high sensitivity and specificity, and allows quantification of parasite DNA. In tests, reproducible quantitative results were obtained over the range of 0.1 ng to 0.1 fg of parasite DNA. Melting curve analysis differentiated between B. bovis and B. bigemina. To assess the performance of the new qPCR procedure it was used to screen for babesiosis in 40 cows and 80 horses. B. bigemina was detected in five cows (three of these were also found to be positive by standard PCR techniques targeting the 18S rRNA gene). In addition, B. bovis was detected in one horse and B. bigemina in two horses using the proposed method, while none was found positive by ribosomal standard PCR. The sequences of the B. bigemina cytochrome b and 18S rRNA genes were completely conserved in isolates from Spain and Argentina, while those of B. bovis showed moderate polymorphism.

Considerations on the determination of the limit of detection and the limit of quantification in one-dimensional and comprehensive two-dimensional gas chromatography.

PubMed

Krupčík, Ján; Májek, Pavel; Gorovenko, Roman; Blaško, Jaroslav; Kubinec, Robert; Sandra, Pat

2015-05-29

Methods based on the blank signal as proposed by IUPAC procedure and on the signal to noise ratio (S/N) as listed in the ISO-11843-1 norm for determination of the limit of detection (LOD) and quantitation (LOQ) in one-dimensional capillary gas chromatography (1D-GC) and comprehensive two-dimensional capillary gas chromatography (CG×GC) are described in detail and compared for both techniques. Flame ionization detection was applied and variables were the data acquisition frequency and, for CG×GC, also the modulation time. It has been stated that LOD and LOQ estimated according to IUPAC might be successfully used for 1D-GC-FID method. Moreover, LOD and LOQ decrease with decrease of data acquisition frequency (DAF). For GC×GC-FID, estimation of LOD by IUPAC gave poor reproducibility of results while for LOQ reproducibility was acceptable (within ±10% rel.). The LOD and LOQ determined by the S/N concept both for 1D-GC-FID and GC×GC-FID methods are ca. three times higher than those values estimated by the standard deviation of the blank. Since the distribution pattern of modulated peaks for any analyte separated by GC×GC is random and cannot be predicted, LOQ and LOD may vary within 30% for 3s modulation time. Concerning sensitivity, 1D-GC-FID at 2Hz and of GC×GC-FID at 50Hz shows a ca. 5 times enhancement of sensitivity in the modulated signal output. Copyright © 2015 Elsevier B.V. All rights reserved.

Thermal Infrared Spectral Band Detection Limits for Unidentified Surface Materials

NASA Technical Reports Server (NTRS)

Kirkland, Laurel E.; Herr, Kenneth C.; Salisbury, John W.

2001-01-01

Infrared emission spectra recorded by airborne or satellite spectrometers can be searched for spectral features to determine the composition of rocks on planetary surfaces. Surface materials are identified by detections of characteristic spectral bands. We show how to define whether to accept an observed spectral feature as a detection when the target material is unknown. We also use remotely sensed spectra measured by the Thermal Emission Spectrometer (TES) and the Spatially Enhanced Broadband Array Spectrograph System to illustrate the importance of instrument parameters and surface properties on band detection limits and how the variation in signal-to-noise ratio with wavelength affects the bands that are most detectable for a given instrument. The spectrometer's sampling interval, spectral resolution, signal-to-noise ratio as a function of wavelength, and the sample's surface properties influence whether the instrument can detect a spectral feature exhibited by a material. As an example, in the 6-13 micrometer wavelength region, massive carbonates exhibit two bands: a very strong, broad feature at approximately 6.5 micrometers and a less intense, sharper band at approximately 11.25 micrometers. Although the 6.5-micrometer band is stronger and broader in laboratory-measured spectra, the 11.25-micrometer band will cause a more detectable feature in TES spectra.

The Power to Detect Linkage Disequilibrium with Quantitative Traits in Selected Samples

PubMed Central

Abecasis, Gonçalo R.; Cookson, William O. C.; Cardon, Lon R.

2001-01-01

Results from power studies for linkage detection have led to many ongoing and planned collections of phenotypically extreme nuclear families. Given the great expense of collecting these families and the imminent availability of a dense diallelic marker map, the families are likely to be used in allelic-association as well as linkage studies. However, optimal selection strategies for linkage may not be equally powerful for association. We examine the power to detect linkage disequilibrium for quantitative traits after phenotypic selection. The results encompass six selection strategies that are in widespread use, including single selection (two designs), affected sib pairs, concordant and discordant pairs, and the extreme-concordant and -discordant design. Selection of sibships on the basis of one extreme proband with high or low trait scores provides as much power as discordant sib pairs but requires the screening and phenotyping of substantially fewer initial families from which to select. Analysis of the role of allele frequencies within each selection design indicates that common trait alleles generally offer the most power, but similarities between the marker- and trait-allele frequencies are much more important than the trait-locus frequency alone. Some of the most widespread selection designs, such as single selection, yield power gains only when both the marker and quantitative trait loci (QTL) are relatively rare in the population. In contrast, discordant pairs and the extreme-proband design provide power for the broadest range of QTL–marker-allele frequency differences. Overall, proband selection from either tail provides the best balance of power, robustness, and simplicity of ascertainment for family-based association analysis. PMID:11349228

Man vs. Machine: A Junior-level Laboratory Exercise Comparing Human and Instrumental Detection Limits

ERIC Educational Resources Information Center

Elias, Ryan J.; Hopfer, Helene; Hofstaedter, Amanda N.; Hayes, John E.

2017-01-01

The human nose is a very sensitive detector and is able to detect potent aroma compounds down to low ng/L levels. These levels are often below detection limits of analytical instrumentation. The following laboratory exercise is designed to compare instrumental and human methods for the detection of volatile odor active compounds. Reference…

THE USE AND LIMITATIONS OF DETECTION AND QUANTITATION LIMITS IN ENVIRONMENTAL ANALYSIS

EPA Science Inventory

Site assessment, remediation and compliance monitoring require the routine determination of the concentration of regulated substances in environmental samples. Each measurement methodology providing the concentration determinations, is required to specify key data quality elemen...

Quantitative analysis of the correlations in the Boltzmann-Grad limit for hard spheres

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

Pulvirenti, M.

2014-12-09

In this contribution I consider the problem of the validity of the Boltzmann equation for a system of hard spheres in the Boltzmann-Grad limit. I briefly review the results available nowadays with a particular emphasis on the celebrated Lanford’s validity theorem. Finally I present some recent results, obtained in collaboration with S. Simonella, concerning a quantitative analysis of the propagation of chaos. More precisely we introduce a quantity (the correlation error) measuring how close a j-particle rescaled correlation function at time t (sufficiently small) is far from the full statistical independence. Roughly speaking, a correlation error of order k, measuresmore » (in the context of the BBKGY hierarchy) the event in which k tagged particles form a recolliding group.« less

A Survey on Pharmacovigilance Activities in ASEAN and Selected Non-ASEAN Countries, and the Use of Quantitative Signal Detection Algorithms.

PubMed

Chan, Cheng Leng; Ang, Pei San; Li, Shu Chuen

2017-06-01

Most Countries have pharmacovigilance (PV) systems in place to monitor the safe use of health products. The process involves the detection and assessment of safety issues from various sources of information, communicating the risk to stakeholders and taking other relevant risk minimization measures. This study aimed to assess the PV status in Association of Southeast Asian Nation (ASEAN) countries, sources for postmarket safety monitoring, methods used for signal detection and the need for a quantitative signal detection algorithm (QSDA). Comparisons were conducted with centres outside ASEAN. A questionnaire was sent to all PV centres in ASEAN countries, as well as seven other countries, from November 2015 to June 2016. The questionnaire was designed to collect information on the status of PV, with a focus on the use of a QSDA. Data were collected from nine ASEAN countries and seven other countries. PV activities were conducted in all these countries, which were at different stages of development. In terms of adverse drug reaction (ADR) reports, the average number received per year ranged from 3 to 50,000 reports for ASEAN countries and from 7000 to 1,103,200 for non-ASEAN countries. Thirty-three percent of ASEAN countries utilized statistical methods to help detect signals from ADR reports compared with 100% in the other non-ASEAN countries. Eighty percent agreed that the development of a QSDA would help in drug signal detection. The main limitation identified was the lack of knowledge and/or lack of resources. Spontaneous ADR reports from healthcare professionals remains the most frequently used source for safety monitoring. The traditional method of case-by-case review of ADR reports prevailed for signal detection in ASEAN countries. As the reports continue to grow, the development of a QSDA would be useful in helping detect safety signals.

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

PubMed

Abdeldaim, Guma M K; Strålin, Kristoffer; Kirsebom, Leif A; Olcén, Per; Blomberg, Jonas; Herrmann, Björn

2009-08-01

A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

Target-Specific Assay for Rapid and Quantitative Detection of Mycobacterium chimaera DNA.

PubMed

Zozaya-Valdés, Enrique; Porter, Jessica L; Coventry, John; Fyfe, Janet A M; Carter, Glen P; Gonçalves da Silva, Anders; Schultz, Mark B; Seemann, Torsten; Johnson, Paul D R; Stewardson, Andrew J; Bastian, Ivan; Roberts, Sally A; Howden, Benjamin P; Williamson, Deborah A; Stinear, Timothy P

2017-06-01

Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium - M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico -predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera . Copyright © 2017 American Society for Microbiology.

Quantitative analysis of professionally trained versus untrained voices.

PubMed

Siupsinskiene, Nora

2003-01-01

The aim of this study was to compare healthy trained and untrained voices as well as healthy and dysphonic trained voices in adults using combined voice range profile and aerodynamic tests, to define the normal range limiting values of quantitative voice parameters and to select the most informative quantitative voice parameters for separation between healthy and dysphonic trained voices. Three groups of persons were evaluated. One hundred eighty six healthy volunteers were divided into two groups according to voice training: non-professional speakers group consisted of 106 untrained voices persons (36 males and 70 females) and professional speakers group--of 80 trained voices persons (21 males and 59 females). Clinical group consisted of 103 dysphonic professional speakers (23 males and 80 females) with various voice disorders. Eighteen quantitative voice parameters from combined voice range profile (VRP) test were analyzed: 8 of voice range profile, 8 of speaking voice, overall vocal dysfunction degree and coefficient of sound, and aerodynamic maximum phonation time. Analysis showed that healthy professional speakers demonstrated expanded vocal abilities in comparison to healthy non-professional speakers. Quantitative voice range profile parameters- pitch range, high frequency limit, area of high frequencies and coefficient of sound differed significantly between healthy professional and non-professional voices, and were more informative than speaking voice or aerodynamic parameters in showing the voice training. Logistic stepwise regression revealed that VRP area in high frequencies was sufficient to discriminate between healthy and dysphonic professional speakers for male subjects (overall discrimination accuracy--81.8%) and combination of three quantitative parameters (VRP high frequency limit, maximum voice intensity and slope of speaking curve) for female subjects (overall model discrimination accuracy--75.4%). We concluded that quantitative voice assessment

Calculation of the detection limits for radionuclides identified in gamma-ray spectra based on post-processing peak analysis results.

PubMed

Korun, M; Vodenik, B; Zorko, B

2018-03-01

A new method for calculating the detection limits of gamma-ray spectrometry measurements is presented. The method is applicable for gamma-ray emitters, irrespective of the influences of the peaked background, the origin of the background and the overlap with other peaks. It offers the opportunity for multi-gamma-ray emitters to calculate the common detection limit, corresponding to more peaks. The detection limit is calculated by approximating the dependence of the uncertainty in the indication on its value with a second-order polynomial. In this approach the relation between the input quantities and the detection limit are described by an explicit expression and can be easy investigated. The detection limit is calculated from the data usually provided by the reports of peak-analyzing programs: the peak areas and their uncertainties. As a result, the need to use individual channel contents for calculating the detection limit is bypassed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantitative blood group typing using surface plasmon resonance.

PubMed

Then, Whui Lyn; Aguilar, Marie-Isabel; Garnier, Gil

2015-11-15

The accurate and reliable typing of blood groups is essential prior to blood transfusion. While current blood typing methods are well established, results are subjective and heavily reliant on analysis by trained personnel. Techniques for quantifying blood group antibody-antigen interactions are also very limited. Many biosensing systems rely on surface plasmon resonance (SPR) detection to quantify biomolecular interactions. While SPR has been widely used for characterizing antibody-antigen interactions, measuring antibody interactions with whole cells is significantly less common. Previous studies utilized SPR for blood group antigen detection, however, showed poor regeneration causing loss of functionality after a single use. In this study, a fully regenerable, multi-functional platform for quantitative blood group typing via SPR detection is achieved by immobilizing anti-human IgG antibody to the sensor surface, which binds to the Fc region of human IgG antibodies. The surface becomes an interchangeable platform capable of quantifying the blood group interactions between red blood cells (RBCs) and IgG antibodies. As with indirect antiglobulin tests (IAT), which use IgG antibodies for detection, IgG antibodies are initially incubated with RBCs. This facilitates binding to the immobilized monolayer and allows for quantitative blood group detection. Using the D-antigen as an example, a clear distinction between positive (>500 RU) and negative (<100 RU) RBCs is achieved using anti-D IgG. Complete regeneration of the anti-human IgG surface is also successful, showing negligible degradation of the surface after more than 100 regenerations. This novel approach is validated with human-sourced whole blood samples to demonstrate an interesting alternative for quantitative blood grouping using SPR analysis. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Quantitative detection of astaxanthin and cantaxanthin in Atlantic salmon by resonance Raman spectroscopy

NASA Astrophysics Data System (ADS)

Ermakov, Igor V.; Ermakova, Maia R.; Gellermann, Werner

2006-02-01

Two major carotenoids species found in salmonids muscle tissues are astaxanthin and cantaxanthin. They are taken up from fish food and are responsible for the attractive red-orange color of salmon filet. Since carotenoids are powerful antioxidants and biomarkers of nutrient consumption, they are thought to indicate fish health and resistance to diseases in fish farm environments. Therefore, a rapid, accurate, quantitative optical technique for measuring carotenoid content in salmon tissues is of economic interest. We demonstrate the possibility of using fast, selective, quantitative detection of astaxanthin and cantaxanthin in salmon muscle tissues, employing resonance Raman spectroscopy. Analyzing strong Raman signals originating from the carbon-carbon double bond stretch vibrations of the carotenoid molecules under blue laser excitation, we are able to characterize quantitatively the concentrations of carotenoids in salmon muscle tissue. To validate the technique, we compared Raman data with absorption measurements of carotenoid extracts in acetone. A close correspondence was observed in absorption spectra for tissue extract in acetone and a pure astaxanthin solution. Raman results show a linear dependence between Raman and absorption data. The proposed technique holds promise as a method of rapid screening of carotenoid levels in fish muscle tissues and may be attractive for the fish farm industry to assess the dietary status of salmon, risk for infective diseases, and product quality control.

Determination of target detection limits in hyperspectral data using band selection and dimensionality reduction

NASA Astrophysics Data System (ADS)

Gross, W.; Boehler, J.; Twizer, K.; Kedem, B.; Lenz, A.; Kneubuehler, M.; Wellig, P.; Oechslin, R.; Schilling, H.; Rotman, S.; Middelmann, W.

2016-10-01

Hyperspectral remote sensing data can be used for civil and military applications to robustly detect and classify target objects. High spectral resolution of hyperspectral data can compensate for the comparatively low spatial resolution, which allows for detection and classification of small targets, even below image resolution. Hyperspectral data sets are prone to considerable spectral redundancy, affecting and limiting data processing and algorithm performance. As a consequence, data reduction strategies become increasingly important, especially in view of near-real-time data analysis. The goal of this paper is to analyze different strategies for hyperspectral band selection algorithms and their effect on subpixel classification for different target and background materials. Airborne hyperspectral data is used in combination with linear target simulation procedures to create a representative amount of target-to-background ratios for evaluation of detection limits. Data from two different airborne hyperspectral sensors, AISA Eagle and Hawk, are used to evaluate transferability of band selection when using different sensors. The same target objects were recorded to compare the calculated detection limits. To determine subpixel classification results, pure pixels from the target materials are extracted and used to simulate mixed pixels with selected background materials. Target signatures are linearly combined with different background materials in varying ratios. The commonly used classification algorithms Adaptive Coherence Estimator (ACE) is used to compare the detection limit for the original data with several band selection and data reduction strategies. The evaluation of the classification results is done by assuming a fixed false alarm ratio and calculating the mean target-to-background ratio of correctly detected pixels. The results allow drawing conclusions about specific band combinations for certain target and background combinations. Additionally

Quantitative characterization of the imaging limits of diffuse low-grade oligodendrogliomas.

PubMed

Gerin, Chloé; Pallud, Johan; Deroulers, Christophe; Varlet, Pascale; Oppenheim, Catherine; Roux, Francois-Xavier; Chrétien, Fabrice; Thomas, Stephen R; Grammaticos, Basile; Badoual, Mathilde

2013-10-01

Supratentorial diffuse low-grade gliomas in adults extend beyond maximal visible MRI-defined abnormalities, and a gap exists between the imaging signal changes and the actual tumor margins. Direct quantitative comparisons between imaging and histological analyses are lacking to date. However, they are of the utmost importance if one wishes to develop realistic models for diffuse glioma growth. In this study, we quantitatively compared the cell concentration and the edema fraction from human histological biopsy samples (BSs) performed inside and outside imaging abnormalities during serial imaging-based stereotactic biopsy of diffuse low-grade gliomas. The cell concentration was significantly higher in BSs located inside (1189 ± 378 cell/mm(2)) than outside (740 ± 124 cell/mm(2)) MRI-defined abnormalities (P = .0003). The edema fraction was significantly higher in BSs located inside (mean, 45% ± 23%) than outside (mean, 5 %± 9%) MRI-defined abnormalities (P < .0001). At borders of the MRI-defined abnormalities, 20% of the tissue surface area was occupied by edema and only 3% by tumor cells. The cycling cell concentration was significantly higher in BSs located inside (10 ± 12 cell/mm(2)), compared with outside (0.5 ± 0.9 cell/mm(2)), MRI-defined abnormalities (P = .0001). We showed that the margins of T2-weighted signal changes are mainly correlated with the edema fraction. In 62.5% of patients, the cycling tumor cell fraction (defined as the ratio of the cycling tumor cell concentration to the total number of tumor cells) was higher at the limits of the MRI-defined abnormalities than closer to the center of the tumor. In the remaining patients, the cycling tumor cell fraction increased towards the center of the tumor.

[THE COMPARATIVE ANALYSIS OF RESULTS OF DETECTION OF CARCINOGENIC TYPES OF HUMAN PAPILLOMA VIRUS BY QUALITATIVE AND QUANTITATIVE TESTS].

PubMed

Kuzmenko, E T; Labigina, A V; Leshenko, O Ya; Rusanov, D N; Kuzmenko, V V; Fedko, L P; Pak, I P

2015-05-01

The analysis of results of screening (n = 3208; sexually active citizen aged from 18 to 59 years) was carried out to detect oncogene types of human papilloma virus in using qualitative (1150 females and 720 males) and quantitative (polymerase chain reaction in real-time (843 females and 115 males) techniques. The human papilloma virus of high oncogene type was detected in 65% and 68.4% of females and in 48.6% and 53% of males correspondingly. Among 12 types of human papilloma virus the most frequently diagnosed was human papilloma virus 16 independently of gender of examined and technique of analysis. In females, under application of qualitative tests rate of human papilloma virus 16 made up to 18.3% (n = 280) and under application of quantitative tests Rte of human papilloma virus made up to 14.9% (n = 126; p ≤ 0.05). Under examination of males using qualitative tests rate of human papilloma virus 16 made up to 8.3% (n = 60) and under application of qualitative tests made up to 12.2% (n = 14; p ≥ 0.05). Under application of qualitative tests rate of detection on the rest ofoncogene types of human papilloma virus varied in females from 3.4% to 8.4% and in males from 1.8% to 5.9%. Under application of qualitative tests to females rate of human papilloma virus with high viral load made up to 68.4%, with medium viral load - 2.85% (n = 24) and with low viral load -0.24% (n = 2). Under application of quantitative tests in males rate of detection of types of human papilloma virus made up to 53% and at that in all high viral load was established. In females, the most of oncogene types of human papilloma virus (except for 31, 39, 59) are detected significantly more often than in males.

Lower reference limits of quantitative cord glucose-6-phosphate dehydrogenase estimated from healthy term neonates according to the clinical and laboratory standards institute guidelines: a cross sectional retrospective study

PubMed Central

2013-01-01

Background Previous studies have reported the lower reference limit (LRL) of quantitative cord glucose-6-phosphate dehydrogenase (G6PD), but they have not used approved international statistical methodology. Using common standards is expecting to yield more true findings. Therefore, we aimed to estimate LRL of quantitative G6PD detection in healthy term neonates by using statistical analyses endorsed by the International Federation of Clinical Chemistry (IFCC) and the Clinical and Laboratory Standards Institute (CLSI) for reference interval estimation. Methods This cross sectional retrospective study was performed at King Abdulaziz Hospital, Saudi Arabia, between March 2010 and June 2012. The study monitored consecutive neonates born to mothers from one Arab Muslim tribe that was assumed to have a low prevalence of G6PD-deficiency. Neonates that satisfied the following criteria were included: full-term birth (37 weeks); no admission to the special care nursery; no phototherapy treatment; negative direct antiglobulin test; and fathers of female neonates were from the same mothers’ tribe. The G6PD activity (Units/gram Hemoglobin) was measured spectrophotometrically by an automated kit. This study used statistical analyses endorsed by IFCC and CLSI for reference interval estimation. The 2.5th percentiles and the corresponding 95% confidence intervals (CI) were estimated as LRLs, both in presence and absence of outliers. Results 207 males and 188 females term neonates who had cord blood quantitative G6PD testing met the inclusion criteria. Method of Horn detected 20 G6PD values as outliers (8 males and 12 females). Distributions of quantitative cord G6PD values exhibited a normal distribution in absence of the outliers only. The Harris-Boyd method and proportion criteria revealed that combined gender LRLs were reliable. The combined bootstrap LRL in presence of the outliers was 10.0 (95% CI: 7.5-10.7) and the combined parametric LRL in absence of the outliers was 11

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...

Characterization of X80 and X100 Microalloyed Pipeline Steel Using Quantitative X-ray Diffraction

NASA Astrophysics Data System (ADS)

Wiskel, J. B.; Li, X.; Ivey, D. G.; Henein, H.

2018-06-01

Quantitative X-ray diffraction characterization of four (4) X80 and three (3) X100 microalloyed steels was undertaken. The effect of through-thickness position, processing parameters, and composition on the measured crystallite size, microstrain, and J index (relative magnitude of crystallographic texture) was determined. Microstructure analysis using optical microscopy, scanning electron microscopy, transmission electron microscopy, and electron-backscattered diffraction was also undertaken. The measured value of microstrain increased with increasing alloy content and decreasing cooling interrupt temperature. Microstructural features corresponding to crystallite size in the X80 steels were both above and below the detection limit for quantitative X-ray diffraction. The X100 steels consistently exhibited microstructure features below the crystallite size detection limit. The yield stress of each steel increased with increasing microstrain. The increase in microstrain from X80 to X100 is also associated with a change in microstructure from predominantly polygonal ferrite to bainitic ferrite.

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

PubMed

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

2008-12-01

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

In vitro bioassays for detecting dioxin-like activity--application potentials and limits of detection, a review.

PubMed

Eichbaum, Kathrin; Brinkmann, Markus; Buchinger, Sebastian; Reifferscheid, Georg; Hecker, Markus; Giesy, John P; Engwall, Magnus; van Bavel, Bert; Hollert, Henner

2014-07-15

Use of in vitro assays as screening tool to characterize contamination of a variety of environmental matrices has become an increasingly popular and powerful toolbox in the field of environmental toxicology. While bioassays cannot entirely substitute analytical methods such as gas chromatography-mass spectrometry (GC-MS), the increasing improvement of cell lines and standardization of bioassay procedures enhance their utility as bioanalytical pre-screening tests prior to more targeted chemical analytical investigations. Dioxin-receptor-based assays provide a holistic characterization of exposure to dioxin-like compounds (DLCs) by integrating their overall toxic potential, including potentials of unknown DLCs not detectable via e.g. GC-MS. Hence, they provide important additional information with respect to environmental risk assessment of DLCs. This review summarizes different in vitro bioassay applications for detection of DLCs and considers the comparability of bioassay and chemical analytically derived toxicity equivalents (TEQs) of different approaches and various matrices. These range from complex samples such as sediments through single reference to compound mixtures. A summary of bioassay derived detection limits (LODs) showed a number of current bioassays to be equally sensitive as chemical methodologies, but moreover revealed that most of the bioanalytical studies conducted to date did not report their LODs, which represents a limitation with regard to low potency samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Predictive inference for best linear combination of biomarkers subject to limits of detection.

PubMed

Coolen-Maturi, Tahani

2017-08-15

Measuring the accuracy of diagnostic tests is crucial in many application areas including medicine, machine learning and credit scoring. The receiver operating characteristic (ROC) curve is a useful tool to assess the ability of a diagnostic test to discriminate between two classes or groups. In practice, multiple diagnostic tests or biomarkers are combined to improve diagnostic accuracy. Often, biomarker measurements are undetectable either below or above the so-called limits of detection (LoD). In this paper, nonparametric predictive inference (NPI) for best linear combination of two or more biomarkers subject to limits of detection is presented. NPI is a frequentist statistical method that is explicitly aimed at using few modelling assumptions, enabled through the use of lower and upper probabilities to quantify uncertainty. The NPI lower and upper bounds for the ROC curve subject to limits of detection are derived, where the objective function to maximize is the area under the ROC curve. In addition, the paper discusses the effect of restriction on the linear combination's coefficients on the analysis. Examples are provided to illustrate the proposed method. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Rapid quantitative analysis of individual anthocyanin content based on high-performance liquid chromatography with diode array detection with the pH differential method.

PubMed

Wang, Huayin

2014-09-01

A new quantitative technique for the simultaneous quantification of the individual anthocyanins based on the pH differential method and high-performance liquid chromatography with diode array detection is proposed in this paper. The six individual anthocyanins (cyanidin 3-glucoside, cyanidin 3-rutinoside, petunidin 3-glucoside, petunidin 3-rutinoside, and malvidin 3-rutinoside) from mulberry (Morus rubra) and Liriope platyphylla were used for demonstration and validation. The elution of anthocyanins was performed using a C18 column with stepwise gradient elution and individual anthocyanins were identified by high-performance liquid chromatography with tandem mass spectrometry. Based on the pH differential method, the high-performance liquid chromatography peak areas of maximum and reference absorption wavelengths of anthocyanin extracts were conducted to quantify individual anthocyanins. The calibration curves for these anthocyanins were linear within the range of 10-5500 mg/L. The correlation coefficients (r(2)) all exceeded 0.9972, and the limits of detection were in the range of 1-4 mg/L at a signal-to-noise ratio ≥5 for these anthocyanins. The proposed quantitative analysis was reproducible with good accuracy of all individual anthocyanins ranging from 96.3 to 104.2% and relative recoveries were in the range 98.4-103.2%. The proposed technique is performed without anthocyanin standards and is a simple, rapid, accurate, and economical method to determine individual anthocyanin contents. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis

PubMed Central

2013-01-01

Background A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. Methods A semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene. Results This study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix. Conclusions The development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to < two hours. Analysis of the PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples. PMID:23433252

Quantitative Detection of Streptococcus pneumoniae in Nasopharyngeal Secretions by Real-Time PCR

PubMed Central

Greiner, Oliver; Day, Philip J. R.; Bosshard, Philipp P.; Imeri, Fatime; Altwegg, Martin; Nadal, David

2001-01-01

Streptococcus pneumoniae is an important cause of community-acquired pneumonia. However, in this setting the diagnostic sensitivity of blood cultures is below 30%. Since during such infections changes in the amounts of S. pneumoniae may also occur in the upper respiratory tract, quantification of these bacteria in nasopharnygeal secretions (NPSs) may offer a suitable diagnostic approach. Real-time PCR offers a sensitive, efficient, and routinely reproducible approach to quantification. Using primers and a fluorescent probe specific for the pneumolysin gene, we were able to detect DNA from serial dilutions of S. pneumoniae cells in which the quantities of DNA ranged from the amounts extracted from 1 to 106 cells. No difference was noted when the same DNA was mixed with DNA extracted from NPSs shown to be deficient of S. pneumoniae following culture, suggesting that this bacterium can be detected and accurately quantitated in clinical samples. DNAs from Haemophilus influenzae, Moraxella catarrhalis, or alpha-hemolytic streptococci other than S. pneumoniae were not amplified or were only weakly amplified when there were ≥106 cells per reaction mixture. When the assay was applied to NPSs from patients with respiratory tract infections, the assay performed with a sensitivity of 100% and a specificity of up to 96% compared to the culture results. The numbers of S. pneumoniae organisms detected by real-time PCR correlated with the numbers detected by semiquantitative cultures. A real-time PCR that targeted the pneumolysin gene provided a sensitive and reliable means for routine rapid detection and quantification of S. pneumoniae present in NPSs. This assay may serve as a tool to study changes in the amounts of S. pneumoniae during lower respiratory tract infections. PMID:11526140

Pushing quantitation limits in micro UHPLC-MS/MS analysis of steroid hormones by sample dilution using high volume injection.

PubMed

Márta, Zoltán; Bobály, Balázs; Fekete, Jenő; Magda, Balázs; Imre, Tímea; Mészáros, Katalin Viola; Szabó, Pál Tamás

2016-09-10

Ultratrace analysis of sample components requires excellent analytical performance in terms of limits of quantitation (LoQ). Micro UHPLC coupling with sensitive tandem mass spectrometry provides state of the art solutions for such analytical problems. Decreased column volume in micro LC limits the injectable sample volume. However, if analyte concentration is extremely low, it might be necessary to inject high sample volumes. This is particularly critical for strong sample solvents and weakly retained analytes, which are often the case when preparing biological samples (protein precipitation, sample extraction, etc.). In that case, high injection volumes may cause band broadening, peak distortion or even elution in dead volume. In this study, we evaluated possibilities of high volume injection onto microbore RP-LC columns, when sample solvent is diluted. The presented micro RP-LC-MS/MS method was optimized for the analysis of steroid hormones from human plasma after protein precipitation with organic solvents. A proper sample dilution procedure helps to increase the injection volume without compromising peak shapes. Finally, due to increased injection volume, the limit of quantitation can be decreased by a factor of 2-5, depending on the analytes and the experimental conditions. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of Goose parvovirus in vivo

PubMed Central

Yang, Jin-Long; Cheng, An-Chun; Wang, Ming-Shu; Pan, Kang-Cheng; Li, Min; Guo, Yu-Fei; Li, Chuan-Feng; Zhu, De-Kang; Chen, Xiao-Yue

2009-01-01

Background Goose parvovirus (GPV) is a Dependovirus associated with latent infection and mortality in geese. Currently, it severely affects geese production worldwide. The objective of this study was to develop a fluorescent quantitative real-time polymerase chain reaction (PCR) (FQ-PCR) assay for fast and accurate quantification of GPV DNA in infected goslings, which can aid in the understanding of the regular distribution pattern and the nosogenesis of GPV in vivo. Results The detection limit of the assay was 2.8 × 101 standard DNA copies, with a sensitivity of 3 logs higher than that of the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intraassay and interassay coefficients of variation. Conclusion The high sensitivity, specificity, simplicity, and reproducibility of the GPV fluorogenic PCR assay, combined with a high throughput, make this method suitable for a broad spectrum of GPV etiology-related applications. PMID:19754946

Quantitation of underivatized branched-chain amino acids in sport nutritional supplements by capillary electrophoresis with direct or indirect UV absorbance detection.

PubMed

Qiu, Jun; Wang, Jinhao; Xu, Zhongqi; Liu, Huiqing; Ren, Jie

2017-01-01

The branched-chain amino acids (BCAAs) including leucine (Leu), isoleucine (Ile) and valine (Val) play a pivotal role in the human body. Herein, we developed capillary electrophoresis (CE) coupled with conventional UV detector to quantify underivatized BCAAs in two kinds of sport nutritional supplements. For direct UV detection at 195 nm, the BCAAs (Leu, two enantiomers of Ile and Val) were separated in a background electrolyte (BGE) consisting of 40.0 mmol/L sodium tetraborate, and 40.0 mmol/L β-cyclodextrin (β-CD) at pH 10.2. In addition, the indirect UV detection at 264 nm was achieved in a BGE of 2.0 mmol/L Na2HPO4, 10.0 mmol/L p-aminosalicylic acid (PAS) as UV absorbing probe, and 40.0 mmol/L β-CD at pH 12.2. The β-CD significantly benefited the isomeric separation of Leu, L- and D-Ile. The optimal conditions allowed the LODs (limit of detections) of direct and indirect UV absorption detection to be tens μmol/L level, which was comparable to the reported CE inline derivatization method. The RSDs (relative standard deviations) of migration time and peak area were less than 0.91% and 3.66% (n = 6). Finally, CE with indirect UV detection method was applied for the quantitation of BCAAs in two commercial sport nutritional supplements, and good recovery and precision were obtained. Such simple CE method without tedious derivatization process is feasible of quality control and efficacy evaluation of the supplemental proteins.

Quantitation of underivatized branched-chain amino acids in sport nutritional supplements by capillary electrophoresis with direct or indirect UV absorbance detection

PubMed Central

Liu, Huiqing; Ren, Jie

2017-01-01

The branched-chain amino acids (BCAAs) including leucine (Leu), isoleucine (Ile) and valine (Val) play a pivotal role in the human body. Herein, we developed capillary electrophoresis (CE) coupled with conventional UV detector to quantify underivatized BCAAs in two kinds of sport nutritional supplements. For direct UV detection at 195 nm, the BCAAs (Leu, two enantiomers of Ile and Val) were separated in a background electrolyte (BGE) consisting of 40.0 mmol/L sodium tetraborate, and 40.0 mmol/L β-cyclodextrin (β-CD) at pH 10.2. In addition, the indirect UV detection at 264 nm was achieved in a BGE of 2.0 mmol/L Na2HPO4, 10.0 mmol/L p-aminosalicylic acid (PAS) as UV absorbing probe, and 40.0 mmol/L β-CD at pH 12.2. The β-CD significantly benefited the isomeric separation of Leu, L- and D-Ile. The optimal conditions allowed the LODs (limit of detections) of direct and indirect UV absorption detection to be tens μmol/L level, which was comparable to the reported CE inline derivatization method. The RSDs (relative standard deviations) of migration time and peak area were less than 0.91% and 3.66% (n = 6). Finally, CE with indirect UV detection method was applied for the quantitation of BCAAs in two commercial sport nutritional supplements, and good recovery and precision were obtained. Such simple CE method without tedious derivatization process is feasible of quality control and efficacy evaluation of the supplemental proteins. PMID:28640882

40 CFR Appendix B to Part 136 - Definition and Procedure for the Determination of the Method Detection Limit-Revision 1.11

Code of Federal Regulations, 2010 CFR

2010-07-01

... calculated method detection limit. To insure that the estimate of the method detection limit is a good...) where: MDL = the method detection limit t(n-1,1- α=.99) = the students' t value appropriate for a 99... Determination of the Method Detection Limit-Revision 1.11 B Appendix B to Part 136 Protection of Environment...

Addition of Adapted Optics towards obtaining a quantitative detection of diabetic retinopathy

NASA Astrophysics Data System (ADS)

Yust, Brian; Obregon, Isidro; Tsin, Andrew; Sardar, Dhiraj

2009-04-01

An adaptive optics system was assembled for correcting the aberrated wavefront of light reflected from the retina. The adaptive optics setup includes a superluminous diode light source, Hartmann-Shack wavefront sensor, deformable mirror, and imaging CCD camera. Aberrations found in the reflected wavefront are caused by changes in the index of refraction along the light path as the beam travels through the cornea, lens, and vitreous humour. The Hartmann-Shack sensor allows for detection of aberrations in the wavefront, which may then be corrected with the deformable mirror. It has been shown that there is a change in the polarization of light reflected from neovascularizations in the retina due to certain diseases, such as diabetic retinopathy. The adaptive optics system was assembled towards the goal of obtaining a quantitative measure of onset and progression of this ailment, as one does not currently exist. The study was done to show that the addition of adaptive optics results in a more accurate detection of neovascularization in the retina by measuring the expected changes in polarization of the corrected wavefront of reflected light.

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

PubMed

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

2012-04-01

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

[Quantitative determination of biogenic amine from Biomphalaria glabrata nervous system by UPLC MS/MS].

PubMed

Tao, Huang; Yun-Hai, Guo; He-Xiang, Liu; Yi, Zhang

2018-04-19

To establish a method for the quantitative determination of serotonin and dopamine in the nervous system of Biomphalaria glabrata by using ultra high performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC MS/MS) . The B. glabrata nervous system was broken in the pure methanol solution after obtaining it by dissecting with microscope. Then, the supernatant containing the target substance after twice high speed centrifugation was got. The extraction was separated on an ACQUITY UPLC BEH Amide column with Waters TQ-XS series mass spectrometry detector, with ESI source and positive electrospray ionization mode when the machine testing. The detection limit of serotonin was 0.03 ng/ml and the limit of quantification was 0.1 ng/ml. The detection limit of dopamine was 0.05 ng/ml and the limit of quantification was 0.15 ng/ml. The recoveries of serotonin ranged from 90.68% to 94.72% over the range of 1 to 40 ng/ml. The recoveries of dopamine ranged from 91.68% to 96.12% over the range of 1.0 ng/ml to 40 ng/ml. The established UPLC MS/MS method is simple, stable and reproducible. It can be used for the quantitative analysis of serotonin and dopamine in the nervous system of B. glabrata snails.

Detection of KIT Genotype in Pigs by TaqMan MGB Real-Time Quantitative Polymerase Chain Reaction.

PubMed

Li, Xiuxiu; Li, Xiaoning; Luo, Rongrong; Wang, Wenwen; Wang, Tao; Tang, Hui

2018-05-01

The dominant white phenotype in domestic pigs is caused by two mutations in the KIT gene: a 450 kb duplication containing the entire KIT gene together with flanking sequences and one splice mutation with a G:A substitution in intron 17. The purpose of this study was to establish a simple, rapid method to determine KIT genotype in pigs. First, to detect KIT copy number variation (CNV), primers for exon 2 of the KIT gene, along with a TaqMan minor groove binder (MGB) probe, were designed. The single-copy gene, estrogen receptor (ESR), was used as an internal control. A real-time fluorescence-based quantitative PCR (FQ-PCR) protocol was developed to accurately detect KIT CNVs. Second, to detect the splice mutation ratio of the G:A substitution in intron 17, a 175 bp region, including the target mutation, was amplified from genomic DNA. Based on the sequence of the resulting amplified fragment, an MGB probe set was designed to detect the ratio of splice mutation to normal using FQ-PCR. A series of parallel amplification curves with the same internal distances were obtained using gradually diluted DNA as templates. The CT values among dilutions were significantly different (p < 0.001) and the coefficients of variation from each dilution were low (from 0.13% to 0.26%). The amplification efficiencies for KIT and ESR were approximately equal, indicating ESR was an appropriate control gene. Furthermore, use of the MGB probe set resulted in detection of the target mutation at a high resolution and stability; standard curves illustrated that the amplification efficiencies of KIT1 (G) and KIT2 (A) were approximately equal (98.8% and 97.2%). In conclusion, a simple, rapid method, with high specificity and stability, for the detection of the KIT genotype in pigs was established using TaqMan MGB probe real-time quantitative PCR.

Quantitative determination of dimethylaminoethanol in cosmetic formulations by nuclear magnetic resonance spectroscopy.

PubMed

Batista, Ivani Aparecida Soares de Andrade; Gonçalves, Maria Inês de Almeida; Singh, Anil Kumar; Hackmann, Erika Rosa Maria Kedor; Santoro, Maria Inês Rocha Miritello

2008-01-01

A nuclear magnetic resonance (NMR) spectroscopic method was validated for the quantitative determination of dimethylaminoethanol (DMAE) in cosmetic formulations. The linearity in the range from 0.5000 to 1.5000 g (DMAE salt/mass maleic acid) presents a correlation coefficient > 0.99 for all DMAE salts. The repeatability (intraday), expressed as relative standard deviation, ranged from 1.08 to 1.44% for samples and 1.31 to 1.88% for raw materials. The detection limit and quantitation limit were 0.0017 and 0.0051 g for DMAE, 0.0018 and 0.0054 g for DMAE bitartrate, and 0.0023 and 0.0071 g for DMAE acetamidobenzoate, respectively. The proposed method is simple, precise, and accurate and can be used in the quality control of raw materials and cosmetic gels containing these compounds as active substances.

Quantitative analysis of wet-heat inactivation in bovine spongiform encephalopathy

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

Matsuura, Yuichi; Ishikawa, Yukiko; Bo, Xiao

2013-03-01

Highlights: ► We quantitatively analyzed wet-heat inactivation of the BSE agent. ► Infectivity of the BSE macerate did not survive 155 °C wet-heat treatment. ► Once the sample was dehydrated, infectivity was observed even at 170 °C. ► A quantitative PMCA assay was used to evaluate the degree of BSE inactivation. - Abstract: The bovine spongiform encephalopathy (BSE) agent is resistant to conventional microbial inactivation procedures and thus threatens the safety of cattle products and by-products. To obtain information necessary to assess BSE inactivation, we performed quantitative analysis of wet-heat inactivation of infectivity in BSE-infected cattle spinal cords. Using amore » highly sensitive bioassay, we found that infectivity in BSE cattle macerates fell with increase in temperatures from 133 °C to 150 °C and was not detected in the samples subjected to temperatures above 155 °C. In dry cattle tissues, infectivity was detected even at 170 °C. Thus, BSE infectivity reduces with increase in wet-heat temperatures but is less affected when tissues are dehydrated prior to the wet-heat treatment. The results of the quantitative protein misfolding cyclic amplification assay also demonstrated that the level of the protease-resistant prion protein fell below the bioassay detection limit by wet-heat at 155 °C and higher and could help assess BSE inactivation. Our results show that BSE infectivity is strongly resistant to wet-heat inactivation and that it is necessary to pay attention to BSE decontamination in recycled cattle by-products.« less

Evaluation of quantitative image analysis criteria for the high-resolution microendoscopic detection of neoplasia in Barrett's esophagus

NASA Astrophysics Data System (ADS)

Muldoon, Timothy J.; Thekkek, Nadhi; Roblyer, Darren; Maru, Dipen; Harpaz, Noam; Potack, Jonathan; Anandasabapathy, Sharmila; Richards-Kortum, Rebecca

2010-03-01

Early detection of neoplasia in patients with Barrett's esophagus is essential to improve outcomes. The aim of this ex vivo study was to evaluate the ability of high-resolution microendoscopic imaging and quantitative image analysis to identify neoplastic lesions in patients with Barrett's esophagus. Nine patients with pathologically confirmed Barrett's esophagus underwent endoscopic examination with biopsies or endoscopic mucosal resection. Resected fresh tissue was imaged with fiber bundle microendoscopy; images were analyzed by visual interpretation or by quantitative image analysis to predict whether the imaged sites were non-neoplastic or neoplastic. The best performing pair of quantitative features were chosen based on their ability to correctly classify the data into the two groups. Predictions were compared to the gold standard of histopathology. Subjective analysis of the images by expert clinicians achieved average sensitivity and specificity of 87% and 61%, respectively. The best performing quantitative classification algorithm relied on two image textural features and achieved a sensitivity and specificity of 87% and 85%, respectively. This ex vivo pilot trial demonstrates that quantitative analysis of images obtained with a simple microendoscope system can distinguish neoplasia in Barrett's esophagus with good sensitivity and specificity when compared to histopathology and to subjective image interpretation.

CdSe/ZnS Quantum Dot-Labeled Lateral Flow Strips for Rapid and Quantitative Detection of Gastric Cancer Carbohydrate Antigen 72-4

NASA Astrophysics Data System (ADS)

Yan, Xinyu; Wang, Kan; Lu, Wenting; Qin, Weijian; Cui, Daxiang; He, Jinghua

2016-03-01

Carbohydrate antigen 72-4 (CA72-4) is an important biomarker associated closely with diagnosis and prognosis of early gastric cancer. How to realize quick, sensitive, specific, and quantitative detection of CA72-4 in clinical specimens has become a great requirement. Herein, we reported a CdSe/ZnS quantum dot-labeled lateral flow test strip combined with a charge-coupled device (CCD)-based reader was developed for rapid, sensitive, and quantitative detection of CA72-4. Two mouse monoclonal antibodies (mAbs) against CA72-4 were employed. One of them was coated as a test line, while another mAb was labeled with quantum dots and coated onto conjugate pad. The goat anti-mouse IgG was immobilized as a control line. After sample was added, a sandwich structure was formed with CA72-4 and these two mAbs. The fluorescent signal from quantum dots (QD)-labeled mAb in sandwich structure was related to the amount of detected CA72-4. A CCD-based reader was used to realize quantitative detection of CA72-4. Results showed that developed QD-labeled lateral flow strips to detect CA72-4 biomarker with the sensitivity of 2 IU/mL and 10 min detection time. One hundred sera samples from clinical patients with gastric cancer and healthy people were used to confirm specificity of this strip method; results showed that established strip method own 100 % reproducibility and 100 % specificity compared with Roche electrochemiluminescence assay results. In conclusion, CdSe/ZnS quantum dot-labeled lateral flow strips for detection of CA72-4 could realize rapid, sensitive, and specific detection of clinical samples and could own great potential in clinical translation in near future.

Diagnostic testing for pandemic influenza in Singapore: a novel dual-gene quantitative real-time RT-PCR for the detection of influenza A/H1N1/2009.

PubMed

Lee, Hong Kai; Lee, Chun Kiat; Loh, Tze Ping; Tang, Julian Wei-Tze; Chiu, Lily; Tambyah, Paul A; Sethi, Sunil K; Koay, Evelyn Siew-Chuan

2010-09-01