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Sample records for rrna-based quantitative detection

  1. Quantitative secondary electron detection

    Science.gov (United States)

    Agrawal, Jyoti; Joy, David C.; Nayak, Subuhadarshi

    2018-05-08

    Quantitative Secondary Electron Detection (QSED) using the array of solid state devices (SSD) based electron-counters enable critical dimension metrology measurements in materials such as semiconductors, nanomaterials, and biological samples (FIG. 3). Methods and devices effect a quantitative detection of secondary electrons with the array of solid state detectors comprising a number of solid state detectors. An array senses the number of secondary electrons with a plurality of solid state detectors, counting the number of secondary electrons with a time to digital converter circuit in counter mode.

  2. Quantitative multiplex detection of pathogen biomarkers

    Energy Technology Data Exchange (ETDEWEB)

    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.

  3. Quantitative multiplex detection of pathogen biomarkers

    Science.gov (United States)

    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.

  4. Limits of qualitative detection and quantitative determination

    International Nuclear Information System (INIS)

    Curie, L.A.

    1976-01-01

    The fact that one can find a series of disagreeing and limiting definitions of the detection limit leads to the reinvestigation of the problems of signal detection and signal processing in analytical and nuclear chemistry. Three cut-off levels were fixed: Lsub(C) - the net signal level (sensitivity of the equipment), above which an observed signal can be reliably recognized as 'detected'; Lsub(D) - the 'true' net signal level, from which one can a priori expect a detection; Lsub(Q) - the level at which the measuring accuracy is sufficient for quantitative determination. Exact definition equations as well as a series of working formulae are given for the general analytical case and for the investigation of radioactivity. As it is assumed that the radioactivity of the Poisson distribution is determined, it is dealt with in such a manner that precise limits can be derived for short-lived and long-lived radionuclides with or without disturbance. The fundamentals are made clear by simple examples for spectrophotometry and radioactivity and by a complicated example for activation analysis in which one must choose between alternative nuclear reactions. (orig./LH) [de

  5. Lesion detection and quantitation of positron emission mammography

    International Nuclear Information System (INIS)

    Qi, Jinyi; Huesman, Ronald H.

    2001-01-01

    A Positron Emission Mammography (PEM) scanner dedicated to breast imaging is being developed at our laboratory. We have developed a list mode likelihood reconstruction algorithm for this scanner. Here we theoretically study the lesion detection and quantitation. The lesion detectability is studied theoretically using computer observers. We found that for the zero-order quadratic prior, the region of interest observer can achieve the performance of the prewhitening observer with a properly selected smoothing parameter. We also study the lesion quantitation using the test statistic of the region of interest observer. The theoretical expressions for the bias, variance, and ensemble mean squared error of the quantitation are derived. Computer simulations show that the theoretical predictions are in good agreement with the Monte Carlo results for both lesion detection and quantitation

  6. Gold nanoparticle immunochromatographic assay for quantitative detection of urinary RBP

    Directory of Open Access Journals (Sweden)

    XU Kuan

    2013-04-01

    Full Text Available A rapid quantitative detection of urinary RBP was established by using nano-gold immunochromatography (sandwich method and trisodium citrate reduction method and a rapid immunochromatographic test strip was developed. Theimmunochromatographic test strip can quantitatively detect RBP within 15 minutes. The detection limit was 150ng/mL and detection range was from 150 to 5000 ng/mL. There were no cross-reactions with others kidney disease markers,such as urinary albumin (ALB,transferrin protein (TRF,β2-microglobulin (β2-MG,urinary fiber connecting protein (FN,and lysozyme (LZM. The results indicate that it is a quick and simple method with strong specificity,high sensitivity,and wide detection range. The rapid detection method will have extensive clinical applications in the early diagnosis of proximal tubular damage,kidney disease,diabetic nephropathy,and process monitoring.

  7. Edge detection versus densitometry for assessing coronary stenting quantitatively

    NARCIS (Netherlands)

    B.H. Strauss (Bradley); Y. Juilliere; B.J.W.M. Rensing (Benno); J.H.C. Reiber (Johan); P.W.J.C. Serruys (Patrick)

    1991-01-01

    textabstractThe optimal method used to analyze quantitatively the immediate angiographic results of coronary stenting in the coronary arteries has not been studied. Accordingly, minimal luminal cross-sectional area was determined by 2 methods, edge detection and densitometry, in 19 patients who

  8. Accounting for imperfect detection in ecology: a quantitative review.

    Science.gov (United States)

    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.

  9. Videodensitometric quantitative angiography after coronary balloon angioplasty, compared to edge-detection quantitative angiography and intracoronary ultrasound imaging

    NARCIS (Netherlands)

    Peters, R. J.; Kok, W. E.; Pasterkamp, G.; von Birgelen, C.; Prins, M. [=Martin H.; Serruys, P. W.

    2000-01-01

    AIMS: To assess the value of videodensitometric quantification of the coronary lumen after angioplasty by comparison to two other techniques of coronary artery lumen quantification. METHODS AND RESULTS: Videodensitometric quantitative angiography, edge detection quantitative angiography and 30 MHz

  10. Immunoliposome-PCR: a generic ultrasensitive quantitative antigen detection system

    Directory of Open Access Journals (Sweden)

    He Junkun

    2012-06-01

    Full Text Available Abstract 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 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 liposomes allows nonspecific DNA in the assay medium to be degraded with DNase I prior to quantification of the encapsulated reporter by PCR, which reduces false-positive results and improves quantitative accuracy. The ability to

  11. Sensitivity of Quantitative Signal Detection in Regards to Pharmacological Neuroenhancement

    Directory of Open Access Journals (Sweden)

    Maximilian Gahr

    2017-01-01

    Full Text Available Pharmacological neuroenhancement (PNE is a form of abuse and has not yet been addressed by methods of pharmacovigilance. In the present study, we tested if quantitative signal detection may be sensitive in regards to PNE. We evaluated the risk of drug abuse and dependence (DAAD related to substances that are known to be used for PNE and divided this group into agents with (methylphenidate and without a known abuse potential outside the field of PNE (atomoxetine, modafinil, acetylcholine esterase inhibitors, and memantine. Reporting odds ratios (RORs were calculated using a case/non-case approach based on global and country-specific drug safety data from the Uppsala Monitoring Centre (UMC. Both control substances (diazepam and lorazepam and methylphenidate were statistically associated with DAAD in all datasets (except methylphenidate in Italy. Modafinil was associated with DAAD in the total dataset (ROR, 2.7 (95% confidence interval (CI, 2.2–3.3, Germany (ROR, 4.6 (95% CI, 1.8–11.5, and the USA (ROR, 2.0 (95% CI, 1.6–2.5. Atomoxetine was associated with DAAD in the total dataset (ROR, 1.3 (95% CI, 1.2–1.5 and in the UK (ROR, 3.3 (95% CI, 1.8–6.1. Apart from memantine, which was associated with DAAD in Germany (ROR, 1.8 (95% CI, 1.0–3.2, no other antidementia drug was associated with DAAD. Quantitative signal detection is suitable to detect agents with a risk for DAAD. Its sensitivity regarding PNE is limited, although atomoxetine and modafinil, which do not have a known abuse potential outside PNE, and no antidementia drugs, whose use in PNE is presumably low, were associated with DAAD in our analysis.

  12. Fast detection of deletion breakpoints using quantitative PCR

    Directory of Open Access Journals (Sweden)

    Gulshara Abildinova

    2016-01-01

    Full Text Available Abstract The routine detection of large and medium copy number variants (CNVs is well established. Hemizygotic deletions or duplications in the large Duchenne muscular dystrophy DMD gene responsible for Duchenne and Becker muscular dystrophies are routinely identified using multiple ligation probe amplification and array-based comparative genomic hybridization. These methods only map deleted or duplicated exons, without providing the exact location of breakpoints. Commonly used methods for the detection of CNV breakpoints include long-range PCR and primer walking, their success being limited by the deletion size, GC content and presence of DNA repeats. Here, we present a strategy for detecting the breakpoints of medium and large CNVs regardless of their size. The hemizygous deletion of exons 45-50 in the DMD gene and the large autosomal heterozygous PARK2 deletion were used to demonstrate the workflow that relies on real-time quantitative PCR to narrow down the deletion region and Sanger sequencing for breakpoint confirmation. The strategy is fast, reliable and cost-efficient, making it amenable to widespread use in genetic laboratories.

  13. Quantitative Alpha Fetoprotein Detection with a Piezoelectric Microcantilever Mass Sensor

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Sang Kyu; Cho, Jong Yun; Jeon, Sang Min; Cha, Hyung Joon; Moon, Won Kyu [Pohang University of Science and Technology, Pohang (Korea, Republic of); Lee, Yeol Ho [Samsung Advanced Institute of Technology, Yongin (Korea, Republic of)

    2011-10-15

    Alpha fetoprotein(AFP), which is serological marker for hepatocellular carcinoma, was quantitatively measured by its normal concentration, 10 ng/ml, with a label-free piezoelectric microcantilever mass sensor. The principle of detection is based on changes in the resonant frequency of the piezoelectric microcantilever before and after target molecules are attached to it, and its resonant frequency is measured electrically using a conductance spectrum. The resonant frequency of the developed sensor is approximately 1.34 MHz and the mass sensitivity is approximately 175 Hz/pg. The sensor has high reliability as mass sensor by reducing the effect of surface stress on resonant frequency due to attached proteins. 'Dip and dry' technique was used to react the sensor with reagents for immobilizing AFP antibody on the sensor and detecting AFP antigen. The measured mass of the detected AFP antigen was 6.02 pg at the concentration of 10 ng/ml, and 10.67 pg at 50 ng/ml when the immunoreaction time was 10 min.

  14. Quantitative Alpha Fetoprotein Detection with a Piezoelectric Microcantilever Mass Sensor

    International Nuclear Information System (INIS)

    Lee, Sang Kyu; Cho, Jong Yun; Jeon, Sang Min; Cha, Hyung Joon; Moon, Won Kyu; Lee, Yeol Ho

    2011-01-01

    Alpha fetoprotein(AFP), which is serological marker for hepatocellular carcinoma, was quantitatively measured by its normal concentration, 10 ng/ml, with a label-free piezoelectric microcantilever mass sensor. The principle of detection is based on changes in the resonant frequency of the piezoelectric microcantilever before and after target molecules are attached to it, and its resonant frequency is measured electrically using a conductance spectrum. The resonant frequency of the developed sensor is approximately 1.34 MHz and the mass sensitivity is approximately 175 Hz/pg. The sensor has high reliability as mass sensor by reducing the effect of surface stress on resonant frequency due to attached proteins. 'Dip and dry' technique was used to react the sensor with reagents for immobilizing AFP antibody on the sensor and detecting AFP antigen. The measured mass of the detected AFP antigen was 6.02 pg at the concentration of 10 ng/ml, and 10.67 pg at 50 ng/ml when the immunoreaction time was 10 min

  15. On the detection of early osteoarthritis by quantitative microscopic imaging

    Science.gov (United States)

    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

  16. Quantitative detection of settled coal dust over green canopy

    Science.gov (United States)

    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

  17. Human lymphocyte polymorphisms detected by quantitative two-dimensional electrophoresis

    International Nuclear Information System (INIS)

    Goldman, D.; Merril, C.R.

    1983-01-01

    A survey of 186 soluble lymphocyte proteins for genetic polymorphism was carried out utilizing two-dimensional electrophoresis of 14 C-labeled phytohemagglutinin (PHA)-stimulated human lymphocyte proteins. Nineteen of these proteins exhibited positional variation consistent with independent genetic polymorphism in a primary sample of 28 individuals. Each of these polymorphisms was characterized by quantitative gene-dosage dependence insofar as the heterozygous phenotype expressed approximately 50% of each allelic gene product as was seen in homozygotes. Patterns observed were also identical in monozygotic twins, replicate samples, and replicate gels. The three expected phenotypes (two homozygotes and a heterozygote) were observed in each of 10 of these polymorphisms while the remaining nine had one of the homozygous classes absent. The presence of the three phenotypes, the demonstration of gene-dosage dependence, and our own and previous pedigree analysis of certain of these polymorphisms supports the genetic basis of these variants. Based on this data, the frequency of polymorphic loci for man is: P . 19/186 . .102, and the average heterozygosity is .024. This estimate is approximately 1/3 to 1/2 the rate of polymorphism previously estimated for man in other studies using one-dimensional electrophoresis of isozyme loci. The newly described polymorphisms and others which should be detectable in larger protein surveys with two-dimensional electrophoresis hold promise as genetic markers of the human genome for use in gene mapping and pedigree analyses

  18. Quantitative Digital Tomosynthesis Mammography for Improved Breast Cancer Detection and Diagnosis

    National Research Council Canada - National Science Library

    Zhang, Yiheng

    2008-01-01

    .... When fully developed, the DTM can provide radiologists improved quantitative, three-dimensional volumetric information of the breast tissue, and assist in breast cancer detection and diagnosis...

  19. Quantitative detection of pathogens in centrifugal microfluidic disks

    Science.gov (United States)

    Koh, Chung-Yan; Schaff, Ulrich Y.; Sommer, Gregory Jon

    2018-02-27

    A system and methods for detection of a nucleic acid including forming a plurality of nucleic acid detection complexes are described, each of the complexes including a nucleic acid analyte, a detection agent and a functionalized probe. The method further including binding the nucleic acid detection complexes to a plurality of functionalized particles in a fluid sample and separating the functionalized particles having the nucleic acid detection complexes bound thereto from the fluid sample using a density media. The nucleic acid analyte is detected by detecting the detection agent.

  20. Quantitative detection of settled dust over green canopy

    Science.gov (United States)

    Brook, Anna

    2016-04-01

    NMF (SS-NMF), 6) Alternating Least-Square (ALS), and 2) Lin's Projected Gradient (LPG). The performance is evaluated on real hyperspectral imagery data via detailed experimental assessment. The study showed that in certain compression tasks content-adapted sparse representation is provided by state-of-the-art solutions. The NMF algorithm estimates endmembers that are used to remove spurious information. If computationally feasible, it should include interaction terms to make the model more flexible. The optimal NMF algorithms, such as ALS and LPG, are assumed to be the simplest methods that achieve the minimum error on the test set. In summary, this work shows that sediment dust can be assessed using airborne HSI data, making it a potentially powerful tool for environmental studies. References Keshava, N., Mustard, J. (2002). Spectral unmixing. IEEE Signal Process. Mag., 19(1), 44-57. Chudnovsky, A., & Ben-Dor, E. (2009). Reflectance spectroscopy as a tool for settled dust monitoring in office environment. International Journal of Environment and Waste Management, 4(1), 32-49. Brook, A. (2014). Quantitative Detection of Settled dust over Green Canopy using Sparse Unmixing of Airborne Hyperspectral Data. IEEE-Whispers 6th Workshop on Hyperspectral Image and Signal Processing: Evolution in Remote Sensing, 2014, Switzerland, 4-8. Keshava, N., Mustard, J. (2002). Spectral unmixing. IEEE Signal Process. Mag., 19(1), 44-57. Bioucas-Dias et al. (2012). Hyperspectral unmixing overview: Geometrical, statistical, and sparse regression-based approaches, IEEE Journal of Selected Topics in Applied Earth Observations and Remote Sensing, 5(2), 354 -379.

  1. Quantitative Indicators for Behaviour Drift Detection from Home Automation Data.

    Science.gov (United States)

    Veronese, Fabio; Masciadri, Andrea; Comai, Sara; Matteucci, Matteo; Salice, Fabio

    2017-01-01

    Smart Homes diffusion provides an opportunity to implement elderly monitoring, extending seniors' independence and avoiding unnecessary assistance costs. Information concerning the inhabitant behaviour is contained in home automation data, and can be extracted by means of quantitative indicators. The application of such approach proves it can evidence behaviour changes.

  2. Quantitative detection of DNA by autocatalytic enlargement of hybridized gold nanoprobes

    DEFF Research Database (Denmark)

    Zhan, Zongrui; Cao, Cuong; Sim, Sang Jun

    2010-01-01

    Quantitative detection of specific viral DNA has become a pressing issue for the earlier clinical diagnosis of viral infectious diseases. Therefore, in this paper, we report a simple, sensitive, and inexpensive quantitative approach for DNA detection based on the autocatalytic Au deposition of go...... to the concentration of the target DNA, could easily be confirmed by a UV–vis scanning spectrophotometer. Limit of detection could be obtained as low as 1.0 fM by this simple method....

  3. Real time quantitative amplification detection on a microarray: towards high multiplex quantitative PCR.

    NARCIS (Netherlands)

    Pierik, A.; Moamfa, M; van Zelst, M.; Clout, D.; Stapert, H.; Dijksman, Johan Frederik; Broer, D.; Wimberger-Friedl, R.

    2012-01-01

    Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that

  4. Real time quantitative amplification detection on a microarray : towards high multiplex quantitative PCR

    NARCIS (Netherlands)

    Pierik, Anke; Boamfa, M.; Zelst, van M.; Clout, D.; Stapert, H.R.; Dijksman, J.F.; Broer, D.J.; Wimberger-Friedl, R.

    2012-01-01

    Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that

  5. Fluorescent chemosensor for detection and quantitation of carbon dioxide gas.

    Science.gov (United States)

    Liu, Yang; Tang, Youhong; Barashkov, Nikolay N; Irgibaeva, Irina S; Lam, Jacky W Y; Hu, Rongrong; Birimzhanova, Dinara; Yu, Yong; Tang, Ben Zhong

    2010-10-13

    CO(2) sensing is of great societal implications, as CO(2) is a component of gas mixtures from many natural and anthropogenic processes with huge impacts on globe climate and human well-being. Herein we report a CO(2) assay scheme over a wide concentration range, utilizing a fluorogen with an aggregation-induced emission feature and a liquid with tunable polarity and viscosity. The CO(2) sensing process is specific, quantitative, and interferent tolerant.

  6. Quantitative Ischemia Detection During Cardiac MR Stress Testing

    National Research Council Canada - National Science Library

    Kraitchman, D

    2001-01-01

    .... During a second ischemic episode, conventional cine wall motion images were acquired. The time from occlusion to the detection of ischemia by each MR technique, as well as ECG ischemic alterations, was determined...

  7. Quantitative Emboli Detection Using Nonlinear Ultrasound Technique, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — We propose to develop a new and innovative method for the detection and classification of emboli flowing into the brain through Carotid arteries, specifically for...

  8. Immunomagnetic nanoparticle based quantitative PCR for rapid detection of Salmonella

    International Nuclear Information System (INIS)

    Bakthavathsalam, Padmavathy; Rajendran, Vinoth Kumar; Saran, Uttara; Chatterjee, Suvro; Ali, Baquir Mohammed Jaffar

    2013-01-01

    We have developed a rapid and sensitive method for immunomagnetic separation (IMS) of Salmonella along with their real time detection via PCR. Silica-coated magnetic nanoparticles were functionalized with carboxy groups to which anti-Salmonella antibody raised against heat-inactivated whole cells of Salmonella were covalently attached. The immuno-captured target cells were detected in beverages like milk and lemon juice by multiplex PCR and real time PCR with a detection limit of 10 4 cfu.mL −1 and 10 3 cfu.mL −1 , respectively. We demonstrate that IMS can be used for selective concentration of target bacteria from beverages for subsequent use in PCR detection. PCR also enables differentiation of Salmonella typhi and Salmonella paratyphi A using a set of four specific primers. In addition, IMS—PCR can be used as a screening tool in the food and beverage industry for the detection of Salmonella within 3–4 h which compares favorably to the time of several days that is needed in case of conventional detection based on culture and biochemical methods. (author)

  9. 16S rRNA based microarray analysis of ten periodontal bacteria in patients with different forms of periodontitis.

    Science.gov (United States)

    Topcuoglu, Nursen; Kulekci, Guven

    2015-10-01

    DNA microarray analysis is a computer based technology, that a reverse capture, which targets 10 periodontal bacteria (ParoCheck) is available for rapid semi-quantitative determination. The aim of this three-year retrospective study was to display the microarray analysis results for the subgingival biofilm samples taken from patient cases diagnosed with different forms of periodontitis. A total of 84 patients with generalized aggressive periodontitis (GAP,n:29), generalized chronic periodontitis (GCP, n:25), peri-implantitis (PI,n:14), localized aggressive periodontitis (LAP,n:8) and refractory chronic periodontitis (RP,n:8) were consecutively selected from the archives of the Oral Microbiological Diagnostic Laboratory. The subgingival biofilm samples were analyzed by the microarray-based identification of 10 selected species. All the tested species were detected in the samples. The red complex bacteria were the most prevalent with very high levels in all groups. Fusobacterium nucleatum was detected in all samples at high levels. The green and blue complex bacteria were less prevalent compared with red and orange complex, except Aggregatibacter actinomycetemcomitas was detected in all LAP group. Positive correlations were found within all the red complex bacteria and between red and orange complex bacteria especially in GCP and GAP groups. Parocheck enables to monitoring of periodontal pathogens in all forms of periodontal disease and can be alternative to other guiding and reliable microbiologic tests. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Quantitative immunoassays for diagnosis and carrier detection in cystic fibrosis

    International Nuclear Information System (INIS)

    Bullock, S.; Hayward, C.; Manson, J.; Brock, D.J.H.; Raeburn, J.A.

    1982-01-01

    Quantitative immunoprecipitation and immunoradiometric assays have been developed for a protein present in the serum of cystic fibrosis homozygotes, and to a lesser extent in the serum of heterozygotes. When tested on a panel of sera from 14 cystic fibrosis patients, 29 heterozygotes and 23 controls, the immunoprecipitation assay allowed correct assignments to be made on 94% of occasions with one batch of antiserum and 95% with another. With the same panel of sera, the immunoradiometric assay allowed 94% correct assignments. It is suggested that such accuracy is the maximum that can be expected in the present state of knowledge of cystic fibrosis. (author)

  11. An electrochemical immunosensor for quantitative detection of ficolin-3

    Science.gov (United States)

    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.

  12. Small Submersible Robust Microflow Cytometer for Quantitative Detection of Phytoplankton, Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — Translume will develop an extremely robust, inexpensive micro flow cytometer (mFCM) for quantitative detection of phytoplankton. This device will be designed to be...

  13. Fluorescence quantitative PCR in detection of HBV DNA

    International Nuclear Information System (INIS)

    Shen Zheng; Li Ming; Shen Xia

    2003-01-01

    Objective: To study the relationship between the serum content of HBV-DNA and expression of serological markers with HBV infection patients. Methods: Serum samples from 375 hepatitis B patients with different clinical status and 70 normal persons were quantitated for HBV-DNA by FQ-PCR. Results: The average of HBV-DNA contents in the patient in the groups of HBsAg (+) and of HBeAg(+) were significantly higher than those in the group of HBsAg(-) and of HBeAg(-). Even in the group of HBeAg negative, high HBV-DNA contents might still be present in both the HBeAg(+) and HBeAg(-) groups. Conclusion: FQ-PCR can be used to monitor the real state of HBV infection, replication and the course of disease

  14. Quantitative detection of microscopic lithium distributions with neutrons

    Energy Technology Data Exchange (ETDEWEB)

    Neri, Giulia; Gernhaeuser, Roman; Lichtinger, Josef; Winkler, Sonja; Seiler, Dominik; Bendel, Michael [Technische Universitaet Muenchen, Physik-Department (Germany); Kunze-Liebhaeuser, Julia; Brumbarov, Jassen; Portenkirchner, Engelbert [Institut fuer Physikalische Chemie, Leopold-Franzens-Universitaet Innsbruck (Austria); Renno, Axel; Rugel, Georg [Helmholtz Zentrum Dresden Rossendorf, Helmholtz-Institut Freiberg fuer Ressourcentechnologie (Germany)

    2016-07-01

    The importance of lithium in the modern industrial society is continuously increasing. Spatially resolved detection of tritium particles from {sup 6}Li(n,α){sup 3}H nuclear reactions is used to reconstruct microscopic lithium distributions. Samples are exposed to a flux of cold neutrons. Emitted charged particles are detected with a PSD. Introducing a pinhole aperture between target and detector, the experimental setup works like a ''camera obscura'', allowing to perform spatially resolved measurements. Tritium detection analysis was successfully used to reconstruct the lithium content in self-organized TiO{sub 2-x}-C and Si/TiO{sub 2-x}-C nanotubes electrochemically lithiated, for the first time. Titanium dioxide nanotubes are a candidate for a safe anode material in lithium-ion batteries. Also lithium distributions in geological samples, so called ''pathfinder-minerals'' containing lithium, like lepidolite from a pegmatite, were analyzed. With this development we present a new precision method using nuclear physics for material science.

  15. Optimization of Quantitative PCR Methods for Enteropathogen Detection

    Science.gov (United States)

    Liu, Jie; Gratz, Jean; Amour, Caroline; Nshama, Rosemary; Walongo, Thomas; Maro, Athanasia; Mduma, Esto; Platts-Mills, James; Boisen, Nadia; Nataro, James; Haverstick, Doris M.; Kabir, Furqan; Lertsethtakarn, Paphavee; Silapong, Sasikorn; Jeamwattanalert, Pimmada; Bodhidatta, Ladaporn; Mason, Carl; Begum, Sharmin; Haque, Rashidul; Praharaj, Ira; Kang, Gagandeep; Houpt, Eric R.

    2016-01-01

    Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen’s extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease. PMID:27336160

  16. Quantitative Laughter Detection, Measurement, and Classification-A Critical Survey.

    Science.gov (United States)

    Cosentino, Sarah; Sessa, Salvatore; Takanishi, Atsuo

    2016-01-01

    The study of human nonverbal social behaviors has taken a more quantitative and computational approach in recent years due to the development of smart interfaces and virtual agents or robots able to interact socially. One of the most interesting nonverbal social behaviors, producing a characteristic vocal signal, is laughing. Laughter is produced in several different situations: in response to external physical, cognitive, or emotional stimuli; to negotiate social interactions; and also, pathologically, as a consequence of neural damage. For this reason, laughter has attracted researchers from many disciplines. A consequence of this multidisciplinarity is the absence of a holistic vision of this complex behavior: the methods of analysis and classification of laughter, as well as the terminology used, are heterogeneous; the findings sometimes contradictory and poorly documented. This survey aims at collecting and presenting objective measurement methods and results from a variety of different studies in different fields, to contribute to build a unified model and taxonomy of laughter. This could be successfully used for advances in several fields, from artificial intelligence and human-robot interaction to medicine and psychiatry.

  17. Optical aptasensors for quantitative detection of small biomolecules: a review.

    Science.gov (United States)

    Feng, Chunjing; Dai, Shuang; Wang, Lei

    2014-09-15

    Aptasensors are aptamer-based biosensors with excellent recognition capability towards a wide range of targets. Specially, there have been ever-growing interests in the development of aptasensors for the detection of small molecules. This phenomenon is contributed to two reasons. On one hand, small biomolecules play an important role in living organisms with many kinds of biological function, such as antiarrhythmic effect and vasodilator activity of adenosine. On the other hand, the concentration of small molecules can be an indicator for disease diagnosis, for example, the concentration of ATP is closely associated with cell injury and cell viability. As a potential analysis tool in the construction of aptasensors, optical analysis has attracted much more interest of researchers due to its high sensitivity, quick response and simple operation. Besides, it promises the promotion of aptasensors in performance toward a new level. Review the development of optical aptasensors for small biomolecules will give readers an overall understanding of its progress and provide some theoretical guidelines for its future development. Hence, we give a mini-review on the advance of optical aptasensors for small biomolecules. This review focuses on recent achievements in the design of various optical aptasensors for small biomolecules, containing fluorescence aptasensors, colorimetric aptasensors, chemiluminescence aptasensors and other optical aptasensors. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  19. Behavior Drift Detection Based on Anomalies Identification in Home Living Quantitative Indicators

    OpenAIRE

    Fabio Veronese; Andrea Masciadri; Sara Comai; Matteo Matteucci; Fabio Salice

    2018-01-01

    Home Automation and Smart Homes diffusion are providing an interesting opportunity to implement elderly monitoring. This is a new valid technological support to allow in-place aging of seniors by means of a detection system to notify potential anomalies. Monitoring has been implemented by means of Complex Event Processing on live streams of home automation data: this allows the analysis of the behavior of the house inhabitant through quantitative indicators. Different kinds of quantitative in...

  20. US detection and classification of hepatic disease: Comparison of quantitative algorithms with clinical readings

    International Nuclear Information System (INIS)

    Insana, M.F.; Garra, B.S.; Shawker, T.H.; Wagner, R.F.; Bradford, M.; Russell, M.A.

    1986-01-01

    A method of quantitative digital analysis of US B-scans is used to differentiate between normal and diseased liver in vivo. The tissue signature is based on five measured parameters: four describe the tissue structure and scattering properties, the fifth is the US attenuation. The patient groups studied included 31 healthy subjects, 97 patients with chronic active hepatitis, 62 with Gaucher disease, and 10 with lymphomas. Receiver operating characteristic curve analysis was used to compare the diagnostic performance of the quantitative method with the clinical reading of trained observers. The quantitative method showed greater diagnostic capability for detecting and classifying diffuse and some focal disease

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2015-01-01

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

  3. Quantitative radioautographic determination of brain tyrosine hydroxylase after direct transfer into nitro-cellulose and immunochemical detection

    International Nuclear Information System (INIS)

    Weissmann, D.; Labatut, R.; Gillon, J.Y.

    1988-01-01

    An improved quantitative immuno chemical determination of tyrosine hydroxylase brain concentrations was designed by using direct transfer into nitro-cellulose from 20 μm thick brain sections followed by immuno-detection and quantitative radioautography [fr

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

    Science.gov (United States)

    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. ...

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

    Science.gov (United States)

    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...

  6. Detection of chromosome abnormalities by quantitative fluorescent PCR in ectopic pregnancies

    NARCIS (Netherlands)

    Goddijn, Mariette; van Stralen, Marja; Schuring-Blom, Heleen; Redeker, Bert; van Leeuwen, Liesbeth; Repping, Sjoerd; Leschot, Nico; van der Veen, Fulco

    2005-01-01

    Objective: To evaluate the potential value of quantitative fluorescent polymerase chain reaction (QF-PCR) in the detection of chromosome abnormalities in ectopic pregnancies. Methods: Seventy chorionic villi samples of ectopic pregnancies were studied by QF-PCR. Primers for chromosomes 16, 21, X and

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  10. Estimation of genetic parameters and detection of quantitative trait loci for metabolites in Danish Holstein milk

    DEFF Research Database (Denmark)

    Buitenhuis, Albert Johannes; Sundekilde, Ulrik; Poulsen, Nina Aagaard

    2013-01-01

    Small components and metabolites in milk are significant for the utilization of milk, not only in dairy food production but also as disease predictors in dairy cattle. This study focused on estimation of genetic parameters and detection of quantitative trait loci for metabolites in bovine milk. F...... for lactic acid to >0.8 for orotic acid and β-hydroxybutyrate. A single SNP association analysis revealed 7 genome-wide significant quantitative trait loci [malonate: Bos taurus autosome (BTA)2 and BTA7; galactose-1-phosphate: BTA2; cis-aconitate: BTA11; urea: BTA12; carnitine: BTA25...

  11. Evaluation of quantitative sacroiliac scintigraphy for the early detection of sacroiliitis

    International Nuclear Information System (INIS)

    Prakash, S.; Malaviya, A.N.; Gopinath, P.G.; Bhargava, S.; Mehra, N.K.

    1983-01-01

    Quantitative sacroliac scintigraphy (QSS) was evaluated for the detection of sacroiliac (SI) joint disease before the appearance of radiographic/changes. QSS with fractional quantitation was done in 13 age- and sexmatched controls and 28 patients with different grades of radiographic sacroliitis. The SI index of each joint was considered separately. The mean SI index values in patients with grade I radiographic sacroiliitis (1.54) and HLA-B27 positive patients with low back pain (1.50) were significantly (P 0.05). Thus a large overlap between the normal and abnormal ranges of sacroiliac ratios limits the utility of quantitative sacroiliac scintigraphy for the early diagnosis of sacroiliac joint disease. (orig.)

  12. Immunoradiometric assay of lipid A: a test for detecting and quantitating endotoxins of various origins

    Energy Technology Data Exchange (ETDEWEB)

    Nolan, J P; Vladutiu, A O; Moreno, D M; Cohen, S A; Camara, D S [State University of New York, Buffalo (USA). School of Medicine

    1982-11-26

    The ability to measure circulating endotoxin in various disease states has been hampered by the lack of a specific and quantitative assay. The test most commonly used has been the Limulus gelation assay, which measures an enzymatic effect of endotoxin rather than the substance itself. Based on a solid-phase immunoradiometric assay previously developed to detect the specific lipopolysaccharide from Escherichia coli 026, a similar assay has been developed for the lipid A moiety of endotoxins. The assay uses rabbit antibodies to lipid A which do not react with ketodeoxyoctonate, myristic or beta-hydroxymyristic acids, and detects lipid A obtained from endotoxins of various origins after acid hydrolysis of lipopolysaccharide. Experiments in rats given exogenous endotoxin suggest that this assay can be useful for quantitation of bacterial endotoxins in serum and for studying the pathophysiology of experimental endotoxemia.

  13. Iridium-191m radionuclide angiocardiography detection and quantitation of left-to-rigth shunts

    International Nuclear Information System (INIS)

    Treves, S.; Fujii, A.; Cheng, C.; Kuruc, A.

    1983-01-01

    The purpose of this study was to determine whether Iridium-191m (Ir-191m) could replace Technetium-99m (Tc-99m) in the detection and quantitation of left-to-right shunts. It was demonstrated that Ir-191m radionuclide angiography is a safe, rapid, and accurate method for the detection and quantitation of left-to-right shunts with very low radiation dose to the patient. It is also possible with this radiotracer to evaluate other aspects of the anatomy and physiology of the circulation such as ventricular function, patency of major vessels, renal and cerebral perfusion. Further improvements on 0s-191 production, generator design and gamma cameras would expand the use of this ultrashort-lived radionuclide

  14. Comparison of qualitative and quantitative approach to prostate MR spectroscopy in peripheral zone cancer detection

    International Nuclear Information System (INIS)

    Klijn, Stijn; De Visschere, Pieter J.; De Meerleer, Gert O.; Villeirs, Geert M.

    2012-01-01

    Objective: To compare the diagnostic performance of a qualitative (pattern recognition) and a quantitative (numerical assessment) approach to magnetic resonance spectroscopy (MRS) in the diagnosis of peripheral zone prostate cancer. Methods: 185 patients (131 with histopathologically proven cancer, 54 normal/benign after at least 12 months follow-up) were prospectively evaluated with qualitative MRS using a 4-point scale between 3/2004 and 1/2008, and retrospectively reassessed using a prototype quantitative postprocessing software in April 2008. Based on pathology and follow-up data, diagnostic performance parameters were calculated. Results: The qualitative and quantitative approaches were concordant in 78.9% (146/185) of cases. The difference between the areas under the ROC curve (0.791 versus 0.772, respectively) was not statistically significant. The sensitivity, specificity and accuracy were 55.7%, 94.4% and 67.0% for the qualitative approach, and 55.0%, 83.3% and 63.2% for the quantitative approach. The sensitivity for high grade tumours (Gleason 4 + 3 or higher) was 85.2% (23/27) for both approaches. All cancers missed on either one approach separately (31/31) and 91% of cancers missed on both approaches together (23/27) were of lower grade (Gleason 3 + 4 or lower). Conclusions: Qualitative and quantitative approaches to MRS yield similar diagnostic results. Discordances in tumour detection only occurred in lower grade cancers.

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

    Science.gov (United States)

    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.

  16. Quantitative electrical detection of immobilized protein using gold nanoparticles and gold enhancement on a biochip

    International Nuclear Information System (INIS)

    Lei, Kin Fong

    2011-01-01

    Electrical detection of the concentration of protein immobilized on a biochip is demonstrated. The concentration of the direct immobilized protein can be determined by the resistance values measured by an ohm-meter directly. Indium tin oxide interdigitated electrodes were utilized as the detection sites on the biochip. Protein, i.e. antibody, of certain concentration was first immobilized on the detection site. Gold nanoparticles were then applied to indicate the immobilized protein. Since the gold nanoparticles were tiny, a detectable electrical signal could not be generated. Hence, a gold enhancement process was performed for signal amplification. Gold nanoparticles were enlarged physically, such that a conductive metal layer was formed on the detection site. The presence and concentration of protein can be determined by the resistance value across the electrode measured by an ohm-meter. An immobilized protein concentration ranging from 50 to 1000 ng ml −1 can be detected quantitatively by the resistance values from 4300 to 1700 Ω. The proposed technique is potentially extended for the detection of immunoassay on the biochip. Since the protocol of the electrical detection does not involve sophisticated equipment, it can therefore be used for the development of a portable immunoassay device

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

    Science.gov (United States)

    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.

  18. Comparison of antigen detection and quantitative PCR in the detection of chlamydial infection in koalas (Phascolarctos cinereus).

    Science.gov (United States)

    Hanger, Jon; Loader, Joanne; Wan, Charles; Beagley, Kenneth W; Timms, Peter; Polkinghorne, Adam

    2013-03-01

    The gold standard method for detecting chlamydial infection in domestic and wild animals is PCR, but the technique is not suited to testing animals in the field when a rapid diagnosis is frequently required. The objective of this study was to compare the results of a commercially available enzyme immunoassay test for Chlamydia against a quantitative Chlamydia pecorum-specific PCR performed on swabs collected from the conjunctival sac, nasal cavity and urogenital sinuses of naturally infected koalas (Phascolarctos cinereus). The level of agreement for positive results between the two assays was low (43.2%). The immunoassay detection cut-off was determined as approximately 400 C. pecorum copies, indicating that the test was sufficiently sensitive to be used for the rapid diagnosis of active chlamydial infections. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  20. Effective Heart Disease Detection Based on Quantitative Computerized Traditional Chinese Medicine Using Representation Based Classifiers

    Directory of Open Access Journals (Sweden)

    Ting Shu

    2017-01-01

    Full Text Available At present, heart disease is the number one cause of death worldwide. Traditionally, heart disease is commonly detected using blood tests, electrocardiogram, cardiac computerized tomography scan, cardiac magnetic resonance imaging, and so on. However, these traditional diagnostic methods are time consuming and/or invasive. In this paper, we propose an effective noninvasive computerized method based on facial images to quantitatively detect heart disease. Specifically, facial key block color features are extracted from facial images and analyzed using the Probabilistic Collaborative Representation Based Classifier. The idea of facial key block color analysis is founded in Traditional Chinese Medicine. A new dataset consisting of 581 heart disease and 581 healthy samples was experimented by the proposed method. In order to optimize the Probabilistic Collaborative Representation Based Classifier, an analysis of its parameters was performed. According to the experimental results, the proposed method obtains the highest accuracy compared with other classifiers and is proven to be effective at heart disease detection.

  1. Quantitative ultrasound imaging detects degenerative changes in articular cartilage surface and subchondral bone

    International Nuclear Information System (INIS)

    Saarakkala, Simo; Laasanen, Mikko S; Jurvelin, Jukka S; Toeyraes, Juha

    2006-01-01

    Previous studies have suggested that quantitative ultrasound imaging could sensitively diagnose degeneration of the articular surface and changes in the subchondral bone during the development of osteoarthrosis (OA). We have recently introduced a new parameter, ultrasound roughness index (URI), for the quantification of cartilage surface roughness, and successfully tested it with normal and experimentally degraded articular surfaces. In this in vitro study, the applicability of URI was tested in bovine cartilage samples with spontaneously developed tissue degeneration. Simultaneously, we studied the sensitivity of quantitative ultrasound imaging to detect degenerative changes in the cartilage-bone interface. For reference, histological degenerative grade of the cartilage samples was determined. Mechanical reference measurements were also conducted. Cartilage surface roughness (URI) was significantly (p < 0.05) higher in histologically degenerated samples with inferior mechanical properties. Ultrasound reflection at the cartilage-bone interface was also significantly (p < 0.05) increased in degenerated samples. Furthermore, it was quantitatively confirmed that ultrasound attenuation in the overlying cartilage significantly affects the measured ultrasound reflection values from the cartilage-bone interface. To conclude, the combined ultrasound measurement of the cartilage surface roughness and ultrasound reflection at the cartilage-bone interface complement each other, and may together enable more sensitive and quantitative diagnosis of early OA or follow up after surgical cartilage repair

  2. Suitability of two-dimensional electrophoretic protein separations for quantitative detection of mutations

    International Nuclear Information System (INIS)

    Taylor, J.; Anderson, N.L.; Anderson, N.G.; Gemmell, A.; Giometti, C.S.; Nance, S.L.; Tollaksen, S.L.

    1986-01-01

    Separation of proteins by two-dimensional electrophoresis (2DE) provides a powerful method for mutagenesis studies, since hundreds of proteins can be monitored simultaneously. In previous mutation studies in which 2DE has been used, only qualitative protein differences were monitored; quantitative protein variations were not evaluated. Although significant differences in protein abundance can be detected by eye, the large number of protein spots present in 2DE patterns together with the large number of individual patterns required for a mutagenesis study would necessitate the use of a computerized analysis system to detect the rare quantitative protein changes indicative of gene deletions or inactivation of genes by point mutations in regulatory genes. A pilot study to search for heritable mutations induced by treatment of mice with either ethylnitrosourea or gamma radiation is underway. Samples are being monitored for quantitative changes that reduce the amount of protein by about 50%. The results of this study indicate that the key methods to improve the application of 2DE to mutation screening are to increase the number of measurable spots (i.e., improve stain sensitivity) and to decrease the spread of values for the volume measurements. Even small improvements in these areas could greatly increase the number of monitorable spots. 9 refs., 4 figs

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

    Science.gov (United States)

    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.

  4. A Highly Sensitive Immunochromatographic Strip Test for Rapid and Quantitative Detection of Saikosaponin d

    Directory of Open Access Journals (Sweden)

    Yue Zhang

    2018-02-01

    Full Text Available A quantitative lateral-flow immunoassay using gold nanoparticles (AuNPs conjugated with a monoclonal antibody (MAb against saikosaponin d (SSd was developed for the analysis of SSd. The AuNPs were prepared in our laboratory. The AuNPs were polyhedral, with an average diameter of approximately 18 nm. We used the conjugation between AuNPs and MAbs against SSd to prepare immunochromatographic strips (ICSs. For the quantitative experiment, the strips with the test results were scanned using a membrane strip reader, and a detection curve (regression equation, y = −0.113ln(x + 1.5451, R2 = 0.983, representing the averages of the scanned data, was obtained. This curve was linear from 96 ng/mL to 150 μg/mL, and the IC50 value was 10.39 μg/mL. In this study, we bring the concept of POCT (point-of-care testing to the measurement of TCM compounds, and this is the first report of quantitative detection of SSd by an ICS.

  5. Rapid and quantitative detection of C-reactive protein based on quantum dots and immunofiltration assay

    Directory of Open Access Journals (Sweden)

    Zhang PF

    2015-09-01

    Full Text Available Pengfei Zhang,1,* Yan Bao,1,* Mohamed Shehata Draz,2,3,* Huiqi Lu,1 Chang Liu,1 Huanxing Han11Center for Translational Medicine, Changzheng Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 2Zhejiang-California International Nanosystems Institute, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China; 3Faculty of Science, Tanta University, Tanta, Egypt*These authors contributed equally to this workAbstract: Convenient and rapid immunofiltration assays (IFAs enable on-site “yes” or “no” determination of disease markers. However, traditional IFAs are commonly qualitative or semi-quantitative and are very limited for the efficient testing of samples in field diagnostics. Here, we overcome these limitations by developing a quantum dots (QDs-based fluorescent IFA for the quantitative detection of C-reactive proteins (CRP. CRP, the well-known diagnostic marker for acute viral and bacterial infections, was used as a model analyte to demonstrate performance and sensitivity of our developed QDs-based IFA. QDs capped with both polyethylene glycol (PEG and glutathione were used as fluorescent labels for our IFAs. The presence of the surface PEG layer, which reduced the non-specific protein interactions, in conjunction with the inherent optical properties of QDs, resulted in lower background signal, increased sensitivity, and ability to detect CRP down to 0.79 mg/L with only 5 µL serum sample. In addition, the developed assay is simple, fast and can quantitatively detect CRP with a detection limit up to 200 mg/L. Clinical test results of our QD-based IFA are well correlated with the traditional latex enhance immune-agglutination aggregation. The proposed QD-based fluorescent IFA is very promising, and potentially will be adopted for multiplexed immunoassay and in field point-of-care test.Keywords: C-reactive proteins, point-of-care test, Glutathione capped QDs, PEGylation

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  8. The level of embryonation influences detection of Ostertagia ostertagi eggs by semi-quantitative PCR

    DEFF Research Database (Denmark)

    Drag, Markus; Höglund, Johan; Nejsum, Peter

    2016-01-01

    The Internal Transcribed Spacer 2 (ITS2) is a candidate diagnostic marker of the pathogenic cattle nematode Ostertagia ostertagi. The aims of this study were: (i) to document and quantify how the development of O. ostertagi eggs affects ITS2 copies under different storage conditions, and (ii......) to suggest optimal storage conditions for faecal samples in a diagnostic pipeline that involves detection and semi-quantification by real-time semi-quantitative polymerase chain reaction (qPCR). Eggs of Ostertagia ostertagi were obtained from fresh faeces and stored at 4 °C or 25 °C under aerobic...

  9. Genome Scan Detects Quantitative Trait Loci Affecting Female Fertility Traits in Danish and Swedish Holstein Cattle

    DEFF Research Database (Denmark)

    Höglund, Johanna Karolina; Guldbrandtsen, B; Su, G

    2009-01-01

    Data from the joint Nordic breeding value prediction for Danish and Swedish Holstein grandsire families were used to locate quantitative trait loci (QTL) for female fertility traits in Danish and Swedish Holstein cattle. Up to 36 Holstein grandsires with over 2,000 sons were genotyped for 416 mic...... for QTL segregating on Bos taurus chromosome (BTA)1, BTA7, BTA10, and BTA26. On each of these chromosomes, several QTL were detected affecting more than one of the fertility traits investigated in this study. Evidence for segregation of additional QTL on BTA2, BTA9, and BTA24 was found...

  10. Quantitative Label-Free Cell Proliferation Tracking with a Versatile Electrochemical Impedance Detection Platform

    DEFF Research Database (Denmark)

    Caviglia, Claudia; Carminati, M; Heiskanen, Arto

    2012-01-01

    optimal detection strategies. Electrochemical Impedance Spectroscopy (EIS) has been used to monitor and compare adhesion of different cell lines. HeLa cells and 3T3 fibroblasts have been cultured for 12 hours on interdigitated electrode arrays integrated into a tailor-made cell culture platform. Both......Since the use of impedance measurements for label-free monitoring of cells has become widespread but still the choice of sensing configuration is not unique though crucial for a quantitative interpretation of data, we demonstrate the application of a novel custom multipotentiostat platform to study...... vertical and coplanar interdigitated sensing configuration approaches have been used and compared on the same cell populations....

  11. A Quantitative PCR Protocol for Detection of Oxyspirura petrowi in Northern Bobwhites (Colinus virginianus).

    Science.gov (United States)

    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.

  12. An effective method for the quantitative detection of porcine endogenous retrovirus in pig tissues.

    Science.gov (United States)

    Zhang, Peng; Yu, Ping; Wang, Wei; Zhang, Li; Li, Shengfu; Bu, Hong

    2010-05-01

    Xenotransplantation shows great promise for providing a virtually limitless supply of cells, tissues, and organs for a variety of therapeutical procedures. However, the potential of porcine endogenous retrovirus (PERV) as a human-tropic pathogen, particularly as a public health risk, is a major concern for xenotransplantation. This study focus on the detection of copy number in various tissues and organs in Banna Minipig Inbreed (BMI) from 2006 to 2007 in West China Hospital, Sichuan University. Real-time quantitative polymerase chain reaction (SYBR Green I) was performed in this study. The results showed that the pol gene had the most copy number in tissues compared with gag, envA, and envB. Our experiment will offer a rapid and accurate method for the detection of the copy number in various tissues and was especially suitable for the selection of tissues or organs in future clinical xenotransplantation.

  13. Quantitative detection of Fusarium spp. and its correlation with fumonisin content in maize from South African subsistence farmers

    NARCIS (Netherlands)

    Waalwijk, C.; Koch, S.H.; Ncube, E.; Allwood, J.; Flett, B.; Vries, de P.M.; Kema, G.H.J.

    2008-01-01

    A quantitative detection tool was developed to enable the monitoring of fumonisin-producing fungi in food and feed commodities. To this end, a quantitative PCR (TaqMan) was developed that targets a conserved region in the polyketide synthase gene fum1, which is involved in the biosynthesis of

  14. Rapid and quantitative detection of C-reactive protein using quantum dots and immunochromatographic test strips

    Directory of Open Access Journals (Sweden)

    Cheng X

    2014-12-01

    Full Text Available Xianglin Cheng,1,* Xu Pu,2,* Pen Jun,3 XiaoBo Zhu,3 Di Zhu,4 Ming Chen1 1Department of Laboratory Medicine, First Affiliated Hospital of Yangtze University, Jingzhou, 2Department of Laboratory Medicine, RenMin Hospital of Wuhan University, Wuhan, 3Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, Hubei, People’s Republic of China; 4Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA *These authors contributed equally to this study and share first authorship Background: Rapid immunochromatographic tests can detect disease markers in 10–15 minutes, which facilitates clinical diagnosis and treatment programs. However, most immunochromatographic tests employ gold nanoparticles as reporters, and these have only moderate sensitivity and act as qualitative methods for analyzing high biomarker concentrations. Methods: In this study, we introduce quantum dots (QDs as fluorescent probes and immunochromatographic strips to develop quantitative fluorescence point-of-care tests (QF-POCT to analyze C-reactive protein (CRP levels. Goat anti-rabbit IgG and rabbit IgG were used as control antibodies, and mouse monoclonal CRP antibody pairs were used for disease marker detection. One monoclonal CRP antibody was conjugated with QDs and served as a signal antibody, and the other monoclonal CRP antibody was dispensed onto the nitrocellulose membrane and served as a capturing antibody. In the presence of CRP, the fluorescence intensity of the monoclonal antibody-CRP-monoclonal antibody sandwich complex captured on the nitrocellulose membrane was determined using the fluorescence strip reader. Results: QF-POCT assays could quantitatively analyze the concentration of CRP in 15 minutes had a detection limit of 0.25 mg/L, and had a wide detection linearity range (0.5–300 mg/L. The intra-assay and interassay

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

    Directory of Open Access Journals (Sweden)

    Jaeyong Yee

    2015-01-01

    Full Text Available 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.

  16. Quantitative analysis of stress thallium-201 studies: comparison of SPET and planar imaging in the detection of CAD

    International Nuclear Information System (INIS)

    Ziada, G.; Hayat, N.; Abdel-Dayem, H.M.; Hassan, I.

    1986-01-01

    The value of thallium-201 tomographic sections in the detection of coronary artery disease is illustrated by comparing visual interpretation (VTS) and quantitative analysis (QTS) with visual planar study (VPS) and quantitative analysis of planar study (QPS), referring to coronary angiography (CA) as the standard technique. It is concluded that visual assessment of single photon emission tomography (VTS) is more valuable than all other techniques (VPS, QPS and QTS) for detecting and localizing coronary artery disease. (UK)

  17. Quantitative detection of nitroxyl upon trapping with glutathione and labeling with a specific fluorogenic reagent.

    Science.gov (United States)

    Johnson, Gail M; Chozinski, Tyler J; Salmon, Debra J; Moghaddam, Alan D; Chen, Hsin Chih; Miranda, Katrina M

    2013-10-01

    Donors of nitroxyl (HNO) have shown promise for treatment of stroke, heart failure, alcoholism and cancer. However, comparing the pharmacological capacities of various donors is difficult without first quantifying the amount of HNO released from each donor. Detection and quantitation of HNO has been complicated by the rapid self-consumption of HNO through irreversible dimerization, poor selectivity of trapping agents against other nitrogen oxides, and/or low sensitivity towards HNO. Here, an assay is described for the trapping of HNO by glutathione (GSH) followed by labeling of GSH with the fluorogenic agent, naphthalene-2,3-dicarboxaldehyde (NDA), and subsequent quantitation by fluorescence difference. The newly developed assay was used to validate the pH-dependence of HNO release from isopropylamine NONOate (IPA/NO), which is a dual donor of HNO and NO at physiological pH. Furthermore, varied assay conditions were utilized to suggest the ratios of the products of the reaction of GSH with HNO. At intracellular concentrations of GSH, the disulfide (GSSG) was the major product, but significant concentrations of glutathione sulfinamide (GS(O)NH₂) were also detected. This suggests that GS(O)NH₂, which is a selective biomarker of HNO, may be produced in concentrations that are amenable to in vivo analysis. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. A Room Temperature Ultrasensitive Magnetoelectric Susceptometer for Quantitative Tissue Iron Detection

    Science.gov (United States)

    Xi, Hao; Qian, Xiaoshi; Lu, Meng-Chien; Mei, Lei; Rupprecht, Sebastian; Yang, Qing X.; Zhang, Q. M.

    2016-07-01

    Iron is a trace mineral that plays a vital role in the human body. However, absorbing and accumulating excessive iron in body organs (iron overload) can damage or even destroy an organ. Even after many decades of research, progress on the development of noninvasive and low-cost tissue iron detection methods is very limited. Here we report a recent advance in a room-temperature ultrasensitive biomagnetic susceptometer for quantitative tissue iron detection. The biomagnetic susceptometer exploits recent advances in the magnetoelectric (ME) composite sensors that exhibit an ultrahigh AC magnetic sensitivity under the presence of a strong DC magnetic field. The first order gradiometer based on piezoelectric and magnetostrictive laminate (ME composite) structure shows an equivalent magnetic noise of 0.99 nT/rt Hz at 1 Hz in the presence of a DC magnetic field of 0.1 Tesla and a great common mode noise rejection ability. A prototype magnetoelectric liver susceptometry has been demonstrated with liver phantoms. The results indicate its output signals to be linearly responsive to iron concentrations from normal iron dose (0.05 mg Fe/g liver phantom) to 5 mg Fe/g liver phantom iron overload (100X overdose). The results here open up many innovative possibilities for compact-size, portable, cost-affordable, and room-temperature operated medical systems for quantitative determinations of tissue iron.

  19. Behavior Drift Detection Based on Anomalies Identification in Home Living Quantitative Indicators

    Directory of Open Access Journals (Sweden)

    Fabio Veronese

    2018-01-01

    Full Text Available Home Automation and Smart Homes diffusion are providing an interesting opportunity to implement elderly monitoring. This is a new valid technological support to allow in-place aging of seniors by means of a detection system to notify potential anomalies. Monitoring has been implemented by means of Complex Event Processing on live streams of home automation data: this allows the analysis of the behavior of the house inhabitant through quantitative indicators. Different kinds of quantitative indicators for monitoring and behavior drift detection have been identified and implemented using the Esper complex event processing engine. The chosen solution permits us not only to exploit the queries when run “online”, but enables also “offline” (re-execution for testing and a posteriori analysis. Indicators were developed on both real world data and on realistic simulations. Tests were made on a dataset of 180 days: the obtained results prove that it is possible to evidence behavior changes for an evaluation of a person’s condition.

  20. Quantitative Surface Chirality Detection with Sum Frequency Generation Vibrational Spectroscopy: Twin Polarization Angle Approach

    International Nuclear Information System (INIS)

    Wei, Feng; Xu, Yanyan; Guo, Yuan; Liu, Shi-lin; Wang, Hongfei

    2009-01-01

    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 polarization 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.

  1. Telomerase Activity Detected by Quantitative Assay in Bladder Carcinoma and Exfoliated Cells in Urine

    Directory of Open Access Journals (Sweden)

    Roberta Fedriga

    2001-01-01

    Full Text Available Early diagnosis is one of the most determining factors for patient survival. The detection of telomerase activity is a potentially promising tool in the diagnosis of bladder and other types of cancer due to the high expression of this enzyme in tumor cells. We carried out a quantitative evaluation of telomerase activity in urine samples in an attempt to determine a cut-off capable of identifying cancer patients. Telomerase activity was quantified by fluorescence TRAP assay in urine from 50 healthy volunteers and in urine and bioptic tumor samples from 56 previously untreated bladder cancer patients and expressed in arbitrary enzymatic units (AEU. Telomerase activity in urine ranged from 0 to 106 AEU (median 0 in healthy donors and from 0 to 282 AEU (median 87 in patients with cancer. A telomerase expression higher than the cut off value determined by receiver operating characteristic (ROC analysis was observed in 78% of cases, regardless of tumor grade and in 71% (15/21 of cases of nonassessable or negative cytology. The quantitative analysis of telomerase activity in urine enabled us to define cut-off values characterized by different sensitivity and specificity. Cytologic and telomerase determination, used sequentially, enabled us to detect about 90% of tumors.

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

    Science.gov (United States)

    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.

  3. Quantitative analysis of elastography images in the detection of breast cancer

    International Nuclear Information System (INIS)

    Landoni, V.; Francione, V.; Marzi, S.; Pasciuti, K.; Ferrante, F.; Saracca, E.; Pedrini, M.; Strigari, L.; Crecco, M.; Di Nallo, A.

    2012-01-01

    Purpose: The aim of this study was to develop a quantitative method for breast cancer diagnosis based on elastosonography images in order to reduce whenever possible unnecessary biopsies. The proposed method was validated by correlating the results of quantitative analysis with the diagnosis assessed by histopathologic exam. Material and methods: 109 images of breast lesions (50 benign and 59 malignant) were acquired with the traditional B-mode technique and with elastographic modality. Images in Digital Imaging and COmmunications in Medicine format (DICOM) were exported into a software, written in Visual Basic, especially developed to perform this study. The lesion was contoured and the mean grey value and softness inside the region of interest (ROI) were calculated. The correlations between variables were investigated and receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic accuracy of the proposed method. Pathologic results were used as standard reference. Results: Both the mean grey value and the softness inside the ROI resulted statistically different at the t test for the two populations of lesions (i.e., benign versus malignant): p < 0.0001. The area under the curve (AUC) was 0.924 (0.834–0.973) and 0.917 (0.826–0.970) for the mean grey value and for the softness respectively. Conclusions: Quantitative elastosonography is a promising ultrasound technique in the detection of breast cancer but large prospective trials are necessary to determine whether quantitative analysis of images can help to overcome some pitfalls of the methodic.

  4. Quantitative detection of Campylobacter jejuni on fresh chicken carcasses by real-time PCR.

    Science.gov (United States)

    Rönner, Anna-Clara; Lindmark, Hans

    2007-06-01

    Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 microl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

  5. Automatic detection and quantitative analysis of cells in the mouse primary motor cortex

    Science.gov (United States)

    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.

  6. Development of a duplex droplet digital PCR assay for absolute quantitative detection of "Candidatus Liberibacter asiaticus".

    Science.gov (United States)

    Selvaraj, Vijayanandraj; Maheshwari, Yogita; Hajeri, Subhas; Chen, Jianchi; McCollum, Thomas Greg; Yokomi, Raymond

    2018-01-01

    Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium "Candidatus Liberibacter asiaticus" (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  9. Quantitative detection of melamine based on terahertz time-domain spectroscopy

    Science.gov (United States)

    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.

  10. Quantitative detection of powdered activated carbon in wastewater treatment plant effluent by thermogravimetric analysis (TGA).

    Science.gov (United States)

    Krahnstöver, Therese; Plattner, Julia; Wintgens, Thomas

    2016-09-15

    For the elimination of potentially harmful micropollutants, powdered activated carbon (PAC) adsorption is applied in many wastewater treatment plants (WWTP). This holds the risk of PAC leakage into the WWTP effluent and desorption of contaminants into natural water bodies. In order to assess a potential PAC leakage, PAC concentrations below several mg/L have to be detected in the WWTP effluent. None of the methods that are used for water analysis today are able to differentiate between activated carbon and solid background matrix. Thus, a selective, quantitative and easily applicable method is still needed for the detection of PAC residues in wastewater. In the present study, a method was developed to quantitatively measure the PAC content in wastewater by using filtration and thermogravimetric analysis (TGA), which is a well-established technique for the distinction between different solid materials. For the sample filtration, quartz filters with a temperature stability up to 950 °C were used. This allowed for sensitive and well reproducible measurements, as the TGA was not affected by the presence of the filter. The sample's mass fractions were calculated by integrating the mass decrease rate obtained by TGA in specific, clearly identifiable peak areas. A two-step TGA heating method consisting of N2 and O2 atmospheres led to a good differentiation between PAC and biological background matrix, thanks to the reduction of peak overlapping. A linear correlation was found between a sample's PAC content and the corresponding peak areas under N2 and O2, the sample volume and the solid mass separated by filtration. Based on these findings, various wastewater samples from different WWTPs were then analyzed by TGA with regard to their PAC content. It was found that, compared to alternative techniques such as measurement of turbidity or total suspended solids, the newly developed TGA method allows for a quantitative and selective detection of PAC concentrations down to 0

  11. Damage detection using piezoelectric transducers and the Lamb wave approach: II. Robust and quantitative decision making

    International Nuclear Information System (INIS)

    Lu, Y; Wang, X; Tang, J; Ding, Y

    2008-01-01

    The propagation of Lamb waves generated by piezoelectric transducers in a one-dimensional structure has been studied comprehensively in part I of this two-paper series. Using the information embedded in the propagating waveforms, we expect to make a decision on whether damage has occurred; however, environmental and operational variances inevitably complicate the problem. To better detect the damage under these variances, we present in this paper a robust and quantitative decision-making methodology involving advanced signal processing and statistical analysis. In order to statistically evaluate the features in Lamb wave propagation in the presence of noise, we collect multiple time series (baseline signals) from the undamaged beam. A combination of the improved adaptive harmonic wavelet transform (AHWT) and the principal component analysis (PCA) is performed on the baseline signals to highlight the critical features of Lamb wave propagation in the undamaged structure. The detection of damage is facilitated by comparing the features of the test signal collected from the test structure (damaged or undamaged) with the features of the baseline signals. In this process, we employ Hotelling's T 2 statistical analysis to first purify the baseline dataset and then to quantify the deviation of the test data vector from the baseline dataset. Through experimental and numerical studies, we systematically investigate the proposed methodology in terms of the detectability (capability of detecting damage), the sensitivity (with respect to damage severity and excitation frequency) and the robustness against noises. The parametric studies also validate, from the signal processing standpoint, the guidelines of Lamb-wave-based damage detection developed in part I

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

    Science.gov (United States)

    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.

  13. Quantitative 3D-KPFM imaging with simultaneous electrostatic force and force gradient detection

    International Nuclear Information System (INIS)

    Collins, L; Rodriguez, B J; Okatan, M B; Li, Q; Kravenchenko, I I; Lavrik, N V; Kalinin, S V; Jesse, S

    2015-01-01

    Kelvin probe force microscopy (KPFM) is a powerful characterization technique for imaging local electrochemical and electrostatic potential distributions and has been applied across a broad range of materials and devices. Proper interpretation of the local KPFM data can be complicated, however, by convolution of the true surface potential under the tip with additional contributions due to long range capacitive coupling between the probe (e.g. cantilever, cone, tip apex) and the sample under test. In this work, band excitation (BE)-KPFM is used to negate such effects. In contrast to traditional single frequency KPFM, multifrequency BE-KPFM is shown to afford dual sensitivity to both the electrostatic force and the force gradient detection, analogous to simultaneous amplitude modulated and frequency modulated KPFM imaging. BE-KPFM is demonstrated on a Pt/Au/SiO x test structure and electrostatic force gradient detection is found to lead to an improved lateral resolution compared to electrostatic force detection. Finally, a 3D-KPFM imaging technique is developed. Force volume (FV) BE-KPFM allows the tip–sample distance dependence of the electrostatic interactions (force and force gradient) to be recorded at each point across the sample surface. As such, FVBE-KPFM provides a much needed pathway towards complete tip–sample capacitive de-convolution in KPFM measurements and will enable quantitative surface potential measurements with nanoscale resolution. (paper)

  14. Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology

    Directory of Open Access Journals (Sweden)

    Ravi Prakash

    2014-12-01

    Full Text Available Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR is facilitated by leveraging droplet microfluidic (DMF system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points. This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs. The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind panels of extracted patient samples with acceptably high PCR efficiency (>94%. The multi-stage qRT-PCR assays on eight panel patient samples were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction.

  15. Tools for the quantitative analysis of sedimentation boundaries detected by fluorescence optical analytical ultracentrifugation.

    Directory of Open Access Journals (Sweden)

    Huaying Zhao

    Full Text Available Fluorescence optical detection in sedimentation velocity analytical ultracentrifugation allows the study of macromolecules at nanomolar concentrations and below. This has significant promise, for example, for the study of systems of high-affinity protein interactions. Here we describe adaptations of the direct boundary modeling analysis approach implemented in the software SEDFIT that were developed to accommodate unique characteristics of the confocal fluorescence detection system. These include spatial gradients of signal intensity due to scanner movements out of the plane of rotation, temporal intensity drifts due to instability of the laser and fluorophores, and masking of the finite excitation and detection cone by the sample holder. In an extensive series of experiments with enhanced green fluorescent protein ranging from low nanomolar to low micromolar concentrations, we show that the experimental data provide sufficient information to determine the parameters required for first-order approximation of the impact of these effects on the recorded data. Systematic deviations of fluorescence optical sedimentation velocity data analyzed using conventional sedimentation models developed for absorbance and interference optics are largely removed after these adaptations, resulting in excellent fits that highlight the high precision of fluorescence sedimentation velocity data, thus allowing a more detailed quantitative interpretation of the signal boundaries that is otherwise not possible for this system.

  16. Molecular diagnosis of urinary tract infections by semi-quantitative detection of uropathogens in a routine clinical hospital setting

    NARCIS (Netherlands)

    A. van der Zee (Anneke); L.D. Roorda (Lieuwe); G. Bosman (Gerda); J.M. Ossewaarde (Jacobus)

    2016-01-01

    textabstractBackground The objective of our study was the development of a semi-quantitative real-time PCR to detect uropathogens. Two multiplex PCR reactions were designed to detect Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Proteus mirabilis, Enterococcus faecalis, and

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  19. Quantitative detection of nitric oxide in exhaled human breath by extractive electrospray ionization mass spectrometry

    Science.gov (United States)

    Pan, Susu; Tian, Yong; Li, Ming; Zhao, Jiuyan; Zhu, Lanlan; Zhang, Wei; Gu, Haiwei; Wang, Haidong; Shi, Jianbo; Fang, Xiang; Li, Penghui; Chen, Huanwen

    2015-03-01

    Exhaled nitric oxide (eNO) is a useful biomarker of various physiological conditions, including asthma and other pulmonary diseases. Herein a fast and sensitive analytical method has been developed for the quantitative detection of eNO based on extractive electrospray ionization mass spectrometry (EESI-MS). Exhaled NO molecules selectively reacted with 2-phenyl-4, 4, 5, 5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) reagent, and eNO concentration was derived based on the EESI-MS response of 1-oxyl-2-phenyl-4, 4, 5, 5-tetramethylimidazoline (PTI) product. The method allowed quantification of eNO below ppb level (~0.02 ppbv) with a relative standard deviation (RSD) of 11.6%. In addition, eNO levels of 20 volunteers were monitored by EESI-MS over the time period of 10 hrs. Long-term eNO response to smoking a cigarette was recorded, and the observed time-dependent profile was discussed. This work extends the application of EESI-MS to small molecules (mass spectrometers. Long-term quantitative profiling of eNO by EESI-MS opens new possibilities for the research of human metabolism and clinical diagnosis.

  20. Quantum dots assisted laser desorption/ionization mass spectrometric detection of carbohydrates: qualitative and quantitative analysis.

    Science.gov (United States)

    Bibi, Aisha; Ju, Huangxian

    2016-04-01

    A quantum dots (QDs) assisted laser desorption/ionization mass spectrometric (QDA-LDI-MS) strategy was proposed for qualitative and quantitative analysis of a series of carbohydrates. The adsorption of carbohydrates on the modified surface of different QDs as the matrices depended mainly on the formation of hydrogen bonding, which led to higher MS intensity than those with conventional organic matrix. The effects of QDs concentration and sample preparation method were explored for improving the selective ionization process and the detection sensitivity. The proposed approach offered a new dimension to the application of QDs as matrices for MALDI-MS research of carbohydrates. It could be used for quantitative measurement of glucose concentration in human serum with good performance. The QDs served as a matrix showed the advantages of low background, higher sensitivity, convenient sample preparation and excellent stability under vacuum. The QDs assisted LDI-MS approach has promising application to the analysis of carbohydrates in complex biological samples. Copyright © 2016 John Wiley & Sons, Ltd.

  1. Detection of Colorectal Cancer by a Quantitative Fluorescence Determination of DNA Amplification in Stool

    Directory of Open Access Journals (Sweden)

    Daniele Calistri

    2004-09-01

    Full Text Available DNA amplification of exfoliated cells in stool repre sents an inexpensive and rapid test, but has only 50% to 60% sensitivity. A new quantitative method, calle( fluorescence long DNA, was developed and validate( in our laboratory on stool obtained from 86 patient., with primary colorectal cancer and from 62 health individuals. It consists of the amplification of stoo DNA with fluorescence primers and the quantification of the amplification using a standard curve. Results are arbitrarily expressed in nanograms. The potential of thi new method compared to the conventional approact was analyzed in a subgroup of 94 individuals (51 patients and 38 healthy volunteers. In the presen series, DNA amplification analysis showed a specific ity of 97% and a sensitivity of only 50%. Conversely fluorescence DNA evaluation, using the best cutoff o 25 ng, showed a sensitivity of about 76% and a spec ificity of 93%. Similar sensitivity was observed regard less of Dukes stage, tumor location, and size, thu., also permitting the detection of early-stage tumors The present study seems to indicate that quantitative fluorescence DNA determination in stool successfully identifies colorectal cancer patients with a sensitivity comparable, if not superior, to that of multiple gene analysis but at a lower cost and in a shorter time.

  2. Monoclonal antibodies for the detection and quantitation of the endogenous plant growth regulator, abscisic acid

    Energy Technology Data Exchange (ETDEWEB)

    Mertens, R.; Weiler, E.W. (Bochum Univ. (Germany, F.R.). Lehrstuhl fuer Pflanzenphysiologie); Deus-Neumann, B. (Muenchen Univ. (Germany, F.R.). Inst. fuer pharmazeutische Biologie)

    1983-08-22

    Monoclonal antibodies (mAB) have been produced which recognize the physiologically active 2-cis-(S)-form of the endogenous plant growth regulator, abscisic acid (ABA). Cross-reaction with the ABA-catabolites, phaseic and dihydrophaseic acid, is negligible, and (R)-ABA, 2-trans-ABA, the ABA-conjugate, ABA-..beta..-D-glucopyranosyl ester, as well as the putative ABA precursor, xanthoxin, are totally unreactive. In addition to being very specific, the mAB exhibit high affinities for 2-cis-(S)-ABA; the K values were 7.9 x 10/sup 9/ l/mol and 3.7 x 10/sup 9/ l/mol for antibodies from two different clones. By mAB-radioimmunoassay (RIA), 4 pg of 2-cis-(S)-ABA (99.5% confidence level) can be detected. mAB-RIA can be used to quantitate ABA directly in unprocessed plant extracts.

  3. Monoclonal antibodies for the detection and quantitation of the endogenous plant growth regulator, abscisic acid

    International Nuclear Information System (INIS)

    Mertens, R.; Weiler, E.W.; Deus-Neumann, B.

    1983-01-01

    Monoclonal antibodies (mAB) have been produced which recognize the physiologically active 2-cis-(S)-form of the endogenous plant growth regulator, abscisic acid (ABA). Cross-reaction with the ABA-catabolites, phaseic and dihydrophaseic acid, is negligible, and (R)-ABA, 2-trans-ABA, the ABA-conjugate, ABA-β-D-glucopyranosyl ester, as well as the putative ABA precursor, xanthoxin, are totally unreactive. In addition to being very specific, the mAB exhibit high affinities for 2-cis-(S)-ABA; the K values were 7.9 x 10 9 l/mol and 3.7 x 10 9 l/mol for antibodies from two different clones. By mAB-radioimmunoassay (RIA), 4 pg of 2-cis-(S)-ABA (99.5% confidence level) can be detected. mAB-RIA can be used to quantitate ABA directly in unprocessed plant extracts. (Auth.)

  4. Detection and quantitation analysis of cocaine and metabolites in fixed liver tissue and formalin solutions.

    Science.gov (United States)

    Cingolani, Mariano; Cippitelli, Marcello; Froldi, Rino; Gambaro, Veniero; Tassoni, Giovanna

    2004-01-01

    This study reports the results of the detection and quantitation of cocaine and its metabolites in liver tissues fixed in formalin and in the formalin solutions in which the same tissues were fixed. Toxicological analyses were performed on formalin-fixed liver samples from four cases of death of cocaine abusers and on formalin solutions (10% buffered, pH 7) in which the samples were preserved. Analyses carried out at the time of autopsy on body fluids and tissues allowed identification of cocaine and the metabolite benzoylecgonine. Liver tissue samples were preserved in formalin solutions for four weeks before analysis. Results only showed the presence of benzoylecgonine in the studied materials. The mean levels of recovery of benzoylecgonine in fixed tissues were 12.31% in liver and 84.47% in formalin from liver. Results indicated that benzoylecgonine has good stability, even in biological specimens subjected to chemical fixation.

  5. Hyperspectral Imaging and SPA-LDA Quantitative Analysis for Detection of Colon Cancer Tissue

    Science.gov (United States)

    Yuan, X.; Zhang, D.; Wang, Ch.; Dai, B.; Zhao, M.; Li, B.

    2018-05-01

    Hyperspectral imaging (HSI) has been demonstrated to provide a rapid, precise, and noninvasive method for cancer detection. However, because HSI contains many data, quantitative analysis is often necessary to distill information useful for distinguishing cancerous from normal tissue. To demonstrate that HSI with our proposed algorithm can make this distinction, we built a Vis-NIR HSI setup and made many spectral images of colon tissues, and then used a successive projection algorithm (SPA) to analyze the hyperspectral image data of the tissues. This was used to build an identification model based on linear discrimination analysis (LDA) using the relative reflectance values of the effective wavelengths. Other tissues were used as a prediction set to verify the reliability of the identification model. The results suggest that Vis-NIR hyperspectral images, together with the spectroscopic classification method, provide a new approach for reliable and safe diagnosis of colon cancer and could lead to advances in cancer diagnosis generally.

  6. Quantitative Detection of Horse Contamination in Cooked Meat Products by ELISA.

    Science.gov (United States)

    Thienes, Cortlandt P; Masiri, Jongkit; Benoit, Lora A; Barrios-Lopez, Brianda; Samuel, Santosh A; Cox, David P; Dobritsa, Anatoly P; Nadala, Cesar; Samadpour, Mansour

    2018-05-01

    Concerns about the contamination of meat products with horse meat and new regulations for the declaration of meat adulterants have highlighted the need for a rapid test to detect horse meat adulteration. To address this need, Microbiologique, Inc., has developed a sandwich ELISA that can quantify the presence of horse meat down to 0.1% (w/w) in cooked pork, beef, chicken, goat, and lamb meats. This horse meat authentication ELISA has an analytical sensitivity of 0.000030 and 0.000046% (w/v) for cooked and autoclaved horse meat, respectively, and an analytical range of quantitation of 0.05-0.8% (w/v) in the absence of other meats. The assay is rapid and can be completed in 1 h and 10 min. Moreover, the assay is specific for cooked horse meat and does not demonstrate any cross-reactivity with xenogeneic cooked meat sources.

  7. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography

    International Nuclear Information System (INIS)

    Laskey, R.A.; Mills, A.D.

    1975-01-01

    Methods which use the scintillator PPO to record film images of 3 H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between redioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by micro-densitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3 H/min or 30 dis. 14 C/min can be detected in a band in a gel in a 24-h exposure. The appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use. (orig.) [de

  8. Quantitative detection of plasma-generated radicals in liquids by electron paramagnetic resonance spectroscopy

    International Nuclear Information System (INIS)

    Tresp, H; Hammer, M U; Winter, J; Reuter, S; Weltmann, K-D

    2013-01-01

    In this paper the qualitative and quantitative detection of oxygen radicals in liquids after plasma treatment with an atmospheric pressure argon plasma jet by electron paramagnetic resonance spectroscopy is investigated. Absolute values for · OH and O 2 ·- radical concentration and their net production rate in plasma-treated liquids are determined without the use of additional scavenging chemicals such as superoxide dismutase (SOD) or mannitol (D-MAN). The main oxygen-centred radical generation in PBS was found to originate from the superoxide radical. It is shown that hidden parameters such as the manufacturer of chemical components could have a big influence on the comparability and reproducibility of the results. Finally, the effect of a shielding gas device for the investigated plasma jet with a shielding gas composition of varying oxygen-to-nitrogen ratio on radical generation after plasma treatment of phosphate-buffered saline solution was investigated. (paper)

  9. A method for accurate detection of genomic microdeletions using real-time quantitative PCR

    Directory of Open Access Journals (Sweden)

    Bassett Anne S

    2005-12-01

    Full Text Available Abstract Background Quantitative Polymerase Chain Reaction (qPCR is a well-established method for quantifying levels of gene expression, but has not been routinely applied to the detection of constitutional copy number alterations of human genomic DNA. Microdeletions or microduplications of the human genome are associated with a variety of genetic disorders. Although, clinical laboratories routinely use fluorescence in situ hybridization (FISH to identify such cryptic genomic alterations, there remains a significant number of individuals in which constitutional genomic imbalance is suspected, based on clinical parameters, but cannot be readily detected using current cytogenetic techniques. Results In this study, a novel application for real-time qPCR is presented that can be used to reproducibly detect chromosomal microdeletions and microduplications. This approach was applied to DNA from a series of patient samples and controls to validate genomic copy number alteration at cytoband 22q11. The study group comprised 12 patients with clinical symptoms of chromosome 22q11 deletion syndrome (22q11DS, 1 patient trisomic for 22q11 and 4 normal controls. 6 of the patients (group 1 had known hemizygous deletions, as detected by standard diagnostic FISH, whilst the remaining 6 patients (group 2 were classified as 22q11DS negative using the clinical FISH assay. Screening of the patients and controls with a set of 10 real time qPCR primers, spanning the 22q11.2-deleted region and flanking sequence, confirmed the FISH assay results for all patients with 100% concordance. Moreover, this qPCR enabled a refinement of the region of deletion at 22q11. Analysis of DNA from chromosome 22 trisomic sample demonstrated genomic duplication within 22q11. Conclusion In this paper we present a qPCR approach for the detection of chromosomal microdeletions and microduplications. The strategic use of in silico modelling for qPCR primer design to avoid regions of repetitive

  10. Dual core quantum dots for highly quantitative ratiometric detection of trypsin activity in cystic fibrosis patients

    Science.gov (United States)

    Castelló Serrano, Iván; Stoica, Georgiana; Matas Adams, Alba; Palomares, Emilio

    2014-10-01

    We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for recessive genetic diseases like human cystic fibrosis. In a screening program in which the goal is to detect disease and also the carrier status, early diagnosis could be of great help.We present herein two colour encoded silica nanospheres (2nanoSi) for the fluorescence quantitative ratiometric determination of trypsin in humans. Current detection methods for cystic fibrosis diagnosis are slow, costly and suffer from false positives. The 2nanoSi proved to be a highly sensitive, fast (minutes), and single-step approach nanosensor for the screening and diagnosis of cystic fibrosis, allowing the quantification of trypsin concentrations in a wide range relevant for clinical applications (25-350 μg L-1). Furthermore, as trypsin is directly related to the development of cystic fibrosis (CF), different human genotypes, i.e. CF homozygotic, CF heterozygotic, and unaffected, respectively, can be determined using our 2nanoSi nanospheres. We anticipate the 2nanoSi system to be a starting point for non-invasive, easy-to-use and cost effective ratiometric fluorescent biomarkers for

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Speicher David J

    2012-09-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusions 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

  14. Quantitative Assessment of Detection Frequency for the INL Ambient Air Monitoring Network

    Energy Technology Data Exchange (ETDEWEB)

    Sondrup, A. Jeffrey [Idaho National Lab. (INL), Idaho Falls, ID (United States); Rood, Arthur S. [Idaho National Lab. (INL), Idaho Falls, ID (United States)

    2014-11-01

    A quantitative assessment of the Idaho National Laboratory (INL) air monitoring network was performed using frequency of detection as the performance metric. The INL air monitoring network consists of 37 low-volume air samplers in 31 different locations. Twenty of the samplers are located on INL (onsite) and 17 are located off INL (offsite). Detection frequencies were calculated using both BEA and ESER laboratory minimum detectable activity (MDA) levels. The CALPUFF Lagrangian puff dispersion model, coupled with 1 year of meteorological data, was used to calculate time-integrated concentrations at sampler locations for a 1-hour release of unit activity (1 Ci) for every hour of the year. The unit-activity time-integrated concentration (TICu) values were calculated at all samplers for releases from eight INL facilities. The TICu values were then scaled and integrated for a given release quantity and release duration. All facilities modeled a ground-level release emanating either from the center of the facility or at a point where significant emissions are possible. In addition to ground-level releases, three existing stacks at the Advanced Test Reactor Complex, Idaho Nuclear Technology and Engineering Center, and Material and Fuels Complex were also modeled. Meteorological data from the 35 stations comprising the INL Mesonet network, data from the Idaho Falls Regional airport, upper air data from the Boise airport, and three-dimensional gridded data from the weather research forecasting model were used for modeling. Three representative radionuclides identified as key radionuclides in INL’s annual National Emission Standards for Hazardous Air Pollutants evaluations were considered for the frequency of detection analysis: Cs-137 (beta-gamma emitter), Pu-239 (alpha emitter), and Sr-90 (beta emitter). Source-specific release quantities were calculated for each radionuclide, such that the maximum inhalation dose at any publicly accessible sampler or the National

  15. Detection of Quantitative Trait Loci Affecting Fat Deposition Traits in Pigs

    Directory of Open Access Journals (Sweden)

    B. H. Choi

    2012-11-01

    Full Text Available Quantitative trait loci (QTL associated with fat deposition traits in pigs are important gene positions in a chromosome that influence meat quality of pork. For QTL study, a three generation resource population was constructed from a cross between Korean native boars and Landrace sows. A total of 240 F2 animals from intercross of F1 were produced. 80 microsatellite markers covering chromosomes 1 to 10 were selected to genotype the resource population. Intervals between adjacent markers were approximately 19 cM. Linkage analysis was performed using CRIMAP software version 2.4 with a FIXED option to obtain the map distances. For QTL analysis, the public web-based software, QTL express (http://www.qtl.cap.ed.ac.uk was used. Two significant and two suggestive QTL were identified on SSC 6, 7, and 8 as affecting body fat and IMF traits. For QTL affecting IMF, the most significant association was detected between marker sw71 and sw1881 on SSC 6, and a suggestive QTL was identified between sw268 and sw205 on SSC8. These QTL accounted for 26.58% and 12.31% of the phenotypic variance, respectively. A significant QTL affecting IMF was detected at position 105 cM between markers sw71 and sw1881 on SSC 6.

  16. Highly sensitive quantitative PCR for the detection and differentiation of Pseudogymnoascus destructans and other Pseudogymnoascus species.

    Science.gov (United States)

    Shuey, Megan M; Drees, Kevin P; Lindner, Daniel L; Keim, Paul; Foster, Jeffrey T

    2014-03-01

    White-nose syndrome is a fungal disease that has decimated bat populations across eastern North America. Identification of the etiologic agent, Pseudogymnoascus destructans (formerly Geomyces destructans), in environmental samples is essential to proposed management plans. A major challenge is the presence of closely related species, which are ubiquitous in many soils and cave sediments and often present in high abundance. We present a dual-probe real-time quantitative PCR assay capable of detecting and differentiating P. destructans from closely related fungi in environmental samples from North America. The assay, based on a single nucleotide polymorphism (SNP) specific to P. destructans, is capable of rapid low-level detection from various sampling media, including sediment, fecal samples, wing biopsy specimens, and skin swabs. This method is a highly sensitive, high-throughput method for identifying P. destructans, other Pseudogymnoascus spp., and Geomyces spp. in the environment, providing a fundamental component of research and risk assessment for addressing this disease, as well as other ecological and mycological work on related fungi.

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

    Directory of Open Access Journals (Sweden)

    Zhonglin Tang

    2010-06-01

    Full Text Available 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, diisopropyl-amine, 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.

  18. Addition of Adapted Optics towards obtaining a quantitative detection of diabetic retinopathy

    Science.gov (United States)

    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.

  19. Toward improved peptide feature detection in quantitative proteomics using stable isotope labeling.

    Science.gov (United States)

    Nilse, Lars; Sigloch, Florian Christoph; Biniossek, Martin L; Schilling, Oliver

    2015-08-01

    Reliable detection of peptides in LC-MS data is a key algorithmic step in the analysis of quantitative proteomics experiments. While highly abundant peptides can be detected reliably by most modern software tools, there is much less agreement on medium and low-intensity peptides in a sample. The choice of software tools can have a big impact on the quantification of proteins, especially for proteins that appear in lower concentrations. However, in many experiments, it is precisely this region of less abundant but substantially regulated proteins that holds the biggest potential for discoveries. This is particularly true for discovery proteomics in the pharmacological sector with a specific interest in key regulatory proteins. In this viewpoint article, we discuss how the development of novel software algorithms allows us to study this region of the proteome with increased confidence. Reliable results are one of many aspects to be considered when deciding on a bioinformatics software platform. Deployment into existing IT infrastructures, compatibility with other software packages, scalability, automation, flexibility, and support need to be considered and are briefly addressed in this viewpoint article. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. Quantitative prediction of perceptual decisions during near-threshold fear detection

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Detection and Analysis of Enamel Cracks by Quantitative Light-induced Fluorescence Technology.

    Science.gov (United States)

    Jun, Mi-Kyoung; Ku, Hye-Min; Kim, Euiseong; Kim, Hee-Eun; Kwon, Ho-Keun; Kim, Baek-Il

    2016-03-01

    The ability to accurately detect tooth cracks and quantify their depth would allow the prediction of crack progression and treatment success. The aim of this in vitro study was to determine the capabilities of quantitative light-induced fluorescence (QLF) technology in the detection of enamel cracks. Ninety-six extracted human teeth were selected for examining naturally existing or suspected cracked teeth surfaces using a photocuring unit. QLF performed with a digital camera (QLF-D) images were used to assess the ability to detect enamel cracks based on the maximum fluorescence loss value (ΔFmax, %), which was then analyzed using the QLF-D software. A histologic evaluation was then performed in which the samples were sectioned and observed with the aid of a polarized light microscope. The relationship between ΔFmax and the histology findings was assessed based on the Spearman rank correlation. The sensitivity and specificity were calculated to evaluate the validity of using QLF-D to analyze enamel inner-half cracks and cracks extending to the dentin-enamel junction. There was a strong correlation between the results of histologic evaluations of enamel cracks and the ΔFmax value, with a correlation coefficient of 0.84. The diagnostic accuracy of QLF-D had a sensitivity of 0.87 and a specificity of 0.98 for enamel inner-half cracks and a sensitivity of 0.90 and a specificity of 1.0 for cracks extending to the dentin-enamel junction. These results indicate that QLF technology would be a useful clinical tool for diagnosing enamel cracks, especially given that this is a nondestructive method. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  5. A SVM-based quantitative fMRI method for resting-state functional network detection.

    Science.gov (United States)

    Song, Xiaomu; Chen, Nan-kuei

    2014-09-01

    Resting-state functional magnetic resonance imaging (fMRI) aims to measure baseline neuronal connectivity independent of specific functional tasks and to capture changes in the connectivity due to neurological diseases. Most existing network detection methods rely on a fixed threshold to identify functionally connected voxels under the resting state. Due to fMRI non-stationarity, the threshold cannot adapt to variation of data characteristics across sessions and subjects, and generates unreliable mapping results. In this study, a new method is presented for resting-state fMRI data analysis. Specifically, the resting-state network mapping is formulated as an outlier detection process that is implemented using one-class support vector machine (SVM). The results are refined by using a spatial-feature domain prototype selection method and two-class SVM reclassification. The final decision on each voxel is made by comparing its probabilities of functionally connected and unconnected instead of a threshold. Multiple features for resting-state analysis were extracted and examined using an SVM-based feature selection method, and the most representative features were identified. The proposed method was evaluated using synthetic and experimental fMRI data. A comparison study was also performed with independent component analysis (ICA) and correlation analysis. The experimental results show that the proposed method can provide comparable or better network detection performance than ICA and correlation analysis. The method is potentially applicable to various resting-state quantitative fMRI studies. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  7. Hypersensitive detection and quantitation of BoNT/A by IgY antibody against substrate linear-peptide.

    Directory of Open Access Journals (Sweden)

    Tao Li

    Full Text Available Botulinum neurotoxin A (BoNT/A, the most acutely poisonous substance to humans known, cleave its SNAP-25 substrate with high specificity. Based on the endopeptidase activity, different methods have been developed to detect BoNT/A, but most lack ideal reproducibility or sensitivity, or suffer from long-term or unwanted interferences. In this study, we developed a simple method to detect and quantitate trace amounts of botulinum neurotoxin A using the IgY antibody against a linear-peptide substrate. The effects of reaction buffer, time, and temperature were analyzed and optimized. When the optimized assay was used to detect BoNT/A, the limit of detection of the assay was 0.01 mouse LD50 (0.04 pg, and the limit of quantitation was 0.12 mouse LD50/ml (0.48 pg. The findings also showed favorable specificity of detecting BoNT/A. When used to detect BoNT/A in milk or human serum, the proposed assay exhibited good quantitative accuracy (88% < recovery < 111%; inter- and intra-assay CVs < 18%. This method of detection took less than 3 h to complete, indicating that it can be a valuable method of detecting BoNT/A in food or clinical diagnosis.

  8. Quantitative Molecular Detection of Putative Periodontal Pathogens in Clinically Healthy and Periodontally Diseased Subjects

    Science.gov (United States)

    Göhler, André; Hetzer, Adrian; Holtfreter, Birte; Geisel, Marie Henrike; Schmidt, Carsten Oliver; Steinmetz, Ivo; Kocher, Thomas

    2014-01-01

    Periodontitis is a multi-microbial oral infection with high prevalence among adults. Putative oral pathogens are commonly found in periodontally diseased individuals. However, these organisms can be also detected in the oral cavity of healthy subjects. This leads to the hypothesis, that alterations in the proportion of these organisms relative to the total amount of oral microorganisms, namely their abundance, rather than their simple presence might be important in the transition from health to disease. Therefore, we developed a quantitative molecular method to determine the abundance of various oral microorganisms and the portion of bacterial and archaeal nucleic acid relative to the total nucleic acid extracted from individual samples. We applied quantitative real-time PCRs targeting single-copy genes of periodontal bacteria and 16S-rRNA genes of Bacteria and Archaea. Testing tongue scrapings of 88 matched pairs of periodontally diseased and healthy subjects revealed a significantly higher abundance of P. gingivalis and a higher total bacterial abundance in diseased subjects. In fully adjusted models the risk of being periodontally diseased was significantly higher in subjects with high P. gingivalis and total bacterial abundance. Interestingly, we found that moderate abundances of A. actinomycetemcomitans were associated with reduced risk for periodontal disease compared to subjects with low abundances, whereas for high abundances, this protective effect leveled off. Moderate archaeal abundances were health associated compared to subjects with low abundances. In conclusion, our methodological approach unraveled associations of the oral flora with periodontal disease, which would have gone undetected if only qualitative data had been determined. PMID:25029268

  9. Sandwich ELISA for quantitative detection of human collagen prolyl 4-hydroxylase

    Directory of Open Access Journals (Sweden)

    Myllyharju Johanna

    2010-06-01

    Full Text Available Abstract Background We describe a method for specific, quantitative and quick detection of human collagen prolyl 4-hydroxylase (C-P4H, the key enzyme for collagen prolyl-4 hydroxylation, in crude samples based on a sandwich ELISA principle. The method is relevant to active C-P4H level monitoring during recombinant C-P4H and collagen production in different expression systems. The assay proves to be specific for the active C-P4H α2β2 tetramer due to the use of antibodies against its both subunits. Thus in keeping with the method C-P4H is captured by coupled to an anti-α subunit antibody magnetic beads and an anti-β subunit antibody binds to the PDI/β subunit of the protein. Then the following holoenzyme detection is accomplished by a goat anti-rabbit IgG labeled with alkaline phosphatase which AP catalyzes the reaction of a substrate transformation with fluorescent signal generation. Results We applied an experimental design approach for the optimization of the antibody concentrations used in the sandwich ELISA. The assay sensitivity was 0.1 ng of C-P4H. The method was utilized for the analysis of C-P4H accumulation in crude cell extracts of E. coli overexpressing C-P4H. The sandwich ELISA signals obtained demonstrated a very good correlation with the detected protein activity levels measured with the standard radioactive assay. The developed assay was applied to optimize C-P4H production in E. coli Origami in a system where the C-P4H subunits expression acted under control by different promoters. The experiments performed in a shake flask fed-batch system (EnBase® verified earlier observations that cell density and oxygen supply are critical factors for the use of the inducer anhydrotetracycline and thus for the soluble C-P4H yield. Conclusions Here we show an example of sandwich ELISA usage for quantifying multimeric proteins. The method was developed for monitoring the amount of recombinant C-P4H tetramer in crude E. coli extracts. Due

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

    Science.gov (United States)

    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.

  11. Droplet Microfluidics Platform for Highly Sensitive and Quantitative Detection of Malaria-Causing Plasmodium Parasites Based on Enzyme Activity Measurement

    DEFF Research Database (Denmark)

    Juul, Sissel; Nielsen, Christine Juul Fælled; Labouriau, Rodrigo

    2012-01-01

    detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite....../μL. Moreover, the setup allowed for detection of Plasmodium parasites in noninvasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide, and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate...

  12. DETECTION OF MALNUTRITION IN PATIENTS UNDERGOING MAINTENANCE HAEMODIALYSIS: A QUANTITATIVE DATA ANALYSIS ON 12 PARAMETERS.

    Science.gov (United States)

    Nafzger, Sonja; Fleury, Lea-Angelica; Uehlinger, Dominik E; Plüss, Petra; Scura, Ninetta; Kurmann, Silvia

    2015-09-01

    Protein-energy-malnutrition (PEM) is common in people with end stage kidney disease (ESKD) undergoing maintenance haemodialysis (MHD) and correlates strongly with mortality. To this day, there is no gold standard for detecting PEM in patients on MHD. The aim of this study was to evaluate if Nutritional Risk Screening 2002 (NRS-2002), handgrip strength measurement, mid-upper arm muscle area (MUAMA), triceps skin fold measurement (TSF), serum albumin, normalised protein catabolic rate (nPCR), Kt/V and eKt/V, dry body weight, body mass index (BMI), age and time since start on MHD are relevant for assessing PEM in patients on MHD. The predictive value of the selected parameters on mortality and mortality or weight loss of more than 5% was assessed. Quantitative data analysis of the 12 parameters in the same patients on MHD in autumn 2009 (n = 64) and spring 2011 (n = 40) with paired statistical analysis and multivariate logistic regression analysis was performed. Paired data analysis showed significant reduction of dry body weight, BMI and nPCR. Kt/Vtot did not change, eKt/v and hand grip strength measurements were significantly higher in spring 2011. No changes were detected in TSF, serum albumin, NRS-2002 and MUAMA. Serum albumin was shown to be the only predictor of death and of the combined endpoint "death or weight loss of more than 5%". We now screen patients biannually for serum albumin, nPCR, Kt/V, handgrip measurement of the shunt-free arm, dry body weight, age and time since initiation of MHD. © 2015 European Dialysis and Transplant Nurses Association/European Renal Care Association.

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

    Science.gov (United States)

    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 (malachite green in various aqueous media, indicating its potential for online real-time monitoring of real life samples.

  14. Network-based group variable selection for detecting expression quantitative trait loci (eQTL

    Directory of Open Access Journals (Sweden)

    Zhang Xuegong

    2011-06-01

    Full Text Available Abstract Background Analysis of expression quantitative trait loci (eQTL aims to identify the genetic loci associated with the expression level of genes. Penalized regression with a proper penalty is suitable for the high-dimensional biological data. Its performance should be enhanced when we incorporate biological knowledge of gene expression network and linkage disequilibrium (LD structure between loci in high-noise background. Results We propose a network-based group variable selection (NGVS method for QTL detection. Our method simultaneously maps highly correlated expression traits sharing the same biological function to marker sets formed by LD. By grouping markers, complex joint activity of multiple SNPs can be considered and the dimensionality of eQTL problem is reduced dramatically. In order to demonstrate the power and flexibility of our method, we used it to analyze two simulations and a mouse obesity and diabetes dataset. We considered the gene co-expression network, grouped markers into marker sets and treated the additive and dominant effect of each locus as a group: as a consequence, we were able to replicate results previously obtained on the mouse linkage dataset. Furthermore, we observed several possible sex-dependent loci and interactions of multiple SNPs. Conclusions The proposed NGVS method is appropriate for problems with high-dimensional data and high-noise background. On eQTL problem it outperforms the classical Lasso method, which does not consider biological knowledge. Introduction of proper gene expression and loci correlation information makes detecting causal markers more accurate. With reasonable model settings, NGVS can lead to novel biological findings.

  15. Lung nodule detection in pediatric chest CT: quantitative relationship between image quality and radiologist performance.

    Science.gov (United States)

    Li, Xiang; Samei, Ehsan; Barnhart, Huiman X; Gaca, Ana Maria; Hollingsworth, Caroline L; Maxfield, Charles M; Carrico, Caroline W T; Colsher, James G; Frush, Donald P

    2011-05-01

    To determine the quantitative relationship between image quality and radiologist performance in detecting small lung nodules in pediatric CT. The study included clinical chest CT images of 30 pediatric patients (0-16 years) scanned at tube currents of 55-180 mA. Calibrated noise addition software was used to simulate cases at three nominal mA settings: 70, 35, and 17.5 mA, resulting in quantum noise of 7-32 Hounsfield Unit (HU). Using a validated nodule simulation technique, lung nodules with diameters of 3-5 mm and peak contrasts of 200-500 HU were inserted into the cases, which were then randomized and rated independently by four experienced pediatric radiologists for nodule presence on a continuous scale from 0 (definitely absent) to 100 (definitely present). The receiver operating characteristic (ROC) data were analyzed to quantify the relationship between diagnostic accuracy (area under the ROC curve, AUC) and image quality (the product of nodule peak contrast and displayed diameter to noise ratio, CDNR display). AUC increased rapidly from 0.70 to 0.87 when CDNR display increased from 60 to 130 mm, followed by a slow increase to 0.94 when CDNR display further increased to 257 mm. For the average nodule diameter (4 mm) and contrast (350 HU), AUC decreased from 0.93 to 0.71 with noise increased from 7 to 28 HU. We quantified the relationship between image quality and the performance of radiologists in detecting lung nodules in pediatric CT. The relationship can guide CT protocol design to achieve the desired diagnostic performance at the lowest radiation dose.

  16. Detection of Ophiocordyceps sinensis in soil by quantitative real-time PCR.

    Science.gov (United States)

    Peng, Qingyun; Zhong, Xin; Lei, Wei; Zhang, Guren; Liu, Xin

    2013-03-01

    Ophiocordyceps sinensis, one of the best known entomopathogenic fungi in traditional Chinese medicine, parasitizes larvae of the moth genus Thitarodes, which lives in soil tunnels. However, little is known about the spatial distribution of O. sinensis in the soil. We established a protocol for DNA extraction, purification, and quantification of O. sinensis in soil with quantitative real-time PCR targeting the internal transcribed spacer region. The method was assessed using 34 soil samples from Tibet. No inhibitory effects in purified soil DNA extracts were detected. The standard curve method for absolute DNA quantification generated crossing point values that were strongly and linearly correlated to the log10 of the initial amount of O. sinensis genomic DNA (r(2) = 0.999) over 7 orders of magnitude (4 × 10(1) to 4 × 10(7) fg). The amplification efficiency and y-intercept value of the standard curve were 1.953 and 37.70, respectively. The amount of O. sinensis genomic DNA decreased with increasing soil depth and horizontal distance from a sclerotium (P protocol is rapid, specific, sensitive, and provides a powerful tool for quantification of O. sinensis from soil.

  17. Rapid detection of Pseudomonas aeruginosa from positive blood cultures by quantitative PCR

    Directory of Open Access Journals (Sweden)

    Cattoir Vincent

    2010-08-01

    Full Text Available Abstract Background Pseudomonas aeruginosa is responsible for numerous bloodstream infections associated with severe adverse outcomes in case of inappropriate initial antimicrobial therapy. The present study was aimed to develop a novel quantitative PCR (qPCR assay, using ecfX as the specific target gene, for the rapid and accurate identification of P. aeruginosa from positive blood cultures (BCs. Methods Over the period August 2008 to June 2009, 100 BC bottles positive for gram-negative bacilli were tested in order to evaluate performances of the qPCR technique with conventional methods as gold standard (i.e. culture and phenotypic identification. Results Thirty-three strains of P. aeruginosa, 53 strains of Enterobactericaeae, nine strains of Stenotrophomonas maltophilia and two other gram-negative species were isolated while 3 BCs were polymicrobial including one mixture containing P. aeruginosa. All P. aeruginosa clinical isolates were detected by qPCR except a single strain in mixed culture. Performances of the qPCR technique were: specificity, 100%; positive predictive value, 100%; negative predictive value, 98.5%; and sensitivity, 97%. Conclusions This reliable technique may offer a rapid (

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

    Science.gov (United States)

    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.

  19. Detection and quantification of Plasmodium falciparum in blood samples using quantitative nucleic acid sequence-based amplification

    NARCIS (Netherlands)

    Schoone, G. J.; Oskam, L.; Kroon, N. C.; Schallig, H. D.; Omar, S. A.

    2000-01-01

    A quantitative nucleic acid sequence-based amplification (QT-NASBA) assay for the detection of Plasmodium parasites has been developed. Primers and probes were selected on the basis of the sequence of the small-subunit rRNA gene. Quantification was achieved by coamplification of the RNA in the

  20. Quantitative PCR Assays for Detecting Loach Minnow (Rhinichthys cobitis and Spikedace (Meda fulgida in the Southwestern United States.

    Directory of Open Access Journals (Sweden)

    Joseph C Dysthe

    Full Text Available Loach minnow (Rhinichthys cobitis and spikedace (Meda fulgida are legally protected with the status of Endangered under the U.S. Endangered Species Act and are endemic to the Gila River basin of Arizona and New Mexico. Efficient and sensitive methods for monitoring these species' distributions are critical for prioritizing conservation efforts. We developed quantitative PCR assays for detecting loach minnow and spikedace DNA in environmental samples. Each assay reliably detected low concentrations of target DNA without detection of non-target species, including other cyprinid fishes with which they co-occur.

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

    Science.gov (United States)

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

    2014-03-01

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

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

    Science.gov (United States)

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

    2016-03-18

    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.

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

    Directory of Open Access Journals (Sweden)

    Pengyu Zhu

    2016-03-01

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

  4. Applications in Bioastronautics and Bioinformatics: Early Radiation Cataracts Detected by Noninvasive, Quantitative, and Remote Means

    Science.gov (United States)

    Ansari, Rafat R.; King, James F.; Giblin, Frank J.

    2000-01-01

    (transparent) to several micrometers (cloudy). Ansari and Datiles have shown that DLS can detect cataracts at least two to three orders of magnitude earlier noninvasively and quantitatively than the best imaging (Scheimpflug) techniques in clinical use today (ref. 3).

  5. Simultaneous quantitative detection of multiple tumor markers with a rapid and sensitive multicolor quantum dots based immunochromatographic test strip.

    Science.gov (United States)

    Wang, Chunying; Hou, Fei; Ma, Yicai

    2015-06-15

    A novel multicolor quantum dots (QDs) based immunochromatographic test strip (ICTS) was developed for simultaneous quantitative detection of multiple tumor markers, by utilizing alpha fetoprotein (AFP) and carcinoembryonic antigen (CEA) as models. The immunosensor could realize simultaneous quantitative detection of tumor markers with only one test line and one control line on the nitrocellulose membrane (NC membrane) due to the introduction of multicolor QDs. In this method, a mixture of mouse anti-AFP McAb and mouse anti-CEA McAb was coated on NC membrane as test line and goat anti-mouse IgG antibody was coated as control line. Anti-AFP McAb-QDs546 conjugates and anti-CEA McAb-QDs620 conjugates were mixed and applied to the conjugate pad. Simultaneous quantitative detection of multiple tumor markers was achieved by detecting the fluorescence intensity of captured QDs labels on test line and control line using a test strip reader. Under the optimum conditions, AFP and CEA could be detected as low as 3 ng/mL and 2 ng/mL in 15 min with a sample volume of 80 μL, and no obvious cross-reactivity was observed. The immunosensor was validated with 130 clinical samples and in which it exhibited high sensitivity (93% for AFP and 87% for CEA) and specificity (94% for AFP and 97% for CEA). The immunosensor also demonstrated high recoveries (87.5-113% for AFP and 90-97.3% for CEA) and low relative standard deviations (RSDs) (2.8-6.2% for AFP and 4.9-9.6% for CEA) when testing spiked human serum. This novel multicolor QDs based ICTS provides an easy and rapid, simultaneous quantitative detecting strategy for point-of-care testing of tumor markers. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  7. Quantitative multiplex assay for simultaneous detection and identification of Indiana and New Jersey serotypes of vesicular stomatitis virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Fernandez, Jovita

    2005-01-01

    In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed......-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating...... probes. The limits of detection ware found to be less than 10 50% tissue culture infective doses/ml for both serotypes. The diagnostic value of the new method was tested with clinical materials from experimentally infected pigs, and it is concluded that the method is a powerful tool for the rapid...

  8. A novel technique with enhanced detection and quantitation of HPV-16 E1- and E2-mediated DNA replication

    International Nuclear Information System (INIS)

    Taylor, Ewan R.; Morgan, Iain M.

    2003-01-01

    Transient DNA replication assays to detect papillomavirus E1/E2-mediated DNA replication have depended upon Southern blotting. This technique is hazardous (radioactive), labour intensive, semiquantitative, and physically limited in the number of samples that can be processed at any one time. We have overcome these problems by developing a real-time PCR protocol for the detection of E1/E2-mediated transient DNA replication. The results demonstrate detection of replication at levels not seen using Southern blotting demonstrating enhanced sensitivity. This technique is also, by definition, highly quantitative. Therefore, the real-time PCR technique is the optimal method for the detection of E1/E2-mediated DNA replication

  9. Detection of Legionella species in environmental water by the quantitative PCR method in combination with ethidium monoazide treatment.

    Science.gov (United States)

    Inoue, Hiroaki; Takama, Tomoko; Yoshizaki, Miwa; Agata, Kunio

    2015-01-01

    We detected Legionella species in 111 bath water samples and 95 cooling tower water samples by using a combination of conventional plate culture, quantitative polymerase chain reaction (qPCR) and qPCR combined with ethidium monoazide treatment (EMA-qPCR) methods. In the case of bath water samples, Legionella spp. were detected in 30 samples by plate culture, in 85 samples by qPCR, and in 49 samples by EMA-qPCR. Of 81 samples determined to be Legionella-negative by plate culture, 56 and 23 samples were positive by qPCR and EMA-qPCR, respectively. Therefore, EMA treatment decreased the number of Legionella-positive bath water samples detected by qPCR. In contrast, EMA treatment had no effect on cooling tower water samples. We therefore expect that EMA-qPCR is a useful method for the rapid detection of viable Legionella spp. from bath water samples.

  10. TaqMan MGB probe fluorescence real-time quantitative PCR for rapid detection of Chinese Sacbrood virus.

    Directory of Open Access Journals (Sweden)

    Ma Mingxiao

    Full Text Available Sacbrood virus (SBV is a picorna-like virus that affects honey bees (Apis mellifera and results in the death of the larvae. Several procedures are available to detect Chinese SBV (CSBV in clinical samples, but not to estimate the level of CSBV infection. The aim of this study was develop an assay for rapid detection and quantification of this virus. Primers and probes were designed that were specific for CSBV structural protein genes. A TaqMan minor groove binder (MGB probe-based, fluorescence real-time quantitative PCR was established. The specificity, sensitivity and stability of the assay were assessed; specificity was high and there were no cross-reactivity with healthy larvae or other bee viruses. The assay was applied to detect CSBV in 37 clinical samples and its efficiency was compared with clinical diagnosis, electron microscopy observation, and conventional RT-PCR. The TaqMan MGB-based probe fluorescence real-time quantitative PCR for CSBV was more sensitive than other methods tested. This assay was a reliable, fast, and sensitive method that was used successfully to detect CSBV in clinical samples. The technology can provide a useful tool for rapid detection of CSBV. This study has established a useful protocol for CSBV testing, epidemiological investigation, and development of animal models.

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

    Science.gov (United States)

    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.

  12. [THE COMPARATIVE ANALYSIS OF RESULTS OF DETECTION OF CARCINOGENIC TYPES OF HUMAN PAPILLOMA VIRUS BY QUALITATIVE AND QUANTITATIVE TESTS].

    Science.gov (United States)

    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.

  13. Detection and characterization of quantitative trait loci for meat quality traits in pigs

    NARCIS (Netherlands)

    Koning, de D.J.; Harlizius, B.; Rattink, A.P.; Groenen, M.A.M.; Brascamp, E.W.; Arendonk, van J.A.M.

    2001-01-01

    In an experimental cross between Meishan and Dutch Large White and Landrace lines, 785 F2 animals with carcass information and their parents were typed for molecular markers covering the entire porcine genome. Linkage was studied between these markers and eight meat quality traits. Quantitative

  14. Absolute quantitation of proteins by Acid hydrolysis combined with amino Acid detection by mass spectrometry

    DEFF Research Database (Denmark)

    Mirgorodskaya, Olga A; Körner, Roman; Kozmin, Yuri P

    2012-01-01

    Amino acid analysis is among the most accurate methods for absolute quantification of proteins and peptides. Here, we combine acid hydrolysis with the addition of isotopically labeled standard amino acids and analysis by mass spectrometry for accurate and sensitive protein quantitation...

  15. Complex pedigree analysis to detect quantitative trait loci in dairy cattle

    NARCIS (Netherlands)

    Bink, M.C.A.M.

    1998-01-01

    In dairy cattle, many quantitative traits of economic importance show phenotypic variation. For breeding purposes the analysis of this phenotypic variation and uncovering the contribution of genetic factors is very important. Usually, the individual gene effects contributing to the

  16. Detection of expression quantitative trait Loci in complex mouse crosses: impact and alleviation of data quality and complex population substructure.

    Science.gov (United States)

    Iancu, Ovidiu D; Darakjian, Priscila; Kawane, Sunita; Bottomly, Daniel; Hitzemann, Robert; McWeeney, Shannon

    2012-01-01

    Complex Mus musculus crosses, e.g., heterogeneous stock (HS), provide increased resolution for quantitative trait loci detection. However, increased genetic complexity challenges detection methods, with discordant results due to low data quality or complex genetic architecture. We quantified the impact of theses factors across three mouse crosses and two different detection methods, identifying procedures that greatly improve detection quality. Importantly, HS populations have complex genetic architectures not fully captured by the whole genome kinship matrix, calling for incorporating chromosome specific relatedness information. We analyze three increasingly complex crosses, using gene expression levels as quantitative traits. The three crosses were an F(2) intercross, a HS formed by crossing four inbred strains (HS4), and a HS (HS-CC) derived from the eight lines found in the collaborative cross. Brain (striatum) gene expression and genotype data were obtained using the Illumina platform. We found large disparities between methods, with concordance varying as genetic complexity increased; this problem was more acute for probes with distant regulatory elements (trans). A suite of data filtering steps resulted in substantial increases in reproducibility. Genetic relatedness between samples generated overabundance of detected eQTLs; an adjustment procedure that includes the kinship matrix attenuates this problem. However, we find that relatedness between individuals is not evenly distributed across the genome; information from distinct chromosomes results in relatedness structure different from the whole genome kinship matrix. Shared polymorphisms from distinct chromosomes collectively affect expression levels, confounding eQTL detection. We suggest that considering chromosome specific relatedness can result in improved eQTL detection.

  17. One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

    Science.gov (United States)

    Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

    2015-03-01

    A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dae-Young [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada)], E-mail: daeyoung.lee@ec.gc.ca; Lauder, Heather; Cruwys, Heather; Falletta, Patricia [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada); Beaudette, Lee A. [Environmental Science and Technology Centre, Environment Canada, 335 River Road South, Ottawa, Ontario, K1A 0H3 (Canada)], E-mail: lee.beaudette@ec.gc.ca

    2008-07-15

    Conventional microbial water quality test methods are well known for their technical limitations, such as lack of direct pathogen detection capacity and low throughput capability. The microarray assay has recently emerged as a promising alternative for environmental pathogen monitoring. In this study, bacterial pathogens were detected in municipal wastewater using a microarray equipped with short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes have been designed against 38 species, 4 genera, and 1 family of pathogens. The detection sensitivity of the microarray for a waterborne pathogen Aeromonas hydrophila was determined to be approximately 1.0% of the total DNA, or approximately 10{sup 3}A. hydrophila cells per sample. The efficacy of the DNA microarray was verified in a parallel study where pathogen genes and E. coli cells were enumerated using real-time quantitative PCR (qPCR) and standard membrane filter techniques, respectively. The microarray and qPCR successfully detected multiple wastewater pathogen species at different stages of the disinfection process (i.e. secondary effluents vs. disinfected final effluents) and at two treatment plants employing different disinfection methods (i.e. chlorination vs. UV irradiation). This result demonstrates the effectiveness of the DNA microarray as a semi-quantitative, high throughput pathogen monitoring tool for municipal wastewater.

  1. Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

    Directory of Open Access Journals (Sweden)

    Sylvia Worbs

    2015-11-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Maria Frølund

    Full Text Available 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.

  5. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR.

    Directory of Open Access Journals (Sweden)

    Yogita Maheshwari

    Full Text Available Droplet digital polymerase chain reaction (ddPCR is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative PCR (qPCR for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.

  6. Highly Sensitive Quantitative PCR for the Detection and Differentiation of Pseudogymnoascus destructans and Other Pseudogymnoascus Species

    OpenAIRE

    Shuey, Megan M.; Drees, Kevin P.; Lindner, Daniel L.; Keim, Paul; Foster, Jeffrey T.

    2014-01-01

    White-nose syndrome is a fungal disease that has decimated bat populations across eastern North America. Identification of the etiologic agent, Pseudogymnoascus destructans (formerly Geomyces destructans), in environmental samples is essential to proposed management plans. A major challenge is the presence of closely related species, which are ubiquitous in many soils and cave sediments and often present in high abundance. We present a dual-probe real-time quantitative PCR assay capable of de...

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

    Science.gov (United States)

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

    2014-01-01

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

  8. Microbial Quality, Safety, and Pathogen Detection by Using Quantitative PCR of Raw Salad Vegetables Sold in Dhanbad City, India.

    Science.gov (United States)

    Mritunjay, Sujeet K; Kumar, Vipin

    2017-01-01

    Consumption of ready-to-eat fresh vegetables has increased worldwide, with a consequent increase in outbreaks caused by foodborne pathogens. In the Indian subcontinent, raw fresh vegetables are usually consumed without washing or other decontamination procedures, thereby leading to new food safety threats. In this study, the microbiological quality and pathogenic profile of raw salad vegetables was evaluated through standard protocols. In total, 480 samples (60 each of eight different salad vegetables) of cucumber, tomato, carrot, coriander, cabbage, beetroot, radish, and spinach were collected from different locations in Dhanbad, a city famous for its coal fields and often called the "Coal Capital of India." The samples were analyzed for total plate count, total coliforms, Escherichia coli , E. coli O157:H7, Listeria monocytogenes , and Salmonella spp. Incidences of pathogens were detected through quantitative PCR subsequent to isolation. Results showed that 46.7% (for total plate counts) and 30% (for total coliforms) of samples were unacceptable for consumption per the Food Safety and Standards Authority of India. Pathogenic microorganisms were detected in 3.7% of total samples. E. coli O157:H7 was detected in three samples of spinach (2) and beetroot ( 1 ); L. monocytogenes was detected in 14 samples of spinach ( 8 ), tomato ( 3 ), cucumber ( 2 ), and radish ( 1 ); and Salmonella spp. were detected in 16 samples of spinach ( 7 ), tomato ( 3 ), beetroot ( 2 ), cucumber ( 2 ), carrot ( 1 ), and radish ( 1 ). Pathogens were not detected in any of the cabbage and coriander samples.

  9. Detection of infectious bronchitis virus with the use of real-time quantitative reverse transcriptase-PCR and correlation with virus detection in embryonated eggs.

    Science.gov (United States)

    Roh, Ha-Jung; Hilt, Deborah A; Jackwood, Mark W

    2014-09-01

    Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays have been used to detect the presence of challenge virus when the efficacy of infectious bronchitis virus (IBV) vaccine against field viruses is being experimentally evaluated. However, federal guidelines for licensing IBV vaccines indicate that challenge-virus detection following vaccination is to be conducted in embryonated eggs. In this study, we examined qRT-PCR data with the use of universal and type-specific primers and probe sets for IBV detection and compared those data with challenge-virus detection in embryonated eggs to determine if the two methods of evaluating vaccine efficacy are comparable. In addition, we tested the qRT-PCR assays on thermocyclers from two different manufacturers. We found the universal IBV primers and probe set to be comparable to challenge-virus detection in embryonated eggs. However, for some IBV types (Mass41 and Conn on the SmartCycler II and Ark, Mass41, Conn, and GA98 on the ABI 7500) the qRT-PCR assay was more sensitive than virus detection in embryonated eggs. This may simply be due to the universal IBV qRT-PCR assay being more sensitive than virus detection in eggs or to the assay detecting nucleic acid from nonviable virus. This finding is important and needs to be considered when evaluating challenge-virus detection for vaccination and challenge studies, because qRT-PCR could potentially identify positive birds that would otherwise be negative by virus detection in embryonated eggs; thus it could lead to a more stringent measure of vaccine efficacy. We also found that the IBV type-specific primers and probe sets designed in this study were in general less sensitive than the universal IBV primers and probe set. Only the Ark-DPI-spedcific assay on the SmartCycler II and the Ark-DPI-, Mass41-, and DE072/GA98- (for detection of GA98 virus only) specific assays on the ABI 7500 were comparable in sensitivity to virus detection in eggs. We

  10. Using rapid-scan EPR to improve the detection limit of quantitative EPR by more than one order of magnitude.

    Science.gov (United States)

    Möser, J; Lips, K; Tseytlin, M; Eaton, G R; Eaton, S S; Schnegg, A

    2017-08-01

    X-band rapid-scan EPR was implemented on a commercially available Bruker ELEXSYS E580 spectrometer. Room temperature rapid-scan and continuous-wave EPR spectra were recorded for amorphous silicon powder samples. By comparing the resulting signal intensities the feasibility of performing quantitative rapid-scan EPR is demonstrated. For different hydrogenated amorphous silicon samples, rapid-scan EPR results in signal-to-noise improvements by factors between 10 and 50. Rapid-scan EPR is thus capable of improving the detection limit of quantitative EPR by at least one order of magnitude. In addition, we provide a recipe for setting up and calibrating a conventional pulsed and continuous-wave EPR spectrometer for rapid-scan EPR. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Improvement of the qualitative and quantitative detection of simultaneously present fluorescent tracers by systematic sample treatment

    International Nuclear Information System (INIS)

    Behrens, H.

    1982-01-01

    The selective instrumental detection of individual fluorescent tracers in mixtures containing further fluorescent dyes is limited by spectral interferences. Therefore additional separations or other suitable procedures have to be included into the analytic technique. With the method described below, the respective tracer to be detected remains with its initial concentration in the sample and is analysed under the appropriate conditions, whereas the interfering tracers are separated or suppressed. The techniques applied for this base on the facts that 1) the fluorescence intensity of the tracers varies differently when the pH-value changes; 2) the tracers show different absorption behaviour and 3) they provide different degrees of light sensitivity. The procedures permit for example to detect uranin when eosin is present in a higher concentration or to detect eosin when amidorhodamin G is present. (orig.) [de

  12. A high-throughput qPCR system for simultaneous quantitative detection of dairy Lactococcus lactis and Leuconostoc bacteriophages

    DEFF Research Database (Denmark)

    Muhammed, Musemma Kedir; Krych, Lukasz; Nielsen, Dennis Sandris

    2017-01-01

    simultaneous quantitative detection of Lc. lactis 936 (now SK1virus), P335, c2 (now C2virus) and Leuconostoc phage groups. Component assays are designed to have high efficiencies and nearly the same dynamic detection ranges, i.e., from 1.1 x 105 to 1.1 x 101 phage genomes per reaction, which corresponds to 9 x......Simultaneous quantitative detection of Lactococcus (Lc.) lactis and Leuconostoc species bacteriophages (phages) has not been reported in dairies using undefined mixed-strain DL-starters, probably due to the lack of applicable methods. We optimized a high-throughput qPCR system that allows...... 107 to 9 x 103 phage particles mL-1 without any additional up-concentrating steps. The amplification efficiencies of the corresponding assays were 100.1±2.6, 98.7±2.3, 101.0±2.3 and 96.2±6.2. The qPCR system was tested on samples obtained from a dairy plant that employed traditional mother...

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

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Xingye; Hu, Jie; Choi, Jane Ru; Huang, Yalin; Wang, Xuemin [The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi' an Jiaotong University, Xi' an, 710049 (China); Bioinspired Engineering and Biomechanics Center (BEBC), Xi' an Jiaotong University, Xi' an, 710049 (China); Lu, Tian Jian, E-mail: tjlu@mail.xjtu.edu.cn [Bioinspired Engineering and Biomechanics Center (BEBC), Xi' an Jiaotong University, Xi' an, 710049 (China); Xu, Feng, E-mail: fengxu@mail.xjtu.edu.cn [The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi' an Jiaotong University, Xi' an, 710049 (China); Bioinspired Engineering and Biomechanics Center (BEBC), Xi' an Jiaotong University, Xi' an, 710049 (China)

    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. - Highlights: • The meter chip is a standalone point-of-care diagnostic tool with visible readouts of quantification results. • A fast and low cost fabrication protocol (~3 min and ~$0.2 per chip) of meter chip was proposed. • The chip may hold the potential for rapid scaning of bovine mastitis in cattle farms for food safety control.

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

    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. - Highlights: • The meter chip is a standalone point-of-care diagnostic tool with visible readouts of quantification results. • A fast and low cost fabrication protocol (~3 min and ~$0.2 per chip) of meter chip was proposed. • The chip may hold the potential for rapid scaning of bovine mastitis in cattle farms for food safety control.

  16. Improved assay to detect Plasmodium falciparum using an uninterrupted, semi-nested PCR and quantitative lateral flow analysis

    Science.gov (United States)

    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 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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  19. Quantitative analysis of arterial flow properties for detection of non-calcified plaques in ECG-gated coronary CT angiography

    Science.gov (United States)

    Wei, Jun; Zhou, Chuan; Chan, Heang-Ping; Chughtai, Aamer; Agarwal, Prachi; Kuriakose, Jean; Hadjiiski, Lubomir; Patel, Smita; Kazerooni, Ella

    2015-03-01

    We are developing a computer-aided detection system to assist radiologists in detection of non-calcified plaques (NCPs) in coronary CT angiograms (cCTA). In this study, we performed quantitative analysis of arterial flow properties in each vessel branch and extracted flow information to differentiate the presence and absence of stenosis in a vessel segment. Under rest conditions, blood flow in a single vessel branch was assumed to follow Poiseuille's law. For a uniform pressure distribution, two quantitative flow features, the normalized arterial compliance per unit length (Cu) and the normalized volumetric flow (Q) along the vessel centerline, were calculated based on the parabolic Poiseuille solution. The flow features were evaluated for a two-class classification task to differentiate NCP candidates obtained by prescreening as true NCPs and false positives (FPs) in cCTA. For evaluation, a data set of 83 cCTA scans was retrospectively collected from 83 patient files with IRB approval. A total of 118 NCPs were identified by experienced cardiothoracic radiologists. The correlation between the two flow features was 0.32. The discriminatory ability of the flow features evaluated as the area under the ROC curve (AUC) was 0.65 for Cu and 0.63 for Q in comparison with AUCs of 0.56-0.69 from our previous luminal features. With stepwise LDA feature selection, volumetric flow (Q) was selected in addition to three other luminal features. With FROC analysis, the test results indicated a reduction of the FP rates to 3.14, 1.98, and 1.32 FPs/scan at sensitivities of 90%, 80%, and 70%, respectively. The study indicated that quantitative blood flow analysis has the potential to provide useful features for the detection of NCPs in cCTA.

  20. Comparative sensitivity of quantitative EEG (QEEG) spectrograms for detecting seizure subtypes.

    Science.gov (United States)

    Goenka, Ajay; Boro, Alexis; Yozawitz, Elissa

    2018-02-01

    To assess the sensitivity of Persyst version 12 QEEG spectrograms to detect focal, focal with secondarily generalized, and generalized onset seizures. A cohort of 562 seizures from 58 patients was analyzed. Successive recordings with 2 or more seizures during continuous EEG monitoring for clinical indications in the ICU or EMU between July 2016 and January 2017 were included. Patient ages ranged from 5 to 64 years (mean = 36 years). There were 125 focal seizures, 187 secondarily generalized and 250 generalized seizures from 58 patients analyzed. Seizures were identified and classified independently by two epileptologists. A correlate to the seizure pattern in the raw EEG was sought in the QEEG spectrograms in 4-6 h EEG epochs surrounding the identified seizures. A given spectrogram was interpreted as indicating a seizure, if at the time of a seizure it showed a visually significant departure from the pre-event baseline. Sensitivities for seizure detection using each spectrogram were determined for each seizure subtype. Overall sensitivities of the QEEG spectrograms for detecting seizures ranged from 43% to 72%, with highest sensitivity (402/562,72%) by the seizure detection trend. The asymmetry spectrogram had the highest sensitivity for detecting focal seizures (117/125,94%). The FFT spectrogram was most sensitive for detecting secondarily generalized seizures (158/187, 84%). The seizure detection trend was the most sensitive for generalized onset seizures (197/250,79%). Our study suggests that different seizure types have specific patterns in the Persyst QEEG spectrograms. Identifying these patterns in the EEG can significantly increase the sensitivity for seizure identification. Copyright © 2018 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

    Krøjgaard, Louise H.; Krogfelt, Karen A.; Albrechtsen, Hans-Jorgen

    2011-01-01

    Background: 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...

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

    Science.gov (United States)

    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.

  3. Detection of nonauthorized genetically modified organisms using differential quantitative polymerase chain reaction: application to 35S in maize.

    Science.gov (United States)

    Cankar, Katarina; Chauvensy-Ancel, Valérie; Fortabat, Marie-Noelle; Gruden, Kristina; Kobilinsky, André; Zel, Jana; Bertheau, Yves

    2008-05-15

    Detection of nonauthorized genetically modified organisms (GMOs) has always presented an analytical challenge because the complete sequence data needed to detect them are generally unavailable although sequence similarity to known GMOs can be expected. A new approach, differential quantitative polymerase chain reaction (PCR), for detection of nonauthorized GMOs is presented here. This method is based on the presence of several common elements (e.g., promoter, genes of interest) in different GMOs. A statistical model was developed to study the difference between the number of molecules of such a common sequence and the number of molecules identifying the approved GMO (as determined by border-fragment-based PCR) and the donor organism of the common sequence. When this difference differs statistically from zero, the presence of a nonauthorized GMO can be inferred. The interest and scope of such an approach were tested on a case study of different proportions of genetically modified maize events, with the P35S promoter as the Cauliflower Mosaic Virus common sequence. The presence of a nonauthorized GMO was successfully detected in the mixtures analyzed and in the presence of (donor organism of P35S promoter). This method could be easily transposed to other common GMO sequences and other species and is applicable to other detection areas such as microbiology.

  4. FDTD-based quantitative analysis of terahertz wave detection for multilayered structures.

    Science.gov (United States)

    Tu, Wanli; Zhong, Shuncong; Shen, Yaochun; Zhou, Qing; Yao, Ligang

    2014-10-01

    Experimental investigations have shown that terahertz pulsed imaging (TPI) is able to quantitatively characterize a range of multilayered media (e.g., biological issues, pharmaceutical tablet coatings, layered polymer composites, etc.). Advanced modeling of the interaction of terahertz radiation with a multilayered medium is required to enable the wide application of terahertz technology in a number of emerging fields, including nondestructive testing. Indeed, there have already been many theoretical analyses performed on the propagation of terahertz radiation in various multilayered media. However, to date, most of these studies used 1D or 2D models, and the dispersive nature of the dielectric layers was not considered or was simplified. In the present work, the theoretical framework of using terahertz waves for the quantitative characterization of multilayered media was established. A 3D model based on the finite difference time domain (FDTD) method is proposed. A batch of pharmaceutical tablets with a single coating layer of different coating thicknesses and different refractive indices was modeled. The reflected terahertz wave from such a sample was computed using the FDTD method, assuming that the incident terahertz wave is broadband, covering a frequency range up to 3.5 THz. The simulated results for all of the pharmaceutical-coated tablets considered were found to be in good agreement with the experimental results obtained using a commercial TPI system. In addition, we studied a three-layered medium to mimic the occurrence of defects in the sample.

  5. Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors

    International Nuclear Information System (INIS)

    Cox, Annie M.; Goodwin, Kelly D.

    2013-01-01

    Highlights: • DNA extraction methods affected measured qPCR target recovery. • Recovery and variability differed, sometimes by more than an order of magnitude. • SCODA did not offer significant improvement with PCR-inhibited seawater. • Aggressive lysis did appear to improve target recovery. • Reliable and affordable correction methods are needed for quantitative PCR. -- Abstract: The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications

  6. Towards quantitative SERS detection of hydrogen cyanide at ppb level for human breath analysis

    DEFF Research Database (Denmark)

    Lauridsen, Rikke Kragh; Rindzevicius, Tomas; Molin, Søren

    2015-01-01

    Lung infections with Pseudomonas aeruginosa (PA) is the most common cause of morbidity and mortality in cystic fibrosis (CF) patients. Due to its ready adaptation to the dehydrated mucosa of CF airways, PA infections tend to become chronic, eventually killing the patient. Hydrogen cyanide (HCN......) at ppb level has been reported to be a PA biomarker. For early PA detection in CF children not yet chronically lung infected a non-invasive Surface-Enhanced Raman Spectroscopy (SERS)-based breath nanosensor is being developed. The triple bond between C and N in cyanide, with its characteristic band...... substrate can be consistently detected under different experimental conditions and up to 9 days after exposure. For detection of lower cyanide concentrations serial dilution experiments using potassium cyanide (KCN) demonstrated cyanide quantification down to 1 μM in solution (corresponding to 18 ppb...

  7. Comparative study on 4 quantitative detection methods of apoptosis induced by radiation

    International Nuclear Information System (INIS)

    Yang Yepeng; Chen Guanying; Zhou Mei; Shen Qinjian; Shen Lei; Zhu Yingbao

    2004-01-01

    Objective: To reveal the capability of 4 apoptosis-detecting methods to discriminate between apoptosis and necrosis and show their respective advantages and shortcomings through comparison of detected results and analysis of detection mechanism. Methods: Four methods, PI staining-flow cytometric detection (P-F method), TUNEL labeling-flow cytometric detection (T-F method), annexing V-FITC/PI vital staining-flow cytometric detection (A-F method) and Hoechst/PI vital staining-fluorescence microscopic observation (H-O method), were used to determine apoptosis and necrosis in human breast cancer MCF-7 cell line induced by γ-rays. Hydroxycamptothecine and sodium azide were used to induce positive controls of apoptosis and necrosis respectively. Results: All 4 methods showed good time-dependent and dose dependent respondence to apoptosis induced by γ-rays and hydroxycamptothecine. Apoptotic cell ratios and curve slopes obtained from P-F method were minimum and, on the contrary, those from T-F method were maximum among these 4 methods. With A-F method and H-O method, two sets of data, apoptosis and necrosis, could be gained respectively and the data gained from these two methods were close to equal. A-F method and H-O method could distinguish necrosis induced by sodium azide from apoptosis while P-F method and T-F method presented false increase of apoptosis. Conclusions: P-F method and T-F method can not discriminate between apoptosis and necrosis. P-F method is less sensitive but more simple, convenient and economical than T-F method. A-F method and H-O method can distinguish necrosis from apoptosis. A-F method is more costly but more quick and reliable than H-O method. H-O method is economical, practical and morphological changes of cells and nucleus can be observed simultaneously with it. (authors)

  8. Detection of occult vertebral fractures by quantitative assessment of bone marrow attenuation values at MDCT

    International Nuclear Information System (INIS)

    Henes, Frank Oliver; Groth, Michael; Kramer, Harald; Schaefer, Christian; Regier, Marc; Derlin, Thorsten; Adam, Gerhard; Bannas, Peter

    2014-01-01

    Objectives: To determine a cut-off value of Hounsfield attenuation units (HU) at multidetector computed tomography (MDCT) for valid and reliable detection of bone marrow oedema (BME) related to occult vertebral fractures. Methods: 36 patients underwent both MDCT and Magnetic Resonance Imaging (MRI) for evaluation of vertebral fractures of the thoracolumbar spine and were included in this retrospective study. Two readers independently assessed HU values at MDCT in a total of 196 vertebrae. Reliability was assessed by intraclass correlation coefficient and Bland–Altman analysis. For each patient we determined the vertebra with the lowest HU value and calculated the HU-difference to each other vertebral body. HU-differences were subjected to receiver operating characteristic (ROC) curve analysis to determine the diagnostic accuracy for detection of BME as determined by MRI, which served as the reference standard. Results of HU-measurements were compared with standard visual evaluation of MDCT. Results: HU measurements demonstrated a high interrater reliability (ICC = 0.984). ROC curve analysis (AUC = 0.978) exhibited an ideal cut-off value of 29.6 HU for detection of BME associated with vertebral fractures with an accuracy of 97.4% as compared to 93.4% accuracy of visual evaluation. Particularly, HU-measurements increased the sensitivity for detection of vertebral fractures from 78.0% to 92.7% due to the detection of 7 of 9 occult fractures that were missed by visual evaluation alone. Conclusions: Assessing bone marrow density by HU measurements using the cut-off of 29.6 HU is a valid and reliable tool for detection of BME related to occult vertebral fractures in MDCT. The introduced technique may allow more accurate treatment decisions and may make further diagnostic work-up with MRI unnecessary

  9. Towards quantitative SERS detection of hydrogen cyanide at ppb level for human breath analysis

    Directory of Open Access Journals (Sweden)

    Rikke Kragh Lauridsen

    2015-09-01

    Full Text Available Lung infections with Pseudomonas aeruginosa (PA is the most common cause of morbidity and mortality in cystic fibrosis (CF patients. Due to its ready adaptation to the dehydrated mucosa of CF airways, PA infections tend to become chronic, eventually killing the patient. Hydrogen cyanide (HCN at ppb level has been reported to be a PA biomarker. For early PA detection in CF children not yet chronically lung infected a non-invasive Surface-Enhanced Raman Spectroscopy (SERS-based breath nanosensor is being developed. The triple bond between C and N in cyanide, with its characteristic band at ∼2133 cm−1, is an excellent case for the SERS-based detection due to the infrequent occurrence of triple bonds in nature. For demonstration of direct HCN detection in the gas phase, a gold-coated silicon nanopillar substrate was exposed to 5 ppm HCN in N2. Results showed that HCN adsorbed on the SERS substrate can be consistently detected under different experimental conditions and up to 9 days after exposure. For detection of lower cyanide concentrations serial dilution experiments using potassium cyanide (KCN demonstrated cyanide quantification down to 1 μM in solution (corresponding to 18 ppb. Lower KCN concentrations of 10 and 100 nM (corresponding to 0.18 and 1.8 ppb produced SERS intensities that were relatively similar to the reference signal. Since HCN concentration in the breath of PA colonized CF children is reported to be ∼13.5 ppb, the detection of cyanide is within the required range. Keywords: Surface-Enhanced Raman Spectroscopy, Hydrogen cyanide, Pseudomonas aeruginosa, Cystic fibrosis, Breath analysis

  10. Detection of occult vertebral fractures by quantitative assessment of bone marrow attenuation values at MDCT

    Energy Technology Data Exchange (ETDEWEB)

    Henes, Frank Oliver, E-mail: f.henes@uke.de [Department of Diagnostic and Interventional Radiology, Center for Radiology and Endoscopy, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg (Germany); Groth, Michael [Department of Diagnostic and Interventional Neuroradiology, Center for Radiology and Endoscopy, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg (Germany); Kramer, Harald [Department of Clinical Radiology, Ludwig-Maximilians-University Hospital Munich, Marchioninistr. 15, 81377 Munich (Germany); Department of Radiology, University of Wisconsin – Madison, Clinical Science Center, 600 Highland Avenue, Madison, WI 53792 (United States); Schaefer, Christian [Department of Trauma-, Hand- and Reconstructive Surgery, Spine Center, Center for Surgical Sciences, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg (Germany); Regier, Marc; Derlin, Thorsten; Adam, Gerhard; Bannas, Peter [Department of Diagnostic and Interventional Radiology, Center for Radiology and Endoscopy, University Medical Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg (Germany)

    2014-01-15

    Objectives: To determine a cut-off value of Hounsfield attenuation units (HU) at multidetector computed tomography (MDCT) for valid and reliable detection of bone marrow oedema (BME) related to occult vertebral fractures. Methods: 36 patients underwent both MDCT and Magnetic Resonance Imaging (MRI) for evaluation of vertebral fractures of the thoracolumbar spine and were included in this retrospective study. Two readers independently assessed HU values at MDCT in a total of 196 vertebrae. Reliability was assessed by intraclass correlation coefficient and Bland–Altman analysis. For each patient we determined the vertebra with the lowest HU value and calculated the HU-difference to each other vertebral body. HU-differences were subjected to receiver operating characteristic (ROC) curve analysis to determine the diagnostic accuracy for detection of BME as determined by MRI, which served as the reference standard. Results of HU-measurements were compared with standard visual evaluation of MDCT. Results: HU measurements demonstrated a high interrater reliability (ICC = 0.984). ROC curve analysis (AUC = 0.978) exhibited an ideal cut-off value of 29.6 HU for detection of BME associated with vertebral fractures with an accuracy of 97.4% as compared to 93.4% accuracy of visual evaluation. Particularly, HU-measurements increased the sensitivity for detection of vertebral fractures from 78.0% to 92.7% due to the detection of 7 of 9 occult fractures that were missed by visual evaluation alone. Conclusions: Assessing bone marrow density by HU measurements using the cut-off of 29.6 HU is a valid and reliable tool for detection of BME related to occult vertebral fractures in MDCT. The introduced technique may allow more accurate treatment decisions and may make further diagnostic work-up with MRI unnecessary.

  11. Quantitative Detection of Combustion Species using Ultra-Violet Diode Lasers

    Science.gov (United States)

    Pilgrim, J. S.; Peterson, K. A.

    2001-01-01

    Southwest Sciences is developing a new microgravity combustion diagnostic based on UV diode lasers. The instrument will allow absolute concentration measurements of combustion species on a variety of microgravity combustion platforms including the Space Station. Our approach uses newly available room temperature UV diode lasers, thereby keeping the instrument compact, rugged and energy efficient. The feasibility of the technique was demonstrated by measurement of CH radicals in laboratory flames. Further progress in fabrication technology of UV diode lasers at shorter wavelengths and higher power will result in detection of transient species in the deeper UV. High sensitivity detection of combustion radicals is provided with wavelength modulation absorption spectroscopy.

  12. Subnanomole detection and quantitation of high specific activity 32P-nucleotides

    International Nuclear Information System (INIS)

    Coniglio, C.; Pappas, G.; Gill, W.J.; Kashdan, M.; Maniscalco, M.

    1991-01-01

    Microbore liquid chromatography utilizes conventional HPLC and ultraviolet detection principles to determine subnanomole mass quantities of biologically significant molecules. This system takes advantage of specifically designed microflow equipment to analyze ultraviolet absorbing species at the picomole range. 32P-labeled nucleotides are examples of compounds routinely used at picomole quantities that are extremely difficult to accurately quantify using standard mass measurement techniques. The procedure described in this paper has the capability of measuring nucleotides down to 10 pmol using commercially available microbore ultraviolet detection equipment. The technique can be used to accurately measure the specific activity of as little as 10 microCi of an aqueous 32P-nucleotide solution

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

    Science.gov (United States)

    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.

  14. Effect of Genital Sampling Site on the Detection and Quantification of Ureaplasma Species with Quantitative Polymerase Chain Reaction during Pregnancy.

    Science.gov (United States)

    Faron, Gilles; Vancutsem, Ellen; Naessens, Anne; Buyl, Ronald; Gucciardo, Leonardo; Foulon, Walter

    2017-01-01

    Objective . This study aimed to compare the qualitative and quantitative reproducibility of quantitative PCR (qPCR) for Ureaplasma species (Ureaplasma spp.) throughout pregnancy and according to the genital sampling site. Study Design . Between 5 and 14 weeks of gestation (T1), vaginal, fornix, and two cervical samples were taken. Sampling was repeated during the 2nd (T2) and 3rd (T3) trimester in randomly selected T1 positive and negative women. Qualitative and quantitative reproducibility were evaluated using, respectively, Cohen's kappa ( κ ) and interclass correlation coefficients (ICC) and repeated measures ANOVA on the log-transformed mean number of DNA copies for each sampling site. Results . During T1, 51/127 women were positive for U. parvum and 8 for U. urealyticum (4 patients for both). Sampling was repeated for 44/55 women at T2 and/or T3; 43 (97.7%) remained positive at the three timepoints. κ ranged between 0.83 and 0.95 and the ICC for cervical samples was 0.86. Conclusions . Colonization by Ureaplasma spp. seems to be very constant during pregnancy and vaginal samples have the highest detection rate.

  15. Quantitative detection of fusion protein rIFN-β-HSA by a sandwich ...

    African Journals Online (AJOL)

    user

    2011-02-14

    Feb 14, 2011 ... (Corning-Costar, NY, USA), urine microalbumin kits were purchased from ... regarded as the standard of rIFN-β-HSA and was stored at −80°C. ... and antibody concentration, sample incubating time and detecting. Zhu et al.

  16. Relationship between Effective Application of Machine Learning and Malware Detection: A Quantitative Study

    Science.gov (United States)

    Enfinger, Kerry Wayne

    2016-01-01

    The number of malicious files present in the public domain continues to rise at a substantial rate. Current anti-malware software utilizes a signature-based method to detect the presence of malicious software. Generating these pattern signatures is time consuming due to malicious code complexity and the need for expert analysis, however, by making…

  17. Development of a real-time quantitative assay for detection of Epstein-Barr virus

    NARCIS (Netherlands)

    Niesters, H. G.; van Esser, J.; Fries, E.; Wolthers, K. C.; Cornelissen, J.; Osterhaus, A. D.

    2000-01-01

    With the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and 10(7) copies of DNA per ml using

  18. Development of a real-time quantitative assay for detection of Epstein-Barr virus

    NARCIS (Netherlands)

    H.G.M. Niesters (Bert); E. Fries; K.C. Wolthers (Katja); A.D.M.E. Osterhaus (Albert); J.J. Cornelissen (Jan); J.W.J. van Esser (Joost)

    2000-01-01

    textabstractWith the use of real-time PCR, we developed and evaluated a rapid, sensitive, specific, and reproducible method for the detection of Epstein-Barr virus (EBV) DNA in plasma samples. This method allowed us to screen plasma and serum samples over a range between 100 and

  19. Explorations of the application of cyanine dyes for quantitative alpha-synuclein detection

    NARCIS (Netherlands)

    Volkova, K.D.; Kovalska, V.B.; Segers-Nolten, Gezina M.J.; Veldhuis, G.; Veldhuis, G.J.; Subramaniam, Vinod; Yarmoluk, S.M.

    2009-01-01

    We examined the practical aspects of using fluorescent mono (T-284) and trimethinecyanine (SH-516) dyes for detecting and quantifying fibrillar α-synuclein (ASN). We studied the interaction of cyanine dyes with fibrillar proteins using fluorescence spectroscopy and atomic force microscopy. The

  20. Explorations of the application of cyanine dyes for quantitative alpha-synuclein detection

    NARCIS (Netherlands)

    Volkova, Kateryna D; Kovalska, V B; Segers-Nolten, G M J; Veldhuis, G.; Subramaniam, V; Yarmoluk, S M

    We examined the practical aspects of using fluorescent mono (T-284) and trimethinecyanine (SH-516) dyes for detecting and quantifying fibrillar alpha-synuclein (ASN). We studied the interaction of cyanine dyes with fibrillar proteins using fluorescence spectroscopy and atomic force microscopy. The

  1. Semi-Quantitative Method for Streptococci Magnetic Detection in Raw Milk

    Directory of Open Access Journals (Sweden)

    Carla Duarte

    2016-04-01

    Full Text Available Bovine mastitis is the most costly disease for dairy farmers and the most frequent reason for the use of antibiotics in dairy cattle; thus, control measures to detect and prevent mastitis are crucial for dairy farm sustainability. The aim of this study was to develop and validate a sensitive method to magnetically detect Streptococcus agalactiae (a Group B streptococci and Streptococcus uberis in raw milk samples. Mastitic milk samples were collected aseptically from 44 cows with subclinical mastitis, from 11 Portuguese dairy farms. Forty-six quarter milk samples were selected based on bacterial identification by conventional microbiology. All samples were submitted to PCR analysis. In parallel, these milk samples were mixed with a solution combining specific antibodies and magnetic nanoparticles, to be analyzed using a lab-on-a-chip magnetoresistive cytometer, with microfluidic sample handling. This paper describes a point of care methodology used for detection of bacteria, including analysis of false positive/negative results. This immunological recognition was able to detect bacterial presence in samples spiked above 100 cfu/mL, independently of antibody and targeted bacteria used in this work. Using PCR as a reference, this method correctly identified 73% of positive samples for streptococci species with an anti-S. agalactiae antibody, and 41% of positive samples for an anti-GB streptococci antibody.

  2. Detection and identification of Rift Valley fever virus in mosquito vectors by quantitative real-time PCR.

    Science.gov (United States)

    Mwaengo, D; Lorenzo, G; Iglesias, J; Warigia, M; Sang, R; Bishop, R P; Brun, A

    2012-10-01

    Diagnostic methods allowing for rapid identification of pathogens are crucial for controlling and preventing dissemination after disease outbreaks as well as for use in surveillance programs. For arboviruses, detection of the presence of virus in their arthropod hosts is important for monitoring of viral activity and quantitative information is useful for modeling of transmission dynamics. In this study, molecular detection of Rift Valley fever virus (RVFV) in mosquito samples from the 2006 to 2007 East African outbreaks was performed using quantitative real-time PCR assay (qRT-PCR). Specific RVFV sequence-based primer/fluorogenic (TaqMan) probe sets were derived from the L and S RNA segments of the virus. Both primer-probe L and S segment-based combinations detected genomic RVFV sequences, with generally comparable levels of sensitivity. Viral loads from three mosquito species, Aedes mcintoshi, Aedes ochraceus and Mansonia uniformis were estimated and significant differences of between 5- and 1000-fold were detected between Ae. mcintoshi and M. uniformis using both the L and S primer-probe-based assays. The genetic relationships of the viral sequences in mosquito samples were established by partial M segment sequencing and assigned to the two previously described viral lineages defined by analysis of livestock isolates obtained during the 2006-2007 outbreak, confirming that similar viruses were present in both the vector and mammalian host. The data confirms the utility of qRT-PCR for identification and initial quantification of virus in mosquito samples during RVFV outbreaks. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Rapid Quantitative Serological Test for Detection of Infection with Mycobacterium leprae, the Causative Agent of Leprosy

    Science.gov (United States)

    Balagon, Marivic F.; Maghanoy, Armi; Orcullo, Florenda M.; Cang, Marjorie; Dias, Ronaldo Ferreira; Collovati, Marco; Reed, Steven G.

    2014-01-01

    Leprosy remains an important health problem in a number of regions. Early detection of infection, followed by effective treatment, is critical to reduce disease progression. New sensitive and specific tools for early detection of infection will be a critical component of an effective leprosy elimination campaign. Diagnosis is made by recognizing clinical signs and symptoms, but few clinicians are able to confidently identify these. Simple tests to facilitate referral to leprosy experts are not widely available, and the correct diagnosis of leprosy is often delayed. In this report, we evaluate the performance of a new leprosy serological test (NDO-LID). As expected, the test readily detected clinically confirmed samples from patients with multibacillary (MB) leprosy, and the rate of positive results declined with bacterial burden. NDO-LID detected larger proportions of MB and paucibacillary (PB) leprosy than the alternative, the Standard Diagnostics leprosy test (87.0% versus 81.7% and 32.3% versus 6.5%, respectively), while also demonstrating improved specificity (97.4% versus 90.4%). Coupled with a new cell phone-based test reader platform (Smart Reader), the NDO-LID test provided consistent, objective test interpretation that could facilitate wider use in nonspecialized settings. In addition, results obtained from sera at the time of diagnosis, versus at the end of treatment, indicated that the quantifiable nature of this system can also be used to monitor treatment efficacy. Taken together, these data indicate that the NDO-LID/Smart Reader system can assist in the diagnosis and monitoring of MB leprosy and can detect a significant number of earlier-stage infections. PMID:24478496

  4. High Specificity of Quantitative Methylation-Specific PCR Analysis for MGMT Promoter Hypermethylation Detection in Gliomas

    Directory of Open Access Journals (Sweden)

    Paola Parrella

    2009-01-01

    Full Text Available Normal brain tissue from 28 individuals and 50 glioma samples were analyzed by real-time Quantitative Methylation-Specific PCR (QMSP. Data from this analysis were compared with results obtained on the same samples by MSP. QMSP analysis demonstrated a statistically significant difference in both methylation level (P=.000009 Mann Whitney Test and frequencies (P=.0000007, Z-test in tumour samples as compared with normal brain tissues. Although QMSP and MSP showed similar sensitivity, the specificity of QMSP analysis was significantly higher (93%; CI95%: 84%–100% as compared with MSP (64%; 95%CI: 46%–82%. Our results suggest that QMSP analysis may represent a powerful tool to identify glioma patients that will benefit from alkylating agents chemotherapy.

  5. Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors.

    Science.gov (United States)

    Cox, Annie M; Goodwin, Kelly D

    2013-08-15

    The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications. Published by Elsevier Ltd.

  6. Detection and quantitative analysis of ferrocyanide and ferricyanide: FY 93 Florida State University Raman spectroscopy report

    Energy Technology Data Exchange (ETDEWEB)

    Mann, C.K.; Vickers, T.J. [Florida State Univ., Tallahassee, FL (United States). Dept. of Chemistry

    1994-10-11

    This report provides a summary of work to develop and investigate the feasibility of using Raman spectroscopy with tank waste materials. It contains Raman spectra from organics, such as ethylenediaminetetraacetic acid (EDTA), hydroxyethylenediaminetetraacteic acid (HEDTA), imino diacetic acid (IDA), kerosene, tributyl phosphate (TBP), acetone and butanol, anticipated to be present in tank wastes and spectra from T-107 real and BY-104 simulant materials. The results of investigating Raman for determining moisture content in tank materials are also presented. A description of software algorithms developed to process Raman spectra from a dispersive grating spectrometer system and an in initial design for a data base to support qualitative and quantitative application of remote Raman sensing with tank wastes.

  7. Detection and quantitative analysis of ferrocyanide and ferricyanide: FY 93 Florida State University Raman spectroscopy report

    International Nuclear Information System (INIS)

    Mann, C.K.; Vickers, T.J.

    1994-01-01

    This report provides a summary of work to develop and investigate the feasibility of using Raman spectroscopy with tank waste materials. It contains Raman spectra from organics, such as ethylenediaminetetraacetic acid (EDTA), hydroxyethylenediaminetetraacteic acid (HEDTA), imino diacetic acid (IDA), kerosene, tributyl phosphate (TBP), acetone and butanol, anticipated to be present in tank wastes and spectra from T-107 real and BY-104 simulant materials. The results of investigating Raman for determining moisture content in tank materials are also presented. A description of software algorithms developed to process Raman spectra from a dispersive grating spectrometer system and an in initial design for a data base to support qualitative and quantitative application of remote Raman sensing with tank wastes

  8. Serial thallium-201 imaging after dipyridamole for coronary disease detection: quantitative analysis using myocardial clearance

    International Nuclear Information System (INIS)

    Okada, R.D.; Dai, Y.H.; Boucher, C.A.; Pohost, G.M.

    1984-01-01

    After dipyridamole, canine studies have demonstrated a slower rate of myocardial thallium-201 clearance from zones distal to a coronary artery stenosis compared to normal zones. To determine if criteria based on canine myocardial thallium-201 clearance rates could be applied clinically, 40 patients with and 26 patients without coronary artery disease (CAD) had serial thallium-201 images obtained for 2 to 5 hours after dipyridamole. Regions of interest were manually placed over six left ventricular segments in two projections for each of three imaging times. The myocardial thallium-201 clearance rate was calculated for each of the six segments and, using the clearance rate criterion found in canine studies, was considered abnormal if less than 6.5%/hr. Using this criterion alone, 22 of 26 patients (85%) without CAD had normal and 30 of 40 patients (75%) with CAD had abnormal myocardial thallium-201 clearance rates. A quantitative analysis of regional inhomogeneity in tracer distribution (normal was greater than or equal to 25% difference between segments) was negative in 24 of 26 patients (92%) without CAD and positive in 20 of 40 patients (50%) with CAD. When both clearance rate and regional inhomogeneity were considered, 21 of 26 patients (81%) without CAD had negative and 36 of 40 patients (90%) with CAD had positive results. Thus, post-dipyridamole myocardial clearance rate criteria derived from canine studies can be applied to clinical thallium imaging. Quantitative analysis of serial thallium-201 images after dipyridamole is optimized by using myocardial thallium-201 clearance rates. Such an approach is independent of regional inhomogeneities in tracer distribution

  9. Diagnostic performance of fecal quantitative real-time polymerase chain reaction for detection of Lawsonia intracellularis–associated proliferative enteropathy in nursery pigs

    DEFF Research Database (Denmark)

    Pedersen, Ken Steen; Stege, Helle; Jensen, Tim Kåre

    2013-01-01

    Quantitative polymerase chain reaction (qPCR) tests for detection and quantification of Lawsonia intracellularis in feces from pigs have been developed. The objective of the current study was to evaluate the diagnostic performance of a fecal qPCR test for detection of nursery pigs with L. intrace......Quantitative polymerase chain reaction (qPCR) tests for detection and quantification of Lawsonia intracellularis in feces from pigs have been developed. The objective of the current study was to evaluate the diagnostic performance of a fecal qPCR test for detection of nursery pigs with L...

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

    Science.gov (United States)

    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

  11. Quantitative detection and biological propagation of scrapie seeding activity in vitro facilitate use of prions as model pathogens for disinfection.

    Directory of Open Access Journals (Sweden)

    Sandra Pritzkow

    Full Text Available Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤10(1- to ≥10(5.5-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological

  12. Quantitative detection of absorbed dose of irradiated dried fruit by ESR spectroscopy method

    International Nuclear Information System (INIS)

    Li Weiming; Ha Yiming; Zhao Yongfu; Zhang Yanli

    2011-01-01

    Sunflower seeds, walnuts, pistachios, and hazelnuts were used as experimental materials which were irradiated at 1.0, 3.0, 5.0 and 10.0 kGy, respectively. The relationships and correlations between ESR signal intensity and irradiation dosages were studied. The results showed that ESR spectra of irradiated samples were obviously different from that of CK, and the ESR signal intensity was positively related with the irradiation dose. After irradiation, the ESR intensity and spectrum shapes all changed and all four samples were clearly identified irradiated or unirradiated. The appearances of the two weak satellite lines which situated left and right to the intense singlet line in walnuts and pistachios proved the existence of cellulose radical. The detection dose limit of irradiated walnut was 1 kGy, and the detection limits of the other three samples were lower than 1 kGy. In conclusion, the ESR method could be used to irradiated. (authors)

  13. Rapid, quantitative and sensitive immunochromatographic assay based on stripping voltammetric detection of a metal ion label

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Fang; Wang, Kaihua; Lin, Yuehe

    2005-10-10

    A novel, sensitive immunochromatographic electrochemical biosensor (IEB) which combines an immunochromatographic strip technique with an electrochemical detection technique is demonstrated. The IEB takes advantages of the speed and low-cost of the conventional immunochromatographic test kits and high-sensitivity of stripping voltammetry. Bismuth ions (Bi3+) have been coupled with the antibody through the bifunctional chelating agent diethylenetriamine pentaacetic acid (DTPA). After immunoreactions, Bi3+ was released and quantified by anodic stripping voltammetry at a built-in single-use screen-printed electrode. As an example for the applications of such novel device, the detection of human chorionic gonadotronphin (HCG) in a specimen was performed. This biosensor provides a more user-friendly, rapid, clinically accurate, and less expensive immunoassay for such analysis in specimens than currently available test kits.

  14. Quantitative defects detection in wind turbine blade using optical infrared thermography

    Energy Technology Data Exchange (ETDEWEB)

    Kwaon, Koo Ahn [School of Aerospace System Engineering, UST, Daejeon (Korea, Republic of); Choi, Man Yong; Park, Hee Sang; Park, Jeong Hak; Huh, Yong Hak; Choi, Won Jai [Safety Measurement Center, Korea Research Institute of Standards and Science, Daejeon (Korea, Republic of)

    2015-02-15

    A wind turbine blade is an important component in wind-power generation, and is generally exposed to harsh environmental conditions. Ultrasonic inspection is mainly used to inspect such blades, but it has been difficult to quantify defect sizes in complicated composite structures. Recently, active infrared thermography has been widely studied for inspecting composite structures, in which thermal energy is applied to an object, and an infrared camera detects the energy emitted from it. In this paper, a calibration method for active optical lock-in thermography is proposed to quantify the size. Inclusion, debonding and wrinkle defects, created in a wind blade for 100 kW wind power generation, were all successfully detected using this method. In particular, a 50.0 mm debonding defect was sized with 98.0% accuracy.

  15. Quantitative detection of glucose level based on radiofrequency patch biosensor combined with volume-fixed structures.

    Science.gov (United States)

    Qiang, Tian; Wang, Cong; Kim, Nam-Young

    2017-12-15

    A concept for characterizing a radiofrequency (RF) patch biosensor combined with volume-fixed structures is presented for timely monitoring of an individual's glucose levels based on frequency variation. Two types of patch biosensors-separately integrated with a backside slot (0.53μL) and a front-side tank (0.70μL) structure-were developed to achieve precise and efficient detection while excluding the effects of interference due to the liquidity, shape, and thickness of the tested glucose sample. A glucose test analyte at different concentrations (50-600mg/dL) was dropped into the volume-fixed structures. It fully interacted with the RF patch electromagnetic field, effectively and sensitively changing the resonance frequency and magnitude of the reflection coefficient. Measurement results based on the resonance frequency showed high sensitivity up to 1.13MHz and 1.97MHz per mg/dL, and low detection limits of 26.54mg/dL and 15.22mg/dL, for the two types of patch biosensors, respectively, as well as a short response time of less than 1s. Excellent reusability of the proposed biosensors was verified through three sets of measurements for each individual glucose sample. Regression analysis revealed a good linear correlation between glucose concentrations and the resonance frequency shift. Moreover, to facilitate a multi-parameter-sensitive detection of glucose, the magnitude of the reflection coefficient was also tested, and it showed a good linear correlation with the glucose concentration. Thus, the proposed approach can be adopted for distinguishing glucose solution levels, and it is a potential candidate for early-stage detection of glucose levels in diabetes patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

    Science.gov (United States)

    Zhang, Jin-Ju; Xu, Jing-Feng; Shen, Yong-Wei; Ma, Shi-Jiao; Zhang, Ting-Ting; Meng, Qing-Lin; Lan, Wen-Jun; Zhang, Chun; Liu, Xiao-Mei

    2017-07-01

    Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  19. Quantitative metabolic imaging using endogenous fluorescence to detect stem cell differentiation

    Science.gov (United States)

    Quinn, Kyle P.; Sridharan, Gautham V.; Hayden, Rebecca S.; Kaplan, David L.; Lee, Kyongbum; Georgakoudi, Irene

    2013-12-01

    The non-invasive high-resolution spatial mapping of cell metabolism within tissues could provide substantial advancements in assessing the efficacy of stem cell therapy and understanding tissue development. Here, using two-photon excited fluorescence microscopy, we elucidate the relationships among endogenous cell fluorescence, cell redox state, and the differentiation of human mesenchymal stem cells into adipogenic and osteoblastic lineages. Using liquid chromatography/mass spectrometry and quantitative PCR, we evaluate the sensitivity of an optical redox ratio of FAD/(NADH + FAD) to metabolic changes associated with stem cell differentiation. Furthermore, we probe the underlying physiological mechanisms, which relate a decrease in the redox ratio to the onset of differentiation. Because traditional assessments of stem cells and engineered tissues are destructive, time consuming, and logistically intensive, the development and validation of a non-invasive, label-free approach to defining the spatiotemporal patterns of cell differentiation can offer a powerful tool for rapid, high-content characterization of cell and tissue cultures.

  20. Quantitative second-harmonic generation imaging to detect osteogenesis imperfecta in human skin samples

    Science.gov (United States)

    Adur, J.; Ferreira, A. E.; D'Souza-Li, L.; Pelegati, V. B.; de Thomaz, A. A.; Almeida, D. B.; Baratti, M. O.; Carvalho, H. F.; Cesar, C. L.

    2012-03-01

    Osteogenesis Imperfecta (OI) is a genetic disorder that leads to bone fractures due to mutations in the Col1A1 or Col1A2 genes that affect the primary structure of the collagen I chain with the ultimate outcome in collagen I fibrils that are either reduced in quantity or abnormally organized in the whole body. A quick test screening of the patients would largely reduce the sample number to be studied by the time consuming molecular genetics techniques. For this reason an assessment of the human skin collagen structure by Second Harmonic Generation (SHG) can be used as a screening technique to speed up the correlation of genetics/phenotype/OI types understanding. In the present work we have used quantitative second harmonic generation (SHG) imaging microscopy to investigate the collagen matrix organization of the OI human skin samples comparing with normal control patients. By comparing fibril collagen distribution and spatial organization, we calculated the anisotropy and texture patterns of this structural protein. The analysis of the anisotropy was performed by means of the two-dimensional Discrete Fourier Transform and image pattern analysis with Gray-Level Co-occurrence Matrix (GLCM). From these results, we show that statistically different results are obtained for the normal and disease states of OI.

  1. Quantitative measurement and analysis for detection and treatment planning of developmental dysplasia of the hip

    Science.gov (United States)

    Liu, Xin; Lu, Hongbing; Chen, Hanyong; Zhao, Li; Shi, Zhengxing; Liang, Zhengrong

    2009-02-01

    Developmental dysplasia of the hip is a congenital hip joint malformation affecting the proximal femurs and acetabulum that are subluxatable, dislocatable, and dislocated. Conventionally, physicians made diagnoses and treatments only based on findings from two-dimensional (2D) images by manually calculating clinic parameters. However, anatomical complexity of the disease and the limitation of current standard procedures make accurate diagnosis quite difficultly. In this study, we developed a system that provides quantitative measurement of 3D clinical indexes based on computed tomography (CT) images. To extract bone structure from surrounding tissues more accurately, the system firstly segments the bone using a knowledge-based fuzzy clustering method, which is formulated by modifying the objective function of the standard fuzzy c-means algorithm with additive adaptation penalty. The second part of the system calculates automatically the clinical indexes, which are extended from 2D to 3D for accurate description of spatial relationship between femurs and acetabulum. To evaluate the system performance, experimental study based on 22 patients with unilateral or bilateral affected hip was performed. The results of 3D acetabulum index (AI) automatically provided by the system were validated by comparison with 2D results measured by surgeons manually. The correlation between the two results was found to be 0.622 (p<0.01).

  2. Quantitative muscle hardness as a noninvasive means for detecting patients at risk of compartment syndromes

    International Nuclear Information System (INIS)

    Steinberg, Bruce; Riel, Ryan; Armitage, Marshal; Berrey, Hudson

    2011-01-01

    The purpose of this project was to study the efficacy of quantitative muscle hardness (QH) curve analysis for noninvasive measurement of muscle compartment interstitial pressure (IMP), and to eliminate the need for a comparison normal QH measurement to determine a pathologic reading. Elevation of IMP may lead to limb compartment syndrome, which may result in irreversible dysfunction, chronic pain and contracture. Two studies were performed by two separate independent examiners on male volunteers, where IMP measurements and QH curves were obtained. QH curves were divided into three parts comparing the third part to the second part using the coefficient of determination (R 2 ). In 205 limb compartments, there were 1432 comparison readings of the IMP versus R 2 . Using receiver operator characteristic curve analysis for all data from both studies, an R 2 cutoff of 0.974 best corresponded to a pathologic IMP of 50 mmHg. For both sets of data and for each compartment tested, the mean IMP values were statistically different (t-test: P < 0.0001) for the group with R 2 values less than 0.974 compared to the group of R 2 values greater than or equal to 0.974. In addition, a pressure prediction model was formulated with a strong overall correlation coefficient of 0.78. The data of this study support that QH analysis is potentially useful for the monitoring of IMP elevation in compartment syndrome

  3. Detection of Campylobacter jejuni in Lizard Faeces from Central Australia Using Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Harriet Whiley

    2016-12-01

    Full Text Available Worldwide, Campylobacter is a significant cause of gastrointestinal illness. It is predominately considered a foodborne pathogen, with human exposure via non-food transmission routes generally overlooked. Current literature has been exploring environmental reservoirs of campylobacteriosis including potential wildlife reservoirs. Given the close proximity between lizards and human habitats in Central Australia, this study examined the presence of Campylobacter jejuni from lizard faeces collected from this region. Of the 51 samples collected, 17 (33% (this included 14/46 (30% wild and 3/5 (60% captive lizard samples were positive for C. jejuni using quantitative PCR (qPCR. This was the first study to investigate the presence of C. jejuni in Australian lizards. This has public health implications regarding the risk of campylobacteriosis from handling of pet reptiles and through cross-contamination or contact with wild lizard faeces. Additionally this has implication for horizontal transmission via lizards of C. jejuni to food production farms. Further research is needed on this environmental reservoir and potential transmission routes to reduce the risk to public health.

  4. Three dimensional quantitative coronary angiography can detect reliably ischemic coronary lesions based on fractional flow reserve.

    Science.gov (United States)

    Chung, Woo-Young; Choi, Byoung-Joo; Lim, Seong-Hoon; Matsuo, Yoshiki; Lennon, Ryan J; Gulati, Rajiv; Sandhu, Gurpreet S; Holmes, David R; Rihal, Charanjit S; Lerman, Amir

    2015-06-01

    Conventional coronary angiography (CAG) has limitations in evaluating lesions producing ischemia. Three dimensional quantitative coronary angiography (3D-QCA) shows reconstructed images of CAG using computer based algorithm, the Cardio-op B system (Paieon Medical, Rosh Ha'ayin, Israel). The aim of this study was to evaluate whether 3D-QCA can reliably predict ischemia assessed by myocardial fractional flow reserve (FFR) < 0.80. 3D-QCA images were reconstructed from CAG which also were evaluated with FFR to assess ischemia. Minimal luminal diameter (MLD), percent diameter stenosis (%DS), minimal luminal area (MLA), and percent area stenosis (%AS) were obtained. The results of 3D-QCA and FFR were compared. A total of 266 patients was enrolled for the present study. FFR for all lesions ranged from 0.57 to 1.00 (0.85 ± 0.09). Measurement of MLD, %DS, MLA, and %AS all were significantly correlated with FFR (r = 0.569, 0609, 0.569, 0.670, respectively, all P < 0.001). In lesions with MLA < 4.0 mm(2), %AS of more than 65.5% had a 80% sensitivity and a 83% specificity to predict FFR < 0.80 (area under curve, AUC was 0.878). 3D-QCA can reliably predict coronary lesions producing ischemia and may be used to guide therapeutic approach for coronary artery disease.

  5. An improved quantitative measurement for thyroid cancer detection based on elastography

    International Nuclear Information System (INIS)

    Ding Jianrui; Cheng, H.D.; Huang Jianhua; Zhang Yingtao; Liu Jiafeng

    2012-01-01

    Objective: To evaluate color thyroid elastograms quantitatively and objectively. Materials and methods: 125 cases (56 malignant and 69 benign) were collected with the HITACHI Vision 900 system (Hitachi Medical System, Tokyo, Japan) and a liner-array-transducer of 6–13 MHz. Standard of reference was cytology (FNA—fine needle aspiration) or histology (core biopsy). The original color thyroid elastograms were transferred from red, green, blue (RGB) color space to hue, saturation, value (HSV) color space. Then, hard area ratio was defined. Finally, a SVM classifier was used to classify thyroid nodules into benign and malignant. The relation between the performance and hard threshold was fully investigated and studied. Results: The classification accuracy changed with the hard threshold, and reached maximum (95.2%) at some values (from 144 to 152). It was higher than strain ratio (87.2%) and color score (83.2%). It was also higher than the one of our previous study (93.6%). Conclusion: The hard area ratio is an important feature of elastogram, and appropriately selected hard threshold can improve classification accuracy.

  6. An automated quantitative DNA image cytometry system detects abnormal cells in cervical cytology with high sensitivity.

    Science.gov (United States)

    Wong, O G; Ho, M W; Tsun, O K; Ng, A K; Tsui, E Y; Chow, J N; Ip, P P; Cheung, A N

    2018-03-26

    To evaluate the performance of an automated DNA-image-cytometry system as a tool to detect cervical carcinoma. Of 384 liquid-based cervical cytology samples with available biopsy follow-up were analyzed by both the Imager System and a high-risk HPV test (Cobas). The sensitivity and specificity of Imager System for detecting biopsy proven high-grade squamous intraepithelial lesion (HSIL, cervical intraepithelial neoplasia [CIN]2-3) and carcinoma were 89.58% and 56.25%, respectively, compared to 97.22% and 23.33% of HPV test but additional HPV 16/18 genotyping increased the specificity to 69.58%. The sensitivity and specificity of the Imager System for predicting HSIL+ (CIN2-3+) lesions among atypical squamous cells of undetermined significance samples were 80.00% and 70.53%, respectively, compared to 100% and 11.58% of HPV test whilst the HPV 16/18 genotyping increased the specificity to 77.89%. Among atypical squamous cells-cannot exclude HSIL, the sensitivity and specificity of Imager System for predicting HSIL+ (CIN2-3+) lesions upon follow up were 82.86% and 33.33%%, respectively, compared to 97.14% and 4.76% of HPV test and the HPV 16/18 genotyping increased the specificity to 19.05%. Among low-grade squamous intraepithelial lesion cases, the sensitivity and specificity of the Imager System for predicting HSIL+ (CIN2-3+) lesions were 66.67% and 35.71%%, respectively, compared to 66.67% and 29.76% of HPV test while HPV 16/18 genotyping increased the specificity to 79.76%. The overall results of imager and high-risk HPV test agreed in 69.43% (268) of all samples. The automated imager system and HPV 16/18 genotyping can enhance the specificity of detecting HSIL+ (CIN2-3+) lesions. © 2018 John Wiley & Sons Ltd.

  7. Quantitative Real-time PCR detection of putrescine-producing Gram-negative bacteria

    Directory of Open Access Journals (Sweden)

    Kristýna Maršálková

    2017-01-01

    Full Text Available Biogenic amines are indispensable components of living cells; nevertheless these compounds could be toxic for human health in higher concentrations. Putrescine is supposed to be the major biogenic amine associated with microbial food spoilage. Development of reliable, fast and culture-independent molecular methods to detect bacteria producing biogenic amines deserves the attention, especially of the food industry in purpose to protect health. The objective of this study was to verify the newly designed primer sets for detection of two inducible genes adiA and speF together in Salmonella enterica and Escherichia coli genome by Real-time PCR. These forenamed genes encode enzymes in the metabolic pathway which leads to production of putrescine in Gram-negative bacteria. Moreover, relative expression of these genes was studied in E. coli CCM 3954 strain using Real-time PCR. In this study, sets of new primers for the detection two inducible genes (speF and adiA in Salmonella enterica and E. coli by Real-time PCR were designed and tested. Amplification efficiency of a Real-time PCR was calculated from the slope of the standard curves (adiA, speF, gapA. An efficiency in a range from 95 to 105 % for all tested reactions was achieved. The gene expression (R of adiA and speF genes in E. coli was varied depending on culture conditions. The highest gene expression of adiA and speF was observed at 6, 24 and 36 h (RadiA ~ 3, 5, 9; RspeF ~11, 10, 9; respectively after initiation of growth of this bacteria in nutrient broth medium enchired with amino acids. The results show that these primers could be used for relative quantification analysis of E. coli.

  8. Visual and quantitative anaysis of cisternography for the detection of cerebrospinal fluid leakage

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Eun Kyoung; Oh, Jin Kyoung; Yoo, Ik Dong; Kim, Dong Hyun; Chung, Yong An [College of Medicine, Incheon St. Mary' s Hospital, The Catholic University of Korea, Incheon (Korea, Republic of); Park, Sonya Young Ju [Dept. of Radiology, College of Medicine, Seoul St. Mary' s Hospital, The Catholic University of Korea, Seoul (Korea, Republic of)

    2017-06-15

    We herein present a case of a 29-year-old man with clear rhinorrhea, which persisted for 8 years following a myringotomy. After cotton pledgets were placed in several different regions of the nasal cavity, cisternography using Tc-99m DTPA was performed to measure the radioactivity of each pledget. Cisternography showed subtle uptake in the nasal cavity. However, intense uptake was detected in the pledget placed in the right eustachian tube orifice, where the pledget:serum count ratio was 10.3:1. The patient underwent duroplasty and cranioplasty, and the rhinorrhea resolved.

  9. Visual and Quantitative Analysis of Cisternography for the Detection of Cerebrospinal Fluid Leakage.

    Science.gov (United States)

    Choi, Eun Kyoung; Oh, Jin Kyoung; Park, Sonya Youngju; Yoo, Ikdong; Kim, Dong-Hyun; Chung, Yong-An

    2017-06-01

    We herein present a case of a 29-year-old man with clear rhinorrhea, which persisted for 8 years following a myringotomy. After cotton pledgets were placed in several different regions of the nasal cavity, cisternography using Tc-99m DTPA was performed to measure the radioactivity of each pledget. Cisternography showed subtle uptake in the nasal cavity. However, intense uptake was detected in the pledget placed in the right eustachian tube orifice, where the pledget:serum count ratio was 10.3:1. The patient underwent duroplasty and cranioplasty, and the rhinorrhea resolved.

  10. SU-F-R-55: Early Detection of Treatment Induced Bone Marrow Injury During Chemoradiation Therapy Using Quantitative CT

    Energy Technology Data Exchange (ETDEWEB)

    Chen, X; Song, Y; Erickson, B; Li, X [Medical College of Wisconsin, Milwaukee, WI (United States)

    2016-06-15

    Purpose: Acute hematologic toxicity associated with bone marrow injury is a common complication of chemoradiation therapy (CRT) for pelvic malignancies. In this work, we investigate the feasibility of using quantitative CT to detect bone marrow injury during CRT. Methods: Daily CTs were acquired during routine CT-guided radiation therapy using a CT-on-rails for 15 cervical cancer patients. All patients treated with a radiation dose of 45.0 to 50.4 Gy in 1.8 Gy/fraction along with chemotherapy. For each patient, the contours of bone marrow were generated in L4, L5 and sacrum on the first daily CT and then populated to other daily CTs by rigid registration using MIM (MIM Software Inc., Cleveland, OH) with manual editing if possible. A series of CT texture parameters, including Hunsfield Unit (HU) histogram, mean HU, entropy, energy, in bone marrow contours were calculated using MATLAB on each daily CT and were correlated with the completed blood counts (CBC) collected weekly for each patient. The correlations were analyzed with Pearson correlation tests. Results: For all patient data analyzed, mean HU in bone marrow decreased during CRT delivery. From the first to the last fraction the average mean HU reduction is 58.1 ± 13.6 HU (P<0.01). This decrease can be observed as early as after first 5 fractions and is strongly associated with the changes of most CBC quantities, such as the reductions of white and blood cell counts (r=0.97, P=0.001). The reduction of HU is spatially varied. Conclusion: Chemoradiation induced bone marrow injury can be detected during the delivery of CRT using quantitative CT. Chemoradiation results in reductions in mean HU, which are strongly associated with the change in the pretrial blood cell counts. Early detection of bone marrow injury with commonly available CT opens a door to improve bone marrow sparing, reducing risk of hematologic toxicity.

  11. A generalized estimating equations approach to quantitative trait locus detection of non-normal traits

    Directory of Open Access Journals (Sweden)

    Thomson Peter C

    2003-05-01

    Full Text Available Abstract To date, most statistical developments in QTL detection methodology have been directed at continuous traits with an underlying normal distribution. This paper presents a method for QTL analysis of non-normal traits using a generalized linear mixed model approach. Development of this method has been motivated by a backcross experiment involving two inbred lines of mice that was conducted in order to locate a QTL for litter size. A Poisson regression form is used to model litter size, with allowances made for under- as well as over-dispersion, as suggested by the experimental data. In addition to fixed parity effects, random animal effects have also been included in the model. However, the method is not fully parametric as the model is specified only in terms of means, variances and covariances, and not as a full probability model. Consequently, a generalized estimating equations (GEE approach is used to fit the model. For statistical inferences, permutation tests and bootstrap procedures are used. This method is illustrated with simulated as well as experimental mouse data. Overall, the method is found to be quite reliable, and with modification, can be used for QTL detection for a range of other non-normally distributed traits.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Sangyong; Jung, Jinwoo [Radiation Research Center for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Kim, Minjeong; Ryu, Sangryeol [Department of Food and Animal Biotechnology, School of Agricultural Biotechnology, Center for Agricultural Biomaterials, Seoul National University, Seoul 151-921 (Korea, Republic of); Kim, Dongho [Radiation Research Center for Biotechnology, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of)], E-mail: fungikim@kaeri.re.kr

    2008-09-15

    Food irradiation is an economically viable technology for inactivating foodborne pathogens, but irradiation can mask pathogens in unhygienically prepared food. The aim of this study was to investigate the effect of irradiation treatment on the detection of Salmonella using real-time PCR. Three commercially available kits were tested, of which the InstaGene Matrix procedure was most effective in preparing template DNA from Salmonella exposed to radiation in broth culture. The minimum level of detection by real-time PCR combined with InstaGene Matrix was 3 log units of Salmonella per milliliter. However, when pure cultures of Salmonella were irradiated at 3 and 5 kGy, the cycle threshold (C{sub T}) increased 1-1.5-fold compared to irradiation at 0 and 1 kGy. This indicated that irradiation treatment may result in an underestimation of bacterial counts due to radiation-induced DNA lesions. We also compared C{sub T} values in inoculated chicken homogenates before and after irradiation, which in this model caused a 1.3-3.3-fold underestimation of bacterial counts with respect to irradiation dose.

  13. Detection of quantitative trait loci for carcass composition traits in pigs

    Directory of Open Access Journals (Sweden)

    Renard Christine

    2002-11-01

    Full Text Available Abstract A quantitative trait locus (QTL analysis of carcass composition data from a three-generation experimental cross between Meishan (MS and Large White (LW pig breeds is presented. A total of 488 F2 males issued from six F1 boars and 23 F1 sows, the progeny of six LW boars and six MS sows, were slaughtered at approximately 80 kg live weight and were submitted to a standardised cutting of the carcass. Fifteen traits, i.e. dressing percentage, loin, ham, shoulder, belly, backfat, leaf fat, feet and head weights, two backfat thickness and one muscle depth measurements, ham + loin and back + leaf fat percentages and estimated carcass lean content were analysed. Animals were typed for a total of 137 markers covering the entire porcine genome. Analyses were performed using a line-cross (LC regression method where founder lines were assumed to be fixed for different QTL alleles and a half/full sib (HFS maximum likelihood method where allele substitution effects were estimated within each half-/full-sib family. Additional analyses were performed to search for multiple linked QTL and imprinting effects. Significant gene effects were evidenced for both leanness and fatness traits in the telomeric regions of SSC 1q and SSC 2p, on SSC 4, SSC 7 and SSC X. Additional significant QTL were identified for ham weight on SSC 5, for head weight on SSC 1 and SSC 7, for feet weight on SSC 7 and for dressing percentage on SSC X. LW alleles were associated with a higher lean content and a lower fat content of the carcass, except for the fatness trait on SSC 7. Suggestive evidence of linked QTL on SSC 7 and of imprinting effects on SSC 6, SSC 7, SSC 9 and SSC 17 were also obtained.

  14. Quantitative structure carcinogenicity relationship for detecting structural alerts in nitroso-compounds

    International Nuclear Information System (INIS)

    Helguera, Aliuska Morales; Cordeiro, M. Natalia D.S.; Perez, Miguel Angel Cabrera; Combes, Robert D.; Gonzalez, Maykel Perez

    2008-01-01

    In this work, Quantitative Structure-Activity Relationship (QSAR) modelling was used as a tool for predicting the carcinogenic potency of a set of 39 nitroso-compounds, which have been bioassayed in male rats by using the oral route of administration. The optimum QSAR model provided evidence of good fit and performance of predicitivity from training set. It was able to account for about 84% of the variance in the experimental activity and exhibited high values of the determination coefficients of cross validations, leave one out and bootstrapping (q 2 LOO = 78.53 and q 2 Boot = 74.97). Such a model was based on spectral moments weighted with Gasteiger-Marsilli atomic charges, polarizability and hydrophobicity, as well as with Abraham indexes, specifically the summation solute hydrogen bond basicity and the combined dipolarity/polarizability. This is the first study to have explored the possibility of combining Abraham solute descriptors with spectral moments. A reasonable interpretation of these molecular descriptors from a toxicological point of view was achieved by means of taking into account bond contributions. The set of relationships so derived revealed the importance of the length of the alkyl chains for determining carcinogenic potential of the chemicals analysed, and were able to explain the difference between mono-substituted and di-substituted nitrosoureas as well as to discriminate between isomeric structures with hydroxyl-alkyl and alkyl substituents in different positions. Moreover, they allowed the recognition of structural alerts in classical structures of two potent nitrosamines, consistent with their biotransformation. These results indicate that this new approach has the potential for improving carcinogenicity predictions based on the identification of structural alerts

  15. Quantitative assessment of hyperspectral imaging in detection of plasmonic nanoparticles: a modified contrast-detail analysis approach

    Science.gov (United States)

    Wang, Jianting; Chen, Yu; Pfefer, T. Joshua

    2016-03-01

    Hyperspectral reflectance imaging (HRI) is an emerging imaging modality being applied for clinical indications such as tissue oximetry, and cancer detection based on endogenous biological constituents including plasmonic nanoparticles. However, there is currently a lack of standardized test methods for objective, quantitative evaluation of HRI system performance. Contrast-detail analysis (CDA) is a phantom-based test method commonly used to evaluate medical imaging devices (e.g., mammography systems) in terms of their lower detection limit. We investigated a modified CDA (mCDA) method to quantify the detectability of gold nanoparticles by HRI systems. Silicone-based turbid phantoms containing micro-fluidic channels were developed for the mCDA tests. Polydimethylsiloxane (PDMS) phantom materials were doped with chromophores and scatterers to achieve biologically relevant optical properties (OPs). Molds were used to produce cylindrical channels of diameters 0.3 to 1.65 mm and depths of 0.2 mm inside the phantoms. Channels were filled with a mixture of hemoglobin and concentrations of gold nanorods (GNR) and measured with our HRI system. The contrast of GNRs was solved with a spectral unmixing algorithm from the reflectance spectra. The lowest detectable concentration was determined as a function of inclusion size and depth and plotted as modified contrast detail curve (mCDC). mCDCs were used to compare the detectabilities of the HRI system with different data processing algorithms. It is demonstrated that our mCDA test method involving turbid microchannel phantoms can help to elucidate the combined performance of imaging devices and plasmonic nanoparticle contrast agents. This approach may be useful for performing clinical trial standardization and device re-calibration, thus ensuring quality control and clinical performance.

  16. Detection and quantitative determination by PIXE of the mutagen Sn2+ in yeast cells

    International Nuclear Information System (INIS)

    Viau, C.M.; Yoneama, M.-L.; Dias, J.F.; Pungartnik, C.; Brendel, M.; Henriques, J.A.P.

    2006-01-01

    The main goal of this work was to determine the concentration of Sn 2+ ions in cells of the yeast Saccharomyces cerevisiae and to correlate their quantity with the genotoxicity of intracellularly accumulated metal ions. The intracellular metal content of yeast cells was determined by PIXE (particle-induced X-ray emission) after cell exposure to SnCl 2 . To that end, a thick target protocol was developed for PIXE analysis. The samples were irradiated with a 2 MeV proton beam, while the induced X-rays were detected with a high-purity germanium detector. The results of the toxicity of SnCl 2 and the PIXE analysis performed with two different yeast strains (haploid and diploid) suggest that the exposure of haploid and diploid yeast to Sn 2+ induces DNA lesions and that the absorption depends on the genetic background of each strain

  17. Use of dew-point detection for quantitative measurement of sweating rate

    Science.gov (United States)

    Brengelmann, G. L.; Mckeag, M.; Rowell, L. B.

    1975-01-01

    A method of measuring sweat rate (SR) based on detection of dew point (DP) is proposed which has advantages that may be attractive to other laboratories concerned with recording SR from selected areas of skin. It is similar to other methods in that dry gas is passed through a capsule which isolates several square centimeters of skin surface. The difference is in the means of determining how much gaseous water is carried off in the effluent moist gas. The DP detector used is free of the drawbacks of previous devices. DP is obtained through the fundamental technique of determining the temperature at which condensate forms on a mirror. Variations in DP are tracked rapidly, and accurately (+ or - 0.8 C nominal, sensitivity + or - 0.05 C) over a wide range ( -40 C to +50 C) without measurable hysteresis. The detector asembly is rugged and readily opened for cleaning and inspection.

  18. Specific antibodies to porcine zona pellucida detected by quantitative radioimmunoassay in both fertile and infertile women

    International Nuclear Information System (INIS)

    Kurachi, H.; Wakimoto, H.; Sakumoto, T.; Aono, T.; Kurachi, K.

    1984-01-01

    The specific radioimmunoassay system was developed for the titration of the antibodies to porcine zona pellucida (ZP) in human sera by using 125 I-labeled purified porcine ZP as antigen, which is known to have cross-reactivity with human ZP. The antibodies in human sera were detected in 3 of 11 (27%) women with unexplained infertility, in 16 of 48 (33%) amenorrheic patients, in 4 of 12 (33%) fertile women, and in 3 of 10 (30%) men. Moreover, antibody titers in infertile women were no higher than those in fertile women and in men. These results seem to suggest that the antibodies in human sera that cross-react with porcine ZP may not be an important factor in causing infertility in women

  19. Improving the competency of dental hygiene students in detecting dental restorations using quantitative light-induced fluorescence technology.

    Science.gov (United States)

    Oh, Hye-Young; Jung, Hoi-In; Lee, Jeong-Woo; de Jong, Elbert de Josselin; Kim, Baek-Il

    2017-03-01

    The purpose of this study was to determine the usefulness of a quantitative light-induced fluorescence (QLF) technology in detecting dental restorations by comparing the detection ability of dental hygiene students between using conventional visual inspection alone and visual inspection combined with QLF technology. The subjects of this study comprised 92 dental hygiene students. The students assigned to the control group only used white-light images to visually assess the mouth environment, while those in the experimental group additionally used fluorescence images. Using the test results of an experienced inspector as a reference value, the agreement between the reference value and the evaluation results of the students in the experimental and control groups was evaluated using Cohen's kappa and the percentage agreement. The subjects were then classified into groups covering three percentage ranges according to the score distribution and agreement values of the three groups were compared. The percentage agreement was calculated according to the type of dental restorations. The mean kappa value was significantly higher in the experimental group than the control group (0.70 vs 0.60, ptechnology increased by 8% more in the middle and bottom percentage groups than in the top percentage group (ptechnology with conventional visual inspections could improve the ability to detect dental restorations and distinguish sound teeth from aesthetic restorations. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Detection and quantitation of circulating tumor cell dynamics by bioluminescence imaging in an orthotopic mammary carcinoma model.

    Directory of Open Access Journals (Sweden)

    Laura Sarah Sasportas

    Full Text Available Circulating tumor cells (CTCs have been detected in the bloodstream of both early-stage and advanced cancer patients. However, very little is know about the dynamics of CTCs during cancer progression and the clinical relevance of longitudinal CTC enumeration. To address this, we developed a simple bioluminescence imaging assay to detect CTCs in mouse models of metastasis. In a 4T1 orthotopic metastatic mammary carcinoma mouse model, we demonstrated that this quantitative method offers sensitivity down to 2 CTCs in 0.1-1mL blood samples and high specificity for CTCs originating from the primary tumor, independently of their epithelial status. In this model, we simultaneously monitored blood CTC dynamics, primary tumor growth, and lung metastasis progression over the course of 24 days. Early in tumor development, we observed low numbers of CTCs in blood samples (10-15 cells/100 µL and demonstrated that CTC dynamics correlate with viable primary tumor growth. To our knowledge, these data represent the first reported use of bioluminescence imaging to detect CTCs and quantify their dynamics in any cancer mouse model. This new assay is opening the door to the study of CTC dynamics in a variety of animal models. These studies may inform clinical decision on the appropriate timing of blood sampling and value of longitudinal CTC enumeration in cancer patients.

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

    Science.gov (United States)

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

    2006-01-01

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

  2. MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

    Science.gov (United States)

    McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

    2016-06-01

    Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

  3. A quantitative method for determining a representative detection limit of the forensic luminol test for latent bloodstains.

    Science.gov (United States)

    Cassidy, Brianna M; Lu, Zhenyu; Martin, Jennifer P; Tazik, Shawna K; Kellogg, Katie W; DeJong, Stephanie A; Belliveau, Elle O; Kilgore, Katherine E; Ervin, Samantha M; Meece-Rayle, Mackenzie; Abraham, Alyssa M; Myrick, Michael L; Morgan, Stephen L

    2017-09-01

    The luminol test has been used for over 60 years by forensic investigators for presumptive identification of blood and visualization of blood splatter patterns. Multiple studies have estimated the limit of detection (LD) for bloodstains when luminol is employed, with results ranging from 100× to 5,000,000× dilute. However, these studies typically have not identified and controlled important experimental variables which may affect the luminol LD for bloodstains. Without control of experimental parameters in the laboratory, variables which affect the potential of presumptive bloodstain test methods remain largely unknown, and comparisons required to establish new, more powerful detection methods are simply impossible. We have developed a quantitative method to determine the relationship between the amount of blood present and its reaction with luminol by measuring, under controlled conditions, the resulting chemiluminescent intensity with a video camera, combined with processing of the digital intensity data. The method resulted in an estimated LD for bloodstains on cotton fabric at ∼200,000× diluted blood with a specific luminol formulation. Although luminol is the focus of this study, the experimental protocol used could be modified to study effects of variables using other blood detection reagents. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Quantitative detection of Staphylococcus aureus and Enterococcus faecalis DNA in blood to diagnose bacteremia in patients in the intensive care unit

    NARCIS (Netherlands)

    Peters, Remco P. H.; van Agtmael, Michiel A.; Gierveld, Sonja; Danner, Sven A.; Groeneveld, A. B. Johan; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

    2007-01-01

    Direct detection of bacterial DNA in blood offers a fast alternative to blood culture and is presumably unaffected by the prior use of antibiotics. We evaluated the performance of two real-time PCR assays for the quantitative detection of Staphylococcus aureus bacteremia and for Enterococcus

  5. HPLC Method for Simultaneous Quantitative Detection of Quercetin and Curcuminoids in Traditional Chinese Medicines

    Directory of Open Access Journals (Sweden)

    Lee Fung Ang

    2014-12-01

    Full Text Available Objectives: Quercetin and curcuminoids are important bioactive compounds found in many herbs. Previously reported high performance liquid chromatography ultraviolet (HPLC-UV methods for the detection of quercetin and curcuminoids have several disadvantages, including unsatisfactory separation times and lack of validation according the standard guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. Methods: A rapid, specific, reversed phase, HPLC-UV method with an isocratic elution of acetonitrile and 2% v/v acetic acid (40% : 60% v/v (pH 2.6 at a flow rate of 1.3 mL/minutes, a column temperature of 35°C, and ultraviolet (UV detection at 370 nm was developed. The method was validated and applied to the quantification of different types of market available Chinese medicine extracts, pills and tablets. Results: The method allowed simultaneous determination of quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin in the concentration ranges of 0.00488 ─ 200 μg/mL, 0.625 ─ 320 μg/mL, 0.07813 ─ 320 μg/mL and 0.03906 ─ 320 μg/mL, respectively. The limits of detection and quantification, respectively, were 0.00488 and 0.03906 μg/mL for quercetin, 0.62500 and 2.50000 μg/mL for bisdemethoxycurcumin, 0.07813 and 0.31250 μg/mL for demethoxycurcumin, and 0.03906 and 0.07813 μg/mL for curcumin. The percent relative intra day standard deviation (% RSD values were 0.432 ─ 0.806 μg/mL, 0.576 ─ 0.723 μg/ mL, 0.635 ─ 0.752 μg/mL and 0.655 ─ 0.732 μg/mL for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively, and those for intra day precision were 0.323 ─ 0.968 μg/mL, 0.805 ─ 0.854 μg/mL, 0.078 ─ 0.844 μg/mL and 0.275 ─ 0.829 μg/mL, respectively. The intra day accuracies were 99.589% ─ 100.821%, 98.588% ─ 101.084%, 9.289% ─ 100.88%, and 98.292% ─ 101.022% for quercetin, bisdemethoxycurcumin

  6. HPLC method for simultaneous quantitative detection of quercetin and curcuminoids in traditional chinese medicines.

    Science.gov (United States)

    Ang, Lee Fung; Yam, Mun Fei; Fung, Yvonne Tan Tze; Kiang, Peh Kok; Darwin, Yusrida

    2014-12-01

    Quercetin and curcuminoids are important bioactive compounds found in many herbs. Previously reported high performance liquid chromatography ultraviolet (HPLC-UV) methods for the detection of quercetin and curcuminoids have several disadvantages, including unsatisfactory separation times and lack of validation according the standard guidelines of the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use. A rapid, specific, reversed phase, HPLC-UV method with an isocratic elution of acetonitrile and 2% v/v acetic acid (40% : 60% v/v) (pH 2.6) at a flow rate of 1.3 mL/minutes, a column temperature of 35°C, and ultraviolet (UV) detection at 370 nm was developed. The method was validated and applied to the quantification of different types of market available Chinese medicine extracts, pills and tablets. The method allowed simultaneous determination of quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin in the concentration ranges of 0.00488 ─ 200 μg/mL, 0.625 ─ 320 μg/mL, 0.07813 ─ 320 μg/mL and 0.03906 ─ 320 μg/mL, respectively. The limits of detection and quantification, respectively, were 0.00488 and 0.03906 μg/mL for quercetin, 0.62500 and 2.50000 μg/mL for bisdemethoxycurcumin, 0.07813 and 0.31250 μg/mL for demethoxycurcumin, and 0.03906 and 0.07813 μg/mL for curcumin. The percent relative intra day standard deviation (% RSD) values were 0.432 ─ 0.806 μg/mL, 0.576 ─ 0.723 μg/mL, 0.635 ─ 0.752 μg/mL and 0.655 ─ 0.732 μg/mL for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively, and those for intra day precision were 0.323 ─ 0.968 μg/mL, 0.805 ─ 0.854 μg/mL, 0.078 ─ 0.844 μg/mL and 0.275 ─ 0.829 μg/mL, respectively. The intra day accuracies were 99.589% ─ 100.821%, 98.588% ─ 101.084%, 9.289% ─ 100.88%, and 98.292% ─ 101.022% for quercetin, bisdemethoxycurcumin, demethoxycurcumin and curcumin, respectively, and the

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

    Science.gov (United States)

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

    2016-07-01

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

  8. Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

    Directory of Open Access Journals (Sweden)

    Yuping Ren

    2017-12-01

    Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

  9. Chromatographic Separation and Visual Detection on Wicking Microfluidic Devices: Quantitation of Cu2+ in Surface, Ground, and Drinking Water.

    Science.gov (United States)

    Bandara, Gayan C; Heist, Christopher A; Remcho, Vincent T

    2018-02-20

    Copper is widely applied in industrial and technological applications and is an essential micronutrient for humans and animals. However, exposure to high environmental levels of copper, especially through drinking water, can lead to copper toxicity, resulting in severe acute and chronic health effects. Therefore, regular monitoring of aqueous copper ions has become necessary as recent anthropogenic activities have led to elevated environmental concentrations of copper. On-site monitoring processes require an inexpensive, simple, and portable analytical approach capable of generating reliable qualitative and quantitative data efficiently. Membrane-based lateral flow microfluidic devices are ideal candidates as they facilitate rapid, inexpensive, and portable measurements. Here we present a simple, chromatographic separation approach in combination with a visual detection method for Cu 2+ quantitation, performed in a lateral flow microfluidic channel. This method appreciably minimizes interferences by incorporating a nonspecific polymer inclusion membrane (PIM) based assay with a "dot-counting" approach to quantification. In this study, hydrophobic polycaprolactone (PCL)-filled glass microfiber (GMF) membranes were used as the base substrate onto which the PIM was evenly dispensed as an array of dots. The devices thus prepared were then selectively exposed to oxygen radicals through a mask to generate a hydrophilic surface path along which the sample was wicked. Using this approach, copper concentrations from 1 to 20 ppm were quantified from 5 μL samples using only visual observation of the assay device.

  10. Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

    Science.gov (United States)

    Kurata, Shinya; Kanagawa, Takahiro; Yamada, Kazutaka; Torimura, Masaki; Yokomaku, Toyokazu; Kamagata, Yoichi; Kurane, Ryuichiro

    2001-01-01

    We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples. PMID:11239011

  11. Quantitative assessment of hepatic and splenic blood flow detected by Tc-99m-Sn colloid liver scintigraphy

    International Nuclear Information System (INIS)

    Narabayashi, Isamu; Nishiyama, Shoji; Sugimura, Kazuro

    1983-01-01

    Quantitative assessment of hepatic and splenic blood flow detected by injecting Tc-99m-Sn colloid as a bolus was performed on 75 patients who were suspected of having liver disease. Using a computer, the hepatic and splenic time-activity curves were analyzed. Hepatic index was calculated as the ratio of arterial to portal blood flow. The peak time of the right kidney was corresponded to the junction of the arterial and portal phases of the hepatic curve. Splenic index was calculated as the ratio of splenic arterial to liver arterial blood flow. Hepatic and splenic indices had elevated in cases of liver cirrhosis and hepatoma than those of normal controls. There was no significant difference in the hepatic and splenic indices among chronic hepatitis, liver metastasis and normal subject. These noninvasive tests for the hepatic and splenic blood flow may be useful in writing a report of liver scintigram because of the added information of the liver. (author)

  12. Species-specific detection and quantification of common barnacle larvae from the Japanese coast using quantitative real-time PCR.

    Science.gov (United States)

    Endo, Noriyuki; Sato, Kana; Matsumura, Kiyotaka; Yoshimura, Erina; Odaka, Yukiko; Nogata, Yasuyuki

    2010-11-01

    Species-specific detection and quantification methods for barnacle larvae using quantitative real-time polymerase chain reaction (qPCR) were developed. Species-specific primers for qPCR were designed for 13 barnacle species in the mitochondrial 12S ribosomal RNA gene region. Primer specificity was examined by PCR using template DNA extracted from each of the 13 barnacle species, other unidentified barnacle species, and field collected zooplankton samples. The resulting PCR products comprised single bands following agarose gel electrophoresis when the templates corresponded to primers. The amplifications were highly species-specific even for the field plankton samples. The field plankton samples were subjected to qPCR assay. The calculated DNA contents for each barnacle species were closely correlated with the number of larvae measured by microscopic examination. The method could be applied to quantify barnacle larvae in natural plankton samples.

  13. Fast quantitative detection of cocaine in beverages using nanoextractive electrospray ionization tandem mass spectrometry.

    Science.gov (United States)

    Hu, Bin; Peng, Xuejiao; Yang, Shuiping; Gu, Haiwei; Chen, Huanwen; Huan, Yanfu; Zhang, Tingting; Qiao, Xiaolin

    2010-02-01

    Without any sample pretreatment, effervescent beverage fluids were manually sprayed into the primary ion plume created by using a nanoelectrospray ionization source for direct ionization, and the analyte ions of interest were guided into an ion trap mass spectrometer for tandem mass analysis. Functional ingredients (e.g., vitamins, taurine, and caffeine, etc.) and spiked impurity (e.g., cocaine) in various beverages, such as Red Bull energy drink, Coco-cola, and Pepsi samples were rapidly identified within 1.5 s. The limit of detection was found to be 7-15 fg (S/N = 3) for cocaine in different samples using the characteristic fragment (m/z 150) observed in the MS(3) experiments. Typical relative standard deviation and recovery of this method were 6.9%-8.6% and 104%-108% for direct analysis of three actual samples, showing that nanoextractive electrospray ionization tandem mass spectrometry is a useful technique for fast screening cocaine presence in beverages. 2010. Published by Elsevier Inc.

  14. Quantitative changes in human epithelial cancers and osteogenesis imperfecta disease detected using nonlinear multicontrast microscopy

    Science.gov (United States)

    Adur, Javier; Pelegati, Vitor B.; de Thomaz, Andre A.; D'Souza-Li, Lilia; Assunção, Maria do Carmo; Bottcher-Luiz, Fátima; Andrade, Liliana A. L. A.; Cesar, Carlos L.

    2012-08-01

    We show that combined multimodal nonlinear optical (NLO) microscopies, including two-photon excitation fluorescence, second-harmonic generation (SHG), third harmonic generation, and fluorescence lifetime imaging microscopy (FLIM) can be used to detect morphological and metabolic changes associated with stroma and epithelial transformation during the progression of cancer and osteogenesis imperfecta (OI) disease. NLO microscopes provide complementary information about tissue microstructure, showing distinctive patterns for different types of human breast cancer, mucinous ovarian tumors, and skin dermis of patients with OI. Using a set of scoring methods (anisotropy, correlation, uniformity, entropy, and lifetime components), we found significant differences in the content, distribution and organization of collagen fibrils in the stroma of breast and ovary as well as in the dermis of skin. We suggest that our results provide a framework for using NLO techniques as a clinical diagnostic tool for human cancer and OI. We further suggest that the SHG and FLIM metrics described could be applied to other connective or epithelial tissue disorders that are characterized by abnormal cells proliferation and collagen assembly.

  15. Direct electrochemistry of dopamine on gold-Agaricus bisporus laccase enzyme electrode: characterization and quantitative detection.

    Science.gov (United States)

    Shervedani, Reza Karimi; Amini, Akbar

    2012-04-01

    Direct electrochemistry of a new laccase enzyme immobilized on gold and its application as a biosensor for dopamine (DA) are investigated by voltammetry and electrochemical impedance spectroscopy. The sensor demonstrated a redox adsorption behavior with E(0') = + 180 mV vs. Ag/AgCl for immobilized Agaricus bisporus laccase (LacAB) enzyme. The MPA platform was assembled on Au with and without utilization of ultrasounds. Excellent results were obtained by using the enzyme electrode fabricated based on MPA assembled with sonication. The LacAB immobilized in this condition showed a large electrocatalytic activity for oxidation of DA. Accordingly, a third-generation (mediator free) biosensor was constructed for DA. The DA concentration could be measured in the linear range of 0.5 to 13.0 and 47.0 to 430.0 μmol L(-1) with correlation coefficients of 0.999 and 0.989, respectively, and a detection limit of 29.0 nmol L(-1). The biosensor was successfully tested for determination of DA in human blood plasma and pharmaceutical samples. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Absorbing Aerosols Above Cloud: Detection, Quantitative Retrieval, and Radiative Forcing from Satellite-based Passive Sensors

    Science.gov (United States)

    Jethva, H.; Torres, O.; Remer, L. A.; Bhartia, P. K.

    2012-12-01

    Light absorbing particles such as carbonaceous aerosols generated from biomass burning activities and windblown dust particles can exert a net warming effect on climate; the strength of which depends on the absorption capacity of the particles and brightness of the underlying reflecting background. When advected over low-level bright clouds, these aerosols absorb the cloud reflected radiation from ultra-violet (UV) to shortwave-IR (SWIR) and makes cloud scene darker-a phenomenon commonly known as "cloud darkening". The apparent "darkening" effect can be seen by eyes in satellite images as well as quantitatively in the spectral reflectance measurements made by space borne sensors over regions where light absorbing carbonaceous and dust aerosols overlay low-level cloud decks. Theoretical radiative transfer simulations support the observational evidence, and further reveal that the strength of the cloud darkening and its spectral signature (or color ratio) between measurements at two wavelengths are a bi-function of aerosol and cloud optical thickness (AOT and COT); both are measures of the total amount of light extinction caused by aerosols and cloud, respectively. Here, we developed a retrieval technique, named as the "color ratio method" that uses the satellite measurements at two channels, one at shorter wavelength in the visible and one at longer wavelength in the shortwave-IR for the simultaneous retrieval of AOT and COT. The present technique requires assumptions on the aerosol single-scattering albedo and aerosol-cloud separation which are supplemented by the Aerosol Robotic Network (AERONET) and space borne CALIOP lidar measurements. The retrieval technique has been tested making use of the near-UV and visible reflectance observations made by the Ozone Monitoring Instrument (OMI) and Moderate Resolution Imaging Spectroradiometer (MODIS) for distinct above-cloud smoke and dust aerosol events observed seasonally over the southeast and tropical Atlantic Ocean

  17. Comparison of Quantitative Wall Motion Analysis and Strain For Detection Of Coronary Stenosis With Three-Dimensional Dobutamine Stress Echocardiography

    Science.gov (United States)

    Parker, Katherine M.; Clark, Alexander P.; Goodman, Norman C.; Glover, David K.; Holmes, Jeffrey W.

    2015-01-01

    Background Quantitative analysis of wall motion from three-dimensional (3D) dobutamine stress echocardiography (DSE) could provide additional diagnostic information not available from qualitative analysis. In this study we compare the effectiveness of 3D fractional shortening (3DFS), a measure of wall motion computed from 3D echocardiography (3DE), to strain and strain rate measured with sonomicrometry for detecting critical stenoses during DSE. Methods Eleven open-chest dogs underwent DSE both with and without a critical stenosis. 3DFS was measured from 3DE images acquired at peak stress. 3DFS was normalized by subtracting average 3DFS during control peak stress (Δ3DFS). Strains in the perfusion defect (PD) were measured from sonomicrometry, and PD size and location were measured with microspheres. Results A Δ3DFS abnormality indicated the presence of a critical stenosis with high sensitivity and specificity (88% and 100%, respectively), and Δ3DFS abnormality size correlated with PD size (R2=0.54). The sensitivity and specificity for Δ3DFS was similar to that for area strain (88%, 100%) and circumferential strain and strain rate (88%, 92% and 88%, 86%, respectively), while longitudinal strain and strain rate were less specific. Δ3DFS correlated significantly with both coronary flow reserve (R2=0.71) and PD size (R2=0.97), while area strain correlated with PD size only (R2=0.67), and other measures were not significantly correlated with flow reserve or PD size. Conclusion Quantitative wall motion analysis using Δ3DFS is effective for detecting critical stenoses during DSE, performing similarly to 3D strain, and provides potentially useful information on the size and location of a perfusion defect. PMID:24815588

  18. Development and validation of a quantitative, high-throughput, fluorescent-based bioassay to detect schistosoma viability.

    Directory of Open Access Journals (Sweden)

    Emily Peak

    2010-07-01

    Full Text Available Schistosomiasis, caused by infection with the blood fluke Schistosoma, is responsible for greater than 200,000 human deaths per annum. Objective high-throughput screens for detecting novel anti-schistosomal targets will drive 'genome to drug' lead translational science at an unprecedented rate. Current methods for detecting schistosome viability rely on qualitative microscopic criteria, which require an understanding of parasite morphology, and most importantly, must be subjectively interpreted. These limitations, in the current state of the art, have significantly impeded progress into whole schistosome screening for next generation chemotherapies.We present here a microtiter plate-based method for reproducibly detecting schistosomula viability that takes advantage of the differential uptake of fluorophores (propidium iodide and fluorescein diacetate by living organisms. We validate this high-throughput system in detecting schistosomula viability using auranofin (a known inhibitor of thioredoxin glutathione reductase, praziquantel and a range of small compounds with previously-described (gambogic acid, sodium salinomycin, ethinyl estradiol, fluoxetidine hydrochloride, miconazole nitrate, chlorpromazine hydrochloride, amphotericin b, niclosamide or suggested (bepridil, ciclopirox, rescinnamine, flucytosine, vinblastine and carbidopa anti-schistosomal activities. This developed method is sensitive (200 schistosomula/well can be assayed, relevant to industrial (384-well microtiter plate compatibility and academic (96-well microtiter plate compatibility settings, translatable to functional genomics screens and drug assays, does not require a priori knowledge of schistosome biology and is quantitative.The wide-scale application of this fluorescence-based bioassay will greatly accelerate the objective identification of novel therapeutic lead targets/compounds to combat schistosomiasis. Adapting this bioassay for use with other parasitic worm species

  19. Comparison of nested PCR and qPCR for the detection and quantitation of BoHV6 DNA.

    Science.gov (United States)

    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.

  20. Nuclear Magnetic Resonance (NMR Study for the Detection and Quantitation of Cholesterol in HSV529 Therapeutic Vaccine Candidate

    Directory of Open Access Journals (Sweden)

    Rahima Khatun

    Full Text Available This study describes the NMR-based method to determine the limit of quantitation (LOQ and limit of detection (LOD of cholesterol, a process-related impurity in the replication-deficient Herpes Simplex Virus (HSV type 2 candidate vaccine HSV529. Three signature peaks from the 1D 1H NMR of a cholesterol reference spectrum were selected for the identification of cholesterol. The LOQ for a cholesterol working standard was found to be 1 μg/mL, and the LOD was found to be 0.1 μg/mL. The identity of cholesterol, separated from the formulation of growth supplement by thin layer chromatography (TLC, was confirmed by 1D 1H NMR and 2D 1H-13C HSQC NMR. The three signature peaks of cholesterol were detected only in a six-times concentrated sample of HSV529 candidate vaccine sample and not in the single dose HSV529 vaccine sample under similar experimental conditions. Taken together, the results demonstrated that NMR is a direct method that can successfully identify and quantify cholesterol in viral vaccine samples, such as HSV529, and as well as in the growth supplement used during the upstream stages of HSV529 manufacturing. Keywords: Herpes simplex virus type 2 (HSV-2, Viral vaccine, NMR, Residuals, LOD and LOQ, TLC, Growth supplement

  1. Detection and quantitative analysis of actin mRNA by in situ hybridization with an oligodeoxynucleotide probe

    International Nuclear Information System (INIS)

    Taneja, K.; Singer, R.

    1987-01-01

    In situ hybridization is a useful method for localizing specific nucleic acid sequences intracellularly and for studying regulation of gene expression. Recently synthetic oligonucleotides have been successfully used as probes in this technique. Since they can be made easily to specific nucleic acid regions, they may be the best approach for analysis of a gene family of highly conserved sequences. They have analyzed these probes for the development of an in situ hybridization method. Oligonucleotides were made to different regions of chick beta-actin mRNA and used for detection of these sequences in a culture of chicken fibroblasts and myoblasts. They found that synthetic DNAs have different efficiencies of hybridization, indicating that not all target sequences are equivalent. They have investigated in detail a particular probe to the actin mRNA coding region and have optimized hybridization parameters. When hybridization was quantitated it was found that an oligonucleotide end labelled with 35 S or 32 P was capable of detecting several thousand messages per cell with a signal-to-noise ratio of 10:1. In situ hybridization confirmed the specificity of the hybridization as well as the background level. Increase in the number of oligonucleotides used should increase the signal-to-noise ratio-proportionately. Under particular circumstances the specificity of oligonucleotides make them an important reagent for in situ hybridization

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

    Science.gov (United States)

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

    2015-07-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  7. Applying quantitative metabolomics based on chemical isotope labeling LC-MS for detecting potential milk adulterant in human milk.

    Science.gov (United States)

    Mung, Dorothea; Li, Liang

    2018-02-25

    There is an increasing demand for donor human milk to feed infants for various reasons including that a mother may be unable to provide sufficient amounts of milk for their child or the milk is considered unsafe for the baby. Selling and buying human milk via the Internet has gained popularity. However, there is a risk of human milk sold containing other adulterants such as animal or plant milk. Analytical tools for rapid detection of adulterants in human milk are needed. We report a quantitative metabolomics method for detecting potential milk adulterants (soy, almond, cow, goat and infant formula milk) in human milk. It is based on the use of a high-performance chemical isotope labeling (CIL) LC-MS platform to profile the metabolome of an unknown milk sample, followed by multivariate or univariate comparison of the resultant metabolomic profile with that of human milk to determine the differences. Using dansylation LC-MS to profile the amine/phenol submetabolome, we could detect an average of 4129 ± 297 (n = 9) soy metabolites, 3080 ± 470 (n = 9) almond metabolites, 4256 ± 136 (n = 18) cow metabolites, 4318 ± 198 (n = 9) goat metabolites, 4444 ± 563 (n = 9) infant formula metabolites, and 4020 ± 375 (n = 30) human metabolites. This high level of coverage allowed us to readily differentiate the six different types of samples. From the analysis of binary mixtures of human milk containing 5, 10, 25, 50 and 75% other type of milk, we demonstrated that this method could be used to detect the presence of as low as 5% adulterant in human milk. We envisage that this method could be applied to detect contaminant or adulterant in other types of food or drinks. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  9. Assessment of quantitative FDG PET data in primary colorectal tumours: which parameters are important with respect to tumour detection?

    International Nuclear Information System (INIS)

    Strauss, Ludwig G.; Pan, Leyun; Dimitrakopoulou-Strauss, Antonia; Klippel, Sven; Schoenleben, Klaus; Haberkorn, Uwe

    2007-01-01

    The impact of quantitative parameters on the differentiation of primary colorectal tumours from normal colon tissue was assessed. Dynamic PET data (DPET) were acquired, and compartment and non-compartment modelling applied. The discriminant power of single parameters and the combination of PET parameters was assessed. All lesions were confirmed by histology. FDG DPET studies were acquired in 22 patients with colorectal tumours prior to surgery. Five of these patients also had liver metastases at the time of the PET study. The SUV 56-60 min p.i. was included in the evaluation. A two-tissue compartment model was applied and the parameters k 1 -k 4 as well as the fractional blood volume (V B ) were obtained. The FDG influx was calculated from the compartment data. Non-compartment modelling was used to calculate the fractal dimension (FD) of the time-activity data. FD, SUV, influx and k 3 were the most important single parameters for lesion differentiation. The highest accuracy was achieved for FD (88.78%). The overall tracer uptake was mainly dependent on k 3 and not on k 1 or V B . The support vector machines (SVM) algorithm was used to predict the classification based on the combination of individual PET parameters. The overall accuracy was 97.3%, with only one false positive case and no false negative results. The analysis of the subgroup of five patients with primary tumours and synchronous metastases revealed no significant differences for the individual PET parameters. However, V B tended to be lower while k 1 and k 2 were higher in patients with synchronous metastases. The SVM classification analysis predicted the presence of metastases based on the PET data of the primary tumour in three of five patients. Quantitative FDG PET studies provide very accurate data for the differentiation of primary colorectal tumours from normal tissue. The use of quantitative data has the advantage that the detection of a colorectal tumour is not primarily dependent on the

  10. Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation

    Directory of Open Access Journals (Sweden)

    Burgos Lorenzo

    2010-07-01

    Full Text Available Abstract Background The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently. Results We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (nptII transgene as well as of an internal control (β-actin, used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the nptII transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the gus marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus. Conclusions We have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot

  11. Development and Evaluation of a PCR and Mass Spectroscopy-based (PCR-MS) Method for Quantitative, Type-specific Detection of Human Papillomavirus

    Science.gov (United States)

    Patel, Divya A.; Shih, Yang-Jen; Newton, Duane W.; Michael, Claire W.; Oeth, Paul A.; Kane, Michael D.; Opipari, Anthony W.; Ruffin, Mack T.; Kalikin, Linda M.; Kurnit, David M.

    2010-01-01

    Knowledge of the central role of high-risk human papillomavirus (HPV) in cervical carcinogenesis, coupled with an emerging need to monitor the efficacy of newly introduced HPV vaccines, warrant development and evaluation of type-specific, quantitative HPV detection methods. In the present study, a prototype PCR and mass spectroscopy (PCR-MS)-based method to detect and quantitate 13 high-risk HPV types is compared to the Hybrid Capture 2 High Risk HPV DNA test (HC2; Digene Corp., Gaithersburg, MD) in 199 cervical scraping samples and to DNA sequencing in 77 cervical tumor samples. High-risk HPV types were detected in 76/77 (98.7%) cervical tumor samples by PCR-MS. Degenerate and type-specific sequencing confirmed the types detected by PCR-MS. In 199 cervical scraping samples, all 13 HPV types were detected by PCR-MS. Eighteen (14.5%) of 124 cervical scraping samples that were positive for high-risk HPV by HC2 were negative by PCR-MS. In all these cases, degenerate DNA sequencing failed to detect any of the 13 high-risk HPV types. Nearly half (46.7%) of the 75 cervical scraping samples that were negative for high-risk HPV by the HC2 assay were positive by PCR-MS. Type-specific sequencing in a subset of these samples confirmed the HPV type detected by PCR-MS. Quantitative PCR-MS results demonstrated that 11/75 (14.7%) samples contained as much HPV copies/cell as HC2-positive samples. These findings suggest that this prototype PCR-MS assay performs at least as well as HC2 for HPV detection, while offering the additional, unique advantages of type-specific identification and quantitation. Further validation work is underway to define clinically meaningful HPV detection thresholds and to evaluate the potential clinical application of future generations of the PCR-MS assay. PMID:19410602

  12. Development and evaluation of a PCR and mass spectroscopy (PCR-MS)-based method for quantitative, type-specific detection of human papillomavirus.

    Science.gov (United States)

    Patel, Divya A; Shih, Yang-Jen; Newton, Duane W; Michael, Claire W; Oeth, Paul A; Kane, Michael D; Opipari, Anthony W; Ruffin, Mack T; Kalikin, Linda M; Kurnit, David M

    2009-09-01

    Knowledge of the central role of high-risk human papillomavirus (HPV) in cervical carcinogenesis, coupled with an emerging need to monitor the efficacy of newly introduced HPV vaccines, warrant development and evaluation of type-specific, quantitative HPV detection methods. In the present study, a prototype PCR and mass spectroscopy (PCR-MS)-based method to detect and quantitate 13 high-risk HPV types is compared to the Hybrid Capture 2 High-Risk HPV DNA test (HC2; Digene Corp., Gaithersburg, MD) in 199 cervical scraping samples and to DNA sequencing in 77 cervical tumor samples. High-risk HPV types were detected in 76/77 (98.7%) cervical tumor samples by PCR-MS. Degenerate and type-specific sequencing confirmed the types detected by PCR-MS. In 199 cervical scraping samples, all 13 HPV types were detected by PCR-MS. Eighteen (14.5%) of 124 cervical scraping samples that were positive for high-risk HPV by HC2 were negative by PCR-MS. In all these cases, degenerate DNA sequencing failed to detect any of the 13 high-risk HPV types. Nearly half (46.7%) of the 75 cervical scraping samples that were negative for high-risk HPV by the HC2 assay were positive by PCR-MS. Type-specific sequencing in a subset of these samples confirmed the HPV type detected by PCR-MS. Quantitative PCR-MS results demonstrated that 11/75 (14.7%) samples contained as much HPV copies/cell as HC2-positive samples. These findings suggest that this prototype PCR-MS assay performs at least as well as HC2 for HPV detection, while offering the additional, unique advantages of type-specific identification and quantitation. Further validation work is underway to define clinically meaningful HPV detection thresholds and to evaluate the potential clinical application of future generations of the PCR-MS assay.

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

    Science.gov (United States)

    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

  14. Using the Quantitative Literacy and Reasoning Assessment (QLRA for Early Detection of Students in Need of Academic Support in Introductory Courses in a Quantitative Discipline: A Case Study

    Directory of Open Access Journals (Sweden)

    Nathan Grawe

    2018-01-01

    Full Text Available As the number of young people attending college has increased, the diversity of college students� educational backgrounds has also risen. Some students enter introductory courses with math anxiety or gaps in their quantitative training that impede their ability to master or even grasp relevant disciplinary content. Too often professors learn of these anxieties and gaps only during the post mortem of the first midterm. By that time, a good portion of a student�s grade is determined and successful recovery may be impossible. During the 2016-17 academic year, the Department of Economics at Carleton College ran a pilot project using the Quantitative Literacy and Reasoning Assessment (QLRA as a pre-course diagnostic tool. Results show that the QLRA predicts student grades even after controlling for other SAT/ACT math scores and overall GPA. This finding suggests that quantitative reasoning is an important input into success in Principles of Economics (both Macro and Micro. When the QLRA alone is used to predict success in a course (as defined by either a grade of C- or better, or a grade of B- or better, we find that we could nearly always pick out students who were on the way to sub-par performance. On the other hand, the tool has a fairly high false positive rate; almost half of students identified as �at risk� based on QLRA performance went on to earn a successful grade in the course. In total, we argue that the QLRA may be a useful and inexpensive early-warning device for introductory courses in economics; it may be worth exploring a similar use of the instrument in other disciplinary settings where introductory courses require quantitative reasoning.

  15. Modification of a Pollen Trap Design To Capture Airborne Conidia of Entomophaga maimaiga and Detection of Conidia by Quantitative PCR.

    Science.gov (United States)

    Bittner, Tonya D; Hajek, Ann E; Liebhold, Andrew M; Thistle, Harold

    2017-09-01

    The goal of this study was to develop effective and practical field sampling methods for quantification of aerial deposition of airborne conidia of Entomophaga maimaiga over space and time. This important fungal pathogen is a major cause of larval death in invasive gypsy moth ( Lymantria dispar ) populations in the United States. Airborne conidia of this pathogen are relatively large (similar in size to pollen), with unusual characteristics, and require specialized methods for collection and quantification. Initially, dry sampling (settling of spores from the air onto a dry surface) was used to confirm the detectability of E. maimaiga at field sites with L. dispar deaths caused by E. maimaiga , using quantitative PCR (qPCR) methods. We then measured the signal degradation of conidial DNA on dry surfaces under field conditions, ultimately rejecting dry sampling as a reliable method due to rapid DNA degradation. We modified a chamber-style trap commonly used in palynology to capture settling spores in buffer. We tested this wet-trapping method in a large-scale (137-km) spore-trapping survey across gypsy moth outbreak regions in Pennsylvania undergoing epizootics, in the summer of 2016. Using 4-day collection periods during the period of late instar and pupal development, we detected variable amounts of target DNA settling from the air. The amounts declined over the season and with distance from the nearest defoliated area, indicating airborne spore dispersal from outbreak areas. IMPORTANCE We report on a method for trapping and quantifying airborne spores of Entomophaga maimaiga , an important fungal pathogen affecting gypsy moth ( Lymantria dispar ) populations. This method can be used to track dispersal of E. maimaiga from epizootic areas and ultimately to provide critical understanding of the spatial dynamics of gypsy moth-pathogen interactions. Copyright © 2017 American Society for Microbiology.

  16. A lab-on-a-chip system with integrated sample preparation and loop-mediated isothermal amplification for rapid and quantitative detection of Salmonella spp. in food samples

    DEFF Research Database (Denmark)

    Sun, Yi; Than Linh, Quyen; Hung, Tran Quang

    2015-01-01

    was capable to detect Salmonella at concentration of 50 cells per test within 40 min. The simple design, together with high level of integration, isothermal amplification, and quantitative analysis of multiple samples in short time will greatly enhance the practical applicability of the LOC system for rapid...... amplification (LAMP) for rapid and quantitative detection of Salmonella spp. in food samples. The whole diagnostic procedures including DNA isolation, isothermal amplification, and real-time detection were accomplished in a single chamber. Up to eight samples could be handled simultaneously and the system...... and usually take a few hours to days to complete. In response to the demand for rapid on line or at site detection of pathogens, in this study, we describe for the first time an eight-chamber lab-on-a-chip (LOC) system with integrated magnetic beads-based sample preparation and loop-mediated isothermal...

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

    Directory of Open Access Journals (Sweden)

    Straube Eberhard

    2010-04-01

    Full Text Available Abstract Background The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. Therefore, optimised DNA extraction methods are critical. The aim of this study was to assess the performance of commercial kits for the extraction of Brucella DNA. Methods Five kits were evaluated using clinical specimens: QIAamp™ DNA Mini Kit (QIAGEN, peqGold™ Tissue DNA Mini Kit (PeqLab, UltraClean™ Tissue and Cells DNA Isolation Kit (MoBio, DNA Isolation Kit for Cells and Tissues (Roche, and NucleoSpin™ Tissue (Macherey-Nagel. DNA yield was determined using a quantitative real-time PCR assay targeting IS711 that included an internal amplification control. Results Kits of QIAGEN and Roche provided the highest amount of DNA, Macherey-Nagel and Peqlab products were intermediate whereas MoBio yielded the lowest amount of DNA. Differences were significant (p Conclusions We observed differences in DNA yield as high as two orders of magnitude for some samples between the best and the worst DNA extraction kits and inhibition was observed occasionally. This indicates that DNA purification may be more relevant than expected when the amount of DNA in tissue is very low.

  18. Lab-on-capillary: a rapid, simple and quantitative genetic analysis platform integrating nucleic acid extraction, amplification and detection.

    Science.gov (United States)

    Fu, Yu; Zhou, Xiaoming; Xing, Da

    2017-12-05

    In this work, we describe for the first time a genetic diagnosis platform employing a polydiallyldimethylammonium chloride (PDDA)-modified capillary and a liquid-based thermalization system for rapid, simple and quantitative DNA analysis with minimal user interaction. Positively charged PDDA is modified on the inner surface of the silicon dioxide capillary by using an electrostatic self-assembly approach that allows the negatively charged DNA to be separated from the lysate in less than 20 seconds. The capillary loaded with the PCR mix is incorporated in the thermalization system, which can achieve on-site real-time PCR. This system is based on the circulation of pre-heated liquids in the chamber, allowing for high-speed thermalization of the capillary and fast amplification. Multiple targets can be simultaneously analysed with multiplex spatial melting. Starting with live Escherichia coli (E. coli) cells in milk, as a realistic sample, the current method can achieve DNA extraction, amplification, and detection within 40 min.

  19. Quantitative analysis of mycosporine-like amino acids in marine algae by capillary electrophoresis with diode-array detection

    Science.gov (United States)

    Hartmann, Anja; Murauer, Adele; Ganzera, Markus

    2017-01-01

    Marine species have evolved a variety of physical or chemical strategies to diminish damage from elevated environmental ultraviolet radiation. Mycosporine-like amino acids, a group of widely distributed small water soluble compounds, are biologically relevant because of their photo-protective potential. In addition, presumed antioxidant and skin protective strategies raise the interest for possible medicinal and cosmetic applications. In this study the first CE method for the quantification of mycosporine-like amino acids in marine species is presented. A borate buffer system consisting of 30 mM sodium tetraborate in water at a pH-value of 10.3 enabled the baseline separation of five MAAs, namely palythine, mycosporine-serinol, asterina-330, shinorine and porphyra-334, in 27 min. Separation voltage, temperature and detection wavelength were 25 kV, 25 °C and 320 nm, respectively. The optimized method was fully validated and applied for the quantitative determination of MAAs in the marine macroalgae Palmaria palmata, Porphyra umbilicalis, and Porphyra sp., as well as the lichen Lichina pygmaea. PMID:28213175

  20. Manufacture and Testing of a High Field Gradient Magnetic Fractionation System for Quantitative Detection of Plasmodium falciparum Gametocytes

    Science.gov (United States)

    Karl, Stephan; Woodward, Robert C.; Davis, Timothy M. E.; St. Pierre, Tim G.

    2010-12-01

    Plasmodium falciparum is the most dangerous of the human malaria parasite species and accounts for millions of clinical episodes of malaria each year in tropical countries. The pathogenicity of Plasmodium falciparum is a result of its ability to infect erythrocytes where it multiplies asexually over 48 h or develops into sexual forms known as gametocytes. If sufficient male and female gametocytes are taken up by a mosquito vector, it becomes infectious. Therefore, the presence and density of gametocytes in human blood is an important indicator of human-to-mosquito transmission of malaria. Recently, we have shown that high field gradient magnetic fractionation improves gametocyte detection in human blood samples. Here we present two important new developments. Firstly we introduce a quantitative approach to replace the previous qualitative method and, secondly, we describe a novel method that enables cost-effective production of the magnetic fractionation equipment required to carry out gametocyte quantification. We show that our custom-made magnetic fractionation equipment can deliver results with similar sensitivity and convenience but for a small fraction of the cost.

  1. Detection of quantitative trait loci causing abnormal spermatogenesis and reduced testis weight in the small testis (Smt) mutant mouse.

    Science.gov (United States)

    Bolor, Hasbaira; Wakasugi, Noboru; Zhao, Wei Dong; Ishikawa, Akira

    2006-04-01

    The small testis (Smt) mutant mouse is characterized by a small testis of one third to one half the size of a normal testis, and its spermatogenesis is mostly arrested at early stages of meiosis, although a small number of spermatocytes at the late prophase of meiosis and a few spermatids can sometimes be seen. We performed quantitative trait locus (QTL) analysis of these spermatogenic traits and testis weight using 221 F2 males obtained from a cross between Smt and MOM (Mus musculus molossinus) mice. At the genome-wide 5% level, we detected two QTLs affecting meiosis on chromosomes 4 and 13, and two QTLs for paired testis weight as a percentage of body weight on chromosomes 4 and X. In addition, we found several QTLs for degenerated germ cells and multinuclear giant cells on chromosomes 4, 7 and 13. Interestingly, for cell degeneration, the QTL on chromosome 13 interacted epistatically with the QTL on chromosome 4. These results reveal polygenic participation in the abnormal spermatogenesis and small testis size in the Smt mutant.

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

    Science.gov (United States)

    Abdeldaim, Guma M K; Strålin, Kristoffer; Kirsebom, Leif A; Olcén, Per; Blomberg, Jonas; Herrmann, Björn

    2009-08-01

    A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10(4) DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

  3. Detection and quantitative determination of heavy metals in electronic cigarette refill liquids using Total Reflection X-ray Fluorescence Spectrometry.

    Science.gov (United States)

    Kamilari, Eleni; Farsalinos, Konstantinos; Poulas, Konstantinos; Kontoyannis, Christos G; Orkoula, Malvina G

    2018-06-01

    Electronic cigarettes are considered healthier alternatives to conventional cigarettes containing tobacco. They produce vapor through heating of the refill liquids (e-liquids) which consist of propylene glycol, vegetable glycerin, nicotine (in various concentrations), water and flavoring agents. Heavy metals may enter the refill liquid during the production, posing a risk for consumer's health due to their toxicity. The objective of the present study was the development of a methodology for the detection and quantitative analysis of cadmium (Cd), lead (Pb), nickel (Ni), copper (Cu), arsenic (As) and chromium (Cr), employing Total Reflection X-Ray Fluorescence Spectroscopy (TXRF) as an alternative technique to ICP-MS or ICP-OES commonly used for this type of analysis. TXRF was chosen due to its advantages, which include short analysis time, promptness, simultaneous multi-element analysis capability and minimum sample preparation, low purchase and operational cost. The proposed methodology was applied to a large number of electronic cigarette liquids commercially available, as well as their constituents, in order to evaluate their safety. TXRF may be a valuable tool for probing heavy metals in electronic cigarette refill liquids to serve for the protection of human health. Copyright © 2018 Elsevier Ltd. All rights reserved.

  4. A versatile method to design stem-loop primer-based quantitative PCR assays for detecting small regulatory RNA molecules.

    Directory of Open Access Journals (Sweden)

    Zsolt Czimmerer

    Full Text Available Short regulatory RNA-s have been identified as key regulators of gene expression in eukaryotes. They have been involved in the regulation of both physiological and pathological processes such as embryonal development, immunoregulation and cancer. One of their relevant characteristics is their high stability, which makes them excellent candidates for use as biomarkers. Their number is constantly increasing as next generation sequencing methods reveal more and more details of their synthesis. These novel findings aim for new detection methods for the individual short regulatory RNA-s in order to be able to confirm the primary data and characterize newly identified subtypes in different biological conditions. We have developed a flexible method to design RT-qPCR assays that are very sensitive and robust. The newly designed assays were tested extensively in samples from plant, mouse and even human formalin fixed paraffin embedded tissues. Moreover, we have shown that these assays are able to quantify endogenously generated shRNA molecules. The assay design method is freely available for anyone who wishes to use a robust and flexible system for the quantitative analysis of matured regulatory RNA-s.

  5. Quantitative analysis of computed tomography images and early detection of cerebral edema for pediatric traumatic brain injury patients: retrospective study.

    Science.gov (United States)

    Kim, Hakseung; Kim, Gwang-dong; Yoon, Byung C; Kim, Keewon; Kim, Byung-Jo; Choi, Young Hun; Czosnyka, Marek; Oh, Byung-Mo; Kim, Dong-Joo

    2014-10-22

    The purpose of this study was to identify whether the distribution of Hounsfield Unit (HU) values across the intracranial area in computed tomography (CT) images can be used as an effective diagnostic tool for determining the severity of cerebral edema in pediatric traumatic brain injury (TBI) patients. CT images, medical records and radiology reports on 70 pediatric patients were collected. Based on radiology reports and the Marshall classification, the patients were grouped as mild edema patients (n=37) or severe edema patients (n=33). Automated quantitative analysis using unenhanced CT images was applied to eliminate artifacts and identify the difference in HU value distribution across the intracranial area between these groups. The proportion of pixels with HU=17 to 24 was highly correlated with the existence of severe cerebral edema (P<0.01). This proportion was also able to differentiate patients who developed delayed cerebral edema from mild TBI patients. A significant difference between deceased patients and surviving patients in terms of the HU distribution came from the proportion of pixels with HU=19 to HU=23 (P<0.01). The proportion of pixels with an HU value of 17 to 24 in the entire cerebral area of a non-enhanced CT image can be an effective basis for evaluating the severity of cerebral edema. Based on this result, we propose a novel approach for the early detection of severe cerebral edema.

  6. Quantitative Detection of ID4 Gene Aberrant Methylation in the Differentiation of Myelodysplastic Syndrome from Aplastic Anemia

    Directory of Open Access Journals (Sweden)

    Mian-Yang Li

    2015-01-01

    Full Text Available Background: The diagnosis of myelodysplastic syndrome (MDS, especially hypoplastic MDS, and MDS with low blast counts or normal karyotype may be problematic. This study characterized ID4 gene methylation in patients with MDS and aplastic anemia (AA. Methods: The methylation status of ID4 was analyzed by bisulfite sequencing polymerase chain reaction (PCR and quantitative real-time methylation-specific PCR (MethyLight PCR in 100 patients with MDS and 31 patients with AA. Results: The MDS group had a higher ID4 gene methylation positivity rate (22.22% and higher methylation levels (0.21 [0-3.79] than the AA group (P < 0.05. Furthermore, there were significant differences between the hypoplastic MDS and AA groups, the MDS with low blast count and the AA groups, and the MDS with normal karyotype and the AA groups. The combination of genetic and epigenetic markers was used in much more patients with MDS (62.5% [35/56] than the use of genetic markers only (51.79% [29/56]. Conclusions: These results showed that the detection of ID4 methylation positivity rates and levels could be a useful biomarker for MDS diagnosis.

  7. Applicability of integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) for the simultaneous detection of the four human enteric enterovirus species in disinfection studies

    Science.gov (United States)

    A newly developed integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method and its applicability in UV disinfection studies is described. This method utilizes a singular cell culture system coupled with four RTqPCR assays to detect infectious serotypes t...

  8. Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs.

    Science.gov (United States)

    Solcà, Manuela da Silva; Guedes, Carlos Eduardo Sampaio; Nascimento, Eliane Gomes; Oliveira, Geraldo Gileno de Sá; dos Santos, Washington Luis Conrado; Fraga, Deborah Bittencourt Mothé; Veras, Patrícia Sampaio Tavares

    2012-03-23

    Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic

  9. Effect of Genital Sampling Site on the Detection and Quantification of Ureaplasma Species with Quantitative Polymerase Chain Reaction during Pregnancy

    OpenAIRE

    Faron, Gilles; Vancutsem, Ellen; Naessens, Anne; Buyl, Ronald; Gucciardo, Leonardo; Foulon, Walter

    2017-01-01

    Objective. This study aimed to compare the qualitative and quantitative reproducibility of quantitative PCR (qPCR) for Ureaplasma species (Ureaplasma spp.) throughout pregnancy and according to the genital sampling site. Study Design. Between 5 and 14 weeks of gestation (T1), vaginal, fornix, and two cervical samples were taken. Sampling was repeated during the 2nd (T2) and 3rd (T3) trimester in randomly selected T1 positive and negative women. Qualitative and quantitative reproducibility wer...

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

    Directory of Open Access Journals (Sweden)

    Ralevski Filip

    2009-12-01

    Full Text Available Abstract Background Accurate laboratory diagnosis of malaria species in returning travelers is paramount in the treatment of this potentially fatal infectious disease. Materials and methods A total of 466 blood specimens from returning travelers to Africa, Asia, and South/Central America with suspected malaria infection were collected between 2007 and 2009 at the reference public health laboratory. These specimens were assessed by reference microscopy, multipex real-time quantitative polymerase chain reaction (QPCR, and two rapid diagnostic immuno-chromatographic tests (ICT in a blinded manner. Key clinical laboratory parameters such as limit of detection (LOD analysis on clinical specimens by parasite stage, inter-reader variability of ICTs, staffing implications, quality assurance and cost analysis were evaluated. Results QPCR is the most analytically sensitive method (sensitivity 99.41%, followed by CARESTART (sensitivity 88.24%, and BINAXNOW (sensitivity 86.47% for the diagnosis of malaria in returning travelers when compared to reference microscopy. However, microscopy was unable to specifically identify Plasmodia spp. in 18 out of 170 positive samples by QPCR. Moreover, the 17 samples that were negative by microscopy and positive by QPCR were also positive by ICTs. Quality assurance was achieved for QPCR by exchanging a blinded proficiency panel with another reference laboratory. The Kappa value of inter-reader variability among three readers for BINAXNOW and CARESTART was calculated to be 0.872 and 0.898 respectively. Serial dilution studies demonstrated that the QPCR cycle threshold correlates linearly with parasitemia (R2 = 0.9746 in a clinically relevant dynamic range and retains a LOD of 11 rDNA copies/μl for P. falciparum, which was several log lower than reference microscopy and ICTs. LOD for QPCR is affected not only by parasitemia but the parasite stage distribution of each clinical specimen. QPCR was approximately 6-fold more

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

    International Nuclear Information System (INIS)

    Huang, S-H; Tsai, M-H; Lin, C-W; Yang, T-C; Chuang, P-H; Tsai, I-S; Lu, H-C; Wan Lei; Lin, Y-J; Lai, C-H

    2008-01-01

    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

  12. Rapid and quantitative detection of the microbial spoilage of meat by fourier transform infrared spectroscopy and machine learning.

    Science.gov (United States)

    Ellis, David I; Broadhurst, David; Kell, Douglas B; Rowland, Jem J; Goodacre, Royston

    2002-06-01

    Fourier transform infrared (FT-IR) spectroscopy is a rapid, noninvasive technique with considerable potential for application in the food and related industries. We show here that this technique can be used directly on the surface of food to produce biochemically interpretable "fingerprints." Spoilage in meat is the result of decomposition and the formation of metabolites caused by the growth and enzymatic activity of microorganisms. FT-IR was exploited to measure biochemical changes within the meat substrate, enhancing and accelerating the detection of microbial spoilage. Chicken breasts were purchased from a national retailer, comminuted for 10 s, and left to spoil at room temperature for 24 h. Every hour, FT-IR measurements were taken directly from the meat surface using attenuated total reflectance, and the total viable counts were obtained by classical plating methods. Quantitative interpretation of FT-IR spectra was possible using partial least-squares regression and allowed accurate estimates of bacterial loads to be calculated directly from the meat surface in 60 s. Genetic programming was used to derive rules showing that at levels of 10(7) bacteria.g(-1) the main biochemical indicator of spoilage was the onset of proteolysis. Thus, using FT-IR we were able to acquire a metabolic snapshot and quantify, noninvasively, the microbial loads of food samples accurately and rapidly in 60 s, directly from the sample surface. We believe this approach will aid in the Hazard Analysis Critical Control Point process for the assessment of the microbiological safety of food at the production, processing, manufacturing, packaging, and storage levels.

  13. Identification of pyrG Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR Detection of Pleurotus ostreatus.

    Science.gov (United States)

    Zheng, Shi; Shan, Luying; Zhuang, Yongliang; Shang, Ying

    2018-03-01

    As a well-known edible fungus rich in nutrients, Pleurotus ostreatus has been used as an alternative to expensive wild edible fungi. Specifically, the fact that using P. ostreatus instead of other expensive wild edible fungi has damaged the rights and interests of consumers. Among the existing methods for detection of food adulteration, the amplification of endogenous reference gene is the most accurate method. However, an ideal endogenous reference gene for P. ostreatus has yet to be developed. In this study, a DNA extraction method for P. ostreatus was optimized, and pyrG was selected as a species-specific gene through sequence alignment. This gene was subsequently subjected to qualitative and quantitative Polymerase Chain Reaction (PCR) assays with 3 different P. ostreatus varieties and 7 other species. A low detection limit of 5 pg/μL was obtained by TaqMan quantitative PCR, and no pyrG amplification product was observed in the 7 other species. No allelic variation was detected in P. ostreatus varieties. These experiments confirmed that pyrG was an ideal endogenous reference gene for the qualitative and real-time quantitative PCR detection of P. ostreatus. This method was also suitable for the examination of processed P. ostreatus samples and determination of adulteration in wild mushrooms. The pyrG gene was chosen as an ideal endogenous reference gene for the qualitative and real-time quantitative PCR detection of P. ostreatus, and the detection limit was 5 pg/μL for the quantification. This method is used not only for raw materials but also for processed P. ostreatus products and other processed mushroom foods. © 2018 Institute of Food Technologists®.

  14. Detection and differentiation of early acute and following age stages of myocardial infarction with quantitative post-mortem cardiac 1.5T MR.

    Science.gov (United States)

    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.

  15. CdSe/ZnS Quantum Dot-Labeled Lateral Flow Strips for Rapid and Quantitative Detection of Gastric Cancer Carbohydrate Antigen 72-4

    Science.gov (United States)

    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.

  16. Cellular phone-based image acquisition and quantitative ratiometric method for detecting cocaine and benzoylecgonine for biological and forensic applications.

    Science.gov (United States)

    Cadle, Brian A; Rasmus, Kristin C; Varela, Juan A; Leverich, Leah S; O'Neill, Casey E; Bachtell, Ryan K; Cooper, Donald C

    2010-01-01

    Here we describe the first report of using low-cost cellular or web-based digital cameras to image and quantify standardized rapid immunoassay strips as a new point-of-care diagnostic and forensics tool with health applications. Quantitative ratiometric pixel density analysis (QRPDA) is an automated method requiring end-users to utilize inexpensive (∼ $1 USD/each) immunotest strips, a commonly available web or mobile phone camera or scanner, and internet or cellular service. A model is described whereby a central computer server and freely available IMAGEJ image analysis software records and analyzes the incoming image data with time-stamp and geo-tag information and performs the QRPDA using custom JAVA based macros (http://www.neurocloud.org). To demonstrate QRPDA we developed a standardized method using rapid immunotest strips directed against cocaine and its major metabolite, benzoylecgonine. Images from standardized samples were acquired using several devices, including a mobile phone camera, web cam, and scanner. We performed image analysis of three brands of commercially available dye-conjugated anti-cocaine/benzoylecgonine (COC/BE) antibody test strips in response to three different series of cocaine concentrations ranging from 0.1 to 300 ng/ml and BE concentrations ranging from 0.003 to 0.1 ng/ml. This data was then used to create standard curves to allow quantification of COC/BE in biological samples. Across all devices, QRPDA quantification of COC and BE proved to be a sensitive, economical, and faster alternative to more costly methods, such as gas chromatography-mass spectrometry, tandem mass spectrometry, or high pressure liquid chromatography. The limit of detection was determined to be between 0.1 and 5 ng/ml. To simulate conditions in the field, QRPDA was found to be robust under a variety of image acquisition and testing conditions that varied temperature, lighting, resolution, magnification and concentrations of biological fluid in a sample. To

  17. Cellular Phone-Based Image Acquisition and Quantitative Ratiometric Method for Detecting Cocaine and Benzoylecgonine for Biological and Forensic Applications

    Directory of Open Access Journals (Sweden)

    Brian A. Cadle

    2010-01-01

    Full Text Available Here we describe the first report of using low-cost cellular or web-based digital cameras to image and quantify standardized rapid immunoassay strips as a new point-of-care diagnostic and forensics tool with health applications. Quantitative ratiometric pixel density analysis (QRPDA is an automated method requiring end-users to utilize inexpensive (~ $1 USD/each immunotest strips, a commonly available web or mobile phone camera or scanner, and internet or cellular service. A model is described whereby a central computer server and freely available IMAGEJ image analysis software records and analyzes the incoming image data with time-stamp and geo-tag information and performs the QRPDA using custom JAVA based macros ( http://www.neurocloud.org . To demonstrate QRPDA we developed a standardized method using rapid immunotest strips directed against cocaine and its major metabolite, benzoylecgonine. Images from standardized samples were acquired using several devices, including a mobile phone camera, web cam, and scanner. We performed image analysis of three brands of commercially available dye-conjugated anti-cocaine/benzoylecgonine (COC/BE antibody test strips in response to three different series of cocaine concentrations ranging from 0.1 to 300 ng/ml and BE concentrations ranging from 0.003 to 0.1 ng/ml. This data was then used to create standard curves to allow quantification of COC/BE in biological samples. Across all devices, QRPDA quantification of COC and BE proved to be a sensitive, economical, and faster alternative to more costly methods, such as gas chromatography-mass spectrometry, tandem mass spectrometry, or high pressure liquid chromatography. The limit of detection was determined to be between 0.1 and 5 ng/ml. To simulate conditions in the field, QRPDA was found to be robust under a variety of image acquisition and testing conditions that varied temperature, lighting, resolution, magnification and concentrations of biological fluid

  18. Simplified and rapid method for extraction of ergosterol from natural samples and detection with quantitative and semi-quantitative methods using thin-layer chromatography

    OpenAIRE

    Larsen, Cand.scient Thomas; Ravn, Senior scientist Helle; Axelsen, Senior Scientist Jørgen

    2004-01-01

    A new and simplified method for extraction of ergosterol (ergoste-5,7,22-trien-3-beta-ol) from fungi in soil and litter was developed using pre-soaking extraction and paraffin oil for recovery. Recoveries of ergosterol were in the range of 94 - 100% depending on the solvent to oil ratio. Extraction efficiencies equal to heat-assisted extraction treatments were obtained with pre-soaked extraction. Ergosterol was detected with thin-layer chromatography (TLC) using fluorodensitometry with a quan...

  19. Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

    Science.gov (United States)

    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.

  20. Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus.

    Science.gov (United States)

    Guionie, O; Toquin, D; Sellal, E; Bouley, S; Zwingelstein, F; Allée, C; Bougeard, S; Lemière, S; Eterradossi, N

    2007-02-01

    Avian metapneumovirus (AMPV) is an important pathogen causing respiratory diseases and egg drops in several avian species. Four AMPV subgroups have been identified. The laboratory diagnosis of AMPV infections relies on serological methods, on labour-intensive virus isolation procedures, and on recently developed subgroup specific reverse transcription PCR (RT-PCR) protocols. In the present study, both the specificity and sensitivity of a commercial real-time reverse transcription PCR (RRT-PCR) for the detection and identification of the four AMPV subgroups were evaluated. Fifteen non-AMPV avian viruses belonging to 7 genera and 32 AMPV belonging to the 4 subgroups were tested. No non-AMPV virus was detected, whereas all AMPV viruses were identified in agreement with their previous molecular and antigenic subgroup assignment. The sensitivity and quantitating ability of the RRT-PCR assay were determined using serial dilutions of RNA derived either from AMPV virus stocks or from runoff transcripts. In all cases, linear dose/responses were observed. The detection limits of the different subgroups ranged from 500 to 5000 RNA copies and from 0.03 to 3.16TCID50/ml. The results were reproducible under laboratory conditions, thus showing that quantitative RRT-PCR is a new and powerful tool for the rapid and sensitive detection, identification and quantitation of AMPVs.

  1. Quantitative detection of caffeine in human skin by confocal Raman spectroscopy--A systematic in vitro validation study.

    Science.gov (United States)

    Franzen, Lutz; Anderski, Juliane; Windbergs, Maike

    2015-09-01

    For rational development and evaluation of dermal drug delivery, the knowledge of rate and extent of substance penetration into the human skin is essential. However, current analytical procedures are destructive, labor intense and lack a defined spatial resolution. In this context, confocal Raman microscopy bares the potential to overcome current limitations in drug depth profiling. Confocal Raman microscopy already proved its suitability for the acquisition of qualitative penetration profiles, but a comprehensive investigation regarding its suitability for quantitative measurements inside the human skin is still missing. In this work, we present a systematic validation study to deploy confocal Raman microscopy for quantitative drug depth profiling in human skin. After we validated our Raman microscopic setup, we successfully established an experimental procedure that allows correlating the Raman signal of a model drug with its controlled concentration in human skin. To overcome current drawbacks in drug depth profiling, we evaluated different modes of peak correlation for quantitative Raman measurements and offer a suitable operating procedure for quantitative drug depth profiling in human skin. In conclusion, we successfully demonstrate the potential of confocal Raman microscopy for quantitative drug depth profiling in human skin as valuable alternative to destructive state-of-the-art techniques. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  3. Detection of quantitative trait loci on chromosomes 1,2,3,12,14,15, X in pigs: performance characteristics

    NARCIS (Netherlands)

    Paixao, D.M.; Carneiro, P.L.S.; Paiva, S.R.; Sousa, K.R.S.; Verardo, L.L.; Braccini Neto, J.; Pinto, A.P.G.; Marubayashi Hidalgo, A.; Nascimento, C.; Périssé, I.V.; Lopes, P.S.; Guimaraes, S.E.F.

    2013-01-01

    The accomplishment of the present study had the objective of mapping Quantitative Trait Loci (QTL) related to performance traits in a F2 pig population developed by mating two Brazilian Piau breed sires with 18 dams from a commercial line (Landrace × Large White × Pietrain). The linkage map for this

  4. A unique charge-coupled device/xenon arc lamp based imaging system for the accurate detection and quantitation of multicolour fluorescence.

    Science.gov (United States)

    Spibey, C A; Jackson, P; Herick, K

    2001-03-01

    In recent years the use of fluorescent dyes in biological applications has dramatically increased. The continual improvement in the capabilities of these fluorescent dyes demands increasingly sensitive detection systems that provide accurate quantitation over a wide linear dynamic range. In the field of proteomics, the detection, quantitation and identification of very low abundance proteins are of extreme importance in understanding cellular processes. Therefore, the instrumentation used to acquire an image of such samples, for spot picking and identification by mass spectrometry, must be sensitive enough to be able, not only, to maximise the sensitivity and dynamic range of the staining dyes but, as importantly, adapt to the ever changing portfolio of fluorescent dyes as they become available. Just as the available fluorescent probes are improving and evolving so are the users application requirements. Therefore, the instrumentation chosen must be flexible to address and adapt to those changing needs. As a result, a highly competitive market for the supply and production of such dyes and the instrumentation for their detection and quantitation have emerged. The instrumentation currently available is based on either laser/photomultiplier tube (PMT) scanning or lamp/charge-coupled device (CCD) based mechanisms. This review briefly discusses the advantages and disadvantages of both System types for fluorescence imaging, gives a technical overview of CCD technology and describes in detail a unique xenon/are lamp CCD based instrument, from PerkinElmer Life Sciences. The Wallac-1442 ARTHUR is unique in its ability to scan both large areas at high resolution and give accurate selectable excitation over the whole of the UV/visible range. It operates by filtering both the excitation and emission wavelengths, providing optimal and accurate measurement and quantitation of virtually any available dye and allows excellent spectral resolution between different fluorophores

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

    Science.gov (United States)

    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 (GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite.

  6. The detection and quantitative analysis of the psychoactive component of Salvia divinorum, salvinorin A, in human biological fluids using liquid chromatography-mass spectrometry.

    Science.gov (United States)

    McDonough, Pamela C; Holler, Justin M; Vorce, Shawn P; Bosy, Thomas Z; Magluilo, Joseph; Past, Marilyn R

    2008-01-01

    Salvia divinorum, a member of the mint plant family, has hallucinogenic properties that have become increasingly sought after by recreational drug users. The main psychoactive component, salvinorin A, has potency comparable to lysergic acid diethylamide. Though still legal to possess in most of the United States and much of Europe, little is known regarding the compound's long-term health effects, addiction liability, and pharmacokinetics. Limited data are available in the scientific literature, and few analytical methods are published for the detection in human biological fluids. These factors contribute to the unfamiliarity of the compound and complicate the method development process necessary to accommodate special requested testing for salvinorin A. A sensitive analytical method for the detection and quantitation of salvinorin A in human biological fluids was developed and validated to resolve analytical shortcomings. The method utilizes a solid-phase extraction technique coupled with liquid chromatography-electrospray ionization mass spectrometry operated in selected ion monitoring mode. The assay has a linear range of 5.0-100 ng/mL with a correlation coefficient of 0.997. The limit of detection and limit of quantitation were experimentally determined as 2.5 and 5.0 ng/mL, respectively. The method has been applied to blood and urine samples successfully and can be used to detect the presence of salvinorin A in forensic testing.

  7. Quantitative Detection of Trace Level Cloxacillin in Food Samples Using Magnetic Molecularly Imprinted Polymer Extraction and Surface-Enhanced Raman Spectroscopy Nanopillars

    DEFF Research Database (Denmark)

    Ashley, Jon; Wu, Kaiyu; Hansen, Mikkel Fougt

    2017-01-01

    There is an increasing demand for rapid, sensitive, and low cost analytical methods to routinely screen antibiotic residues in food products. Conventional detection of antibiotics involves sample preparation by liquid-liquid or solid-phase extraction, followed by analysis using liquid...... with surface-enhanced Raman spectroscopy (SERS)-based detection for quantitative analysis of cloxacillin in pig serum. MMIP microspheres were synthesized using a core-shell technique. The large loading capacity and high selectivity of the MMIP microspheres enabled efficient extraction of cloxacillin, while...... using an internal standard. By coherently combining MMIP extraction and silicon nanopillar-based SERS biosensor, good sensitivity toward cloxacillin was achieved. The detection limit was 7.8 pmol. Cloxacillin recoveries from spiked pig plasma samples were found to be more than 80%....

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

    Science.gov (United States)

    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.

  9. Using rapid scan EPR to improve the detection limit of quantitative EPR by more than one order of magnitude

    OpenAIRE

    Möser, J.; Lips, K.; Tseytlin, M.; Eaton, G.; Eaton, S.; Schnegg, A

    2017-01-01

    X band rapid scan EPR was implemented on a commercially available Bruker ELEXSYS E580 spectrometer. Room temperature rapid scan and continuous wave EPR spectra were recorded for amorphous silicon powder samples. By comparing the resulting signal intensities the feasibility of performing quantitative rapid scan EPR is demonstrated. For different hydrogenated amorphous silicon samples, rapid scan EPR results in signal to noise improvements by factors between 10 and 50. Rapid scan EPR is thus ca...

  10. Cellular Phone-Based Image Acquisition and Quantitative Ratiometric Method for Detecting Cocaine and Benzoylecgonine for Biological and Forensic Applications

    OpenAIRE

    Cadle, Brian A.; Rasmus, Kristin C.; Varela, Juan A.; Leverich, Leah S.; O’Neill, Casey E.; Bachtell, Ryan K.; Cooper, Donald C.

    2010-01-01

    Here we describe the first report of using low-cost cellular or web-based digital cameras to image and quantify standardized rapid immunoassay strips as a new point-of-care diagnostic and forensics tool with health applications. Quantitative ratiometric pixel density analysis (QRPDA) is an automated method requiring end-users to utilize inexpensive (~ $1 USD/each) immunotest strips, a commonly available web or mobile phone camera or scanner, and internet or cellular service. A model is descri...

  11. Noninvasive radioisotopic technique for detection of platelet deposition in mitral valve prostheses and quantitation of visceral microembolism in dogs

    International Nuclear Information System (INIS)

    Dewanjee, M.K.; Fuster, V.; Rao, S.A.; Forshaw, P.L.; Kaye, M.P.

    1983-01-01

    A noninvasive technique has been developed in the dog model for imaging, with a gamma camera, the platelet deposition on Bjoerk-Shiley mitral valve prostheses early postoperatively. At 25 hours after implantation of the prosthesis and 24 hours after intravenous administration of 400 to 500 microCi of platelets labeled with indium-111, the platelet deposition in the sewing ring and perivalvular cardiac tissue can be clearly delineated in a scintiphotograph. An in vitro technique was also developed for quantitation of visceral microemboli in brain, lungs, kidneys, and other tissues. Biodistribution of the labeled platelets was quantitated, and the tissue/blood radioactivity ratio was determined in 22 dogs in four groups: unoperated normal dogs, sham-operated dogs, prosthesis-implanted dogs, and prosthesis-implanted dogs treated with dipyridamole before and aspirin and dipyridamole immediately after operation. Fifteen to 20% of total platelets were consumed as a consequence of the surgical procedure. On quantitation, we found that platelet deposition on the components of the prostheses was significantly reduced in prosthesis-implanted animals treated with dipyridamole and aspirin when compared with prosthesis-implanted, untreated dogs. All prosthesis-implanted animals considered together had a twofold to fourfold increase in tissue/blood radioactivity ratio in comparison with unoperated and sham-operated animals, an indication that the viscera work as filters and trap platelet microemboli that are presumably produced in the region of the mitral valve prostheses. In the dog model, indium-111-labeled platelets thus provide a sensitive marker for noninvasive imaging of platelet deposition on mechanical mitral valve prostheses, in vitro evaluation of platelet microembolism in viscera, in vitro quantitation of surgical consumption of platelets, and evaluation of platelet-inhibitor drugs

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

    Science.gov (United States)

    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.

  13. Development and validation of quantitative PCR for detection of Terrapene herpesvirus 1 utilizing free-ranging eastern box turtles (Terrapene carolina carolina).

    Science.gov (United States)

    Kane, Lauren P; Bunick, David; Abd-Eldaim, Mohamed; Dzhaman, Elena; Allender, Matthew C

    2016-06-01

    Diseases that affect the upper respiratory tract (URT) in chelonians have been well described as a significant contributor of morbidity and mortality. Specifically, herpesviruses are common pathogens in captive chelonians worldwide, but their importance on free-ranging populations is less well known. Historical methods for the diagnosis of herpesvirus infections include virus isolation and conventional PCR. Real-time PCR has become an essential tool for detection and quantitation of many pathogens, but has not yet been developed for herpesviruses in box turtles. Two quantitative real-time TaqMan PCR assays, TerHV58 and TerHV64, were developed targeting the DNA polymerase gene of Terrapene herpesvirus 1 (TerHV1). The assay detected a viral DNA segment cloned within a plasmid with 10-fold serial dilutions from 1.04 × 10(7) to 1.04 × 10(1) viral copies per reaction. Even though both primers had acceptable levels of efficiency and variation, TerHV58 was utilized to test clinical samples based on less variation and increased efficiency. This assay detected as few as 10 viral copies per reaction and should be utilized in free-ranging and captive box turtles to aid in the characterization of the epidemiology of this disease. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Integrating qualitative and quantitative characterization of traditional Chinese medicine injection by high-performance liquid chromatography with diode array detection and tandem mass spectrometry.

    Science.gov (United States)

    Xie, Yuan-yuan; Xiao, Xue; Luo, Juan-min; Fu, Chan; Wang, Qiao-wei; Wang, Yi-ming; Liang, Qiong-lin; Luo, Guo-an

    2014-06-01

    The present study aims to describe and exemplify an integrated strategy of the combination of qualitative and quantitative characterization of a multicomponent mixture for the quality control of traditional Chinese medicine injections with the example of Danhong injection (DHI). The standardized chemical profile of DHI has been established based on liquid chromatography with diode array detection. High-performance liquid chromatography coupled with time-of-flight mass spectrometry and high-performance liquid chromatography with electrospray multistage tandem ion-trap mass spectrometry have been developed to identify the major constituents in DHI. The structures of 26 compounds including nucleotides, phenolic acids, and flavonoid glycosides were identified or tentatively characterized. Meanwhile, the simultaneous determination of seven marker constituents, including uridine, adenosine, danshensu, protocatechuic aldehyde, p-coumaric acid, rosmarinic acid, and salvianolic acid B, in DHI was performed by multiwavelength detection based on high-performance liquid chromatography with diode array detection. The integrated qualitative and quantitative characterization strategy provided an effective and reliable pattern for the comprehensive and systematic characterization of the complex traditional Chinese medicine system. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

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

    2002-01-01

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

  16. A neutral glyoxal gel electrophoresis method for the detection and semi-quantitation of DNA single-strand breaks.

    Science.gov (United States)

    Pachkowski, Brian; Nakamura, Jun

    2013-01-01

    Single-strand breaks are among the most prevalent lesions found in DNA. Traditional electrophoretic methods (e.g., the Comet assay) used for investigating these lesions rely on alkaline conditions to denature DNA prior to electrophoresis. However, the presence of alkali-labile sites in DNA can result in the introduction of additional single-strand breaks upon alkali treatment during DNA sample processing. Herein, we describe a neutral glyoxal gel electrophoresis assay which is based on alkali-free DNA denaturation and is suitable for qualitative and semi-quantitative analyses of single-strand breaks in DNA isolated from different organisms.

  17. Effectiveness of medicines authentication technology to detect counterfeit, recalled and expired medicines: a two-stage quantitative secondary care study

    Science.gov (United States)

    Naughton, Bernard; Roberts, Lindsey; Dopson, Sue; Chapman, Stephen; Brindley, David

    2016-01-01

    Objectives To identify the authentication and detection rate of serialised medicines using medicines authentication technology. Design and intervention 4192 serialised medicines were entered into a hospital dispensary over two separate 8-week stages in 2015. Medicines were authenticated using secure external database cross-checking, triggered by the scanning of a two-dimensional data matrix with a unit specific 12-digit serial code. 4% of medicines included were preprogrammed with a message to identify the product as either expired, pack recalled, product recalled or counterfeit. Setting A site within a large UK National Health Service teaching hospital trust. Participants Accredited checking staff, pharmacists and dispensers in a pharmacy department. Primary outcome measures Authentication and detection rate of counterfeit expired and recalled medicines. Results The operational detection rate of counterfeit, recalled and expired medicines scanned as a combined group was 81.4% (stage 1 (S1)) and 87% (stage 2 (S2)). The technology's technical detection rate (TDR) was 100%; however, not all medicines were scanned and of those that were scanned not all that generated a warning message were quarantined. Owing to an operational authentication rate (OAR) of 66.3% (over both stages), only 31.8% of counterfeit medicines, 58% of recalled drugs and 64% of expired medicines were detected as a proportion of those entered into the study. Response times (RTs) of 152 ms (S1) and 165 ms (S2) were recorded, meeting the falsified medicines directive-mandated 300 ms limit. Conclusions TDRs and RTs were not a limiting factor in this study. The suboptimal OAR poses significant quality and safety issues with this detection approach. Authentication at the checking stage, however, demonstrated higher OARs. There is a need for further qualitative research to establish the reasons for less than absolute authentication and detection rates in the hospital environment to improve this

  18. Quantitative detection of the tumor-associated antigen large external antigen in colorectal cancer tissues and cells using quantum dot probe

    Directory of Open Access Journals (Sweden)

    Wang S

    2016-01-01

    Full Text Available Shuo Wang, Wanming Li, Dezheng Yuan, Jindan Song, Jin Fang Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, Shenyang, People’s Republic of China Abstract: The large external antigen (LEA is a cell surface glycoprotein that has been proven to be highly expressed in colorectal cancer (CRC as a tumor-associated antigen. To evaluate and validate the relationship between LEA expression and clinical characteristics of CRC with high efficiency, LEA expression levels were detected in 85 tissue blocks from CRC patients by quantum dot-based immunohistochemistry (QD-IHC combined with imaging quantitative analysis using quantum dots with a 605 nm emission wavelength (QD605 conjugated to an ND-1 monoclonal antibody against LEA as a probe. Conventional IHC was performed in parallel for comparison. Both QD-IHC and conventional IHC showed that LEA was specifically expressed in CRC, but not in non-CRC tissues, and high LEA expression was significantly associated with a more advanced T-stage (P<0.05, indicating that LEA is likely to serve as a CRC prognostic marker. Compared with conventional IHC, receiver operating characteristic analysis revealed that QD-IHC possessed higher sensitivity, resulting in an increased positive detection rate of CRC, from 70.1% to 89.6%. In addition, a simpler operation, objective analysis of results, and excellent repeatability make QD-IHC an attractive alternative to conventional IHC in clinical practice. Furthermore, to explore whether the QD probes can be utilized to quantitatively detect living cells or single cells, quantum dot-based immunocytochemistry (QD-ICC combined with imaging quantitative analysis was developed to evaluate LEA expression in several CRC cell lines. It was demonstrated that QD-ICC could also predict the correlation between LEA expression and the T-stage characteristics of

  19. Detection and quantitation of benzo[a]pyrene-DNA adducts in brain and liver tissues of Beluga whales (Delphinapterus leucas) from the St. Lawrence and Mackenzie Estuaries

    International Nuclear Information System (INIS)

    Shugart, L.R.

    1988-01-01

    It should be noted that there are few analytical techniques available for the detection and quantitation of chemical adducts in the DNA of living organisms. The reasons for this are: the analytical technique often has to accommodate the unique chemical and/or physical properties of the individual chemical or its metabolite; the percentage of total chemical that becomes most of the parent compound is usually detoxified and excreted; not all adducts that form between the genotoxic agent and DNA are stable or are involved in the development of subsequent deleterious events in the organism; and the amount of DNA available for analysis is often quite limited. 16 refs., 1 tab

  20. Quantitative analysis of flavonols, flavones, and flavanones in fruits, vegetables and beverages by high-performance liquid chromatography with photo-diode array and mass spectrometric detection

    DEFF Research Database (Denmark)

    Justesen, U.; Knuthsen, Pia; Leth, Torben

    1998-01-01

    after acid hydrolysis of freeze-dried food material. Identification was based on retention time, UV and mass spectra by comparison with commercial standards, and the UV peak areas were used for quantitation of the flavonoid contents. Examples of HPLC-MS analyses of orange pulp, tomato, and apple......A high-performance liquid chromatographic (HPLC) separation method viith photo-diode array (PDA) and mass spectrometric (MS) detection was developed to determine and quantify flavonols, flavones, and flavanones in fruits, vegetables and beverages. The compounds were analysed as aglycones, obtained...

  1. Fluorography--limitations on its use for the quantitative detection of 3H- and 14-C-labeled proteins in polyacrylamide gels

    International Nuclear Information System (INIS)

    Harding, C.R.; Scott, I.R.

    1983-01-01

    The suitability of fluorography for the detection of 3 H- and 14 C-labeled proteins on polyacrylamide gradient gels has been investigated. If was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration

  2. Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection

    International Nuclear Information System (INIS)

    Zapparoli, Giada V; Jorissen, Robert N; Hewitt, Chelsee A; McBean, Michelle; Westerman, David A; Dobrovic, Alexander

    2013-01-01

    The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3′dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. We showed that the addition of the 3′dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10 -4 per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a

  3. A Rapid, Onsite, Ultrasensitive Melamine Quantitation Method for Protein Beverages Using Time-Resolved Fluorescence Detection Paper.

    Science.gov (United States)

    Li, Guanghua; Wang, Du; Zhou, Aijun; Sun, Yimin; Zhang, Qi; Poapolathep, Amnart; Zhang, Li; Fan, Zhiyong; Zhang, Zhaowei; Li, Peiwu

    2018-05-02

    To ensure protein beverage safety and prevent illegal melamine use to artificially increase protein content, a rapid, onsite, ultrasensitive detection method for melamine must be developed because melamine is detrimental to human health and life. Herein, an ultrasensitive time-resolved fluorescence detection paper (TFDP) was developed to detect melamine in protein beverages within 15 min using a one-step sample preparation. The lower limits of detection were 0.89, 0.94, and 1.05 ng/mL, and the linear ranges were 2.67-150, 2.82-150, and 3.15-150 ng/mL (R2>0.982) for peanut, walnut, and coconut beverages, respectively. The recovery rates were 85.86-110.60% with a coefficient of variation beverage samples, the TFDP and ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) results were consistent. This method is a promising alternative for rapid, onsite detection of melamine in beverages.

  4. Two quantitative real-time PCR assays for the detection of penaeid shrimp and blue crab, crustacean shellfish allergens.

    Science.gov (United States)

    Eischeid, Anne C; Kim, Bang-hyun; Kasko, Sasha M

    2013-06-19

    Food allergen detection methods must be able to specifically detect minute quantities of an allergenic food in a complex food matrix. One technique that can be used is real-time PCR. For the work described here, real-time PCR assays were developed to detect penaeid shrimp and blue crab, crustacean shellfish allergens. The method was tested using shrimp meat and crab meat spiked into several types of foods, including canned soups, deli foods, meat, seafood, and prepared seafood products. Foods were spiked with either shrimp or crab at levels ranging from 0.1 to 10⁶ parts per million (ppm) and analyzed either raw or cooked by a variety of methods. Real-time PCR data were used to generate linear standard curves, and assays were evaluated with respect to linear range and reaction efficiency. Results indicate that both assays performed well in a variety of food types. High reaction efficiencies were achieved across a linear range of 6-8 orders of magnitude. Limits of detection were generally between 0.1 and 1 ppm. Cooking methods used to simulate thermal processing of foods had little effect on assay performance. This work demonstrates that real-time PCR can be a valuable tool in the detection of crustacean shellfish.

  5. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

    Science.gov (United States)

    McCutchen-Maloney, Sandra L.

    2002-01-01

    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  6. Quantitative analysis of the self-absorption and reemission effects on the emission spectrum of photoluminescence in right-angle excitation—detection configuration

    International Nuclear Information System (INIS)

    Wang Zhen-Hua; Wu Yu-E; Zhang Xin-Zheng; Yun Zhi-Qiang; Li Wei; Xu Jing-Jun

    2013-01-01

    A theoretical approach based on differential radiative transport is proposed to quantitatively analyze the self-absorption and reemission effects on the emission spectrum for right angle excitation—detection photoluminescence measurements, and the wavelength dependence of the reemission effect is taken into account. Simulations and experiments are performed using rhodamine 6G solutions in ethanol as model samples. It is shown that the self-absorption effect is the dominant effect on the detected spectrum by inducing pseudo red-shift and reducing total intensity; whereas the reemission effect partly compensates for signal decrease and also results in an apparent signal gain at the wavelengths without absorption. Both effects decrease with the decrease in the sample concentration and the propagation distance of the emission light inside the sample. We therefore suggest that diluted solutions are required for accurate photoluminescence spectrum measurements and photoluminescence-based measurements

  7. Quantitative colorimetry of atherosclerotic plaque using the L*a*b* color space during angioscopy for the detection of lipid cores underneath thin fibrous caps.

    Science.gov (United States)

    Ishibashi, Fumiyuki; Yokoyama, Shinya; Miyahara, Kengo; Dabreo, Alexandra; Weiss, Eric R; Iafrati, Mark; Takano, Masamichi; Okamatsu, Kentaro; Mizuno, Kyoichi; Waxman, Sergio

    2007-12-01

    Yellow plaques seen during angioscopy are thought to represent lipid cores underneath thin fibrous caps (LCTCs) and may be indicative of vulnerable sites. However, plaque color assessment during angioscopy has been criticized because of its qualitative nature. The purpose of the present study was to test the ability of a quantitative colorimetric system to measure yellow color intensity of atherosclerotic plaques during angioscopy and to characterize the color of LCTCs. Using angioscopy and a quantitative colorimetry system based on the L*a*b* color space [L* describes brightness (-100 to +100), b* describes blue to yellow (-100 to +100)], the optimal conditions for measuring plaque color were determined in three flat standard color samples and five artificial plaque models in cylinder porcine carotid arteries. In 88 human tissue samples, the colorimetric characteristics of LCTCs were then evaluated. In in-vitro samples and ex-vivo plaque models, brightness L* between 40 and 80 was determined to be optimal for acquiring b* values, and the variables unique to angioscopy in color perception did not impact b* values after adjusting for brightness L* by manipulating light or distance. In ex-vivo human tissue samples, b* value >/=23 (35.91 +/- 8.13) with L* between 40 and 80 was associated with LCTCs (fibrous caps colorimetry. High yellow color intensity, determined by this system, was associated with LCTCs. Quantitative colorimetry during angioscopy may be used for detection of LCTCs, which may be markers of vulnerability.

  8. Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR.

    Science.gov (United States)

    Malandraki, Ioanna; Beris, Despoina; Isaioglou, Ioannis; Olmos, Antonio; Varveri, Christina; Vassilakos, Nikon

    2017-01-01

    A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10-4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.

  9. The application of diode laser colorimetry coupled with fiber optic dipping probe for quantitative detection of a protein

    International Nuclear Information System (INIS)

    Kim, Sung Ho; Yoo, Jong Shin

    1996-01-01

    The in-situ, simple and inexpensive analysis of protein was achieved by the portable diode laser absorption spectrometry, which consisted of visible diode laser, photodiode, optical fiber and dipping probe. It gives comparable detection limit to the use of conventional UV/Vis spectrometer for the determination of protein by Lowry method.

  10. Detection of cerebral arterial gas embolism using regional cerebral oxygen saturation, quantitative electroencephalography, and brain oxygen tension in the swine

    NARCIS (Netherlands)

    Weenink, R. P.; Hollmann, M. W.; Stevens, M. F.; Kager, J.; van Gulik, T. M.; van Hulst, R. A.

    2014-01-01

    Cerebral air emboli occur as a complication of invasive medical procedures. The sensitivity of cerebral monitoring methods for the detection of air emboli is not known. This study investigates the utility of electroencephalography and non-invasively measured cerebral oxygen saturation in the

  11. Partial validation of a TaqMan real-time quantitative PCR for the detection of ranaviruses

    DEFF Research Database (Denmark)

    Stilwell, Natalie K.; Whittington, Richard J.; Hick, Paul M.

    2018-01-01

    Ranaviruses are globally emerging pathogens negatively impacting wild and cultured fish, amphibians, and reptiles. Although conventional and diagnostic real-time PCR (qPCR) assays have been developed to detect ranaviruses, these assays often have not been tested against the known diversity of ran...

  12. Detection of hydrogen peroxide-producing Lactobacillus species in the vagina: a comparison of culture and quantitative PCR among HIV-1 seropositive women

    Directory of Open Access Journals (Sweden)

    Balkus Jennifer E

    2012-08-01

    Full Text Available Abstract Background The presence of hydrogen peroxide (H2O2 producing Lactobacillus in the vagina may play a role in controlling genital HIV-1 shedding. Sensitive molecular methods improve our ability to characterize the vaginal microbiota; however, they cannot characterize phenotype. We assessed the concordance of H2O2-producing Lactobacillus detected by culture with quantitative PCR (qPCR detection of Lactobacillus species commonly assumed to be H2O2-producers. Methods Samples were collected as part of a prospective cohort study of HIV-1 seropositive US women. Cervicovaginal lavage specimens were tested for L. crispatus and L. jensenii using 16S rRNA gene qPCR assays. Vaginal swabs were cultured for Lactobacillus and tested for H2O2-production. We calculated a kappa statistic to assess concordance between culture and qPCR. Results Culture and qPCR results were available for 376 visits from 57 women. Lactobacilli were detected by culture at 308 (82% visits, of which 233 of 308 (76% produced H2O2. L. crispatus and/or L. jensenii were detected at 215 (57% visits. Concordance between detection of L. crispatus and/or L. jensenii by qPCR and H2O2-producing Lactobacillus by culture was 75% (kappa = 0.45. Conclusions Among HIV-1 seropositive women, there was a moderate level of concordance between H2O2-producing Lactobacillus detected by culture and the presence of L. crispatus and/or L. jensenii by qPCR. However, one-quarter of samples with growth of H2O2-producing lactobacilli did not have L. crispatus or L. jensenii detected by qPCR. This discordance may be due to the presence of other H2O2-producing Lactobacillus species.

  13. A Survey on Pharmacovigilance Activities in ASEAN and Selected Non-ASEAN Countries, and the Use of Quantitative Signal Detection Algorithms.

    Science.gov (United States)

    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.

  14. Rapid and quantitative detection of zoonotic influenza A virus infection utilizing coumarin-derived dendrimer-based fluorescent immunochromatographic strip test (FICT).

    Science.gov (United States)

    Yeo, Seon-Ju; Huong, Dinh Thi; Hong, Nguyen Ngoc; Li, Chun-Ying; Choi, Kyunghan; Yu, Kyoungsik; Choi, Du-Young; Chong, Chom-Kyu; Choi, Hak Soo; Mallik, Shyam Kumar; Kim, Hak Sung; Sung, Haan Woo; Park, Hyun

    2014-01-01

    Great efforts have been made to develop robust signal-generating fluorescence materials which will help in improving the rapid diagnostic test (RDT) in terms of sensitivity and quantification. In this study, we developed coumarin-derived dendrimer-based fluorescent immunochromatographic strip test (FICT) assay with enhanced sensitivity as a quantitative diagnostic tool in typical RDT environments. The accuracy of the proposed FICT was compared with that of dot blot immunoassay techniques and conventional RDTs. Through conjugation of coumarin-derived dendrimers with latex beads, fluorescent emission covering broad output spectral ranges was obtained which provided a distinct advantage of easy discrimination of the fluorescent emission of the latex beads with a simple insertion of a long-pass optical filter away from the excitation wavelength. The newly developed FICT assay was able to detect 100 ng/10 μL of influenza A nucleoprotein (NP) antigen within 5 minutes, which corresponded to 2.5-fold higher sensitivity than that of the dot blot immunoassay or conventional RDTs. Moreover, the FICT assay was confirmed to detect at least four avian influenza A subtypes (H5N3, H7N1, H7N7, and H9N2). On applying the FICT to the clinical swab samples infected with respiratory viruses, our FICT assay was confirmed to differentiate influenza H1N1 infection from other respiratory viral diseases. These data demonstrate that the proposed FICT assay is able to detect zoonotic influenza A viruses with a high sensitivity, and it enables the quantitation of the infection intensity by providing the numerical diagnostic values; thus demonstrating enhanced detectability of influenza A viruses.

  15. The combination of quantitative PCR and western blot detecting CP4-EPSPS component in Roundup Ready soy plant tissues and commercial soy-related foodstuffs.

    Science.gov (United States)

    Xiao, Xiao; Wu, Honghong; Zhou, Xinghu; Xu, Sheng; He, Jian; Shen, Wenbiao; Zhou, Guanghong; Huang, Ming

    2012-06-01

    With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods

  16. Rapid quantitative analysis of individual anthocyanin content based on high-performance liquid chromatography with diode array detection with the pH differential method.

    Science.gov (United States)

    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.

  17. Quantitative method for the detection and localization of quantum-limited events from radionuclides in cells and tissue sections by computer-enhanced video microscopy

    International Nuclear Information System (INIS)

    Pressman, N.J.; Frost, J.K.; Gupta, P.K.; Showers, R.L.; Gill, G.W.; Cook, D.L.; Frost, J.K. Jr.; Traub, R.K.

    1987-01-01

    Cellular dynamics often involve extremely low concentrations of biologically active substances, which can be radiolabeled and detected, localized and quantitated by autoradiography. The latter may require exposures from a few days to many months. The objective of this research was to demonstrate the feasibility of reducing this long period of data collection by one to two orders of magnitude, while maintaining or improving the spatial resolution and localization in tissues and the quantitative characteristics inherent in autoradiography. A mathematical model describing the complete system was generated using energy partition calculations to estimate photon production via scintillant per H3 beta particle emission and to estimate the subsequent photon capture based upon imaging system parameters and microscope geometry. Calculations showed that, typically, a single tritium beta particle produces a maximum of 5.8 X 10(3) photons. A photon-limited camera and microscope imaging system were selected and optimized in conjunction with a specially developed physical scintillation model. Results showed that the number of detected photoevents increases monotonically with both signal integration time and, independently, with the concentration of the radionuclide. Consequently, this work demonstrates that video microscopy imaging methods can spatially and temporally quantify very low concentrations of radiolabeled substances and can reduce data acquisition times

  18. Quantitative in vivo detection of brain cell death after hypoxia ischemia using the lipid peak at 1.3 ppm of proton magnetic resonance spectroscopy in neonatal rats.

    Science.gov (United States)

    Ahn, So Yoon; Yoo, Hye Soo; Lee, Jang Hoon; Sung, Dong Kyung; Jung, Yu Jin; Sung, Se In; Lim, Keun Ho; Chang, Yun Sil; Lee, Jung Hee; Kim, Ki Soo; Park, Won Soon

    2013-07-01

    This study was performed to determine the accuracy of proton magnetic spectroscopy ((1)H-MRS) lipid peak as a noninvasive tool for quantitative in vivo detection of brain cell death. Seven day-old Sprague Dawley rats were subjected to 8% oxygen following a unilateral carotid artery ligation. For treatment, cycloheximide was given immediately after hypoxic ischemia (HI). Lipid peak was measured using (1)H-MRS at 24 hr after HI, and then brains were harvested for fluorocytometric analyses with annexin V/propidium iodide (PI) and fluorescent probe JC-1, and for adenosine-5'-triphosphate (ATP) and lactate. Increased lipid peak at 1.3 ppm measured with (1)H-MRS, apoptotic and necrotic cells, and loss of mitochondrial membrane potential (ΔΨ) at 24 hr after HI were significantly improved with cycloheximide treatment. Significantly reduced brain ATP and increased lactate levels observed at 24 hr after HI showed a tendency to improve without statistical significance with cycloheximide treatment. Lipid peak at 1.3 ppm showed significant positive correlation with both apoptotic and necrotic cells and loss of ΔΨ, and negative correlation with normal live cells. Lipid peak at 1.3 ppm measured by (1)H-MRS might be a sensitive and reliable diagnostic tool for quantitative in vivo detection of brain cell death after HI.

  19. Assessment of Intrathecal Free Light Chain Synthesis: Comparison of Different Quantitative Methods with the Detection of Oligoclonal Free Light Chains by Isoelectric Focusing and Affinity-Mediated Immunoblotting.

    Science.gov (United States)

    Zeman, David; Kušnierová, Pavlína; Švagera, Zdeněk; Všianský, František; Byrtusová, Monika; Hradílek, Pavel; Kurková, Barbora; Zapletalová, Olga; Bartoš, Vladimír

    2016-01-01

    We aimed to compare various methods for free light chain (fLC) quantitation in cerebrospinal fluid (CSF) and serum and to determine whether quantitative CSF measurements could reliably predict intrathecal fLC synthesis. In addition, we wished to determine the relationship between free kappa and free lambda light chain concentrations in CSF and serum in various disease groups. We analysed 166 paired CSF and serum samples by at least one of the following methods: turbidimetry (Freelite™, SPAPLUS), nephelometry (N Latex FLC™, BN ProSpec), and two different (commercially available and in-house developed) sandwich ELISAs. The results were compared with oligoclonal fLC detected by affinity-mediated immunoblotting after isoelectric focusing. Although the correlations between quantitative methods were good, both proportional and systematic differences were discerned. However, no major differences were observed in the prediction of positive oligoclonal fLC test. Surprisingly, CSF free kappa/free lambda light chain ratios were lower than those in serum in about 75% of samples with negative oligoclonal fLC test. In about a half of patients with multiple sclerosis and clinically isolated syndrome, profoundly increased free kappa/free lambda light chain ratios were found in the CSF. Our results show that using appropriate method-specific cut-offs, different methods of CSF fLC quantitation can be used for the prediction of intrathecal fLC synthesis. The reason for unexpectedly low free kappa/free lambda light chain ratios in normal CSFs remains to be elucidated. Whereas CSF free kappa light chain concentration is increased in most patients with multiple sclerosis and clinically isolated syndrome, CSF free lambda light chain values show large interindividual variability in these patients and should be investigated further for possible immunopathological and prognostic significance.

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

    Science.gov (United States)

    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; PPCR Kit. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. [Qualitative and quantitative diagnostic performance of 320-slice computed tomography for detecting coronary artery disease with respect to atherosclerotic plaque characteristics].

    Science.gov (United States)

    Li, Suhua; Liu, Jinlai; Peng, Long; Dong, Ruimin; Wu, Huilan; Wang, Chenlin; Ni, Qiongqiong; Luo, Yanting; Zhu, Jieming; Chen, Lin

    2014-10-28

    To investigate qualitatively and quantitatively the diagnostic performance of 320-slice CT for detection of coronary artery disease with respect to different atherosclerotic plaque characteristics. A retrospective search was performed for inpatients underwent both coronary CT and further coronary angiography (CAG) from December 1, 2008 to December 31, 2012. The diagnostic performance of 320-slice CTA for detecting significant stenosis ( ≥ 50% diameter) with respect to atherosclerotic plaque characteristics were analyzed by calculating sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, kappa index (κ), and area under the receiver operating characteristic curve (AUC). Chi-square test was used to evaluate whether there were significant differences of the true-case frequency (true positive + true negative) and false-case frequency (false positive + false negative) among groups. Bland-Altman analysis was used to determine limits of agreement between CTA and CAG. A total of 454 patients and 6 779 segments were analyzed. Diagnostic accuracy was higher in non-calcified segments; whereas they decreased in the presence of both mild-moderately and heavily calcified plaques. Excellent agreement (κ = 0.810) between CT and CAG was observed for non-calcified segments, while good agreement was observed for both mild-moderately (κ = 0.701) and heavily calcified segments (κ = 0.750). Both mild-moderate (P = 0.000) and heavy (P = 0.000) calcification decreased the true-case frequency and increased the false-case frequency when compared to non-calcification. There were no significant underestimation or overestimation for non-calcified (P = 0.087) and mild-moderately calcified (P = 0.704) segments, while there was significant overestimation for heavily calcified segments (P = 0.001). Great qualitative and quantitative diagnostic performances of 320-slice CT were observed in non-calcified coronary segments. However, qualitative

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

    Science.gov (United States)

    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.

  3. Provider report of the existence of detection and care of perinatal depression: quantitative evidence from public obstetric units in Mexico

    Directory of Open Access Journals (Sweden)

    Filipa de Castro

    2016-07-01

    Full Text Available Objective. To provide evidence on perinatal mental healthcare in Mexico. Materials and methods. Descriptive and bivariate analyses of data from a cross-sectional probabilistic survey of 211 public obstetric units. Results. Over half (64.0% of units offer mental healthcare; fewer offer perinatal depression (PND detection (37.1% and care (40.3%. More units had protocols/guidelines for PND detection and for care, respectively, in Mexico City-Mexico state (76.7%; 78.1% than in Southern (26.5%; 36.4%, Northern (27.3%; 28.1% and Central Mexico (50.0%; 52.7%. Conclusion. Protocols and provider training in PND, implementation of brief screening tools and psychosocial interventions delivered by non-clinical personnel are needed.      DOI: http://dx.doi.org/10.21149/spm.v58i4.8028

  4. Provider report of the existence of detection and care of perinatal depression: quantitative evidence from public obstetric units in Mexico.

    Science.gov (United States)

    Castro, Filipa de; Place, Jean Marie; Allen-Leigh, Betania; Rivera-Rivera, Leonor; Billings, Deborah

    2016-08-01

    To provide evidence on perinatal mental healthcare in Mexico. Descriptive and bivariate analyses of data from a cross-sectional probabilistic survey of 211 public obstetric units. Over half (64.0%) of units offer mental healthcare; fewer offer perinatal depression (PND) detection (37.1%) and care (40.3%). More units had protocols/guidelines for PND detection and for care, respectively, in Mexico City-Mexico state (76.7%; 78.1%) than in Southern (26.5%; 36.4%), Northern (27.3%; 28.1%) and Central Mexico (50.0%; 52.7%). Protocols and provider training in PND, implementation of brief screening tools and psychosocial interventions delivered by non-clinical personnel are needed.

  5. Quantitative determination of the dopamine agonist lisuride in plasma using high-performance liquid chromatography with fluorescence detection.

    Science.gov (United States)

    Wolthers, B G; Verhagen Kamerbeek, W D; van Beusekom, C M; Elshof, F; de Ruyter Buitenhuis, A W; Brunt, E P; Lakke, J P

    1993-12-08

    An HPLC method for the determination of lisuride hydrogen maleate in plasma is described. After addition of ergotamine tartrate as internal standard, plasma is extracted with diethyl ether. Following evaporation of the solvent and redissolving in methanol the extract is injected on a silica HPLC column and lisuride is monitored by fluorescence detection using an excitation wavelength of 322 nm and an emission wavelength of 405 nm. The method is sufficiently accurate and precise with a detection limit of 20 pg/ml lisuride in plasma. The usefulness of the method is demonstrated by measurements of lisuride levels after oral intake of a 0.6 mg dose of the drug by a healthy male volunteer, showing a peak level of 1266 pg/ml, 45 min after intake.

  6. Detection limits in the chromatographic element trace analysis - quantitative TLC, HPLC and GC with the example of beryllium acetylacetonate

    International Nuclear Information System (INIS)

    Schwedt, G.

    1981-01-01

    Chromatographic analyses of beryllium acetylacetonate are carried out in synthetic solutions within the nano- and picogram range of beryllium. For thin-layer chromatography (TLC) normal and silanized silica gel is used, for high-performance liquid chromatography (HPLC) silica gel of 7 μm particles, for gas chromatography (GC) silicone SE-30 as stationary phase. Visual evaluation and remission measurements in TLC, UV-254 nm absorption measurements in HPLC and measurements with a FID in GC are employed for the determination of the calibration curves. A calibration curve through the origin and a detection limit of 150 pg Be determinable form are received by HPLC only. For trace analyses by GC a new definition of a detection limit for the evaluation of substance peaks on a solvent tailing is suggested. (orig.) [de

  7. Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR.

    Science.gov (United States)

    Witte, Anna Kristina; Fister, Susanne; Mester, Patrick; Schoder, Dagmar; Rossmanith, Peter

    2016-11-01

    Fast and reliable pathogen detection is an important issue for human health. Since conventional microbiological methods are rather slow, there is growing interest in detection and quantification using molecular methods. The droplet digital polymerase chain reaction (ddPCR) is a relatively new PCR method for absolute and accurate quantification without external standards. Using the Listeria monocytogenes specific prfA assay, we focused on the questions of whether the assay was directly transferable to ddPCR and whether ddPCR was suitable for samples derived from heterogeneous matrices, such as foodstuffs that often included inhibitors and a non-target bacterial background flora. Although the prfA assay showed suboptimal cluster formation, use of ddPCR for quantification of L. monocytogenes from pure bacterial cultures, artificially contaminated cheese, and naturally contaminated foodstuff was satisfactory over a relatively broad dynamic range. Moreover, results demonstrated the outstanding detection limit of one copy. However, while poorer DNA quality, such as resulting from longer storage, can impair ddPCR, internal amplification control (IAC) of prfA by ddPCR, that is integrated in the genome of L. monocytogenes ΔprfA, showed even slightly better quantification over a broader dynamic range. Graphical Abstract Evaluating the absolute quantification potential of ddPCR targeting Listeria monocytogenes prfA.

  8. Automatic reference selection for quantitative EEG interpretation: identification of diffuse/localised activity and the active earlobe reference, iterative detection of the distribution of EEG rhythms.

    Science.gov (United States)

    Wang, Bei; Wang, Xingyu; Ikeda, Akio; Nagamine, Takashi; Shibasaki, Hiroshi; Nakamura, Masatoshi

    2014-01-01

    EEG (Electroencephalograph) interpretation is important for the diagnosis of neurological disorders. The proper adjustment of the montage can highlight the EEG rhythm of interest and avoid false interpretation. The aim of this study was to develop an automatic reference selection method to identify a suitable reference. The results may contribute to the accurate inspection of the distribution of EEG rhythms for quantitative EEG interpretation. The method includes two pre-judgements and one iterative detection module. The diffuse case is initially identified by pre-judgement 1 when intermittent rhythmic waveforms occur over large areas along the scalp. The earlobe reference or averaged reference is adopted for the diffuse case due to the effect of the earlobe reference depending on pre-judgement 2. An iterative detection algorithm is developed for the localised case when the signal is distributed in a small area of the brain. The suitable averaged reference is finally determined based on the detected focal and distributed electrodes. The presented technique was applied to the pathological EEG recordings of nine patients. One example of the diffuse case is introduced by illustrating the results of the pre-judgements. The diffusely intermittent rhythmic slow wave is identified. The effect of active earlobe reference is analysed. Two examples of the localised case are presented, indicating the results of the iterative detection module. The focal and distributed electrodes are detected automatically during the repeating algorithm. The identification of diffuse and localised activity was satisfactory compared with the visual inspection. The EEG rhythm of interest can be highlighted using a suitable selected reference. The implementation of an automatic reference selection method is helpful to detect the distribution of an EEG rhythm, which can improve the accuracy of EEG interpretation during both visual inspection and automatic interpretation. Copyright © 2013 IPEM

  9. Quantitative Analysis of Humectants in Tobacco Products Using Gas Chromatography (GC with Simultaneous Mass Spectrometry (MSD and Flame Ionization Detection (FID

    Directory of Open Access Journals (Sweden)

    Rainey CL

    2014-12-01

    Full Text Available This paper describes the modification of an existing gas chromatographic (GC method to incorporate simultaneous mass spectrometric (MSD and flame ionization detection (FID into the analysis of tobacco humectants. Glycerol, propylene glycol, and triethylene glycol were analyzed in tobacco labeled as roll-your-own (RYO, cigar, cigarette, moist snuff, and hookah tobacco. Tobacco was extracted in methanol containing 1,3-butanediol (internal standard, filtered, and separated on a 15 m megabore DB-Wax column. Post-column flow was distributed using a microfluidic splitter between the MSD and FID for simultaneous detection. The limits of detection for the FID detector were 0.5 μg/mL (propylene glycol and triethylene glycol and 0.25 μg/mL (glycerol with a linear range of 2-2000 μg/mL (propylene glycol and triethylene glycol and 1-4000 μg/mL (glycerol. The limits of detection for the MSD detector were 2 μg/mL (propylene glycol and triethylene glycol and 4 μg/mL (glycerol with a linear range of 20-2000 μg/mL (propylene glycol and triethylene glycol and 40-4000 μg/mL (glycerol. Significant improvement in the sensitivity of the MSD can be achieved by employing selective ion monitoring (SIM detection mode. Although a high degree of correlation was observed between the results from FID and MSD analyses, marginal chromatographic resolution between glycerol and triethylene glycol limits the applicability of FID to samples containing low levels of both of these humectants. Utilizing MSD greatly improves the reliability of quantitative results because compensation for inadequate chromatographic resolution can be accomplished with mass selectivity in detection.

  10. Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins.

    Science.gov (United States)

    Mueller, Jenna L; Harmany, Zachary T; Mito, Jeffrey K; Kennedy, Stephanie A; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G; Willett, Rebecca M; Brown, J Quincy; Ramanujam, Nimmi

    2013-01-01

    To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features. TISSUE EXCISED FROM A GENETICALLY ENGINEERED MOUSE MODEL OF SARCOMA WAS IMAGED USING A SUBCELLULAR RESOLUTION MICROENDOSCOPE AFTER TOPICAL APPLICATION OF A FLUORESCENT ANATOMICAL CONTRAST AGENT: acriflavine. An algorithm based on sparse component analysis (SCA) and the circle transform (CT) was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma. Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity). For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach. The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.

  11. Pt-MWCNT modified carbon electrode strip for rapid and quantitative detection of H2O2 in food

    Directory of Open Access Journals (Sweden)

    Tai-Cheng Chou

    2018-04-01

    Full Text Available A single-use screen-printed carbon electrode strip was designed and fabricated. Nanohybrids, prepared by deposition of platinum (Pt nanoparticles on multi-wall carbon nanotube (MWCNT, was modified on the surface of screen-printed carbon electrode for the development of a fast, sensitive and cost-effective hydrogen peroxide (H2O2 detection amperometric sensor strip. With Pt-MWCNT nanohybrids surface modification, current generated in response to H2O2 by the screen-printed carbon electrode strip was enhanced 100 fold with an applied potential of 300 mV. Quality of as-prepared electrode strip was assured by the low coefficient of variation (CV (<5% of currents measured at 5 s. Three linear detection ranges with sensitivity of 75.2, 120.7, and 142.8 μA mM−1 cm−2 were observed for H2O2 concentration in the range of 1–15 mM, 0.1–1 mM, and 10–100 μM, respectively. The lowest H2O2 concentration could be measured by the as-prepared strip was 10 μM. H2O2 levels in green tea infusion and pressed Tofu could be rapidly detected with results comparable to that measured by ferrous oxidation xylenol orange (FOX assay and peroxidase colorimetric method. Keywords: Platinum-multi-wall carbon nanotube (Pt-MWCNT, Disposable carbon electrode, Hydrogen peroxide (H2O2, Amperometric sensor

  12. A longitudinal evaluation of performance of automated BCR-ABL1 quantitation using cartridge-based detection system.

    Science.gov (United States)

    Enjeti, Anoop; Granter, Neil; Ashraf, Asma; Fletcher, Linda; Branford, Susan; Rowlings, Philip; Dooley, Susan

    2015-10-01

    An automated cartridge-based detection system (GeneXpert; Cepheid) is being widely adopted in low throughput laboratories for monitoring BCR-ABL1 transcript in chronic myelogenous leukaemia. This Australian study evaluated the longitudinal performance specific characteristics of the automated system.The automated cartridge-based system was compared prospectively with the manual qRT-PCR-based reference method at SA Pathology, Adelaide, over a period of 2.5 years. A conversion factor determination was followed by four re-validations. Peripheral blood samples (n = 129) with international scale (IS) values within detectable range were selected for assessment. The mean bias, proportion of results within specified fold difference (2-, 3- and 5-fold), the concordance rate of major molecular remission (MMR) and concordance across a range of IS values on paired samples were evaluated.The initial conversion factor for the automated system was determined as 0.43. Except for the second re-validation, where a negative bias of 1.9-fold was detected, all other biases fell within desirable limits. A cartridge-specific conversion factor and efficiency value was introduced and the conversion factor was confirmed to be stable in subsequent re-validation cycles. Concordance with the reference method/laboratory at >0.1-≤10 IS was 78.2% and at ≤0.001 was 80%, compared to 86.8% in the >0.01-≤0.1 IS range. The overall and MMR concordance were 85.7% and 94% respectively, for samples that fell within ± 5-fold of the reference laboratory value over the entire period of study.Conversion factor and performance specific characteristics for the automated system were longitudinally stable in the clinically relevant range, following introduction by the manufacturer of lot specific efficiency values.

  13. A quantitative method to detect explosives and selected semivolatiles in soil samples by Fourier transform infrared spectroscopy

    International Nuclear Information System (INIS)

    Clapper-Gowdy, M.; Dermirgian, J.; Robitaille, G.

    1995-01-01

    This paper describes a novel Fourier transform infrared (FTIR) spectroscopic method that can be used to rapidly screen soil samples from potentially hazardous waste sites. Samples are heated in a thermal desorption unit and the resultant vapors are collected and analyzed in a long-path gas cell mounted in a FTIR. Laboratory analysis of a soil sample by FTIR takes approximately 10 minutes. This method has been developed to identify and quantify microgram concentrations of explosives in soil samples and is directly applicable to the detection of selected volatile organics, semivolatile organics, and pesticides

  14. Quantitative analysis and demonstration of modified triple-branch signal detection scheme for SAC-OCDMA systems

    Science.gov (United States)

    Chen, Fujun; Feng, Gang; Zhang, Siwei

    2016-10-01

    The triple-branch signal detection (TBSD) scheme can eliminate multiple-user interference (MUI) without fixed in-phase cross-correlation (IPCC) stipulation in the spectral-amplitude-coding optical code division multiple access (SACOCDMA) systems. In this paper, we modify the traditional TBSD scheme and theoretically analyze the principle of the MUI elimination. Then, the bit-error-rate (BER) performance of the modified TBSD scheme is investigated under multiple transmission rates. The results show that the modified TBSD employing the codes with unfixed IPCC can achieve simultaneous optical code recognition and MUI elimination in the SAC-OCDMA.

  15. Quantitative investigation of a novel small field of view hybrid gamma camera (HGC) capability for sentinel lymph node detection

    Science.gov (United States)

    Lees, John E; Bugby, Sarah L; Jambi, Layal K; Perkins, Alan C

    2016-01-01

    Objective: The hybrid gamma camera (HGC) has been developed to enhance the localization of radiopharmaceutical uptake in targeted tissues during surgical procedures such as sentinel lymph node (SLN) biopsy. To assess the capability of the HGC, a lymph node contrast (LNC) phantom was constructed to simulate medical scenarios of varying radioactivity concentrations and SLN size. Methods: The phantom was constructed using two clear acrylic glass plates. The SLNs were simulated by circular wells of diameters ranging from 10 to 2.5 mm (16 wells in total) in 1 plate. The second plate contains four larger rectangular wells to simulate tissue background activity surrounding the SLNs. The activity used to simulate each SLN ranged between 4 and 0.025 MBq. The activity concentration ratio between the background and the activity injected in the SLNs was 1 : 10. The LNC phantom was placed at different depths of scattering material ranging between 5 and 40 mm. The collimator-to-source distance was 120 mm. Image acquisition times ranged from 60 to 240 s. Results: Contrast-to-noise ratio analysis and full-width-at-half-maximum (FWHM) measurements of the simulated SLNs were carried out for the images obtained. Over the range of activities used, the HGC detected between 87.5 and 100% of the SLNs through 20 mm of scattering material and 75–93.75% of the SLNs through 40 mm of scattering material. The FWHM of the detected SLNs ranged between 11.93 and 14.70 mm. Conclusion: The HGC is capable of detecting low accumulation of activity in small SLNs, indicating its usefulness as an intraoperative imaging system during surgical SLN procedures. Advances in knowledge: This study investigates the capability of a novel small-field-of-view (SFOV) HGC to detect low activity uptake in small SLNs. The phantom and procedure described are inexpensive and could be easily replicated and applied to any SFOV camera, to provide a comparison between systems with clinically relevant

  16. Detection and Quantification of the Entomopathogenic Fungal Endophyte Beauveria bassiana in Plants by Nested and Quantitative PCR.

    Science.gov (United States)

    Garrido-Jurado, Inmaculada; Landa, Blanca B; Quesada-Moraga, Enrique

    2016-01-01

    The described protocol allows detecting as low as 10 fg the entomopathogenic fungal endophyte Beauveria bassiana in host plants by using a two-step nested PCR with the ITS1F/ITS4 and BB.fw and BB.rv primer pairs. On the other hand, a qPCR protocol using BB.fw and BB.rv primers is also available allowing the quantification of up to 26 fg of B. bassiana DNA per 20 ng of leaf DNA.

  17. High-Performance Liquid Chromatography (HPLC)-Based Detection and Quantitation of Cellular c-di-GMP.

    Science.gov (United States)

    Petrova, Olga E; Sauer, Karin

    2017-01-01

    The modulation of c-di-GMP levels plays a vital role in the regulation of various processes in a wide array of bacterial species. Thus, investigation of c-di-GMP regulation requires reliable methods for the assessment of c-di-GMP levels and turnover. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis has become a commonly used approach to accomplish these goals. The following describes the extraction and HPLC-based detection and quantification of c-di-GMP from Pseudomonas aeruginosa samples, a procedure that is amenable to modifications for the analysis of c-di-GMP in other bacterial species.

  18. Quantitative performance of E-Scribe warehouse in detecting quality issues with digital annotated ECG data from healthy subjects.

    Science.gov (United States)

    Sarapa, Nenad; Mortara, Justin L; Brown, Barry D; Isola, Lamberto; Badilini, Fabio

    2008-05-01

    The US Food and Drug Administration recommends submission of digital electrocardiograms in the standard HL7 XML format into the electrocardiogram warehouse to support preapproval review of new drug applications. The Food and Drug Administration scrutinizes electrocardiogram quality by viewing the annotated waveforms and scoring electrocardiogram quality by the warehouse algorithms. Part of the Food and Drug Administration warehouse is commercially available to sponsors as the E-Scribe Warehouse. The authors tested the performance of E-Scribe Warehouse algorithms by quantifying electrocardiogram acquisition quality, adherence to QT annotation protocol, and T-wave signal strength in 2 data sets: "reference" (104 digital electrocardiograms from a phase I study with sotalol in 26 healthy subjects with QT annotations by computer-assisted manual adjustment) and "test" (the same electrocardiograms with an intentionally introduced predefined number of quality issues). The E-Scribe Warehouse correctly detected differences between the 2 sets expected from the number and pattern of errors in the "test" set (except for 1 subject with QT misannotated in different leads of serial electrocardiograms) and confirmed the absence of differences where none was expected. E-Scribe Warehouse scores below the threshold value identified individual electrocardiograms with questionable T-wave signal strength. The E-Scribe Warehouse showed satisfactory performance in detecting electrocardiogram quality issues that may impair reliability of QTc assessment in clinical trials in healthy subjects.

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

    International Nuclear Information System (INIS)

    Gokahmetoglu, S.; Deniz, E.

    2007-01-01

    To compare the real-time (RT) and qualitative (Q) polymerase chain reaction (PCR) assays for detection of Cytomegalovirus (CMV) DNA. The study took place in the Department of Microbiology, Erciyes University, Kayseri and in Iontek Laboratory, Istanbul, Turkey, from August to December 2006. One hundred and seven clinical specimens from 67 patients were included in the study. Cytomegalovirus DNA was investigated using RT-PCR kit (Fluorion Iontek, Turkey) and Q-PCR kit (Fluorion Iontek, Turkey). Deoxyribonucleic acid sequencing was applied to the samples that yielded discrepant results in both assays. Mac Nema's Chi Square test was used for statistical analysis. Of the specimens, 27 were found positive with both assays: 9 with only RT-PCR, and 11 with only Q-PCR assay. Both assays were found negative in 60 of the specimens. There was a good agreement between the 2 assays in 87(81.3%) of the specimens. There was no statistical significant difference between the assays (p>0.05). Two of the 11 samples that RT-PCR negative Q-PCR positive, and 3 of 9 samples that RT-PCR positive Q-PCR negative were found to be CMV DNA positive by DNA sequencing. A good level of concordance between RT-PCR and Q-PCR assays for CMV DNA detection has been found. (author)

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

    Science.gov (United States)

    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.

  1. Alterations of the cerebellum and basal ganglia in bipolar disorder mood states detected by quantitative T1ρ mapping.

    Science.gov (United States)

    Johnson, Casey P; Christensen, Gary E; Fiedorowicz, Jess G; Mani, Merry; Shaffer, Joseph J; Magnotta, Vincent A; Wemmie, John A

    2018-01-07

    Quantitative mapping of T1 relaxation in the rotating frame (T1ρ) is a magnetic resonance imaging technique sensitive to pH and other cellular and microstructural factors, and is a potentially valuable tool for identifying brain alterations in bipolar disorder. Recently, this technique identified differences in the cerebellum and cerebral white matter of euthymic patients vs healthy controls that were consistent with reduced pH in these regions, suggesting an underlying metabolic abnormality. The current study built upon this prior work to investigate brain T1ρ differences across euthymic, depressed, and manic mood states of bipolar disorder. Forty participants with bipolar I disorder and 29 healthy control participants matched for age and gender were enrolled. Participants with bipolar disorder were imaged in one or more mood states, yielding 27, 12, and 13 imaging sessions in euthymic, depressed, and manic mood states, respectively. Three-dimensional, whole-brain anatomical images and T1ρ maps were acquired for all participants, enabling voxel-wise evaluation of T1ρ differences between bipolar mood state and healthy control groups. All three mood state groups had increased T1ρ relaxation times in the cerebellum compared to the healthy control group. Additionally, the depressed and manic groups had reduced T1ρ relaxation times in and around the basal ganglia compared to the control and euthymic groups. The study implicated the cerebellum and basal ganglia in the pathophysiology of bipolar disorder and its mood states, the roles of which are relatively unexplored. These findings motivate further investigation of the underlying cause of the abnormalities, and the potential role of altered metabolic activity in these regions. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  2. Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins.

    Directory of Open Access Journals (Sweden)

    Jenna L Mueller

    Full Text Available To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features.TISSUE EXCISED FROM A GENETICALLY ENGINEERED MOUSE MODEL OF SARCOMA WAS IMAGED USING A SUBCELLULAR RESOLUTION MICROENDOSCOPE AFTER TOPICAL APPLICATION OF A FLUORESCENT ANATOMICAL CONTRAST AGENT: acriflavine. An algorithm based on sparse component analysis (SCA and the circle transform (CT was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma.Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity. For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach.The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.

  3. Quantitation of underivatized branched-chain amino acids in sport nutritional supplements by capillary electrophoresis with direct or indirect UV absorbance detection.

    Science.gov (United States)

    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.

  4. Quantitation of underivatized branched-chain amino acids in sport nutritional supplements by capillary electrophoresis with direct or indirect UV absorbance detection.

    Directory of Open Access Journals (Sweden)

    Jun Qiu

    Full Text Available 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.

  5. Qualitative and quantitative analysis of milk for the detection of adulteration by Laser Induced Breakdown Spectroscopy (LIBS).

    Science.gov (United States)

    Moncayo, S; Manzoor, S; Rosales, J D; Anzano, J; Caceres, J O

    2017-10-01

    The present work focuses on the development of a fast and cost effective method based on Laser Induced Breakdown Spectroscopy (LIBS) to the quality control, traceability and detection of adulteration in milk. Two adulteration cases have been studied; a qualitative analysis for the discrimination between different milk blends and quantification of melamine in adulterated toddler milk powder. Principal Component Analysis (PCA) and neural networks (NN) have been used to analyze LIBS spectra obtaining a correct classification rate of 98% with a 100% of robustness. For the quantification of melamine, two methodologies have been developed; univariate analysis using CN emission band and multivariate calibration NN model obtaining correlation coefficient (R 2 ) values of 0.982 and 0.999 respectively. The results of the use of LIBS technique coupled with chemometric analysis are discussed in terms of its potential use in the food industry to perform the quality control of this dairy product. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Hairpin stabilized fluorescent silver nanoclusters for quantitative detection of NAD+ and monitoring NAD+/NADH based enzymatic reactions.

    Science.gov (United States)

    Jain, Priyamvada; Chakma, Babina; Patra, Sanjukta; Goswami, Pranab

    2017-03-01

    A set of 90 mer long ssDNA candidates, with different degrees of cytosine (C-levels) (% and clusters) was analyzed for their function as suitable Ag-nanocluster (AgNC) nucleation scaffolds. The sequence (P4) with highest C-level (42.2%) emerged as the only candidate supporting the nucleation process as evident from its intense fluorescence peak at λ 660 nm . Shorter DNA subsets derived from P4 with only stable hairpin structures could support the AgNC formation. The secondary hairpin structures were confirmed by PAGE, and CD studies. The number of base pairs in the stem region also contributes to the stability of the hairpins. A shorter 29 mer sequence (Sub 3) (ΔG = -1.3 kcal/mol) with 3-bp in the stem of a 7-mer loop conferred highly stable AgNC. NAD + strongly quenched the fluorescence of Sub 3-AgNC in a concentration dependent manner. Time resolved photoluminescence studies revealed the quenching involves a combined static and dynamic interaction where the binding constant and number of binding sites for NAD + were 0.201 L mol -1 and 3.6, respectively. A dynamic NAD + detection range of 50-500 μM with a limit of detection of 22.3 μM was discerned. The NAD + mediated quenching of AgNC was not interfered by NADH, NADP + , monovalent and divalent ions, or serum samples. The method was also used to follow alcohol dehydrogenase and lactate dehydrogenase catalyzed physiological reactions in a turn-on and turn-off assay, respectively. The proposed method with ssDNA-AgNC could therefore be extended to monitor other NAD + /NADH based enzyme catalyzed reactions in a turn-on/turn-off approach. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Rapid and quantitative detection of the microbial spoilage in milk using Fourier transform infrared spectroscopy and chemometrics.

    Science.gov (United States)

    Nicolaou, Nicoletta; Goodacre, Royston

    2008-10-01

    Microbiological safety plays a very significant part in the quality control of milk and dairy products worldwide. Current methods used in the detection and enumeration of spoilage bacteria in pasteurized milk in the dairy industry, although accurate and sensitive, are time-consuming. FT-IR spectroscopy is a metabolic fingerprinting technique that can potentially be used to deliver results with the same accuracy and sensitivity, within minutes after minimal sample preparation. We tested this hypothesis using attenuated total reflectance (ATR), and high throughput (HT) FT-IR techniques. Three main types of pasteurized milk - whole, semi-skimmed and skimmed - were used and milk was allowed to spoil naturally by incubation at 15 degrees C. Samples for FT-IR were obtained at frequent, fixed time intervals and pH and total viable counts were also recorded. Multivariate statistical methods, including principal components-discriminant function analysis and partial least squares regression (PLSR), were then used to investigate the relationship between metabolic fingerprints and the total viable counts. FT-IR ATR data for all milks showed reasonable results for bacterial loads above 10(5) cfu ml(-1). By contrast, FT-IR HT provided more accurate results for lower viable bacterial counts down to 10(3) cfu ml(-1) for whole milk and, 4 x 10(2) cfu ml(-1) for semi-skimmed and skimmed milk. Using FT-IR with PLSR we were able to acquire a metabolic fingerprint rapidly and quantify the microbial load of milk samples accurately, with very little sample preparation. We believe that metabolic fingerprinting using FT-IR has very good potential for future use in the dairy industry as a rapid method of detection and enumeration.

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

    Science.gov (United States)

    Kolm, Claudia; Martzy, Roland; Brunner, Kurt; Mach, Robert L; Krska, Rudolf; Heinze, Georg; Sommer, Regina; Reischer, Georg H; Farnleitner, Andreas H

    2017-06-20

    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.

  9. Optimisation of a quantitative polymerase chain reaction-based strategy for the detection and quantification of human herpesvirus 6 DNA in patients undergoing allogeneic haematopoietic stem cell transplantation

    Directory of Open Access Journals (Sweden)

    Miriam YH Ueda

    2015-06-01

    Full Text Available Human herpesvirus 6 (HHV-6 may cause severe complications after haematopoietic stem cell transplantation (HSCT. Monitoring this virus and providing precise, rapid and early diagnosis of related clinical diseases, constitute essential measures to improve outcomes. A prospective survey on the incidence and clinical features of HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the impact of this infection on HSCT outcome remains unclear. A rapid test based on real-time quantitative polymerase chain reaction (qPCR has been optimised to screen and quantify clinical samples for HHV-6. The detection step was based on reaction with TaqMan® hydrolysis probes. A set of previously described primers and probes have been tested to evaluate efficiency, sensitivity and reproducibility. The target efficiency range was 91.4% with linearity ranging from 10-106 copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of plasma. The qPCR assay developed in the present study was simple, rapid and sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this test may be useful as a practical tool to help elucidate the clinical relevance of HHV-6 infection and reactivation in different scenarios and to determine the need for surveillance.

  10. Detection and quantitation of trace phenolphthalein (in pharmaceutical preparations and in forensic exhibits) by liquid chromatography-tandem mass spectrometry, a sensitive and accurate method.

    Science.gov (United States)

    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.

  11. SU-E-QI-14: Quantitative Variogram Detection of Mild, Unilateral Disease in Elastase-Treated Rats

    Energy Technology Data Exchange (ETDEWEB)

    Jacob, R [Pacific Northwest National Laboraory, Richland, WA (United States); Carson, J [Texas Advanced Computing Center, Austin, TX (United States)

    2014-06-15

    Purpose: Determining the presence of mild or early disease in the lungs can be challenging and subjective. We present a rapid and objective method for evaluating lung damage in a rat model of unilateral mild emphysema based on a new approach to heterogeneity assessment. We combined octree decomposition (used in three-dimensional (3D) computer graphics) with variograms (used in geostatistics to assess spatial relationships) to evaluate 3D computed tomography (CT) lung images for disease. Methods: Male, Sprague-Dawley rats (232 ± 7 g) were intratracheally dosed with 50 U/kg of elastase dissolved in 200 μL of saline to a single lobe (n=6) or with saline only (n=5). After four weeks, 3D micro-CT images were acquired at end expiration on mechanically ventilated rats using prospective gating. Images were masked, and lungs were decomposed to homogeneous blocks of 2×2×2, 4×4×4, and 8×8×8 voxels using octree decomposition. The spatial variance – the square of the difference of signal intensity – between all pairs of the 8×8×8 blocks was calculated. Variograms – graphs of distance vs. variance - were made, and data were fit to a power law and the exponent determined. The mean HU values, coefficient of variation (CoV), and the emphysema index (EI) were calculated and compared to the variograms. Results: The variogram analysis showed that significant differences between groups existed (p<0.01), whereas the mean HU (p=0.07), CoV (p=0.24), and EI (p=0.08) did not. Calculation time for the variogram for a typical 1000 block decomposition was ∼6 seconds, and octree decomposition took ∼2 minutes. Decomposing the images prior to variogram calculation resulted in a ∼700x decrease in time as compared to other published approaches. Conclusions: Our results suggest that the approach combining octree decomposition and variogram analysis may be a rapid, non-subjective, and sensitive imaging-based biomarker for quantitative characterization of lung disease.

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

    Science.gov (United States)

    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.

  13. Detection of quantitative trait loci in Danish Holstein cattle affecting clinical mastitis, somatic cell score, udder conformation traits, and assessment of associated effects on milk yield

    DEFF Research Database (Denmark)

    Lund, M S; Guldbrandtsen, B; Buitenhuis, A J

    2008-01-01

    The aim of this study was to 1) detect QTL across the cattle genome that influence the incidence of clinical mastitis and somatic cell score (SCS) in Danish Holsteins, and 2) characterize these QTL for pleiotropy versus multiple linked quantitative trait loci (QTL) when chromosomal regions...... affecting clinical mastitis were also affecting other traits in the Danish udder health index or milk production traits. The chromosomes were scanned using a granddaughter design where markers were typed for 19 to 34 grandsire families and 1,373 to 2,042 sons. A total of 356 microsatellites covering all 29...... autosomes were used in the scan. Among the across-family regression analyses, 16 showed chromosome-wide significance for the primary traits incidence of clinical mastitis in first (CM1), second (CM2), and third (CM3) lactations, and SCS. Regions of chromosomes 5, 6, 9, 11, 15, and 26 were found to affect CM...

  14. Quantitation of Pyrrole-Imidazole Polyamide in Rat Plasma by High-Performance Liquid Chromatography Coupled with UV Detection

    Directory of Open Access Journals (Sweden)

    Tomonori Kamei

    2012-01-01

    Full Text Available A simple and robust method using high-performance liquid chromatography with UV detection was developed and validated for the determination of six pyrrole-imidazole (PI polyamides (HN.49, TGF-β1f, TGF-β1t, HN.50f, HN.50t, and LOX-1 in rat plasma. After the plasma proteins were precipitated with methanol containing phenacetin as an internal standard, the analytes were separated on a Luna C18 (2 (5 μm, 4.6×150 mm. Calibration curves were linear over the range of 0.5 to 200 μg/mL for HN.49, 0.25 to 200 μg/mL for TGF-β1f, TGF-β1t, HN.50t, and LOX-1, 1 to 200 μg/mL for HN.50f in rat plasma. The inter- and intraday precision were below 15%, and the accuracy was within 15% at the quality controls. The validated method was successfully applied to sample analysis for the pharmacokinetic study.

  15. Microfluidic paper-based analytical devices for potential use in quantitative and direct detection of disease biomarkers in clinical analysis.

    Science.gov (United States)

    Lim, Wei Yin; Goh, Boon Tong; Khor, Sook Mei

    2017-08-15

    Clinicians, working in the health-care diagnostic systems of developing countries, currently face the challenges of rising costs, increased number of patient visits, and limited resources. A significant trend is using low-cost substrates to develop microfluidic devices for diagnostic purposes. Various fabrication techniques, materials, and detection methods have been explored to develop these devices. Microfluidic paper-based analytical devices (μPADs) have gained attention for sensing multiplex analytes, confirming diagnostic test results, rapid sample analysis, and reducing the volume of samples and analytical reagents. μPADs, which can provide accurate and reliable direct measurement without sample pretreatment, can reduce patient medical burden and yield rapid test results, aiding physicians in choosing appropriate treatment. The objectives of this review are to provide an overview of the strategies used for developing paper-based sensors with enhanced analytical performances and to discuss the current challenges, limitations, advantages, disadvantages, and future prospects of paper-based microfluidic platforms in clinical diagnostics. μPADs, with validated and justified analytical performances, can potentially improve the quality of life by providing inexpensive, rapid, portable, biodegradable, and reliable diagnostics. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Development of a quantitative PCR assay for rapid detection of Lactobacillus plantarum and Lactobacillus fermentum in cocoa bean fermentation.

    Science.gov (United States)

    Schwendimann, Livia; Kauf, Peter; Fieseler, Lars; Gantenbein-Demarchi, Corinne; Miescher Schwenninger, Susanne

    2015-08-01

    To monitor dominant species of lactic acid bacteria during cocoa bean fermentation, i.e. Lactobacillus plantarum and Lactobacillus fermentum, a fast and reliable culture-independent qPCR assay was developed. A modified DNA isolation procedure using a commercial kit followed by two species-specific qPCR assays resulted in 100% sensitivity for L. plantarum and L. fermentum. Kruskal-Wallis and post-hoc analyses of data obtained from experiments with cocoa beans that were artificially spiked with decimal concentrations of L. plantarum and L. fermentum strains allowed the calculation of a regression line suitable for the estimation of both species with a detection limit of 3 to 4 Log cells/g cocoa beans. This process was successfully tested for efficacy through the analyses of samples from laboratory-scale cocoa bean fermentations with both the qPCR assay and a culture-dependent method which resulted in comparable results. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Enhancing the response of CALUX and CAFLUX cell bioassays for quantitative detection of dioxin-like compounds

    Science.gov (United States)

    ZHAO, Bin; BASTON, David S.; KHAN, Elaine; SORRENTINO, Claudio; DENISON, Michael S.

    2011-01-01

    Reporter genes produce a protein product in transfected cells that can be easily measured in intact or lysed cells and they have been extensively used in numerous basic and applied research applications. Over the past 10 years, reporter gene assays have been widely accepted and used for analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like compounds in various types of matrices, such as biological, environmental, food and feed samples, given that high-resolution instrumental analysis techniques are impractical for large-scale screening analysis. The most sensitive cell-based reporter gene bioassay systems developed are the mechanism-based CALUX (Chemically Activated Luciferase Expression) and CAFLUX (Chemically Activated Fluorescent Expression) bioassays, which utilize recombinant cell lines containing stably transfected dioxin (AhR)-responsive firefly luciferase or enhanced green fluorescent protein (EGFP) reporter genes, respectively. While the current CALUX and CAFLUX bioassays are very sensitive, increasing their lower limit of sensitivity, magnitude of response and dynamic range for chemical detection would significantly increase their utility, particularly for those samples that contain low levels of dioxin-like HAHs (i.e., serum). In this study, we report that the addition of modulators of cell signaling pathways or modification of cell culture conditions results in significant improvement in the magnitude and overall responsiveness of the existing CALUX and CAFLUX cell bioassays. PMID:21394221

  18. Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization.

    Science.gov (United States)

    Pauletti, G; Godolphin, W; Press, M F; Slamon, D J

    1996-07-04

    Amplification and overexpression of the HER-2/neu gene occurs in 25-30% of human breast cancers. This genetic alteration is associated with a poor clinical prognosis in women with either node negative or node positive breast cancers. The initial studies testing this association were somewhat controversial and this controversy was due in large part to significant heterogeneity in both the methods and/or reagents used in testing archival material for the presence of the alteration. These methods included a number of solid matrix blotting techniques for DNA, RNA and protein as well as immunohistochemistry. Fluorescence in situ hybridization (FISH) represents the newest methodologic approach for testing for this genetic alteration. In this study, FISH is compared to Southern, Northern and Western blot analyses as well as immunohistochemistry in a large cohort of archival human breast cancer specimens. FISH was found to be superior to all other methodologies tested in assessing formalin fixed, paraffin embedded material for HER-2/neu amplification. The results from this study also confirm that overexpression of HER-2/neu rarely occurs in the absence of gene amplification in breast cancer (approximately 3% of cases). This method of analysis is rapid, reproducible and extremely reliable in detecting presence of HER-2/neu gene amplification and should have clinical utility.

  19. Rapid and High-Throughput Detection and Quantitation of Radiation Biomarkers in Human and Nonhuman Primates by Differential Mobility Spectrometry-Mass Spectrometry

    Science.gov (United States)

    Chen, Zhidan; Coy, Stephen L.; Pannkuk, Evan L.; Laiakis, Evagelia C.; Hall, Adam B.; Fornace, Albert J.; Vouros, Paul

    2016-10-01

    Radiation exposure is an important public health issue due to a range of accidental and intentional threats. Prompt and effective large-scale screening and appropriate use of medical countermeasures (MCM) to mitigate radiation injury requires rapid methods for determining the radiation dose. In a number of studies, metabolomics has identified small-molecule biomarkers responding to the radiation dose. Differential mobility spectrometry-mass spectrometry (DMS-MS) has been used for similar compounds for high-throughput small-molecule detection and quantitation. In this study, we show that DMS-MS can detect and quantify two radiation biomarkers, trimethyl-L-lysine (TML) and hypoxanthine. Hypoxanthine is a human and nonhuman primate (NHP) radiation biomarker and metabolic intermediate, whereas TML is a radiation biomarker in humans but not in NHP, which is involved in carnitine synthesis. They have been analyzed by DMS-MS from urine samples after a simple strong cation exchange-solid phase extraction (SCX-SPE). The dramatic suppression of background and chemical noise provided by DMS-MS results in an approximately 10-fold reduction in time, including sample pretreatment time, compared with liquid chromatography-mass spectrometry (LC-MS). DMS-MS quantitation accuracy has been verified by validation testing for each biomarker. Human samples are not yet available, but for hypoxanthine, selected NHP urine samples (pre- and 7-d-post 10 Gy exposure) were analyzed, resulting in a mean change in concentration essentially identical to that obtained by LC-MS (fold-change 2.76 versus 2.59). These results confirm the potential of DMS-MS for field or clinical first-level rapid screening for radiation exposure.

  20. The development of 1,3-diphenylisobenzofuran as a highly selective probe for the detection and quantitative determination of hydrogen peroxide.

    Science.gov (United States)

    Żamojć, Krzysztof; Zdrowowicz, Magdalena; Rudnicki-Velasquez, Paweł Błażej; Krzymiński, Karol; Zaborowski, Bartłomiej; Niedziałkowski, Paweł; Jacewicz, Dagmara; Chmurzyński, Lech

    2017-01-01

    1,3-Diphenylisobenzofuran (DPBF) has been developed as a selective probe for the detection and quantitative determination of hydrogen peroxide in samples containing different reactive nitrogen and oxygen species (RNOS). DPBF is a fluorescent probe which, for almost 20 years, was believed to react in a highly specific manner toward some reactive oxygen species (ROS) such as singlet oxygen and hydroxy, alkyloxy or alkylperoxy radicals. Under the action of these individuals DPBF has been rapidly transformed to 1,2-dibenzoylbenzene (DBB). In order to check if DPBF can act as a unique indicator of the total amount of different RNOS, as well as oxidative stress caused by an overproduction of these individuals, a series of experiments was carried out, in which DPBF reacted with peroxynitrite anion, superoxide anion, hydrogen peroxide, hypochlorite anion, and anions commonly present under biological conditions, namely nitrite and nitrate. In all cases, except for hydrogen peroxide, the product of the reaction is DBB. Only under the action of H 2 O 2 9-hydroxyanthracen-10(9H)-one (oxanthrone) is formed. This product has been identified with the use of fluorescence spectroscopy, NMR spectroscopy, high performance liquid chromatography coupled with mass spectrometry, infrared spectroscopy, elemental analysis, and cyclic voltammetry (CV). A linear relationship was found between a decrease in the fluorescence intensity of DPBF and the concentration of hydrogen peroxide in the range of concentrations of 0.196-3.941 mM. DPBF responds to hydrogen peroxide in a very specific way with the limits of detection and quantitation of 88 and 122.8 μM, respectively. The kinetics of the reaction between DBBF and H 2 O 2 was also studied.

  1. [Detecting HB-1 Expression Level in Bone Marrow of Acute Leukemia Patients by Real-Time Fluorescence Quantitative RT-PCR].

    Science.gov (United States)

    Wang, Qing-Yun; Li, Yuan; Ji, Li; Liang, Ze-Yin; Liu, Wei; Ren, Han-Yun; Qiu, Zhi-Xiang

    2018-02-01

    To investigate the expression level of HB-1 gene in patients with acute lymphoblastic leukemia (ALL) and the significance of HB-1 gene in monitoring of minimal residual disease (MRD). The method of real-time fluorescence quantitative RT-PCR (Taqman probe) was established to detect the expression levels of HB-1 gene; then the sensitivity, specificity and repeatability of this assay were evaluated and verified. The HB-1 gene expression levels in bone marrow of 183 cases of ALL, 70 cases of acute myeloid leukemias (AML), 52 cases of non-malignant hematologic diseases and 24 healthy hematopoietic stem cell donors were detected. The correlation of HB-1 level with diagnosis and relapse was analyzed by detecting bone marrow samples of 33 B-ALL. The sensitivity of this assay reached the 10 -4 level. The coefficient of variation for inter-batch and inter-tube of HB-1 were 6.79% and 4.80%, respectively. It was found that HB-1 gene specifically expressed in acute B lymphoblastic leukemia. The median expression levels of HB-1 gene in newly diagnosed and relapsed B-ALL patients were statistically significantly higher than those in ALL in complete remission(CR), newly diagnosed T-ALL, newly diagnosed AML, non-malignant hematologic diseases, and healthy hematopoietic stem cell donors(33.0% vs 0.68%, 0.07%, 0.02%, 0.58% and 0, respectively) (P0.05). The expression level of HB-1 gene declined sharply when B-ALL patients reached complete remission (0-7.99%, with median level 0.68%), but increased when relapsed (7.69%, 8.08% and 484.0% in 3 relapsed samples), which was in accordance with results of flow cytometry. HB-1 gene specifically expressed in acute B lymphoblastic leukemia cells. The established real-time fluorescence quantitative RT-PCR assay shows good sensitivity, specificity and repeatability, thus, can be used as a biological marker in the clinical detection, monitoring MRD and predicting of early relapse for B-ALL patients.

  2. Precipitation evidences on X-Band Synthetic Aperture Radar imagery: an approach for quantitative detection and estimation

    Science.gov (United States)

    Mori, Saverio; Marzano, Frank S.; Montopoli, Mario; Pulvirenti, Luca; Pierdicca, Nazzareno

    2017-04-01

    Spaceborne synthetic aperture radars (SARs) operating at L-band and above are nowadays a well-established tool for Earth remote sensing; among the numerous civil applications we can indicate flood areas detection and monitoring, earthquakes analysis, digital elevation model production, land use monitoring and classification. Appealing characteristics of this kind of instruments is the high spatial resolution ensured in almost all-weather conditions and with a reasonable duty cycle and coverage. This result has achieved by the by the most recent generation of SAR missions, which moreover allow polarimetric observation of the target. Nevertheless, atmospheric clouds, in particular the precipitating ones, can significantly affect the signal backscattered from the ground surface (e.g. Ferrazzoli and Schiavon, 1997), on both amplitude and phase, with effects increasing with the operating frequency. In this respect, proofs are given by several recent works (e.g. Marzano et al., 2010, Baldini et al., 2014) using X-Band SAR data by COSMO-SkyMed (CSK) and TerraSAR-X (TSX) missions. On the other hand, this sensitivity open interesting perspectives towards the SAR observation, and eventually quantification, of precipitations. In this respect, a proposal approach for X-SARs precipitation maps production and cloud masking arise from our work. Cloud masking allows detection of precipitation compromised areas. Respect precipitation maps, satellite X-SARs offer the unique possibility to ingest within flood forecasting model precipitation data at the catchment scale. This aspect is particularly innovative, even if work has been done the late years, and some aspects need to still address. Our developed processing framework allows, within the cloud masking stage, distinguishing flooded areas, precipitating clouds together with permanent water bodies, all appearing dark in the SAR image. The procedure is mainly based on image segmentation techniques and fuzzy logic (e.g. Pulvirenti et

  3. A propidium monoazide–quantitative PCR method for the detection and quantification of viable Enterococcus faecalis in large-volume samples of marine waters

    KAUST Repository

    Salam, Khaled W.; El-Fadel, Mutasem E.; Barbour, Elie K.; Saikaly, Pascal

    2014-01-01

    The development of rapid detection assays of cell viability is essential for monitoring the microbiological quality of water systems. Coupling propidium monoazide with quantitative PCR (PMA-qPCR) has been successfully applied in different studies for the detection and quantification of viable cells in small-volume samples (0.25-1.00 mL), but it has not been evaluated sufficiently in marine environments or in large-volume samples. In this study, we successfully integrated blue light-emitting diodes for photoactivating PMA and membrane filtration into the PMA-qPCR assay for the rapid detection and quantification of viable Enterococcus faecalis cells in 10-mL samples of marine waters. The assay was optimized in phosphate-buffered saline and seawater, reducing the qPCR signal of heat-killed E. faecalis cells by 4 log10 and 3 log10 units, respectively. Results suggest that high total dissolved solid concentration (32 g/L) in seawater can reduce PMA activity. Optimal PMA-qPCR standard curves with a 6-log dynamic range and detection limit of 102 cells/mL were generated for quantifying viable E. faecalis cells in marine waters. The developed assay was compared with the standard membrane filter (MF) method by quantifying viable E. faecalis cells in seawater samples exposed to solar radiation. The results of the developed PMA-qPCR assay did not match that of the standard MF method. This difference in the results reflects the different physiological states of E. faecalis cells in seawater. In conclusion, the developed assay is a rapid (∼5 h) method for the quantification of viable E. faecalis cells in marine recreational waters, which should be further improved and tested in different seawater settings. © 2014 Springer-Verlag Berlin Heidelberg.

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

    Science.gov (United States)

    Psifidi, Androniki; Dovas, Chrysostomos; Banos, Georgios

    2011-01-01

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

  5. A propidium monoazide–quantitative PCR method for the detection and quantification of viable Enterococcus faecalis in large-volume samples of marine waters

    KAUST Repository

    Salam, Khaled W.

    2014-08-23

    The development of rapid detection assays of cell viability is essential for monitoring the microbiological quality of water systems. Coupling propidium monoazide with quantitative PCR (PMA-qPCR) has been successfully applied in different studies for the detection and quantification of viable cells in small-volume samples (0.25-1.00 mL), but it has not been evaluated sufficiently in marine environments or in large-volume samples. In this study, we successfully integrated blue light-emitting diodes for photoactivating PMA and membrane filtration into the PMA-qPCR assay for the rapid detection and quantification of viable Enterococcus faecalis cells in 10-mL samples of marine waters. The assay was optimized in phosphate-buffered saline and seawater, reducing the qPCR signal of heat-killed E. faecalis cells by 4 log10 and 3 log10 units, respectively. Results suggest that high total dissolved solid concentration (32 g/L) in seawater can reduce PMA activity. Optimal PMA-qPCR standard curves with a 6-log dynamic range and detection limit of 102 cells/mL were generated for quantifying viable E. faecalis cells in marine waters. The developed assay was compared with the standard membrane filter (MF) method by quantifying viable E. faecalis cells in seawater samples exposed to solar radiation. The results of the developed PMA-qPCR assay did not match that of the standard MF method. This difference in the results reflects the different physiological states of E. faecalis cells in seawater. In conclusion, the developed assay is a rapid (∼5 h) method for the quantification of viable E. faecalis cells in marine recreational waters, which should be further improved and tested in different seawater settings. © 2014 Springer-Verlag Berlin Heidelberg.

  6. Quantitation of myocardial blood flow and myocardial flow reserve with {sup 99m}Tc-sestamibi dynamic SPECT/CT to enhance detection of coronary artery disease

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Bailing [University of Missouri-Columbia, Nuclear Science and Engineering Institute, Columbia, MO (United States); Chen, Fu-Chung; Chen, Chien-Cheng [Show Chwan Memorial Hospital, Section of Cardiology, Department of Internal Medicine, Changhua (China); Wu, Tao-Cheng [Taipei Veterans General Hospital, Section of Cardiology, Department of Internal Medicine, Taipei (China); Huang, Wen-Sheng [Changhua Christian Hospital, Department of Medical Research and Department of Nuclear Medicine, Changhua (China); Hou, Po-Nien [Chang Bing Show Chwan Memorial Hospital, Department of Nuclear Medicine, Lukong Town, Changhua Shien (China); Hung, Guang-Uei [Chang Bing Show Chwan Memorial Hospital, Department of Nuclear Medicine, Lukong Town, Changhua Shien (China); Central Taiwan University of Science and Technology, Department of Medical Imaging and Radiological Science, Taichung (China); China Medical University, Department of Biomedical Imaging and Radiological Science, Taichung (China)

    2014-12-15

    Conventional dual-head single photon emission computed tomography (SPECT)/CT systems capable of fast dynamic SPECT (DySPECT) imaging have a potential for flow quantitation. This study introduced a new method to quantify myocardial blood flow (MBF) and myocardial flow reserve (MFR) with DySPECT scan and evaluated the diagnostic performance of detecting coronary artery disease (CAD) compared with perfusion using invasive coronary angiography (CAG) as the reference standard. This study included 21 patients with suspected or known CAD who had received DySPECT, ECG-gated SPECT (GSPECT), and CAG (13 with ≥50 % stenosis in any vessel; non-CAD group: 8 with patent arteries or <50 % stenosis). DySPECT and GSPECT scans were performed on a widely used dual-head SPECT/CT scanner. The DySPECT imaging protocol utilized 12-min multiple back-and-forth gantry rotations during injections of {sup 99m}Tc-sestamibi (MIBI) tracer at rest or dipyridamole-stress stages. DySPECT images were reconstructed with full physical corrections and converted to the physical unit of becquerels per milliliter. Stress MBF (SMBF), rest MBF (RMBF), and MFR were quantified by a one-tissue compartment flow model using time-activity curves derived from DySPECT images. Perfusion images were processed for GSPECT scan and interpreted to obtain summed stress score (SSS) and summed difference score (SDS). Receiver-operating characteristic (ROC) analyses were conducted to evaluate the diagnostic performance of flow and perfusion. Using the criteria of ≥50 % stenosis as positive CAD, areas under the ROC curve (AUCs) of flow assessment were overall significantly greater than those of perfusion. For patient-based analysis, AUCs for MFR, SMBF, SSS, and SDS were 0.91 ± 0.07, 0.86 ± 0.09, 0.64 ± 0.12, and 0.59 ± 0.13. For vessel-based analysis, AUCs for MFR, SMBF, SSS, and SDS were 0.81 ± 0.05, 0.76 ± 0.06, 0.62 ± 0.07, and 0.56 ± 0.08, respectively. The preliminary data suggest that MBF quantitation with a

  7. Quantitation of myocardial blood flow and myocardial flow reserve with 99mTc-sestamibi dynamic SPECT/CT to enhance detection of coronary artery disease

    International Nuclear Information System (INIS)

    Hsu, Bailing; Chen, Fu-Chung; Chen, Chien-Cheng; Wu, Tao-Cheng; Huang, Wen-Sheng; Hou, Po-Nien; Hung, Guang-Uei

    2014-01-01

    Conventional dual-head single photon emission computed tomography (SPECT)/CT systems capable of fast dynamic SPECT (DySPECT) imaging have a potential for flow quantitation. This study introduced a new method to quantify myocardial blood flow (MBF) and myocardial flow reserve (MFR) with DySPECT scan and evaluated the diagnostic performance of detecting coronary artery disease (CAD) compared with perfusion using invasive coronary angiography (CAG) as the reference standard. This study included 21 patients with suspected or known CAD who had received DySPECT, ECG-gated SPECT (GSPECT), and CAG (13 with ≥50 % stenosis in any vessel; non-CAD group: 8 with patent arteries or 99m Tc-sestamibi (MIBI) tracer at rest or dipyridamole-stress stages. DySPECT images were reconstructed with full physical corrections and converted to the physical unit of becquerels per milliliter. Stress MBF (SMBF), rest MBF (RMBF), and MFR were quantified by a one-tissue compartment flow model using time-activity curves derived from DySPECT images. Perfusion images were processed for GSPECT scan and interpreted to obtain summed stress score (SSS) and summed difference score (SDS). Receiver-operating characteristic (ROC) analyses were conducted to evaluate the diagnostic performance of flow and perfusion. Using the criteria of ≥50 % stenosis as positive CAD, areas under the ROC curve (AUCs) of flow assessment were overall significantly greater than those of perfusion. For patient-based analysis, AUCs for MFR, SMBF, SSS, and SDS were 0.91 ± 0.07, 0.86 ± 0.09, 0.64 ± 0.12, and 0.59 ± 0.13. For vessel-based analysis, AUCs for MFR, SMBF, SSS, and SDS were 0.81 ± 0.05, 0.76 ± 0.06, 0.62 ± 0.07, and 0.56 ± 0.08, respectively. The preliminary data suggest that MBF quantitation with a conventional SPECT/CT system and the flow quantitation method is a clinically effective approach to enhance CAD detection. (orig.)

  8. Quantitative radiography

    International Nuclear Information System (INIS)

    Brase, J.M.; Martz, H.E.; Waltjen, K.E.; Hurd, R.L.; Wieting, M.G.

    1986-01-01

    Radiographic techniques have been used in nondestructive evaluation primarily to develop qualitative information (i.e., defect detection). This project applies and extends the techniques developed in medical x-ray imaging, particularly computed tomography (CT), to develop quantitative information (both spatial dimensions and material quantities) on the three-dimensional (3D) structure of solids. Accomplishments in FY 86 include (1) improvements in experimental equipment - an improved microfocus system that will give 20-μm resolution and has potential for increased imaging speed, and (2) development of a simple new technique for displaying 3D images so as to clearly show the structure of the object. Image reconstruction and data analysis for a series of synchrotron CT experiments conducted by LLNL's Chemistry Department has begun

  9. Quantitative detection of epstein-barr virus DNA in cerebrospinal fluid and blood samples of patients with relapsing-remitting multiple sclerosis.

    Directory of Open Access Journals (Sweden)

    Clementina E Cocuzza

    Full Text Available The presence of Epstein-Barr Virus (EBV DNA in cerebrospinal fluid (CSF and peripheral blood (PB samples collected from 55 patients with clinical and radiologically-active relapsing-remitting MS (RRMS and 51 subjects with other neurological diseases was determined using standardized commercially available kits for viral nucleic acid extraction and quantitative EBV DNA detection. Both cell-free and cell-associated CSF and PB fractions were analyzed, to distinguish latent from lytic EBV infection. EBV DNA was detected in 5.5% and 18.2% of cell-free and cell-associated CSF fractions of patients with RRMS as compared to 7.8% and 7.8% of controls; plasma and peripheral blood mononuclear cells (PBMC positivity rates were 7.3% and 47.3% versus 5.8% and 31.4%, respectively. No significant difference in median EBV viral loads of positive samples was found between RRMS and control patients in all tested samples. Absence of statistically significant differences in EBV positivity rates between RRMS and control patients, despite the use of highly sensitive standardized methods, points to the lack of association between EBV and MS disease activity.

  10. Simultaneous quantitative determination of six active components in traditional Chinese medicinal preparation Cerebralcare Granule® by RP-HPLC coupled with diode array detection for quality control.

    Science.gov (United States)

    Wang, Xiang-yang; Ma, Xiao-hui; Li, Wei; Chu, Yang; Guo, Jia-hua; Zhou, Shui-ping; Zhu, Yong-hong

    2014-09-01

    A simple, accurate and reliable method for the simultaneous separation and determination of six active components (protocatechuic acid, chlorogenic acid, caffeic acid, paeoniflorin, ferulic acid and rosmarinic acid) in traditional Chinese medicinal preparation Cerebralcare Granule(®) (CG) was developed using reverse-phase high-performance liquid chromatography coupled with diode array detector detection. The chromatographic separation was performed on a Hypersil GOLD C18 column with aqueous formic acid (0.1%, v/v) and acetonitrile as mobile phase at a flow rate of 0.2 ml/min at 30 °C. Because of the different UV characteristics of these components, change detection wavelength method was used for quantitative analysis. All of the analytes showed good linearity (r > 0.9992). The established method showed good precision and relative standard deviations (%) for intra-day and inter-day variations of 0.15-1.81 and 0.11-1.98%, respectively. The validated method was successfully applied to the simultaneously determination of six active components in CG from different batches. © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

    Science.gov (United States)

    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.

  12. Quantitative analysis of the eight major compounds in the Samsoeum using a high-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometer

    Science.gov (United States)

    Weon, Jin Bae; Yang, Hye Jin; Lee, Bohyoung; Ma, Jin Yeul; Ma, Choong Je

    2015-01-01

    Background: Samsoeum was traditionally used for treatment of a respiratory disease. Objective: The simultaneous determination of eight major compounds, ginsenoside Rg3, caffeic acid, puerarin, costunolide, hesperidin, naringin, glycyrrhizin, and 6-gingerol in the Samsoeum using a high-performance liquid chromatography (HPLC) coupled with diode array detection (DAD) and an electrospray ionization mass spectrometer was developed for an accurate and reliable quality assessment. Materials and Methods: Eight compounds were qualitative identified based on their mass spectra and by comparing with standard compounds and quantitative analyzed by HPLC-DAD. Separation of eight compounds was carried out on a LUNA C18 column (S-5 μm, 4.6 mm i.d. ×250 mm) with gradient elution composed of acetonitrile and 0.1% trifluoroacetic acid. Results: The data showed good linearity (R2 > 0.9996). The limits of detection and the limits of quantification were <0.53 μg and 1.62 μg, respectively. Inter- and Intra-day precisions (expressed as relative standard deviation values) were within 1.94% and 1.91%, respectively. The recovery of the method was in the range of 94.24–107.90%. Conclusion: The established method is effective and could be applied to quality control of Samsoeum. PMID:25829771

  13. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan PCR Assay

    Directory of Open Access Journals (Sweden)

    Hua-Ying Fu

    2016-01-01

    Full Text Available Ratoon stunting disease (RSD of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx. A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR and a fluorogenic probe (Pat1-QP targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7% of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174 were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174 were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  14. Comparison of culture versus quantitative real-time polymerase chain reaction for the detection of Taylorella equigenitalis in field samples from naturally infected horses in Canada and Germany.

    Science.gov (United States)

    Nadin-Davis, Susan; Knowles, Margaret K; Burke, Teresa; Böse, Reinhard; Devenish, John

    2015-07-01

    A quantitative real-time polymerase chain reaction method (qPCR) was developed and tested for the detection of Taylorella equigenitalis. It was shown to have an analytical sensitivity of 5 colony-forming units (CFU) of T. equigenitalis when applied to the testing of culture swabs that mimicked field samples, and a high analytical specificity in not reacting to 8 other commensal bacterial species associated with horses. As designed, it could also differentiate specifically between T. equigenitalis and T. asinigenitalis. The qPCR was compared to standard culture in a study that included 45 swab samples from 6 horses (1 stallion, 5 mares) naturally infected with T. equigenitalis in Canada, 39 swab samples from 5 naturally infected stallions in Germany, and 311 swab samples from 87 culture negative horses in Canada. When the comparison was conducted on an individual sample swab basis, the qPCR had a statistical sensitivity and specificity of 100% and 96.4%, respectively, and 100% and 99.1% when the comparison was conducted on a sample set basis. A comparison was also made on 203 sample swabs from the 5 German stallions taken over a span of 4 to 9 mo following antibiotic treatment. The qPCR was found to be highly sensitive and at least as good as culture in detecting the presence of T. equigenitalis in post-treatment samples. The work demonstrates that the qPCR assay described here can potentially be used to detect the presence of T. equigenitalis directly from submitted sample swabs taken from infected horses and also for determining T. equigenitalis freedom following treatment.

  15. Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea in soil and fecal samples

    Directory of Open Access Journals (Sweden)

    Durant Jean-Francois

    2012-12-01

    Full Text Available Abstract Background Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis and/or Toxocara cati (T. cati, two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. Methods A duplex quantitative real-time PCR (2qPCR targeting the ribosomal RNA gene internal transcribed spacer (ITS2 has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum and Parascaris equorum (P. equorum. The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. Results 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. Conclusion The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

  16. Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea) in soil and fecal samples.

    Science.gov (United States)

    Durant, Jean-Francois; Irenge, Leonid M; Fogt-Wyrwas, Renata; Dumont, Catherine; Doucet, Jean-Pierre; Mignon, Bernard; Losson, Bertrand; Gala, Jean-Luc

    2012-12-07

    Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis) and/or Toxocara cati (T. cati), two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. A duplex quantitative real-time PCR (2qPCR) targeting the ribosomal RNA gene internal transcribed spacer (ITS2) has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum) and Parascaris equorum (P. equorum). The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD) in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

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

    Science.gov (United States)

    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

  18. Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

    Science.gov (United States)

    Ghosh, Prakash; Khan, Md. Anik Ashfaq; Duthie, Malcolm S.; Vallur, Aarthy C.; Picone, Alessandro; Howard, Randall F.; Reed, Steven G.

    2017-01-01

    Sustained elimination of Visceral Leishmaniasis (VL) requires the reduction and control of parasite reservoirs to minimize the transmission of Leishmania donovani infection. A simple, reproducible and definitive diagnostic procedure is therefore indispensable for the early and accurate detection of parasites in VL, Relapsed VL (RVL) and Post Kala-azar Dermal Leishmaniasis (PKDL) patients, all of whom are potential reservoirs of Leishmania parasites. To overcome the limitations of current diagnostic approaches, a novel quantitative real-time polymerase chain reaction (qPCR) method based on Taqman chemistry was devised for the detection and quantification of L. donovani in blood and skin. The diagnostic efficacy was evaluated using archived peripheral blood buffy coat DNA from 40 VL, 40 PKDL, 10 RVL, 20 cured VL, and 40 cured PKDL along with 10 tuberculosis (TB) cases and 80 healthy endemic controls. Results were compared to those obtained using a Leishmania-specific nested PCR (Ln-PCR). The real time PCR assay was 100% (95% CI, 91.19–100%) sensitive in detecting parasite genomes in VL and RVL samples and 85.0% (95% CI, 70.16–94.29%) sensitive for PKDL samples. In contrast, the sensitivity of Ln-PCR was 77.5% (95% CI, 61.55–89.16%) for VL samples, 100% (95%CI, 69.15–100%) for RVL samples, and 52.5% (95% CI, 36.13–68.49%) for PKDL samples. There was significant discordance between the two methods with the overall sensitivity of the qPCR assay being considerably higher than Ln-PCR. None of the assay detected L. donovani DNA in buffy coats from cured VL cases, and reduced infectious burdens were demonstrated in cured PKDL cases who remained positive in 7.5% (3/40) and 2.5% (1/40) cases by real-time PCR and Ln-PCR, respectively. Both assays were 100% (95% CI, 95.98–100) specific with no positive signals in either endemic healthy control or TB samples. The real time PCR assay we developed offers a molecular tool for accurate detection of circulating L

  19. Tunable and Linker Free Nanogaps in Core-Shell Plasmonic Nanorods for Selective and Quantitative Detection of Circulating Tumor Cells by SERS

    KAUST Repository

    Zhang, Yang; Yang, Peng; Madathumpady Abubaker, Habeeb Muhammed; Alsaiari, Shahad K.; Moosa, Basem; AlMalik, Abdulaziz; Kumar, Anjli; Ringe, Emilie; Khashab, Niveen M.

    2017-01-01

    Controlling the size, number, and shape of nanogaps in plasmonic nanostructures is of significant importance for the development of novel quantum plasmonic devices and quantitative sensing techniques such as surface-enhanced Raman scattering (SERS). Here, we introduce a new synthetic method based on coordination interactions and galvanic replacement to prepare core-shell plasmonic nanorods with tunable enclosed nanogaps. Decorating Au nanorods with Raman reporters that strongly coordinate Ag+ ions (e.g., 4-mercaptopyridine) afforded uniform nucleation sites to form a sacrificial Ag shell. Galvanic replacement of the Ag shell by HAuCl4 resulted in Au-AgAu core-shell structure with a uniform intra-nanoparticle gap. The size (length and width) and morphology of the core-shell plasmonic nanorods as well as the nanogap size depends on the concentration of the coordination complexes formed between Ag+ ions and 4-mercaptopyridine. Moreover, encapsulating Raman reporters within the nanogaps afforded an internal standard for sensitive and quantitative SERS analysis. To test the applicability, core-shell plasmonic nanorods were functionalized with aptamers specific to circulating tumor cells such as MCF-7 (Michigan Cancer Foundation-7, breast cancer cell line). This system could selectively detect as low as 20 MCF-7 cells in a blood mimicking fluid employing SERS. The linking DNA duplex on core-shell plasmonic nanorods can also intercalate hydrophobic drug molecules such as Doxorubicin, thereby increasing the versatility of this sensing platform to include drug delivery. Our synthetic method offers the possibility of developing multifunctional SERS-active materials with a wide range of applications including bio sensing, imaging and therapy.

  20. Tunable and Linker Free Nanogaps in Core-Shell Plasmonic Nanorods for Selective and Quantitative Detection of Circulating Tumor Cells by SERS

    KAUST Repository

    Zhang, Yang

    2017-10-09

    Controlling the size, number, and shape of nanogaps in plasmonic nanostructures is of significant importance for the development of novel quantum plasmonic devices and quantitative sensing techniques such as surface-enhanced Raman scattering (SERS). Here, we introduce a new synthetic method based on coordination interactions and galvanic replacement to prepare core-shell plasmonic nanorods with tunable enclosed nanogaps. Decorating Au nanorods with Raman reporters that strongly coordinate Ag+ ions (e.g., 4-mercaptopyridine) afforded uniform nucleation sites to form a sacrificial Ag shell. Galvanic replacement of the Ag shell by HAuCl4 resulted in Au-AgAu core-shell structure with a uniform intra-nanoparticle gap. The size (length and width) and morphology of the core-shell plasmonic nanorods as well as the nanogap size depends on the concentration of the coordination complexes formed between Ag+ ions and 4-mercaptopyridine. Moreover, encapsulating Raman reporters within the nanogaps afforded an internal standard for sensitive and quantitative SERS analysis. To test the applicability, core-shell plasmonic nanorods were functionalized with aptamers specific to circulating tumor cells such as MCF-7 (Michigan Cancer Foundation-7, breast cancer cell line). This system could selectively detect as low as 20 MCF-7 cells in a blood mimicking fluid employing SERS. The linking DNA duplex on core-shell plasmonic nanorods can also intercalate hydrophobic drug molecules such as Doxorubicin, thereby increasing the versatility of this sensing platform to include drug delivery. Our synthetic method offers the possibility of developing multifunctional SERS-active materials with a wide range of applications including bio sensing, imaging and therapy.

  1. Quantitation of HBV cccDNA in anti-HBc-positive liver donors by droplet digital PCR: a new tool to detect occult infection.

    Science.gov (United States)

    Caviglia, Gian Paolo; Abate, Maria Lorena; Tandoi, Francesco; Ciancio, Alessia; Amoroso, Antonio; Salizzoni, Mauro; Saracco, Giorgio Maria; Rizzetto, Mario; Romagnoli, Renato; Smedile, Antonina

    2018-04-02

    The accurate diagnosis of occult HBV infection (OBI) requires the demonstration of HBV DNA in liver biopsies of HBsAg-negative subjects. However, in clinical practice a latent OBI is deduced by the finding of the antibody to the HB-core antigen (anti-HBc). We investigated the true prevalence of OBI and the molecular features of intrahepatic HBV in anti-HBc-positive subjects. The livers of 100 transplant donors (median age 68.2 years; 64 males, 36 females) positive for anti-HBc at standard serologic testing, were examined for total HBV DNA by nested-PCR and for the HBV covalently closed circular DNA (HBV cccDNA) with an in-house droplet digital PCR assay (ddPCR) (Linearity: R 2 = 0.9998; lower limit of quantitation and detection of 2.4 and 0.8 copies/10 5 cells, respectively). A true OBI status was found in 52% (52/100) of the subjects and cccDNA was found in 52% (27/52) of the OBI-positive, with a median 13 copies/10 5 cells (95% confidence interval 5-25). Using an assay specific for anti-HBc of IgG class, the median antibody level was significantly higher in HBV cccDNA-positive than negative donors (5.7 [3.6-9.7] vs. 17.0 [7.0-39.2] COI, p = 0.007). By multivariate analysis, an anti-HBc IgG value above a 4.4 cut-off index (COI) was associated with the finding of intrahepatic HBV cccDNA (OR = 8.516, p = 0.009); a lower value ruled out its presence with a negative predictive value of 94.6%. With a new in-house ddPCR-based method, intrahepatic HBV cccDNA was detectable in quantifiable levels in about half of the OBI cases examined. The titer of anti-HBc IgG may be a useful surrogate to predict the risk of OBI reactivation in immunosuppressed patients. The covalently closed circular DNA (cccDNA) form of the Hepatitis B virus (HBV) sustains the persistence of the virus even after decades of resolution of the florid infection (Occult HBV infection=OBI). In the present study we developed an highly sensitive method based on droplet digital PCR technology for the detection

  2. Qualitative and quantitative analysis of pyrolysis oil by gas chromatography with flame ionization detection and comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry.

    Science.gov (United States)

    Sfetsas, Themistoklis; Michailof, Chrysa; Lappas, Angelos; Li, Qiangyi; Kneale, Brian

    2011-05-27

    Pyrolysis oils have attracted a lot of interest, as they are liquid energy carriers and general sources of chemicals. In this work, gas chromatography with flame ionization detector (GC-FID) and two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) techniques were used to provide both qualitative and quantitative results of the analysis of three different pyrolysis oils. The chromatographic methods and parameters were optimized and solvent choice and separation restrictions are discussed. Pyrolysis oil samples were diluted in suitable organic solvent and were analyzed by GC×GC-TOFMS. An average of 300 compounds were detected and identified in all three samples using the ChromaToF (Leco) software. The deconvoluted spectra were compared with the NIST software library for correct matching. Group type classification was performed by use of the ChromaToF software. The quantification of 11 selected compounds was performed by means of a multiple-point external calibration curve. Afterwards, the pyrolysis oils were extracted with water, and the aqueous phase was analyzed both by GC-FID and, after proper change of solvent, by GC×GC-TOFMS. As previously, the selected compounds were quantified by both techniques, by means of multiple point external calibration curves. The parameters of the calibration curves were calculated by weighted linear regression analysis. The limit of detection, limit of quantitation and linearity range for each standard compound with each method are presented. The potency of GC×GC-TOFMS for an efficient mapping of the pyrolysis oil is undisputable, and the possibility of using it for quantification as well has been demonstrated. On the other hand, the GC-FID analysis provides reliable results that allow for a rapid screening of the pyrolysis oil. To the best of our knowledge, very few papers have been reported with quantification attempts on pyrolysis oil samples using GC×GC-TOFMS most of which make use of the

  3. Development and Use of a Real-Time Quantitative PCR Method for Detecting and Quantifying Equol-Producing Bacteria in Human Faecal Samples and Slurry Cultures

    Directory of Open Access Journals (Sweden)

    Lucía Vázquez

    2017-06-01

    Full Text Available This work introduces a novel real-time quantitative PCR (qPCR protocol for detecting and quantifying equol-producing bacteria. To this end, two sets of primers targeting the dihydrodaidzein reductase (ddr and tetrahydrodaidzein reductase (tdr genes, which are involved in the synthesis of equol, were designed. The primers showed high specificity and sensitivity when used to examine DNA from control bacteria, such as Slackia isoflavoniconvertens, Slackia equolifaciens, Asaccharobacter celatus, Adlercreutzia equolifaciens, and Enterorhabdus mucosicola. To demonstrate the validity and reliability of the protocol, it was used to detect and quantify equol-producing bacteria in human faecal samples and their derived slurry cultures. These samples were provided by 18 menopausal women under treatment of menopause symptoms with a soy isoflavone concentrate, among whom three were known to be equol-producers given the prior detection of the molecule in their urine. The tdr gene was detected in the faeces of all these equol-producing women at about 4–5 log10 copies per gram of faeces. In contrast, the ddr gene was only amplified in the faecal samples of two of these three women, suggesting the presence in the non-amplified sample of reductase genes unrelated to those known to be involved in equol formation and used for primer design in this study. When tdr and ddr were present in the same sample, similar copy numbers of the two genes were recorded. However, no significant increase in the copy number of equol-related genes along isoflavone treatment was observed. Surprisingly, positive amplification for both tdr and ddr genes was obtained in faecal samples and derived slurry cultures from two non-equol producing women, suggesting the genes could be non-functional or the daidzein metabolized to other compounds in samples from these two women. This novel qPCR tool provides a technique for monitoring gut microbes that produce equol in humans. Monitoring equol

  4. Establishment of real time allele specific locked nucleic acid quantitative PCR for detection of HBV YIDD (ATT mutation and evaluation of its application.

    Directory of Open Access Journals (Sweden)

    Yongbin Zeng

    Full Text Available BACKGROUND: Long-term use of nucleos(tide analogues can increase risk of HBV drug-resistance mutations. The rtM204I (ATT coding for isoleucine is one of the most important resistance mutation sites. Establishing a simple, rapid, reliable and highly sensitive assay to detect the resistant mutants as early as possible is of great clinical significance. METHODS: Recombinant plasmids for HBV YMDD (tyrosine-methionine-aspartate-aspartate and YIDD (tyrosine-isoleucine-aspartate-aspartate were constructed by TA cloning. Real time allele specific locked nucleic acid quantitative PCR (RT-AS-LNA-qPCR with SYBR Green I was established by LNA-modified primers and evaluated with standard recombinant plasmids, clinical templates (the clinical wild type and mutant HBV DNA mixture and 102 serum samples from nucleos(tide analogues-experienced patients. The serum samples from a chronic hepatitis B (CHB patient firstly received LMV mono therapy and then switched to LMV + ADV combined therapy were also dynamically analyzed for 10 times. RESULTS: The linear range of the assay was between 1×10(9 copies/μl and 1 × 10(2 copies/μl. The low detection limit was 1 × 10(1 copies/μl. Sensitivity of the assay were 10(-6, 10(-4 and 10(-2 in the wild-type background of 1 × 10(9 copies/μl, 1 × 10(7 copies/μl and 1 × 10(5 copies/μl, respectively. The sensitivity of the assay in detection of clinical samples was 0.03%. The complete coincidence rate between RT-AS-LNA-qPCR and direct sequencing was 91.2% (93/102, partial coincidence rate was 8.8% (9/102, and no complete discordance was observed. The two assays showed a high concordance (Kappa = 0.676, P = 0.000. Minor variants can be detected 18 weeks earlier than the rebound of HBV DNA load and alanine aminotransferase level. CONCLUSIONS: A rapid, cost-effective, high sensitive, specific and reliable method of RT-AS-LNA-qPCR with SYBR Green I for early and absolute quantification of HBV YIDD (ATT coding for isoleucine

  5. Breath-hold [68Ga]DOTA-TOC PET/CT in neuroendocrine tumors: detection of additional lesions and effects on quantitative parameters.

    Science.gov (United States)

    Zirnsak, Mariana; Bärwolf, Robert; Freesmeyer, Martin

    2016-11-08

    Respiratory motion during PET/CT acquisition generates artifacts in the form of breath-related blurring, which influences the lesion detectability and diagnostic accuracy. The goal of this study was to verify whether breath-hold [68Ga]DOTA-TOC PET/CT (bhPET) allows detection of additional foci compared to free-breathing PET/CT (fbPET), and to assess the impact of breath-holding on standard uptake values (SUV) and isocontoured volume (Vic40) in patients with neuroendocrine tumors (NET). Patients with NET (n=39) were included in this study. BhPET and fbPET characteristics of 96 lesions were compared, and correlated with standard contrast-enhanced (ce) CT and MRI for lesion verification. Quantitative parameters SUV (max and mean) and Vic40 were assessed for both methods and evaluated by linear regression and Spearman's correlation. The impact of lesion size, localization and time interval between investigations was also analyzed. bhPET identified one additional metastasis not seen at fbPET but visible at ceMRI. Another additional bhPET focus did not have a morphological correlate. At bhPET, the SUVmax and SUVmean proved significantly higher and the Vic40 significantly lower than at fbPET. Lesion size, localization and time intervals did not impact significantly on SUV or Vic40. Currently, routine use of breath-hold [68Ga]DOTA-TOC PET/CT cannot be recommended as only one additional lesion was identified. Therefore, bhPET has currently no indication in patients with NET. If technical improvements regarding PET/CT scanner sensitivity are available, bhPET should be reevaluated in the future.

  6. Quantitative detection of RO2 radicals and other products from cyclohexene ozonolysis with ammonium-CI3-TOF and acetate-CI-API-TOF

    Science.gov (United States)

    Hansel, A.; Scholz, W.; Mentler, B.; Fischer, L.; Berndt, T.

    2017-12-01

    The performance of the novel ammonium-CI3-TOF utilizing NH4+ adduct ion chemistry to measure quantitatively first generation oxidized product molecules (OMs) as well as highly oxidized organic molecules (HOMs) was investigated for the first time. The gas-phase ozonolysis of cyclohexene served as a test system in order to evaluate the capability of the detection systems. Experiments have been carried out in the TROPOS free-jet flow system at close to atmospheric conditions. Product ion signals were simultaneously observed by the ammonium-CI3-TOF and the acetate-CI-API-TOF. Both instruments are in remarkable good agreement within a factor of two for HOMs. For OMs not containing an OOH group the acetate technique can considerably underestimate OM concentrations by 2-3 orders of magnitude. First steps of cyclohexene ozonolysis generate ten different (m/z product peaks) main products comprising 92% of observed OMs. The remaining 8% are distributed over several (m/z peaks) minor products that can be attributed to HOMs, predominately to highly oxidized RO2 radicals. Summing up, observed ammonium-CI3-TOF products yield 4.9 x 109 molecules cm-³ in excellent agreement with the amount of reacted cyclohexene of 5.0 x 109 molecules cm-³ for reactant concentrations of [O3] = 2.25 x 1012 molecules cm-³ and [cyclohexene] = 2.0 x 1012 molecules cm-³ and a reaction time of 7.9 s. NH4+ adduct ion chemistry based CIMS techniques offer a unique opportunity for complete detection of the whole product distribution, and consequently, for a much better understanding of atmospheric oxidation processes.

  7. Multislice quantitative computed tomography allows early detection of bone mineral density alterations induced by atherogenic diet in a growing rat experimental model

    International Nuclear Information System (INIS)

    Gubert, M.J.; Monforte, F.; Calo, C.; Lylyk, P.; Friedman, M.F.; Gamba, C.A.

    2012-01-01

    Purpose. To demonstrate the utility of Multislice Quantitative Computed Tomography (MS-QCT) in the early detection of mandibular bone mineral density (BMD) alterations induced by an atherogenic diet in a growing rat experimental model. Materials and Methods. Male weanling Wistar rats (n =16) were divided by body weight (Wt) into 2 groups: control (C) and experimental (E), with no significant differences in the mean initial Wt (p>0.05). C was fed rodent stock diet ad libitum, and E an atherogenic diet for 3 weeks (3w). Zoometry (body weight and length) and diet intake (g/100g rat/day) were monitored. At 3 w in serum (mg/dL) lipidlipoprotein profile was studied: total cholesterol (t-C), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), non-HDL cholesterol (non-HDL-C) and MSQCT (Philips 64 CT, quantified with the eFilm Workstation 2.1) in seven mandibular areas (MA): n. 1 to 4: from chin to mandibular foramen, n. 5: coronoid process, n. 6: condylar process, n. 7: angular process. Statistics: Pearson's correlation between BMD in each MA and serum t-C. p 0.05). Correlation coefficients (r) and their significance levels (p) were relevant in 5/7 MA. MA1:-0.580 (p=0.019), MA2:-0.709 (p=0.002), MA3:-0.635 (p=0.008), MA5:-0.674 (p=0.004), MA6:-0.564 (p=0.023). Conclusions. These results suggest that MS-QCT is an imaging diagnostic method that allows the early detection of mandible bone architecture alterations induced by an atherogenic diet. Inverse correlation between BMD and t-C would indicate an association between an atherogenic diet intake and potential temporomandibular disorders. (authors)

  8. Detection of Viral Hemorrhagic Septicemia Virus by Quantitative Reverse Transcription Polymerase Chain Reaction from Two Fish Species at Two Sites in Lake Superior

    Science.gov (United States)

    Cornwell, Emily R.; Eckerlin, Geofrey E.; Getchell, Rodman G.; Groocock, Geoffrey H.; Thompson, Tarin M.; Batts, William N.; Casey, Rufina N.; Kurath, Gael; Winton, James R.; Bowser, Paul R.; Bain, Mark B.; Casey, James W.

    2011-01-01

    Viral hemorrhagic septicemia virus (VHSV) was first detected in the Laurentian Great Lakes in 2005 during a mortality event in the Bay of Quinte, Lake Ontario. Subsequent analysis of archived samples determined that the first known isolation of VHSV in the Laurentian Great Lakes was from a muskellunge Esox masquinongy collected in Lake St. Clair in 2003. By the end of 2008, mortality events and viral isolations had occurred in all of the Laurentian Great Lakes except Lake Superior. In 2009, a focused disease surveillance program was designed to determine whether VHSV was also present in Lake Superior. In this survey, 874 fish from 7 sites along the U.S. shoreline of Lake Superior were collected during June 2009. Collections were focused on nearshore species known to be susceptible to VHSV. All fish were dissected individually by using aseptic techniques and were tested for the presence of VHSV genetic material by use of a quantitative reverse transcription (qRT) polymerase chain reaction (PCR) targeting the viral nucleoprotein gene. Seventeen fish from two host species at two different sites tested positive at low levels for VHSV. All attempts to isolate virus in cell culture were unsuccessful. However, the presence of viral RNA was confirmed independently in five fish by using a nested PCR that targeted the glycoprotein (G) gene. Partial G gene sequences obtained from three fish were identical to the corresponding sequence from the original 2003 VHSV isolate (MI03) from muskellunge. These detections represent the earliest evidence for the presence of VHSV in Lake Superior and illustrate the utility of the highly sensitive qRT-PCR assay for disease surveillance in aquatic animals.

  9. Investigation of solid phase sorbents for the pre- concentration of pads from aqueous medium and their quantitation by high performance liquid chromatography-UV detection

    International Nuclear Information System (INIS)

    Waqar, F.; Jan, S.; Muhammad, B.; Ahmad, S.; Riaz, M.; Akram, N.

    2005-01-01

    A solid phase extraction method was optimized for the pre-concentration of polyaromatic hydrocarbons (PAHs) in water samples. Graphite powder and Lab scale locally synthesized styrene divinylbenzene (SDVB) Copolymer were used as sorbents for the extraction of PAHs and compared with commercially used C18 solid phase extraction cartridge (SPE). Various parameters were optimized to evaluate the extraction efficiencies, the best results were obtained by proper conditioning of extraction cartridges and desorption with suitable solvent. Percentage recoveries were enhanced by rinsing the sample bottles with acetonitrile and combining the rinse with the sample extract. Quantitative analysis was performed by High performance Liquid chromatography (HPLC) with UV detection. Many other parameters, including optimization of mobile phase, selection of HPLC Columns, sample-loading flow rate on extraction cartridge and weight of sorbent were performed to get optimal results. Percent recoveries obtained with synthesized copolymer were comparable with commercial cartridge, while graphite powder showed excellent retention but very poor recoveries. Obtained recoveries of selected PAHs were ranged from 80-87% with relative standard deviation <6%. Developed method was applied for the analysis of drinking water samples(author)

  10. [Cloning and sequence analysis of the DHBV genome of the brown ducks in Guilin region and establishment of the quantitative method for detecting DHBV].

    Science.gov (United States)

    Su, He-Ling; Huang, Ri-Dong; He, Song-Qing; Xu, Qing; Zhu, Hua; Mo, Zhi-Jing; Liu, Qing-Bo; Liu, Yong-Ming

    2013-03-01

    Brown ducks carrying DHBV were widely used as hepatitis B animal model in the research of the activity and toxicity of anti-HBV dugs. Studies showed that the ratio of DHBV carriers in the brown ducks in Guilin region was relatively high. Nevertheless, the characters of the DHBV genome of Guilin brown duck remain unknown. Here we report the cloning of the genome of Guilin brown duck DHBV and the sequence analysis of the genome. The full length of the DHBV genome of Guilin brown duck was 3 027bp. Analysis using ORF finder found that there was an ORF for an unknown peptide other than S-ORF, PORF and C-ORF in the genome of the DHBV. Vector NTI 8. 0 analysis revealed that the unknown peptide contained a motif which binded to HLA * 0201. Aligning with the DHBV sequences from different countries and regions indicated that there were no obvious differences of regional distribution among the sequences. A fluorescence quantitative PCR for detecting DHBV was establishment based on the recombinant plasmid pGEM-DHBV-S constructed. This study laid the groundwork for using Guilin brown duck as a hepatitis B animal model.

  11. Quantitative detection and typing of hepatitis D virus in human serum by real-time polymerase chain reaction and melting curve analysis.

    Science.gov (United States)

    Hofmann, Joerg; Frenzel, Katrin; Minh, Bui Q; von Haeseler, Arndt; Edelmann, Anke; Ross, Stefan R; Berg, Thomas; Krüger, Detlev H; Meisel, Helga

    2010-06-01

    Hepatitis D virus (HDV) infection is an important etiologic agent of fulminant hepatitis and may aggravate the clinical course of chronic hepatitis B infection resulting in cirrhosis and liver failure. This report describes the establishment of a real-time reverse transcriptase polymerase chain reaction method that allows the quantitative detection of HDV-1 and HDV-3 with a sensitivity in a linear range of 2 x 10(3) to 10(8) copies/mL. Additionally, the new assay provides the opportunity to distinguish HDV-1 from HDV-3 by a subsequent melting curve analysis, an important option because these HDV types are highly associated with severe clinical outcome. The results of the melting curve analysis of 42 HDV sequences obtained in this study and the phylogenetic analysis based on 139 full-length sequences from GenBank were consistent and showed that all sequences described here cluster within the HDV-1 clade. Therefore, this assay is useful for monitoring of antiviral treatment and molecular epidemiologic studies of HDV distribution. Copyright 2010 Elsevier Inc. All rights reserved.

  12. The objective of this program is to develop innovative DNA detection technologies to achieve fast microbial community assessment. The specific approaches are (1) to develop inexpensive and reliable sequence-proof hybridization DNA detection technology (2) to develop quantitative DNA hybridization technology for microbial community assessment and (3) to study the microbes which have demonstrated the potential to have nuclear waste bioremediation

    International Nuclear Information System (INIS)

    Chen, Chung H.

    2004-01-01

    The objective of this program is to develop innovative DNA detection technologies to achieve fast microbial community assessment. The specific approaches are (1) to develop inexpensive and reliable sequence-proof hybridization DNA detection technology (2) to develop quantitative DNA hybridization technology for microbial community assessment and (3) to study the microbes which have demonstrated the potential to have nuclear waste bioremediation

  13. Effect of Box-Cox transformation on power of Haseman-Elston and maximum-likelihood variance components tests to detect quantitative trait Loci.

    Science.gov (United States)

    Etzel, C J; Shete, S; Beasley, T M; Fernandez, J R; Allison, D B; Amos, C I

    2003-01-01

    Non-normality of the phenotypic distribution can affect power to detect quantitative trait loci in sib pair studies. Previously, we observed that Winsorizing the sib pair phenotypes increased the power of quantitative trait locus (QTL) detection for both Haseman-Elston (HE) least-squares tests [Hum Hered 2002;53:59-67] and maximum likelihood-based variance components (MLVC) analysis [Behav Genet (in press)]. Winsorizing the phenotypes led to a slight increase in type 1 error in H-E tests and a slight decrease in type I error for MLVC analysis. Herein, we considered transforming the sib pair phenotypes using the Box-Cox family of transformations. Data were simulated for normal and non-normal (skewed and kurtic) distributions. Phenotypic values were replaced by Box-Cox transformed values. Twenty thousand replications were performed for three H-E tests of linkage and the likelihood ratio test (LRT), the Wald test and other robust versions based on the MLVC method. We calculated the relative nominal inflation rate as the ratio of observed empirical type 1 error divided by the set alpha level (5, 1 and 0.1% alpha levels). MLVC tests applied to non-normal data had inflated type I errors (rate ratio greater than 1.0), which were controlled best by Box-Cox transformation and to a lesser degree by Winsorizing. For example, for non-transformed, skewed phenotypes (derived from a chi2 distribution with 2 degrees of freedom), the rates of empirical type 1 error with respect to set alpha level=0.01 were 0.80, 4.35 and 7.33 for the original H-E test, LRT and Wald test, respectively. For the same alpha level=0.01, these rates were 1.12, 3.095 and 4.088 after Winsorizing and 0.723, 1.195 and 1.905 after Box-Cox transformation. Winsorizing reduced inflated error rates for the leptokurtic distribution (derived from a Laplace distribution with mean 0 and variance 8). Further, power (adjusted for empirical type 1 error) at the 0.01 alpha level ranged from 4.7 to 17.3% across all tests

  14. Detection of quantitative trait loci controlling grain zinc concentration using Australian wild rice, Oryza meridionalis, a potential genetic resource for biofortification of rice.

    Science.gov (United States)

    Ishikawa, Ryo; Iwata, Masahide; Taniko, Kenta; Monden, Gotaro; Miyazaki, Naoya; Orn, Chhourn; Tsujimura, Yuki; Yoshida, Shusaku; Ma, Jian Feng; Ishii, Takashige

    2017-01-01

    Zinc (Zn) is one of the essential mineral elements for both plants and humans. Zn deficiency in human is one of the major causes of hidden hunger, a serious health problem observed in many developing countries. Therefore, increasing Zn concentration in edible part is an important issue for improving human Zn nutrition. Here, we found that an Australian wild rice O. meridionalis showed higher grain Zn concentrations compared with cultivated and other wild rice species. The quantitative trait loci (QTL) analysis was then performed to identify the genomic regions controlling grain Zn levels using backcross recombinant inbred lines derived from O. sativa 'Nipponbare' and O. meridionalis W1627. Four QTLs responsible for high grain Zn were detected on chromosomes 2, 9, and 10. The QTL on the chromosome 9 (named qGZn9), which showed the largest effect on grain Zn concentration was confirmed with the introgression line, which had a W1627 chromosomal segment covering the qGZn9 region in the genetic background of O. sativa 'Nipponbare'. Fine mapping of this QTL resulted in identification of two tightly linked loci, qGZn9a and qGZn9b. The candidate regions of qGZn9a and qGZn9b were estimated to be 190 and 950 kb, respectively. Furthermore, we also found that plants having a wild chromosomal segment covering qGZn9a, but not qGZn9b, is associated with fertility reduction. qGZn9b, therefore, provides a valuable allele for breeding rice with high Zn in the grains.

  15. Quantitation of itopride in human serum by high-performance liquid chromatography with fluorescence detection and its application to a bioequivalence study.

    Science.gov (United States)

    Singh, Sonu Sundd; Jain, Manish; Sharma, Kuldeep; Shah, Bhavin; Vyas, Meghna; Thakkar, Purav; Shah, Ruchy; Singh, Shriprakash; Lohray, Brajbhushan

    2005-04-25

    A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm x 4.6 mm, 5 microm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31-3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17-9.50%. Serum samples containing itopride were stable for 180.0 days at -70+/-5 degrees C and for 24.0 h at ambient temperature (25+/-5 degrees C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.

  16. Detection of quantitative trait loci controlling grain zinc concentration using Australian wild rice, Oryza meridionalis, a potential genetic resource for biofortification of rice.

    Directory of Open Access Journals (Sweden)

    Ryo Ishikawa

    Full Text Available Zinc (Zn is one of the essential mineral elements for both plants and humans. Zn deficiency in human is one of the major causes of hidden hunger, a serious health problem observed in many developing countries. Therefore, increasing Zn concentration in edible part is an important issue for improving human Zn nutrition. Here, we found that an Australian wild rice O. meridionalis showed higher grain Zn concentrations compared with cultivated and other wild rice species. The quantitative trait loci (QTL analysis was then performed to identify the genomic regions controlling grain Zn levels using backcross recombinant inbred lines derived from O. sativa 'Nipponbare' and O. meridionalis W1627. Four QTLs responsible for high grain Zn were detected on chromosomes 2, 9, and 10. The QTL on the chromosome 9 (named qGZn9, which showed the largest effect on grain Zn concentration was confirmed with the introgression line, which had a W1627 chromosomal segment covering the qGZn9 region in the genetic background of O. sativa 'Nipponbare'. Fine mapping of this QTL resulted in identification of two tightly linked loci, qGZn9a and qGZn9b. The candidate regions of qGZn9a and qGZn9b were estimated to be 190 and 950 kb, respectively. Furthermore, we also found that plants having a wild chromosomal segment covering qGZn9a, but not qGZn9b, is associated with fertility reduction. qGZn9b, therefore, provides a valuable allele for breeding rice with high Zn in the grains.

  17. Quantitative characterization of gold nanoparticles by field-flow fractionation coupled online with light scattering detection and inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Schmidt, Bjørn; Loeschner, Katrin; Hadrup, Niels; Mortensen, Alicja; Sloth, Jens J; Koch, Christian Bender; Larsen, Erik H

    2011-04-01

    An analytical platform coupling asymmetric flow field-flow fractionation (AF(4)) with multiangle light scattering (MALS), dynamic light scattering (DLS), and inductively coupled plasma mass spectrometry (ICPMS) was established and used for separation and quantitative determination of size and mass concentration of nanoparticles (NPs) in aqueous suspension. Mixtures of three polystyrene (PS) NPs between 20 and 100 nm in diameter and mixtures of three gold (Au) NPs between 10 and 60 nm in diameter were separated by AF(4). The geometric diameters of the separated PS NPs and the hydrodynamic diameters of the Au and PS NPs were determined online by MALS and DLS, respectively. The three separated Au NPs were quantified by ICPMS and recovered at 50-95% of the injected masses, which ranged between approximately 8-80 ng of each nanoparticle size. Au NPs adhering to the membrane in the separation channel was found to be a major cause for incomplete recoveries. The lower limit of detection (LOD) ranged between 0.02 ng Au and 0.4 ng Au, with increasing LOD by increasing nanoparticle diameter. The analytical platform was applied to characterization of Au NPs in livers of rats, which were dosed with 10 nm, 60 nm, or a mixture of 10 and 60 nm nanoparticles by intravenous injection. The homogenized livers were solubilized in tetramethylammonium hydroxide (TMAH), and the recovery of Au NPs from the livers amounted to 86-123% of their total Au content. In spite of successful stabilization with bovine serum albumin even in alkaline medium, separation of the Au NPs by AF(4) was not possible due to association with undissolved remains of the alkali-treated liver tissues as demonstrated by electron microscopy images.

  18. Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis.

    Science.gov (United States)

    Abdeldaim, Guma M K; Strålin, Kristoffer; Korsgaard, Jens; Blomberg, Jonas; Welinder-Olsson, Christina; Herrmann, Björn

    2010-12-03

    Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients. The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis) in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 10⁵ genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively.In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both bacteria. The PCR provides increased

  19. Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis

    Directory of Open Access Journals (Sweden)

    Welinder-Olsson Christina

    2010-12-01

    Full Text Available Abstract Background Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR for detection of S. pneumoniae (9802 gene fragment, H. influenzae (omp P6 gene and N. meningitidis (ctrA gene. The method was evaluated on bronchoalveolar lavage (BAL samples from 156 adults with lower respiratory tract infection (LRTI and 31 controls, and on 87 cerebrospinal fluid (CSF samples from meningitis patients. Results The analytical sensitivity was not affected by using a combined mixture of reagents and a combined DNA standard (S. pneumoniae/H. influenzae/N. meningitidis in single tubes. By blood- and BAL-culture and S. pneumoniae urinary antigen test, S. pneumoniae and H. influenzae were aetiological agents in 21 and 31 of the LTRI patients, respectively. These pathogens were identified by qmPCR in 52 and 72 of the cases, respectively, yielding sensitivities and specificities of 95% and 75% for S. pneumoniae, and 90% and 65% for H. influenzae, respectively. When using a cut-off of 105 genome copies/mL for clinical positivity the sensitivities and specificities were 90% and 80% for S. pneumoniae, and 81% and 85% for H. influenzae, respectively. Of 44 culture negative but qmPCR positive for H. influenzae, 41 were confirmed by fucK PCR as H. influenzae. Of the 103 patients who had taken antibiotics prior to sampling, S. pneumoniae and H. influenzae were identified by culture in 6% and 20% of the cases, respectively, and by the qmPCR in 36% and 53% of the cases, respectively. In 87 CSF samples S. pneumoniae and N. meningitidis were identified by culture and/or 16 S rRNA in 14 and 10 samples and by qmPCR in 14 and 10 samples, respectively, giving a sensitivity of 100% and a specificity of 100% for both

  20. Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley.

    Directory of Open Access Journals (Sweden)

    Jing Cai

    Full Text Available Hulless barley (Hordeum vulgare L. var. nudum. hook. f. has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes-ACT (Actin, E2 (Ubiquitin conjugating enzyme 2, TUBα (Alpha-tubulin, TUBβ6 (Beta-tubulin 6, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase, EF-1α (Elongation factor 1-alpha, SAMDC (S-adenosylmethionine decarboxylase, PKABA1 (Gene for protein kinase HvPKABA1, PGK (Phosphoglycerate kinase, and HSP90 (Heat shock protein 90-were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUBβ6, E2, TUBα, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUBα and HSP90 showed peak stability under salt stress, TUBα and EF-1α were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression

  1. A novel approach to the quantitative detection of anabolic steroids in bovine muscle tissue by means of a hybrid quadrupole time-of-flight-mass spectrometry instrument.

    Science.gov (United States)

    Bussche, Julie Vanden; Decloedt, Anneleen; Van Meulebroek, Lieven; De Clercq, Nathalie; Lock, Stephen; Stahl-Zeng, Jianru; Vanhaecke, Lynn

    2014-09-19

    In recent years, the analysis of veterinary drugs and growth-promoting agents has shifted from target-oriented procedures, mainly based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-MS), towards accurate mass full scan MS (such as Time-of-Flight (ToF) and Fourier Transform (FT)-MS). In this study, the performance of a hybrid analysis instrument (i.e. UHPLC-QuadrupoleTime-of-Flight-MS (QqToF-MS)), able to exploit both full scan HR and MS/MS capabilities within a single analytical platform, was evaluated for confirmatory analysis of anabolic steroids (gestagens, estrogens including stilbenes and androgens) in meat. The validation data was compared to previously obtained results (CD 2002/657/EC) for QqQ-MS and single stage Orbitrap-MS. Additionally, a fractional factorial design was used to shorten and optimize the sample extraction. Validation according to CD 2002/657/EC demonstrated that steroid analysis using QqToF has a higher competing value towards QqQ-MS in terms of selectivity/specificity, compared to single stage Orbitrap-MS. While providing excellent linearity, based on lack-of-fit calculations (F-test, α=0.05 for all steroids except 17β-ethinylestradiol: α=0.01), the sensitivity of QqToF-MS proved for 61.8% and 85.3% of the compounds more sensitive compared to QqQ-MS and Orbitrap-MS, respectively. Indeed, the CCα values, obtained upon ToF-MS/MS detection, ranged from 0.02 to 1.74μgkg(-1) for the 34 anabolic steroids, while for QqQ-MS and Orbitrap-MS values ranged from 0.04 to 0.88μgkg(-1) and from 0.07 to 2.50μgkg(-1), respectively. Using QqToF-MS and QqQ-MS, adequate precision was obtained as relative standard deviations for repeatability and within-laboratory reproducibility, were below 20%. In case of Orbitrap-MS, some compounds (i.e. some estrogens) displayed poor precision, which was possibly caused by some lack of sensitivity at lower concentrations and the absence of MRM-like experiments. Overall, it can be

  2. Selection of appropriate reference genes for the detection of rhythmic gene expression via quantitative real-time PCR in Tibetan hulless barley.

    Science.gov (United States)

    Cai, Jing; Li, Pengfei; Luo, Xiao; Chang, Tianliang; Li, Jiaxing; Zhao, Yuwei; Xu, Yao

    2018-01-01

    Hulless barley (Hordeum vulgare L. var. nudum. hook. f.) has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes-ACT (Actin), E2 (Ubiquitin conjugating enzyme 2), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), EF-1α (Elongation factor 1-alpha), SAMDC (S-adenosylmethionine decarboxylase), PKABA1 (Gene for protein kinase HvPKABA1), PGK (Phosphoglycerate kinase), and HSP90 (Heat shock protein 90)-were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUBβ6, E2, TUBα, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUBα and HSP90 showed peak stability under salt stress, TUBα and EF-1α were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression analysis

  3. Comparison between the gold standard DXA with calcaneal quantitative ultrasound based-strategy (QUS) to detect osteoporosis in an HIV infected cohort.

    Science.gov (United States)

    Quiros Roldan, Eugenia; Brianese, Nigritella; Raffetti, Elena; Focà, Emanuele; Pezzoli, Maria Chiara; Bonito, Andrea; Ferraresi, Alice; Lanza, Paola; Porcelli, Teresa; Castelli, Francesco

    Osteoporosis represents one of the most frequent comorbidity among HIV patients. The current standard method for osteoporosis diagnosis is dual-energy X-ray absorptiometry. Calcaneal quantitative ultrasound can provide information about bone quality. The aims of this study are to compare these two methods and to evaluate their ability to screen for vertebral fracture. This cross-sectional study was conducted in HIV patients attending the Clinic of Infectious and Tropical Diseases of Brescia during 2014 and who underwent lumbar/femoral dual-energy X-ray absorptiometry, vertebral fracture assessment and calcaneal quantitative ultrasound. The assessment of osteoporosis diagnostic accuracy was performed for calcaneal quantitative ultrasound and for vertebral fracture comparing them with dual-energy X-ray absorptiometry. We enrolled 73 patients and almost 48% of them had osteoporosis with at least one of the method used. Vertebral fracture were present in 27.4%. Among patients with normal bone measurements, we found vertebral fracture in proportion between 10% and 30%. If we used calcaneal quantitative ultrasound method and/or X-ray as screening, the percentages of possible savable dual-energy X-ray absorptiometry ranged from 12% to 89% and misclassification rates ranged from 0 to 24.6%. A combined strategy, calcaneal quantitative ultrasound and X-Ray, identified 67% of patients with low risk of osteoporosis, but 16.4% of patients were misclassified. We observed that patients with osteoporosis determined by calcaneal quantitative ultrasound and/or dual-energy X-ray absorptiometry have higher probability to undergo vertebral fracture, but neither of them can be used for predicting vertebral fracture. Use of calcaneal quantitative ultrasound for screening is a reasonable alternative of dual-energy X-ray absorptiometry since our study confirm that none strategy is clearly superior, but both screen tools must be always completed with X-ray. Copyright © 2017 Sociedade

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

    Science.gov (United States)

    Yankson, Kweku K.; Steck, Todd R.

    2009-01-01

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

  5. Alternative models for detection of quantitative trait loci (QTL) for growth and carcass traits in pigs chromosomes 4, 5 and 7

    NARCIS (Netherlands)

    Moraes Gonçalves, de T.; Nunes de Oliveira, H.; Bovenhuis, H.; Bink, M.C.A.M.; Arendonk, van J.A.M.

    2005-01-01

    Genome scans can be used to identify chromosomal regions and eventually genes that control quantitative traits (QTL) of economic importance. In an experimental cross between Meishan (male) and Dutch Large White and Landrace lines (female), 298 F1 and 831 F2 animals were evaluated for intramuscular

  6. Detection of novel quantitative trait loci for cutaneous melanoma by genome-wide scan in the MeLiM swine model

    Czech Academy of Sciences Publication Activity Database

    Du, Z. Q.; Vincent-Naulleau, S.; Gilbert, H.; Vignoles, F.; Créchet, F.; Shimogiri, T.; Yasue, H.; Leplat, J. J.; Bouet, S.; Gruand, J.; Horák, Vratislav; Milan, D.; Le Roy, P.; Geffrotin, C.

    2006-01-01

    Roč. 120, - (2006), s. 303-320 ISSN 0020-7136 Institutional research plan: CEZ:AV0Z50450515 Keywords : swine melanoma * quantitative trait loci * MC1R Subject RIV: FD - Oncology ; Hematology Impact factor: 4.693, year: 2006

  7. A New Diagnostic system for Ultra Sensitive and Specific Detection and Quantitation of “Candidatus Liberibacter asiaticus”, the Bacterium Associated with Citrus Huanglongbing

    Science.gov (United States)

    In this study, an ultra sensitive and quantitative diagnostic system for “Candidatus Liberibacter asiaticus” was developed. This system adapts a nested PCR and Taq-Man PCR in a single closed tube. The procedure involves two steps of PCR using the species specific outer and inner primer pairs. Differ...

  8. Detection of prostate cancer in peripheral zone: comparison of MR diffusion tensor imaging, quantitative dynamic contrast-enhanced MRI, and the two techniques combined at 3.0 T.

    Science.gov (United States)

    Li, Chunmei; Chen, Min; Li, Saying; Zhao, Xuna; Zhang, Chen; Luo, Xiaojie; Zhou, Cheng

    2014-03-01

    Previous studies have shown that the diagnostic accuracy for prostate cancer improved with diffusion tensor imaging (DTI) or quantitative dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) only. However, the efficacy of combined DTI and quantitative DCE-MRI in detecting prostate cancer at 3.0 T is still indeterminate. To investigate the utility of diffusion tensor imaging (DTI), quantitative DCE-MRI, and the two techniques combined at 3.0 T in detecting prostate cancer of the peripheral zone (PZ). DTI and DCE-MRI of 33 patients was acquired prior to prostate biopsy. Regions of interest (ROIs) were drawn according to biopsy zones which were apex, mid-gland, and base on each side of the PZ. Apparent diffusion coefficient (ADC), fractional anisotropy (FA), volume transfer constant (K(trans)), and rate constant (kep) values of cancerous sextants and non-cancerous sextants in PZ were calculated. Logistic regression models were generated for DTI, DCE-MRI, and DTI + DCE-MRI. Receiver-operating characteristic (ROC) curves were used to compare the ability of these models to differentiate cancerous sextants from non-cancerous sextants of PZ. There were significant differences in the ADC, FA, K(trans), and kep values between cancerous sextants and non-cancerous sextants in PZ (P < 0.0001, P < 0.0001, P < 0.0001, and P < 0.0001, respectively). The area under curve (AUC) for DTI + DCE-MRI was significantly greater than that for either DTI (0.93 vs. 0.86, P = 0.0017) or DCE-MRI (0.93 vs. 0.84, P = 0.0034) alone. The combination of DTI and quantitative DCE-MRI has better diagnostic performance in detecting prostate cancer of the PZ than either technique alone.

  9. Quantitative research.

    Science.gov (United States)

    Watson, Roger

    2015-04-01

    This article describes the basic tenets of quantitative research. The concepts of dependent and independent variables are addressed and the concept of measurement and its associated issues, such as error, reliability and validity, are explored. Experiments and surveys – the principal research designs in quantitative research – are described and key features explained. The importance of the double-blind randomised controlled trial is emphasised, alongside the importance of longitudinal surveys, as opposed to cross-sectional surveys. Essential features of data storage are covered, with an emphasis on safe, anonymous storage. Finally, the article explores the analysis of quantitative data, considering what may be analysed and the main uses of statistics in analysis.

  10. Touch-down reverse transcriptase-PCR detection of IgV(H) rearrangement and Sybr-Green-based real-time RT-PCR quantitation of minimal residual disease in patients with chronic lymphocytic leukemia.

    Science.gov (United States)

    Peková, Sona; Marková, Jana; Pajer, Petr; Dvorák, Michal; Cetkovský, Petr; Schwarz, Jirí

    2005-01-01

    Patients with chronic lymphocytic leukemia (CLL) can relapse even after aggressive therapy and autografts. It is commonly assumed that to prevent relapse the level of minimal residual disease (MRD) should be as low as possible. To evaluate MRD, highly sensitive quantitative assays are needed. The aim of the study was to develop a robust and sensitive method for detection of the clonal immunoglobulin heavy-chain variable (IgV(H)) rearrangement in CLL and to introduce a highly sensitive and specific methodology for MRD monitoring in patients with CLL who undergo intensive treatment. As a prerequisite for MRD detection, touch-down reverse transcriptase (RT)-PCR using degenerate primers were used for the diagnostic identification of (H) gene rearrangement(s). For quantitative MRD detection in 18 patients, we employed a real-time RT-PCR assay (RQ-PCR) making use of patient-specific primers and the cost-saving Sybr-Green reporter dye (SG). For precise calibration of RQ-PCR, patient-specific IgV(H) sequences were cloned. Touch-down RT-PCR with degenerate primers allowed the successful detection of IgV(H) clonal rearrangement(s) in 252 of 257 (98.1%) diagnostic samples. Biallelic rearrangements were found in 27 of 252 (10.7%) cases. Degenerate primers used for the identification of clonal expansion at diagnosis were not sensitive enough for MRD detection. In contrast, our RQ-PCR assay using patient-specific primers and SG reached the sensitivity of 10(-)(6). We demonstrated MRD in each patient tested, including four of four patients in complete remission following autologous hematopoietic stem cell transplantation (HSCT) and three of three following allogeneic 'mini'-HSCT. Increments in MRD might herald relapse; aggressive chemotherapy could induce molecular remission. Our touch-down RT-PCR has higher efficiency to detect clonal IgV(H) rearrangements including the biallelic ones. MRD quantitation of IgV(H) expression using SG-based RQ-PCR represents a highly specific

  11. Quantitative habitability.

    Science.gov (United States)

    Shock, Everett L; Holland, Melanie E

    2007-12-01

    A framework is proposed for a quantitative approach to studying habitability. Considerations of environmental supply and organismal demand of energy lead to the conclusions that power units are most appropriate and that the units for habitability become watts per organism. Extreme and plush environments are revealed to be on a habitability continuum, and extreme environments can be quantified as those where power supply only barely exceeds demand. Strategies for laboratory and field experiments are outlined that would quantify power supplies, power demands, and habitability. An example involving a comparison of various metabolisms pursued by halophiles is shown to be well on the way to a quantitative habitability analysis.

  12. Enhanced reliability and accuracy for field deployable bioforensic detection and discrimination of Xylella fastidiosa subsp. pauca, causal agent of citrus variegated chlorosis using razor ex technology and TaqMan quantitative PCR.

    Science.gov (United States)

    Ouyang, Ping; Arif, Mohammad; Fletcher, Jacqueline; Melcher, Ulrich; Ochoa Corona, Francisco Manuel

    2013-01-01

    A reliable, accurate and rapid multigene-based assay combining real time quantitative PCR (qPCR) and a Razor Ex BioDetection System (Razor Ex) was validated for detection of Xylella fastidiosa subsp. pauca (Xfp, a xylem-limited bacterium that causes citrus variegated chlorosis [CVC]). CVC, which is exotic to the United States, has spread through South and Central America and could significantly impact U.S. citrus if it arrives. A method for early, accurate and sensitive detection of Xfp in plant tissues is needed by plant health officials for inspection of products from quarantined locations, and by extension specialists for detection, identification and management of disease outbreaks and reservoir hosts. Two sets of specific PCR primers and probes, targeting Xfp genes for fimbrillin and the periplasmic iron-binding protein were designed. A third pair of primers targeting the conserved cobalamin synthesis protein gene was designed to detect all possible X. fastidiosa (Xf) strains. All three primer sets detected as little as 1 fg of plasmid DNA carrying X. fastidiosa target sequences and genomic DNA of Xfp at as little as 1 - 10 fg. The use of Razor Ex facilitates a rapid (about 30 min) in-field assay capability for detection of all Xf strains, and for specific detection of Xfp. Combined use of three primer sets targeting different genes increased the assay accuracy and broadened the range of detection. To our knowledge, this is the first report of a field-deployable rapid and reliable bioforensic detection and discrimination method for a bacterial phytopathogen based on multigene targets.

  13. Quantitative Trait Loci in Inbred Lines

    NARCIS (Netherlands)

    Jansen, R.C.

    2001-01-01

    Quantitative traits result from the influence of multiple genes (quantitative trait loci) and environmental factors. Detecting and mapping the individual genes underlying such 'complex' traits is a difficult task. Fortunately, populations obtained from crosses between inbred lines are relatively

  14. Express immunochromatographic detection of antibodies against Brucella abortus in cattle sera based on quantitative photometric registration and modulated cut-off level.

    Science.gov (United States)

    Sotnikov, Dmitriy V; Byzova, Nadezhda A; Zherdev, Anatoly V; Eskendirova, Saule Z; Baltin, Kairat K; Mukanov, Kasim K; Ramankulov, Erlan M; Sadykhov, Elchin G; Dzantiev, Boris B

    2015-01-01

    An immunochromatographic test system was developed for rapid detection of the levels of specific IgG antibodies to Brucella abortus lipopolysaccharide, as a tool for diagnosis of brucellosis in cattle. The pilot test strips were examined using blood sera from sick (78 samples) and healthy (35 samples) cows. The results obtained by immunochromatographic assay, using a portable optical densitometer for digital video detection, correlate well with the results obtained by immunoenzyme assay and are in agreement with the results of the disease diagnosis. The new test system allows detection of antibodies within 10 min and can be proposed as an alternative to the methods available for serodiagnosis of brucellosis.

  15. 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.

    Science.gov (United States)

    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.

  16. Quantitative Finance

    Science.gov (United States)

    James, Jessica

    2017-01-01

    Quantitative finance is a field that has risen to prominence over the last few decades. It encompasses the complex models and calculations that value financial contracts, particularly those which reference events in the future, and apply probabilities to these events. While adding greatly to the flexibility of the market available to corporations and investors, it has also been blamed for worsening the impact of financial crises. But what exactly does quantitative finance encompass, and where did these ideas and models originate? We show that the mathematics behind finance and behind games of chance have tracked each other closely over the centuries and that many well-known physicists and mathematicians have contributed to the field.

  17. Detection and quantitation of twenty-seven cytokines, chemokines and growth factors pre- and post-high abundance protein depletion in human plasma

    Directory of Open Access Journals (Sweden)

    Seong-Beom Ahn

    2014-06-01

    Full Text Available Cytokines, chemokines and growth factors (CCGFs in human plasma are analyzed for identification of biomarkers. However concentrations of CCGFs are very low; it is difficult to identify and quantify low abundance proteins in the presence of the high abundance proteins (HAPs unless HAPs are removed prior to analysis. However, there is a concern that the low abundance proteins such as CCGFs may also be removed during the HAP depletion process. In this study, we have examined whether or not depletion of the HAPs enhances detection of the CCGFs by immuno-assays. Top 14 HAPs were depleted from 10 healthy volunteers’ plasma using MARS-14 immuno-depletion column and a total of 27 CCGFs were analyzed by bead-based multiplexed immuno-assay. All 27 CCGFs were detected in neat plasma (NP, 25 were detected in flow through fraction (FT and 21 were detected in bound protein (BP fraction. Concentrations of 22 CCGFs were significantly higher in NP compared to FT and BP. Only one CCGF had higher concentration in FT compared to NP. The remaining 2 CCGFs were not different between NP and FT. It was counter-productive for the detection of 24 CCGFs after HAP removal, primarily due to post-depletion protein precipitation and/or re-suspension of pellets.

  18. Enteroviruses in blood of patients with type 1 diabetes detected by integrated cell culture and reverse transcription quantitative real-time PCR.

    Science.gov (United States)

    Alidjinou, Enagnon Kazali; Sane, Famara; Lefevre, Christine; Baras, Agathe; Moumna, Ilham; Engelmann, Ilka; Vantyghem, Marie-Christine; Hober, Didier

    2017-11-01

    Enteroviruses (EV) have been associated with type 1 diabetes (T1D), but EV RNA detection has been reported in only a small proportion of T1D patients. We studied whether integrated cell culture and reverse transcription real-time PCR could improve EV detection in blood samples from patients with T1D. Blood was collected from 13 patients with T1D. The presence of EV RNA in blood was investigated by using real-time RT-PCR. In addition, plasma and white blood cells (WBC) were inoculated to BGM and Vero cell line cultures. Culture supernatants and cells collected on day 7 and day 14 were tested for EV RNA by real-time RT-PCR. Enterovirus identification was performed through sequencing of the VP4/VP2 region. Enterovirus RNA was detected in blood by using real-time RT-PCR in only one out of 13 patients. The detection of EV RNA in cultures inoculated with clinical samples (plasma and/or WBC) gave positive results in five other patients. The viral loads were low, ranging from 45 to 4420 copies/ng of total RNA. One isolate was successfully identified as coxsackievirus B1. Integrated cell culture and reverse transcription real-time PCR can improve the detection rate of EV in blood samples of patients with T1D and can be useful to investigate further the relationship between EV and the disease.

  19. Quantitative detection of the potato cyst nematode, Globodera pallida, and the beet cyst nematode, Heterodera schachtii, using Real-Time PCR with SYBR green I dye.

    Science.gov (United States)

    Madani, Mehrdad; Subbotin, Sergei A; Moens, Maurice

    2005-04-01

    The potato cyst nematode Globodera pallida and the beet cyst nematode Heterodera schachtii are major nematode pests in world agriculture. Precise identification and knowledge about the number of nematodes in field soil are necessary to develop effective integrated pest control. Here we report the results of the Real-Time PCR assay for the rapid detection and quantification of G. pallida and H. schachtii. Using species specific primers and SYBR green I dye, we were able to detect a single second stage juvenile of cyst forming nematodes in samples. The specificity of the reaction was confirmed by the lack of amplification of DNAs from other Heterodera or Globodera species. Validation tests showed a rather high correlation between real numbers of second stage juveniles in a sample and expected numbers detected by Real-Time PCR. Reasons for observed differences in sensitivity and reliability of quantification detection for two species as well as other problems of Real-Time PCR are discussed. The Real-Time PCR assay with SYBR green I dye targeting fragments of the ITS-rDNA provided a sensitive means for the rapid and simultaneous detection and quantification of juveniles of these pests.

  20. Detection of hemodynamic impairment using magnetic resonance angiography in patients with internal carotid artery stenoocclusive disease. Comparison with quantitative brain perfusion single-photon emission computed tomography

    International Nuclear Information System (INIS)

    Hirooka, Ryonoshin; Ogasawara, Kuniaki

    2008-01-01

    Cerebrovascular reactivity (CVR) to acetazolamideis a key parameter in determining the severity of hemodynamic impairment in patients with major cerebral artery occlusive disease. The aim of the present study is to validate the accuracy of magnetic resonance angiography (MRA) for detecting hemodynamic impairment by correlating detectability of the middle cerebral artery obtained by MRA with CVR measured by single-photon emission computed tomography (SPECT) in patients with internal carotid artery (ICA) occlusive disease. Ninety-four patients with chronic ICA occlusion underwent single slab three-dimensional time-of-flight MRA and SPECT. SPECT-CVR was calculated by measured cerebral blood flow before and after acetazolamide challenge. CVR was significantly lower in patients without detection of any portion (M1, M2 or M3) of the MCA than in those with detection of all portions. When SPECT-CVR lower than the mean- 2 standard deviation (SD) obtained in normal subjects was defined as reduced and the SPECT-CVR was assumed as the true determinant of hemodynamic impairment, MRA provided 92% sensitivity and 73% specificity, with 96% negative predictive value for detecting patients with reduced CVR. The present MRA method is effective for the identification of patients with hemodynamic impairment. (author)

  1. Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

    Science.gov (United States)

    Böttcher, S; Ritgen, M; Pott, C; Brüggemann, M; Raff, T; Stilgenbauer, S; Döhner, H; Dreger, P; Kneba, M

    2004-10-01

    The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.

  2. Quantitative and Label-Free Detection of Protein Kinase A Activity Based on Surface-Enhanced Raman Spectroscopy with Gold Nanostars.

    Science.gov (United States)

    He, Shuai; Kyaw, Yi Mon Ei; Tan, Eddie Khay Ming; Bekale, Laurent; Kang, Malvin Wei Cherng; Kim, Susana Soo-Yeon; Tan, Ivan; Lam, Kong-Peng; Kah, James Chen Yong

    2018-04-26

    The activity of extracellular protein kinase A (PKA) is known to be a biomarker for cancer. However, conventional PKA assays based on colorimetric, radioactive, and fluorometric techniques suffer from intensive labeling-related preparations, background interference, photobleaching, and safety concerns. While surface-enhanced Raman spectroscopy (SERS)-based assays have been developed for various enzymes to address these limitations, their use in probing PKA activity is limited due to subtle changes in the Raman spectrum with phosphorylation. Here, we developed a robust colloidal SERS-based scheme for label-free quantitative measurement of PKA activity using gold nanostars (AuNS) as a SERS substrate functionalized with bovine serum albumin (BSA)-kemptide (Kem) bioconjugate (AuNS-BSA-Kem), where BSA conferred colloidal stability and Kem is a high-affinity peptide substrate for PKA. By performing principle component analysis (PCA) on the SERS spectrum, we identified two Raman peaks at 725 and 1395 cm -1 , whose ratiometric intensity change provided a quantitative measure of Kem phosphorylation by PKA in vitro and allowed us to distinguish MDA-MB-231 and MCF-7 breast cancer cells known to overexpress extracellular PKA catalytic subunits from noncancerous human umbilical vein endothelial cells (HUVEC) based on their PKA activity in cell culture supernatant. The outcome demonstrated potential application of AuNS-BSA-Kem as a SERS probe for cancer screening based on PKA activity.

  3. Quantitation of fumonisin B1 and B2 in feed using FMOC pre-column derivatization with HPLC and fluorescence detection.

    Science.gov (United States)

    Smith, Lori L; Francis, Kyle A; Johnson, Joseph T; Gaskill, Cynthia L

    2017-11-01

    Pre-column derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) was determined to be effective for quantitation of fumonisins B 1 and B 2 in feed. Liquid-solid extraction, clean-up using immunoaffinity solid phase extraction chromatography, and FMOC-derivatization preceded analysis by reverse phase HPLC with fluorescence. Instrument response was unchanged in the presence of matrix, indicating no need to use matrix-matched calibrants. Furthermore, high method recoveries indicated calibrants do not need to undergo clean-up to account for analyte loss. Established method features include linear instrument response from 0.04-2.5µg/mL and stable derivatized calibrants over 7days. Fortified cornmeal method recoveries from 0.1-30.0μg/g were determined for FB 1 (75.1%-109%) and FB 2 (96.0%-115.2%). Inter-assay precision ranged from 1.0%-16.7%. Method accuracy was further confirmed using certified reference material. Inter-laboratory comparison with naturally-contaminated field corn demonstrated equivalent results with conventional derivatization. These results indicate FMOC derivatization is a suitable alternative for fumonisins B 1 and B 2 quantitation in corn-based feeds. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Glycosyltransferases as marker genes for the quantitative polymerase chain reaction-based detection of circulating tumour cells from blood samples of patients with breast cancer undergoing adjuvant therapy.

    Science.gov (United States)

    Kölbl, Alexandra C; Hiller, Roman A; Ilmer, Mathias; Liesche, Friederike; Heublein, Sabine; Schröder, Lennard; Hutter, Stefan; Friese, Klaus; Jeschke, Udo; Andergassen, Ulrich

    2015-08-01

    Altered glycosylation is a predominant feature of tumour cells; it serves for cell adhesion and detachment, respectively, and facilitates the immune escape of these cells. Therefore changes in the expression of glycosyltransferase genes could help to identify circulating tumour cells (CTCs) in the blood samples of cancer patients using a quantitative polymerase chain reaction (PCR) approach. Blood samples of healthy donors were inoculated with certain numbers of established breast cancer cell line cells, thus creating a model system. These samples were analysed by quantitative PCR for the expression of six different glycosyltransferase genes. The three genes with the best results in the model system were consecutively applied to samples from adjuvant breast cancer patients and of healthy donors. FUT3 and GALNT6 showed the highest increase in relative expression, while GALNT6 and ST3GAL3 were the first to reach statistically significant different ∆CT-values comparing the sample with and without addition of tumour cells. These three genes were applied to patient samples, but did not show any significant results that may suggest the presence of CTCs in the blood. Although the relative expression of some of the glycosyltransferase genes exhibited reasonable results in the model system, their application to breast cancer patient samples will have to be further improved, e.g. by co-analysis of patient blood samples by gold-standard methods.

  5. Quantitative detection of mass concentration of sand-dust storms via wind-profiling radar and analysis of Z- M relationship

    Science.gov (United States)

    Wang, Minzhong; Ming, Hu; Ruan, Zheng; Gao, Lianhui; Yang, Di

    2018-02-01

    With the aim to achieve quantitative monitoring of sand-dust storms in real time, wind-profiling radar is applied to monitor and study the process of four sand-dust storms in the Tazhong area of the Taklimakan Desert. Through evaluation and analysis of the spatial-temporal distribution of reflectivity factor, it is found that reflectivity factor ranges from 2 to 18 dBz under sand-dust storm weather. Using echo power spectrum of radar vertical beams, sand-dust particle spectrum and sand-dust mass concentration at the altitude of 600 ˜ 1500 m are retrieved. This study shows that sand-dust mass concentration reaches 700 μg/m3 under blowing sand weather, 2000 μg/m3 under sand-dust storm weather, and 400 μg/m3 under floating dust weather. The following equations are established to represent the relationship between the reflectivity factor and sand-dust mass concentration: Z = 20713.5 M 0.995 under floating dust weather, Z = 22988.3 M 1.006 under blowing sand weather, and Z = 24584.2 M 1.013 under sand-dust storm weather. The retrieval results from this paper are almost consistent with previous monitoring results achieved by former researchers; thus, it is implied that wind-profiling radar can be used as a new reference device to quantitatively monitor sand-dust storms.

  6. MR-perfusion (MRP) and diffusion-weighted imaging (DWI) in prostate cancer: Quantitative and model-based gadobenate dimeglumine MRP parameters in detection of prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Scherr, M.K., E-mail: michael.scherr@med.uni-muenchen.de [Institute of Clinical Radiology, University of Munich, Munich (Germany); Seitz, M. [Department of Urology, University of Munich, Munich (Germany); Mueller-Lisse, U.G. [Institute of Clinical Radiology, University of Munich, Munich (Germany); Ingrisch, M. [Josef Lissner Laboratory for Biomedical Imaging, Institute of Clinical Radiology, University of Munich, Munich (Germany); Reiser, M.F. [Institute of Clinical Radiology, University of Munich, Munich (Germany); Mueller-Lisse, U.L. [Department of Urology, University of Munich, Munich (Germany)

    2010-12-15

    Background: Various MR methods, including MR-spectroscopy (MRS), dynamic, contrast-enhanced MRI (DCE-MRI), and diffusion-weighted imaging (DWI) have been applied to improve test quality of standard MRI of the prostate. Purpose: To determine if quantitative, model-based MR-perfusion (MRP) with gadobenate dimeglumine (Gd-BOPTA) discriminates between prostate cancer, benign tissue, and transitional zone (TZ) tissue. Material and methods: 27 patients (age, 65 {+-} 4 years; PSA 11.0 {+-} 6.1 ng/ml) with clinical suspicion of prostate cancer underwent standard MRI, 3D MR-spectroscopy (MRS), and MRP with Gd-BOPTA. Based on results of combined MRI/MRS and subsequent guided prostate biopsy alone (17/27), biopsy and radical prostatectomy (9/27), or sufficient negative follow-up (7/27), maps of model-free, deconvolution-based mean transit time (dMTT) were generated for 29 benign regions (bROIs), 14 cancer regions (cROIs), and 18 regions of transitional zone (tzROIs). Applying a 2-compartment exchange model, quantitative perfusion analysis was performed including as parameters: plasma flow (PF), plasma volume (PV), plasma mean transit time (PMTT), extraction flow (EFL), extraction fraction (EFR), interstitial volume (IV) and interstitial mean transit time (IMTT). Two-sided T-tests (significance level p < 0.05) discriminated bROIs vs. cROIs and cROIs vs. tzROIs, respectively. Results: PMTT discriminated best between bROIs (11.8 {+-} 3.0 s) and cROIs (24.3 {+-} 9.6 s) (p < 0.0001), while PF, PV, PS, EFR, IV, IMTT also differed significantly (p 0.00002-0.0136). Discrimination between cROIs and tzROIs was insignificant for all parameters except PV (14.3 {+-} 2.5 ml vs. 17.6 {+-} 2.6 ml, p < 0.05). Conclusions: Besides MRI, MRS and DWI quantitative, 2-compartment MRP with Gd-BOPTA discriminates between prostate cancer and benign tissue with several parameters. However, distinction of prostate cancer and TZ does not appear to be reliable.

  7. Immunoglobulin kappa deleting element rearrangements in precursor-B acute lymphoblastic leukemia are stable targets for detection of minimal residual disease by real-time quantitative PCR

    NARCIS (Netherlands)

    van der Velden, V. H. J.; Willemse, M. J.; van der Schoot, C. E.; Hählen, K.; van Wering, E. R.; van Dongen, J. J. M.

    2002-01-01

    Immunoglobulin gene rearrangements are used as PCR targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). We Investigated the occurrence of monoclonal immunoglobulin kappa-deleting element (IGK-Kde) rearrangements by Southern blotting and PCR/heteroduplex

  8. A combination of baiting and different PCR formats, including measurement of real-time quantitative fluorescence, for the detection of Phytophthora fragariae in strawberry plants

    NARCIS (Netherlands)

    Bonants, P.J.M.; Gent-Pelzer, van M.P.E.; Hooftman, R.; Cooke, D.E.L.; Guy, D.C.; Duncan, J.M.

    2004-01-01

    Phytophthora fragariae, the cause of strawberry red stele disease, is a quarantine pathogen in Europe. Detecting low levels of infection requires sensitive and specific methods. In the past, Dutch and English inspection services have used bait plants to test strawberry propagation stocks destined

  9. Quantitative 16S rDNA-targeted polymerase chain reaction and oligonucleotide hybridization for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere

    NARCIS (Netherlands)

    Rosado, A.S.; Seldin, L.; Wolters, A.; Elsas, van J.D.

    1996-01-01

    A molecular method for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere was developed. The system consisted of polymerase chain reaction (PCR) amplification of part of the variable V1 to V4 regions of the 16S ribosomal RNA gene, followed by hybridization with a specific

  10. Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of Foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; de Stricker, K.

    2003-01-01

    Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting...

  11. Olfaction-based Detection Distance: A Quantitative Analysis of How Far Away Dogs Recognize Tortoise Odor and Follow It to Source

    Directory of Open Access Journals (Sweden)

    Cindee Valentin

    2008-03-01

    Full Text Available The use of detector dogs has been demonstrated to be effective and safe for finding Mojave desert tortoises and provides certain advantages over humans in field surveys. Unlike humans who rely on visual cues for target identification, dogs use primarily olfactory cues and can therefore locate targets that are not visually obvious. One of the key benefits of surveying with dogs is their efficiency at covering ground and their ability to detect targets from long distances. Dogs may investigate potential targets using visual cues but confirm the presence of a target based on scent. Everything that emits odor does so via vapor-phase molecules and the components comprising a particular scent are carried primarily though bulk movement of the atmosphere. It is the ability to search for target odor and then go to its source that makes dogs ideal for rapid target recognition in the field setting. Using tortoises as targets, we quantified distances that dogs detected tortoise scent, followed it to source, and correctly identified tortoises as targets. Detection distance data were collected during experimental trials with advanced global positioning system (GPS technology and then analyzed using geographic information system (GIS modeling techniques. Detection distances ranged from 0.5 m to 62.8 m for tortoises on the surface. We did not observe bias with tortoise size, age class, sex or the degree to which tortoises were handled prior to being found by the dogs. The methodology we developed to quantify olfaction-based detection distance using dogs can be applied to other targets that dogs are trained to find.

  12. MR-perfusion (MRP) and diffusion-weighted imaging (DWI) in prostate cancer: quantitative and model-based gadobenate dimeglumine MRP parameters in detection of prostate cancer.

    Science.gov (United States)

    Scherr, M K; Seitz, M; Müller-Lisse, U G; Ingrisch, M; Reiser, M F; Müller-Lisse, U L

    2010-12-01

    Various MR methods, including MR-spectroscopy (MRS), dynamic, contrast-enhanced MRI (DCE-MRI), and diffusion-weighted imaging (DWI) have been applied to improve test quality of standard MRI of the prostate. To determine if quantitative, model-based MR-perfusion (MRP) with gadobenate dimeglumine (Gd-BOPTA) discriminates between prostate cancer, benign tissue, and transitional zone (TZ) tissue. 27 patients (age, 65±4 years; PSA 11.0±6.1 ng/ml) with clinical suspicion of prostate cancer underwent standard MRI, 3D MR-spectroscopy (MRS), and MRP with Gd-BOPTA. Based on results of combined MRI/MRS and subsequent guided prostate biopsy alone (17/27), biopsy and radical prostatectomy (9/27), or sufficient negative follow-up (7/27), maps of model-free, deconvolution-based mean transit time (dMTT) were generated for 29 benign regions (bROIs), 14 cancer regions (cROIs), and 18 regions of transitional zone (tzROIs). Applying a 2-compartment exchange model, quantitative perfusion analysis was performed including as parameters: plasma flow (PF), plasma volume (PV), plasma mean transit time (PMTT), extraction flow (EFL), extraction fraction (EFR), interstitial volume (IV) and interstitial mean transit time (IMTT). Two-sided T-tests (significance level pMRP with Gd-BOPTA discriminates between prostate cancer and benign tissue with several parameters. However, distinction of prostate cancer and TZ does not appear to be reliable. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  13. Quantitative fluorescence-polymerase chain reaction assay for the detection of the duplication of the Charcot Marie Tooth disease type 1A critical region.

    Science.gov (United States)

    De Toffol, Simona; Bellone, Emilia; Dulcetti, Francesca; Ruggeri, Anna Maria; Maggio, Pietro Paolo; Pulimeno, Maria Rosaria; Mandich, Paola; Maggi, Federico; Simoni, Giuseppe; Grati, Francesca Romana

    2010-04-01

    Charcot Marie Tooth (CMT) syndrome is the most common hereditary peripheral neuropathy, with an incidence of about 1 in 2500. The subtype 1A (CMT1A) is caused by a tandem duplication of a 1.5-Mb region encompassing the PMP22 gene. Conventional short tandem repeat (STR) analysis can reveal this imbalance if a triallelic pattern, defining with certainty the presence of duplication, is present. In case of duplication with a biallelic pattern, it can only indicate a semiquantitative dosage of the fluorescence intensity ratio of the two fragments. In this study we developed a quantitative fluorescence-PCR using seven highly informative STRs within the CMT1A critical region that successfully disclosed or excluded the presence of the pathogenic imbalance in a cohort of 60 samples including 40 DNAs from samples with the CMT1A duplication previously characterized with two different molecular approaches, and 20 diagnostic samples from 10 members of a five-generation pedigree segregating CMT1A, 8 unrelated cases and 2 prenatal samples. The application of the quantitative fluorescence-PCR using STRs located in the critical region could be a reliable method to evaluate the presence of the PMP22 duplication for the diagnosis and classification of hereditary neuropathies in asymptomatic subjects with a family history of inherited neuropathy, in prenatal samples in cases with one affected parent, and in unrelated patients with a sporadic demyelinating neuropathy with clinical features resembling CMT (i.e., pes cavus with hammer toes) or with conduction velocities in the range of CMT1A.

  14. Binding assays for the quantitative detection of P. brevis polyether neurotoxins in biological samples and antibodies as therapeutic aids for polyether marine intoxication. Annual report, 1 December 1987-30 November 1988

    Energy Technology Data Exchange (ETDEWEB)

    Baden, D.G.

    1988-12-15

    The polyether lipid-soluble toxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) can be detected using two separate types of specific binding reaction. Using tritiated PbTx-3 as a specific probe for binding to voltage-dependent sodium channels in rat brain synaptosomes or to specific polyclonal antibodies, binding equilibria and displacement by unlabeled brevetoxins were compared. Labeled toxin can be displaced in a competitive manner by any of the other 5 naturally-occurring toxins; the quantitative displacement ability of each appears to reflect individual potency in fish bioassay. A comparison of ED50 in Radioimmunoassay and ED50 in synaptosome binding assay indicates that the former assay is useful for detection of toxins which possess the structural backbone of PbTx-3, the immunizing hapten. Thus, the two assays have quantitative applicability; the sodium channel with respect to potency and the antibodies with respect to structure. Microtiter plate assays utilizing each specific brevetoxin binding component and enzyme-linked toxin hapten have been successful and indicate a general applicability of colorimetric prototypes. There, is however, considerable manipulation required to decrease non-specific binding of the hydrophobic toxin-enzyme complex to the plates. Preliminary studies aimed at producing monoclonal antibodies have been explored using brevetoxins linked to keyhole limpet hemocyanin.

  15. SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes.

    Science.gov (United States)

    Tien, Wei-Ping; Lim, Gareth; Yeo, Gladys; Chiang, Suzanna Nicole; Chong, Chee-Seng; Ng, Lee-Ching; Hapuarachchi, Hapuarachchige Chanditha

    2017-09-19

    The monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures. We developed a rapid, sensitive and specific real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the detection of ZIKV in field-caught mosquitoes. The primers and PCR cycling conditions were optimized to minimize non-specific amplification due to cross-reactivity with the genomic material of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex tritaeniorhynchus, Culex sitiens and Anopheles sinensis, as well as accompanying microbiota. The performance of the assay was further evaluated with a panel of flaviviruses and alphaviruses as well as in field-caught Ae. aegypti mosquitoes confirmed to be positive for ZIKV. As compared to a probe-based assay, the newly developed assay demonstrated 100% specificity and comparable detection sensitivity for ZIKV in mosquitoes. Being a SYBR Green-based method, the newly-developed assay is cost-effective and easy to adapt, thus is applicable to large-scale vector surveillance activities in endemic countries, including those with limited resources and expertise. The amplicon size (119 bp) also allows sequencing to confirm the virus type. The primers flank relatively conserved regions of ZIKV genome, so that, the assay is able to detect genetically diverse ZIKV strains. Our findings, therefore, testify the potential use of the newly-developed assay in vector surveillance programmes for ZIKV in endemic regions.

  16. Quantitative PCR Detection and Characterisation of Human Adenovirus, Rotavirus and Hepatitis A Virus in Discharged Effluents of Two Wastewater Treatment Facilities in the Eastern Cape, South Africa.

    Science.gov (United States)

    Adefisoye, Martins Ajibade; Nwodo, Uchechukwu U; Green, Ezekiel; Okoh, Anthony Ifeanyin

    2016-12-01

    The occurrence of enteric viruses in reclaimed wastewater, their removal by efficient treatment processes and the public health hazards associated with their release into the environments are of great significance in environmental microbiology. In this study, TaqMan-based real-time polymerase chain reaction (qPCR) was used to assess the prevalence of human adenovirus (HAdV), rotavirus (RV) and hepatitis A virus (HAV) in the final effluents of two wastewater treatment plants in the Eastern Cape Province, South Africa, over a twelve-month sampling period. The correlation between the concentrations of viruses in the effluents samples and faecal coliform (FC) densities were assessed as to validate the use of FC as microbiological indicator in water quality assessment. HAdV was detected in 62.5 % (30/48) of the samples with concentrations ranging between 8.4 × 10 1 and 1.0 × 10 5 genome copies/L while HAV and RV were only detected at concentrations below the set detection limits. FCs densities ranged from 1 to 2.7 × 10 4 CFU/100 ml. Adenovirus species HAdV-B (serotype 2) and HAdV-F (serotype 41) were detected in 86.7 % (26/30) and 6.7 % (2/30) of the HAdV-positive samples, respectively. No consistent seasonal trend was observed in HAdV concentrations, however, increased concentrations of HAdV were generally observed in the winter months. Also, there was no correlation between the occurrence of HAdV and FC at both the treatment plants. The persistent occurrence of HAdV in the discharged treated effluents points to the potential public health risk through the release of HAdV into the receiving watersheds, and the possibility of their transmission to human population.

  17. Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus by a Quantitative PCR (qPCR Assay.

    Directory of Open Access Journals (Sweden)

    Susan I Jarvi

    Full Text Available The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that causes human eosinophilic meningitis and ocular angiostrongyliasis characteristic of rat lungworm (RLW disease. Definitive diagnosis is made by finding and identifying A. cantonensis larvae in the cerebral spinal fluid or by using a custom immunological or molecular test. This study was conducted to determine if genomic DNA from A. cantonensis is detectable by qPCR in the blood or tissues of experimentally infected rats. F1 offspring from wild rats were subjected to experimental infection with RLW larvae isolated from slugs, then blood or tissue samples were collected over multiple time points. Blood samples were collected from 21 rats throughout the course of two trials (15 rats in Trial I, and 6 rats in Trial II. In addition to a control group, each trial had two treatment groups: the rats in the low dose (LD group were infected by approximately 10 larvae and the rats in the high dose (HD group were infected with approximately 50 larvae. In Trial I, parasite DNA was detected in cardiac bleed samples from five of five LD rats and five of five HD rats at six weeks post-infection (PI, and three of five LD rats and five of five HD rats from tail tissue. In Trial II, parasite DNA was detected in peripheral blood samples from one of two HD rats at 53 minutes PI, one of two LD rats at 1.5 hours PI, one of two HD rats at 18 hours PI, one of two LD rats at five weeks PI and two of two at six weeks PI, and two of two HD rats at weeks five and six PI. These data demonstrate that parasite DNA can be detected in peripheral blood at various time points throughout RLW infection in rats.

  18. Detection of Angiostrongylus cantonensis in the Blood and Peripheral Tissues of Wild Hawaiian Rats (Rattus rattus) by a Quantitative PCR (qPCR) Assay.

    Science.gov (United States)

    Jarvi, Susan I; Pitt, William C; Farias, Margaret E; Shiels, Laura; Severino, Michael G; Howe, Kathleen M; Jacquier, Steven H; Shiels, Aaron B; Amano, Karis K; Luiz, Blaine C; Maher, Daisy E; Allison, Maureen L; Holtquist, Zachariah C; Scheibelhut, Neil T

    2015-01-01

    The nematode Angiostrongylus cantonensis is a rat lungworm, a zoonotic pathogen that causes human eosinophilic meningitis and ocular angiostrongyliasis characteristic of rat lungworm (RLW) disease. Definitive diagnosis is made by finding and identifying A. cantonensis larvae in the cerebral spinal fluid or by using a custom immunological or molecular test. This study was conducted to determine if genomic DNA from A. cantonensis is detectable by qPCR in the blood or tissues of experimentally infected rats. F1 offspring from wild rats were subjected to experimental infection with RLW larvae isolated from slugs, then blood or tissue samples were collected over multiple time points. Blood samples were collected from 21 rats throughout the course of two trials (15 rats in Trial I, and 6 rats in Trial II). In addition to a control group, each trial had two treatment groups: the rats in the low dose (LD) group were infected by approximately 10 larvae and the rats in the high dose (HD) group were infected with approximately 50 larvae. In Trial I, parasite DNA was detected in cardiac bleed samples from five of five LD rats and five of five HD rats at six weeks post-infection (PI), and three of five LD rats and five of five HD rats from tail tissue. In Trial II, parasite DNA was detected in peripheral blood samples from one of two HD rats at 53 minutes PI, one of two LD rats at 1.5 hours PI, one of two HD rats at 18 hours PI, one of two LD rats at five weeks PI and two of two at six weeks PI, and two of two HD rats at weeks five and six PI. These data demonstrate that parasite DNA can be detected in peripheral blood at various time points throughout RLW infection in rats.

  19. Evaluation and validation of a real-time PCR assay for detection and quantitation of human adenovirus 14 from clinical samples.

    Directory of Open Access Journals (Sweden)

    David Metzgar

    Full Text Available In 2007, the Centers for Disease Control and Prevention (CDC reported that Human adenovirus type 14 (HAdV-14 infected 106 military personnel and was responsible for the death of one U.S. soldier at Lackland Air Force Base in Texas. Identification of the responsible adenovirus, which had not previously been seen in North America and for which rapid diagnostic tools were unavailable, required retrospective analysis at reference laboratories. Initial quarantine measures were also reliant on relatively slow traditional PCR analysis at other locations. To address this problem, we developed a real-time PCR assay that detects a 225 base pair sequence in the HAdV-14a hexon gene. Fifty-one oropharyngeal swab specimens from the Naval Health Research Center, San Diego, CA and Advanced Diagnostic Laboratory, Lackland AFB, TX were used to validate the new assay. The described assay detected eight of eight and 19 of 19 confirmed HAdV-14a clinical isolates in two separate cohorts from respiratory disease outbreaks. The real-time PCR assay had a wide dynamic range, detecting from 10(2 to 10(7 copies of genomic DNA per reaction. The assay did not cross-react with other adenoviruses, influenza, respiratory syncytial virus, or common respiratory tract bacteria. The described assay is easy to use, sensitive and specific for HAdV-14a in clinical throat swab specimens, and very rapid since turnaround time is less than four hours to obtain an answer.

  20. Quantitative lymphography

    International Nuclear Information System (INIS)

    Mostbeck, A.; Lofferer, O.; Kahn, P.; Partsch, H.; Koehn, H.; Bialonczyk, Ch.; Koenig, B.

    1984-01-01

    Labelled colloids and macromolecules are removed lymphatically. The uptake of tracer in the regional lymphnodes is a parameter of lymphatic flow. Due to great variations in patient shape - obesity, cachexia - and accompanying variations in counting efficiencies quantitative measurements with reasonable accuracy have not been reported to date. A new approach to regional absorption correction is based on the combination of transmission and emission scans for each patient. The transmission scan is used for calculation of an absorption correction matrix. Accurate superposition of the correction matrix and the emission scan is achieved by computing the centers of gravity of point sources and - in the case of aligning opposite views - by cross correlation of binary images. In phantom studies the recovery was high (98.3%) and the coefficient of variation of repeated measurement below 1%. In patient studies a standardized stress is a prerequisite for reliable and comparable results. Discrimination between normals (14.3 +- 4.2D%) and patients with lymphedema (2.05 +- 2.5D%) was highly significant using praefascial lymphography and sc injection. Clearence curve analysis of the activities at the injection site, however, gave no reliable data for this purpose. In normals, the uptake in lymphnodes after im injection is by one order of magnitude lower then the uptake after sc injection. The discrimination between normals and patients with postthromboic syndrome was significant. Lymphography after ic injection was in the normal range in 2/3 of the patients with lymphedema and is therefore of no diagnostic value. The difference in uptake after ic and sc injection demonstrated for the first time by our quantitative method provides new insights into the pathophysiology of lymphedema and needs further investigation. (Author)

  1. Quantitative determination of regorafenib and its two major metabolites in human plasma with high-performance liquid chromatography and ultraviolet detection.

    Science.gov (United States)

    Fujita, Kazuma; Miura, Masatomo; Shibata, Hiroyuki

    2016-10-01

    A simple, highly sensitive and specific high-performance liquid chromatography (HPLC) method was developed for the simultaneous quantitation of regorafenib, N-oxidemetabolite (M-2) and the desmethyl N-oxide metabolite (M-5) in human plasma. Regorafenib, M-2, M-5 and the internal standard sorafenib were separated using a mobile phase of 0.5% KH2 PO4 (pH 3.5)-acetonitrile (30:70, v/v), on a Capcell PAK MG II column at a flow rate of 0.5 mL/min and measurement at UV 260 nm. The lower limits of quantification for regorafenib, M-2 and M-5 were 10 ng/mL for each analyte. A procedure using solid-phase extraction required only a small amount of plasma (100 μL) for one analysis while providing high extraction recovery (>81% for all compounds) and good selectivity. Coefficients of variation for intra- and inter-day assays were HPLC assay is suitable for clinical pharmacokinetic studies of regorafenib. The present HPLC method is currently in use for our observational studies of patients under treatment. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Comparison of serum pools and oral fluid samples for detection of porcine circovirus type 2 by quantitative real-time PCR in finisher pigs

    DEFF Research Database (Denmark)

    Nielsen, Gitte Blach; Nielsen, Jens Peter; Haugegaard, John

    2018-01-01

    Porcine circovirus type 2 (PCV2) diagnostics in live pigs often involves pooled serum and/or oral fluid samples for group-level determination of viral load by quantitative real-time polymerase chain reaction (qPCR). The purpose of the study was to compare the PCV2 viral load determined by q......PCR of paired samples at the pen level of pools of sera (SP) from 4 to 5 pigs and the collective oral fluid (OF) from around 30 pigs corresponding to one rope put in the same pen. Pigs in pens of 2 finishing herds were sampled by cross-sectional (Herd 1) and cross-sectional with follow-up (Herd 2) study designs....... In Herd 1, 50 sample pairs consisting of SP from 4 to 5 pigs and OF from around 23 pigs were collected. In Herd 2, 65 sample pairs consisting of 4 (SP) and around 30 (OF) pigs were collected 4 times at 3-week intervals. A higher proportion of PCV2-positive pens (86% vs. 80% and 100% vs. 91%) and higher...

  3. Determination of the spectral dependence of reduced scattering and quantitative second-harmonic generation imaging for detection of fibrillary changes in ovarian cancer

    Science.gov (United States)

    Campbell, Kirby R.; Tilbury, Karissa B.; Campagnola, Paul J.

    2015-03-01

    Here, we examine ovarian cancer extracellular matrix (ECM) modification by measuring the wavelength dependence of optical scattering measurements and quantitative second-harmonic generation (SHG) imaging metrics in the range of 800-1100 nm in order to determine fibrillary changes in ex vivo normal ovary, type I, and type II ovarian cancer. Mass fractals of the collagen fiber structure is analyzed based on a power law correlation function using spectral dependence measurements of the reduced scattering coefficient μs' where the mass fractal dimension is related to the power. Values of μs' are measured using independent methods of determining the values of μs and g by on-axis attenuation measurements using the Beer-Lambert Law and by fitting the angular distribution of scattering to the Henyey-Greenstein phase function, respectively. Quantitativespectral SHG imaging on the same tissues determines FSHG/BSHG creation ratios related to size and harmonophore distributions. Both techniques probe fibril packing order, but the optical scattering probes structures of sizes from about 50-2000 nm where SHG imaging - although only able to resolve individual fibers - builds contrast from the assembly of fibrils. Our findings suggest that type I ovarian tumor structure has the most ordered collagen fibers followed by normal ovary then type II tumors showing the least order.

  4. Production of monoclonal antibodies and development of a quantitative immuno-polymerase chain reaction assay to detect and quantify recombinant Glutathione S-transferase.

    Science.gov (United States)

    Abud, J E; Luque, E H; Ramos, J G; Rodriguez, H A

    2017-07-01

    GST-tagged proteins are important tools for the production of recombinant proteins. Removal of GST tag from its fusion protein, frequently by harsh chemical treatments or proteolytic methods, is often required. Thus, the monitoring of the proteins in tag-free form requires a significant effort to determine the remnants of GST during purification process. In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and an immuno-polymerase chain reaction (IPCR) assay, both specific for detection of recombinant GST (rGST). rGST was expressed in Escherichia coli JM109, using a pGEX4T-3 vector, and several anti-rGST monoclonal antibodies were generated using hybridoma technology. Two of these were rationally selected as capture and detection antibodies, allowing the development of a sandwich ELISA with a limit of detection (LOD) of 0.01 μg/ml. To develop the rGST-IPCR assay, we selected "Universal-IPCR" format, comprising the biotin-avidin binding as the coupling system. In addition, the rGST-IPCR was developed in standard PCR tubes, and the surface adsorption of antibodies on PCR tubes, the optimal neutravidin concentrations, the generation of a reporter DNA and the concentration effect were studied and determined. Under optimized assay conditions, the rGST-IPCR assay provided a 100-fold increase in the LOD as well as an expanded working range, in comparison with rGST-ELISA. The proposed method exhibited great potentiality for application in several fields in which measurement of very low levels of GST is necessary, and might provide a model for other IPCR assays. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Rapid extraction and quantitative detection of the herbicide diuron in surface water by a hapten-functionalized carbon nanotubes based electrochemical analyzer.

    Science.gov (United States)

    Sharma, Priyanka; Bhalla, Vijayender; Tuteja, Satish; Kukkar, Manil; Suri, C Raman

    2012-05-21

    A solid phase extraction micro-cartridge containing a non-polar polystyrene absorbent matrix was coupled with an electrochemical immunoassay analyzer (EIA) and used for the ultra-sensitive detection of the phenyl urea herbicide diuron in real samples. The EIA was fabricated by using carboxylated carbon nanotubes (CNTs) functionalized with a hapten molecule (an amine functionalized diuron derivative). Screen printed electrodes (SPE) were modified with these haptenized CNTs and specific in-house generated anti diuron antibodies were used for bio-interface development. The immunodetection was realized in a competitive electrochemical immunoassay format using alkaline phosphatase labeled secondary anti-IgG antibody. The addition of 1-naphthyl phosphate substrate resulted in the production of an electrochemically active product, 1-naphthol, which was monitored by using differential pulse voltammetry (DPV). The assay exhibited excellent sensitivity and specificity having a dynamic response range of 0.01 pg mL(-1) to 10 μg mL(-1) for diuron with a limit of detection of around 0.1 pg mL(-1) (n = 3) in standard water samples. The micro-cartridge coupled hapten-CNTs modified SPE provided an effective and efficient electrochemical immunoassay for the real-time monitoring of pesticides samples with a very high degree of sensitivity.

  6. Quantitative assessment of bone marrow attenuation values at MDCT: An objective tool for the detection of bone bruise related to occult sacral insufficiency fractures

    Energy Technology Data Exchange (ETDEWEB)

    Henes, F.O.; Groth, M.; Bley, T.A.; Regier, M.; Ittrich, H.; Adam, G.; Bannas, P. [University Medical Center Hamburg-Eppendorf, Department of Diagnostic and Interventional Radiology, Hamburg (Germany); Nuechtern, J.V. [University Medical Center Hamburg-Eppendorf, Department of Trauma, Hand and Reconstructive Surgery, Hamburg (Germany); Treszl, A. [University Medical Center Hamburg-Eppendorf, Center for Experimental Medicine, Department of Medical Biometry and Epidemiology, Hamburg (Germany)

    2012-10-15

    To prove the feasibility of using Hounsfield attenuation values at MDCT to detect bone bruises related to sacral insufficiency fractures. Twenty-two patients with acute sacrum trauma and no fracture findings at MDCT were included in our prospective study. Two observers independently reviewed CTs regarding visual signs of bone bruises in 132 defined regions of the sacral alae. Interobserver agreement was tested by {kappa} statistics. Subsequently, HU values were obtained in the same regions, and attenuation differences between the two sides were calculated. Validity and reliability were assessed by intraclass correlation coefficient and Bland-Altman analysis. HU differences were subjected to ROC curve analysis to determine sensitivity, specificity, PPV and NPV. MRI served as standard reference. MRI revealed 19 regions with bone bruises and associated sacral insufficiency fractures. HU measurements demonstrated good validity and reliability (r = 0.989). ROC curve analysis exhibited an ideal cutoff value of 35.7 HU density difference between affected and non-affected regions. Visual evaluation revealed moderate agreement ({kappa} = 0.48); diagnostic accuracy was inferior to objective evaluation. Assessment of differences in bone marrow density by HU measurements is an objective and reliable tool for detection of bone bruises associated with occult sacral insufficiency fractures. (orig.)

  7. Detection of gene copy number aberrations in mantle cell lymphoma by a single quantitative multiplex PCR assay: clinicopathological relevance and prognosis value.

    Science.gov (United States)

    Jardin, Fabrice; Picquenot, Jean-Michel; Parmentier, Françoise; Ruminy, Philippe; Cornic, Marie; Penther, Dominique; Bertrand, Philippe; Lanic, Hélène; Cassuto, Ophélie; Humbrecht, Catherine; Lemasle, Emilie; Wautier, Agathe; Bastard, Christian; Tilly, Hervé

    2009-09-01

    The t(11;14)(q13;q32) is the hallmark of mantle cell lymphoma (MCL). Additional genetic alterations occur in the majority of cases. This study aimed to design a polymerase chain reaction (PCR) assay to determine the incidence and relevance of recurrent gene copy number aberrations in this disease. Forty-two MCL cases with frozen- or paraffin-embedded (FFPE) tissues were selected. Three different quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF) assays were designed to simultaneously analyse eight genes (CDKN2A, RB1, ATM, CDK2, TP53, MYC, CDKN1B, MDM2), to analyse the 9p21 locus (CDKN2A/CDKN2B) and FFPE tissues. Gains of MYC, CDK2, CDKN1B, and MDM2 were observed in 10% of cases. Losses of RB1, CDKN2A, ATM or TP53 were observed in 38%, 31%, 24% and 10% of cases, respectively. Analysis of the 9p21 locus indicated that, in most cases, tumours displayed a complete inactivation of p14(ARF)/p15I(NK4B)/p16I(NK4A). CDKN2A and MYC aberrations were associated with a high MCL international prognostic index (MIPI). CDK2/MDM2 gains and CDKN2A/TP53 losses correlated with an unfavourable outcome. PCR experiments with frozen and FFPE-tissues indicated that our approach is valid in a routine diagnostic setting, providing a powerful tool that could be used for patient stratification in combination with MIPI in future clinical trials.

  8. Quantitative real-time RT-PCR and chromogenic in situ hybridization: precise methods to detect HER-2 status in breast carcinoma

    Directory of Open Access Journals (Sweden)

    Soares Fernando A

    2009-03-01

    Full Text Available Abstract Background HER-2 gene testing has become an integral part of breast cancer patient diagnosis. The most commonly used assay in the clinical setting for evaluating HER-2 status is immunohistochemistry (IHC and fluorescence in situ hybridization (FISH. These procedures permit correlation between HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas. Methods To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. Results The concordance rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1+ immunostaining cases, which showed high-level amplification (15.4% and HER-2 transcript overexpression (20%. Moreover, 2+ immunostaining cases presented nonamplified status (50% by CISH and HER-2 downexpression (38.5% by qRT-PCR. In general, concordance occurred between qRT-PCR and CISH results. A high concordance was observed between CISH/qRT-PCR and FISH. Comparisons with clinicopathological data revealed a significant association between HER-2 downexpression and the involvement of less than four lymph nodes (P = 0.0350. Conclusion Based on these findings, qRT-PCR was more precise and reproducible than IHC. Furthermore, CISH was revealed as an alternative and useful procedure for investigating amplifications involving the HER-2 gene.