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Sample records for assaying quantitative

  1. Ideal target organism for quantitative bactericidal assays.

    PubMed Central

    Hooke, A M; Oeschger, M P; Zeligs, B J; Bellanti, J A

    1978-01-01

    We have developed a target organism which permits quantitative bactericidal assays. The organism is an Escherichia coli mutant which cannot grow at the temperature of the assay (37 degrees C), but retains full colony-forming potential for subsequent quantitation at 25 degrees C. We show that quantitative data on the bactericidal capacity of polymorphonuclear leukocytes and alveolar macrophages can be obtained when this mutant is used as a target. The procedure used to generate the strain is described in detail and should be applicable to many bacterial species. Characterization of the properties of the mutant indicates that it has a strong potential for use in other in vivo and in vitro investigations of host responses to microbial invasion. PMID:352943

  2. Photoelectric AFP quantitative detection of immunochromatographic assay strip

    NASA Astrophysics Data System (ADS)

    Du, Min; Jiang, Hai-yan

    2006-09-01

    An immunochromatographic assay (ICA) system for quantitative determination of analyte in sample is described in this paper. The designed system not only quantitatively detect target analytes in sample but also enhance detection precision in immunochromatographic assay technique.

  3. A quantitative assay for intercellular aggregation

    NASA Technical Reports Server (NTRS)

    Neelamegham, S.; Zygourakis, K.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    In an earlier communication (Munn et al., J Immunol. Methods 166: 11-25, 1993), we presented the initial development of a quantitative assay for monitoring the rates of cellular aggregation based on digital image processing and video microscopy. This study describes some important enhancements and modifications to the procedure. A new index is introduced to characterize the three-dimensional morphology of the aggregates. This index is based on temporal changes in the projected area of the cells and cell aggregates during the course of the experiment. By drawing an analogy with the kinetic theory of gases, we have also introduced a procedure to normalize for variations in cell seeding density among different experiments. In addition, the image analysis technique has been improved by introducing a background subtraction algorithm to remove illumination defects and an adaptive segmentation procedure. These improvements allowed us to completely automate the image analysis procedure, thus minimizing user intervention and improving the reproducibility of the measurements. The enhanced visual assay is evaluated using some recent results from our studies on homotypic lymphocyte aggregation.

  4. A quantitative assay for intercellular aggregation

    NASA Technical Reports Server (NTRS)

    Neelamegham, S.; Zygourakis, K.; McIntire, L. V. (Principal Investigator)

    1997-01-01

    In an earlier communication (Munn et al., J Immunol. Methods 166: 11-25, 1993), we presented the initial development of a quantitative assay for monitoring the rates of cellular aggregation based on digital image processing and video microscopy. This study describes some important enhancements and modifications to the procedure. A new index is introduced to characterize the three-dimensional morphology of the aggregates. This index is based on temporal changes in the projected area of the cells and cell aggregates during the course of the experiment. By drawing an analogy with the kinetic theory of gases, we have also introduced a procedure to normalize for variations in cell seeding density among different experiments. In addition, the image analysis technique has been improved by introducing a background subtraction algorithm to remove illumination defects and an adaptive segmentation procedure. These improvements allowed us to completely automate the image analysis procedure, thus minimizing user intervention and improving the reproducibility of the measurements. The enhanced visual assay is evaluated using some recent results from our studies on homotypic lymphocyte aggregation.

  5. Rapid Quantitative Serological Assay of Staphylococcal Enterotoxin B

    PubMed Central

    Weirether, Francis J.; Lewis, Evelyn E.; Rosenwald, Albert J.; Lincoln, Ralph E.

    1966-01-01

    A simple, rapid method, based on the Oudin single diffusion technique, is described for the quantitative assay of staphylococcal enterotoxin B. The method yields reproducible results without close control of such assay variables as temperature, antiserum dilution, and assay time, provided that the ionic strength is maintained above 0.2 n sodium chloride equivalent. PMID:4959985

  6. A Rapid and Quantitative Recombinase Activity Assay

    USDA-ARS?s Scientific Manuscript database

    We present here a comparison between the recombinase systems FLP-FRT and Cre-loxP. A transient excision based dual luciferase expression assay is used for its rapid and repeatable nature. The detection system was designed within an intron to remove the remaining recombinase recognition site and no...

  7. Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

    Cancer.gov

    The NCI CPTAC program is applying its expertise in quantitative proteomics to develop assays for RAS pathway proteins. Targets include key phosphopeptides that should increase our understanding of how the RAS pathway is regulated.

  8. A quantitative comet infection assay for influenza virus

    PubMed Central

    Lindsay, Stephen M.; Timm, Andrea; Yin, John

    2011-01-01

    Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

  9. A comparison of protein quantitation assays for biopharmaceutical applications.

    PubMed

    Noble, J E; Knight, A E; Reason, A J; Di Matola, A; Bailey, M J A

    2007-10-01

    Dye-based protein determination assays are widely used to estimate protein concentration, however various reports suggest that the response is dependent on the composition and sequence of the protein, limiting confidence in the resulting concentration estimates. In this study a diverse set of model proteins representing various sizes of protein and covalent modifications, some typical of biopharmaceuticals have been used to assess the utility of dye-based protein concentration assays. The protein concentration assays (Bicinchoninic acid (BCA), Bradford, 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA), DC, Fluorescamine and Quant-i) were compared to the 'gold standard' assay, quantitative amino acid analysis (AAA). The assays that displayed the lowest variability between proteins, BCA and DC, also generated improved estimates when BSA was used as a standard, when compared to AAA derived concentrations. Assays read out by absorbance tended to display enhanced robustness and repeatability, whereas the fluorescence based assays had wider quantitation ranges and lower limits of detection. Protein modification, in the form of glycosylation and PEGylation, and the addition of excipients, were found to affect the estimation of protein concentration for some of the assays when compared to the unmodified protein. We discuss the suitability and limitations of the selected assays for the estimation of protein concentration in biopharmaceutical applications.

  10. A Nanogram Level Colloidal Gold Single Reagent Quantitative Protein Assay

    PubMed Central

    Harrison, Gerald; Haffey, Patrick; Golub, Ellis E.

    2008-01-01

    We have developed a nanogram level quantitative protein assay based on the binding of colloidal gold to proteins adhered to nitrocellulose paper. The protein-gold complex produces a purple color proportional to the amount of protein present, and the intensity of the stain is quantified by densitometry. Typical assays require minimal starting material (10 – 20 μl) containing 2 – 5 μg protein. A small volume (2 μl) of protein solution is applied to nitrocellulose paper in a grid array, and dried. The nitrocellulose is incubated in colloidal gold suspension with gentle agitation (4–16 h), rinsed with water and scanned. Densitometric analysis of the scanned images allows quantitation of the unknown sample protein concentration by comparison with protein standards placed on the same nitrocellulose grid. The assay requires significantly less sample than conventional protein assays. In this report, the Golddots assay is calibrated against weighed protein samples, and compared with the Pierce Micro BCA protein assay kit. In addition, the assay is evaluated with several known proteins with different physical properties. PMID:18539124

  11. Optimized conditions for a quantitative SELDI TOF MS protein assay.

    PubMed

    Lomas, Lee; Clarke, Charlotte H; Thulasiraman, Vanitha; Fung, Eric

    2012-01-01

    The development of peptide/protein analyte assays for the purpose of diagnostic tests is driven by multiple factors, including sample availability, required throughput, and quantitative reproducibility. Laser Desorption/ionization mass spectrometry methods (LDI-MS) are particularly well suited for both peptide and protein characterization, and combining chromatographic surfaces directly onto the MS probe in the form of surface enhanced laser desorption/ionization (SELDI)-biochips has improved the reproducibility of analyte detection and provided effective relative quantitation. Here, we provide methods for developing reproducible SELDI-based assays by providing a complex artificial protein matrix background within the sample to be analyzed that allows for a common and reproducible ionization background as well as internal normalization standards. Using this approach, quantitative assays can be developed with CVs typically less than 10% across assays and days. Although the method has been extensively and successfully implemented in association with a protein matrix from E. coli, any other source for the complex protein matrix can be considered as long as it adheres to a set of conditions including the following: (1) the protein matrix must not provide interferences with the analyte to be detected, (2) the protein matrix must be sufficiently complex such that a majority of ion current generated from the desorption of the sample comes from the complex protein matrix, and (3) specific and well-resolved protein matrix peaks must be present within the mass range of the analyte of interest for appropriate normalization.

  12. Quantitative Fissile Assay In Used Fuel Using LSDS System

    NASA Astrophysics Data System (ADS)

    Lee, YongDeok; Jeon, Ju Young; Park, Chang-Je

    2017-09-01

    A quantitative assay of isotopic fissile materials (U235, Pu239, Pu241) was done at Korea Atomic Energy Research Institute (KAERI), using lead slowing down spectrometer (LSDS). The optimum design of LSDS was performed based on economics, easy maintenance and assay effectiveness. LSDS system consists of spectrometer, neutron source, detection and control. LSDS system induces fissile fission and fast neutrons are collected at fission chamber. The detected signal has a direct relation to the mass of existing fissile isotopes. Many current commercial assay technologies have a limitation in direct application on isotopic fissile assay of spent fuel, except chemical analysis. In the designed system, the fissile assay model was setup and the correction factor for self-shield was obtained. The isotopic fissile content assay was performed by changing the content of Pu239. Based on the fuel rod, the isotopic content was consistent with 2% uncertainty for Pu239. By applying the covering (neutron absorber), the effective shielding was obtained and the activation was calculated on the target. From the assay evaluation, LSDS technique is very powerful and direct to analyze the isotopic fissile content. LSDS is applicable for nuclear fuel cycle and spent fuel management for safety and economics. Additionally, an accurate fissile content will contribute to the international transparency and credibility on spent fuel.

  13. Optimization of a Quantitative Micro-neutralization Assay

    PubMed Central

    Lin, Yipu; Gu, Yan; McCauley, John W.

    2016-01-01

    The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells. PMID:28060291

  14. Quantitative turbidimetric assay of enzymatic gram-negative bacteria lysis.

    PubMed

    Levashov, Pavel A; Sedov, Sergey A; Shipovskov, Stepan; Belogurova, Natalia G; Levashov, Andrey V

    2010-03-01

    In this Technical Note, the quantitative turbidimetric assay for determination of the bacteriolytic activity of enzymes with gram-negative bacteria is proposed. The reactivity of hen white-egg lysozyme toward gram-negative E. coli intact cells was studied. It was found that the highest lysis rate occurred at pH 8.9 in the system containing 0.03 M NaCl. The mechanism of the reaction is discussed and applied for the quantitative evaluation of the reaction rate. The proposed method enables fast, reliable, and reproducible analysis of bacteriolytic activity of lysozyme with gram-negative bacteria.

  15. Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    PubMed Central

    Langel, Ülo

    2016-01-01

    Utilizing the phenomenon of nucleases exposing oligonucleotide phosphate backbones to phosphatases we present a novel quantitative method for kinetics of nuclease catalysis. Inorganic phosphate released from nuclease products by phosphatases could be quantified in real-time by a fluorescent sensor of inorganic phosphate. Two different nucleases were employed, showing the versatility of this assay for multiple turnover label-free nuclease studies. PMID:27101307

  16. Field-based multiplex and quantitative assay platforms for diagnostics

    NASA Astrophysics Data System (ADS)

    Venkatasubbarao, Srivatsa; Dixon, C. Edward; Chipman, Russell; Scherer, Axel; Beshay, Manal; Kempen, Lothar U.; Chandra Sekhar, Jai Ganesh; Yan, Hong; Puccio, Ava; Okonkwo, David; McClain, Stephen; Gilbert, Noah; Vyawahare, Saurabh

    2011-06-01

    The U.S. military has a continued interest in the development of handheld, field-usable sensors and test kits for a variety of diagnostic applications, such as traumatic brain injury (TBI) and infectious diseases. Field-use presents unique challenges for biosensor design, both for the readout unit and for the biological assay platform. We have developed robust biosensor devices that offer ultra-high sensitivity and also meet field-use needs. The systems under development include a multiplexed quantitative lateral flow test strip for TBI diagnostics, a field test kit for the diagnosis of pathogens endemic to the Middle East, and a microfluidic assay platform with a label-free reader for performing complex biological automated assays in the field.

  17. Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity.

    PubMed

    Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A; Bradford, William D; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S; Li, Rong

    2015-03-30

    Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein-based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. Copyright © 2015 Zhu et al.

  18. Single-Cell Based Quantitative Assay of Chromosome Transmission Fidelity

    PubMed Central

    Zhu, Jin; Heinecke, Dominic; Mulla, Wahid A.; Bradford, William D.; Rubinstein, Boris; Box, Andrew; Haug, Jeffrey S.; Li, Rong

    2015-01-01

    Errors in mitosis are a primary cause of chromosome instability (CIN), generating aneuploid progeny cells. Whereas a variety of factors can influence CIN, under most conditions mitotic errors are rare events that have been difficult to measure accurately. Here we report a green fluorescent protein−based quantitative chromosome transmission fidelity (qCTF) assay in budding yeast that allows sensitive and quantitative detection of CIN and can be easily adapted to high-throughput analysis. Using the qCTF assay, we performed genome-wide quantitative profiling of genes that affect CIN in a dosage-dependent manner and identified genes that elevate CIN when either increased (icCIN) or decreased in copy number (dcCIN). Unexpectedly, qCTF screening also revealed genes whose change in copy number quantitatively suppress CIN, suggesting that the basal error rate of the wild-type genome is not minimized, but rather, may have evolved toward an optimal level that balances both stability and low-level karyotype variation for evolutionary adaptation. PMID:25823586

  19. Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.

    PubMed

    Li, R; Kast, J

    2017-01-01

    Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics. © 2017 Elsevier Inc. All rights reserved.

  20. Quantitative Validation and Quality Control of Pyrosequencing® Assays.

    PubMed

    Lehmann, Ulrich

    2015-01-01

    Pyrosequencing(®) offers the opportunity to quantify the amount of nucleotides incorporated during the sequencing reaction. This enables the precise and sensitive quantification of DNA methylation, allele-specific expression, or mutation load. However, only a few studies addressed the question how precise and sensitive Pyrosequencing really is. Many publications claiming precise measurement (with two decimal places) fail to demonstrate performance figures like precision or reproducibility. And most publications reporting the detection (and quantification) of very weak signals by Pyrosequencing do omit to report the technical sensitivity of the assay used (i.e., limit of blank, limit of detection, limit of quantification).Therefore, this chapter aims at providing (1) an introduction to important basic concepts defining quantitative analytical assays, (2) an outline of a minimal set of validation measurements every study employing Pyrosequencing should include, especially if values close to the analytical threshold are reported.

  1. A quantitative assay for telomere protection in Saccharomyces cerevisiae.

    PubMed Central

    DuBois, Michelle L; Haimberger, Zara W; McIntosh, Martin W; Gottschling, Daniel E

    2002-01-01

    Telomeres are the protective ends of linear chromosomes. Telomeric components have been identified and described by their abilities to bind telomeric DNA, affect telomere repeat length, participate in telomeric DNA replication, or modulate transcriptional silencing of telomere-adjacent genes; however, their roles in chromosome end protection are not as well defined. We have developed a genetic, quantitative assay in Saccharomyces cerevisiae to measure whether various telomeric components protect chromosome ends from homologous recombination. This "chromosomal cap" assay has revealed that the telomeric end-binding proteins, Cdc13p and Ku, both protect the chromosome end from homologous recombination, as does the ATM-related kinase, Tel1p. We propose that Cdc13p and Ku structurally inhibit recombination at telomeres and that Tel1p regulates the chromosomal cap, acting through Cdc13p. Analysis with recombination mutants indicated that telomeric homologous recombination events proceeded by different mechanisms, depending on which capping component was compromised. Furthermore, we found that neither telomere repeat length nor telomeric silencing correlated with chromosomal capping efficiency. This capping assay provides a sensitive in vivo approach for identifying the components of chromosome ends and the mechanisms by which they are protected. PMID:12136006

  2. In vivo osteogenesis assay: a rapid method for quantitative analysis.

    PubMed

    Dennis, J E; Konstantakos, E K; Arm, D; Caplan, A I

    1998-08-01

    A quantitative in vivo osteogenesis assay is a useful tool for the analysis of cells and bioactive factors that affect the amount or rate of bone formation. There are currently two assays in general use for the in vivo assessment of osteogenesis by isolated cells: diffusion chambers and porous calcium phosphate ceramics. Due to the relative ease of specimen preparation and reproducibility of results, the porous ceramic assay was chosen for the development of a rapid method for quantitating in vivo bone formation. The ceramic cube implantation technique consists of combining osteogenic cells with 27-mm3 porous calcium phosphate ceramics, implanting the cell-ceramic composites subcutaneously into an immuno-tolerant host, and, after 2-6 weeks, harvesting and preparing the ceramic implants for histologic analysis. A drawback to the analysis of bone formation within these porous ceramics is that the entire cube must be examined to find small foci of bone present in some samples; a single cross-sectional area is not representative. For this reason, image analysis of serial sections from ceramics is often prohibitively time-consuming. Two alternative scoring methodologies were tested and compared to bone volume measurements obtained by image analysis. The two subjective scoring methods were: (1) Bone Scale: the amount of bone within pores of the ceramic implant is estimated on a scale of 0-4 based on the degree of bone fill (0=no bone, 1=up to 25%, 2=25 to 75%, 4=75 to 100% fill); and (2) Percentage Bone: the amount of bone is estimated by determining the percentage of ceramic pores which contain bone. Every tenth section of serially sectioned cubes was scored by each of these methods under double-blind conditions, and the Bone Scale and Percentage Bone results were directly compared to image analysis measurements from identical samples. Correlation coefficients indicate that the Percentage Bone method was more accurate than the Bone Scale scoring method. The Bone Scale

  3. Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution.

    PubMed

    Jones, Laurie J; Haugland, Richard P; Singer, Victoria L

    2003-04-01

    We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 micrograms/mL protein with a standard fluorometer, offering a broad, dynamic quantitation range and improved sensitivity relative to absorption-based protein solution assays. The protein-to-protein variability of the NanoOrange assay was comparable to those of standard assays, including Lowry, bicinchoninic acid, and Bradford procedures. We also found that the NanoOrange assay is useful for detecting relatively small proteins or large peptides, such as aprotinin and insulin. The assay was somewhat sensitive to the presence of several common contaminants found in protein preparations such as salts and detergents; however, it was insensitive to the presence of reducing agents, nucleic acids, and free amino acids. The simple assay protocol is suitable for automation. Samples are briefly heated in the presence of dye in a detergent-containing diluent, allowed to cool to room temperature, and fluorescence is measured using 485-nm excitation and 590-nm emission wavelengths. Therefore, the NanoOrange assay is well suited for use with standard fluorescence microplate readers, fluorometers, and some laser scanners.

  4. An evaluation of protein assays for quantitative determination of drugs.

    PubMed

    Williams, Katherine M; Arthur, Sarah J; Burrell, Gillian; Kelly, Fionnuala; Phillips, Darren W; Marshall, Thomas

    2003-07-31

    We have evaluated the response of six protein assays [the biuret, Lowry, bicinchoninic acid (BCA), Coomassie Brilliant Blue (CBB), Pyrogallol Red-Molybdate (PRM), and benzethonium chloride (BEC)] to 21 pharmaceutical drugs. The drugs evaluated were analgesics (acetaminophen, aspirin, codeine, methadone, morphine and pethidine), antibiotics (amoxicillin, ampicillin, gentamicin, neomycin, penicillin G and vancomycin), antipsychotics (chlorpromazine, fluphenazine, prochlorperazine, promazine and thioridazine) and water-soluble vitamins (ascorbic acid, niacinamide, pantothenic acid and pyridoxine). The biuret, Lowry and BCA assays responded strongly to most of the drugs tested. The PRM assay gave a sensitive response to the aminoglycoside antibiotics (gentamicin and neomycin) and the antipsychotic drugs. In contrast, the CBB assay showed little response to the aminoglycosides and gave a relatively poor response with the antipsychotics. The BEC assay did not respond significantly to the drugs tested. The response of the protein assays to the drugs was further evaluated by investigating the linearity of the response and the combined response of drug plus protein. The results are discussed with reference to drug interference in protein assays and the development of new methods for the quantification of drugs in protein-free solution.

  5. A Quantitative Electrochemiluminescence Assay for Clostridium perfringens alpha toxin

    DTIC Science & Technology

    2006-08-10

    Clostridium perfringens alpha toxin , Gerald A. Merrill a,b,¤, Victor R. Rivera b, Dwayne D. Neal b, Charles Young c, Mark A. Poli b a Department of...format electrochemiluminescence assay for identifying and assaying Clostridium perfringens alpha toxin. Biotinylated antibodies to C. perfringens alpha...matrix eVects. © 2006 Elsevier Inc. All rights reserved. Keywords: Electrochemiluminescence (ECL); Immunoassay; Clostridium perfringens ; Alpha toxin

  6. Serum free light chain quantitative assays: Dilemma of a biomarker.

    PubMed

    Cigliana, Giovanni; Gulli, Francesca; Napodano, Cecilia; Pocino, Krizia; De Santis, Elena; Colacicco, Luigi; Cordone, Iole; Conti, Laura; Basile, Umberto

    2017-04-26

    Serum free light chains detection assays are consistently meeting greater interest for the diagnosis and monitoring of monoclonal gammopathies and plasma cell dyscrasias. Nowadays, there are neither standardized methods nor reference material for the determination of free light chains; for this reason, it is important to compare two different assays used in clinical laboratory. We evaluated 300 serum samples from patients with B-cell disorders and compared the analytical performances of both assay. Each test was assayed on both testing platforms (Siemens Dade Behring BN II Nephelometer and SPAPLUS by The Binding Site). κ/λ ratios were determined and compared. Results were analyzed by Passing-Bablok and Bland-Altman plots to evaluate comparability of the two techniques and to determine bias. The reproducibility of both assays is acceptable, reaching minimum and desirable analytical goals derived from biological variability. However, values are not interchangeable between systems. This study shows that the two systems do not allow results to be transferred from one method to the other even if they display good agreement. Our study highlights the importance of elaborating an international standard for free light chains quantification in order to offer homogeneous results as well as guarantee harmonization of values among laboratories. Moreover, the assays should be validated in specific patient groups to determine that they are clinically fit for purpose. © 2017 Wiley Periodicals, Inc.

  7. Calibration of qualitative HBsAg assay results for quantitative HBsAg monitoring.

    PubMed

    Gunning, Hans; Adachi, Dena; Tang, Julian W

    2014-10-01

    Evidence is accumulating that quantitative hepatitis B surface antigen monitoring may be useful in managing patients with chronic HBV infection on certain treatment regimens. Based on these results with the Abbott Architect qualitative and quantitative HBsAg assays, it seems feasible to convert qualitative to quantitative HBsAg values for this purpose. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Quantitative CrAssphage PCR Assays for Human Fecal ...

    EPA Pesticide Factsheets

    Environmental waters are monitored for fecal pollution to protect public health and water resources. Traditionally, general fecal indicator bacteria are used; however, they cannot distinguish human fecal waste from pollution from other animals. Recently, a novel bacteriophage, crAssphage, was discovered by metagenomic data mining and reported to be abundant in and closely associated with human fecal waste. To confirm bioinformatic predictions, 384 primer sets were designed along the length of the crAssphage genome. Based upon initial screening, two novel crAssphage qPCR assays (CPQ_056 and CPQ_064) were designed and evaluated in reference fecal samples and water matrices. The assays exhibited high specificities (98.6%) when tested against a large animal fecal reference library and were highly abundant in raw sewage and sewage impacted water samples. In addition, CPQ_056 and CPQ_064 assay performance was compared to HF183/BacR287 and HumM2 methods in paired experiments. Findings confirm viral crAssphage qPCR assays perform at a similar level to well established bacterial human-associated fecal source identification technologies. These new viral based assays could become important water quality management and research tools. To inform the public.

  9. A Quantitative High Throughput Assay for Identifying Gametocytocidal Compounds

    PubMed Central

    Tanaka, Takeshi Q.; Dehdashti, Seameen J.; Nguyen, Dac-Trung; McKew, John C.; Zheng, Wei; Williamson, Kim C.

    2013-01-01

    Current antimalarial drug treatment does not effectively kill mature Plasmodium falciparum gametocytes, the parasite stage responsible for malaria transmission from human to human via a mosquito. Consequently, following standard therapy malaria can still be transmitted for over a week after the clearance of asexual parasites. A new generation of malaria drugs with gametocytocidal properties, or a gametocytocidal drug that could be used in combinational therapy with currently available antimalarials, is needed to control the spread of the disease and facilitate eradication efforts. We have developed a 1,536-well gametocyte viability assay for the high throughput screening of large compound collections to identify novel compounds with gametocytocidal activity. The signal-to-basal ratio and Z′-factor for this assay were 3.2-fold and 0.68, respectively. The IC50 value of epoxomicin, the positive control compound, was 1.42 ± 0.09 nM that is comparable to previously reported values. This miniaturized assay significantly reduces the number of gametocytes required for the alamarBlue viability assay, and enables high throughput screening for lead discovery efforts. Additionally, the screen does not require a specialized parasite line, gametocytes from any strain, including field isolates, can be tested. A pilot screen utilizing the commercially available LOPAC library, consisting of 1,280 known compounds, revealed two selective gametocytocidal compounds having 54 and 7.8-fold gametocytocidal selectivity in comparison to their cell cytotoxicity effect against the mammalian SH-SY5Y cell line. PMID:23454872

  10. Comparison of Droplet Digital PCR and Quantitative PCR Assays for Quantitative Detection of Xanthomonas citri Subsp. citri

    PubMed Central

    Yin, Youping; Wang, Zhongkang

    2016-01-01

    Droplet digital polymerase chain reaction (ddPCR) is a novel molecular biology technique providing absolute quantification of target nucleic acids without the need for an external calibrator. Despite its emerging applications in medical diagnosis, there are few reports of its use for the detection of plant pathogens. This work was designed to assess the diagnosis potential of the ddPCR for absolute quantitative detection of Xanthomonas citri subsp. citri, a quarantine plant pathogenic bacterium that causes citrus bacterial canker in susceptible Citrus species. We transferred an established quantitative PCR (qPCR) assay for citrus bacterial canker diagnosis directly to the ddPCR format and compared the performance of the two methods. The qPCR assay has a broader dynamic range compared to the ddPCR assay and the ddPCR assay has a significantly higher degree of sensitivity compared to the qPCR assay. The influence of PCR inhibitors can be reduced considerably in the ddPCR assay because the collection of end-point fluorescent signals and the counting of binomial events (positive or negative droplets) are associated with a Poisson algorithm. The ddPCR assay also shows lower coefficient of variation compared to the qPCR assay especially in low target concentration. The linear association of the measurements by ddPCR and qPCR assays is strong (Pearson correlation = 0.8633; P<0.001). Receiver operating characteristic analysis indicates the ddPCR methodology is a more robust approach for diagnosis of citrus bacterial canker. In summary, the results demonstrated that the ddPCR assay has the potential for the quantitative detection of X. citri subsp. citri with high precision and accuracy as compared with the results from qPCR assay. Further studies are required to evaluate and validate the value of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

  11. A monoclonal antibody-based VZV glycoprotein E quantitative assay and its application on antigen quantitation in VZV vaccine.

    PubMed

    Liu, Jian; Zhu, Rui; Ye, Xiangzhong; Yang, Lianwei; Wang, Yongmei; Huang, Yanying; Wu, Jun; Wang, Wei; Ye, Jianghui; Li, Yimin; Zhao, Qinjian; Zhu, Hua; Cheng, Tong; Xia, Ningshao

    2015-06-01

    Varicella-zoster virus (VZV) is a highly infectious agent that causes varicella and herpes zoster (HZ), which may be associated with severe neuralgia. Vaccination is the most effective way to reduce the burden of the diseases. VZV glycoprotein E (gE) is the major and most immunogenic membrane protein that plays important roles in vaccine efficacy. A quantitative assay for gE content is desirable for the VZV vaccine process monitoring and product analysis. In this study, 70 monoclonal antibodies (mAbs) were obtained after immunizing mice with purified recombinant gE (rgE). The collection of mAbs was well-characterized, and a pair of high-affinity neutralization antibodies (capture mAb 4A2 and detection mAb 4H10) was selected to establish a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) to quantify the native and recombinant gE. The detection limit of this assay was found to be 1.95 ng/mL. Furthermore, a reasonably good correlation between the gE content (as measured by the mAb-based quantitative ELISA) and the virus titer (as measured by the "gold standard" plaque assay) was observed when both assays were performed for tracking the kinetics of virus growth during cell culture. A total of 16 batches of lyophilized VZV vaccine were tested using the newly developed quantitative ELISA and classical plaque assay, demonstrating reasonably good correlation between gE content and virus titer. Therefore, this mAb-based gE quantitative assay serves as a rapid, stable, and sensitive method for monitoring viral antigen content, one additional quantitative method for VZV vaccine process and product characterization. This quantitative ELISA may also serve as a complementary method for virus titering.

  12. Colorimetric assays for quantitative analysis and screening of epoxide hydrolase activity.

    PubMed

    Cedrone, F; Bhatnagar, T; Baratti, Jacques C

    2005-12-01

    Focusing on directed evolution to tailor enzymes as usable biocatalysts for fine chemistry, we have studied in detail several colorimetric assays for quantitative analysis of epoxide hydrolase (EH) activity. In particular, two assays have been optimized to characterize variants issued from the directed evolution of the EH from Aspergillus niger. Assays described in this paper are sufficiently reliable for quantitative screening of EH activity in microtiter plates and are low cost alternatives to GC or MS analysis. Moreover, they are usable for various epoxides and not restricted to a type of substrate, such as those amenable to assay by UV absorbancy. They can be used to assay EH activity on any epoxide and to directly assay enantioselectivity when both (R) and (S) substrates are available. The advantages and drawbacks of these two methods to assay EH activity of a large number of natural samples are summarized.

  13. A quantitative technical quality assay method for electrocardiograms.

    PubMed

    Sheffield, L T; Roitman, D I; Kansal, S

    1978-07-01

    A method is presented for quantitative assessment of ECG technical quality to permit sensitive detection of performance trends of individual technicians or entire ECG laboratories. The common flaws in ECG recording technic are those which introduce ultra-low frequency artifact or baseline shift, powerline artifact, myographic and intermediate frequency artifact, clipping of waveforms, disconnection or reversal of electrodes and general faults of protocol adherence. Procedures for quantitating these attributes are presented and explained in order to arrive at a mean figure of merit for each ECG recording. Use of such a method can aid ECG technicians' understanding of the important features of tracing quality and provide an objective basis for the recognition of outstanding performance, on one hand, or the need for remedial training, on the other.

  14. Improved assay for quantitating adherence of ruminal bacteria to cellulose.

    PubMed Central

    Rasmussen, M A; White, B A; Hespell, R B

    1989-01-01

    A quantitative technique suitable for the determination of adherence of ruminal bacteria to cellulose was developed. This technique employs adherence of cells to cellulose disks and alleviates the problem of nonspecific cell entrapment within cellulose particles. By using this technique, it was demonstrated that the adherence of Ruminococcus flavefaciens FD1 to cellulose was inhibited by formaldehyde, methylcellulose, and carboxymethyl cellulose. Adherence was unaffected by acid hydrolysates of methylcellulose, glucose, and cellobiose. PMID:2782879

  15. A novel quantitative immunomagnetic reduction assay for Nervous necrosis virus.

    PubMed

    Yang, Shieh Yueh; Wu, Jen Leih; Tso, Chun Hsi; Ngou, Fang Huar; Chou, Hsin Yiu; Nan, Fan Hua; Horng, Herng Er; Lu, Ming Wei

    2012-09-01

    Rapid, sensitive, and automatic detection platforms are among the major approaches of controlling viral diseases in aquaculture. An efficient detection platform permits the monitoring of pathogen spread and helps to enhance the economic benefits of commercial aquaculture. Nervous necrosis virus (NNV), the cause of viral encephalopathy and retinopathy, is among the most devastating aquaculture viruses that infect marine fish species worldwide. In the present study, a highly sensitive magnetoreduction assay was developed for detecting target biomolecules with a primary focus on NNV antigens. A standard curve of the different NNV concentrations that were isolated from infected Malabar grouper (Epinephelus malabaricus) was established before experiments were conducted. The test solution was prepared by homogeneous dispersion of magnetic nanoparticles coated with rabbit anti-NNV antibody. The magnetic nanoparticles in the solution were oscillated by magnetic interaction with multiple externally applied, alternating current magnetic fields. The assay's limit of detection was approximately 2 × 10(1) TCID(50)/ml for NNV. Moreover, the immunomagnetic reduction readings for other aquatic viruses (i.e., 1 × 10(7) TCID(50)/ml for Infectious pancreatic necrosis virus and 1 × 10(6.5) TCID(50)/ml for grouper iridovirus) were below the background noise in the NNV solution, demonstrating the specificity of the new detection platform.

  16. Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

    EPA Science Inventory

    Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

  17. Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

    EPA Science Inventory

    Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

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

    EPA Science Inventory

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

  19. Smartphone based visual and quantitative assays on upconversional paper sensor.

    PubMed

    Mei, Qingsong; Jing, Huarong; Li, You; Yisibashaer, Wuerzha; Chen, Jian; Nan Li, Bing; Zhang, Yong

    2016-01-15

    The integration of smartphone with paper sensors recently has been gain increasing attentions because of the achievement of quantitative and rapid analysis. However, smartphone based upconversional paper sensors have been restricted by the lack of effective methods to acquire luminescence signals on test paper. Herein, by the virtue of 3D printing technology, we exploited an auxiliary reusable device, which orderly assembled a 980nm mini-laser, optical filter and mini-cavity together, for digitally imaging the luminescence variations on test paper and quantitative analyzing pesticide thiram by smartphone. In detail, copper ions decorated NaYF4:Yb/Tm upconversion nanoparticles were fixed onto filter paper to form test paper, and the blue luminescence on it would be quenched after additions of thiram through luminescence resonance energy transfer mechanism. These variations could be monitored by the smartphone camera, and then the blue channel intensities of obtained colored images were calculated to quantify amounts of thiram through a self-written Android program installed on the smartphone, offering a reliable and accurate detection limit of 0.1μM for the system. This work provides an initial demonstration of integrating upconversion nanosensors with smartphone digital imaging for point-of-care analysis on a paper-based platform.

  20. Evaluation of a quantitative plasma PCR plate assay for detecting cytomegalovirus infection in marrow transplant recipients.

    PubMed Central

    Gallez-Hawkins, G M; Tegtmeier, B R; ter Veer, A; Niland, J C; Forman, S J; Zaia, J A

    1997-01-01

    A plasma PCR test, using a nonradioactive PCR plate assay, was evaluated for detection of human cytomegalovirus reactivation. This assay was compared to Southern blotting and found to perform well. As a noncompetitive method of quantitation, it was similar to a competitive method for detecting the number of genome copies per milliliter of plasma in marrow transplant recipients. This is a technically simplified assay with potential for adaptation to automation. PMID:9041438

  1. Qualitative and quantitative assays for flagellum-mediated chemotaxis.

    PubMed

    Darias, José Antonio Reyes; García-Fontana, Cristina; Lugo, Andrés Corral; Rico-Jiménez, Miriam; Krell, Tino

    2014-01-01

    A primary driving force during bacterial evolution was the capacity to access compounds necessary for growth and survival. Since the species of the genus Pseudomonas are characterized by metabolic versatility, these bacteria have developed chemotactic behaviors towards a wide range of different compounds. The specificity of a chemotactic response is determined by the chemoreceptor, which is at the beginning of the signaling cascade and to which chemoattractants and chemorepellents bind. The number of chemoreceptor genes of Pseudomonas species is significantly higher than the average number in motile bacteria. Although some of the receptors have been annotated with a function, the cognate signal molecules for the majority of them still need to be identified. Different qualitative and quantitative methods are presented that can be used to study flagellum-mediated taxis.

  2. Rapid method for protein quantitation by Bradford assay after elimination of the interference of polysorbate 80.

    PubMed

    Cheng, Yongfeng; Wei, Haiming; Sun, Rui; Tian, Zhigang; Zheng, Xiaodong

    2016-02-01

    Bradford assay is one of the most common methods for measuring protein concentrations. However, some pharmaceutical excipients, such as detergents, interfere with Bradford assay even at low concentrations. Protein precipitation can be used to overcome sample incompatibility with protein quantitation. But the rate of protein recovery caused by acetone precipitation is only about 70%. In this study, we found that sucrose not only could increase the rate of protein recovery after 1 h acetone precipitation, but also did not interfere with Bradford assay. So we developed a method for rapid protein quantitation in protein drugs even if they contained interfering substances. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Highly Rapid Amplification-Free and Quantitative DNA Imaging Assay

    PubMed Central

    Klamp, Tobias; Camps, Marta; Nieto, Benjamin; Guasch, Francesc; Ranasinghe, Rohan T.; Wiedemann, Jens; Petrášek, Zdeněk; Schwille, Petra; Klenerman, David; Sauer, Markus

    2013-01-01

    There is an urgent need for rapid and highly sensitive detection of pathogen-derived DNA in a point-of-care (POC) device for diagnostics in hospitals and clinics. This device needs to work in a ‘sample-in-result-out’ mode with minimum number of steps so that it can be completely integrated into a cheap and simple instrument. We have developed a method that directly detects unamplified DNA, and demonstrate its sensitivity on realistically sized 5 kbp target DNA fragments of Micrococcus luteus in small sample volumes of 20 μL. The assay consists of capturing and accumulating of target DNA on magnetic beads with specific capture oligonucleotides, hybridization of complementary fluorescently labeled detection oligonucleotides, and fluorescence imaging on a miniaturized wide-field fluorescence microscope. Our simple method delivers results in less than 20 minutes with a limit of detection (LOD) of ~5 pM and a linear detection range spanning three orders of magnitude. PMID:23677392

  4. Quantitative data analysis methods for bead-based DNA hybridization assays using generic flow cytometry platforms.

    PubMed

    Corrie, S R; Lawrie, G A; Battersby, B J; Ford, K; Rühmann, A; Koehler, K; Sabath, D E; Trau, M

    2008-05-01

    Bead-based assays are in demand for rapid genomic and proteomic assays for both research and clinical purposes. Standard quantitative procedures addressing raw data quality and analysis are required to ensure the data are consistent and reproducible across laboratories independent of flow platform. Quantitative procedures have been introduced spanning raw histogram analysis through to absolute target quantitation. These included models developed to estimate the absolute number of sample molecules bound per bead (Langmuir isotherm), relative quantitative comparisons (two-sided t-tests), and statistical analyses investigating the quality of raw fluorescence data. The absolute target quantitation method revealed a concentration range (below probe saturation) of Cy5-labeled synthetic cytokeratin 19 (K19) RNA of c.a. 1 x 10(4) to 500 x 10(4) molecules/bead, with a binding constant of c.a. 1.6 nM. Raw hybridization frequency histograms were observed to be highly reproducible across 10 triplex assay replicates and only three assay replicates were required to distinguish overlapping peaks representing small sequence mismatches. This study provides a quantitative scheme for determining the absolute target concentration in nucleic acid hybridization reactions and the equilibrium binding constants for individual probe/target pairs. It is envisaged that such studies will form the basis of standard analytical procedures for bead-based cytometry assays to ensure reproducibility in inter- and intra-platform comparisons of data between laboratories. (c) 2008 International Society for Advancement of Cytometry.

  5. A novel multi-walled carbon nanotube-based antibody conjugate for quantitative and semi-quantitative lateral flow assays.

    PubMed

    Sun, Wenjuan; Hu, Xiaolong; Liu, Jia; Zhang, Yurong; Lu, Jianzhong; Zeng, Libo

    2017-10-01

    In this study, the multi-walled carbon nanotubes (MWCNTs) were applied in lateral flow strips (LFS) for semi-quantitative and quantitative assays. Firstly, the solubility of MWCNTs was improved using various surfactants to enhance their biocompatibility for practical application. The dispersed MWCNTs were conjugated with the methamphetamine (MET) antibody in a non-covalent manner and then manufactured into the LFS for the quantitative detection of MET. The MWCNTs-based lateral flow assay (MWCNTs-LFA) exhibited an excellent linear relationship between the values of test line and MET when its concentration ranges from 62.5 to 1500 ng/mL. The sensitivity of the LFS was evaluated by conjugating MWCNTs with HCG antibody and the MWCNTs conjugated method is 10 times more sensitive than the one conjugated with classical colloidal gold nanoparticles. Taken together, our data demonstrate that MWCNTs-LFA is a more sensitive and reliable assay for semi-quantitative and quantitative detection which can be used in forensic analysis.

  6. Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models.

    PubMed

    Ku, Hyung-Keun; Lim, Hyuk-Min; Oh, Kyong-Hwa; Yang, Hyo-Jin; Jeong, Ji-Seon; Kim, Sook-Kyung

    2013-03-01

    The Bradford assay is a simple method for protein quantitation, but variation in the results between proteins is a matter of concern. In this study, we compared and normalized quantitative values from two models for protein quantitation, where the residues in the protein that bind to anionic Coomassie Brilliant Blue G-250 comprise either Arg and Lys (Method 1, M1) or Arg, Lys, and His (Method 2, M2). Use of the M2 model yielded much more consistent quantitation values compared with use of the M1 model, which exhibited marked overestimations against protein standards. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Analytical and clinical performance of a new molecular assay for Epstein-Barr virus DNA quantitation.

    PubMed

    Hübner, Margit; Bozic, Michael; Konrad, Petra M; Grohs, Katharina; Santner, Brigitte I; Kessler, Harald H

    2015-02-01

    Quantitation of EBV DNA has been shown to be a useful tool to identify and monitor patients with immunosuppression and high risk for EBV-associated disease. In this study, the analytical and clinical performance of the new Realquality RS-EBV Kit (AB Analitica, Padova, Italy) was investigated. The clinical performance was compared to that of the EBV R-gene (bioMerieux, Varilhes, France) assay. When the accuracy of the new assay was tested, all results except of one were found to be within ±0.5log10 unit of the expected panel results. Determination of linearity showed a quasilinear curve, the between day imprecision ranged from 18% to 88% and the within run imprecision from 16% to 53%. When 96 clinical EDTA whole blood samples were tested, 77 concordant and 19 discordant results were obtained. When the results for the 69 samples quantifiable with both assays were compared, the new assay revealed a mean 0.31log10 unit higher measurement. The new assay proved to be suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory. The variation between quantitative results obtained by the assays used in this study reinforces the use of calibrators traceable to the existing international WHO standard making different assays better comparable.

  8. Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

    PubMed Central

    Farris, M. Heath; Ford, Kara A.; Doyle, Richard C.

    2016-01-01

    Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest. PMID:27166738

  9. Development of a quantitative fluorescence-based ligand-binding assay

    PubMed Central

    Breen, Conor J.; Raverdeau, Mathilde; Voorheis, H. Paul

    2016-01-01

    A major goal of biology is to develop a quantitative ligand-binding assay that does not involve the use of radioactivity. Existing fluorescence-based assays have a serious drawback due to fluorescence quenching that accompanies the binding of fluorescently-labeled ligands to their receptors. This limitation of existing fluorescence-based assays prevents the number of cellular receptors under investigation from being accurately measured. We have developed a method where FITC-labeled proteins bound to a cell surface are proteolyzed extensively to eliminate fluorescence quenching and then the fluorescence of the resulting sample is compared to that of a known concentration of the proteolyzed FITC-protein employed. This step enables the number of cellular receptors to be measured quantitatively. We expect that this method will provide researchers with a viable alternative to the use of radioactivity in ligand binding assays. PMID:27161290

  10. Evaluation of various real-time reverse transcription quantitative PCR assays for norovirus detection.

    PubMed

    Yoo, Ju Eun; Lee, Cheonghoon; Park, SungJun; Ko, GwangPyo

    2017-02-01

    Human noroviruses are widespread and contagious viruses causing nonbacterial gastroenteritis. Real-time reverse transcription quantitative PCR (real-time RT-qPCR) is currently the gold standard for sensitive and accurate detection for these pathogens and serves as a critical tool in outbreak prevention and control. Different surveillance teams, however, may use different assays and variability in specimen conditions may lead to disagreement in results. Furthermore, the norovirus genome is highly variable and continuously evolving. These issues necessitate the re-examination of the real-time RT-qPCR's robustness in the context of accurate detection as well as the investigation of practical strategies to enhance assay performance. Four widely referenced real-time RT-qPCR assays (Assay A-D) were simultaneously performed to evaluate characteristics such as PCR efficiency, detection limit, as well as sensitivity and specificity with RT-PCR, and to assess the most accurate method for detecting norovirus genogroups I and II. Overall, Assay D was evaluated to be the most precise and accurate assay in this study. A Zen internal quencher, which decreases nonspecific fluorescence during the PCR reaction, was added to Assay D's probe which further improved assay performance. This study compared several detection assays for noroviruses and an improvement strategy based on such comparisons provided useful characterizations of a highly optimized real-time RT-qPCR assay for norovirus detection.

  11. Quantitative assay of glycocalyx produced by viridans group streptococci that cause endocarditis.

    PubMed Central

    Dall, L; Herndon, B

    1989-01-01

    A quantitative method to determine glycocalyx production by strains of viridans group streptococci from patients with endocarditis is presented. There is good correlation between this new tryptophan quantitative assay and qualitative assays employing polysaccharide stains (ruthenium red, periodic acid-Schiff, and Cellufluor) or the Molisch test. The quantification of the glycocalyx production in glucose substrate in vitro by viridans group streptococci correlates with the size of cardiac vegetation and ease of antimicrobial sterilization in experimental endocarditis. The relationship of in vitro quantification of glycocalyx to maintenance of infection, morbidity of infection, and antimicrobial treatment is discussed. PMID:2476465

  12. Quantitative assay of glycocalyx produced by viridans group streptococci that cause endocarditis.

    PubMed

    Dall, L; Herndon, B

    1989-09-01

    A quantitative method to determine glycocalyx production by strains of viridans group streptococci from patients with endocarditis is presented. There is good correlation between this new tryptophan quantitative assay and qualitative assays employing polysaccharide stains (ruthenium red, periodic acid-Schiff, and Cellufluor) or the Molisch test. The quantification of the glycocalyx production in glucose substrate in vitro by viridans group streptococci correlates with the size of cardiac vegetation and ease of antimicrobial sterilization in experimental endocarditis. The relationship of in vitro quantification of glycocalyx to maintenance of infection, morbidity of infection, and antimicrobial treatment is discussed.

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  15. Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays.

    PubMed

    Jung, Se-Hui; Kong, Deok-Hoon; Park, Seoung-Woo; Kim, Young-Myeong; Ha, Kwon-Soo

    2012-08-21

    Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

  16. Improved TLC Bioautographic Assay for Qualitative and Quantitative Estimation of Tyrosinase Inhibitors in Natural Products.

    PubMed

    Zhou, Jinge; Tang, Qingjiu; Wu, Tao; Cheng, Zhihong

    2017-03-01

    TLC bioautography for tyrosinase inhibitors has made recent progress; however, an assay with a relative low consumption of enzyme and quantitative capability would greatly advance the efficacy of related TLC bioautographic assays. An improved TLC bioautographic assay for detecting tyrosinase inhibitors was developed and validated in this study. L-DOPA (better water-solubility than L-tyrosine) was used as the substrate instead of reported L-tyrosine. The effects of enzyme and substrate concentrations, reaction temperatures and times, and pH values of the reaction system as well as different plate types on the TLC bioautographic assay were optimised. The quantitative analysis was conducted by densitometric scanning of spot areas, and expressed as the relative tyrosinase inhibitory capacity (RTIC) using a positive control (kojic acid) equivalent. The limit of detection (LOD) of this assay was 1.0 ng for kojic acid. This assay has acceptable accuracy (101.73-102.90%), intra- and inter-day, and intra- and inter-plate precisions [relative standard deviation (RSD), less than 7.0%], and ruggedness (RSD, less than 3.5%). The consumption of enzyme (75 U/mL) is relatively low. Two tyrosinase inhibitory compounds including naringenin and 1-O-β-D-glucopyranosyl-4-allylbenzene have been isolated from Rhodiola sacra guided by this TLC bioautographic assay. Our improved assay is a relatively low-cost, sensitive, and quantitative method compared to the reported TLC bioautographic assays. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  17. Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay[S

    PubMed Central

    Pais de Barros, Jean-Paul; Gautier, Thomas; Sali, Wahib; Adrie, Christophe; Choubley, Hélène; Charron, Emilie; Lalande, Caroline; Le Guern, Naig; Deckert, Valérie; Monchi, Mehran; Quenot, Jean-Pierre; Lagrost, Laurent

    2015-01-01

    Quantitation of plasma lipopolysaccharides (LPSs) might be used to document Gram-negative bacterial infection. In the present work, LPS-derived 3-hydroxymyristate was extracted from plasma samples with an organic solvent, separated by reversed phase HPLC, and quantitated by MS/MS. This mass assay was combined with the limulus amebocyte lysate (LAL) bioassay to monitor neutralization of LPS activity in biological samples. The described HPLC/MS/MS method is a reliable, practical, accurate, and sensitive tool to quantitate LPS. The combination of the LAL and HPLC/MS/MS analyses provided new evidence for the intrinsic capacity of plasma lipoproteins and phospholipid transfer protein to neutralize the activity of LPS. In a subset of patients with systemic inflammatory response syndrome, with documented infection but with a negative plasma LAL test, significant amounts of LPS were measured by the HPLC/MS/MS method. Patients with the highest plasma LPS concentration were more severely ill. HPLC/MS/MS is a relevant method to quantitate endotoxin in a sample, to assess the efficacy of LPS neutralization, and to evaluate the proinflammatory potential of LPS in vivo. PMID:26023073

  18. Quantitative, Fluorogenic Probe PCR Assay for Detection of Human Herpesvirus 8 DNA in Clinical Specimens

    PubMed Central

    Stamey, Felicia R.; Patel, Mitesh M.; Holloway, Brian P.; Pellett, Philip E.

    2001-01-01

    A quantitative, fluorescence-based PCR assay (TaqMan-based system) was developed for detection of human herpesvirus 8 (HHV-8) DNA in clinical specimens. Primers and probes chosen from each of five 10-kb segments from the unique region of the HHV-8 genome were evaluated for sensitivity with dilution series of DNA extracted from a cell line (BCBL-1) that harbors HHV-8 DNA. Although several of the primer-probe sets performed similarly with BCBL-1 DNA that had been diluted in water, their performance differed when target DNA was diluted in a constant background of uninfected cell DNA, an environment more relevant to their intended use. The two best primer-probe combinations were specific for HHV-8 relative to the other known human herpesviruses and herpesvirus saimiri, a closely related gammaherpesvirus of nonhuman primates. PCRs included an enzymatic digestion step to eliminate PCR carryover and an exogenous internal positive control that enabled discrimination of false-negative from true-negative reactions. The new assays were compared to conventional PCR assays for clinical specimens (saliva, rectal brushings, rectal swab specimens, peripheral blood lymphocytes, semen, and urine) from human immunodeficiency virus-positive patients with or without Kaposi's sarcoma. In all instances, the new assays agreed with each other and with the conventional PCR system. In addition, the quantitative results obtained with the new assays were in good agreement both for duplicate reactions in the same assay and between assays. PMID:11574569

  19. Quantitative, fluorogenic probe PCR assay for detection of human herpesvirus 8 DNA in clinical specimens.

    PubMed

    Stamey, F R; Patel, M M; Holloway, B P; Pellett, P E

    2001-10-01

    A quantitative, fluorescence-based PCR assay (TaqMan-based system) was developed for detection of human herpesvirus 8 (HHV-8) DNA in clinical specimens. Primers and probes chosen from each of five 10-kb segments from the unique region of the HHV-8 genome were evaluated for sensitivity with dilution series of DNA extracted from a cell line (BCBL-1) that harbors HHV-8 DNA. Although several of the primer-probe sets performed similarly with BCBL-1 DNA that had been diluted in water, their performance differed when target DNA was diluted in a constant background of uninfected cell DNA, an environment more relevant to their intended use. The two best primer-probe combinations were specific for HHV-8 relative to the other known human herpesviruses and herpesvirus saimiri, a closely related gammaherpesvirus of nonhuman primates. PCRs included an enzymatic digestion step to eliminate PCR carryover and an exogenous internal positive control that enabled discrimination of false-negative from true-negative reactions. The new assays were compared to conventional PCR assays for clinical specimens (saliva, rectal brushings, rectal swab specimens, peripheral blood lymphocytes, semen, and urine) from human immunodeficiency virus-positive patients with or without Kaposi's sarcoma. In all instances, the new assays agreed with each other and with the conventional PCR system. In addition, the quantitative results obtained with the new assays were in good agreement both for duplicate reactions in the same assay and between assays.

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

    PubMed

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

    2008-07-01

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

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

    ERIC Educational Resources Information Center

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

    2012-01-01

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

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

    ERIC Educational Resources Information Center

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

    2012-01-01

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

  3. A Multiplexed, Probe-Based Quantitative PCR Assay for DNA of Phytophthora sojae

    USDA-ARS?s Scientific Manuscript database

    Phytophthora sojae (Kaufm. & Gerd.) causes seed rot, pre- and post-emergence damping off, and sometimes foliar blight in soybean (Glycine max). Crop loss may approach 100% with susceptible cultivars. We report here the development of a unique quantitative PCR assay specific to DNA of P. sojae, and a...

  4. A sandwich assay for quantitative detection of transcription factors in cell lysate.

    PubMed

    Fang, Zhiyuan; Zhang, Wenjuan; Ge, Chenchen; Liu, Jie; Lie, Puchang; Zeng, Lingwen

    2012-09-21

    A double-stranded DNA (dsDNA) mediated sandwich assay was developed for quantitative detection of transcription factors. The detection limit for human recombinant c-jun protein is 2.5 ng, and for c-jun protein the limit is as low as 0.625 μg of cell lysate.

  5. Microplate assay for quantitation of neutral lipids in extracts from microalgae.

    PubMed

    Higgins, Brendan T; Thornton-Dunwoody, Alexander; Labavitch, John M; VanderGheynst, Jean S

    2014-11-15

    Lipid quantitation is widespread in the algae literature, but popular methods such as gravimetry, gas chromatography and mass spectrometry (GC-MS), and Nile red cell staining suffer drawbacks, including poor quantitation of neutral lipids, expensive equipment, and variable results among algae species, respectively. A high-throughput microplate assay was developed that uses Nile red dye to quantify neutral lipids that have been extracted from algae cells. Because the algal extracts contained pigments that quenched Nile red fluorescence, a mild bleach solution was used to destroy pigments, resulting in a nearly linear response for lipid quantities in the range of 0.75 to 40 μg. Corn oil was used as a standard for quantitation, although other vegetable oils displayed a similar response. The assay was tested on lipids extracted from three species of Chlorella and resulted in close agreement with triacylglycerol (TAG) levels determined by thin layer chromatography. The assay was found to more accurately measure algal lipids conducive to biodiesel production and nutrition applications than the widely used gravimetric assay. Assay response was also consistent among different species, in contrast to Nile red cell staining procedures.

  6. Radioimmunofocus assay for quantitation of hepatitis A virus in cell cultures.

    PubMed Central

    Lemon, S M; Binn, L N; Marchwicki, R H

    1983-01-01

    A new method is described for the quantitation of hepatitis A virus in cell cultures, based on the immune autoradiographic detection of foci of infected cells (radioimmunofoci) developing beneath an agarose overlay 14 days after the inoculation of petri dish cultures of continuous African green monkey kidney cells (BS-C-1). The number of foci developing in each culture was linearly related to the dose of hepatitis A virus (either HM-175 or PA-21 strain) inoculated. Focus development was prevented by prior incubation of virus with specific antisera, and the specificity of the radiolabeled antibody reaction was confirmed in competitive blocking experiments. This new assay method retains many of the advantages of conventional plaque assays for virus. Compared with existing end-dilution methods for the quantitation of hepatitis A virus, the radioimmunofocus assay offers greatly improved accuracy and comparable sensitivity, yet is relatively rapid and highly conservative of reagents. Images PMID:6306048

  7. Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay.

    PubMed

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-08-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87(N). Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

  8. Quantitative assay of diphtherial toxin and of immunologically cross-reacting proteins by reversed passive hemagglutination.

    PubMed Central

    Holmes, R K; Perlow, R B

    1975-01-01

    A reversed passive hemagglutination (RPHA) assay for diptherial toxin has been developed. Antitoxic antibodies were isolated from commercially available equine diptherial antitoxin by immunoabsorption using highly purified diphtherial toxin covalently linked to Sepharose 4B. Formalinized, tanned sheep erythrocytes sensitized with the purified antitoxic antibodies are specifically agglutinated by diphtherial toxin but are not agglutinated by extracellular antigens of Corynebacterium diptheriae that are unrelated to toxin. The RPHA assay described can detect less than 20 pg of diphtherial toxin and is comparable in sensitivity to intracutaneous tests for toxin. The RPHA assay was shown to be at least 1,000 times more sensitive than quantitative immunological assays for diptherial toxin performed by single radial immunodiffusion or by one-dimensional double diffusion in agar gels. Fragment A prepared from purified diphtherial toxin and nontoxic mutant proteins that cross-react immunologically with toxin can be assayed directly by RPHA, but the sensitivity of the assay for these proteins is less than for native diphtherial toxin. Inhibition of RPHA was also shown to be a sensitive quantitative method for measuring diptherial antitoxin in vitro. Images PMID:54339

  9. Analysis of JC virus DNA replication using a quantitative and high-throughput assay.

    PubMed

    Shin, Jong; Phelan, Paul J; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A

    2014-11-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication.

  10. A fluorescence-based quantitative real-time PCR assay for accurate Pocillopora damicornis species identification

    NASA Astrophysics Data System (ADS)

    Thomas, Luke; Stat, Michael; Evans, Richard D.; Kennington, W. Jason

    2016-09-01

    Pocillopora damicornis is one of the most extensively studied coral species globally, but high levels of phenotypic plasticity within the genus make species identification based on morphology alone unreliable. As a result, there is a compelling need to develop cheap and time-effective molecular techniques capable of accurately distinguishing P. damicornis from other congeneric species. Here, we develop a fluorescence-based quantitative real-time PCR (qPCR) assay to genotype a single nucleotide polymorphism that accurately distinguishes P. damicornis from other morphologically similar Pocillopora species. We trial the assay across colonies representing multiple Pocillopora species and then apply the assay to screen samples of Pocillopora spp. collected at regional scales along the coastline of Western Australia. This assay offers a cheap and time-effective alternative to Sanger sequencing and has broad applications including studies on gene flow, dispersal, recruitment and physiological thresholds of P. damicornis.

  11. Quantitative methylene blue decolourisation assays as rapid screening tools for assessing the efficiency of catalytic reactions.

    PubMed

    Kruid, Jan; Fogel, Ronen; Limson, Janice Leigh

    2017-05-01

    Identifying the most efficient oxidation process to achieve maximum removal of a target pollutant compound forms the subject of much research. There exists a need to develop rapid screening tools to support research in this area. In this work we report on the development of a quantitative assay as a means for identifying catalysts capable of decolourising methylene blue through the generation of oxidising species from hydrogen peroxide. Here, a previously described methylene blue test strip method was repurposed as a quantitative, aqueous-based spectrophotometric assay. From amongst a selection of metal salts and metallophthalocyanine complexes, monitoring of the decolourisation of the cationic dye methylene blue (via Fenton-like and non-Fenton oxidation reactions) by the assay identified the following to be suitable oxidation catalysts: CuSO4 (a Fenton-like catalyst), iron(II)phthalocyanine (a non-Fenton oxidation catalyst), as well as manganese(II) phthalocyanine. The applicability of the method was examined for the removal of bisphenol A (BPA), as measured by HPLC, during parallel oxidation experiments. The order of catalytic activity was identified as FePc > MnPc > CuSO4 for both BPA and MB. The quantitative MB decolourisation assay may offer a rapid method for screening a wide range of potential catalysts for oxidation processes.

  12. Enzyme-linked immunosorbent assay for quantitation of attachment and ingestion stages of bacterial phagocytosis.

    PubMed Central

    Athamna, A; Ofek, I

    1988-01-01

    Research on phagocytosis of bacteria is often hampered by the inability to distinguish quantitatively between bacteria that have been ingested by phagocytic cells and those which are attached to the surface of the cells. A method using the enzyme-linked immunosorbent assay technique to simply and accurately measure the rate of bacterial ingestion by phagocytic cells is described. The method is based on the ability of antibacterial antibodies to bind to bacteria attached to but not internalized by phagocytic cells. The attached bacteria were quantitated by enzyme-linked immunosorbent assay. Compared with the number of bacteria at zero time (17 bacteria attached per phagocyte) only 10 to 20% of the bacteria remained attached to phagocytic cells after incubation for 30 min at 37 degrees C. The decrease in detected attached bacteria at 37 degrees C was due to internalization of the bacteria by phagocytic cells, since upon disruption of the monolayer, most of the ingested bacteria were recovered, and at 4 degrees C, most of the bacteria remained extracellularly attached. The proposed attachment and ingestion assay is easy to perform, allows the detection of specific attachment of test bacteria, and provides objective quantitation of attached and ingested bacteria. Most importantly, the assay allows testing of ingestion rates of bacteria under many variables on the same day. PMID:2893805

  13. A new TLC bioautographic assay for qualitative and quantitative estimation of lipase inhibitors.

    PubMed

    Tang, Jihe; Zhou, Jinge; Tang, Qingjiu; Wu, Tao; Cheng, Zhihong

    2016-01-01

    Lipase inhibitory assays based on TLC bioautography have made recent progress; however, an assay with greater substrate specificity and quantitative capabilities would advance the efficacy of this particular bioassay. To address these limitations, a new TLC bioautographic assay for detecting lipase inhibitors was developed and validated in this study. The new TLC bioautographic assay was based on reaction of lipase with β-naphthyl myristate and the subsequent formation of the purple dye between β-naphthol and Fast Blue B salt (FBB). The relative lipase inhibitory capacity (RLIC) was determined by a TLC densitometry with fluorescence detection, expressed as orlistat equivalents in millimoles on a per sample weight basis. Six pure compounds and three natural extracts were evaluated for their potential lipase inhibitory activities by this TLC bioautographic assay. The β-naphthyl myristate as the substrate improved the detection sensitivity and specificity significantly. The limit of detection (LOD) of this assay was 0.01 ng for orlistat, the current treatment for obesity. This assay has acceptable accuracy (92.07-105.39%), intra-day and inter-day precisions [relative standard deviation (RSD), 2.64-4.40%], as well as intra-plate and inter-plate precisions (RSD, 1.8-4.9%). The developed method is rapid, simple, stable, and specific for screening and estimation of the potential lipase inhibitors. Copyright © 2015 John Wiley & Sons, Ltd.

  14. A quantitative infection assay for human type I, II, and III interferon antiviral activities

    PubMed Central

    2013-01-01

    Background Upon virus infection, cells secrete a diverse group of antiviral molecules that signal proximal cells to enter into an antiviral state, slowing or preventing viral spread. These paracrine signaling molecules can work synergistically, so measurement of any one antiviral molecule does not reflect the total antiviral activity of the system. Results We have developed an antiviral assay based on replication inhibition of an engineered fluorescent vesicular stomatitis virus reporter strain on A549 human lung epithelial cells. Our assay provides a quantitative functional readout of human type I, II, and III interferon activities, and it provides better sensitivity, intra-, and inter-assay reproducibility than the traditional crystal violet based assay. Further, it eliminates cell fixation, rinsing, and staining steps, and is inexpensive to implement. Conclusions A dsRed2-strain of vesicular stomatitis virus that is sensitive to type I, II, and III interferons was used to develop a convenient and sensitive assay for interferon antiviral activity. We demonstrate use of the assay to quantify the kinetics of paracrine antiviral signaling from human prostate cancer (PC3) cells in response to viral infection. The assay is applicable to high-throughput screening for anti-viral compounds as well as basic studies of cellular antiviral signaling. PMID:23829314

  15. Lactate as a Novel Quantitative Measure of Viability in Schistosoma mansoni Drug Sensitivity Assays

    PubMed Central

    Howe, Stephanie; Zöphel, Dorina; Subbaraman, Harini; Unger, Clemens; Held, Jana; Engleitner, Thomas; Hoffmann, Wolfgang H.

    2014-01-01

    Whole-organism compound sensitivity assays are a valuable strategy in infectious diseases to identify active molecules. In schistosomiasis drug discovery, larval-stage Schistosoma allows the use of a certain degree of automation in the screening of compounds. Unfortunately, the throughput is limited, as drug activity is determined by manual assessment of Schistosoma viability by microscopy. To develop a simple and quantifiable surrogate marker for viability, we targeted glucose metabolism, which is central to Schistosoma survival. Lactate is the end product of glycolysis in human Schistosoma stages and can be detected in the supernatant. We assessed lactate as a surrogate marker for viability in Schistosoma drug screening assays. We thoroughly investigated parameters of lactate measurement and performed drug sensitivity assays by applying schistosomula and adult worms to establish a proof of concept. Lactate levels clearly reflected the viability of schistosomula and correlated with schistosomulum numbers. Compounds with reported potencies were tested, and activities were determined by lactate assay and by microscopy. We conclude that lactate is a sensitive and simple surrogate marker to be measured to determine Schistosoma viability in compound screening assays. Low numbers of schistosomula and the commercial availability of lactate assay reagents make the assay particularly attractive to throughput approaches. Furthermore, standardization of procedures and quantitative evaluation of compound activities facilitate interassay comparisons of potencies and, thus, concerted drug discovery approaches. PMID:25487803

  16. A quantitative in vitro assay for the evaluation of phototoxic potential of topically applied materials.

    PubMed

    Tenenbaum, S; DiNardo, J; Morris, W E; Wolf, B A; Schnetzinger, R W

    1984-10-01

    A quantitative in vitro method for phototoxic evaluation of chemicals has been developed and validated. The assay uses Saccharomyces cerevisiae, seeded in an agar overlay on top of a plate count agar base. 8-Methoxy psoralen is used as a reference standard against which materials are measured. Activity is quantified by cytotoxicity measured as zones of inhibition. Several known phototoxins (heliotropine, lyral, phantolid, and bergamot oil) and photoallergens (6-methyl coumarin and musk ambrette) are used to validate the assay. An excellent correlation is observed between in vivo studies employing Hartley albino guinea pigs and the in vitro assay for several fragrance raw materials and other chemicals. The in vitro assay exhibits a greater sensitivity from 2-500 fold. For three fragrance oils, the in vitro assay detects low levels of photobiological activity while the in vivo assay is negative. Although the in vitro assay does not discriminate between phototoxins and photoallergens, it can be used for screening of raw materials so that reduction in animal usage can be achieved while maintaining the protection of the consumer.

  17. Quantitative analysis of cell proliferation by a dye dilution assay: Application to cell lines and cocultures.

    PubMed

    Chung, Soobin; Kim, Seol-Hee; Seo, Yuri; Kim, Sook-Kyung; Lee, Ji Youn

    2017-04-04

    Cell proliferation represents one of the most fundamental processes in biological systems, thus the quantitative analysis of cell proliferation is important in many biological applications such as drug screening, production of biologics, and assessment of cytotoxicity. Conventional proliferation assays mainly quantify cell number based on a calibration curve of a homogeneous cell population, and therefore are not applicable for the analysis of cocultured cells. Moreover, these assays measure cell proliferation indirectly, based on cellular metabolic activity or DNA content. To overcome these shortcomings, a dye dilution assay employing fluorescent cell tracking dyes that are retained within cells was applied and was diluted proportionally by subsequent cell divisions. Here, it was demonstrated that this assay could be implemented to quantitatively analyze the cell proliferation of different types of cell lines, and to concurrently analyze the proliferation of two types of cell lines in coculture by utilizing cell tracking dyes with different spectral characteristics. The mean division time estimated by the dye dilution assay is compared with the population doubling time obtained from conventional methods and values from literature. Additionally, dye transfer between cocultured cells was investigated and it was found that it is a characteristic of the cells rather than a characteristic of the dye. It was suggested that this method can be easily combined with other flow cytometric analyses of cellular properties, providing valuable information on cell status under diverse conditions. © 2017 International Society for Advancement of Cytometry.

  18. Development of Multiplexed Real-Time Quantitative PCR Assay for Detecting Human Adenoviruses

    PubMed Central

    Huang, Meei-Li; Nguy, Long; Ferrenberg, James; Boeckh, Michael; Cent, Anne; Corey, Lawrence

    2008-01-01

    Adenoviruses (AdV) have been associated with a wide variety of human disease and are increasingly recognized as viral pathogens that can cause significant morbidity and mortality in immunocompromised patients. Early detection of AdV DNA in plasma and sterile fluids has been shown to be useful for identifying patients at risk for invasive AdV disease. Due to the large number of existing Adv types, few real-time quantitative AdV PCR assays published effectively cover all AdV types. We designed a series of AdV PCR primers and probes and empirically multiplexed them into two separate real-time PCR assays to quantitatively detect all 49 serotypes of human AdV (Types 1-49) available from ATCC. We then subsequently multiplexed all the primers and probes into one reaction. The sensitivity of these assays was determined to be less than 10 copies per reaction (500 copies/ml plasma). In a retrospective evaluation we detected all 84 clinical AdV isolates isolated in cell culture from patients undergoing hematopoietic stem cell transplant (HSCT) between 1981 and 1987. Prospective analysis of 46 consecutive clinical samples submitted for adenovirus testing showed greater sensitivity and equal specificity of the AdV PCR than viral culture. This real time PCR assay allows rapid, sensitive and specific quantification of all currently defined adenoviruses into either two or one multiplex assay for clinical samples. PMID:18707838

  19. Quantitative Molecular Assay for Fingerprinting Microbial Communities of Wastewater and Estrogen-Degrading Consortia

    PubMed Central

    Yu, Chang-Ping; Ahuja, Rajiv; Sayler, Gary; Chu, Kung-Hui

    2005-01-01

    A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, an Escherichia coli strain (gram negative), a Pseudomonas fluorescens strain (gram negative), and a Bacillus thuringiensis strain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17α-estradiol, 17β-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats. PMID:15746346

  20. Quantitative fluorometric assay for the measurement of endo-1,4-β-glucanase.

    PubMed

    Mangan, D; McCleary, B V; Liadova, A; Ivory, R; McCormack, N

    2014-08-18

    There is a growing demand for research tools to aid the scientific community in the search for improved cellulase enzymes for the biofuel industry. In this work, we describe a novel fluorometric assay for cellulase (endo-1,4-β-glucanase) which is based on the use of 4,6-O-benzylidene-4-methylumbelliferyl-β-cellotrioside (BzMUG3) in the presence of an ancillary β-glucosidase. This assay can be used quantitatively over a reasonable linear range, or qualitatively as a solution screening tool which may find extensive use in the area of metagenomics.

  1. Western blot assay for quantitative and qualitative antigen detection in vaccine development.

    PubMed

    Kumar, Sanjai; Zheng, Hong; Mahajan, Babita; Kozakai, Yukiko; Morin, Merribeth; Locke, Emily

    2014-05-01

    Immunological methods for quantitative measurement, antigenic characterization, and monitoring the stability of active immunogenic component(s) are a critical need in the vaccine development process. This unit describes an enhanced chemiluminescence-based western blot for quantitative detection of Plasmodium falciparum circumsporozoite protein (PfCSP), a major malaria candidate vaccine antigen. The most salient features of this assay are its high sensitivity and reproducibility; it can reliably detect ∼5 to 10 pg PfCSP expressed on native parasites or recombinantly expressed in Escherichia coli. Although described for a specific vaccine antigen, this assay should be applicable for any antigen-antibody combination for which relevant detection reagents are available. Detailed stepwise experimental procedures and methods for data acquisition and analysis are described. Copyright © 2014 John Wiley & Sons, Inc.

  2. Quantitative assay of photoinduced DNA strand breaks by real-time PCR.

    PubMed

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-05

    Real-time PCR (qPCR) - a modern methodology primarily used for studying gene expression has been employed for the quantitative assay of an important class of DNA damage - single strand breaks. These DNA lesions which may lead to highly cytotoxic double strand breaks were quantified in a model system where double stranded DNA was sensitized to UV photons by labeling with 5-bromo-2'-deoxyuridine. The amount of breaks formed due to irradiation with several doses of 320nm photons was assayed by two independent methods: LC-MS and qPCR. A very good agreement between the relative damage measured by the two completely different analytical tools proves the applicability of qPCR for the quantitative analysis of SSBs. Our results suggest that the popularity of the hitherto underestimated though accurate and site-specific technique of real-time PCR may increase in future DNA damage studies. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    SciTech Connect

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2014-11-15

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. - Highlights: • Development of a high-throughput screening assay for JCV DNA replication using C33A cells. • Evidence that T-ag fails to accumulate in the nuclei of established glioma cell lines. • Evidence that NF-1 directly promotes JCV DNA replication in C33A cells. • Proof-of-concept that the HTS assay can be used to identify pharmacological inhibitor of JCV DNA replication.

  4. A Data Analysis Pipeline Accounting for Artifacts in Tox21 Quantitative High-Throughput Screening Assays.

    PubMed

    Hsieh, Jui-Hua; Sedykh, Alexander; Huang, Ruili; Xia, Menghang; Tice, Raymond R

    2015-08-01

    A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise-filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC(50) (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds.

  5. Rapid, specific and quantitative assays for the detection of the endophytic bacterium Methylobacterium mesophilicum in plants.

    PubMed

    Lacava, P T; Li, W B; Araújo, W L; Azevedo, J L; Hartung, J S

    2006-06-01

    Xylella fastidiosa is a xylem-limited bacterium that causes citrus variegated chlorosis disease in sweet orange. There is evidence that X. fastidiosa interacts with endophytic bacteria present in the xylem of sweet orange, and that these interactions, particularly with Methylobacterium mesophilicum, may affect disease progress. However, these interactions cannot be evaluated in detail until efficient methods for detection and enumeration of these bacteria in planta are developed. We have previously developed standard and quantitative PCR-based assays specific for X. fastidiosa using the LightCycler system [Li, W.B., Pria Jr., L.P.M.W.D., X. Qin, and J.S. Hartung, 2003. Presence of Xylella fastidiosa in sweet orange fruit and seeds and its transmission to seedlings. Phytopathology 93:953-958.], and now report the development of both standard and quantitative PCR assays for M. mesophilicum. The assays are specific for M. mesophilicum and do not amplify DNA from other species of Methylobacterium or other bacteria commonly associated with citrus or plant tissue. Other bacteria tested included Curtobacterium flaccumfaciens, Pantoea agglomerans, Enterobacter cloacae, Bacillus sp., X. fastidiosa, Xanthomonas axonopodis pv. citri, and Candidatus Liberibacter asiaticus. We have demonstrated that with these methods we can quantitatively monitor the colonization of xylem by M. mesophilicum during the course of disease development in plants artificially inoculated with both bacteria.

  6. Small unilamellar vesicles as reagents: a chemically defined, quantitative assay for lectins

    SciTech Connect

    Rando, R.R.

    1981-01-01

    Samll unilamellar vesicles containing synthetic glycolipids can be prepared. These vesicles are aggregated by the appropriate lectin (Orr et al., 1979; Rando and Bangerter, 1979; Slama and Rando, 1980). It is shown here that extent of aggregation of these vesicles as measured by light scattering at 360 nm, is, under certain conditions, linear with amount of lectin added. This forms the basis of a rapid and simple quantitative assay for lectins using the modified vesicles as a defined chemical substrate. The assay is sensitive to lectin concentrations in the low ..mu..g range. The assay is applied here to studies on concanavalin A, Ricinus communis agglutinin and the ..cap alpha..-fucosyl binding lectin from Ulex europaeus (Type I).

  7. Quantitative determination of triglyceride by photoactivated CdSe/ZnS quantum dots through fluorescence assay.

    PubMed

    Huang, Chin-Ping; Li, Yaw-Kuen; Chen, Teng-Ming

    2008-07-01

    The quantitative detection of triglycerides is an important issue for health inspection of metabolic disorders and for food and oil-refining industries. Many methods have been designed to approach this target, in which multiple reactions catalyzed by enzymes are normally coupled consecutively. In this study, we demonstrated a simple assay system containing lipase and photoactivated luminescent CdSe/ZnS quantum dots (QDs) for the quantitative detection of triglycerides. Photoactivated CdSe/ZnS QDs function as a sensitive "indicator" to reveal the minute acidity change of the assay system resulting from the enzymatic hydrolysis of triglycerides. By controlling the initial buffer condition of the assay system at 5, 10, or 20 mM phosphate buffer at pH 8.0, respectively, the quenching ratio of the QDs fluorescence intensity monitored at the maximum photoluminescence showed a linear correlation with the concentration of the examined triglyceride in the range of 0.02-6, 0.2-10, or 2-20 mM, respectively. The assay system also provides a convenient way to estimate triglyceride concentration by visualizing the color change of the QDs fluorescence. As compared to most of the existing methods, the system reported herein possessed many advantages, including simplicity, low cost, high flexibility, and high sensitivity. Furthermore, no complicated chemical modification or enzyme immobilization is needed.

  8. [Comparison of two commercial molecular assays for quantitative measurement of hepatitis B viral DNA].

    PubMed

    Zalewska, Małgorzata; Domagała, Małgorzata; Gładysz, Andrzej

    2003-12-01

    The detection and quantification of hepatitis B virus (HBV) genomes appear to be the most reliable method for monitoring HBV infection and assessing responses to antiviral treatment. For quantitative determination of HBV viremia molecular biology-based assays are used. The aim of this study was to compare and evaluate the performance of two HBV DNA detection and quantification commercial assays: hybrid-capture Digene Hybrid Capture HBV DNA assays and based on competitive polymerase chain reaction (PCR) Cobas Amplicor HBV Monitor Roche Diagnostics. Reproducibility, linearity, sensitivity were determined with 2-fold dilution series of high-titers samples and with 113 sera samples from patients with chronic HBV infection. Within-run and between-run coefficients of variation ranged from 2.4-9.7% for hybrid-capture and from 3.7-15% for PCR-based Monitor. The hybrid-capture and PCR Monitor assays appeared to be linear throughout their range of quantification: 5-2000 pg/ml and 2 x 10(2)-2 x 10(5) copies/ml respectively. The HBV DNA units used in the two assays were not comparable. Hybrid-capture and Monitor gave concordant results with 87 (82.1%) of 106 samples. The assays were both positive with 79 (74.5%) samples and were negative in 7 (7.5%) cases. Hybrid-capture and Monitor gave discordant results in 17 (17.9%) cases. The Monitor Assay was positive in 13 (61.9%) of the 21 samples negative in hybrid-capture. The competitive PCR-based Monitor assay appear to be significantly more sensitive but slightly less reproducible than the hybrid-capture. In the group of patients with seroconversion to anti-HBe PCR method should be used for measurement of viral load. In the presence of HBe antigen concentration of HBV DNA may be tested by hybrid-capture assay. Also these two assays may be used in complementary fashion in the management of HBV infected patients. It seems reasonable to use a hybrid-capture assay first, because its linear range of quantification is extended to high

  9. Development of a Quantitative Real-Time PCR Assay for Detection of Mycoplasma genitalium

    PubMed Central

    Svenstrup, Helle Friis; Jensen, Jørgen Skov; Björnelius, Eva; Lidbrink, Peter; Birkelund, Svend; Christiansen, Gunna

    2005-01-01

    Mycoplasma genitalium is known to cause nonchlamydial, nongonococcal urethritis in men and to be associated with pelvic inflammatory disease in women. Specific and sensitive PCR methods are needed for diagnosis of this bacterium because it is very difficult to culture from patient samples. To determine the bacterial load in patients' specimens, a quantitative real-time LightCycler PCR was developed. The housekeeping gene gap encoding glyceraldehyde-3-phosphate dehydrogenase was chosen as the target gene. The assay could consistently detect five genome copies per reaction. To evaluate the PCR, we tested 246 selected urethral swab samples from men attending a clinic for sexually transmitted diseases. Eighty-two of the samples were found positive for M. genitalium by a conventional 16S rRNA gene PCR assay, whereas 164 samples were randomly chosen among those tested negative. Of the positive samples, 78 (95.1%) were found positive, whereas 6 (3.7%) of the negatives were found positive by the LightCycler assay. The patient samples were also tested with a quantitative TaqMan assay, and the bacterial load was compared to the LightCycler results. A good linear correlation between the LightCycler and the TaqMan assays was found with a correlation coefficient of 0.89 and a slope of 0.99. Significantly more M. genitalium-positive men had urethritis, discharge, and dysuria than had M. genitalium-negative men. The M. genitalium DNA load in samples from patients with urethritis was significantly higher than in samples from those without (61 and 2.9 copies/μl, respectively [P = 0.0005]). This assay may prove useful in the monitoring of treatment and for optimizing sample preparation methods. PMID:16000423

  10. Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay.

    PubMed

    Liu, Zongxiao; Teng, Yong; Liu, Hong; Jiang, Yulin; Xie, Xiayang; Li, Huifang; Lv, Jiangqiang; Gao, Longying; He, Junqiang; Shi, Xiujie; Tian, Feiyan; Yang, Jingshun; Xie, Congxin

    2008-04-01

    Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.

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

    PubMed Central

    Richards-Kortum, Rebecca

    2015-01-01

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

  12. Real-time PCR assay for rapid qualitative and quantitative detection of Entamoeba histolytica.

    PubMed

    Orosz, Erika; Perkátai, Katalin; Kapusinszky, Beatrix; Farkas, Agnes; Kucsera, István

    2012-12-01

    Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

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

    PubMed

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

    2015-03-30

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

  14. Quantitative rRNA-targeted solution-based hybridization assay using peptide nucleic acid molecular beacons.

    PubMed

    Li, Xu; Morgenroth, Eberhard; Raskin, Lutgarde

    2008-12-01

    The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.

  15. BactQuant: An enhanced broad-coverage bacterial quantitative real-time PCR assay

    PubMed Central

    2012-01-01

    Background Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our analysis of a diverse collection of 16 S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay—BactQuant—for quantifying 16 S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant. Methods The aligned core set of 4,938 16 S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines. Results A bacterial TaqMan® qPCR assay targeting a 466 bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively. Conclusions The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions. PMID:22510143

  16. A quantitative and high-throughput assay of human papillomavirus DNA replication.

    PubMed

    Gagnon, David; Fradet-Turcotte, Amélie; Archambault, Jacques

    2015-01-01

    Replication of the human papillomavirus (HPV) double-stranded DNA genome is accomplished by the two viral proteins E1 and E2 in concert with host DNA replication factors. HPV DNA replication is an established model of eukaryotic DNA replication and a potential target for antiviral therapy. Assays to measure the transient replication of HPV DNA in transfected cells have been developed, which rely on a plasmid carrying the viral origin of DNA replication (ori) together with expression vectors for E1 and E2. Replication of the ori-plasmid is typically measured by Southern blotting or PCR analysis of newly replicated DNA (i.e., DpnI digested DNA) several days post-transfection. Although extremely valuable, these assays have been difficult to perform in a high-throughput and quantitative manner. Here, we describe a modified version of the transient DNA replication assay that circumvents these limitations by incorporating a firefly luciferase expression cassette in cis of the ori. Replication of this ori-plasmid by E1 and E2 results in increased levels of firefly luciferase activity that can be accurately quantified and normalized to those of Renilla luciferase expressed from a control plasmid, thus obviating the need for DNA extraction, digestion, and analysis. We provide a detailed protocol for performing the HPV type 31 DNA replication assay in a 96-well plate format suitable for small-molecule screening and EC50 determinations. The quantitative and high-throughput nature of the assay should greatly facilitate the study of HPV DNA replication and the identification of inhibitors thereof.

  17. A quantitative ELISA assay for the fragile x mental retardation 1 protein.

    PubMed

    Iwahashi, Christine; Tassone, Flora; Hagerman, Randi J; Yasui, Dag; Parrott, George; Nguyen, Danh; Mayeur, Greg; Hagerman, Paul J

    2009-07-01

    Non-coding (CGG-repeat) expansions in the fragile X mental retardation 1 (FMR1) gene result in a spectrum of disorders involving altered neurodevelopment (fragile X syndrome), neurodegeneration (late-onset fragile X-associated tremor/ataxia syndrome), or primary ovarian insufficiency. While reliable and quantitative assays for the number of CGG repeats and FMR1 mRNA levels are now available, there has been no scalable, quantitative assay for the FMR1 protein (FMRP) in non-transformed cells. Using a combination of avian and murine antibodies to FMRP, we developed a sensitive and highly specific sandwich enzyme-linked immunosorbent assay (ELISA) for FMRP in peripheral blood lymphocytes. This ELISA method is capable of quantifying FMRP levels throughout the biologically relevant range of protein concentrations and is specific for the intact FMRP protein. Moreover, the ELISA is well-suited for replicate protein determinations across serial dilutions in non-transformed cells and is readily scalable for large sample numbers. The FMRP ELISA is potentially a powerful tool in expanding our understanding of the relationship between FMRP levels and the various FMR1-associated clinical phenotypes.

  18. Rapid and Quantitative Assay of Amyloid-Seeding Activity in Human Brains Affected with Prion Diseases

    PubMed Central

    Takatsuki, Hanae; Satoh, Katsuya; Sano, Kazunori; Fuse, Takayuki; Nakagaki, Takehiro; Mori, Tsuyoshi; Ishibashi, Daisuke; Mihara, Ban; Takao, Masaki; Iwasaki, Yasushi; Yoshida, Mari; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 1010/g brain (values varies 108.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06–0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission. PMID:26070208

  19. Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes.

    PubMed

    Ding, Xiaokang; Yang, Kun-Lin

    2015-01-07

    A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.

  20. A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC).

    PubMed

    Orton, Dennis J; Doucette, Alan A

    2013-03-15

    As an alternative to direct UV absorbance measurements, estimation of total protein concentration is typically conducted through colorimetric reagent assays. However, for protein-limited applications, the proportion of the sample sacrificed to the assay becomes increasingly significant. This work demonstrates a method for quantitation of protein samples with high recovery. Temperature programmed liquid chromatography (TPLC) with absorbance detection at 214nm permits accurate estimation of total protein concentration from samples containing as little as 0.75μg. The method incorporates a temperature gradient from 25 to 80°C to facilitate elution of total protein into a single fraction. Analyte recovery, as measured from 1 and 10μg protein extracts of Escherichia coli, is shown to exceed 93%. Extinction coefficients at 214nm were calculated across the human proteome, providing a relative standard deviation of 21% (versus 42% at 280nm), suggesting absorbance values at 214nm provide a more consistent measure of protein concentration. These results translate to a universal protein detection strategy exhibiting a coefficient of variation below 10%. Together with the sensitivity and tolerance to contaminants, TPLC with UV detection is a favorable alternative to colorimetric assay for total protein quantitation, particularly in sample-limited applications.

  1. Development of a Quantitative Bead Capture Assay for Soluble IL-7 Receptor Alpha in Human Plasma

    PubMed Central

    Faucher, Sylvie; Crawley, Angela M.; Decker, Wendy; Sherring, Alice; Bogdanovic, Dragica; Ding, Tao; Bergeron, Michele; Angel, Jonathan B.; Sandstrom, Paul

    2009-01-01

    Background IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Rα (CD127) and common γ (CD132) chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. Previous reports showed the usefulness of CD127 as a prognostic marker in viral infections such as HIV, CMV, EBV and HCV. A soluble CD127 (sCD127) is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. Measuring sCD127 is important to define its role and may complement existing markers used in lymphopenic disease management. We describe a new quantitative assay for the measurement of sCD127 in plasma and report sCD127 concentrations in healthy adults. Methodology/Principal Findings We developed a quantitative bead-based sCD127 capture assay. Polyclonal CD127-specific antibodies were chosen for capture and a biotinylated monoclonal anti-CD127 antibody was selected for detection. The assay can detect native sCD127 and recombinant sCD127 which served as the calibrator. The analytical performance of the assay was characterized and the concentration and stability of plasma sCD127 in healthy adults was determined. The assay's range was 3.2–1000 ng/mL. The concentration of plasma sCD127 was 164±104 ng/mL with over a log variation between subjects. Individual sCD127 concentrations remained stable when measured serially during a period of up to one year. Conclusions/Significance This is the first report on the quantification of plasma sCD127 in a population of healthy adults. Soluble CD127 plasma concentrations remained stable over time in a given individual and sCD127 immunoreactivity was resistant to repeated freeze-thaw cycles. This quantitative sCD127 assay is a valuable tool for defining the potential role of sCD127 in lymphopenic diseases. PMID:19690616

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

    PubMed Central

    Zhang, Pengfei; Bao, Yan; Draz, Mohamed Shehata; Lu, Huiqi; Liu, Chang; Han, Huanxing

    2015-01-01

    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. PMID:26491289

  3. Naked-eye quantitative aptamer-based assay on paper device.

    PubMed

    Zhang, Yun; Gao, Dong; Fan, Jinlong; Nie, Jinfang; Le, Shangwang; Zhu, Wenyuan; Yang, Jiani; Li, Jianping

    2016-04-15

    This work initially describes the design of low-cost, naked-eye quantitative aptamer-based assays by using microfluidic paper-based analytical device (μPAD). Two new detection motifs are proposed for quantitative μPAD measurement without using external electronic readers, which depend on the length of colored region in a strip-like μPAD and the number of colorless detection microzones in a multi-zone μPAD. The length measuring method is based on selective color change of paper from colorless to blue-black via formation of iodine-starch complex. The counting method is conducted on the basis of oxidation-reduction reaction between hydrogen peroxide and potassium permanganate. Their utility is well demonstrated with sensitive, specific detection of adenosine as a model analyte with the naked eye in buffer samples and undiluted human serum. These equipment-free quantitative methods proposed thus hold great potential for the development of more aptamer-based assays that are simple, cost-efficient, portable, and user-friendly for various point-of-care applications particularly in resource-constrained environments.

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

    PubMed

    Zhang, Pengfei; Bao, Yan; Draz, Mohamed Shehata; Lu, Huiqi; Liu, Chang; Han, Huanxing

    2015-01-01

    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.

  5. Analysis of JC virus DNA replication using a quantitative and high-throughput assay

    PubMed Central

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

    2015-01-01

    Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. PMID:25155200

  6. Multiple reaction monitoring and multiple reaction monitoring cubed based assays for the quantitation of apolipoprotein F.

    PubMed

    Kumar, Abhinav; Gangadharan, Bevin; Zitzmann, Nicole

    2016-10-15

    Apolipoprotein F (APO-F) is a novel low abundance liver fibrosis biomarker and its concentration decreases in human serum and plasma across liver fibrosis stages. Current antibody based assays for APO-F suffer from limitations such as unspecific binding, antibody availability and undetectable target if the protein is degraded; and so an antibody-free assay has the potential to be a valuable diagnostic tool. We report an antibody-free, rapid, sensitive, selective and robust LC-MS/MS (MRM and MRM(3)) method for the detection and quantitation of APO-F in healthy human plasma. With further analysis of clinical samples, this LC-MS based method could be established as the first ever antibody-free biomarker assay for liver fibrosis. We explain the use of Skyline software for peptide selection and the creation of a reference library to aid in true peak identification of endogenous APO-F peptides in digests of human plasma without protein or peptide enrichment. Detection of a glycopeptide using MRM-EPI mode and reduction of interferences using MRM3 are explained. The amount of APO-F in human plasma from a healthy volunteer was determined to be 445.2ng/mL, the coefficient of variation (CV) of precision for 20 injections was <12% and the percentage error of each point along the calibration curve was calculated to be <8%, which is in line with the assay requirements for clinical samples.

  7. Quantitative Microplate-Based Growth Assay for Determination of Antifungal Susceptibility of Histoplasma capsulatum Yeasts

    PubMed Central

    Goughenour, Kristie D.; Balada-Llasat, Joan-Miquel

    2015-01-01

    Standardized methodologies for determining the antifungal susceptibility of fungal pathogens is central to the clinical management of invasive fungal disease. Yeast-form fungi can be tested using broth macrodilution and microdilution assays. Reference procedures exist for Candida species and Cryptococcus yeasts; however, no standardized methods have been developed for testing the antifungal susceptibility of yeast forms of the dimorphic systemic fungal pathogens. For the dimorphic fungal pathogen Histoplasma capsulatum, susceptibility to echinocandins differs for the yeast and the filamentous forms, which highlights the need to employ Histoplasma yeasts, not hyphae, in antifungal susceptibility tests. To address this, we developed and optimized methodology for the 96-well microtiter plate-based measurement of Histoplasma yeast growth in vitro. Using optical density, the assay is quantitative for fungal growth with a dynamic range greater than 30-fold. Concentration and assay reaction time parameters were also optimized for colorimetric (MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] reduction) and fluorescent (resazurin reduction) indicators of fungal vitality. We employed this microtiter-based assay to determine the antifungal susceptibility patterns of multiple clinical isolates of Histoplasma representing different phylogenetic groups. This methodology fulfills a critical need for the ability to monitor the effectiveness of antifungals on Histoplasma yeasts, the morphological form present in mammalian hosts and, thus, the form most relevant to disease. PMID:26246483

  8. Indirect enzyme-linked immunosorbent assay for the quantitative estimation of lysergic acid diethylamide in urine.

    PubMed

    Kerrigan, S; Brooks, D E

    1998-05-01

    A new antibody to lysergic acid diethylamide (LSD) was used to develop a novel indirect ELISA for the quantification of drug in urine. Evaluation of the new assay with the commercially available LSD ELISA (STC Diagnostics) shows improved performance. The test requires 50 microL of urine, which is used to measure concentrations of drug in the microg/L to ng/L range. The limit of detection was 8 ng/L compared with 85 ng/L in the commercial assay, and analytical recoveries were 98-106%. Our test detected 0.1 microg/L of LSD in urine with an intraassay CV of 2.4% (n = 8) compared with 6.0% for a 0.5 microg/L sample in the commercial assay (n = 20). The upper and lower limits of quantification were estimated to be 7 microg/L and 50 ng/L, respectively. Specificity was evaluated by measuring the extent of cross-reactivity with 24 related substances. Drug determination using the new assay offers both improved sensitivity and precision compared with existing methods, thus facilitating the preliminary quantitative estimation of LSD in urine at lower concentrations with a greater degree of certainty.

  9. Impedance Analysis of Colloidal Gold Nanoparticles in Chromatography Paper for Quantitation of an Immunochromatographic Assay.

    PubMed

    Hori, Fumitaka; Harada, Yuji; Kuretake, Tatsumi; Uno, Shigeyasu

    2016-01-01

    A detection method of gold nanoparticles in chromatography paper has been developed for a simple, cost-effective and reliable quantitation of immunochromatographic strip test. The time courses of the solution resistance in chromatography paper with the gold nanoparticles solution are electrochemically measured by chrono-impedimetry. The dependence of the solution resistance on the concentration of gold nanoparticles has been successfully observed. The main factor to increase the solution resistance may be obstruction of the ion transport due to the presence of gold nanoparticles. The existence of gold nanoparticles with 1.92 × 10(9) particles/mL in an indistinctly-colored chromatography paper is also identified by a solution resistance measurement. This indicates that the solution resistance assay has the potential to lower the detection limit of the conventional qualitative assay.

  10. Quantitative measurement of fathead minnow vitellogenin mRNA using hybridization protection assays.

    PubMed

    Thomas-Jones, Emma; Walkley, Neal; Morris, Ceri; Kille, Peter; Cryer, Jennifer; Weeks, Ian; Woodhead, J Stuart

    2003-05-01

    We have developed a novel test system for the quantitative assessment of gene transcription. The procedure involves the use of chemiluminescent-labeled oligonucleotide probes in a hybridization protection assay (HPA) format. We have used this technology to measure changes in vitellogenin mRNA to demonstrate the impact of estrogen exposure in the juvenile fathead minnow (Pimephales promelas). Marked changes in mRNA expression were observed in response to intraperitoneal injection of 17beta-estradiol demonstrating the utility of this technique for the identification and monitoring of toxic responses to xenobiotics.

  11. Applications and challenges in using LC-MS/MS assays for quantitative doping analysis.

    PubMed

    Wang, Zhanliang; Lu, Jianghai; Zhang, Yinong; Tian, Ye; Yuan, Hong; Xu, Youxuan

    2016-06-01

    LC-MS/MS is useful for qualitative and quantitative analysis of 'doped' biological samples from athletes. LC-MS/MS-based assays at low-mass resolution allow fast and sensitive screening and quantification of targeted analytes that are based on preselected diagnostic precursor-product ion pairs. Whereas LC coupled with high-resolution/high-accuracy MS can be used for identification and quantification, both have advantages and challenges for routine analysis. Here, we review the literature regarding various quantification methods for measuring prohibited substances in athletes as they pertain to World Anti-Doping Agency regulations.

  12. Importance of Purity Evaluation and the Potential of Quantitative 1H NMR as a Purity Assay

    PubMed Central

    2015-01-01

    In any biomedical and chemical context, a truthful description of chemical constitution requires coverage of both structure and purity. This qualification affects all drug molecules, regardless of development stage (early discovery to approved drug) and source (natural product or synthetic). Purity assessment is particularly critical in discovery programs and whenever chemistry is linked with biological and/or therapeutic outcome. Compared with chromatography and elemental analysis, quantitative NMR (qNMR) uses nearly universal detection and provides a versatile and orthogonal means of purity evaluation. Absolute qNMR with flexible calibration captures analytes that frequently escape detection (water, sorbents). Widely accepted structural NMR workflows require minimal or no adjustments to become practical 1H qNMR (qHNMR) procedures with simultaneous qualitative and (absolute) quantitative capability. This study reviews underlying concepts, provides a framework for standard qHNMR purity assays, and shows how adequate accuracy and precision are achieved for the intended use of the material. PMID:25295852

  13. Timed ELISA: an alternative approach to quantitative enzyme-linked immunosorbent assay.

    PubMed

    Muller, R

    1997-10-01

    As the uses for ELISA (enzyme-linked immunosorbent assay) increase, so does the need for a quantitative procedure that does not require a spectrophotometer or other expensive equipment. 'Timed ELISA' employs an 'iodine clock' as the final step such that quantitative measurements may be made using a stopwatch. Catalase, coupled to the primary antibody, reduces the concentration of H2O2 available to generate iodine in the clock reaction. Iodine stains the starch component blue, but catalase prolongs the time taken for the change in colour to be observed. After the time delay occurs the transition to full colour development is extremely rapid (< 1 s) at all analyte concentrations, allowing clear definition of the end point. The performance of Timed ELISA is similar to that obtained using a horseradish peroxidase-conjugated system employing the customary spectrophotometric determination.

  14. Quantitative dynamic nuclear polarization-NMR on blood plasma for assays of drug metabolism.

    PubMed

    Lerche, Mathilde H; Meier, Sebastian; Jensen, Pernille R; Hustvedt, Svein-Olaf; Karlsson, Magnus; Duus, Jens Ø; Ardenkjaer-Larsen, Jan H

    2011-01-01

    Analytical platforms for the fast detection, identification and quantification of circulating drugs with a narrow therapeutic range are vital in clinical pharmacology. As a result of low drug concentrations, analytical tools need to provide high sensitivity and specificity. Dynamic nuclear polarization-NMR (DNP-NMR) in the form of the hyperpolarization-dissolution method should afford the sensitivity and spectral resolution for the direct detection and quantification of numerous isotopically labeled circulating drugs and their metabolites in single liquid-state NMR transients. This study explores the capability of quantitative in vitro DNP-NMR to assay drug metabolites in blood plasma. The lower limit of detection for the anti-epileptic drug (13)C-carbamazepine and its pharmacologically active metabolite (13)C-carbamazepine-10,11-epoxide is 0.08 µg/mL in rabbit blood plasma analyzed by single-scan (13)C DNP-NMR. An internal standard is used for the accurate quantification of drug and metabolite. Comparison of quantitative DNP-NMR data with an established analytical method (liquid chromatography-mass spectrometry) yields a Pearson correlation coefficient r of 0.99. Notably, all DNP-NMR determinations were performed without analyte derivatization or sample purification other than plasma protein precipitation. Quantitative DNP-NMR is an emerging methodology which requires little sample preparation and yields quantitative data with high sensitivity for therapeutic drug monitoring. Copyright © 2010 John Wiley & Sons, Ltd.

  15. A parallel and quantitative cell migration assay using a novel multi-well-based device.

    PubMed

    Quan, Qianghua; Zhang, Shuwen; Wang, Xudong; Ouyang, Qi; Wang, Yugang; Yang, Gen; Luo, Chunxiong

    2016-12-01

    Cell migration assays for different chemical environments are important for both scientists and clinicians searching for new therapeutics. In this study, we developed a multi-well-based microfluidic chip that has multiple units for different conditions. In each unit, cells can be patterned and then released to observe their migration. Automatic image analysis and model-based data processing were developed to describe the integrated cell migration assay precisely and quickly. As a demonstration, the migration behaviors of two types of cells in eight chemical conditions were studied. The results showed that supplementation with transforming growth factor-β(TGF-β) significantly promoted the migration of MCF-7 and MCF-10 A cells compared to several growth factors, such as Epidermal Growth Factor(EGF) and basic fibroblast growth factor(bFGF), as well as a control sample. Cells can migrate particularly fast with two or more mixed supplementary factors, such as TGF-β + bFGF + EGF, which indicated a synergy effect. Thus, this chip could be used to quantitatively observe cancer cell migration and demonstrated great potential for use in quantitative migration studies and chemical screening.

  16. Flow Cytometry and Transplantation-Based Quantitative Assays for Satellite Cell Self-Renewal and Differentiation.

    PubMed

    Arpke, Robert W; Kyba, Michael

    2016-01-01

    In response to muscle damage, satellite cells proliferate and undertake both differentiation and self-renewal, generating new functional muscle tissue and repopulating this new muscle with stem cells for future injury responses. For many questions relating to the physiological regulation of satellite cells, quantitative readouts of self-renewal and differentiation can be very useful. There is a particular need for a quantitative assay for satellite cell self-renewal that does not rely solely upon sectioning, staining and counting cells in sections. In this chapter, we provide detailed methods for quantifying the self-renewal and differentiation potential of a given population of satellite cells using an assay involving transplantation into injured, regenerating muscle together with specific markers for donor cell identity and state of differentiation. In particular, using the Pax7-ZsGreen transgene as a marker of satellite cell state, self-renewal can be quantified by FACS on transplanted muscle to actually count the total number of resident satellite cells at time points following transplantation.

  17. Quantitative human chorionic gonadotropin analysis. I. Comparison of an immunoradiometric assay and a radioimmunoassay

    SciTech Connect

    Shapiro, A.I.; Wu, T.F.; Ballon, S.C.; Lamb, E.J.

    1984-01-01

    An immunoradiometric assay (IRMA) for the quantitative analysis of human chorionic gonadotropin (hCG) was evaluated for specificity, sensitivity, accuracy and precision. The results were compared with those of the conventional radioimmunoassay (RIA) used in our laboratory. The IRMA is a solid-phase, double-antibody immunoassay that sandwiches the intact hCG molecule between the two antibodies. It has specificity, accuracy, and precision which are similar to those of the RIA. The RIA is based upon the assumptions that the antigenicity of the tracer is not altered by the iodination process and that the antibody reacts equally with all of the antigens, including the radiolabeled antigen. The IRMA does not use radiolabeled antigens and thus is free of the assumptions made in the conventional RIA. The IRMA may be more accurate at the lower limits of the assay because it does not require logarithmic transformations. Since the IRMA does not measure the free beta-subunit of hCG, it cannot be endorsed as the sole technique to quantitate hCG in patients with gestational trophoblastic neoplasia until the significance of the free beta-subunit in these patients is determined.

  18. A CAPS-based binding assay provides semi-quantitative validation of protein-DNA interactions.

    PubMed

    Xie, Yongyao; Zhang, Yaling; Zhao, Xiucai; Liu, Yao-Guang; Chen, Letian

    2016-02-15

    Investigation of protein-DNA interactions provides crucial information for understanding the mechanisms of gene regulation. Current methods for studying protein-DNA interactions, such as DNaseI footprinting or gel shift assays, involve labeling DNA with radioactive or fluorescent tags, making these methods costly, laborious, and potentially damaging to the environment. Here, we describe a novel cleaved amplified polymorphic sequence (CAPS)-based binding assay (CBA), which is a label-free method that can simplify the semi-quantitative validation of protein-DNA interactions. The CBA tests the interaction between a protein and its target DNA, based on the CAPS pattern produced due to differences in the accessibility of a restriction endonuclease site (intrinsic or artificial) in amplified DNA in the presence and absence of the protein of interest. Thus, the CBA can produce a semi-quantitative readout of the interaction strength based on the dose of the binding protein. We demonstrate the principle and feasibility of CBA using B3, MADS3 proteins and the corresponding RY or CArG-box containing DNAs.

  19. A quantitative proteomics-based competition binding assay to characterize pITAM-protein interactions.

    PubMed

    Hu, Lianghai; Yang, Li; Lipchik, Andrew M; Geahlen, Robert L; Parker, Laurie L; Tao, W Andy

    2013-05-21

    Characterization of ligand-protein binding is of crucial importance in drug discovery. Classical competition binding assays measure the binding of a labeled ligand in the presence of various concentrations of unlabeled ligand and typically use single purified proteins. Here, we introduce a high-throughput approach to study ligand-protein interactions by coupling competition binding assays with mass spectrometry-based quantitative proteomics. With the use of a phosphorylated immunoreceptor tyrosine-based activation motif (pITAM) peptide as a model, we characterized pITAM-interacting partners in human lymphocytes. The shapes of competition binding curves of various interacting partners constructed in a single set of quantitative proteomics experiments reflect relative affinities for the pITAM peptide. This strategy can provide an efficient approach to distinguish specific interacting partners, including two signaling kinases possessing tandem SH2 domains, SYK and ZAP-70, as well as other SH2 domain-containing proteins such as CSK and PI3K, from contaminants and to measure relative binding affinities of multiple proteins in a single experiment.

  20. A quantitative evaluation of cell migration by the phagokinetic track motility assay.

    PubMed

    Nogalski, Maciej T; Chan, Gary C T; Stevenson, Emily V; Collins-McMillen, Donna K; Yurochko, Andrew D

    2012-12-04

    Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles(1,2,3). Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread(4,5). Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell(6,7,8). However, many biological questions about cell migration remain unanswered. The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers. The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip(9,10). With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells(11,12), fibroblasts(9), neutrophils(13), skeletal muscle cells(14

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

    USDA-ARS?s Scientific Manuscript database

    A new TaqMan real-time PCR assay was developed for the simultaneous quantitative detection of two seedborne maize pathogens in a single assay. Pantoea stewartii subsp. stewartii (Pnss) (syn. Erwinia stewartii) is the causal agent of Stewart's bacterial wilt and leaf blight of maize. Stewart's wilt i...

  2. Detection and quantitation of cellulase by Congo red staining of substrates in a cup-plate diffusion assay.

    PubMed

    Carder, J H

    1986-02-15

    A simple, inexpensive, and sensitive cellulase assay which allows simultaneous enzyme quantitation for many samples has been developed. Radial diffusion of enzyme into substrate incorporated in an agar gel produces zones of hydrolysis which can be visualized by staining undegraded substrate with Congo red dye. Comparisons were made between this and other commonly used cellulase assays based on accuracy, sensitivity, and ease of use.

  3. Enzyme-linked immunosorbent assay for detection and quantitation of capsular antigen of Haemophilus influenzae type b.

    PubMed Central

    Crosson, F J; Winkelstein, J A; Moxon, E R

    1978-01-01

    An enzyme-linked immunosorbent assay was developed to detect the presence of the ribose-ribitol phosphate capsular antigen of Haemophilus influenzae type b in laboratory and clinical specimens. The assay is simple, sensitive, specific, and quantitative and should prove to be of value in the diagnosis and management of H. influenzae infections. PMID:310425

  4. Migration and interaction tracking for quantitative analysis of phagocyte-pathogen confrontation assays.

    PubMed

    Brandes, Susanne; Dietrich, Stefanie; Hünniger, Kerstin; Kurzai, Oliver; Figge, Marc Thilo

    2017-02-01

    Invasive fungal infections are emerging as a significant health risk for humans. The innate immune system is the first line of defense against invading micro-organisms and involves the recruitment of phagocytes, which engulf and kill pathogens, to the site of infection. To gain a quantitative understanding of the interplay between phagocytes and fungal pathogens, live-cell imaging is a modern approach to monitor the dynamic process of phagocytosis in time and space. However, this requires the processing of large amounts of video data that is tedious to be performed manually. Here, we present a novel framework, called AMIT (algorithm for migration and interaction tracking), that enables automated high-throughput analysis of multi-channel time-lapse microscopy videos of phagocyte-pathogen confrontation assays. The framework is based on our previously developed segmentation and tracking framework for non-rigid cells in brightfield microscopy (Brandes et al., 2015). We here present an advancement of this framework to segment and track different cell types in different video channels as well as to track the interactions between different cell types. For the confrontation assays of polymorphonuclear neutrophils (PMNs) and Candida glabrata considered in this work, the main focus lies on the correct detection of phagocytic events. To achieve this, we introduced different PMN states and a state-transition model that represents the basic principles of phagocyte-pathogen interactions. The framework is validated by a direct comparison of the automatically detected phagocytic activity of PMNs to a manual analysis and by a qualitative comparison with previously published analyses (Duggan et al., 2105; Essig et al., 2015). We demonstrate the potential of our algorithm by comprehensive quantitative and multivariate analyses of confrontation assays involving human PMNs and the fungus C. glabrata.

  5. Virological Diagnosis of Herpes Simplex Virus 1 Esophagitis by Quantitative Real-Time PCR Assay

    PubMed Central

    Jazeron, Jean-François; Barbe, Coralie; Frobert, Emilie; Renois, Fanny; Talmud, Déborah; Brixi-Benmansour, Hedia; Brodard, Véronique; Andréoletti, Laurent; Diebold, Marie-Danièle

    2012-01-01

    Herpes simplex virus 1 (HSV-1) esophagitis diagnosis is routinely based on the endoscopic findings confirmed by histopathological examination of the esophagitis lesions. Virological diagnosis is not systematically performed and restricted to viral culture or to qualitative PCR assay from esophagitis biopsy specimens. The aim of this study was to assess the interest of quantitative real-time PCR assay in HSV-1 esophagitis diagnosis by comparing the results obtained to those of histological examination associated with immunohistochemical staining, which is considered the “gold standard.” From 53 esophagitis biopsy specimens, the PCR assay detected HSV-1 in 18 of 19 histologically proven to have herpetic esophagitis and in 9 of 34 that had esophagitis related to other causes, demonstrating sensitivity, specificity, positive predictive value, and negative predictive value of 94.7%, 73%, 66.7%, and 96%, respectively. Interestingly, HSV-1 was not detected in 16 specimens without the histological aspect of esophagitis. The viral loads normalized per μg of total extracted DNA in each biopsy specimen detected positive by HSV PCR were then compared and appeared to be significantly higher in histopathologically positive herpetic esophagitis (median = 2.9 × 106 ± 1.1 × 108) than in histopathologically negative herpetic esophagitis (median = 3.1 × 103 ± 6.2 × 103) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 104 copies would allow an HSV-1 esophagitis diagnosis with a sensitivity and specificity of 83.3% and 100%, respectively. In conclusion, this work demonstrated that HSV quantitative PCR results for paraffin-embedded esophageal tissue was well correlated to histopathological findings for an HSV-1 esophagitis diagnosis and could be diagnostic through viral load assessment when histopathological results are missing or uncertain. PMID:22170921

  6. Quantitative PCR assays to survey the bovine adenovirus levels in environmental samples.

    PubMed

    Wong, K; Xagoraraki, I

    2010-08-01

    Previous studies suggested bovine adenoviruses (BAdVs) could be used as cattle faecal indicators. The main aim of this study was to survey the levels of BAdV in environmental samples using quantitative polymerase chain reaction (qPCR). Two qPCR assays were developed to identify and quantify BAdVs in environmental samples. BAdVs were detected in all dairy manure, and in most cases, the sample concentrations were around 10(3)-10(4) copies ml(-1). Farm tile drainage samples were also detected, but the concentrations were about 1-3 log(10) lower than the BAdV concentrations in the manure samples. The genome equivalent copy (GEC) levels of BAdV and the phage forming unit (PFU) levels of somatic phage in manure samples were comparable. Four of twenty individual cattle faeces were positive with concentrations similar to that found in the manure samples. Sequencing results confirmed the presence of BAdV in the environmental samples, and phylogenetic analysis indicated that BAdV 2 and 4 were the most prevalent serotypes in all the manure samples tested. The qPCR assays developed in this study showed higher sensitivity in detecting BAdV 1 and 2 than the previous published nested assay. The high levels of BAdV in the environmental samples may suggest it could be used for bovine faecal indicator. The significant levels of BAdV in the drainage samples may indicate the potential of surface water pollution by the manure applied to farm fields. This is the first study that reports the quantitative level of BAdV in environmental samples. These results could be useful when it comes to determining whether BAdV could be utilized as a bovine faecal indicator.

  7. A Caco-2 cell-based quantitative antioxidant activity assay for antioxidants.

    PubMed

    Wan, Hongxia; Liu, Dong; Yu, Xiangying; Sun, Haiyan; Li, Yan

    2015-05-15

    A Caco-2 cell-based antioxidant activity (CAA) assay for quantitative evaluation of antioxidants was developed by optimizing seeding density and culture time of Caco-2 cells, incubation time and concentration of fluorescent probe (2',7'-dichlorofluorescin diacetate, DCFH-DA), incubation way and incubation time of antioxidants (pure phytochemicals) and DCFH-DA with cells, and detection time of fluorescence. Results showed that the CAA assay was of good reproducibility and could be used to evaluate the antioxidant activity of antioxidants at the following conditions: seeding density of 5 × 10(4)/well, cell culture time of 24h, co-incubation of 60 μM DCFH-DA and pure phytochemicals with Caco-2 cells for 20 min and fluorescence recorded for 90 min. Additionally, a significant correlation was observed between CAA values and rat plasma ORAC values following the intake of antioxidants for selected pure phytochemicals (R(2) = 0.815, p < 0.01), demonstrating the good biological relevance of CAA assay.

  8. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions

    PubMed Central

    Vickers, Timothy A.; Crooke, Stanley T.

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  9. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

    PubMed

    Vickers, Timothy A; Crooke, Stanley T

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells.

  10. Detection of yellow fever virus: a comparison of quantitative real-time PCR and plaque assay.

    PubMed

    Bae, Hi-Gung; Nitsche, Andreas; Teichmann, Anette; Biel, Stefan S; Niedrig, Matthias

    2003-06-30

    Yellow fever virus quantitation is performed routinely by cultivation of virus containing samples using susceptible cells. Counting of the resulting plaques provides a marker for the number of infectious particles present in the sample. This assay usually takes up to 5 days before results are obtained and must be carried out under L2 or L3 laboratory conditions, depending on the yellow fever virus strain used. For clinical diagnosis of yellow fever virus infections the cell culture-based approach takes too long and is of limited practical relevance. Recently, due to its considerable sensitivity, PCR has become a promising method for virus detection. However, whilst PCR can detect virus-specific nucleic acids, it does not allow conclusions to be drawn regarding the infectious potential of the virus detected. Nonetheless, for diagnostic purposes, a rapid, specific and sensitive virus PCR is preferable. Therefore, two independent yellow fever virus-specific real-time PCR assays were established and compared the viral RNA loads to the results of a traditional plaque assay. The estimated ratio of yellow fever virus genomes to infectious particles was between 1000:1 and 5000:1; both approaches displayed a comparable precision of <45%. A significant correlation between genome number as determined by real-time PCR and the corresponding number of plaques in paired samples was found with a Pearson coefficient of correlation of r=0.88 (P<0.0001).

  11. A smartphone-readable barcode assay for the detection and quantitation of pesticide residues.

    PubMed

    Guo, Juan; Wong, Jessica X H; Cui, Caie; Li, Xiaochun; Yu, Hua-Zhong

    2015-08-21

    In this paper, we present a smartphone-readable barcode assay for the qualitative detection of methyl parathion residues, a toxic organophosphorus pesticide that is popularly used in agriculture worldwide. The detection principle is based on the irreversible inhibition of the enzymatic activity of acetylcholinesterase (AchE) by methyl parathion; AchE catalytically hydrolyzes acetylthiocholine iodine to thiocholine that in turn dissociates dithiobis-nitrobenzoate to produce a yellow product (deprotonated thio-nitrobenzoate). The yellow intensity of the product was confirmed to be inversely dependent on the concentration of the pesticide. We have designed a barcode-formatted assay chip by using a PDMS (polydimethylsiloxane) channel plate (as the reaction reservoir), situated under a printed partial barcode, to complete the whole barcode such that it can be directly read by a barcode scanning app installed on a smartphone. The app is able to qualitatively present the result of the pesticide test; the absence or a low concentration of methyl parathion results in the barcode reading as "-", identifying the test as negative for pesticides. Upon obtaining a positive result (the app reads a "+" character), the captured image can be further analyzed to quantitate the methyl parathion concentration in the sample. Besides the portability and simplicity, this mobile-app based colorimetric barcode assay compares favorably with the standard spectrophotometric method.

  12. Development, validation and quantitative assessment of an enzymatic assay suitable for small molecule screening and profiling: A case-study.

    PubMed

    Sancenon, Vicente; Goh, Wei Hau; Sundaram, Aishwarya; Er, Kai Shih; Johal, Nidhi; Mukhina, Svetlana; Carr, Grant; Dhakshinamoorthy, Saravanakumar

    2015-06-01

    The successful discovery and subsequent development of small molecule inhibitors of drug targets relies on the establishment of robust, cost-effective, quantitative, and physiologically relevant in vitro assays that can support prolonged screening and optimization campaigns. The current study illustrates the process of developing and validating an enzymatic assay for the discovery of small molecule inhibitors using alkaline phosphatase from bovine intestine as model target. The assay development workflow includes an initial phase of optimization of assay materials, reagents, and conditions, continues with a process of miniaturization and automation, and concludes with validation by quantitative measurement of assay performance and signal variability. The assay is further evaluated for dose-response and mechanism-of-action studies required to support structure-activity-relationship studies. Emphasis is placed on the most critical aspects of assay optimization and other relevant considerations, including the technology, assay materials, buffer constituents, reaction conditions, liquid handling equipment, analytical instrumentation, and quantitative assessments. Examples of bottlenecks encountered during assay development and strategies to address them are provided.

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

    PubMed

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

    2017-06-01

    Mycobacterium chimaera is an opportunistic environmental mycobacterium belonging to the Mycobacterium avium-M. intracellulare complex. Although most commonly associated with pulmonary disease, there has been growing awareness of invasive M. chimaera infections following cardiac surgery. Investigations suggest worldwide spread of a specific M. chimaera clone, associated with contaminated hospital heater-cooler units used during the surgery. Given the global dissemination of this clone, its potential to cause invasive disease, and the laboriousness of current culture-based diagnostic methods, there is a pressing need to develop rapid and accurate diagnostic assays specific for M. chimaera Here, we assessed 354 mycobacterial genome sequences and confirmed that M. chimaera is a phylogenetically coherent group. In silico comparisons indicated six DNA regions present only in M. chimaera We targeted one of these regions and developed a TaqMan quantitative PCR (qPCR) assay for M. chimaera with a detection limit of 100 CFU/ml in whole blood spiked with bacteria. In vitro screening against DNA extracted from 40 other mycobacterial species and 22 bacterial species from 21 diverse genera confirmed the in silico-predicted specificity for M. chimaera Screening 33 water samples from heater-cooler units with this assay highlighted the increased sensitivity of PCR compared to culture, with 15 of 23 culture-negative samples positive by M. chimaera qPCR. We have thus developed a robust molecular assay that can be readily and rapidly deployed to screen clinical and environmental specimens for M. chimaera. Copyright © 2017 American Society for Microbiology.

  14. Development and evaluation of a quantitative PCR assay targeting sandhill crane (Grus canadensis) fecal pollution.

    PubMed

    Ryu, Hodon; Lu, Jingrang; Vogel, Jason; Elk, Michael; Chávez-Ramírez, Felipe; Ashbolt, Nicholas; Santo Domingo, Jorge

    2012-06-01

    While the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no methods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gene fecal clone libraries. Using these sequences, a novel crane quantitative PCR (Crane1) assay was developed, and its applicability as a microbial source tracking (MST) assay was evaluated by determining its host specificity and detection ability in environmental waters. Bacteria from crane excreta were dominated by bacilli and proteobacteria, with a notable paucity of sequences homologous to Bacteroidetes and Clostridia. The Crane1 marker targeted a dominant clade of unclassified Lactobacillales sequences closely related to Catellicoccus marimammalium. The host distribution of the Crane1 marker was relatively high, being positive for 69% (66/96) of the crane excreta samples tested. The assay also showed high host specificity, with 95% of the nontarget fecal samples (i.e., n = 553; 20 different free-range hosts) being negative. Of the presumed crane-impacted water samples (n = 16), 88% were positive for the Crane1 assay, whereas none of the water samples not impacted by cranes were positive (n = 165). Bayesian statistical models of the Crane1 MST marker demonstrated high confidence in detecting true-positive signals and a low probability of false-negative signals from environmental water samples. Altogether, these data suggest that the newly developed marker could be used in environmental monitoring studies to study crane fecal pollution dynamics.

  15. Development and Evaluation of a Quantitative PCR Assay Targeting Sandhill Crane (Grus canadensis) Fecal Pollution

    PubMed Central

    Ryu, Hodon; Lu, Jingrang; Vogel, Jason; Elk, Michael; Chávez-Ramírez, Felipe; Ashbolt, Nicholas

    2012-01-01

    While the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no methods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gene fecal clone libraries. Using these sequences, a novel crane quantitative PCR (Crane1) assay was developed, and its applicability as a microbial source tracking (MST) assay was evaluated by determining its host specificity and detection ability in environmental waters. Bacteria from crane excreta were dominated by bacilli and proteobacteria, with a notable paucity of sequences homologous to Bacteroidetes and Clostridia. The Crane1 marker targeted a dominant clade of unclassified Lactobacillales sequences closely related to Catellicoccus marimammalium. The host distribution of the Crane1 marker was relatively high, being positive for 69% (66/96) of the crane excreta samples tested. The assay also showed high host specificity, with 95% of the nontarget fecal samples (i.e., n = 553; 20 different free-range hosts) being negative. Of the presumed crane-impacted water samples (n = 16), 88% were positive for the Crane1 assay, whereas none of the water samples not impacted by cranes were positive (n = 165). Bayesian statistical models of the Crane1 MST marker demonstrated high confidence in detecting true-positive signals and a low probability of false-negative signals from environmental water samples. Altogether, these data suggest that the newly developed marker could be used in environmental monitoring studies to study crane fecal pollution dynamics. PMID:22492437

  16. Development of Conventional and Real-Time Quantitative PCR Assays for Diagnosis and Monitoring of Scabies

    PubMed Central

    Wong, Samson S. Y.; Poon, Rosana W. S.; Chau, Sandy; Wong, Sally C. Y.; To, Kelvin K. W.; Cheng, Vincent C. C.; Fung, Kitty S. C.

    2015-01-01

    Scabies remains the most prevalent, endemic, and neglected ectoparasitic infestation globally and can cause institutional outbreaks. The sensitivity of routine microscopy for demonstration of Sarcoptes scabiei mites or eggs in skin scrapings is only about 50%. Except for three studies using conventional or two-tube nested PCR on a small number of cases, no systematic study has been performed to improve the laboratory diagnosis of this important infection. We developed a conventional and a real-time quantitative PCR (qPCR) assay based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of S. scabiei. The cox1 gene is relatively well conserved, with its sequence having no high levels of similarity to the sequences of other human skin mites, pathogenic zoonotic mites, or common house dust mite species. This mitochondrial gene is also present in large quantities in arthropod cells, potentially improving the sensitivity of a PCR-based assay. In our study, both assays were specific and were more sensitive than microscopy in diagnosing scabies, with positive and negative predictive values of 100%. The S. scabiei DNA copy number in the microscopy-positive specimens was significantly higher than that in the microscopy-negative specimens (median S. scabiei DNA copy number, 3.604 versus 2.457 log10 copies per reaction; P = 0.0213). In the patient with crusted scabies, the qPCR assay performed on lesional skin swabs instead of scrapings revealed that the parasite DNA load took about 2 weeks to become negative after treatment. The utility of using lesional skin swabs as an alternative sample for diagnosis of scabies by PCR should be further evaluated. PMID:25903566

  17. Development of Conventional and Real-Time Quantitative PCR Assays for Diagnosis and Monitoring of Scabies.

    PubMed

    Wong, Samson S Y; Poon, Rosana W S; Chau, Sandy; Wong, Sally C Y; To, Kelvin K W; Cheng, Vincent C C; Fung, Kitty S C; Yuen, K Y

    2015-07-01

    Scabies remains the most prevalent, endemic, and neglected ectoparasitic infestation globally and can cause institutional outbreaks. The sensitivity of routine microscopy for demonstration of Sarcoptes scabiei mites or eggs in skin scrapings is only about 50%. Except for three studies using conventional or two-tube nested PCR on a small number of cases, no systematic study has been performed to improve the laboratory diagnosis of this important infection. We developed a conventional and a real-time quantitative PCR (qPCR) assay based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of S. scabiei. The cox1 gene is relatively well conserved, with its sequence having no high levels of similarity to the sequences of other human skin mites, pathogenic zoonotic mites, or common house dust mite species. This mitochondrial gene is also present in large quantities in arthropod cells, potentially improving the sensitivity of a PCR-based assay. In our study, both assays were specific and were more sensitive than microscopy in diagnosing scabies, with positive and negative predictive values of 100%. The S. scabiei DNA copy number in the microscopy-positive specimens was significantly higher than that in the microscopy-negative specimens (median S. scabiei DNA copy number, 3.604 versus 2.457 log10 copies per reaction; P = 0.0213). In the patient with crusted scabies, the qPCR assay performed on lesional skin swabs instead of scrapings revealed that the parasite DNA load took about 2 weeks to become negative after treatment. The utility of using lesional skin swabs as an alternative sample for diagnosis of scabies by PCR should be further evaluated. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. Quantitative methods of measuring the sensitivity of the mouse sperm morphology assay

    SciTech Connect

    Moore, D.H.; Bennett, D.E.; Kranzler, D.; Wyrobek, A.J.

    1982-09-01

    In this study murine sperm were subjected to graded doses of X irradiation (0 to 120 rad) to determine whether quantitative measurements made on enlarged photographs of the sperm heads are related to radiation dose. We found that the Mahalanobis distance statistic, when used to measure distance in a multivariate space from a control group of measurements, could be used to classify sperm as normal or abnormal. The percent classified as abnormal by this method was found to be linearly related to dose. The results suggest that sensitivity of the murine sperm assay can be improved by selecting an optimal set of measurements. This improvement can reduce the doubling dose from approximately 70 rad to 10 to 15 rad while keeping the percentage of abnormal sperm in control mice at 3%, equal to the current visual method.

  19. Quantitative real-time PCR assay for Clostridium septicum in poultry gangrenous dermatitis associated samples.

    PubMed

    Neumann, A P; Dunham, S M; Rehberger, T G; Siragusa, G R

    2010-08-01

    Clostridium septicum is a spore-forming anaerobe frequently implicated in cases of gangrenous dermatitis (GD) and other spontaneously occurring myonecrotic infections of poultry. Although C. septicum is readily cultured from diseased tissues it can be difficult to enumerate due to its tendency to swarm over the surface of agar plates. In this study a quantitative real-time PCR assay was developed in order to more accurately measure the levels of C. septicum in healthy as well as GD associated poultry samples. The assay was specifically designed to target the C. septicum alpha toxin gene, csa, which is, to our knowledge, carried by all strains of C. septicum and has been shown to be essential for virulence. Genomic DNAs from a diverse collection of bacterial species, including closely related Clostridium chauvoei, Clostridium carnis, Clostridium tertium as well as several strains of Clostridium perfringens, all failed to produce a positive reaction. An approximate reproducible limit of detection in spiked extracts of at least 10(3) cfu/g of C. septicum was observed for a variety of different sample types. C. septicum levels in broiler chicken field samples estimated from the results of qPCR were statistically correlated to culture based enumerations obtained from those same tissues.

  20. Comparison of immunodiffusion techniques with standard complement fixation assay for quantitation of coccidioidal antibodies.

    PubMed Central

    Wieden, M A; Galgiani, J N; Pappagianis, D

    1983-01-01

    Quantitative immunodiffusion (QID) and complement fixation (CF) methods were compared for their agreement in detecting coccidioidal antibodies. For these studies, we assayed 719 sera from 181 patients with coccidioidomycosis. Over 60% of the specimens had CF results of 1:2 to 1:256. A total of 43 patients had five or more specimens obtained over periods of between 1 and 8 years. The QID method, as originally performed, agreed within a twofold dilution of the CF titer in 191 of 267 sera (71.5%). Modification of QID by repeated filling of the antigen and serum wells improved agreement to 84.7% (383 of 452 sera). The degree of CF titer change in patients over time periods was more closely matched by the modified than by the original QID method. Discrepancies between the CF and QID methods appeared not to be due to a subpopulation of patients. QID measurement of coccidioidal antibodies may be a useful substitute for the CF assay in certain clinical laboratories. Images PMID:6415091

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

    NASA Astrophysics Data System (ADS)

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-05-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85-112% (R2 = 0.962-0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants.

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

    PubMed Central

    Zhang, An Ni; Mao, Yanping; Zhang, Tong

    2016-01-01

    We designed novel quantitative real-time polymerase chain reaction (qPCR) primers for the polyphosphate kinase 1 (ppk1) gene, targeting eight individual “Candidatus Accumulibacter” (referred to as Accumulibacter) clades. An evaluation of primer sets was conducted regarding the coverage, specificity, and PCR efficiency. (i) All primer sets were designed to cover all available sequences of the target clade. (ii) The phylogenetic analysis of the sequences retrieved from the qPCR products by each primer set demonstrated a high level of specificity. (iii) All calibration curves presented high PCR efficiencies in the range of 85–112% (R2 = 0.962–0.998). In addition, the possible interference of non-target amplicons was individually examined using the qPCR assay for 13 Accumulibacter clades, which were either undetected or showed negligible detection. With the primers designed by other research groups, a highly selective and sensitive qPCR-based method was developed to quantify all Accumulibacter clades, with the exception of Clade IE, in one assay, which enables more comprehensive insights into the community dynamics. The applicability to environmental samples was demonstrated by profiling the Accumulibacter clades in activated sludge samples of nine full-scale wastewater treatment plants. PMID:27142574

  3. A revisited folding reporter for quantitative assay of protein misfolding and aggregation in mammalian cells.

    PubMed

    Gregoire, Simpson; Kwon, Inchan

    2012-10-01

    Protein misfolding and aggregation play important roles in many physiological processes. These include pathological protein aggregation in neurodegenerative diseases and biopharmaceutical protein aggregation during production in mammalian cells. To develop a simple non-invasive assay for protein misfolding and aggregation in mammalian cells, the folding reporter green fluorescent protein (GFP) system, originally developed for bacterial cells, was evaluated. As a folding reporter, GFP was fused to the C-terminus of a panel of human copper/zinc superoxide dismutase (SOD1) mutants with varying misfolding/aggregation propensities. Flow cytometric analysis of transfected HEK293T and NSC-34 cells revealed that the mean fluorescence intensities of the cells expressing GFP fusion of SOD1 variants exhibited an inverse correlation with the misfolding/aggregation propensities of the four SOD1 variants. Our results support the hypothesis that the extent of misfolding/aggregation of a target protein in mammalian cells can be quantitatively estimated by measuring the mean fluorescence intensity of the cells expressing GFP fusion. The assay method developed herein will facilitate the understanding of aggregation process of SOD1 variants and the identification of aggregation inhibitors. The method also has great promise for misfolding/aggregation studies of other proteins in mammalian cells. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. A Quantitative Microtiter Assay for Sialylated Glycoform Analyses Using Lectin Complexes

    PubMed Central

    Srinivasan, Karunya; Washburn, Nathaniel; Sipsey, Sandra F.; Meccariello, Robin; Meador, James W.; Ling, Leona E.; Manning, Anthony M.; Kaundinya, Ganesh V.

    2015-01-01

    Fidelity of glycan structures is a key requirement for biotherapeutics, with carbohydrates playing an important role for therapeutic efficacy. Comprehensive glycan profiling techniques such as liquid chromatography (LC) and mass spectrometry (MS), while providing detailed description of glycan structures, require glycan cleavage, labeling, and paradigms to deconvolute the considerable data sets they generate. On the other hand, lectins as probes on microarrays have recently been used in orthogonal approaches for in situ glycoprofiling but require analyte labeling to take advantage of the capabilities of automated microarray readers and data analysis they afford. Herein, we describe a lectin-based microtiter assay (lectin–enzyme-linked immunosorbent assay [ELISA]) to quantify terminal glycan moieties, applicable to in vitro and in-cell glycan-engineered Fc proteins as well as intact IgGs from intravenous immunoglobulin (IVIG), a blood product containing pooled polyvalent IgG antibodies extracted from plasma from healthy human donors. We corroborate our findings with industry-standard LC-MS profiling. This “customizable” ELISA juxtaposes readouts from multiple lectins, focusing on a subset of glycoforms, and provides the ability to discern single- versus dual-arm glycosylation while defining levels of epitopes at sensitivities comparable to MS. Extendable to other biologics, this ELISA can be used stand-alone or complementary to MS for quantitative glycan analysis. PMID:25851037

  5. A quantitative assay for assessing the effects of DNA lesions on transcription.

    PubMed

    You, Changjun; Dai, Xiaoxia; Yuan, Bifeng; Wang, Jin; Wang, Jianshuang; Brooks, Philip J; Niedernhofer, Laura J; Wang, Yinsheng

    2012-10-01

    Most mammalian cells in nature are quiescent but actively transcribing mRNA for normal physiological processes; thus, it is important to investigate how endogenous and exogenous DNA damage compromises transcription in cells. Here we describe a new competitive transcription and adduct bypass (CTAB) assay to determine the effects of DNA lesions on the fidelity and efficiency of transcription. Using this strategy, we demonstrate that the oxidatively induced lesions 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) and the methylglyoxal-induced lesion N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) strongly inhibited transcription in vitro and in mammalian cells. In addition, cdA and cdG, but not N(2)-CEdG, induced transcriptional mutagenesis in vitro and in vivo. Furthermore, when located on the template DNA strand, all examined lesions were primarily repaired by transcription-coupled nucleotide excision repair in mammalian cells. This newly developed CTAB assay should be generally applicable for quantitatively assessing how other DNA lesions affect DNA transcription in vitro and in cells.

  6. Development of quantitative and high-throughput assays of polyomavirus and papillomavirus DNA replication.

    PubMed

    Fradet-Turcotte, Amélie; Morin, Geneviève; Lehoux, Michaël; Bullock, Peter A; Archambault, Jacques

    2010-03-30

    Polyoma- and papillomaviruses genome replication is initiated by the binding of large T antigen (LT) and of E1 and E2, respectively, at the viral origin (ori). Replication of an ori-containing plasmid occurs in cells transiently expressing these viral proteins and is typically quantified by Southern blotting or PCR. To facilitate the study of SV40 and HPV31 DNA replication, we developed cellular assays in which transient replication of the ori-plasmid is quantified using a firefly luciferase gene located in cis to the ori. Under optimized conditions, replication of the SV40 and HPV31 ori-plasmids resulted in a 50- and 150-fold increase in firefly luciferase levels, respectively. These results were validated using replication-defective mutants of LT, E1 and E2 and with inhibitors of DNA replication and cell-cycle progression. These quantitative and high-throughput assays should greatly facilitate the study of SV40 and HPV31 DNA replication and the identification of small-molecule inhibitors of this process.

  7. Quantitative determination of VEGF165 in cell culture medium by aptamer sandwich based chemiluminescence assay.

    PubMed

    Shan, Siwen; He, Ziyi; Mao, Sifeng; Jie, Mingsha; Yi, Linglu; Lin, Jin-Ming

    2017-08-15

    In this work, we have developed a sensitive and selective chemiluminescence (CL) assay for vascular endothelial growth factor (VEGF165) quantitative detection based on two specific VEGF165 binding aptamers (Apt). VEGF is a predominant biomarker in cancer angiogenesis, and sensitive detection method of VEGF are highly demanded for both academic study and clinical diagnosis of multiple cancers. In our experiment, VEGF165 was captured in a sandwich structure assembled by two binding aptamers, one capture aptamer was immobilized on streptavidin-coated magnetic beads (MBs) and another VEGF-binding aptamer was labeled by biotin for further phosphatase conjunction. After Apt-VEGF-Apt sandwich was formed on MBs surface, alkaline phosphatase (ALP) was modified to the second aptamer to catalyze CL reaction. By applying 4-methoxy-4-(3-phosphatephenyl)-spiro-(1,2-dioxetane-3,2-adamantane) (AMPPD) as CL substrate, strong signal intensity was achieved. VEGF165 content as low as 1ng/mL was detected in standard spiked samples by our assay, and linear range of working curve was confirmed from 1 to 20ng/mL. Then our method was successfully applied for cell culture medium analysis and on-chip hypoxic HepG2-HUVEC co-culture model study with excellent accuracy equal to ELISA Kit. Our developed assay demonstrated an outstanding performance in VEGF165 quantification and may be further extended to clinical testing of important biomarkers as well as probing microchip cell culture model. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Quantitative, competitive PCR assay for HIV-1 using a microplate-based detection system.

    PubMed

    Guenthner, P C; Hart, C E

    1998-05-01

    We have developed a quantitative competitive PCR (QC-PCR) assay in a microplate format for quantifying human immunodeficiency virus Type 1 (HIV-1) DNA or RNA in a broad range of source materials. Our QC-PCR assay is a modification of technique originally described by Piatak et al. (1993), which is based on the presence of a competitive internal standard containing an internal 80-bp deletion of HIV-1 gag target sequence. For improved detection and quantification of the wild-type and internal-standard PCR products in a microplate format, we introduced a non-HIV, 31-bp insert into the internal standard as a probe hybridization site that does not cross-hybridize with wild-type HIV-1 products. By using a primer pair in which one primer is biotinylated, QC-PCRs can be bound to a streptavidin-coated microplate, denatured and probed with a digoxigenin (Dig)-labeled, wild-type or internal-standard probe. The hybridized Dig-labeled probes are detected with an anti-Dig antibody conjugated to detector molecules for luminometry (aequorin) or optical densitometry (peroxidase), yielding results that are quantifiable over the range of 100-10,000 copies of HIV gag. Tested source materials for HIV-1 DNA or RNA quantification include plasma, vaginal lavage and cultured cells. The application of the QC-PCR assay using the microplate format affords a convenient and cost-effective method for quantifying HIV-1 proviral and viral loads from a variety of body fluids, cells and tissues.

  9. Quantum-dot submicrobead-based immunochromatographic assay for quantitative and sensitive detection of zearalenone.

    PubMed

    Duan, Hong; Chen, Xuelan; Xu, Wei; Fu, Jinhua; Xiong, Yonghua; Wang, Andrew

    2015-01-01

    Mycotoxin pollutants are commonly related to cereal products and cause fatal threats in food safety, and therefore require simple and sensitive detection. In this work, quantum-dot (QD) submicrobeads (QBs) were synthesized by encapsulating CdSe/ZnS QDs using the microemulsion technique. The resultant QBs, with approximately 2800 times brighter luminescence than the corresponding QDs, were explored as novel fluorescent probes in the immunochromatographic assay (ICA) for sensitive and quantitative detection of zearalenone (ZEN) in corns. Various parameters that influenced the sensitivity and stability of QB-based ICA (QB-ICA) were investigated and optimized. The optimal QB-ICA exhibits good dynamic linear detection for ZEN over the range of 0.125 ng/mL to 10 ng/mL with a median inhibitory concentration of 1.01±0.09 ng/mL (n=3). The detection limits for ZEN in a standard solution and real corn sample (dilution ratio of 1:30) are 0.0625 ng/mL and 3.6 µg/kg, respectively, which is much better than that of a previously reported gold nanoparticle-based ICA method. Forty-six natural corn samples are assayed using both QB-ICA and enzyme-linked immunosorbent assay. The two methods show a highly significant correlation (R(2)=0.92). Nine ZEN-contaminated samples were further confirmed with liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the QB-ICA results also exhibited good agreement with LC-MS/MS method. In brief, this work demonstrates that QB-ICA is capable of rapid, sensitive screening of toxins in food analysis, and shows great promise for point-of-care testing of other analytes.

  10. A quantitative method for the measurement of membrane affinity by polydiacetylene-based colorimetric assay.

    PubMed

    Zheng, Feng; Wu, Zheng; Chen, Yihua

    2012-01-15

    The measurement of membrane affinity is an important early screening step during drug discovery. However, classical methods for membrane affinity measurement are tedious and difficult to implement in high-throughput screening. This article describes a quantitative method for the measurement of membrane affinity by colorimetric assay based on polydiacetylene (PDA) sensors. Prepared lipid/PDA chromatic vesicles were used to model cell membranes. By measuring the colorimetric response of the chromatic vesicles when drug-membrane interactions occurred, membrane affinity constant K(b) could be calculated using a simple quantitative model. Under optimized preparation conditions, the calculated log(K(b)) values exhibited an in-batch relative standard deviation (RSD) of less than 4% and a between-batch RSD of less than 8% for all three reference compounds. The logarithm of K(b) of the six β-blockers exhibited excellent linear correlation with the logarithm of the liposome/water partition coefficient (K(m)) with R(2)=0.9793. For neutral compounds, the log(K(b)) of n-fatty alcohols correlated with the logarithm of the n-octanol/water partition coefficient (K(oct)) with a linear correlation coefficient R(2)=0.9833. This work provides a simple, convenient, and reproducible method for the rapid measurement of membrane affinity and presents important implications for high-throughput screening. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Characterization of a method for quantitating food consumption for mutation assays in Drosophila

    SciTech Connect

    Thompson, E.D.; Reeder, B.A.; Bruce, R.D. )

    1991-01-01

    Quantitation of food consumption is necessary when determining mutation responses to multiple chemical exposures in the sex-linked recessive lethal assay in Drosophila. One method proposed for quantitating food consumption by Drosophila is to measure the incorporation of 14C-leucine into the flies during the feeding period. Three sources of variation in the technique of Thompson and Reeder have been identified and characterized. First, the amount of food consumed by individual flies differed by almost 30% in a 24 hr feeding period. Second, the variability from vial to vial (each containing multiple flies) was around 15%. Finally, the amount of food consumed in identical feeding experiments performed over the course of 1 year varied nearly 2-fold. The use of chemical consumption values in place of exposure levels provided a better means of expressing the combined mutagenic response. In addition, the kinetics of food consumption over a 3 day feeding period for exposures to cyclophosphamide which produce lethality were compared to non-lethal exposures. Extensive characterization of lethality induced by exposures to cyclophosphamide demonstrate that the lethality is most likely due to starvation, not chemical toxicity.

  12. Quantitative Analysis of Small-Subunit rRNA Genes in Mixed Microbial Populations via 5′-Nuclease Assays

    PubMed Central

    Suzuki, Marcelino T.; Taylor, Lance T.; DeLong, Edward F.

    2000-01-01

    Few techniques are currently available for quantifying specific prokaryotic taxa in environmental samples. Quantification of specific genotypes has relied mainly on oligonucleotide hybridization to extracted rRNA or intact rRNA in whole cells. However, low abundance and cellular rRNA content limit the application of these techniques in aquatic environments. In this study, we applied a newly developed quantitative PCR assay (5′-nuclease assay, also known as TaqMan) to quantify specific small-subunit (SSU) rRNA genes (rDNAs) from uncultivated planktonic prokaryotes in Monterey Bay. Primer and probe combinations for quantification of SSU rDNAs at the domain and group levels were developed and tested for specificity and quantitative reliability. We examined the spatial and temporal variations of SSU rDNAs from Synechococcus plus Prochlorococcus and marine Archaea and compared the results of the quantitative PCR assays to those obtained by alternative methods. The 5′-nuclease assays reliably quantified rDNAs over at least 4 orders of magnitude and accurately measured the proportions of genes in artificial mixtures. The spatial and temporal distributions of planktonic microbial groups measured by the 5′-nuclease assays were similar to the distributions estimated by quantitative oligonucleotide probe hybridization, whole-cell hybridization assays, and flow cytometry. PMID:11055900

  13. Mass spectrometry assay as an alternative to the enzyme-linked immunosorbent assay test for biomarker quantitation in ecotoxicology: application to vitellogenin in Crustacea (Gammarus fossarum).

    PubMed

    Simon, Romain; Jubeaux, Guillaume; Chaumot, Arnaud; Lemoine, Jérôme; Geffard, Olivier; Salvador, Arnaud

    2010-07-30

    Vitellogenin (Vg) is a widespread biomarker for measuring exposure to endocrine disruptors. Vg quantification is usually done by using the ELISA test (enzyme-linked immunosorbent assay). Since this test is specific to a target protein, it can rarely be used with other species due to low cross-reactivity across species. Therefore alternative analytical methods have to be considered as the development of a specific and sensitive ELISA test for each new target is time-consuming and may prove unsuccessful. This paper focuses on the development of a quantitative assay by liquid chromatography tandem mass spectrometry (LC-MS/MS) of vitelogenin in an invertebrate (Gammarus fossarum) for which no ELISA kit is available. The linearity of the method was within the concentration range 2.5-25,000pg/mL and the limit of detection was estimated at 0.75pg/mL of Vg. This method has been demonstrated to be an alternative to existing immunological methods for quantifying Vg in invertebrates due to its greater sensitivity, specificity and reproducibility (intra- and inter-assay below 15%). This assay was applied for Vg determination in female G. fossarum following exposure to a known endocrine disruptor, methyl farnesoate, in crustaceans. The availability of a quantitative G. fossarum LC-MS/MS assay should open the way for further studies to evaluate xenoestrogen effects in aquatic male G. fossarum.

  14. Development of candidate reference reagent for HIV-1 RNA and comparison analysis for different HIV-1 RNA quantitative assay.

    PubMed

    Park, Borae G; Park, Ae Ja; Choi, Jee-Hye; Park, Jina; Kim, Sung Soon; Wang, Jin-Sook; Kee, Mee Kyung; Choi, Ju-yeon

    2011-09-01

    Human immunodeficiency virus type-1 (HIV-1) RNA viral load is a surrogate marker that is routinely used to determine indications for, and monitor the effectiveness of HIV-1 treatment. We developed three reagents for potential use in routine quality control of HIV-1 RNA quantitative assays. In this report, we compare the stability of these re-agents in storage and compare their performance in three different HIV-1 RNA quantitative assays. The candidate reagents were derived from readily available pre-existing reagents and examined for stability at different storage temperatures. They were compared in three commercially available HIV-1 RNA quantitative assays: the Cobas TaqMan HIV-1 Test (Cobas TaqMan), the RealTime HIV-1 Assay (Abbott RealTime), and the NucliSens EasyQ HIV-1 Assay v1.1 (NucliSens EasyQ). The candidate reagent derived from an HIV culture supernatant (candidate CS) was the most stable of the three candidates and showed good reproducibility. Candidate CS yielded the highest HIV-1 titer of the three candidates in the Cobas TaqMan assay and the lowest HIV-1 titer and stability of the three candidates in the NucliSens EasyQ system. The candidate CS is the most appropriate of the three candidate reagents for quantitative testing of HIV-1 RNA. This working reagent should be useful for use in routine calibration for quality control in centers with limited financial resources. The Cobas TaqMan assay tended to yield higher viral load results than the other assays when used with our three candidate reagents.

  15. Comparison of colorimetric assays with quantitative amino acid analysis for protein quantification of Generalized Modules for Membrane Antigens (GMMA).

    PubMed

    Rossi, Omar; Maggiore, Luana; Necchi, Francesca; Koeberling, Oliver; MacLennan, Calman A; Saul, Allan; Gerke, Christiane

    2015-01-01

    Genetically induced outer membrane particles from Gram-negative bacteria, called Generalized Modules for Membrane Antigens (GMMA), are being investigated as vaccines. Rapid methods are required for estimating the protein content for in-process assays during production. Since GMMA are complex biological structures containing lipid and polysaccharide as well as protein, protein determinations are not necessarily straightforward. We compared protein quantification by Bradford, Lowry, and Non-Interfering assays using bovine serum albumin (BSA) as standard with quantitative amino acid (AA) analysis, the most accurate currently available method for protein quantification. The Lowry assay has the lowest inter- and intra-assay variation and gives the best linearity between protein amount and absorbance. In all three assays, the color yield (optical density per mass of protein) of GMMA was markedly different from that of BSA with a ratio of approximately 4 for the Bradford assay, and highly variable between different GMMA; and approximately 0.7 for the Lowry and Non-Interfering assays, highlighting the need for calibrating the standard used in the colorimetric assay against GMMA quantified by AA analysis. In terms of a combination of ease, reproducibility, and proportionality of protein measurement, and comparability between samples, the Lowry assay was superior to Bradford and Non-Interfering assays for GMMA quantification.

  16. Multicolor bioluminescence boosts malaria research: quantitative dual-color assay and single-cell imaging in Plasmodium falciparum parasites.

    PubMed

    Cevenini, Luca; Camarda, Grazia; Michelini, Elisa; Siciliano, Giulia; Calabretta, Maria Maddalena; Bona, Roberta; Kumar, T R Santha; Cara, Andrea; Branchini, Bruce R; Fidock, David A; Roda, Aldo; Alano, Pietro

    2014-09-02

    New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z' factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z' factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing D-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level.

  17. Serial Quantitative PCR Assay for Detection, Species Discrimination, and Quantification of Leishmania spp. in Human Samples▿

    PubMed Central

    Weirather, Jason L.; Jeronimo, Selma M. B.; Gautam, Shalini; Sundar, Shyam; Kang, Mitchell; Kurtz, Melissa A.; Haque, Rashidul; Schriefer, Albert; Talhari, Sinésio; Carvalho, Edgar M.; Donelson, John E.; Wilson, Mary E.

    2011-01-01

    The Leishmania species cause a variety of human disease syndromes. Methods for diagnosis and species differentiation are insensitive and many require invasive sampling. Although quantitative PCR (qPCR) methods are reported for leishmania detection, no systematic method to quantify parasites and determine the species in clinical specimens is established. We developed a serial qPCR strategy to identify and rapidly differentiate Leishmania species and quantify parasites in clinical or environmental specimens. SYBR green qPCR is mainly employed, with corresponding TaqMan assays for validation. The screening primers recognize kinetoplast minicircle DNA of all Leishmania species. Species identification employs further qPCR set(s) individualized for geographic regions, combining species-discriminating probes with melt curve analysis. The assay was sufficient to detect Leishmania parasites, make species determinations, and quantify Leishmania spp. in sera, cutaneous biopsy specimens, or cultured isolates from subjects from Bangladesh or Brazil with different forms of leishmaniasis. The multicopy kinetoplast DNA (kDNA) probes were the most sensitive and useful for quantification based on promastigote standard curves. To test their validity for quantification, kDNA copy numbers were compared between Leishmania species, isolates, and life stages using qPCR. Maxicircle and minicircle copy numbers differed up to 6-fold between Leishmania species, but the differences were smaller between strains of the same species. Amastigote and promastigote leishmania life stages retained similar numbers of kDNA maxi- or minicircles. Thus, serial qPCR is useful for leishmania detection and species determination and for absolute quantification when compared to a standard curve from the same Leishmania species. PMID:22042830

  18. Electron paramagnetic resonance method for the quantitative assay of ketoconazole in pharmaceutical preparations.

    PubMed

    Morsy, Mohamed A; Sultan, Salah M; Dafalla, Hatim

    2009-08-15

    In this study, electron paramagnetic resonance (EPR) is used, for the first time, as an analytical tool for the quantitative assay of ketoconazole (KTZ) in drug formulations. The drug was successfully characterized by the prominent signals by two radical species produced as a result of its oxidation with 400 microg/mL cerium(IV) in 0.10 mol dm(-3) sulfuric acid. The EPR signal of the reaction mixture was measured in eight capillary tubes housed in a 4 mm EPR sample tube. The radical stability was investigated by obtaining multi-EPR scans of each KTZ sample solution at time intervals of 2.5 min of the reaction mixing time. The plot of the disappearance of the radical species show that the disappearance is apparently of zero order. The zero-time intercept of the EPR signal amplitude, which should be proportional to the initial radical concentration, is linear in the sample concentration in the range between 100 and 400 microg/mL, with a correlation coefficient, r, of 0.999. The detection limit was determined to be 11.7 +/- 2.5 microg/mL. The method newly adopted was fully validated following the United States Pharmacopeia (USP) monograph protocol in both the generic and the proprietary forms. The method is very accurate, such that we were able to measure the concentration at confidence levels of 99.9%. The method was also found to be suitable for the assay of KTZ in its tablet and cream pharmaceutical preparations, as no interferences were encountered from excipients of the proprietary drugs. High specificity, simplicity, and rapidity are the merits of the present method compared to the previously reported methods.

  19. Botulinum Neurotoxins: Qualitative and Quantitative Analysis Using the Mouse Phrenic Nerve Hemidiaphragm Assay (MPN).

    PubMed

    Bigalke, Hans; Rummel, Andreas

    2015-11-25

    The historical method for the detection of botulinum neurotoxin (BoNT) is represented by the mouse bioassay (MBA) measuring the animal survival rate. Since the endpoint of the MBA is the death of the mice due to paralysis of the respiratory muscle, an ex vivo animal replacement method, called mouse phrenic nerve (MPN) assay, employs the isolated N. phrenicus-hemidiaphragm tissue. Here, BoNT causes a dose-dependent characteristic decrease of the contraction amplitude of the indirectly stimulated muscle. Within the EQuATox BoNT proficiency 13 test samples were analysed using the MPN assay by serial dilution to a bath concentration resulting in a paralysis time within the range of calibration curves generated with BoNT/A, B and E standards, respectively. For serotype identification the diluted samples were pre-incubated with polyclonal anti-BoNT/A, B or E antitoxin or a combination of each. All 13 samples were qualitatively correctly identified thereby delivering superior results compared to single in vitro methods like LFA, ELISA and LC-MS/MS. Having characterized the BoNT serotype, the final bath concentrations were calculated using the calibration curves and then multiplied by the respective dilution factor to obtain the sample concentration. Depending on the source of the BoNT standards used, the quantitation of ten BoNT/A containing samples delivered a mean z-score of 7 and of three BoNT/B or BoNT/E containing samples z-scores <2, respectively.

  20. Bacteroides gingivalis-Actinomyces viscosus cohesive interactions as measured by a quantitative binding assay

    SciTech Connect

    Schwarz, S.; Ellen, R.P.; Grove, D.A.

    1987-10-01

    There is limited evidence, mostly indirect, to suggest that the adherence of Bacteroides gingivalis to teeth may be enhanced by the presence of gram-positive dental plaque bacteria like Actinomyces viscosus. The purpose of this study was to carry out direct quantitative assessments of the cohesion of B gingivalis and A. viscosus by using an in vitro assay modeled on the natural sequence in which these two species colonize the teeth. The assay allowed comparisons to be made of the adherence of /sup 3/H-labeled B. gingivalis 2561 and 381 to saliva-coated hydroxyapatite beads (S-HA) and A. viscosus WVU627- or T14V-coated S-HA (actinobeads) in equilibrium and kinetics binding studies. A series of preliminary binding studies with 3H-labeled A. viscosus and parallel studies by scanning electron microscopy with unlabeled A. viscosus were conducted to establish a protocol by which actinobeads suitable for subsequent Bacteroides adherence experiments could be prepared. By scanning electron microscopy, the actinobeads had only small gaps of exposed S-HA between essentially irreversibly bound A. viscosus cells. Furthermore, B. gingivalis cells appeared to bind preferentially to the Actinomyces cells instead of the exposed S-HA. B. gingivalis binding to both S-HA and actinobeads was saturable with at least 2 X 10(9) to 3 X 10(9) cells per ml, and equilibrium with saturating concentrations was reached within 10 to 20 min. B. gingivalis always bound in greater numbers to the actinobeads than to S-HA. These findings provide direct measurements supporting the concept that cohesion with dental plaque bacteria like A. viscosus may foster the establishment of B. gingivalis on teeth by enhancing its adherence.

  1. A rapid colorimetric assay for the quantitation of the viability of free-living larvae of nematodes in vitro.

    PubMed

    James, Catherine E; Davey, Mary W

    2007-09-01

    With increasing drug resistance in gastrointestinal parasites, identification of new anthelmintics is essential. The non-parasitic nematode Caenorhabditis elegans is used extensively as a model to identify drug targets and potential novel anthelmintics because it can be readily cultured in vitro. Traditionally, the assessment of worm viability has relied on labour-intensive developmental and behavioral assays. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide-formazan (MTT-formazan) colorimetric assay uses metabolic activity as a marker of viability in mammalian cell culture systems and has been applied for use with filarial nematodes. In the present study, this assay has been optimized and validated to rapidly assess the viability of C. elegans after drug treatment. Living, but not dead, C. elegans take up MTT and reduce it to the blue formazan, providing visual, qualitative, and quantitative assessment of viability. MTT at a concentration of 5 mg/ml with 3 h incubation was optimal for detecting changes in viability with drug treatment. We have applied this assay to quantitate the effects of ivermectin and short-chain alcohols on the viability of C. elegans. This assay is also applicable to first-stage larvae of the parasitic nematode Haemonchus contortus. The advantage of this assay is the rapid quantitation in screening drugs to identify potential anthelmintics.

  2. Development of stable isotope dilution assays for the quantitation of Amadori compounds in foods.

    PubMed

    Meitinger, Michael; Hartmann, Sandra; Schieberle, Peter

    2014-06-04

    During thermal processing of foods, reducing carbohydrates and amino acids may form 1-amino-1-desoxyketoses named Amadori rearrangement products after the Italian chemist Mario Amadori. Although these compounds are transient intermediates of the Maillard reaction, they are often used as suitable markers to measure the extent of a thermal food processing, such as for spray-dried milk or dried fruits. Several methods are already available in the literature for their quantitation, but measurements are often done with external calibration without addressing losses during the workup procedure. To cope with this challenge, stable isotope dilution assays in combination with LC-MS/MS were developed for the glucose-derived Amadori products of the seven amino acids valine, leucine, isoleucine, phenylalanine, tyrosine, methionine, and histidine using the respective synthesized [(13)C6]-labeled isotopologues as internal standards. The quantitation of the analytes added to a model matrix showed a very good sensitivity with the lowest limits of detection for the Amadori compound of phenylalanine of 0.1 μg/kg starch and 0.2 μg/kg oil, respectively. Also, the standard deviation measured in, for example, wheat beer was only ±2% for this analyte. Application of the method to several foods showed the highest concentrations of the Amadori product of valine in unroasted cocoa (342 mg/kg) as well as in dried bell pepper (3460 mg/kg). In agreement with literature data, drying of foods led to the formation of Amadori products, whereas they were degraded during roasting of, for example, coffee or cocoa. The study presents for the first time results on concentrations of the Amadori compounds of tyrosine and histidine in foods.

  3. Comparison of Quantitation of Cytomegalovirus DNA by Real-Time PCR in Whole Blood with the Cytomegalovirus Antigenemia Assay

    PubMed Central

    Kwon, Seonhee; Jung, Bo Kyeung; Ko, Sun-Young; Lee, Chang Kyu

    2015-01-01

    Background Quantitation of cytomegalovirus (CMV) DNA using real-time PCR has been utilized for monitoring CMV infection. However, the CMV antigenemia assay is still the 'gold standard' assay. There are only a few studies in Korea that compared the efficacy of use of real-time PCR for quantitation of CMV DNA in whole blood with the antigenemia assay, and most of these studies have been limited to transplant recipients. Method 479 whole blood samples from 79 patients, falling under different disease groups, were tested by real-time CMV DNA PCR using the Q-CMV real-time complete kit (Nanogen Advanced Diagnostic S.r.L., Italy) and CMV antigenemia assay (CINA Kit, ArgeneBiosoft, France), and the results were compared. Repeatedly tested patients were selected and their charts were reviewed for ganciclovir therapy. Results The concordance rate of the two assays was 86.4% (Cohen's kappa coefficient value=0.659). Quantitative correlation between the two assays was a moderate (r=0.5504, P<0.0001). Among 20 patients tested repeatedly with the two assays, 13 patients were transplant recipients and treated with ganciclovir. Before treatment, CMV was detected earlier by real-time CMV DNA PCR than the antigenemia assay, with a median difference of 8 days. After treatment, the antigenemia assay achieved negative results earlier than real-time CMV DNA PCR with a median difference of 10.5 days. Conclusions Q-CMV real-time complete kit is a useful tool for early detection of CMV infection in whole blood samples in transplant recipients. PMID:25553288

  4. A Quantitative Toxicogenomics Assay for High-throughput and Mechanistic Genotoxicity Assessment and Screening of Environmental Pollutants.

    PubMed

    Lan, Jiaqi; Gou, Na; Rahman, Sheikh Mokhles; Gao, Ce; He, Miao; Gu, April Z

    2016-03-15

    The ecological and health concern of mutagenicity and carcinogenicity potentially associated with an overwhelmingly large and ever-increasing number of chemicals demands for cost-effective and feasible method for genotoxicity screening and risk assessment. This study proposed a genotoxicity assay using GFP-tagged yeast reporter strains, covering 38 selected protein biomarkers indicative of all the seven known DNA damage repair pathways. The assay was applied to assess four model genotoxic chemicals, eight environmental pollutants and four negative controls across six concentrations. Quantitative molecular genotoxicity end points were derived based on dose response modeling of a newly developed integrated molecular effect quantifier, Protein Effect Level Index (PELI). The molecular genotoxicity end points were consistent with multiple conventional in vitro genotoxicity assays, as well as with in vivo carcinogenicity assay results. Further more, the proposed genotoxicity end point PELI values quantitatively correlated with both comet assay in human cell and carcinogenicity potency assay in mice, providing promising evidence for linking the molecular disturbance measurements to adverse outcomes at a biological relevant level. In addition, the high-resolution DNA damaging repair pathway alternated protein expression profiles allowed for chemical clustering and classification. This toxicogenomics-based assay presents a promising alternative for fast, efficient and mechanistic genotoxicity screening and assessment of drugs, foods, and environmental contaminants.

  5. Quantitative lateral flow strip assays as User-Friendly Tools To Detect Biomarker Profiles For Leprosy

    PubMed Central

    van Hooij, Anouk; Tjon Kon Fat, Elisa M.; Richardus, Renate; van den Eeden, Susan J. F.; Wilson, Louis; de Dood, Claudia J.; Faber, Roel; Alam, Korshed; Richardus, Jan Hendrik; Corstjens, Paul L. A. M.; Geluk, Annemieke

    2016-01-01

    Leprosy is a debilitating, infectious disease caused by Mycobacterium leprae. Despite the availability of multidrug therapy, transmission is unremitting. Thus, early identification of M. leprae infection is essential to reduce transmission. The immune response to M. leprae is determined by host genetics, resulting in paucibacillary (PB) and multibacillary (MB) leprosy associated with dominant cellular or humoral immunity, respectively. This spectral pathology of leprosy compels detection of immunity to M. leprae to be based on multiple, diverse biomarkers. In this study we have applied quantitative user friendly lateral flow assays (LFAs) for four immune markers (anti-PGL-I antibodies, IL-10, CCL4 and IP-10) for whole blood samples from a longitudinal BCG vaccination field-trial in Bangladesh. Different biomarker profiles, in contrast to single markers, distinguished M. leprae infected from non-infected test groups, patients from household contacts (HHC) and endemic controls (EC), or MB from PB patients. The test protocol presented in this study merging detection of innate, adaptive cellular as well as humoral immunity, thus provides a convenient tool to measure specific biomarker profiles for M. leprae infection and leprosy utilizing a field-friendly technology. PMID:27682181

  6. Broadband reflectance spectroscopy for establishing a quantitative metric of vascular leak using the Miles assay

    NASA Astrophysics Data System (ADS)

    McMurdy, John; Reichner, Jonathan; Mathews, Zara; Markey, Mary; Intwala, Sunny; Crawford, Gregory

    2009-09-01

    Monitoring the physiological effects of biological mediators on vascular permeability is important for identifying potential targets for antivascular leak therapy. This therapy is relevant to treatments for pulmonary edema and other disorders. Current methods of quantifying vascular leak are in vitro and do not allow repeated measurement of the same animal. Using an in vivo diffuse reflectance optical method allows pharmacokinetic analysis of candidate antileak molecules. Here, vascular leak is assessed in mice and rats by using the Miles assay and introducing irritation both topically using mustard oil and intradermally using vascular endothelial growth factor (VEGF). The severity of the leak is assessed using broadband diffuse reflectance spectroscopy with a fiber reflectance probe. Postprocessing techniques are applied to extract an artificial quantitative metric of leak from reflectance spectra at vascular leak sites on the skin of the animal. This leak metric is calculated with respect to elapsed time from irritation in both mustard oil and VEGF treatments on mice and VEGF treatments on rats, showing a repeatable increase in leak metric with leak severity. Furthermore, effects of pressure on the leak metric are observed to have minimal effect on the reflectance spectra, while spatial positioning showed spatially nonuniform leak sites.

  7. Real-time quantitative PCR assay for monitoring of nervous necrosis virus infection in grouper aquaculture.

    PubMed

    Kuo, Hsiao-Che; Wang, Ting-Yu; Chen, Peng-Peng; Chen, Young-Mao; Chuang, Hui-Ching; Chen, Tzong-Yueh

    2011-03-01

    Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r(2) = 0.99) between threshold cycle (C(T)) and RNA quantities, which allowed identification of infected groupers by the C(T) value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture.

  8. A Guide to Quantitative Biomarker Assay Development using Whispering Gallery Mode Biosensors.

    PubMed

    Robison, Heather M; Bailey, Ryan C

    2017-09-14

    Whispering gallery mode (WGM) sensors are a class of powerful analytical techniques defined by the measurement of changes in the local refractive index at or near the sensor surface. When functionalized with target-specific capture agents, analyte binding can be measured with very low limits of detection. There are many geometric manifestations of WGM sensors, with chip-integrated silicon photonic devices first commercialized because of the robust, wafer-scale device fabrication, facile optical interrogation, and amenability to the creation of multiplexed sensor arrays. Using these arrays, a number of biomolecular targets have been detected in both label-free and label-enhanced assay formats. For example, sub-picomolar detection limits for multiple cytokines were achieved using an enzymatically enhanced sandwich immunoassay that showed high analyte specificity suitable for detection in complex, clinical matrices. This protocol describes a generalizable approach for the development of quantitative, multiplexed immunoassays using silicon photonic microrings as an example WGM platform. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

  9. Are extraction methods in quantitative assays of pharmacopoeia monographs exhaustive? A comparison with pressurized liquid extraction.

    PubMed

    Basalo, Carlos; Mohn, Tobias; Hamburger, Matthias

    2006-10-01

    The extraction methods in selected monographs of the European and the Swiss Pharmacopoeia were compared to pressurized liquid extraction (PLE) with respect to the yield of constituents to be dosed in the quantitative assay for the respective herbal drugs. The study included five drugs, Belladonnae folium, Colae semen, Boldo folium, Tanaceti herba and Agni casti fructus. They were selected to cover different classes of compounds to be analyzed and different extraction methods to be used according to the monographs. Extraction protocols for PLE were optimized by varying the solvents and number of extraction cycles. In PLE, yields > 97 % of extractable analytes were typically achieved with two extraction cycles. For alkaloid-containing drugs, the addition of ammonia prior to extraction significantly increased the yield and reduced the number of extraction cycles required for exhaustive extraction. PLE was in all cases superior to the extraction protocol of the pharmacopoeia monographs (taken as 100 %), with differences ranging from 108 % in case of parthenolide in Tanaceti herba to 343 % in case of alkaloids in Boldo folium.

  10. Rapid Detection and Quantitation of BRAF Mutations in Hairy Cell Leukemia Using a Sensitive Pyrosequencing Assay

    PubMed Central

    Verma, Shalini; Greaves, Wesley O.; Ravandi, Farhad; Reddy, Neelima; Bueso-Ramos, Carlos E.; O’Brien, Susan; Thomas, Deborah A.; Kantarjian, Hagop; Medeiros, L. Jeffrey; Luthra, Rajyalakshmi; Patel, Keyur P.

    2017-01-01

    BRAF proto-oncogene is an important mediator of cell proliferation and survival signals via the RAS-RAF-MEK-ERK signaling pathway. BRAF p.V600E mutation was recently described as a molecular marker of hairy cell leukemia (HCL). We developed and validated a pyrosequencing-based approach that covers BRAF mutational hotspots in exons 11 (codon 468) and 15 (codons 595 to 600) and detects BRAF mutations at an analytical sensitivity of 5%. We screened 16 unenriched archived bone marrow aspirate samples from patients with diagnosis of HCL (n=12) and hairy cell leukemia-variant (HCL-v) (n=4) using pyrosequencing. BRAF p.V600E mutation was present in all HCL cases and absent in all HCL-v. Our data support the recent finding that BRAF p.V600E mutation is universally present in HCL. Moreover, our pyrosequencing based assay provides a convenient, rapid, sensitive and quantitative tool for the detection of BRAF p.V600E mutations in HCL in clinical setting. PMID:22706871

  11. A quantitative multiplexed mass spectrometry assay for studying the kinetic of residue-specific histone acetylation.

    PubMed

    Kuo, Yin-Ming; Henry, Ryan A; Andrews, Andrew J

    2014-12-01

    Histone acetylation is involved in gene regulation and, most importantly, aberrant regulation of histone acetylation is correlated with major human diseases. Although many lysine acetyltransferases (KATs) have been characterized as being capable of acetylating multiple lysine residues on histones, how different factors such as enzyme complexes or external stimuli (e.g. KAT activators or inhibitors) alter KAT specificity remains elusive. In order to comprehensively understand how the homeostasis of histone acetylation is maintained, a method that can quantitate acetylation levels of individual lysines on histones is needed. Here we demonstrate that our mass spectrometry (MS)-based method accomplishes this goal. In addition, the high throughput, high sensitivity, and high dynamic range of this method allows for effectively and accurately studying steady-state kinetics. Based on the kinetic parameters from in vitro enzymatic assays, we can determine the specificity and selectivity of a KAT and use this information to understand what factors influence histone acetylation. These approaches can be used to study the enzymatic mechanisms of histone acetylation as well as be adapted to other histone modifications. Understanding the post-translational modification of individual residues within the histones will provide a better picture of chromatin regulation in the cell.

  12. Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays

    PubMed Central

    Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J. L.; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

    2016-01-01

    Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download. PMID:26863543

  13. Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays.

    PubMed

    Kimura, Yasumasa; Soma, Takahiro; Kasahara, Naoko; Delobel, Diane; Hanami, Takeshi; Tanaka, Yuki; de Hoon, Michiel J L; Hayashizaki, Yoshihide; Usui, Kengo; Harbers, Matthias

    2016-01-01

    Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.

  14. Quantitation of Rotundone in Grapefruit (Citrus paradisi) Peel and Juice by Stable Isotope Dilution Assay.

    PubMed

    Nakanishi, Akira; Fukushima, Yusuke; Miyazawa, Norio; Yoshikawa, Keisuke; Maeda, Tomoko; Kurobayashi, Yoshiko

    2017-06-21

    Aroma extract dilution analyses of the aromas of peels and juices of white and pink grapefruits revealed that rotundone, responsible for peppery, spicy, and woody odors, was detected for the first time at high flavor dilution factors of 256-1024. In both juices, rotundone was detected at the highest flavor dilution factor of 1024. Rotundone in grapefruits was quantitated by a stable isotope dilution assay with a newly synthesized deuterium-labeled internal standard, rotundone-d2,3: its levels were 2180 and 1920 ng/kg in white and pink grapefruit peels and 29.6 and 49.8 ng/kg in white and pink grapefruit juices, respectively. On the basis of these results, sensory analysis was performed to assess the effects of rotundone on a white grapefruit juice aroma reconstitute. This sensory analysis revealed that rotundone does not impart a woody odor or affect any of the existing attributes, but increases various attributes, thus confirming that rotundone is indispensable for the aroma of grapefruit juice.

  15. Diagnostic value of a "wide-range" quantitative nested real-time PCR assay for varicella zoster virus myelitis.

    PubMed

    Takahashi, Teruyuki; Tamura, Masato; Takasu, Toshiaki

    2013-11-01

    Myelitis is one of the rarest neurological complications of varicella zoster virus (VZV) infection. In this study, the authors remodeled the "wide-range" quantitative nested real-time (QNRT) polymerase chain reaction (PCR) assay to quantitatively detect a small amount of VZV-DNA in cerebrospinal fluid (CSF). For use as a specific internal control "calibrator," an original mutation-VZV (MZ) plasmid was developed. The initial copy number of VZV-DNA in CSF specimens was measured by the amplification rate of the MZ-plasmid. For 17 consecutive CSF specimens collected from three elderly patients with VZV myelitis, the diagnostic value of the wide-range QNRT-PCR assay was evaluated and compared with other conventional PCR assays and enzyme immunoassay (EIA). The MZ-plasmid demonstrated statistically uniform amplifications (F=1.016) against a wide range (1-100,000) of copy numbers of mimic VZV-DNA. The wide-range QNRT-PCR assay quantitatively and rapidly (within 48 hr) detected 5,863, 3,052, 958, and 6,721 copies/ml of VZV-DNA in the CSF specimens collected from all patients in the acute phase. Additionally, there was a significant difference (*P=0.023) in the copy number of VZV-DNA between before and after acyclovir treatment. Other conventional single PCR assays all revealed negative results, but were nevertheless time-consuming (7 days). The IgG EIA-value for VZV was continually elevated throughout the clinical course of all patients. The MZ-plasmid was thus regarded as an appropriate "calibrator" in the wide-range QNRT-PCR assay. This assay is a novel, rapid, accurate, quantitative, and highly sensitive technique, and will contribute as a reliable and useful clinical examination for the rapid diagnosis of VZV infection to central nervous system. © 2013 Wiley Periodicals, Inc.

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

    PubMed Central

    Larsen, Hans Henrik; Kovacs, Joseph A.; Stock, Frida; Vestereng, Vibeke H.; Lundgren, Bettina; Fischer, Steven H.; Gill, Vee J.

    2002-01-01

    A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r = 0.99) over 6 log values for standards containing ≥5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments. PMID:12149363

  17. Evaluation of assays for the identification and quantitation of muconic acid, a benzene metabolite in human urine

    SciTech Connect

    Bartczak, A.; Kline, S.A.; Yu, R.; Weisel, C.P.; Goldstein, B.D.; Witz, G.; Bechtold, W.E.

    1994-12-31

    Muconic acid (MA) is a urinary metabolite of benzene and has been used as a biomarker of exposure to benzene in humans exposed to levels as low as 1 ppm. We have modified a high-pressure liquid chromatography (HPLC) based assay for urinary MA by the use of a diode array detector. This modification increases the specificity of the HPLC-based assay by identifying false positives. In addition, we have developed a gas chromatography (GC) based assay that uses a flame ionization detector (GC-FID). Both assays identified and quantified MA in human urine at concentrations greater than 40-50 ng/ml. Assay precision was within 10% relative standard deviation for MA concentrations above 90 ng/ml using the HPLC assay and above 40 ng/ml using the GC-FID assay. Quantitative accuracy of the assays was evaluated by determining MA in human urine samples using both methods and also a gas chromatography-mass spectrometry (GC-MS) procedure. Numerical correlation among the three assays was good at MA concentrations above 100 ng/ml. 26 refs., 3 figs., 2 tabs.

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

    EPA Science Inventory

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

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

    USDA-ARS?s Scientific Manuscript database

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

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

    EPA Science Inventory

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

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

    USDA-ARS?s Scientific Manuscript database

    Real-time quantitative PCR assays that target the human-associated HF183 bacterial cluster have been found to be some of the top performing methods for the characterization of human fecal pollution in ambient surface waters. The United States Environmental Protection Agency is planning to conduct a ...

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  4. The rapid quantitation of the filamentous blue-green alga plectonema boryanum by the luciferase assay for ATP

    NASA Technical Reports Server (NTRS)

    Bush, V. N.

    1974-01-01

    Plectonema boryanum is a filamentous blue green alga. Blue green algae have a procaryotic cellular organization similar to bacteria, but are usually obligate photoautotrophs, obtaining their carbon and energy from photosynthetic mechanism similar to higher plants. This research deals with a comparison of three methods of quantitating filamentous populations: microscopic cell counts, the luciferase assay for ATP and optical density measurements.

  5. QUANTITATIVE ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETERMINATION OF POLYCHLORINATED BIPHENYLS IN ENVIRONMENTAL SOIL AND SEDIMENT SAMPLES

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of Aroclors 1242, 1248, 1254, and 1260 in soil and sediments was developed and its performance compared with that of gas chromatography (GC). The detection limits for Aroclors 1242 and 1248 in soil ar...

  6. QUANTITATIVE ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETERMINATION OF POLYCHLORINATED BIPHENYLS IN ENVIRONMENTAL SOIL AND SEDIMENT SAMPLES

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of Aroclors 1242, 1248, 1254, and 1260 in soil and sediments was developed and its performance compared with that of gas chromatography (GC). The detection limits for Aroclors 1242 and 1248 in soil ar...

  7. Multi-laboratory comparison of quantitative PCR assays for detection and quantification of Fusarium virguliforme from soybean roots and soil

    USDA-ARS?s Scientific Manuscript database

    Accurate identification and quantification of Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, within root tissue and soil are important tasks. Several quantitative PCR (qPCR) assays have been developed but there are no reports comparing their use in sensitive and specific...

  8. Multilaboratory Comparison of Quantitative PCR Assays for Detection and Quantification of Fusarium virguliforme from Soybean Roots and Soil.

    PubMed

    Kandel, Yuba R; Haudenshield, James S; Srour, Ali Y; Islam, Kazi Tariqul; Fakhoury, Ahmad M; Santos, Patricia; Wang, Jie; Chilvers, Martin I; Hartman, Glen L; Malvick, Dean K; Floyd, Crystal M; Mueller, Daren S; Leandro, Leonor F S

    2015-12-01

    The ability to accurately detect and quantify Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, in samples such as plant root tissue and soil is extremely valuable for accurate disease diagnoses and to address research questions. Numerous quantitative real-time polymerase chain reaction (qPCR) assays have been developed for this pathogen but their sensitivity and specificity for F. virguliforme have not been compared. In this study, six qPCR assays were compared in five independent laboratories using the same set of DNA samples from fungi, plants, and soil. Multicopy gene-based assays targeting the ribosomal DNA intergenic spacer (IGS) or the mitochondrial small subunit (mtSSU) showed relatively high sensitivity (limit of detection [LOD] = 0.05 to 5 pg) compared with a single-copy gene (FvTox1)-based assay (LOD = 5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (100%) and two IGS assays being slightly less specific (95 to 96%). Another IGS assay targeting four SDS-causing fusaria showed lower specificity (70%), while the two mtSSU assays were lowest (41 and 47%). An IGS-based assay showed consistently highest sensitivity (LOD = 0.05 pg) and specificity and inclusivity above 94% and, thus, is suggested as the most useful qPCR assay for F. virguliforme diagnosis and quantification. However, specificity was also above 94% in two other assays and their selection for diagnostics and research will depend on objectives, samples, and materials used. These results will facilitate both fundamental and disease management research pertinent to SDS.

  9. A rapid quantitative assay for the detection of mammalian heparanase activity.

    PubMed Central

    Freeman, C; Parish, C R

    1997-01-01

    Heparan sulphate (HS) is an important component of the extracellular matrix and the vasculature basal laminar which functions as a barrier to the extravasation of metastatic and inflammatory cells. Cleavage of HS by endoglycosidase or heparanase activity produced by invading cells may assist in the disassembly of the extracellular matrix and basal laminar, and thereby facilitate cell migration. Heparanase activity has previously been shown to be related to the metastatic potential of murine and human melanoma cell lines [Nakajima, Irimura and Nicolson (1988) J. Cell. Biochem. 36, 157-167]. To determine heparanase activity, porcine mucosal HS was partially de-N-acetylated and re-N-acetylated with [3H]acetic anhydride to yield a radiolabelled substrate. This procedure prevented the masking of, or possible formation of, new heparanase-sensitive cleavage sites as has been observed with previous methods of radiolabelling. Heparanase activity in a variety of tissues and cell homogenates including human platelets, colonic carcinoma cells, umbilical vein endothelial cells and rat mammary adenocarcinoma cells (both metastatic and non-metastatic variants) and liver homogenates all degraded the substrate in a stepwise fashion from 18.5 to approximately 13, 8 and finally to 4.5 kDa fragments, as assessed by gel-filtration analysis, confirming the substrate as suitable for the detection of heparanase activity present in a variety of cells and tissues. A rapid quantitative assay was developed with the HS substrate using a novel method for separating degradation products from the substrate by taking advantage of the decreased affinity of the heparanase-cleaved products for the HS-binding plasma protein chicken histidine-rich glycoprotein (cHRG). Incubation mixtures were applied to cHRG-Sepharose columns, with unbound material corresponding to heparanase-degradation products. Heparanase activity was determined for a variety of human, rat and murine cell and tissue homogenates. The

  10. Measuring stem cell frequency in epidermis: A quantitative in vivo functional assay for long-term repopulating cells

    NASA Astrophysics Data System (ADS)

    Schneider, T. E.; Barland, C.; Alex, A. M.; Mancianti, M. L.; Lu, Y.; Cleaver, J. E.; Lawrence, H. J.; Ghadially, R.

    2003-09-01

    Epidermal stem cells play a central role in tissue homeostasis, wound repair, tumor initiation, and gene therapy. A major impediment to the purification and molecular characterization of epidermal stem cells is the lack of a quantitative assay for cells capable of long-term repopulation in vivo, such as exists for hematopoietic cells. The tremendous strides made in the characterization and purification of hematopoietic stem cells have been critically dependent on the availability of competitive transplantation assays, because these assays permit the accurate quantitation of long-term repopulating cells in vivo. We have developed an analogous functional assay for epidermal stem cells, and have measured the frequency of functional epidermal stem cells in interfollicular epidermis. These studies indicate that cells capable of long-term reconstitution of a squamous epithelium reside in the interfollicular epidermis. We find that the frequency of these long-term repopulating cells is 1 in 35,000 total epidermal cells, or in the order of 1 in 104 basal epidermal cells, similar to that of hematopoietic stem cells in the bone marrow, and much lower than previously estimated in epidermis. Furthermore, these studies establish a novel functional assay that can be used to validate immunophenotypic markers and enrichment strategies for epidermal stem cells, and to quantify epidermal stem cells in various keratinocyte populations. Thus further studies using this type of assay for epidermis should aid in the progress of cutaneous stem cell-targeted gene therapy, and in more basic studies of epidermal stem cell regulation and differentiation.

  11. Analytical and Clinical Validation of the Immulite 1000 hCG Assay for Quantitative Analysis in Urine

    PubMed Central

    Cate, Frances L.; Moffett, Courtney; Gronowski, Ann M.; Grenache, David G.; Hartmann, Katherine E.; Woodworth, Alison

    2013-01-01

    Background The Siemens Immulite hCG assay detects all major hCG variants in serum. Currently, this assay is only FDA approved for qualitative measurement of hCG in urine. Methods Complete validation of the hCG assay in urine was performed on the Siemens Immulite 1000 immunoassay platform. Reference intervals were established for females <55 y, females ≥55 y, and males 20–70 y. Results The limit of quantitation was 2.0 IU/l. The Immulite hCG assay was precise for measuring hCG in urine from pregnant patients with intra- and inter-assay imprecision of <11% CV. The assay was linear over a dynamic range of 2–2600 IU/l and 2–3500 IU/l for hCG and hCGβ respectively. The assay was non-linear for hCGβcf. No hook effect was observed at concentrations up to 1,200,000 pmol/l, for hCGβ or hCGβcf. The reference intervals were <2.0 IU/l for males, <2.2 IU/l for females <55 y, and <12.2 IU/l for females ≥55 y. Conclusion The Immulite 1000 hCG assay can accurately quantify hCG in urine. PMID:23470427

  12. Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture

    USGS Publications Warehouse

    Elliott, D.G.; Applegate, L.J.; Murray, A.L.; Purcell, M.K.; McKibben, C.L.

    2013-01-01

    No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

  13. Quantitative measurement of serum hepatitis B surface antigen using an immunoradiometric assay in chronic hepatitis B.

    PubMed

    Kwon, Hyun-Woo; Lee, Ho-Young; Kim, Seog Gyun; Kim, Won; Jung, Yong Jin; Kang, Keon Wook; Chung, June-Key; Lee, Myung Chul; Lee, Dong Soo

    2011-03-01

    Measurement of serum hepatitis B virus surface antigen (HBsAg) levels is important for the management of chronic hepatitis B patients in terms of monitoring response to antiviral therapy. This study aimed to evaluate the diagnostic performance of a new diagnostic kit, which quantitatively measures serum HBsAg level using an immunoradiometric assay (IRMA)-based method. Measurements were compared with those obtained using a chemiluminescent microparticle immunoassay (CMIA)-based method. The blood samples of 96 patients with chronic hepatitis B were used in this study. Copy numbers of serum hepatitis B virus (HBV) DNA were determined in 23 of these samples. The correlation between and the concordance of IRMA and CMIA results were determined using Pearson's correlation coefficients. P values of 0.05 were considered to be statistically significant throughout. Laboratory diagnoses based on IRMA were wholly in agreement with those based on CMIA. Furthermore, serum HBsAg levels by IRMA were found to be highly correlated with those determined by CMIA (correlation coefficient R (2) = 0.838, p < 0.001). Serum HBsAg level and serum HBV DNA copies were found to be linearly related by both methods (R (2) = 0.067, p = 0.316 by IRMA, and R (2) = 0.101, p = 0.215 by CMIA). The diagnostic performance of the investigated IRMA method of determining HBsAg levels was found to be comparable with that of a CMIA-based method in chronic hepatitis B patients.

  14. Laboratory Assay of Brood Care for Quantitative Analyses of Individual Differences in Honey Bee (Apis mellifera) Affiliative Behavior

    PubMed Central

    Shpigler, Hagai Y.; Robinson, Gene E.

    2015-01-01

    Care of offspring is a form of affiliative behavior that is fundamental to studies of animal social behavior. Insects do not figure prominently in this topic because Drosophila melanogaster and other traditional models show little if any paternal or maternal care. However, the eusocial honey bee exhibits cooperative brood care with larvae receiving intense and continuous care from their adult sisters, but this behavior has not been well studied because a robust quantitative assay does not exist. We present a new laboratory assay that enables quantification of group or individual honey bee brood “nursing behavior” toward a queen larva. In addition to validating the assay, we used it to examine the influence of the age of the larva and the genetic background of the adult bees on nursing performance. This new assay also can be used in the future for mechanistic analyses of eusociality and comparative analyses of affilative behavior with other animals. PMID:26569402

  15. A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir

    PubMed Central

    Fun, Axel; Mok, Hoi Ping; Wills, Mark R.; Lever, Andrew M.

    2017-01-01

    Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4+ T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4+ T cells from whole PBMC is expedited and achieved in 3 hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity. PMID:28233807

  16. A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.

    PubMed

    Fun, Axel; Mok, Hoi Ping; Wills, Mark R; Lever, Andrew M

    2017-02-24

    Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4(+) T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4(+) T cells from whole PBMC is expedited and achieved in 3 hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity.

  17. Laboratory Assay of Brood Care for Quantitative Analyses of Individual Differences in Honey Bee (Apis mellifera) Affiliative Behavior.

    PubMed

    Shpigler, Hagai Y; Robinson, Gene E

    2015-01-01

    Care of offspring is a form of affiliative behavior that is fundamental to studies of animal social behavior. Insects do not figure prominently in this topic because Drosophila melanogaster and other traditional models show little if any paternal or maternal care. However, the eusocial honey bee exhibits cooperative brood care with larvae receiving intense and continuous care from their adult sisters, but this behavior has not been well studied because a robust quantitative assay does not exist. We present a new laboratory assay that enables quantification of group or individual honey bee brood "nursing behavior" toward a queen larva. In addition to validating the assay, we used it to examine the influence of the age of the larva and the genetic background of the adult bees on nursing performance. This new assay also can be used in the future for mechanistic analyses of eusociality and comparative analyses of affilative behavior with other animals.

  18. Inhibitory Activity of Human Immunodeficiency Virus Aspartyl Protease Inhibitors against Encephalitozoon intestinalis Evaluated by Cell Culture-Quantitative PCR Assay

    PubMed Central

    Menotti, Jean; Santillana-Hayat, Maud; Cassinat, Bruno; Sarfati, Claudine; Derouin, Francis; Molina, Jean-Michel

    2005-01-01

    Immune reconstitution might not be the only factor contributing to the low prevalence of microsporidiosis in human immunodeficiency virus (HIV)-infected patients treated with protease inhibitors, as these drugs may exert a direct inhibitory effect against fungi and protozoa. In this study, we developed a cell culture-quantitative PCR assay to quantify Encephalitozoon intestinalis growth in U-373-MG human glioblastoma cells and used this assay to evaluate the activities of six HIV aspartyl protease inhibitors against E. intestinalis. A real-time quantitative PCR assay targeted the E. intestinalis small-subunit rRNA gene. HIV aspartyl protease inhibitors were tested over serial concentrations ranging from 0.2 to 10 mg/liter, with albendazole used as a control. Ritonavir, lopinavir, and saquinavir were able to inhibit E. intestinalis growth, with 50% inhibitory concentrations of 1.5, 2.2, and 4.6 mg/liter, respectively, whereas amprenavir, indinavir, and nelfinavir had no inhibitory effect. Pepstatin A, a reference aspartyl protease inhibitor, could also inhibit E. intestinalis growth, suggesting that HIV protease inhibitors may act through the inhibition of an E. intestinalis-encoded aspartyl protease. These results showed that some HIV protease inhibitors can inhibit E. intestinalis growth at concentrations that are achievable in vivo and that the real-time quantitative PCR assay that we used is a valuable tool for the in vitro assessment of the activities of drugs against E. intestinalis. PMID:15917534

  19. Evaluation of a PCR assay for quantitation of Rickettsia rickettsii and closely related spotted fever group rickettsiae.

    PubMed

    Eremeeva, Marina E; Dasch, Gregory A; Silverman, David J

    2003-12-01

    A spotted fever rickettsia quantitative PCR assay (SQ-PCR) was developed for the detection and enumeration of Rickettsia rickettsii and other closely related spotted fever group rickettsiae. The assay is based on fluorescence detection of SYBR Green dye intercalation in a 154-bp fragment of the rOmpA gene during amplification by PCR. As few as 5 copies of the rOmpA gene of R. rickettsii can be detected. SQ-PCR is suitable for quantitation of R. rickettsii and 10 other genotypes of spotted fever group rickettsiae but not for R. akari, R. australis, R. bellii, or typhus group rickettsiae. The sensitivity of SQ-PCR was comparable to that of a plaque assay using centrifugation for inoculation. The SQ-PCR assay was applied successfully to the characterization of rickettsial stock cultures, the replication of rickettsiae in cell culture, the recovery of rickettsial DNA following different methods of extraction, and the quantitation of rickettsial loads in infected animal tissues, clinical samples, and ticks.

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

    PubMed

    Green, Hyatt C; Haugland, Richard A; Varma, Manju; Millen, Hana T; Borchardt, Mark A; Field, Katharine G; Walters, William A; Knight, R; Sivaganesan, Mano; Kelty, Catherine A; Shanks, Orin C

    2014-05-01

    Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters.

  1. Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

    PubMed

    Sangesland, Maya; Atwood-Moore, Angela; Rai, Sudhir K; Levin, Henry L

    2016-01-01

    Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

  2. DEVELOPMENT OF SEMI-QUANTITATIVE PCR ASSAYS FOR THE DETECTION AND ENUMERATION OF GAMBIERDISCUS SPECIES (GONYAULACALES, DINOPHYCEAE)(1).

    PubMed

    Vandersea, Mark W; Kibler, Steven R; Holland, William C; Tester, Patricia A; Schultz, Thomas F; Faust, Maria A; Holmes, Michael J; Chinain, Mirelle; Wayne Litaker, R

    2012-08-01

    Ciguatera fish poisoning (CFP) is a serious health problem in tropical regions and is caused by the bioaccumulation of lipophilic toxins produced by dinoflagellates in the genus Gambierdiscus. Gambierdiscus species are morphologically similar and are difficult to distinguish from one another even when using scanning electron microscopy. Improved identification and detection methods that are sensitive and rapid are needed to identify toxic species and investigate potential distribution and abundance patterns in relation to incidences of CFP. This study presents the first species-specific, semi-quantitative polymerase chain reaction (qPCR) assays that can be used to address these questions. These assays are specific for five Gambierdiscus species and one undescribed ribotype. The assays utilized a SYBR green format and targeted unique sequences found within the SSU, ITS, and the D1/D3 LSU ribosomal domains. Standard curves were constructed using known concentrations of cultured cells and 10-fold serial dilutions of rDNA PCR amplicons containing the target sequence for each specific assay. Assay sensitivity and accuracy were tested using DNA extracts purified from known concentrations of multiple Gambierdiscus species. The qPCR assays were used to assess Gambierdiscus species diversity and abundance in samples collected from nearshore areas adjacent to Ft. Pierce and Jupiter, Florida USA. The results indicated that the practical limit of detection for each assay was 10 cells per sample. Most interestingly, the qPCR analysis revealed that as many as four species of Gambierdiscus were present in a single macrophyte sample.

  3. Improved HF183 Quantitative Real-Time PCR Assay for Characterization of Human Fecal Pollution in Ambient Surface Water Samples

    PubMed Central

    Green, Hyatt C.; Haugland, Richard A.; Varma, Manju; Millen, Hana T.; Borchardt, Mark A.; Field, Katharine G.; Walters, William A.; Knight, R.; Sivaganesan, Mano; Kelty, Catherine A.

    2014-01-01

    Quantitative real-time PCR (qPCR) assays that target the human-associated HF183 bacterial cluster within members of the genus Bacteroides are among the most widely used methods for the characterization of human fecal pollution in ambient surface waters. In this study, we show that a current TaqMan HF183 qPCR assay (HF183/BFDrev) routinely forms nonspecific amplification products and introduce a modified TaqMan assay (HF183/BacR287) that alleviates this problem. The performance of each qPCR assay was compared in head-to-head experiments investigating limits of detection, analytical precision, predicted hybridization to 16S rRNA gene sequences from a reference database, and relative marker concentrations in fecal and sewage samples. The performance of the modified HF183/BacR287 assay is equal to or improves upon that of the original HF183/BFDrev assay. In addition, a qPCR chemistry designed to combat amplification inhibition and a multiplexed internal amplification control are included. In light of the expanding use of PCR-based methods that rely on the detection of extremely low concentrations of DNA template, such as qPCR and digital PCR, the new TaqMan HF183/BacR287 assay should provide more accurate estimations of human-derived fecal contaminants in ambient surface waters. PMID:24610857

  4. Strand-Specific Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Measurement of Arenavirus Genomic and Antigenomic RNAs

    PubMed Central

    Haist, Kelsey; Ziegler, Christopher; Botten, Jason

    2015-01-01

    Arenaviruses are bi-segmented, single-stranded RNA viruses that cause significant human disease. The manner in which they regulate the replication of their genome is not well-understood. This is partly due to the absence of a highly sensitive assay to measure individual species of arenavirus replicative RNAs. To overcome this obstacle, we designed a quantitative reverse transcription (RT)-PCR assay for selective quantitation of each of the lymphocytic choriomeningitis virus (LCMV) genomic or antigenomic RNAs. During the course of assay design, we identified a nonspecific priming phenomenon whereby, in the absence of an RT primer, cDNAs complementary to each of the LCMV replicative RNA species are generated during RT. We successfully circumvented this nonspecific priming event through the use of biotinylated primers in the RT reaction, which permitted affinity purification of primer-specific cDNAs using streptavidin-coated magnetic beads. As proof of principle, we used the assay to map the dynamics of LCMV replication at acute and persistent time points and to determine the quantities of genomic and antigenomic RNAs that are incorporated into LCMV particles. This assay can be adapted to measure total S or L segment-derived viral RNAs and therefore represents a highly sensitive diagnostic platform to screen for LCMV infection in rodent and human tissue samples and can also be used to quantify virus-cell attachment. PMID:25978311

  5. A mechanistic analysis of the quantitation of α-hydroxy ketones by the bicinchoninic acid assay.

    PubMed

    Weiser, Jennifer R; Ricapito, Nicole G; Yueh, Alice; Weiser, Ellen L; Putnam, David

    2012-11-15

    A new class of compounds amenable to quantification by the bicinchoninic acid (BCA) assay was identified, allowing an expansion of compounds quantifiable within the assay's capacity. In this article, we demonstrate that compounds containing the α-hydroxy ketone structure are easily measured under standard BCA assay conditions. A nonchromophore analyte containing the α-hydroxy ketone structure, 1,3-dihydroxypropan-2-one (commonly known as dihydroxyacetone), and various structural derivatives were explored on an equimolar basis in the BCA assay. Combined with earlier studies exploring α-hydroxy ketones within copper oxidation systems, the data support the mechanism of this class of compound's ability to enolize through an enediol intermediate to generate a strong signal in the BCA assay. This new quantification technique also highlights the potential for α-hydroxy ketones to interfere with other analytes quantified by the BCA assay.

  6. A semi-universal assay platform to quantitate vaccines with potential applications for biotherapeutics.

    PubMed

    Roman, Jeanette; Bouaraphan, Silikhone; Verch, Thorsten

    2016-12-01

    Biologics development often requires multiple immunoassays to evaluate both assay reagents and potential drug candidates resulting in extensive analytical development. We developed a semi-universal, 5-layer platform assay on Gyrolab using secondary antispecies or anti-isotype-specific capture and detection antibodies. We applied the assay to several multivalent vaccines. Method performance exhibited a median accuracy of 110%, reproducibility of 9% CV and intermediate precision of 11% CV. System suitability criteria were met for 92.5% of the samples and only one out of 31 replicate samples exhibited a %CV greater than 20%. The semi-universal Gyrolab assay allowed assay development without reagent labeling. The format could also be translated into a plate-based assay.

  7. An assay to measure the affinity of proteins for microtubules by quantitative fluorescent microscopy.

    PubMed

    Graczyk, Beth; Davis, Trisha N

    2011-03-15

    We report a fluorescence-based assay for measuring the affinity of microtubule binding proteins for microtubules. The affinity of any fluorescently tagged protein for taxol-stabilized microtubules can be measured with this assay. We describe the assay and provide a detailed protocol. Using this assay, we found that the affinity of the Dam1 complex for microtubules is decreased by the presence of free unpolymerized tubulin and is sensitive to the salt concentration in the binding buffer. These effects may account for the previous differences in binding affinities reported. Copyright © 2010 Elsevier Inc. All rights reserved.

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

    PubMed Central

    Carim, Kellie J.; Paroz, Yvette M.; McKelvey, Kevin S.; Young, Michael K.; Schwartz, Michael K.

    2016-01-01

    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. PMID:27583576

  9. Development of a high-throughput quantitative assay for detecting herpes simplex virus DNA in clinical samples.

    PubMed

    Ryncarz, A J; Goddard, J; Wald, A; Huang, M L; Roizman, B; Corey, L

    1999-06-01

    We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 10(8) copies of HSV DNA/20 microl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the

  10. Development of a High-Throughput Quantitative Assay for Detecting Herpes Simplex Virus DNA in Clinical Samples

    PubMed Central

    Ryncarz, Alexander J.; Goddard, James; Wald, Anna; Huang, Meei-Li; Roizman, Bernard; Corey, Lawrence

    1999-01-01

    We have developed a high-throughput, semiautomated, quantitative fluorescence-based PCR assay to detect and type herpes simplex virus (HSV) DNA in clinical samples. The detection assay, which uses primers to the type-common region of HSV glycoprotein B (gB), was linear from <10 to 108 copies of HSV DNA/20 μl of sample. Among duplicate samples in reproducibility runs, the assay showed less than 5% variability. We compared the fluorescence-based PCR assay with culture and gel-based liquid hybridization system with 335 genital tract specimens from HSV type 2 (HSV-2)-seropositive persons attending a research clinic and 380 consecutive cerebrospinal fluid (CSF) samples submitted to a diagnostic virology laboratory. Among the 162 culture-positive genital tract specimens, TaqMan PCR was positive for 157 (97%) specimens, whereas the quantitative-competitive PCR was positive for 144 (89%) specimens. Comparisons of the mean titer of HSV DNA detected by the two assays revealed that the mean titer detected by the gel-based system was slightly higher (median, 1 log). These differences in titers were in part related to the fivefold difference in the amount of HSV DNA used in the amplicon standards with the two assays. Among the 380 CSF samples, 42 were positive by both assays, 13 were positive only by the assay with the agarose gel, and 3 were positive only by the assay with the fluorescent probe. To define the subtype of HSV DNA detected in the screening assay, we also designed one set of primers which amplifies the gG regions of both types of HSV and probes which are specific to either HSV-1 (gG1) or HSV-2 (gG2). These probes were labeled with different fluorescent dyes (6-carboxyfluorescein for gG2 and 6-hexachlorofluorescein for gG1) to enable detection in a single PCR. In mixing experiments the probes discriminated the correct subtype in mixtures with up to a 7-log-higher concentration of the opposite subtype. The PCR typing results showed 100% concordance with the results

  11. Clinical performance of a new hepatitis B surface antigen quantitative assay with automatic dilution.

    PubMed

    Liu, Ta-Wei; Yeh, Ming-Lun; Huang, Chung-Feng; Lin, I-Ling; Huang, Jee-Fu; Dai, Chia-Yen; Chen, Yao-Li; Chuang, Wan-Long; Yu, Ming-Lung

    2015-01-01

    Hepatitis B virus surface antigen (HBsAg) levels reflect disease status and can predict the clinical response to antiviral treatment; however, the emergence of HBsAg mutant strains has become a challenge. The Abbott HBsAg quantification assay provides enhanced detection of HBsAg and HBsAg mutants. We aimed to evaluate the performance of the Abbott HBsAg quantification assay with automatic sample dilutions (shortened as automatic Architect assay), compared with the Abbott HBsAg quantification assay with manual sample dilutions (shortened as manual Architect assay) and the Roche HBsAg quantification assay with automatic sample dilutions (shortened as Elecsys). A total of 130 sera samples obtained from 87 hepatitis B virus (HBV)-infected patients were collected to assess the correlation between the automatic and manual Architect assays. Among the 87 patients, 41 provided 42 sera samples to confirm the linearity and reproducibility of the automatic Architect assay, and find out the correlation among the Elecsys and two Architect assays. The coefficients of variation (0.44-9.53%) and R(2) = 0.996-1, which were both determined using values obtained from the automatic Architect assay, showed good reproducibility and linearity. Results of the two Architect assays demonstrated a feasible correlation (n = 130 samples; R = 0.898, p < 0.01). With regard to subgroups, correlations between the two Architect assays were better in the hepatitis B e antigen (HBeAg)-negative group (HBeAg-negative group vs. HBeAg-positive group: R = 0.885 vs. R = 0.865, both p < 0.01) and low HBV DNA group (low DNA group vs. high DNA group: R = 0.886 vs. R = 0.844, both p < 0.01). Significant correlations were also found between the results of the Elecsys and Architect assays (R > 0.93 in all cases). In conclusion, the correlation between the automatic and manual dilution Architect assays was feasible, particularly in the HBeAg-negative and low DNA groups. With lower labor costs and less human error

  12. Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizers.

    PubMed

    Villegas, Eric N; Augustine, Swinburne A J; Villegas, Leah Fohl; Ware, Michael W; See, Mary Jean; Lindquist, H D Alan; Schaefer, Frank W; Dubey, J P

    2010-06-01

    Toxoplasma gondii oocysts are highly resistant to many chemical sanitizers. Methods used to determine oocyst infectivity have relied primarily on mouse, chicken, and feline bioassays. Although considered gold standards, they only provide a qualitative assessment of oocyst viability. In this study, two alternative approaches were developed to quantitate viable T. gondii oocysts following treatment with several common sanitizers. The first is a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay targeting the ACT1 and SporoSAG genes to enumerate viable T. gondii oocysts. RT-qPCR C(T) values between Wescodyne(R), acidified ethanol, or heat treated oocysts were not significantly different as compared with untreated controls. By contrast, treatment with formalin or Clorox(R) resulted in a 2-log(10) reduction in C(T) values. An in vitro T. gondii oocyst plaque assay (TOP-assay) was also developed to measure oocyst viability. This assay used a combination of bead milling and bile digestion, followed by culturing the excysted sporozoites in a confluent fibroblast cell monolayer. Results showed that no significant reduction in sporozoite viability was detected in acidified ethanol or Wescodyne(R) treated oocysts while at least a 2-log(10) reduction in plaques formed was observed with Clorox(R) treated oocysts. Moreover, formalin or heat treatment of oocysts resulted in at least a 5-log(10) reduction in plaques formed. This study demonstrates that an mRNA-based PCR viability assay targeting the ACT1 or SporoSAG genes is a relatively rapid technique compared to in vitro and in vivo assays. In addition, the TOP-assay proved very effective and sensitive at quantifying oocyst viability when compared with animal bioassays.

  13. Targeted proteomic assays for quantitation of proteins identified by proteogenomic analysis of ovarian cancer

    DOE PAGES

    Song, Ehwang; Gao, Yuqian; Wu, Chaochao; ...

    2017-07-19

    Here, mass spectrometry (MS) based targeted proteomic methods such as selected reaction monitoring (SRM) are becoming the method of choice for preclinical verification of candidate protein biomarkers. The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute has investigated the standardization and analytical validation of the SRM assays and demonstrated robust analytical performance on different instruments across different laboratories. An Assay Portal has also been established by CPTAC to provide the research community a resource consisting of large set of targeted MS-based assays, and a depository to share assays publicly, providing that assays meet the guidelines proposed bymore » CPTAC. Herein, we report 98 SRM assays covering 70 candidate protein biomarkers previously reported as associated with ovarian cancer that have been thoroughly characterized according to the CPTAC Assay Characterization Guidance Document. The experiments, methods and results for characterizing these SRM assays for their MS response, repeatability, selectivity, stability, and reproducible detection of endogenous analytes are described in detail.« less

  14. A competitive and reversible deactivation approach to catalysis-based quantitative assays

    PubMed Central

    Koide, Kazunori; Tracey, Matthew P.; Bu, Xiaodong; Jo, Junyong; Williams, Michael J.; Welch, Christopher J.

    2016-01-01

    Catalysis-based signal amplification makes optical assays highly sensitive and widely useful in chemical and biochemical research. However, assays must be fine-tuned to avoid signal saturation, substrate depletion and nonlinear performance. Furthermore, once stopped, such assays cannot be restarted, limiting the dynamic range to two orders of magnitude with respect to analyte concentrations. In addition, abundant analytes are difficult to quantify under catalytic conditions due to rapid signal saturation. Herein, we report an approach in which a catalytic reaction competes with a concomitant inactivation of the catalyst or consumption of a reagent required for signal generation. As such, signal generation proceeds for a limited time, then autonomously and reversibly stalls. In two catalysis-based assays, we demonstrate restarting autonomously stalled reactions, enabling accurate measurement over five orders of magnitude, including analyte levels above substrate concentration. This indicates that the dynamic range of catalysis-based assays can be significantly broadened through competitive and reversible deactivation. PMID:26891765

  15. Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients.

    PubMed

    Watzinger, F; Suda, M; Preuner, S; Baumgartinger, R; Ebner, K; Baskova, L; Niesters, H G M; Lawitschka, A; Lion, T

    2004-11-01

    A panel of 23 real-time PCR assays based on TaqMan technology has been developed for the detection and monitoring of 16 different viruses and virus families including human polyomaviruses BK virus and JC virus, human herpesviruses 6, 7, and 8, human adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory syncytial virus. The test systems presented have a broad dynamic range and display high sensitivity, reproducibility, and specificity. Moreover, the assays allow precise quantification of viral load in a variety of clinical specimens. The ability to use uniform PCR conditions for all assays permits simultaneous processing and detection of many different viruses, thus economizing the diagnostic work. Our observations based on more than 50,000 assays reveal the potential of the real-time PCR tests to facilitate early diagnosis of infection and to monitor the kinetics of viral proliferation and the response to treatment. We demonstrate that, in immunosuppressed patients with invasive virus infections, surveillance by the assays described may permit detection of increasing viral load several days to weeks prior to the onset of clinical symptoms. In virus infections for which specific treatment is available, the quantitative PCR assays presented provide reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies.

  16. Development and application of a quantitative real-time PCR assay to detect feline leukemia virus RNA.

    PubMed

    Torres, Andrea N; O'Halloran, Kevin P; Larson, Laurie J; Schultz, Ronald D; Hoover, Edward A

    2008-05-15

    We previously defined four categories of feline leukemia virus (FeLV) infection, designated as abortive, regressive, latent, and progressive. To determine if detectable viral DNA is transcriptionally active in the absence of antigenemia, we developed and validated a real-time viral RNA qPCR assay. This assay proved to be highly sensitive, specific, reproducible, and allowed reliable quantitation. We then applied this methodology, together with real-time DNA qPCR and p27 capsid antigen capture ELISA, to examine cats challenged with FeLV. We found that circulating viral RNA and DNA levels were highly correlated and the assays were almost in perfect agreement. This indicates that the vast majority of viral DNA is transcriptionally active, even in the absence of antigenemia. The real-time qPCR assays are more sensitive than the most commonly used FeLV diagnostic assay, the p27 capsid antigen capture ELISA. Application of qPCR assays may add greater depth in understanding of FeLV-host relationships.

  17. Characterization of vitellogenin gene expression in round goby (Neogobius melanostomus) using a quantitative polymerase chain reaction assay.

    PubMed

    Bowley, Lucas A; Alam, Farhana; Marentette, Julie R; Balshine, Sigal; Wilson, Joanna Y

    2010-12-01

    A growing concern over endocrine disruption in aquatic species has prompted the development of molecular assays to monitor environmental impacts. This study describes the development of quantitative polymerase chain reaction (qPCR) assays to characterize the expression of two vitellogenin (Vtg) genes in the invasive round goby (Neogobius melanostomus). Fragments from the 18SrRNA (housekeeping gene), Vtg II, and Vtg III genes were cloned and sequenced. The qPCR assays were developed to detect hepatic Vtg expression in goby. The assays detected induction of both Vtg genes in nonreproductive males following a two-week laboratory exposure to 17β-estradiol (≥1 mg/kg i.p. injection). The assays were applied to goby from Hamilton Harbour, Lake Ontario (Canada), including those from sites where feminization and intersex of goby has been documented. Both Vtg genes had significantly higher expression in females compared to males. Male reproductive goby adopt either parental or sneaker tactics; Vtg II expression was higher in sneaker than in parental males but parental and nonreproductive males did not differ from each other. The Vtg III expression was significantly higher in sneaker males followed by parental males and nonreproductive males, respectively. The Vtg II and III expression in nonreproductive males was elevated in the contaminated site with documented intersex. This assay provides an important tool for the use of an invasive species in monitoring endocrine disruption in the Great Lakes region.

  18. Improved Methods for Capture, Extraction, and Quantitative Assay of Environmental DNA from Asian Bigheaded Carp (Hypophthalmichthys spp.)

    PubMed Central

    Turner, Cameron R.; Miller, Derryl J.; Coyne, Kathryn J.; Corush, Joel

    2014-01-01

    Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species. PMID:25474207

  19. Improved methods for capture, extraction, and quantitative assay of environmental DNA from Asian bigheaded carp (Hypophthalmichthys spp.).

    PubMed

    Turner, Cameron R; Miller, Derryl J; Coyne, Kathryn J; Corush, Joel

    2014-01-01

    Indirect, non-invasive detection of rare aquatic macrofauna using aqueous environmental DNA (eDNA) is a relatively new approach to population and biodiversity monitoring. As such, the sensitivity of monitoring results to different methods of eDNA capture, extraction, and detection is being investigated in many ecosystems and species. One of the first and largest conservation programs with eDNA-based monitoring as a central instrument focuses on Asian bigheaded carp (Hypophthalmichthys spp.), an invasive fish spreading toward the Laurentian Great Lakes. However, the standard eDNA methods of this program have not advanced since their development in 2010. We developed new, quantitative, and more cost-effective methods and tested them against the standard protocols. In laboratory testing, our new quantitative PCR (qPCR) assay for bigheaded carp eDNA was one to two orders of magnitude more sensitive than the existing endpoint PCR assays. When applied to eDNA samples from an experimental pond containing bigheaded carp, the qPCR assay produced a detection probability of 94.8% compared to 4.2% for the endpoint PCR assays. Also, the eDNA capture and extraction method we adapted from aquatic microbiology yielded five times more bigheaded carp eDNA from the experimental pond than the standard method, at a per sample cost over forty times lower. Our new, more sensitive assay provides a quantitative tool for eDNA-based monitoring of bigheaded carp, and the higher-yielding eDNA capture and extraction method we describe can be used for eDNA-based monitoring of any aquatic species.

  20. Ultrasensitive and quantitative gold nanoparticle-based immunochromatographic assay for detection of ochratoxin A in agro-products.

    PubMed

    Majdinasab, Marjan; Sheikh-Zeinoddin, Mahmoud; Soleimanian-Zad, Sabihe; Li, Peiwu; Zhang, Qi; Li, Xin; Tang, Xiaoqian

    2015-01-01

    In most cases of mycotoxin detection, quantitation is critical while immunochromatographic strip tests are qualitative in nature. Moreover, the sensitivity of this technique is questioned. In order to overcome these limitations, an ultrasensitive and quantitative immunochromatographic assay (ICA) for rapid and sensitive quantitation of ochratoxin A (OTA) was developed. The assay was based on a competitive format and its sensitivity was improved by using a sensitive and selective OTA monoclonal antibody (OTA-mAb). The visible ICA results were obtained within 15 min, and in addition to visual examination, they were read by the rapid color intensity portable strip reader. The visual and computational detection limits (vLOD and cLOD, respectively) for ochratoxin A were 0.2 and 0.25 ng mL(-1), respectively. These values were lower than those reported by previous studies in a range 5-2500 folds. For validation, contaminated samples including wheat, maize, rice and soybean were assayed by ICA and a standard high performance liquid chromatography (HPLC). The results were in good agreement for both ICA and HPLC methods. The average recoveries of the HPLC were in the range 72-120% while the ICA values were from 76 to 104%, confirming the accuracy and sensitivity of this method. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Robust in vitro assay system for quantitative analysis of parasitic root-knot nematode infestation using Lotus japonicus.

    PubMed

    Amin, Arshana N N; Hayashi, Shuhei; Bartlem, Derek G

    2014-08-01

    Root-knot nematodes are sedentary endoparasites that induce permanent infestation sites inside the roots of a broad range of crop plants. The development of effective control strategies require understanding the root-knot nematode parasitic process, however, the key molecular determinants for host manipulation during infestation remain elusive. One limiting factor has been the lack of a standardized conventional method for quantitative measurement of host parasitism by root-knot nematodes, particularly one that enables efficient downstream analyses and is free from other biological sources of variability. We report here a robust, highly reproducible system for quantitative analysis of all stages of root-knot nematode infestation using the legume Lotus japonicus as the plant host. This system provides a high quality nematode inoculum that maintains consistency in juvenile age and viability even between independently prepared populations. An optimized root transformation protocol was also developed for L. japonicus to facilitate downstream molecular studies in conjunction with the quantitative assay. Hairy root transformation efficiencies up to 91% were achieved. Root-knot nematodes formed egg masses at the root surface of both intact plants and transgenic hairy root cultures within eight weeks, confirming the assay conditions support an efficient completion of the infestation cycle. The in vitro assay system described here is compatible with other plant hosts and will benefit agricultural biotechnology research as it now enables specific high-throughput screening of nematode resistance traits together with subsequent mechanistic elucidation of the causative factors.

  2. TaqMan real-time quantitative PCR assay for detection of fluoroquinolone-resistant Neisseria gonorrhoeae.

    PubMed

    Zhao, LiHong; Zhao, ShuPing

    2012-12-01

    It is noted that more than 99 % of fluoroquinolone resistance in Neisseria gonorrhoeae (QRNG) specimens have been shown to have the mutation of Ser91/Phe in the gyrA gene. In order to detect QRNG isolates as quickly as possible, the real-time TaqMan quantitative PCR assay was established for detection of the point mutation of Ser91/Phe in gyrA gene. The standard curve was generated automatically on ABI Prism PE7500. The correlation coefficient (r) of the standard curve was -0.9984 (R(2) = 0.9968), indicating a quietly precise log-linear relationship between the concentration of target DNA and the Ct value. Presently, correlated, cultured antimicrobial susceptibility testing of N. gonorrhoeae isolates continues to be the gold standard method for the detection of antimicrobial resistance. Comparison to the correlated, cultured antimicrobial susceptibility testing, the sensitivity and specificity of the established TaqMan assay for the detection of the QRNG specimens were 100 and 99 %, respectively. The TaqMan assay also allows for rapid detection of QRNG isolates without complex laboratory techniques. Therefore, real-time TaqMan quantitative PCR assay is a rapid, simple, highly sensitive, highly specific, and easy-to-perform method for the detection of the QRNG specimens. It can be applied as a quick screening method for QRNG isolates to help clinical determination of optimal treatment prescription.

  3. A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

    PubMed

    Gatto, Francesca; Cassina, Giulia; Broccolo, Francesco; Morreale, Giuseppe; Lanino, Edoardo; Di Marco, Eddi; Vardas, Efthiya; Bernasconi, Daniela; Buttò, Stefano; Principi, Nicola; Esposito, Susanna; Scarlatti, Gabriella; Lusso, Paolo; Malnati, Mauro S

    2011-12-01

    Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.

  4. Fluorescence-based quantitative scratch wound healing assay demonstrating the role of MAPKAPK-2/3 in fibroblast migration.

    PubMed

    Menon, Manoj B; Ronkina, Natalia; Schwermann, Jessica; Kotlyarov, Alexey; Gaestel, Matthias

    2009-12-01

    The scratch wound healing assay is a sensitive method to characterize cell proliferation and migration, but it is difficult to be quantitatively evaluated. Therefore, we developed an infrared fluorescence detection-based real-time assay for sensitive and accurate quantification of cell migration in vitro. The method offers sensitivity, simplicity, and the potential for integration into automated large-scale screening studies. A live cell staining lipophilic tracer-1,1'-dioctadecyl-3,3,3',3'-tetramethyl indotricarbocyanine iodide (DiR)-is used for accurate imaging of wound closure in a simple 96-well scratch assay. Scratches are made on prestained confluent cell monolayers using a pipette tip and scanned at different time intervals using a fluorescent scanner. Images are analyzed using Image J software and the migration index is calculated. Effect of cell number, time after scratch and software settings are analyzed. The method is validated by showing concentration- and time-dependent effects of cytochalasin-D on fibroblast migration. Using this assay, we quantitatively evaluate the role of the MAPK-activated protein kinases MK2 and MK3 in fibroblast migration. First, the migratory phenotype of MK2-deficient MEFs is analyzed in a retroviral rescue model. In addition, migration of MK2/3-double-deficient cells is determined and the ability of MK3 to rescue cell migration in MK2/3-double-deficient fibroblasts is demonstrated.

  5. Performance Assessment of Human and Cattle Associated Quantitative Real-time PCR Assays - slides

    EPA Science Inventory

    The presentation overview is (1) Single laboratory performance assessment of human- and cattle associated PCR assays and (2) A Field Study: Evaluation of two human fecal waste management practices in Ohio watershed.

  6. Performance Assessment of Human and Cattle Associated Quantitative Real-time PCR Assays - slides

    EPA Science Inventory

    The presentation overview is (1) Single laboratory performance assessment of human- and cattle associated PCR assays and (2) A Field Study: Evaluation of two human fecal waste management practices in Ohio watershed.

  7. Diagnostic significance and clinical impact of quantitative assays for diagnosis of human cytomegalovirus infection/disease in immunocompromised patients.

    PubMed

    Gerna, G; Percivalle, E; Baldanti, F; Sarasini, A; Zavattoni, M; Furione, M; Torsellini, M; Revello, M G

    1998-07-01

    In recent years several assays have been developed for quantitation of human cytomegalovirus (HCMV) in blood of immunocompromised (transplanted and AIDS) patients. It is currently agreed that the only reliable indication of the degree of dissemination of HCMV infection/disease is the measurement of HCMV in blood. Diagnosis of HCMV end-organ disease (organ localizations) often does not benefit from quantitation of virus in blood, but requires detection and quantification of virus in samples taken locally. The most important and clinically useful diagnostic assays for HCMV quantitation in blood are: i) viremia, quantifying infectious HCMV carried by peripheral blood leukocytes (PBL); ii) pp65-antigenemia, quantifying the number of PBL positive for HCMV pp65 in the nucleus; iii) circulating cytomegalic endothelial cell (CEC) viremia (CEC-viremia) measuring the number of circulating CEC carrying infectious HCMV (during the antigenemia assay); iv) leuko- and plasma-DNAemia, quantifying the number of HCMV genome equivalents present in PBL or plasma, respectively, by quantitative polymerase chain reaction (Q-PCR). Other less widely used assays are: i) determination of immediate early and late gene transcripts (mRNA) to detect active viral infection; ii) in situ hybridization to detect viral nucleic acid (DNA or RNA) in tissue sections or cell smears; iii) in situ PCR to detect a low DNA copy number in single cells. Monitoring of HCMV infection/disease in transplant recipients and AIDS patients has established threshold values for different assays above which HCMV-related clinical symptoms are likely to appear. These values are approximately 10 for viremia, 100 for antigenemia and 1,000 GE for leukoDNAemia, and are valid for both solid organ and bone marrow transplant recipients as well as AIDS patients, whereas presence of even a single circulating CEC is sufficient to suggest the presence of a disseminated HCMV infection with potential organ involvement. Monitoring of

  8. A versatile electrowetting-based digital microfluidic platform for quantitative homogeneous and heterogeneous bio-assays

    NASA Astrophysics Data System (ADS)

    Vergauwe, Nicolas; Witters, Daan; Ceyssens, Frederik; Vermeir, Steven; Verbruggen, Bert; Puers, Robert; Lammertyn, Jeroen

    2011-05-01

    Electrowetting-on-dielectric (EWOD) lab-on-a-chip systems have already proven their potential within a broad range of bio-assays. Nevertheless, research on the analytical performance of those systems is limited, yet crucial for a further breakthrough in the diagnostic field. Therefore, this paper presents the intrinsic possibilities of an EWOD lab-on-a-chip as a versatile platform for homogeneous and heterogeneous bio-assays with high analytical performance. Both droplet dispensing and splitting cause variations in droplet size, thereby directly influencing the assay's performance. The extent to which they influence the performance is assessed by a theoretical sensitivity analysis, which allows the definition of a basic framework for the reduction of droplet size variability. Taking advantage of the optimized droplet manipulations, both homogeneous and heterogeneous bio-assays are implemented in the EWOD lab-on-a-chip to demonstrate the analytical capabilities and versatility of the device. A fully on-chip enzymatic assay is realized with high analytical performance. It demonstrates the promising capabilities of an EWOD lab-on-a-chip in food-related and medical applications, such as nutritional and blood analyses. Further, a magnetic bio-assay for IgE detection using superparamagnetic nanoparticles is presented whereby the nanoparticles are used as solid carriers during the bio-assay. Crucial elements are the precise manipulation of the superparamagnetic nanoparticles with respect to dispensing and separation. Although the principle of using nano-carriers is demonstrated for protein detection, it can be easily extended to a broader range of bio-related applications like DNA sensing. In heterogeneous bio-assays the chip surface is actively involved during the execution of the bio-assay. Through immobilization of specific biological compounds like DNA, proteins and cells a reactive chip surface is realized, which enhances the bio-assay performance. To demonstrate

  9. NEW TARGET AND CONTROL ASSAYS FOR QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ANALYSIS OF ENTEROCOCCI IN WATER

    EPA Science Inventory

    Enterococci are frequently monitored in water samples as indicators of fecal pollution. Attention is now shifting from culture based methods for enumerating these organisms to more rapid molecular methods such as QPCR. Accurate quantitative analyses by this method requires highly...

  10. NEW TARGET AND CONTROL ASSAYS FOR QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ANALYSIS OF ENTEROCOCCI IN WATER

    EPA Science Inventory

    Enterococci are frequently monitored in water samples as indicators of fecal pollution. Attention is now shifting from culture based methods for enumerating these organisms to more rapid molecular methods such as QPCR. Accurate quantitative analyses by this method requires highly...

  11. Japanese reference panel of blood specimens for evaluation of hepatitis C virus RNA and core antigen quantitative assays.

    PubMed

    Murayama, Asako; Sugiyama, Nao; Watashi, Koichi; Masaki, Takahiro; Suzuki, Ryosuke; Aizaki, Hideki; Mizuochi, Toshiaki; Wakita, Takaji; Kato, Takanobu

    2012-06-01

    An accurate and reliable quantitative assay for hepatitis C virus (HCV) is essential for measuring viral propagation and the efficacy of antiviral therapy. There is a growing need for domestic reference panels for evaluation of clinical assay kits because the performance of these kits may vary with region-specific genotypes or polymorphisms. In this study, we established a reference panel by selecting 80 donated blood specimens in Japan that tested positive for HCV. Using this panel, we quantified HCV viral loads using two HCV RNA kits and five core antigen (Ag) kits currently available in Japan. The data from the two HCV RNA assay kits showed excellent correlation. All RNA titers were distributed evenly across a range from 3 to 7 log IU/ml. Although the data from the five core Ag kits also correlated with RNA titers, the sensitivities of individual kits were not sufficient to quantify viral load in all samples. As calculated by the correlation with RNA titers, the theoretical lower limits of detection by these core Ag assays were higher than those for the detection of RNA. Moreover, in several samples in our panel, core Ag levels were underestimated compared to RNA titers. Sequence analysis in the HCV core region suggested that polymorphisms at amino acids 47 to 49 of the core Ag were responsible for this underestimation. The panel established in this study will be useful for estimating the quality of currently available and upcoming HCV assay kits; such quality control is essential for clinical usage of these kits.

  12. Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes.

    PubMed

    Zweitzig, Daniel R; Riccardello, Nichol M; Sodowich, Bruce I; O'Hara, S Mark

    2012-08-01

    During the past 50 years, in vitro measurement of DNA polymerase activity has become an essential molecular biology tool. Traditional methods used to measure DNA polymerase activity in vitro are undesirable due to the usage of radionucleotides. Fluorescence-based DNA polymerase assays have been developed; however, they also suffer from various limitations. Herein we present a rapid, highly sensitive and quantitative assay capable of measuring DNA polymerase extension activity from purified enzymes or directly from microbial lysates. When tested with purified DNA polymerase, the assay detected as little as 2 × 10(-11)U of enzyme (∼ 50 molecules), while demonstrating excellent linearity (R(2)=0.992). The assay was also able to detect endogenous DNA polymerase extension activity down to less than 10 colony forming units (cfu) of input Gram-positive or Gram-negative bacteria when coupled to bead mill lysis while maintaining an R(2)=0.999. Furthermore, preliminary evidence presented here suggests that DNA polymerase extension activity is an indicator of microbial viability, as demonstrated by the reproducibly strong concordance between assay signal and bacterial colony formation. Together, the innovative methodology described here represents a significant advancement toward sensitive detection of potentially any microorganism containing active DNA polymerase within a given sample matrix.

  13. Development of a SYBR Green quantitative polymerase chain reaction assay for rapid detection and quantification of infectious laryngotracheitis virus.

    PubMed

    Mahmoudian, Alireza; Kirkpatrick, Naomi C; Coppo, Mauricio; Lee, Sang-Won; Devlin, Joanne M; Markham, Philip F; Browning, Glenn F; Noormohammadi, Amir H

    2011-06-01

    Infectious laryngotracheitis is an acute viral respiratory disease of chickens with a worldwide distribution. Sensitive detection of the causative herpesvirus is particularly important because it can persist in the host at a very low copy number and be transmitted to other birds. Quantification of viral genome copy number is also useful for clinical investigations and experimental studies. In the study presented here, a quantitative polymerase chain reaction (qPCR) assay was developed using SYBR Green chemistry and the viral gene UL15a to detect and quantify infectious laryngotracheitis virus (ILTV) in ILTV-inoculated chicken embryos or naturally infected birds. The specificity of the assay was confirmed using a panel of viral and bacterial pathogens of poultry. The sensitivity of the assay was compared with two conventional PCR assays, virus titration and an antigen-detecting enzyme-linked immunosorbent assay. The qPCR developed in this study was highly sensitive and specific, and has potential for quantification of ILTV in tissues from naturally and experimentally infected birds and embryos.

  14. Development and evaluation of a real-time PCR assay for the quantitative detection of Theileria annulata in cattle

    PubMed Central

    2012-01-01

    Background The tick-borne apicomplexan bovine parasite Theileria annulata is endemic in many tropical and temperate areas, including Minorca (Balearic Islands, Spain). Real-time PCR is widely used for the detection of piroplasms but quantification is not commonly considered. Results We developed a real-time quantitative PCR (qPCR) assay for the detection and quantification of T. annulata that included an internal amplification control (IAC) to monitor for the presence of potential inhibitors. Specificity, sensitivity, precision, linear range and PCR efficiency were calculated and different methods for transformation of quantification cycle (Cq) values into quantities (Q) were evaluated. The assay was able to detect (100% probability) and quantify (linear response) 100 gene copies, and clinical sensitivity was set at 10 T. annulata per μl of blood. The assay was then validated on 141 bovine blood samples analyzed in parallel by a Luminex® suspension array, showing the utility of the qPCR assay developed here for the detection and quantification of the parasite in field conditions. Once validated it was used to monitor T. annulata parasitaemia throughout a year in 8 carrier animals from a farm in Minorca. Conclusions The developed qPCR assay offers a reliable and simple way to quantify T. annulata infection loads, which could prove crucial in studying the role of carrier animals as a source of the infection, or assessing the efficacy of treatment and control measures. PMID:22889141

  15. Rapid Molecular Assays for Specific Detection and Quantitation of Loa loa Microfilaremia

    PubMed Central

    Fink, Doran L.; Kamgno, Joseph; Nutman, Thomas B.

    2011-01-01

    Background Accurate diagnosis of Loa loa infection is essential to the success of mass drug administration efforts to eliminate onchocerciasis and lymphatic filariasis, due to the risk of fatal encephalopathic reactions to ivermectin occurring among highly microfilaremic Loa-infected individuals living in areas co-endemic for multiple filarial species. Methodology/Principal Findings From a pool of over 1,800 L. loa microfilaria (mf) expressed sequence tags, 18 candidate L. loa mf-specific PCR targets were identified. Real-time PCR (qPCR) assays were developed for two targets (LLMF72 and LLMF269). The qPCR assays were highly specific for L. loa compared with related filariae and also highly sensitive, with detection limits of 0.1 pg genomic DNA, or 1% of DNA extracted from normal blood spiked with a single L. loa microfilaria. Using various DNA extraction methods with dried blood spots obtained from Cameroonian subjects with parasitologically proven loiasis, the LLMF72 qPCR assay successfully estimated mf burden in 65 of 68 samples (50–96,000 mf/mL by microscopy), including all 12 samples subjected to a simple 10-minute boiling extraction. Additionally, the assay detected low-level microfilaremia among 5 of 16 samples from patients thought to be amicrofilaremic by microscopy. Conclusions/Significance This novel, rapid, highly sensitive and specific qPCR assay is an important step forward in the laboratory diagnosis of L. loa infection. PMID:21912716

  16. Comparison of NucliSens and Roche Monitor Assays for Quantitation of Levels of Human Immunodeficiency Virus Type 1 RNA in Plasma

    PubMed Central

    Dyer, John R.; Pilcher, Christopher D.; Shepard, Robin; Schock, Jody; Eron, Joseph J.; Fiscus, Susan A.

    1999-01-01

    We compared the performance of Organon Teknika’s NucliSens and Roche Diagnostic Systems’ Monitor quantitative human immunodeficiency type 1 RNA assays. Both had similar linearity and sensitivity over most of the dynamic range of the assays, although the Monitor assay was superior at the low range of RNA values while the NucliSens assay was more consistent at higher RNA values. NucliSens generally showed less interassay variability. PMID:9889240

  17. Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins ▿

    PubMed Central

    Carney, Paul J.; Lipatov, Aleksandr S.; Monto, Arnold S.; Donis, Ruben O.; Stevens, James

    2010-01-01

    During the initial pandemic influenza H1N1 virus outbreak, assays such as hemagglutination inhibition and microneutralization provided important information on the relative protection afforded by the population's cross-reactivity from prior infections and immunizations with seasonal vaccines. However, these assays continue to be limited in that they are difficult to automate for high throughput, such as in pandemic situations, as well as to standardize between labs. Thus, new technologies are being sought to improve standardization, reliability, and throughput by using chemically defined reagents rather than whole cells and virions. We now report the use of a cell-free and label-free flu antibody biosensor assay (f-AbBA) for influenza research and diagnostics that utilizes recombinant hemagglutinin (HA) in conjunction with label-free biolayer interferometry technology to measure biomolecular interactions between the HA and specific anti-HA antibodies or sialylated ligands. We evaluated f-AbBA to determine anti-HA antibody binding activity in serum or plasma to assess vaccine-induced humoral responses. This assay can reveal the impact of antigenic difference on antibody binding to HA and also measure binding to different subtypes of HA. We also show that the biosensor assay can measure the ability of HA to bind a model sialylated receptor-like ligand. f-AbBA could be used in global surveillance laboratories since preliminary tests on desiccated HA probes showed no loss of activity after >2 months in storage at room temperature, indicating that the same reagent lots could be used in different laboratories to minimize interlaboratory assay fluctuation. Future development of such reagents and similar technologies may offer a robust platform for future influenza surveillance activities. PMID:20660137

  18. Assaying estrogenicity by quantitating the expression levels of endogenous estrogen-regulated genes.

    PubMed

    Jørgensen, M; Vendelbo, B; Skakkebaek, N E; Leffers, H

    2000-05-01

    Scientific evidence suggests that humans and wildlife species may experience adverse health consequences from exposure to environmental chemicals that interact with the endocrine system. Reliable short-term assays are needed to identify hormone-disrupting chemicals. In this study we demonstrate that the estrogenic activity of a chemical can be evaluated by assaying induction or repression of endogenous estrogen-regulated "marker genes" in human breast cancer MCF-7 cells. We included four marker genes in the assay--pS2, transforming growth factor beta3 (TGFbeta3), monoamine oxidase A, and [alpha]1-antichymotrypsin--and we evaluated estrogenic activity for 17beta-estradiol (E(2)), diethylstilbestrol, [alpha]-zearalanol, nonylphenol, genistein, methoxychlor, endosulphan, o,p-DDE, bisphenol A, dibutylphthalate, 4-hydroxy tamoxifen, and ICI 182.780. All four marker genes responded strongly to the three high-potency estrogens (E(2), diethylstilbestrol, and [alpha]-zearalanol), whereas the potency of the other chemicals was 10(3)- to 10(6)-fold lower than that of E(2). There were some marker gene-dependent differences in the relative potencies of the tested chemicals. TGFbeta3 was equally sensitive to the three high-potency estrogens, whereas the sensitivity to [alpha]-zearalanol was approximately 10-fold lower than the sensitivity to E(2) and diethylstilbestrol when assayed with the other three marker genes. The potency of nonylphenol was equal to that of genistein when assayed with pS2 and TGFbeta3, but 10- to 100-fold higher/lower with monoamine oxidase A and [alpha]1-antichymotrypsin, respectively. The results are in agreement with results obtained by other methods and suggest that an assay based on endogenous gene expression may offer an attractive alternative to other E-SCREEN methods.

  19. A radiometric assay for bacterial growth detection and quantitative antibiotic testing

    SciTech Connect

    Boonkitticharoen, V.; Kirchner, P.T.; Ehrhardt, J.C.

    1984-01-01

    Buddemeyer's two-compartment radiometric assay for bacterial growth using respired C-14 carbon dioxide promised major advantages over other available methods, but limitations of the technique have restricted its application. Through a systemic study of relevant physical and chemical factors the authors sought to improve the assay for earlier detection of bacterial growth and to extend its use to measurement of antibiotic drug susceptibility and potency. A 35-fold improvement in count rate response was achieved by a) reversing growth and detector chambers to permit rigorous agitation, b) increasing NaOH quantity and using a supersaturated PPO solution, and c) adding detergent to stabilize NaOH-PPO contact. Bacterial growth may be detected as early as 1/2 hour after inoculation. For rapidly growing bacteria the growth rate constant is defined as the slope of the growth curve (log count rate vs. time). The validity of the growth behavior was verified by measuring growth at several inoculum sizes over 3 orders of magnitude using standard strains of S. aureus and E. coli. The growth rate constant proved to be independent of inoculum size. To test the merit of the system as an antibiotic assay, E. coli were exposed to doses of spectinomycin hydrochloride in the range which yielded a nonlinear dose-response relation by a turbidity assay. The test, however, showed a linear relation between growth rate constant and antibiotic dose. The results clearly indicate the radiometric growth rate assay to be a rapid, valid and objective assay for bacterial growth and antibiotic sensitivity.

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

    PubMed

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

    2012-10-01

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

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

    PubMed

    Berzofsky, Ronald N.

    1995-01-01

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

  2. Antibody performance in ChIP-sequencing assays: From quality scores of public data sets to quantitative certification.

    PubMed

    Mendoza-Parra, Marco-Antonio; Saravaki, Vincent; Cholley, Pierre-Etienne; Blum, Matthias; Billoré, Benjamin; Gronemeyer, Hinrich

    2016-01-01

    We have established a certification system for antibodies to be used in chromatin immunoprecipitation assays coupled to massive parallel sequencing (ChIP-seq). This certification comprises a standardized ChIP procedure and the attribution of a numerical quality control indicator (QCi) to biological replicate experiments. The QCi computation is based on a universally applicable quality assessment that quantitates the global deviation of randomly sampled subsets of ChIP-seq dataset with the original genome-aligned sequence reads. Comparison with a QCi database for >28,000 ChIP-seq assays were used to attribute quality grades (ranging from 'AAA' to 'DDD') to a given dataset. In the present report we used the numerical QC system to assess the factors influencing the quality of ChIP-seq assays, including the nature of the target, the sequencing depth and the commercial source of the antibody.  We have used this approach specifically to certify mono and polyclonal antibodies obtained from Active Motif directed against the histone modification marks H3K4me3, H3K27ac and H3K9ac for ChIP-seq. The antibodies received the grades AAA to BBC ( www.ngs-qc.org). We propose to attribute such quantitative grading of all antibodies attributed with the label "ChIP-seq grade".

  3. Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis

    PubMed Central

    Smith, William L.; Chadwick, Sean G.; Toner, Geoffrey; Mordechai, Eli; Adelson, Martin E.; Aguin, Tina J.; Sobel, Jack D.

    2016-01-01

    Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease: Gardnerella vaginalis, Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the order Clostridiales), Megasphaera phylotype 1 or 2, Lactobacillus iners, Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus jensenii. We generated a logistic regression model that identified G. vaginalis, A. vaginae, and Megasphaera phylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion of Lactobacillus spp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV. PMID:26818677

  4. Construction of a taste-blind medaka fish and quantitative assay of its preference-aversion behavior.

    PubMed

    Aihara, Y; Yasuoka, A; Iwamoto, S; Yoshida, Y; Misaka, T; Abe, K

    2008-11-01

    In vertebrates, the taste system provides information used in the regulation of food ingestion. In mammals, each cell group within the taste buds expresses either the T1R or the T2R taste receptor for preference-aversion discrimination. However, no such information is available regarding fish. We developed a novel system for quantitatively assaying taste preference-aversion in medaka fish. In this study, we prepared fluorescently labeled foods with fine cavities designed to retain tastants until they were bitten by the fish. The subjects were fed food containing a mixture of amino acids and inosine monophosphate (AN food), denatonium benzoate (DN food) or no tastant (NT food), and the amounts of ingested food were measured by fluorescence microscopy. Statistical analysis of the fluorescence intensities yielded quantitative measurements of AN food preference and DN food aversion. We then generated a transgenic fish expressing dominant-negative Galpha(i2) both in T1R-expressing and in T2R-expressing cells. The feeding assay revealed that the transgenic fish was unable to show a preference for AN food and an aversion to DN food. The assay system was useful for evaluating taste-blind behaviors, and the results indicate that the two taste signaling pathways conveying preferable and aversive taste information are conserved in fish as well as in mammals.

  5. [Comparison of direct immune-fluorescent assay and real-time quantitative PCR in detecting the Hantavirus].

    PubMed

    Yu, Peng-bo; Li, Shen; Wei, Jing; Ma, Chang-an; Lu, Xiao-ling; DU, Shui-quan; Guan, Lu-yuan; Zheng, Yuan; Dong, Jian-hua; Ma, Chao-feng; Wang, Jing-jun

    2013-04-01

    To compare the differences between the direct immuno-fluorescent assay (DFA) and real-time quantitative PCR in detecting the Hantavirus (HV) in rat lungs. From April to October in 2012, a total of 479 rats were caught by mouse-trap in residential or wild areas in Huxian, Jingyang, and Meixian of Shaanxi province, where haemorrhagic fever with renal syndrome (HFRS) was highly prevalent. The rats were dissected to take the two lungs, one was frozen and applied immuno-fluorescent assay to detect HV antigen while the other one was extracted its RNA and detected HV nucleic acid by real-time quantitative PCR. Then we compared the positive rate of the two methods. Out of the 479 rats, 105 were caught from residential areas and the other 374 were caught from wild areas. Among the 105 rats caught from residential areas, no HV were detected out neither by DFA nor by real-time quantitative PCR. Among the 374 wild rats, 13.1% (49/374) were detected HV positive by DFA and 14.7% (55/374) were detected HV positive by real-time quantitative PCR. The difference showed no statistical significance (χ(2) = 0.402, P = 0.526). When detecting each lung sample, the HV positive rate was 10.2% (49/479) under the detection by DFA while the HV positive rate was 11.5% (55/479) under the detection by real-time quantitative PCR. The difference had no statistical significance (χ(2) = 1.286, P = 0.257) and the consistency coefficient was 68.2% under the paired chi-square test analysis, which showed high consistency (u = 11.759, P < 0.05). The sensitivity of real-time quantitative PCR to detect HV was 77.6% (38/49) comparing with DFA as standard, and the specificity was 96.1% (413/430). Out of the 9 suspected HV positive sample detected by DFA, 6 were confirmed positive by real-time quantitative PCR and 3 were denied. Compared with the DFA, real-time quantitative PCR could also be used to detect the infection of HV in rats, and the result might be more stable.

  6. Bayer Immuno 1 PSA Assay: an automated, ultrasensitive method to quantitate total PSA in serum.

    PubMed

    Morris, D L; Dillon, P W; Very, D L; Ng, P; Kish, L; Goldblatt, J L; Bruzek, D J; Chan, D W; Ahmed, M S; Witek, D; Fritsche, H A; Smith, C; Schwartz, D; Schwartz, M K; Noteboom, J L; Vessella, R L; Yeung, K K; Allard, W J

    1998-01-01

    The Bayer Immuno 1 PSA Assay measures total PSA in human serum and demonstrates excellent performance with an interassay CV < or = 3.4% and a biological detection limit of 0.03 microgram/L. No significant interference from common hormonal and chemotherapeutic drugs, kallikrein, prostatic acid phosphatase, and trypsin, or elevated levels of total bilirubin, hemoglobin, triglycerides, and IgG was observed. The 95th percentile values for healthy individuals increased with age from 3.0 micrograms/L for males 50-59 years and 3.3 micrograms/L for males 60-69 years, to 4.6 micrograms/L for males > or = 70 years. Clinical studies with retrospective samples demonstrated correspondence between serial measurements of PSA and clinical outcome for 98% of 159 prostate cancer patients. Clinical sensitivity for patients with clinical evidence of disease, untreated at the time of specimen draw, increased with increasing stage from 77.5-100%. Specificity of 60-70% for BPH and other benign urogenital diseases was consistent with previous findings. Bayer Immuno 1 PSA Assay values for 2131 specimens from healthy subjects and patients with prostate cancer, BPH, and other malignant and nonmalignant diseases correlated well with the Abbott IMx PSA Assay over the range 0.0-6,238 micrograms/L (Y = 1.10 x + 0.02). The Bayer Immuno 1 PSA Assay provides automated ultrasensitive, precise, and equimolar measurement of total PSA in human serum.

  7. A Call for Nominations of Quantitative High-Throughput Screening Assays from Relevant Human Toxicity Pathways

    EPA Science Inventory

    The National Research Council of the United States National Academies of Science has recently released a document outlining a long-range vision and strategy for transforming toxicity testing from largely whole animal-based testing to one based on in vitro assays. “Toxicity Testin...

  8. Commercially available antibodies can be applied in quantitative multiplexed peptide immunoaffinity enrichment targeted mass spectrometry assays

    PubMed Central

    Schoenherr, Regine M.; Zhao, Lei; Ivey, Richard G.; Voytovich, Uliana J.; Kennedy, Jacob; Yan, Ping; Lin, Chenwei; Whiteaker, Jeffrey R.; Paulovich, Amanda G.

    2016-01-01

    Immunoaffinity enrichment of peptides coupled to multiple reaction monitoring-mass spectrometry (immuno-MRM) enables highly specific, sensitive, and precise quantification of peptides and post-translational modifications. Major obstacles to developing a large number of immuno-MRM assays are the poor availability of monoclonal antibodies (mAbs) validated for immunoaffinity enrichment of peptides and the cost and lead time of developing the antibodies de novo. Although many thousands of mAbs are commercially offered, few have been tested for application to immunoaffinity enrichment of peptides. In this study we tested the success rate of using commercially available mAbs for peptide immuno-MRM assays. We selected 105 commercial mAbs (76 targeting non-modified “pan” epitopes, 29 targeting phosphorylation) to proteins associated with the DNA damage response network. We found that 8 of the 76 pan (11%) and 5 of the 29 phospho-specific mAbs (17%) captured tryptic peptides (detected by LC-MS/MS) of their protein targets from human cell lysates. Seven of these mAbs were successfully used to configure and analytically characterize immuno-MRM assays. By applying selection criteria upfront, the results indicate that a screening success rate of up to 24% is possible, establishing the feasibility of screening a large number of catalog antibodies to provide readily-available assay reagents. PMID:27094115

  9. A Call for Nominations of Quantitative High-Throughput Screening Assays from Relevant Human Toxicity Pathways

    EPA Science Inventory

    The National Research Council of the United States National Academies of Science has recently released a document outlining a long-range vision and strategy for transforming toxicity testing from largely whole animal-based testing to one based on in vitro assays. “Toxicity Testin...

  10. Characterization of Diversity in Toxicity Mechanism Using In Vitro Cytotoxicity Assays in Quantitative High Throughput Screening

    PubMed Central

    Huang, Ruili; Southall, Noel; Cho, Ming-Hsuang; Xia, Menghang; Inglese, James; Austin, Christopher P.

    2009-01-01

    Assessing the potential health risks of environmental chemical compounds is an expensive undertaking which has motivated the development of new alternatives to traditional in vivo toxicological testing. One approach is to stage the evaluation, beginning with less expensive and higher throughput in vitro testing before progressing to more definitive trials. In vitro testing can be used to generate a hypothesis about a compound's mechanism of action, which can then be used to design an appropriate in vivo experiment. Here we begin to address the question of how to design such a battery of in vitro cell-based assays by combining data from two different types of assays, cell viability and caspase activation, with the aim of elucidating mechanism of action. Because caspase activation is a transient event during apoptosis, it is not possible to design a single end-point assay protocol that would identify all instances of compound-induced caspase activation. Nevertheless, useful information about compound mechanism of action can be obtained from these assays in combination with cell viability data. Unsupervised clustering in combination with Dunn's cluster validity index is a robust method for identifying mechanisms of action without requiring any a priori knowledge about mechanisms of toxicity. The performance of this clustering method is evaluated by comparing the clustering results against literature annotations of compound mechanisms. PMID:18281954

  11. A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell level.

    PubMed

    Koh, Siang-Boon; Mascalchi, Patrice; Rodriguez, Esther; Lin, Yao; Jodrell, Duncan I; Richards, Frances M; Lyons, Scott K

    2017-01-15

    The fluorescence ubiquitination-based cell cycle indicator (FUCCI) is a powerful tool for use in live cells but current FUCCI-based assays have limited throughput in terms of image processing and quantification. Here, we developed a lentiviral system that rapidly introduced FUCCI transgenes into cells by using an all-in-one expression cassette, FastFUCCI. The approach alleviated the need for sequential transduction and characterisation, improving labelling efficiency. We coupled the system to an automated imaging workflow capable of handling large datasets. The integrated assay enabled analyses of single-cell readouts at high spatiotemporal resolution. With the assay, we captured in detail the cell cycle alterations induced by antimitotic agents. We found that treated cells accumulated at G2 or M phase but eventually advanced through mitosis into the next interphase, where the majority of cell death occurred, irrespective of the preceding mitotic phenotype. Some cells appeared viable after mitotic slippage, and a fraction of them subsequently re-entered S phase. Accordingly, we found evidence that targeting the DNA replication origin activity sensitised cells to paclitaxel. In summary, we demonstrate the utility of the FastFUCCI assay for quantifying spatiotemporal dynamics and identify its potential in preclinical drug development. © 2017. Published by The Company of Biologists Ltd.

  12. A rapid, quantitative assay for measuring alkaline phosphatase activity in osteoblastic cells in vitro.

    PubMed

    Sabokbar, A; Millett, P J; Myer, B; Rushton, N

    1994-10-01

    Alkaline phosphatase (ALP) is the most widely recognized biochemical marker for osteoblast activity. Although its precise function is poorly understood, it is believed to play a role in skeletal mineralization. The aim of this study was to develop an assay suitable for measuring the activity of this enzyme in microtiter plate format. Using the well-characterized osteoblast-like cell line Saos-2, this paper describes an optimized biochemical assay suitable for measuring ALP activity in tissue culture samples. We have determined that a p-nitrophenyl phosphate substrate concentration of 9 mM provides highest enzyme activities. We have found that cell concentration, and hence enzyme concentration, affects both the kinetics and precision of the assay. We also tested several methods of enzyme solubilization and found that freeze-thawing the membrane fractions twice at -70 degrees C/37 degrees C or freeze-thawing once with sonication yielded highest enzyme activities. The activity of the enzyme decreased by 10% after 7 days storage. This assay provides a sensitive and reproducible method that is ideally suited for measuring ALP activity in isolated osteoblastic cells, although sample preparation and storage can influence results.

  13. A comparison of quantitative-competitive and realtime PCR assays using an identical target sequence to detect Epstein-Barr virus viral load in the peripheral blood.

    PubMed

    Xu, Shushen; Green, Michael; Kingsley, Laurence; Webber, Steven; Rowe, David

    2006-11-01

    Monitoring the load of Epstein-Barr virus (EBV) in the peripheral blood by quantitative PCR has been accepted as a useful tool for predicting the onset of EBV related diseases, confirming an EBV disease diagnosis and following the response to treatment interventions. In the present study, the use of a realtime polymerase chain reaction (rt-PCR) assay developed for unpurified cell preparations was examined and the results of the realtime assay were compared to an EBV quantitative-competitive PCR assay (QC-PCR). Both assays use the same target sequence and the same method for determining the standard value for the copy number of EBV genomes present. A comparison of 572 PCR results reveals that the realtime assay gave 5-10-fold higher values than the QC-PCR. Fifty-one results (8.9%) were discordant between the two sets of data. The most commonly encountered discordant result was detection of low amounts of EBV DNA by the rt-PCR assay that were not detected in specimens by QC-PCR. The two assays had a high degree of correlation across the range of load detection allowing clinically relevant threshold values determined in the QC-PCR assay to be inferred for the rt-PCR assay. External normalization of the rt-PCR assay was determined to be an important tool for monitoring the quality and/or quantity of human DNA in the starting material. rt-PCR assays with unpurified cell lysates compare favorably with quantitative-competitive assays and when normalized offer real advantages in specimen preparation, assay manipulations and reproducibility over both quantitative-competitive assays and realtime assays that require purified nucleic acid inputs.

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

    PubMed

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

    2016-03-01

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

  15. INTERNAL AMPLIFICATION CONTROL FOR USE IN QUANTITATIVE POLYMERASE CHAIN REACTION FECAL INDICATOR BACTERIA ASSAYS

    EPA Science Inventory

    Quantitative polymerase chain reaction (QPCR) can be used as a rapid method for detecting fecal indicator bacteria. Because false negative results can be caused by PCR inhibitors that co-extract with the DNA samples, an internal amplification control (IAC) should be run with eac...

  16. INTERNAL AMPLIFICATION CONTROL FOR USE IN QUANTITATIVE POLYMERASE CHAIN REACTION FECAL INDICATOR BACTERIA ASSAYS

    EPA Science Inventory

    Quantitative polymerase chain reaction (QPCR) can be used as a rapid method for detecting fecal indicator bacteria. Because false negative results can be caused by PCR inhibitors that co-extract with the DNA samples, an internal amplification control (IAC) should be run with eac...

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

    USDA-ARS?s Scientific Manuscript database

    Citrus huanglongbing (HLB), or citrus greening, is a serious and industry-limiting disease. Preliminary diagnoses can be made through visual symptoms, and greater certainty can be achieved through quantitative real-time PCR (qPCR). Several qPCR procedures are available including those by designed by...

  18. Development of species-, strain- and antibiotic biosynthesis-specific quantitative PCR assays for Pantoea agglomerans as tools for biocontrol monitoring.

    PubMed

    Braun-Kiewnick, Andrea; Lehmann, Andreas; Rezzonico, Fabio; Wend, Chris; Smits, Theo H M; Duffy, Brion

    2012-09-01

    Pantoea agglomerans is a cosmopolitan plant epiphytic bacterium that includes some of the most effective biological antagonists against the fire blight pathogen Erwinia amylovora, a major threat to pome fruit production worldwide. Strain E325 is commercially available as Bloomtime Biological™ in the USA and Canada. New quantitative PCR (qPCR) assays were developed for species- and strain -specific detection in the environment, and for detection of indigenous strains carrying the biocontrol antibacterial peptide biosynthesis gene paaA. The qPCR assays were highly specific, efficient and sensitive, detecting fewer than three cells per reaction or 700 colony forming units per flower, respectively. The qPCR assays were tested on field samples, giving first indications to the incidence of P. agglomerans E325 related strains, total P. agglomerans and pantocin A producing bacteria in commercial orchards. These assays will facilitate monitoring the environmental behavior of biocontrol P. agglomerans after orchard application for disease protection, proprietary strain-tracking, and streamlined screening for discovery of new biocontrol strains.

  19. [Detection of Plasmodium falciparum by using magnetic nanoparticles separation-based quantitative real-time PCR assay].

    PubMed

    Wang, Fei; Tian, Yin; Yang, Jing; Sun, Fu-Jun; Sun, Ning; Liu, Bi-Yong; Tian, Rui; Ge, Guang-Lu; Zou, Ming-qiang; Deng, Cong-liang; Liu, Yi

    2014-10-01

    To establish a magnetic nanoparticles separation-based quantitative real-time PCR (RT-PCR) assay for fast and accurate detection of Plasmodium falciparum and providing a technical support for improving the control and prevention of imported malaria. According to the conserved sequences of the P. falciparum genome 18SrRNA, the species-specific primers and probe were designed and synthetized. The RT-PCR was established by constructing the plasmid standard, fitting the standard curve and using magnetic nanoparticles separation. The sensitivity and specificity of the assay were evaluated. The relationship between the threshold cycle (Ct) and logarithm of initial templates copies was linear over a range of 2.5 x 10(1) to 2.5 x 10(8) copies/μl (R2 = 0.999). Among 13 subjects of entry frontier, a P. falciparum carrier with low load was detected by using the assay and none was detected with the conventional examinations (microscopic examinations and rapid tests). This assay shows a high sensitivity in detection of P. falciparum, with rapid and accurate characteristics, and is especially useful in diagnosis of P. falciparum infectors with low parasitaemia at entry-exit frontier ports.

  20. A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR

    PubMed Central

    Maier, Helena J.; Van Borm, Steven; Young, John R.; Fife, Mark

    2016-01-01

    Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology. PMID:27537060

  1. A Versatile Panel of Reference Gene Assays for the Measurement of Chicken mRNA by Quantitative PCR.

    PubMed

    Staines, Karen; Batra, Ambalika; Mwangi, William; Maier, Helena J; Van Borm, Steven; Young, John R; Fife, Mark; Butter, Colin

    2016-01-01

    Quantitative real-time PCR assays are widely used for the quantification of mRNA within avian experimental samples. Multiple stably-expressed reference genes, selected for the lowest variation in representative samples, can be used to control random technical variation. Reference gene assays must be reliable, have high amplification specificity and efficiency, and not produce signals from contaminating DNA. Whilst recent research papers identify specific genes that are stable in particular tissues and experimental treatments, here we describe a panel of ten avian gene primer and probe sets that can be used to identify suitable reference genes in many experimental contexts. The panel was tested with TaqMan and SYBR Green systems in two experimental scenarios: a tissue collection and virus infection of cultured fibroblasts. GeNorm and NormFinder algorithms were able to select appropriate reference gene sets in each case. We show the effects of using the selected genes on the detection of statistically significant differences in expression. The results are compared with those obtained using 28s ribosomal RNA, the present most widely accepted reference gene in chicken work, identifying circumstances where its use might provide misleading results. Methods for eliminating DNA contamination of RNA reduced, but did not completely remove, detectable DNA. We therefore attached special importance to testing each qPCR assay for absence of signal using DNA template. The assays and analyses developed here provide a useful resource for selecting reference genes for investigations of avian biology.

  2. Development of a quantitative PCR assay for measurement of trichechid herpesvirus 1 load in the Florida manatee ( Trichechus manatus latirostris).

    PubMed

    Ferrante, Jason A; Cortés-Hinojosa, Galaxia; Archer, Linda L; Wellehan, James F X

    2017-07-01

    Trichechid herpesvirus 1 (TrHV-1) is currently the only known herpesvirus in any sirenian. We hypothesized that stress may lead to recrudescence of TrHV-1 in manatees, thus making TrHV-1 a potential biomarker of stress. We optimized and validated a TrHV-1 real-time quantitative probe hybridization PCR (qPCR) assay that was used to quantify TrHV-1 in manatee peripheral blood mononuclear cells (PBMCs). Average baseline TrHV-1 loads in a clinically healthy wild Florida manatee ( Trichechus manatus latirostris) population ( n = 42) were 40.9 ± SD 21.2 copies/100 ng DNA; 19 of 42 manatees were positive. TrHV-1 loads were significantly different between the 2 field seasons ( p < 0.025). This optimized and validated qPCR assay may be used as a tool for further research into TrHV-1 in Florida manatees.

  3. Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay.

    PubMed

    Ummarino, Simone; Mozzon, Massimo; Zamporlini, Federica; Amici, Adolfo; Mazzola, Francesca; Orsomando, Giuseppe; Ruggieri, Silverio; Raffaelli, Nadia

    2017-04-15

    Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time.

  4. Quantitative Profiling of Protein Tyrosine Kinases in Human Cancer Cell Lines by Multiplexed Parallel Reaction Monitoring Assays*

    PubMed Central

    Kim, Hye-Jung; Lin, De; Lee, Hyoung-Joo; Li, Ming; Liebler, Daniel C.

    2016-01-01

    Protein tyrosine kinases (PTKs) play key roles in cellular signal transduction, cell cycle regulation, cell division, and cell differentiation. Dysregulation of PTK-activated pathways, often by receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. In this study, we have developed a parallel reaction monitoring-based assay for quantitative profiling of 83 PTKs. The assay detects 308 proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We implemented the assay in four cell models: 1) a comparison of proliferating versus epidermal growth factor-stimulated A431 cells, 2) a comparison of SW480Null (mutant APC) and SW480APC (APC restored) colon tumor cell lines, and 3) a comparison of 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility (11–18) or acquired resistance (11–18R) to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. We observed distinct PTK expression changes that were induced by stimuli, genomic features or drug resistance, which were consistent with previous reports. However, most of the measured expression differences were novel observations. For example, acquired resistance to erlotinib in the 11–18 cell model was associated not only with previously reported up-regulation of MET, but also with up-regulation of FLK2 and down-regulation of LYN and PTK7. Immunoblot analyses and shotgun proteomics data were highly consistent with parallel reaction monitoring data. Multiplexed parallel reaction monitoring assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations. Data are available through Proteome eXchange Accession PXD002706. PMID:26631510

  5. Evaluation of quantitative assays for the identification of direct signal transducer and activator of transcription 3 (STAT3) inhibitors

    PubMed Central

    Furtek, Steffanie L.; Matheson, Christopher J.; Backos, Donald S.; Reigan, Philip

    2016-01-01

    In many forms of cancer the signal transducer and activator of transcription 3 (STAT3) transcription factor remains constitutively active, driving cancer survival and progression. The critical role of STAT3 in tumorigenesis has prompted a campaign of drug discovery programs to identify small molecules that disrupt the function of STAT3, with more recent efforts focusing on direct STAT3 inhibition. There are two target binding sites for direct STAT3 inhibitors: the SH2 dimerization domain and the DNA-binding domain. An in vitro fluorescence polarization assay, using recombinant STAT3 protein, has successfully identified compounds that target the SH2 domain; however, no assay has been reported to identify inhibitors that bind the DNA-binding domain. The lack of such a quantitative assay has limited the identification and development of STAT3 DNA-binding domain inhibitors. Here, we report a modified DNA-binding ELISA to incorporate recombinant STAT3 protein to evaluate small molecules that prevent STAT3-DNA binding. The concomitant use of the ELISA and fluorescence polarization assay enables the classification of direct STAT3 inhibitors by their site of action. Our data provide further support that niclosamide inhibits STAT3 through interaction with the DNA-binding domain. Furthermore, the ELISA can support medicinal chemistry efforts by identifying DNA-binding domain inhibitors and allowing the determination of an IC50 value, supporting the ranking of inhibitors and development of structure-activity relationships. Therefore, we propose a tandem evaluation approach to identify small molecules that target the SH2 domain or the DNA-binding domain of STAT3, which allows for quantitative evaluation of candidate STAT3 inhibitors. PMID:27793003

  6. Evaluation of quantitative assays for the identification of direct signal transducer and activator of transcription 3 (STAT3) inhibitors.

    PubMed

    Furtek, Steffanie L; Matheson, Christopher J; Backos, Donald S; Reigan, Philip

    2016-11-22

    In many forms of cancer the signal transducer and activator of transcription 3 (STAT3) transcription factor remains constitutively active, driving cancer survival and progression. The critical role of STAT3 in tumorigenesis has prompted a campaign of drug discovery programs to identify small molecules that disrupt the function of STAT3, with more recent efforts focusing on direct STAT3 inhibition. There are two target binding sites for direct STAT3 inhibitors: the SH2 dimerization domain and the DNA-binding domain. An in vitro fluorescence polarization assay, using recombinant STAT3 protein, has successfully identified compounds that target the SH2 domain; however, no assay has been reported to identify inhibitors that bind the DNA-binding domain. The lack of such a quantitative assay has limited the identification and development of STAT3 DNA-binding domain inhibitors. Here, we report a modified DNA-binding ELISA to incorporate recombinant STAT3 protein to evaluate small molecules that prevent STAT3-DNA binding. The concomitant use of the ELISA and fluorescence polarization assay enables the classification of direct STAT3 inhibitors by their site of action. Our data provide further support that niclosamide inhibits STAT3 through interaction with the DNA-binding domain. Furthermore, the ELISA can support medicinal chemistry efforts by identifying DNA-binding domain inhibitors and allowing the determination of an IC50 value, supporting the ranking of inhibitors and development of structure-activity relationships. Therefore, we propose a tandem evaluation approach to identify small molecules that target the SH2 domain or the DNA-binding domain of STAT3, which allows for quantitative evaluation of candidate STAT3 inhibitors.

  7. A sensitive liquid chromatography/mass spectrometry-based assay for quantitation of amino-containing moieties in lipid A

    PubMed Central

    Kalhorn, Thomas F.; Kiavand, Anahita; Cohen, Ilana E.; Nelson, Amanda K.; Ernst, Robert K.

    2009-01-01

    A novel sensitive liquid chromatography/mass spectrometry-based assay was developed for the quantitation of aminosugars, including 2-amino-2-deoxyglucose (glucosamine, GlcN), 2-amino-2-deoxygalactose (galactosamine, GalN), and 4-amino-4-deoxyarabinose (aminoarabinose, AraN), and for ethanolamine (EtN), present in lipid A. This assay enables the identification and quantitation of all amino-containing moieties present in lipopolysaccharide or lipid A from a single sample. The method was applied to the analysis of lipid A (endotoxin) isolated from a variety of biosynthetic and regulatory mutants of Salmonella enterica serovar Typhimurium and Francisella tularensis subspecies novicida. Lipid A is treated with trifluoroacetic acid to liberate and deacetylate individual aminosugars and mass tagged with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, which reacts with primary and secondary amines. The derivatives are separated using reversed-phase chromatography and analyzed using a single quadrupole mass spectrometer to detect quantities as small as 20 fmol. GalN was detected only in Francisella and AraN only in Salmonella, while GlcN was detected in lipid A samples from both species of bacteria. Additionally, we found an approximately 10-fold increase in the level of AraN in lipid A isolated from Salmonella grown in magnesium-limited versus magnesium-replete conditions. Salmonella with defined mutations in lipid A synthesis and regulatory genes were used to further validate the assay. Salmonella with null mutations in the phoP, pmrE, and prmF genes were unable to add AraN to their lipid A, while Salmonella with constitutively active phoP and pmrA exhibited AraN modification of lipid A even in the normally repressive magnesium-replete growth condition. The described assay produces excellent repeatability and reproducibility for the detection of amino-containing moieties in lipid A from a variety of bacterial sources. PMID:19130491

  8. QIAD assay for quantitating a compound’s efficacy in elimination of toxic Aβ oligomers

    PubMed Central

    Brener, Oleksandr; Dunkelmann, Tina; Gremer, Lothar; van Groen, Thomas; Mirecka, Ewa A.; Kadish, Inga; Willuweit, Antje; Kutzsche, Janine; Jürgens, Dagmar; Rudolph, Stephan; Tusche, Markus; Bongen, Patrick; Pietruszka, Jörg; Oesterhelt, Filipp; Langen, Karl-Josef; Demuth, Hans-Ulrich; Janssen, Arnold; Hoyer, Wolfgang; Funke, Susanne A.; Nagel-Steger, Luitgard; Willbold, Dieter

    2015-01-01

    Strong evidence exists for a central role of amyloid β-protein (Aβ) oligomers in the pathogenesis of Alzheimer’s disease. We have developed a fast, reliable and robust in vitro assay, termed QIAD, to quantify the effect of any compound on the Aβ aggregate size distribution. Applying QIAD, we studied the effect of homotaurine, scyllo-inositol, EGCG, the benzofuran derivative KMS88009, ZAβ3W, the D-enantiomeric peptide D3 and its tandem version D3D3 on Aβ aggregation. The predictive power of the assay for in vivo efficacy is demonstrated by comparing the oligomer elimination efficiency of D3 and D3D3 with their treatment effects in animal models of Alzheimer´s disease. PMID:26394756

  9. A quantitative study of optical mapping surfaces by atomic force microscopy and restriction endonuclease digestion assays.

    PubMed

    Reed, J; Singer, E; Kresbach, G; Schwartz, D C

    1998-05-15

    Many new techniques in biomolecular chemistry and genomic analysis require the immobilization of molecular reagents on specially prepared surfaces. However, the process of molecular fixation often interferes with or precludes the use of standard in vitro biochemical assays. Optical mapping is an emergent technology for genomic analysis which relies on the biochemical activity of DNA fixed to silanized glass surfaces. Optical mapping surfaces have been shown to be compatible with restriction endonucleases and a variety of DNA polymerases. The essential properties of biochemically active surfaces are poorly understood in most of the current technologies which utilize molecular fixation, including optical mapping. The purpose of this study is to use the powerful technique of atomic force microscopy, in combination with informative enzymatic assays, to correlate biochemical activity with microscopic surface structure. The results presented provide meaningful insight into the effect of surface preparation on the biochemical accessibility of surface-bound molecules. Novel analysis which may facilitate the automation of optical mapping is presented.

  10. Duplex Quantitative PCR Assay for Detection of Haemophilus influenzae That Distinguishes Fucose- and Protein D-Negative Strains

    PubMed Central

    de Gier, Camilla; Pickering, Janessa L.; Richmond, Peter C.; Thornton, Ruth B.

    2016-01-01

    We have developed a specific Haemophilus influenzae quantitative PCR (qPCR) that also identifies fucose-negative and protein D-negative strains. Analysis of 100 H. influenzae isolates, 28 Haemophilus haemolyticus isolates, and 14 other bacterial species revealed 100% sensitivity (95% confidence interval [CI], 96% to 100%) and 100% specificity (95% CI, 92% to 100%) for this assay. The evaluation of 80 clinical specimens demonstrated a strong correlation between semiquantitative culture and the qPCR (P < 0.001). PMID:27335148

  11. Comparison between ELISA and gel-filtration assay for the quantitation of airway mucins.

    PubMed

    Shin, C Y; Kang, S J; Kim, K C; Ko, K H

    1998-06-01

    In this study, we developed immunoassay methods for the more convenient and effective detection of rat tracheal mucin and the results were compared with those of [3H]glucosamine based gel-filtration method. A monoclonal anti-rat tracheal mucin antibody, mAbRT03, which specifically recognizes rat tracheal mucins, was used throughout in this study. To induce mucin secretion, varying concentrations of ATP (0-2 mM) were applied to the primary rat tracheal surface epithelial (RTSE) cell culture which had been metabolically radiolabeled with [3H]glucosamine and the secretion of mucin was analyzed both by the immunoassay and the gel-filtration chromatography methods. For the immunoassay, the following two procedures were employed. 1) Simple ELISA; the culture spent media were directly coated onto the assay plate and the immunoreactivity with mAbRT03 was assessed from the standard curve generated with the purified rat mucin. 2) Inhibition ELISA; A known amount of the purified rat mucin was coated onto the assay plate and then ATP-stimulated culture spent media were added to inhibit the immunoreactivity with mAbRT03. The contents of mucin in the sample were calculated from the standard inhibition curve which was generated with the purified rat mucin. The assay results obtained from the immunoassays were identical with those from the gel-filtration methods. The present result indicates that ELISA can be substituted for the laborious, time-consuming gel-filtration assay in studying the regulation of airway mucin release from cultured airway epithelial cells.

  12. Importance of purity evaluation and the potential of quantitative ¹H NMR as a purity assay.

    PubMed

    Pauli, Guido F; Chen, Shao-Nong; Simmler, Charlotte; Lankin, David C; Gödecke, Tanja; Jaki, Birgit U; Friesen, J Brent; McAlpine, James B; Napolitano, José G

    2014-11-26

    In any biomedical and chemical context, a truthful description of chemical constitution requires coverage of both structure and purity. This qualification affects all drug molecules, regardless of development stage (early discovery to approved drug) and source (natural product or synthetic). Purity assessment is particularly critical in discovery programs and whenever chemistry is linked with biological and/or therapeutic outcome. Compared with chromatography and elemental analysis, quantitative NMR (qNMR) uses nearly universal detection and provides a versatile and orthogonal means of purity evaluation. Absolute qNMR with flexible calibration captures analytes that frequently escape detection (water, sorbents). Widely accepted structural NMR workflows require minimal or no adjustments to become practical ¹H qNMR (qHNMR) procedures with simultaneous qualitative and (absolute) quantitative capability. This study reviews underlying concepts, provides a framework for standard qHNMR purity assays, and shows how adequate accuracy and precision are achieved for the intended use of the material.

  13. Total protein quantitation using the bicinchoninic acid assay and gradient elution moving boundary electrophoresis.

    PubMed

    Kralj, Jason G; Munson, Matthew S; Ross, David

    2014-07-01

    We investigated the ability of gradient elution moving boundary electrophoresis (GEMBE) with capacitively coupled contactless conductivity detection (C(4) D) to assay total protein concentration using the bicinchoninic acid (BCA) reaction. We chose this format because GEMBE-C(4) D behaves as a concentration dependent detection system, unlike optical methods that also rely on pathlength (due to Beer's law). This system tolerates proteins well compared with other capillary electrophoretic methods, allowing the capillary to be reused without coatings or additional hydroxide wash steps. The typical reaction protocol was modified by reducing the pH slightly from 11.25 to 9.4, which enabled elimination of tartrate from the reagents. We estimated that copper (I) could be detected at approximately 3.0 μmol/L, which agrees with similar GEMBE and CZE systems utilizing C(4) D. Under conditions similar to the BCA "micro method" assay, we determined the LOD for three common proteins (insulin, BSA, and bovine gamma globulin) and found that they agree well with the existing spectroscopic detection methods. Further, we investigated how long reaction times impact the LOD and found that the conversion was proportional to log(time). This indicated that little sensitivity is gained by extending the reaction past 1 h. Hence, GEMBE provides an alternative platform for total protein assays while maintaining the excellent sensitivity of the optical-based methods.

  14. Quantitative colorimetric assay for total protein applied to the red wine Pinot noir.

    PubMed

    Smith, Mark R; Penner, Mike H; Bennett, Samuel E; Bakalinsky, Alan T

    2011-07-13

    A standard method for assaying protein in red wine is currently lacking. The method described here is based on protein precipitation followed by dye binding quantification. Improvements over existing approaches include minimal sample processing prior to protein precipitation with cold trichloroacetic acid/acetone and quantification based on absorbance relative to a commercially available standard representative of proteins likely to be found in wine, the yeast mannoprotein invertase. The precipitation method shortened preparation time relative to currently published methods and the mannoprotein standard yielded values comparable to those obtained by micro-Kjeldahl analysis. The assay was used to measure protein in 48 Pinot noir wines from 6 to 32 years old. The protein content of these wines was found to range from 50 to 102 mg/L with a mean value of 70 mg/L. The availability of a simple and relatively rapid procedure for assaying protein provides a practical tool to quantify a wine component that has been overlooked in routine analyses of red wines.

  15. Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay.

    PubMed

    Huerta, Leonor; Lamoyi, Edmundo; Báez-Saldaña, Armida; Larralde, Carlos

    2002-02-01

    In vitro fusion of transfected cells expressing the human immunodeficiency virus (HIV) envelope proteins gp120/gp41, with target cells expressing CD4, and a suitable chemokine coreceptor is used widely to investigate the mechanisms of molecular recognition and membrane fusion involved in the entry of the HIV genome into cells and in syncytia formation. We developed an assay that uses two different fluorescent lipophilic probes to single label each reacting cell population and flow cytometry to quantify the extent of cellular fusion after coculture. Fused cells are detected as double-fluorescent particles in this assay, therefore permitting measurement of their proportion in the total cell population. The time course and extent of HIV-glycoprotein-related cellular fusion, the optimal cell ratio, the size and cell composition of the fusion products, and the inhibition of fusion caused by soluble CD4 and anti-CXCR4 antibody 12G5 were determined. The assay was applied to measure fusion between gp120/gp41 and CD4-expressing cells growing as monolayers (HeLa/CHO fusion), as well as to suspension lymphocyte cultures (Jurkat/Jurkat fusion). The method's simple technical and minimal cell-invasive procedures, as well as its non-ambiguous automatic numerical quantification should be useful for the study of factors influencing cell-cell fusion. Copyright 2002 Wiley-Liss, Inc.

  16. Comparison of various assays to quantitate macrophage activation by biological response modifiers

    SciTech Connect

    Schultz, R.M.; Nanda, S.; Altom, M.G.

    1984-01-01

    Macrophages treated with various compounds that enhance host antitumor resistance exhibit measurable changes in metabolism, function, and surface antigens. In this study, murine peptone-induced peritoneal macrophages were stimulated in vitro by bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP), and poly I.poly C. They were subsequently compared in their ability to release superoxide and act as tumoristatic and tumoricidal effector cells. Superoxide generation was assayed by the reduction of ferricytochrome C. All three compounds failed to induce significant O/sub 2/- release, unless the cells were also treated with phorbol myristate acetate (PMA). MDP was most active in potentiating the PMA response. In the tumor growth inhibition assay, cytostatic activity was comparable for all three compounds and did not exceed 32 percent. The combination of subthreshold levels of these compounds and hybridoma-derived MAF acted synergistically to induce potent cytostatic activity. In the chromium release assay, LPS and poly I.poly C rendered macrophages cytolytic for P815 target cells at concentrations greater than or equal to 1 microgram/ml. In contrast, significant cytolysis was observed with MDP only at 100 micrograms/ml. Defining precisely the effect of various biological response modifiers on several parameters of macrophage function may facilitate use of these agents in cancer therapy.

  17. Improved PCR assay for the species-specific identification and quantitation of Legionella pneumophila in water.

    PubMed

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

    2015-11-01

    Legionellosis outbreak is a major global health care problem. However, current Legionella risk assessments may be compromised by uncertainties in Legionella detection methods, infectious dose, and strain infectivity. These limitations may place public health at significant risk, leading to significant monetary losses in health care. However, there are still unmet needs for its rapid identification and monitoring of legionellae in water systems. Therefore, in the present study, a primer set was designed based on a LysR-type transcriptional regulator (LTTR) family protein gene of Legionella pneumophila subsp. pneumophila str. Philadelphia 1 because it was found that this gene is structurally diverse among species through BLAST searches. The specificity of the primer set was evaluated using genomic DNA from 6 strains of L. pneumophila, 5 type strains of other related Legionella species, and other 29 reference pathogenic bacteria. The primer set used in the PCR assay amplified a 264-bp product for only targeted six strains of L. pneumophila. The assay was also able to detect at least 1.39 × 10(3) copies/μl of cloned amplified target DNA using purified DNA or 7.4 × 10(0) colony-forming unit per reaction when using calibrated cell suspension. In addition, the sensitivity and specificity of this assay were confirmed by successful detection of Legionella pneumophila in environmental water samples.

  18. Development of MRM-based assays for the absolute quantitation of plasma proteins.

    PubMed

    Kuzyk, Michael A; Parker, Carol E; Domanski, Dominik; Borchers, Christoph H

    2013-01-01

    Multiple reaction monitoring (MRM), sometimes called selected reaction monitoring (SRM), is a directed tandem mass spectrometric technique performed on to triple quadrupole mass spectrometers. MRM assays can be used to sensitively and specifically quantify proteins based on peptides that are specific to the target protein. Stable-isotope-labeled standard peptide analogues (SIS peptides) of target peptides are added to enzymatic digests of samples, and quantified along with the native peptides during MRM analysis. Monitoring of the intact peptide and a collision-induced fragment of this peptide (an ion pair) can be used to provide information on the absolute peptide concentration of the peptide in the sample and, by inference, the concentration of the intact protein. This technique provides high specificity by selecting for biophysical parameters that are unique to the target peptides: (1) the molecular weight of the peptide, (2) the generation of a specific fragment from the peptide, and (3) the HPLC retention time during LC/MRM-MS analysis. MRM is a highly sensitive technique that has been shown to be capable of detecting attomole levels of target peptides in complex samples such as tryptic digests of human plasma. This chapter provides a detailed description of how to develop and use an MRM protein assay. It includes sections on the critical "first step" of selecting the target peptides, as well as optimization of MRM acquisition parameters for maximum sensitivity of the ion pairs that will be used in the final method, and characterization of the final MRM assay.

  19. A filter microplate assay for quantitative analysis of DNA binding proteins using fluorescent DNA.

    PubMed

    Yang, William C; Swartz, James R

    2011-08-15

    We present a rapid method for quantifying the apparent DNA binding affinity and capacity of recombinant transcription factors (TFs). We capture His6-tagged TFs using nickel-nitrilotriacetic acid (Ni-NTA) agarose and incubate the immobilized TFs with fluorescently labeled cognate DNA probes. After washing, the strength of the fluorescence signal indicates the extent of DNA binding. The assay was validated using two pluripotency-regulating TFs: SOX2 and NANOG. Using competitive binding analysis with nonlabeled competitor DNA, we show that SOX2 and NANOG specifically bind to their consensus sequences. We also determined the apparent affinity of SOX2 and NANOG for their consensus sequences to be 54.2±9 and 44.0±6nM, respectively, in approximate agreement with literature values. Our assay does not require radioactivity, but radioactively labeling the TFs enables the measurement of absolute amounts of immobilized SOX2 and NANOG and, hence, a DNA-to-protein binding ratio. SOX2 possesses a 0.95 DNA-to-protein binding ratio, whereas NANOG possesses a 0.44 ratio, suggesting that most of the SOX2 and approximately half of the NANOG are competent for DNA binding. Alternatively, the NANOG dimer may be capable of binding only one DNA target. This flexible DNA binding assay enables the analysis of crude or purified samples with or without radioactivity.

  20. A versatile quantitation platform based on platinum nanoparticles incorporated volumetric bar-chart chip for highly sensitive assays.

    PubMed

    Wang, Yuzhen; Zhu, Guixian; Qi, Wenjin; Li, Ying; Song, Yujun

    2016-11-15

    Platinum nanoparticles incorporated volumetric bar-chart chip (PtNPs-V-Chip) is able to be used for point-of-care tests by providing quantitative and visualized readout without any assistance from instruments, data processing, or graphic plotting. To improve the sensitivity of PtNPs-V-Chip, hybridization chain reaction was employed in this quantitation platform for highly sensitive assays that can detect as low as 16 pM Ebola Virus DNA, 0.01ng/mL carcinoembryonic antigen (CEA), and the 10 HER2-expressing cancer cells. Based on this amplified strategy, a 100-fold decrease of detection limit was achieved for DNA by improving the number of platinum nanoparticle catalyst for the captured analyte. This quantitation platform can also distinguish single base mismatch of DNA hybridization and observe the concentration threshold of CEA. The new strategy lays the foundation for this quantitation platform to be applied in forensic analysis, biothreat detection, clinical diagnostics and drug screening.

  1. Quantitative imaging assay for NF-kappaB nuclear translocation in primary human macrophages.

    PubMed

    Noursadeghi, Mahdad; Tsang, Jhen; Haustein, Thomas; Miller, Robert F; Chain, Benjamin M; Katz, David R

    2008-01-01

    Quantitative measurement of NF-kappaB nuclear translocation is an important research tool in cellular immunology. Established methodologies have a number of limitations, such as poor sensitivity, high cost or dependence on cell lines. Novel imaging methods to measure nuclear translocation of transcriptionally active components of NF-kappaB are being used but are also partly limited by the need for specialist imaging equipment or image analysis software. Herein we present a method for quantitative detection of NF-kappaB rel A nuclear translocation, using immunofluorescence microscopy and the public domain image analysis software ImageJ that can be easily adopted for cellular immunology research without the need for specialist image analysis expertise and at low cost. The method presented here is validated by demonstrating the time course and dose response of NF-kappaB nuclear translocation in primary human macrophages stimulated with LPS, and by comparison with a commercial NF-kappaB activation reporter cell line.

  2. Detection and Quantitation of T-2 Mycotoxin Using a Simplified Protein Synthesis Inhibition Assay.

    DTIC Science & Technology

    1983-07-18

    chemical agents. It is presently being used in studies of mycotoxin mecha- ism of action and approaches toward in vivo neutralization of the toxic effects... neutralization of the toxic effects of mycotoxins . 3 Introduction Numerous approaches have been used in the past for the detection and quantitation of...study would be the srsarch for a means of in vivo neutralization of mycotoxin effects using antibodies .. ’ chemical agents by first testing for

  3. Apparatus and method for quantitative assay of generic transuranic wastes from nuclear reactors

    DOEpatents

    Caldwell, John T.; Kunz, Walter E.; Atencio, James D.

    1984-01-01

    A combination of passive and active neutron measurements which yields quantitative information about the isotopic composition of transuranic wastes from nuclear power or weapons material manufacture reactors is described. From the measurement of prompt and delayed neutron emission and the incidence of two coincidentally emitted neutrons from induced fission of fissile material in the sample, one can quantify .sup.233 U, .sup.235 U and .sup.239 Pu isotopes in waste samples. Passive coincidence counting, including neutron multiplicity measurement and determination of the overall passive neutron flux additionally enables the separate quantitative evaluation of spontaneous fission isotopes such as .sup.240 Pu, .sup.244 Cm and .sup.252 Cf, and the spontaneous alpha particle emitter .sup.241 Am. These seven isotopes are the most important constituents of wastes from nuclear power reactors and once the mass of each isotope present is determined by the apparatus and method of the instant invention, the overall alpha particle activity can be determined to better than 1 nCi/g from known radioactivity data. Therefore, in addition to the quantitative analysis of the waste sample useful for later reclamation purposes, the alpha particle activity can be determined to decide whether "permanent" low-level burial is appropriate for the waste sample.

  4. Apparatus and method for quantitative assay of generic transuranic wastes from nuclear reactors

    DOEpatents

    Caldwell, J.T.; Kunz, W.E.; Atencio, J.D.

    1982-03-31

    A combination of passive and active neutron measurements which yields quantitative information about the isotopic composition of transuranic wastes from nuclear power or weapons material manufacture reactors is described. From the measurement of prompt and delayed neutron emission and the incidence of two coincidentally emitted neutrons from induced fission of fissile material in the sample, one can quantify /sup 233/U, /sup 235/U and /sup 239/Pu isotopes in waste samples. Passive coincidence counting, including neutron multiplicity measurement and determination of the overall passive neutron flux additionally enables the separate quantitative evaluation of spontaneous fission isotopes such as /sup 240/Pu, /sup 244/Cm and /sup 252/Cf, and the spontaneous alpha particle emitter /sup 241/Am. These seven isotopes are the most important constituents of wastes from nuclear power reactors and once the mass of each isotope present is determined by the apparatus and method of the instant invention, the overall alpha particle activity can be determined to better than 1 nCi/g from known radioactivity data. Therefore, in addition to the quantitative analysis of the waste sample useful for later reclamation purposes, the alpha particle activity can be determined to decide whether permanent low-level burial is appropriate for the waste sample.

  5. Quantitative activation suppression assay to evaluate human bone marrow-derived mesenchymal stromal cell potency.

    PubMed

    Salem, Bahey; Miner, Samantha; Hensel, Nancy F; Battiwalla, Minoo; Keyvanfar, Keyvan; Stroncek, David F; Gee, Adrian P; Hanley, Patrick J; Bollard, Catherine M; Ito, Sawa; Barrett, A John

    2015-12-01

    With the increasing use of cell therapies involving immune modulatory cells, there is a need for a simple standardized method to evaluate and compare the suppressive potency of different cell products. We used the Karpas 299 (K299) cell line as the reference suppressor cell to develop a standardized suppression assay to quantify the immune-modulatory capacity of bone marrow-derived mesenchymal stromal cells (BM-MSCs). Healthy donor CD4 T cells were co-cultured with the K299 cell line or with third-party BM-MSCs. After stimulation with anti-CD3/CD28 beads, CD154 activation and proliferation of CD4 T cells were measured to calculate suppression. The K299 cell line reproducibly suppressed both the activation and proliferation of healthy donor CD4 T cells in a dose-dependent manner. A rapid (16-h) assay that was based on activation-suppression was selected for development. In replicate testing, there was an inherent variability of suppression of 11% coefficient of variation between different responder T cells. Suppression by BM-MSCs on different responders correlated with suppression by K299. We therefore used K299 suppression as the reference to define suppression potency of BM-MSCs in K299 Suppression Units. We found that inter-donor variability, passage number, method of manufacture and exposure of BM-MSCs to steroids or interferon-γ all affected BM-MSC potency of suppression. This method provides a platform for standardizing suppressor function to facilitate comparisons between laboratories and for use as a cell product release assay. Published by Elsevier Inc.

  6. Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

    PubMed

    Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R

    2015-11-17

    Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

  7. Effects of Ureaplasma diversum on bovine oviductal explants: quantitative measurement using a calmodulin assay.

    PubMed Central

    Smits, B; Rosendal, S; Ruhnke, H L; Plante, C; O'Brien, P J; Miller, R B

    1994-01-01

    Calmodulin (CAM) acts as an intracellular regulator of calcium, an important mediator of many cell processes. We used the CAM assay and electron microscopy to investigate the effects of Ureaplasma diversum on bovine oviductal explants obtained aseptically from slaughtered cows. A stock suspension of U. diversum (treated specimens) and sterile broth (controls) was added to replicates of cultured explants and incubated at 38 degrees C in an atmosphere of 5.5% CO2 for 48 hours. Explants were examined for ciliary activity, extracellular CAM loss, and for histological and ultrastructural changes. Explants and their culture media were examined for changes in CAM concentration. All experiments were replicated three times. In addition, U. diversum, medium and broth were assayed for CAM content. The concentrations of CAM in explants and media changed significantly (p < 0.05) in samples which were inoculated with U. diversum when compared to controls. The controls and infected specimens did not differ histologically or ultrastructurally, but U. diversum was seen to be closely associated with infected explant tissue. In view of this close affinity it is assumed the loss of CAM from the oviductal cells was causally related, but this was not proven. The failure to show cell membrane injury on light and electron microscopic examination was probably related to the short duration of the experiment and may only point out the sensitivity of the CAM assay in detecting early cell membrane injury. Compromise in characteristics of the medium to support both, the viability of oviductal cells and U. diversum limited the experimental time to 48 hours.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:8004536

  8. A pyrosequencing assay for the quantitative methylation analysis of the PCDHB gene cluster, the major factor in neuroblastoma methylator phenotype.

    PubMed

    Banelli, Barbara; Brigati, Claudio; Di Vinci, Angela; Casciano, Ida; Forlani, Alessandra; Borzì, Luana; Allemanni, Giorgio; Romani, Massimo

    2012-03-01

    Epigenetic alterations are hallmarks of cancer and powerful biomarkers, whose clinical utilization is made difficult by the absence of standardization and of common methods of data interpretation. The coordinate methylation of many loci in cancer is defined as 'CpG island methylator phenotype' (CIMP) and identifies clinically distinct groups of patients. In neuroblastoma (NB), CIMP is defined by a methylation signature, which includes different loci, but its predictive power on outcome is entirely recapitulated by the PCDHB cluster only. We have developed a robust and cost-effective pyrosequencing-based assay that could facilitate the clinical application of CIMP in NB. This assay permits the unbiased simultaneous amplification and sequencing of 17 out of 19 genes of the PCDHB cluster for quantitative methylation analysis, taking into account all the sequence variations. As some of these variations were at CpG doublets, we bypassed the data interpretation conducted by the methylation analysis software to assign the corrected methylation value at these sites. The final result of the assay is the mean methylation level of 17 gene fragments in the protocadherin B cluster (PCDHB) cluster. We have utilized this assay to compare the methylation levels of the PCDHB cluster between high-risk and very low-risk NB patients, confirming the predictive value of CIMP. Our results demonstrate that the pyrosequencing-based assay herein described is a powerful instrument for the analysis of this gene cluster that may simplify the data comparison between different laboratories and, in perspective, could facilitate its clinical application. Furthermore, our results demonstrate that, in principle, pyrosequencing can be efficiently utilized for the methylation analysis of gene clusters with high internal homologies.

  9. Development of a quantitative polymerase chain reaction assay for detection of Kudoa septempunctata in olive flounder (Paralichthys olivaceus).

    PubMed

    Harada, Tetsuya; Kawai, Takao; Sato, Hiroshi; Yokoyama, Hiroshi; Kumeda, Yuko

    2012-05-15

    Kudoa septempunctata is a newly identified myxosporean parasite that infects the trunk muscles of olive flounder (Paralichthys olivaceus) and a causative agent of the increasing number of foodborne gastroenteritis outbreaks with unknown etiology which have occurred in Japan over the last few years. Here, we developed a quantitative polymerase chain reaction (QPCR) assay for the detection of K. septempunctata 18S rDNA in olive flounder muscle tissue samples. Additionally, we compared the relative efficacy of four DNA extraction methods, including two commercial kits, and assessed intrafish variability in the distribution of K. septempunctata spores in flounder using this QPCR method in order to establish a more accurate quantitative measurement. Our QPCR assay displayed high sensitivity, specificity, and reproducibility, and had good correlation with a microscopic detection method. Our data also indicated that the DNeasy® Blood & Tissue Kit was more efficient method for the extraction of K. septempunctata DNA than the three other methods (heating, alkaline lysis, and FastDNA® SPIN Kit method). We believe that our method would be useful for investigating foodborne outbreaks caused by K. septempunctata and for the monitoring and quantification of this parasite in retail or aquacultured olive flounders to prevent such outbreaks. Copyright © 2012 Elsevier B.V. All rights reserved.

  10. Rapid and quantitative detection of 4(5)-methylimidazole in caramel colours: A novel fluorescent-based immunochromatographic assay.

    PubMed

    Wu, Xinlan; Huang, Minghui; Yu, Shujuan; Kong, Fansheng

    2016-01-01

    A novel fluorescence-based immunochromatographic assay (ICA) for rapid detecting 4(5)-methylimidazole (4-MI) is presented in this study. In our work, the conjugates of fluorescent microspheres (FMs) and 4-MI monoclonal antibody were used as probe for ICA. Under optimal conditions, a standard curve of ICA-based detection of 4-MI was developed, linear detection ranged from 0.50 to 32.0 mg/L. The cross-reactivities were observed less than 3.93% by detecting 6 selected structural analogues of 4-MI. The recoveries of 4-MI in caramels detection were ranged from 82.85% to 102.31%, with the coefficient of variation (n = 3) below 9.06%. Quantitative comparison of the established fluorescence-based ICA with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) analysis of real caramel colour samples indicated a good correlation among the methods. Therefore, our developed fluorescence-based ICA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of 4-MI in food safety control.

  11. Systematic study of substance P analogs. I. Evaluation of peptides synthesized by the multipin method for quantitative receptor binding assay.

    PubMed

    Wang, J X; Bray, A M; Dipasquale, A J; Maeji, N J; Geysen, H M

    1993-10-01

    The multipin peptide synthesis technique, a method for simultaneous multiple peptide synthesis, was developed for large-scale screening of oligopeptides [Geysen et al. (1984) Proc. Natl. Acad. Sci. USA, 81, 3998-4002]. A modification of the technique allows the peptides assembled on polyethylene pins to be cleaved in their native amide form and reconstituted into physiologically compatible solutions. In this study, the suitability of these peptides for quantitative receptor binding assay was evaluated. Substance P and 18 analogs, including a set of N-terminal truncated substance P and a set of naturally occurring substance P analogs, were synthesized by the multipin methods. An average yield of 20 +/- 3 nmol of peptide per pin was obtained. The purity of the peptides was estimated to be ca. 90%. The binding activities of these peptides were determined in a competition assay against 125I-BHSP binding to a rat brain synaptosome preparation. The rank order of the affinities of these peptides depicted a typical pharmacological profile of central NK1 receptor. The IC50 values obtained were also in good agreement with data reported by other groups using similar experimental conditions, except that bulk synthesized peptides were used. This study demonstrates that the peptides synthesized with the multipin technique are suitable for quantitative receptor studies, particularly for a high-volume screening of bioactive peptides.

  12. A fluorescent assay to quantitatively measure in vitro acyl CoA:diacylglycerol acyltransferase activity.

    PubMed

    McFie, Pamela J; Stone, Scot J

    2011-09-01

    Triacylglycerols (TG) are the major storage form of energy in eukaryotic organisms and are synthesized primarily by acyl CoA:1,2-diacylglycerol acyltransferase (DGAT) enzymes. In vitro DGAT activity has previously been quantified by measuring the incorporation of either radiolabeled fatty acyl CoA or diacylglycerol (DG) into TG. We developed a modified acyltransferase assay using a fluorescent fatty acyl CoA substrate to accurately quantify in vitro DGAT activity. In the modified assay, radioactive fatty acyl CoA is replaced with fluorescent NBD-palmitoyl CoA, which is used as a substrate by DGAT with DG to produce NBD-TG. After extraction with organic solvents and separation by thin layer chromatography, NBD-TG formation can be detected and accurately quantified using a fluorescent imaging system. We demonstrate that this method can be adapted to detect other acyltransferase activities. Because NBD-palmitoyl CoA is commercially available at a much lower cost compared with radioactive acyl CoA substrates, it is a more economical alternative to radioactive tracers. In addition, the exposure of laboratory personnel to radioactivity is greatly reduced.

  13. Immunoradiometric assay for quantitation of Dirofilaria immitis antigen in dogs with heartworm infections

    SciTech Connect

    Hamilton, R.G.; Scott, A.L.

    1984-10-01

    An immunoradiometric assay (IRMA) was developed, optimized, and validated for detection of parasite-specific antigen in sera from hosts with filarial infections, using Dirofilaria immitis in dogs as a model. The precision, reproducibility, and parallelism of the IRMA were examined, using precision profile analysis. The IRMA had acceptable precision and reproducibility (less than 15% intra-assay coefficient of variation (CV)) over a working range of 10 to 2000 ng of D immitis-antigen (AG)/ml. The IRMA parallelism (agreement between dilutions) was acceptable (less than 10% interdilutional CV) with laboratory-spiked D immitis AG sera containing no D immitis-antibody (AB). However, it was not acceptable (greater than 20% interdilutional CV) for analysis of sera from naturally infected dogs containing D immitis AB, probably due to dissociation of immune-complexed AG with increasing serum dilution. Nonparallelism limited the accuracy of binding data interpolation from the standard curve. Specificity of the IRMA was enhanced by preabsorption of the radiolabeled detection antibody with Toxocara canis AG before use. Varying amounts of D immitis AG (22 to 1000 ng/ml) were detected in 42% (20/48) of microfilaremic dogs. The presence of AG-specific AB at concentrations as low as 1 microgram/ml reduced the ability of the IRMA to detect D immitis AG. Factors that influence the accuracy and sensitivity of immunoassays for circulating filarial antigens are discussed.

  14. High-Throughput Electrophoretic Mobility Shift Assays for Quantitative Analysis of Molecular Binding Reactions

    PubMed Central

    2015-01-01

    We describe a platform for high-throughput electrophoretic mobility shift assays (EMSAs) for identification and characterization of molecular binding reactions. A photopatterned free-standing polyacrylamide gel array comprised of 8 mm-scale polyacrylamide gel strips acts as a chassis for 96 concurrent EMSAs. The high-throughput EMSAs was employed to assess binding of the Vc2 cyclic-di-GMP riboswitch to its ligand. In optimizing the riboswitch EMSAs on the free-standing polyacrylamide gel array, three design considerations were made: minimizing sample injection dispersion, mitigating evaporation from the open free-standing polyacrylamide gel structures during electrophoresis, and controlling unit-to-unit variation across the large-format free-standing polyacrylamide gel array. Optimized electrophoretic mobility shift conditions allowed for 10% difference in mobility shift baseline resolution within 3 min. The powerful 96-plex EMSAs increased the throughput to ∼10 data/min, notably more efficient than either conventional slab EMSAs (∼0.01 data/min) or even microchannel based microfluidic EMSAs (∼0.3 data/min). The free-standing polyacrylamide gel EMSAs yielded reliable quantification of molecular binding and associated mobility shifts for a riboswitch–ligand interaction, thus demonstrating a screening assay platform suitable for riboswitches and potentially a wide range of RNA and other macromolecular targets. PMID:25233437

  15. A novel quantitative kinase assay using bacterial surface display and flow cytometry.

    PubMed

    Henriques, Sónia Troeira; Thorstholm, Louise; Huang, Yen-Hua; Getz, Jennifer A; Daugherty, Patrick S; Craik, David J

    2013-01-01

    The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli) to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development.

  16. Development of a rapid semi-quantitative immunochromatographic assay for serum hepatocyte growth factor and its usefulness in acute liver failure.

    PubMed

    Uto, Hirofumi; Ido, Akio; Kusumoto, Kazunori; Hasuike, Satoru; Nagata, Kenji; Hayashi, Katsuhiro; Yamagishi, Toshiya; Gohda, Eiichi; Tsubouchi, Hirohito

    2005-12-01

    Measurement of serum human hepatocyte growth factor (HGF) by enzyme-linked immunosorbent assay (ELISA) is useful for the early diagnosis and prediction of prognosis of patients with acute liver failure (ALF). This ELISA methodology, however, is neither rapid nor convenient for use at the bedside. In this study, we have developed a rapid semi-quantitative immunochromatographic (IC) assay and evaluated its usefulness in assessing patients with acute hepatic injury. Only 100mul of serum is required; the assay can be easily completed in 20min. The values obtained using this novel assay correlated well with the values obtained using the standard ELISA protocol. In addition, the values obtained in the IC assay correlated with clinical course; increased serum HGF levels were associated with an increased frequency of ALF and death. These results indicate that this rapid semi-quantitative IC assay for HGF is useful for the early diagnosis of ALF and prediction of clinical outcome in acute hepatic injury.

  17. A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection

    SciTech Connect

    Pierson, Theodore C. . E-mail: piersontc@mail.nih.gov; Sanchez, Melissa D.; Puffer, Bridget A.; Ahmed, Asim A.; Geiss, Brian J.; Valentine, Laura E.; Altamura, Louis A.; Diamond, Michael S.; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu

    2006-03-01

    West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo. In this study, we describe a novel approach for measuring antibody-mediated neutralization of WNV infection using virus-like particles that measure infection as a function of reporter gene expression. These reporter virus particles (RVPs) are produced by complementation of a sub-genomic replicon with WNV structural proteins provided in trans using conventional DNA expression vectors. The precision and accuracy of this approach stem from an ability to measure the outcome of the interaction between antibody and viral antigens under conditions that satisfy the assumptions of the law of mass action as applied to virus neutralization. In addition to its quantitative strengths, this approach allows the production of WNV RVPs bearing the prM-E proteins of different WNV strains and mutants, offering considerable flexibility for the study of the humoral immune response to WNV in vitro. WNV RVPs are capable of only a single round of infection, can be used under BSL-2 conditions, and offer a rapid and quantitative approach for detecting virus entry and its inhibition by neutralizing antibody.

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

    PubMed Central

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

    2011-01-01

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

  19. Quantitative microplate assay for studying mesenchymal stromal cell-induced neuropoiesis.

    PubMed

    Aizman, Irina; McGrogan, Michael; Case, Casey C

    2013-03-01

    Transplanting mesenchymal stromal cells (MSCs) or their derivatives in a neurodegenerative environment is believed to be beneficial because of the trophic support, migratory guidance, and neurogenic stimuli they provide. There is a growing need for in vitro models of mesenchymal-neural cell interactions to enable identification of mediators of the MSC activity and quantitative assessment of neuropoietic potency of MSC preparations. Here, we characterize a microplate-format coculture system in which primary embryonic rat cortex cells are directly cocultured with human MSCs on cell-derived extracellular matrix (ECM) in the absence of exogenous growth factors. In this system, expression levels of the rat neural stem/early progenitor marker nestin, as well as neuronal and astrocytic markers, directly depended on MSC dose, whereas an oligodendrogenic marker exhibited a biphasic MSC-dose response, as measured using species-specific quantitative reverse transcription-polymerase chain reaction in total cell lysates and confirmed using immunostaining. Both neural cell proliferation and differentiation contributed to the MSC-mediated neuropoiesis. ECM's heparan sulfate proteoglycans were essential for the growth of the nestin-positive cell population. Neutralization studies showed that MSC-derived fibroblast growth factor 2 was a major and diffusible inducer of rat nestin, whereas MSC-derived bone morphogenetic proteins (BMPs), particularly, BMP4, were astrogenesis mediators, predominantly acting in a coculture setting. This system enables analysis of multifactorial MSC-neural cell interactions and can be used for elucidating the neuropoietic potency of MSCs and their derivative preparations.

  20. A Novel Qualitative and Quantitative Biofilm Assay Based on 3D Soft Tissue.

    PubMed

    Hakonen, Bodil; Lönnberg, Linnea K; Larkö, Eva; Blom, Kristina

    2014-01-01

    The lack of predictable in vitro methods to analyze antimicrobial activity could play a role in the development of resistance to antibiotics. Current used methods analyze planktonic cells but for the method to be clinically relevant, biofilm in in vivo like conditions ought to be studied. Hence, our group has developed a qualitative and quantitative method with in vivo like 3D tissue for prediction of antimicrobial activity in reality. Devices (wound dressings) were applied on top of Pseudomonas aeruginosa inoculated Muller-Hinton (MH) agar or 3D synthetic soft tissues (SST) and incubated for 24 hours. The antibacterial activity was then analyzed visually and by viable counts. On MH agar two out of three silver containing devices showed zone of inhibitions (ZOI) and on SST, ZOI were detected for all three. Corroborating results were found upon evaluating the bacterial load in SST and shown to be silver concentration dependent. In conclusion, a novel method was developed combining visual rapid screening and quantitative evaluation of the antimicrobial activity in both tissue and devices. It uses tissue allowing biofilm formation thus mimicking reality closely. These conditions are essential in order to predict antimicrobial activity of medical devices in the task to prevent device related infections.

  1. Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.

    PubMed

    Vu, Hong; Shulenin, Sergey; Grolla, Allen; Audet, Jonathan; He, Shihua; Kobinger, Gary; Unfer, Robert C; Warfield, Kelly L; Aman, M Javad; Holtsberg, Frederick W

    2016-02-01

    The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA's), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in Escherichia coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Quantitative analysis of energy metabolic pathways in MCF-7 breast cancer cells by selected reaction monitoring assay.

    PubMed

    Drabovich, Andrei P; Pavlou, Maria P; Dimitromanolakis, Apostolos; Diamandis, Eleftherios P

    2012-08-01

    To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells.

  3. Quantitative Serology Assays for Determination of Antibody Responses to Ebola Virus Glycoprotein and Matrix Protein in Nonhuman Primates and Humans

    PubMed Central

    Vu, Hong; Shulenin, Sergey; Grolla, Allen; Audet, Jonathan; He, Shihua; Kobinger, Gary; Unfer, Robert C.; Warfield, Kelly L.; Aman, M. Javad; Holtsberg, Frederick W.

    2016-01-01

    The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA’s), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in E. coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals. PMID:26681387

  4. Development and Validation of a Highly Accurate Quantitative Real-Time PCR Assay for Diagnosis of Bacterial Vaginosis.

    PubMed

    Hilbert, David W; Smith, William L; Chadwick, Sean G; Toner, Geoffrey; Mordechai, Eli; Adelson, Martin E; Aguin, Tina J; Sobel, Jack D; Gygax, Scott E

    2016-04-01

    Bacterial vaginosis (BV) is the most common gynecological infection in the United States. Diagnosis based on Amsel's criteria can be challenging and can be aided by laboratory-based testing. A standard method for diagnosis in research studies is enumeration of bacterial morphotypes of a Gram-stained vaginal smear (i.e., Nugent scoring). However, this technique is subjective, requires specialized training, and is not widely available. Therefore, a highly accurate molecular assay for the diagnosis of BV would be of great utility. We analyzed 385 vaginal specimens collected prospectively from subjects who were evaluated for BV by clinical signs and Nugent scoring. We analyzed quantitative real-time PCR (qPCR) assays on DNA extracted from these specimens to quantify nine organisms associated with vaginal health or disease:Gardnerella vaginalis,Atopobium vaginae, BV-associated bacteria 2 (BVAB2, an uncultured member of the orderClostridiales),Megasphaeraphylotype 1 or 2,Lactobacillus iners,Lactobacillus crispatus,Lactobacillus gasseri, andLactobacillus jensenii We generated a logistic regression model that identifiedG. vaginalis,A. vaginae, andMegasphaeraphylotypes 1 and 2 as the organisms for which quantification provided the most accurate diagnosis of symptomatic BV, as defined by Amsel's criteria and Nugent scoring, with 92% sensitivity, 95% specificity, 94% positive predictive value, and 94% negative predictive value. The inclusion ofLactobacillusspp. did not contribute sufficiently to the quantitative model for symptomatic BV detection. This molecular assay is a highly accurate laboratory tool to assist in the diagnosis of symptomatic BV.

  5. Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR.

    PubMed

    Teferedegne, Belete; Lewis, Andrew M; Peden, Keith; Murata, Haruhiko

    2013-01-01

    A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN) is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN) in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses). The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours). In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID(50)). Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses.

  6. Development of a Neutralization Assay for Influenza Virus Using an Endpoint Assessment Based on Quantitative Reverse-Transcription PCR

    PubMed Central

    Teferedegne, Belete; Lewis, Andrew M.; Peden, Keith; Murata, Haruhiko

    2013-01-01

    A microneutralization assay using an ELISA-based endpoint assessment (ELISA-MN) is widely used to measure the serological response to influenza virus infection and vaccination. We have developed an alternative microneutralization assay for influenza virus using a quantitative reverse transcription PCR-based endpoint assessment (qPCR-MN) in order to improve upon technical limitations associated with ELISA-MN. For qPCR-MN, infected MDCK-London cells in 96-well cell-culture plates are processed with minimal steps such that resulting samples are amenable to high-throughput analysis by downstream one-step quantitative reverse transcription PCR (qRT-PCR; SYBR Green chemistry with primers targeting a conserved region of the M1 gene of influenza A viruses). The growth curves of three recent vaccine strains demonstrated that the qRT-PCR signal detected at 6 hours post-infection reflected an amplification of at least 100-fold over input. Using ferret antisera, we have established the feasibility of measuring virus neutralization at 6 hours post-infection, a duration likely confined to a single virus-replication cycle. The neutralization titer for qPCR-MN was defined as the highest reciprocal serum dilution necessary to achieve a 90% inhibition of the qRT-PCR signal; this endpoint was found to be in agreement with ELISA-MN using the same critical reagents in each assay. qPCR-MN was robust with respect to assay duration (6 hours vs. 12 hours). In addition, qPCR-MN appeared to be compliant with the Percentage Law (i.e., virus neutralization results appear to be consistent over an input virus dose ranging from 500 to 12,000 TCID50). Compared with ELISA-MN, qPCR-MN might have inherent properties conducive to reducing intra- and inter-laboratory variability while affording suitability for automation and high-throughput uses. Finally, our qRT-PCR-based approach may be broadly applicable to the development of neutralization assays for a wide variety of viruses. PMID:23437084

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

    PubMed

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

    2015-07-01

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

  8. Quantitative determination of recombinant bovine somatotropin in commercial shrimp feed using a competitive enzyme-linked immunosorbent assay.

    PubMed

    Munro, James L; Boon, Virginia A

    2010-02-10

    Recombinant bovine somatotropin (rbST), also known as growth hormone, is used to enhance production and development of animals within the agriculture and aquaculture industries. Its use is controversial because of its potential effects on human and animal health. To screen for rbST in shrimp feed, a competitive enzyme-linked immunosorbent assay (ELISA) with an inhibition step was developed. Sample and rbST antibody (rabbit anti-rbST) were incubated at room temperature for 30 min. Subsequently, this competitive reaction was transferred to a microplate coated with rbST, using goat antirabbit IgG linked with horseradish peroxidise as the secondary antibody. Substrates for peroxidise were added, and the absorbance at 410 nm was determined. The applicability of the method was assessed using rbST extracted from "spiked" shrimp feed samples. The assay was reproducible and linear with R(2) values greater than 0.98 over the standard curve range of 20-500 microg/g. The intra- and interday precisions expressed as relative standard deviations were 3.4 and 5.3%, respectively. The mean recovery from 15 spiked feed samples was 105%. This assay will be a valuable tool for quantitative detection of rbST by both governments and commercial companies and can be modified for other types of feed.

  9. A quantitative reverse transcriptase polymerase chain reaction-based assay to detect carcinoma cells in peripheral blood.

    PubMed Central

    Helfrich, W.; ten Poele, R.; Meersma, G. J.; Mulder, N. H.; de Vries, E. G.; de Leij, L.; Smit, E. F.

    1997-01-01

    The presence of tumour cells in the circulation may predict disease recurrence and metastasis. To improve on existing methods of cytological or immunocytological detection, we have developed a sensitive and quantitative technique for the detection of carcinoma cells in blood, using the reverse transcriptase polymerase chain reaction (RT-PCR) identifying transcripts of the pancarcinoma-associated tumour marker EGP-2 (KSA or 17-1A antigen). The amount of EGP2 mRNA was quantified using an internal recombinant competitor RNA standard with known concentration and which is both reversely transcribed and co-amplified in the same reaction, allowing for a reliable assessment of the initial amount of EGP2 mRNA in the sample. Calibration studies, seeding blood with MCF-7 breast carcinoma cells, showed that the assay can detect ten tumour cells among 1.0 x 10(6) leucocytes. The PCR assay revealed that normal bone marrow expresses low levels of EGP2 mRNA, although immunocytochemistry with the anti-EGP2 MAb MOC31 could not identify any positively stained cell. Analyses using this RT-PCR assay may prove to have applications to the assessment of circulating tumour cells in clinical samples. Images Figure 3 Figure 4 PMID:9218728

  10. Quantitative High Throughput Screening Using a Live Cell cAMP Assay Identifies Small Molecule Agonists of the TSH Receptor

    PubMed Central

    Titus, Steve; Neumann, Susanne; Zheng, Wei; Southall, Noel; Michael, Sam; Klumpp, Carleen; Yasgar, Adam; Shinn, Paul; Thomas, Craig J.; Inglese, Jim; Gershengorn, Marvin C.; Austin, Christopher P.

    2009-01-01

    The thyroid stimulating hormone receptor (TSHR) belongs to the glycoprotein hormone receptor subfamily of seven-transmembrane spanning receptors. TSHR is expressed in thyroid follicular cells and is activated by TSH, which regulates growth and function of these cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small molecule agonist of the TSHR is available. To screen for novel TSHR agonists, we miniaturized a cell-based cAMP assay into 1536-well plate format. This assay uses a HEK293 cell line stably expressing the TSHR and a cyclic nucleotide gated ion channel (CNG), which functions as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal HTRF cAMP-based assay. 49 compounds in several structural classes have been confirmed as small molecule TSHR agonists that will serve as starting compounds for chemical optimization and studies of thyroid physiology in health and disease. PMID:18216391

  11. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

    PubMed

    Kanthaswamy, S; Premasuthan, A; Ng, J; Satkoski, J; Goyal, V

    2012-03-01

    In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan(®) quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human-dog-cat origin with as little as 0.4 pg of human and cat nuclear DNA, respectively, and 4.0 pg of dog nuclear DNA.

  12. Quantitative enzyme-linked immunosorbent assay for determination of polychlorinated biphenyls in environmental soil and sediment samples.

    PubMed

    Johnson, J C; Van Emon, J M

    1996-01-01

    An enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of Aroclors 1242, 1248, 1254, and 1260 in soil and sediments was developed and its performance compared with that of gas chromatography (GC). The detection limits for Aroclors 1242 and 1248 in soil are 10.5 and 9 ng/g, respectively. The assay linear dynamic range is 50-1333 ng/g. Cross-reactivity of the assay with 37 structurally related potential cocontaminants in environmental soil samples was examined; none of the chlorinated anisoles, benzenes, or phenols exhibited >3% cross-reactivity, with <0.1% cross-reactivity being the norm. Soil spike recoveries of 107% and 104% were obtained for Aroclors 1242 and 1248, respectively, for a spike level of 5 mg/kg, with corresponding relative standard deviations of 14% and 17%. One hundred forty-eight environmental soil, sediment, and paper pulp samples, obtained from two EPA listed Superfund sites, were analyzed by ELISA and standard GC methods. Samples were extracted for ELISA analysis by shaking with methanol. Additional extractions of the same samples were performed either with supercritical carbon dioxide or by Soxhlet extraction with methanol. ELISA results for both the supercritical fluid and the Soxhlet extracts were in close agreement with the GC results, while the ELISA results for the methanol shake extracts were not. The data for the environmental samples demonstrated the capability of the ELISA to provide accurate results and reinforced the dependence of any detection method, including ELISA, on appropriate extraction procedures.

  13. Development of a Chip Assay and Quantitative PCR for Detecting Microcystin Synthetase E Gene Expression ▿ †

    PubMed Central

    Sipari, Hanna; Rantala-Ylinen, Anne; Jokela, Jouni; Oksanen, Ilona; Sivonen, Kaarina

    2010-01-01

    The chip and quantitative real-time PCR (qPCR) assays were optimized to study the expression of microcystin biosynthesis genes (mcy) with RNA samples extracted from cyanobacterial strains and environmental water samples. Both microcystin-producing Anabaena and Microcystis were identified in Lake Tuusulanjärvi samples. Microcystis transcribed the mcyE genes throughout the summer of 2006, while expression by Anabaena became evident later in August and September. Active mcyE gene expression was also detectable when microcystin concentrations were very low. Detection of Anabaena mcyE transcripts by qPCR, as well as certain cyanobacterial 16S rRNAs with the chip assay, showed slightly reduced sensitivity compared with the DNA analyses. In contrast, even groups undetectable or present in low quantities as determined by microscopy could be identified with the chip assay from DNA samples. The methods introduced add to the previously scarce repertoire of applications for mcy expression profiling in environmental samples and enable in situ studies of regulation of microcystin synthesis in response to environmental factors. PMID:20400558

  14. Optimization in multidimensional gas chromatography applying quantitative analysis via a stable isotope dilution assay.

    PubMed

    Schmarr, Hans-Georg; Slabizki, Petra; Legrum, Charlotte

    2013-08-01

    Trace level analyses in complex matrices benefit from heart-cut multidimensional gas chromatographic (MDGC) separations and quantification via a stable isotope dilution assay. Minimization of the potential transfer of co-eluting matrix compounds from the first dimension ((1)D) separation into the second dimension separation requests narrow cut-windows. Knowledge about the nature of the isotope effect in the separation of labeled and unlabeled compounds allows choosing conditions resulting in at best a co-elution situation in the (1)D separation. Since the isotope effect strongly depends on the interactions of the analytes with the stationary phase, an appropriate separation column polarity is mandatory for an isotopic co-elution. With 3-alkyl-2-methoxypyrazines and an ionic liquid stationary phase as an example, optimization of the MDGC method is demonstrated and critical aspects of narrow cut-window definition are discussed.

  15. A Quantitative Fluorescence-Based Assay for Assessing LIM Domain-Peptide Interactions.

    PubMed

    Robertson, Neil O; Shah, Manan; Matthews, Jacqueline M

    2016-10-10

    We have developed Förster resonance energy transfer (FRET)-based experiments for measuring the binding affinity, off-rates, and inferred on-rates for interactions between a family of transcriptional regulators and their intrinsically disordered binding partners. It was difficult to evaluate these interactions previously, as the transcriptional regulators are obligate binding proteins that aggregate in the absence of a binding partner. The assays rely on fusion constructs where binding domains are linked by a flexible tether containing a specific protease site, with fluorescent proteins at either end that display FRET when the complex is formed. Loss of FRET is monitored after cutting the tether followed by dilution or competition with a non-fluorescent peptide. These methods allowed a wide range of binding affinities (10(-9) -10(-5)  m) to be determined. Our data indicate that interactions of closely related proteins can have surprisingly different binding properties.

  16. A quantitative enzyme-linked immunosorbent assay for quantification of secretory immunoglobulin A in serum.

    PubMed

    Pérez-Griera, Jaime; Andreu-Ballester, Juan Carlos; Hueso Zarandieta, Alejandro; García de la Asunción, José; Masquefa Bondia, Silvia

    2017-01-01

    Secretory immunoglobulin A (SIgA) in serum is possibly the best index of SIgA presence in mucosal secretions in digestive tract and the mirror of its immunologic barrier against many pathogenic aggressions. The measurement of salivary SigA alone may be affected by total salivary secretion and its final concentration in the gland lumen is probably not useful as an appropriate index of mucosal secretions in the digestive tract. The usefulness of the determination of SigA against various epitopes in serum from patients with various autoimmune disease has been demonstrated. The aetiology of many digestive related disorders could be triggered by an alteration of mucose SIgA barrier. The determination of Igs is important for different liver diseases and specifically the SIgA in autoimmune diseases such as rheumatoid arthritis. We developed an easy and efficient immunologic assay to quantify SIgA in serum samples.

  17. Comparison between Culture and a Multiplex Quantitative Real-Time Polymerase Chain Reaction Assay Detecting Ureaplasma urealyticum and U. parvum

    PubMed Central

    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. PMID:25047036

  18. Analytical characteristics and comparative evaluation of Aptima HCV quant Dx assay with the Abbott RealTime HCV assay and Roche COBAS AmpliPrep/COBAS TaqMan HCV quantitative test v2.0.

    PubMed

    Worlock, A; Blair, D; Hunsicker, M; Le-Nguyen, T; Motta, C; Nguyen, C; Papachristou, E; Pham, J; Williams, A; Vi, M; Vinluan, B; Hatzakis, A

    2017-04-04

    The Aptima HCV Quant Dx assay (Aptima assay) is a fully automated quantitative assay on the Panther® system. This assay is intended for confirmation of diagnosis and monitoring of HCV RNA in plasma and serum specimens. The purpose of the testing described in this paper was to evaluate the performance of the Aptima assay. The analytical sensitivity, analytical specificity, precision, and linearity of the Aptima assay were assessed. The performance of the Aptima assay was compared to two commercially available HCV assays; the Abbott RealTime HCV assay (Abbott assay, Abbott Labs Illinois, USA) and the Roche COBAS Ampliprep/COBAS Taqman HCV Quantitative Test v2.0 (Roche Assay, Roche Molecular Systems, Pleasanton CA, USA). The 95% Lower Limit of Detection (LoD) of the assay was determined from dilutions of the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) and HCV positive clinical specimens in HCV negative human plasma and serum. Probit analysis was performed to generate the 95% predicted detection limits. The Lower Limit of Quantitation (LLoQ) was established for each genotype by diluting clinical specimens and the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) in HCV negative human plasma and serum. Specificity was determined using 200 fresh and 536 frozen HCV RNA negative clinical specimens including 370 plasma specimens and 366 serum specimens. Linearity for genotypes 1 to 6 was established by diluting armored RNA or HCV positive clinical specimens in HCV negative serum or plasma from 8.08 log IU/mL to below 1 log IU/mL. Precision was tested using a 10 member panel made by diluting HCV positive clinical specimens or spiking armored RNA into HCV negative plasma and serum. A method comparison was conducted against the Abbott assay using 1058 clinical specimens and against the Roche assay using 608 clinical specimens from HCV infected patients. In addition, agreement between the Roche assay and the Aptima assay using specimens with low

  19. Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual determinations of ergot contamination

    PubMed Central

    Comte, Alexia; Gräfenhan, Tom; Links, Matthew G.; Hemmingsen, Sean M.

    2017-01-01

    We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes. PMID:28257512

  20. A quantitative polymerase chain reaction assay for rapid detection of 9 pathogens directly from stools of travelers with diarrhea.

    PubMed

    Antikainen, Jenni; Kantele, Anu; Pakkanen, Sari H; Lääveri, Tinja; Riutta, Jukka; Vaara, Martti; Kirveskari, Juha

    2013-10-01

    Every year, 80 million tourists traveling to tropical and subtropical areas contract traveler's diarrhea (TD). Forty percent to 80% of cases are caused by bacteria, yet clinical diagnostic tests are available to identify only a few of the strains that cause TD. We aimed to develop a quantitative polymerase chain reaction (qPCR) assay to identify all major pathogens in stool samples. We developed a low-cost, high-throughput, multiplex qPCR assay for simultaneous detection of 9 bacterial pathogens in stool samples: Salmonella, Yersinia, Campylobacter, and Vibrio cholerae, as well as Shigella or enteroinvasive Escherichia coli, enterohemorrhagic E coli, enterotoxigenic E coli (ETEC), enteroaggregative E coli (EAEC), and enteropathogenic E coli (EPEC). The assay was validated using positive (n = 245) and negative (n = 243) control strains, as well as preselected positive and negative stool samples. In addition, stool samples were collected from 96 returning travelers with TD. The findings were compared with those from routine diagnostic tests. The assay detected the bacterial strains with 100% sensitivity and specificity, compared with results from the reference tests. Of all stool samples collected from travelers with TD, EPEC was found in 47%, EAEC in 46%, ETEC in 22%, enterohemorrhagic E coli in 7%, Campylobacter in 6%, Shigella or enteroinvasive E coli in 2%, and Salmonella in 2%. Multiple pathogens were found in 37% of all samples. We developed a low-cost, high-throughput qPCR assay for use in routine diagnostic analysis and research. It detects the pathogenic bacteria most commonly associated with TD in stool samples with 100% sensitivity and specificity, compared with reference methods. The assay requires 4 hours, whereas current detection methods require 1 to 7 days. At least 1 TD pathogen was identified in stool samples from 76% of returning travelers, whereas conventional methods found a pathogen in only 17%. The most commonly detected bacteria were EPEC

  1. Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array.

    PubMed

    McKendry, Rachel; Zhang, Jiayun; Arntz, Youri; Strunz, Torsten; Hegner, Martin; Lang, Hans Peter; Baller, Marko K; Certa, Ulrich; Meyer, Ernst; Güntherodt, Hans-Joachim; Gerber, Christoph

    2002-07-23

    We report a microarray of cantilevers to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomechanical bending, which is detected optically in situ. Differential measurements including reference cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3' and 5' overhangs. Our experiments suggest that the nanomechanical motion originates from predominantly steric hindrance effects and depends on the concentration of DNA molecules in solution. We show that cantilever arrays can be used to investigate the thermodynamics of biomolecular interactions mechanically, and we have found that the specificity of the reaction on a cantilever is consistent with solution data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concentration in solution of 75 nM.

  2. Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array

    PubMed Central

    McKendry, Rachel; Zhang, Jiayun; Arntz, Youri; Strunz, Torsten; Hegner, Martin; Lang, Hans Peter; Baller, Marko K.; Certa, Ulrich; Meyer, Ernst; Güntherodt, Hans-Joachim; Gerber, Christoph

    2002-01-01

    We report a microarray of cantilevers to detect multiple unlabeled biomolecules simultaneously at nanomolar concentrations within minutes. Ligand-receptor binding interactions such as DNA hybridization or protein recognition occurring on microfabricated silicon cantilevers generate nanomechanical bending, which is detected optically in situ. Differential measurements including reference cantilevers on an array of eight sensors can sequence-specifically detect unlabeled DNA targets in 80-fold excess of nonmatching DNA as a background and discriminate 3′ and 5′ overhangs. Our experiments suggest that the nanomechanical motion originates from predominantly steric hindrance effects and depends on the concentration of DNA molecules in solution. We show that cantilever arrays can be used to investigate the thermodynamics of biomolecular interactions mechanically, and we have found that the specificity of the reaction on a cantilever is consistent with solution data. Hence cantilever arrays permit multiple binding assays in parallel and can detect femtomoles of DNA on the cantilever at a DNA concentration in solution of 75 nM. PMID:12119412

  3. Re-examination of chromogenic quantitative assays for determining flavonoid content.

    PubMed

    Ho, Yu-Chi; Yu, Hui-Tzu; Su, Nan-Wei

    2012-03-14

    Flavonoids in plants have gained worldwide attention because of their benefits for human health. This study compared three analytical procedures commonly used for determining flavonoid content in plant samples in terms of chromogenic relationships and the reaction products of different flavonoid structures by means of using flavonoid standards with flavone, flavonol, flavanone, flavanol, and isoflavone and analytes such as phenolic acids commonly found in plant extracts. Procedure A produced a stable color reaction between 3-hydroxy-4-keto-flavonoids (flavonols) and 5-hydroxyflavones and was highly sensitive. Procedure B produced color reactions among most of the flavonoids, but the reaction products had different colors and faded over time. Procedure B also produced a color reaction with caffeic and chlorogenic acid. Procedure C was the most sensitive. It produced a color reaction and, like procedure A, could be used to quantify flavonols and 5-hydroxyflavones, but also showed color reaction toward caffeic and chlorogenic acid. On the basis of the results, the current three procedures are not satisfactory for determining all of the types of flavonoid. Two issues needed to be clarified before a promising determination of flavonoid content could be performed with chromogenic assays. The first is a survey of the literature to screen the possible predominant component of flavonoid in analytes. The other is guided by the predominant flavonoid; a promising calibration curve for flavonoid detection can be established on the basis of the selection of an appropriate method and a chemical standard with an equivalent dose response to the predominant flavonoid.

  4. Statistical inference for the within-device precision of quantitative measurements in assay validation.

    PubMed

    Liu, Jen-Pei; Lu, Li-Tien; Liao, C T

    2009-09-01

    Intermediate precision is one of the most important characteristics for evaluation of precision in assay validation. The current methods for evaluation of within-device precision recommended by the Clinical Laboratory Standard Institute (CLSI) guideline EP5-A2 are based on the point estimator. On the other hand, in addition to point estimators, confidence intervals can provide a range for the within-device precision with a probability statement. Therefore, we suggest a confidence interval approach for assessment of the within-device precision. Furthermore, under the two-stage nested random-effects model recommended by the approved CLSI guideline EP5-A2, in addition to the current Satterthwaite's approximation and the modified large sample (MLS) methods, we apply the technique of generalized pivotal quantities (GPQ) to derive the confidence interval for the within-device precision. The data from the approved CLSI guideline EP5-A2 illustrate the applications of the confidence interval approach and comparison of results between the three methods. Results of a simulation study on the coverage probability and expected length of the three methods are reported. The proposed method of the GPQ-based confidence intervals is also extended to consider the between-laboratories variation for precision assessment.

  5. A Quantitative Flurometric Assay for the Measurement of Antibody to Pasteurella haemolytica in Cattle

    PubMed Central

    Confer, A.W.; Fox, J.C.; Newman, P.R.; Lawson, G.W.; Corstvet, R.E.

    1983-01-01

    A rapid, simple fluorometric method is described for measuring antibody to Pasteurella haemolytica in sera of cattle. Various antigen preparations were compared for the test including live, formalin-killed and phenol-killed P. haemolytica. A preparation composed of formalin-killed organisms from a 22 hour culture gave consistent results and was used in the studies. The test was reproduciable with percent coefficients of variation for fluorescent signal unit values on ten or more replicate samples ranging from 5.7 to 28.0. Sera from calves vaccinated by aerosol exposure to live P. haemolytica had up to a five-fold increase in antibody titer as measured by the flurometric method test during a 21 day period. Fluorometric method titers were comparable to those obtained by the indirect bacterial agglutination test. There was no seroconversion to P. haemolytica in calves vaccinated by aerosol exposure of P. multocida. The major advantages of the fluorometric method test over conventional methods are that the assay does not require serial dilutions of serum samples and thus limits time and effort to determine antibody titers. PMID:6339016

  6. Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions.

    PubMed

    Ream, Jennifer A; Lewis, L Kevin; Lewis, Karen A

    2016-10-15

    Interactions between proteins and nucleic acids are frequently analyzed using electrophoretic mobility shift assays (EMSAs). This technique separates bound protein:nucleic acid complexes from free nucleic acids by electrophoresis, most commonly using polyacrylamide gels. The current study utilizes recent advances in agarose gel electrophoresis technology to develop a new EMSA protocol that is simpler and faster than traditional polyacrylamide methods. Agarose gels are normally run at low voltages (∼10 V/cm) to minimize heating and gel artifacts. In this study we demonstrate that EMSAs performed using agarose gels can be run at high voltages (≥20 V/cm) with 0.5 × TB (Tris-borate) buffer, allowing for short run times while simultaneously yielding high band resolution. Several parameters affecting band and image quality were optimized for the procedure, including gel thickness, agarose percentage, and applied voltage. Association of the siRNA-binding protein p19 with its target RNA was investigated using the new system. The agarose gel and conventional polyacrylamide gel methods generated similar apparent binding constants in side-by-side experiments. A particular advantage of the new approach described here is that the short run times (5-10 min) reduce opportunities for dissociation of bound complexes, an important concern in non-equilibrium nucleic acid binding experiments.

  7. Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay

    PubMed Central

    Shen, Shu-Min; Chou, Ming-Yuan; Ji, Wen-Tsai; Hsu, Tsui-Kang; Tsai, Hsiu-Feng; Huang, Yu-Li; Chiu, Yi-Chou; Kao, Erl-Shyh; Kao, Po-Min; Fan, Cheng-Wei

    2015-01-01

    Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 102 and 3.3 × 105 cells/l in river water and 72.1–5.7 × 106 cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors. PMID:26184706

  8. Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay.

    PubMed

    Shen, Shu-Min; Chou, Ming-Yuan; Hsu, Bing-Mu; Ji, Wen-Tsai; Hsu, Tsui-Kang; Tsai, Hsiu-Feng; Huang, Yu-Li; Chiu, Yi-Chou; Kao, Erl-Shyh; Kao, Po-Min; Fan, Cheng-Wei

    2015-07-01

    Legionella spp. are common in various natural and man-made aquatic environments. Recreational hot spring is frequently reported as an infection hotspot because of various factors such as temperature and humidity. Although polymerase chain reaction (PCR) had been used for detecting Legionella, several inhibitors such as humic substances, calcium, and melanin in the recreational spring water may interfere with the reaction thus resulting in risk underestimation. The purpose of this study was to compare the efficiencies of conventional and Taqman quantitative PCR (qPCR) on detecting Legionella pneumophila in spring facilities and in receiving water. In the results, Taqman PCR had much better efficiency on specifying the pathogen in both river and spring samples. L. pneumophila was detected in all of the 27 river water samples and 45 of the 48 hot spring water samples. The estimated L. pneumophela concentrations ranged between 1.0 × 10(2) and 3.3 × 10(5) cells/l in river water and 72.1-5.7 × 10(6) cells/l in hot spring water. Total coliforms and turbidity were significantly correlated with concentrations of L. pneumophila in positive water samples. Significant difference was also found in water temperature between the presence/absence of L. pneumophila. Our results suggest that conventional PCR may be not enough for detecting L. pneumophila particularly in the aquatic environments full of reaction inhibitors.

  9. LC-MS assay for quantitative determination of cardio glycoside in human blood samples.

    PubMed

    Frommherz, L; Köhler, H; Brinkmann, B; Lehr, M; Beike, J

    2008-03-01

    A method is described for liquid chromatography-mass spectrometry analysis of the cardio glycosides digoxin and digitoxin in biological samples. The method was optimized for use in the forensic field and, therefore, comprises the determination from whole blood and tissue samples. Sample cleanup by solid phase extraction (SPE) on a functionalized polymeric phase was sufficient to limit matrix suppression to <10% for all analytes. Chromatographic separation was achieved using an RP-8 column. Detection of the cardio glycosides was performed with electrospray ionization in the positive mode. The system was run in single ion monitoring mode, measuring the sodium adducts (M + Na)+ of the analyte and of the internal standard, respectively. The method was fully validated for the analysis of blood samples and was also successfully applied in forensic cases. The method was accurate and precise over a linear concentration range up to 50 ng/g blood. Lower limit of quantitation was 0.2 ng/g for digoxin and 2 ng/g for digitoxin, respectively. As deuterated analyte was used as internal standard, we also present a new microwave-enhanced method for the fast preparation of the labelled analyte within 20 min.

  10. Dual enzyme activities assay by quantitative electrospray ionization quadrupole-time-of-flight mass spectrometry.

    PubMed

    Cai, Tingting; Zhang, Li; Wang, Haoyang; Zhang, Jing; Wang, Rong; Zhang, Yurong; Guo, Yinlong

    2012-01-01

    A practical and rapid method based on electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-ToF MS) was developed for detecting activities of both acetylcholinesterase IAChEI and glutathione S-transferase (GST). The simultaneous study of these two enzyme activities is significant for studying human bio-functions, especially for those who take in toxic compounds and have a risk of disease. Here, the enzyme activities were represented by the conversion of enzymatic substrates and determined by quantitatively analyzing enzymatic substrates. Different internal standards were used to quantify each enzymatic substrate and the good linearity of calibration curves demonstrated the feasibility of the internal standards. The Michaelis-Menten constants (Km) of both GST and AChE were measured by this method and were consistent with values previously reported. Furthermore, we applied this approach to detect GST and AChE activities of whole bloods from four deceased and healthy people. The variation in enzyme activity was in accord with information from gas chromatography mass spectrometry [GC/MS). The screening of AChE and GST provided reliable results and strong forensic evidence. This method offers an alternative choice for detecting enzyme activities and is anticipated to have wide applications in pharmaceutical research and prevention in toxic compounds.

  11. Quantitative analysis of lab-to-lab variability in Caco-2 permeability assays.

    PubMed

    Lee, Jong Bong; Zgair, Atheer; Taha, Dhiaa A; Zang, Xiaowei; Kagan, Leonid; Kim, Tae Hwan; Kim, Min Gi; Yun, Hwi-Yeol; Fischer, Peter M; Gershkovich, Pavel

    2017-05-01

    In this study, Caco-2 permeability results from different laboratories were compared. Six different sets of apparent permeability coefficient (Papp) values reported in the literature were compared to experimental Papp obtained in our laboratory. The differences were assessed by determining the root mean square error (RMSE) values between the datasets, which reached levels as high as 0.581 for the training set compounds, i.e. ten compounds with known effective human permeability (Peff). The consequences of these differences in Papp for prediction of oral drug absorption were demonstrated by introducing the Papp into the absorption and pharmacokinetics simulation software application GastroPlus™ for prediction of the fraction absorbed (Fa) in humans using calibrated "user-defined permeability models". The RMSE were calculated to assess the differences between the simulated Fa and experimental values reported in the literature. The RMSE for Fa simulated with the permeability model calibrated using experimental Papp from our laboratory was 0.128. When the calibration was performed using Papp from literature datasets, the RMSE values for Fa were higher in all cases except one. This study shows quantitative lab-to-lab variability of Caco-2 permeability results and the potential consequences this can have in the use of these results for predicting intestinal absorption of drugs. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Hessian-based quantitative image analysis of host-pathogen confrontation assays.

    PubMed

    Cseresnyes, Zoltan; Kraibooj, Kaswara; Figge, Marc Thilo

    2017-09-15

    Host-fungus interactions have gained a lot of interest in the past few decades, mainly due to an increasing number of fungal infections that are often associated with a high mortality rate in the absence of effective therapies. These interactions can be studied at the genetic level or at the functional level via imaging. Here, we introduce a new image processing method that quantifies the interaction between host cells and fungal invaders, for example, alveolar macrophages and the conidia of Aspergillus fumigatus. The new technique relies on the information content of transmitted light bright field microscopy images, utilizing the Hessian matrix eigenvalues to distinguish between unstained macrophages and the background, as well as between macrophages and fungal conidia. The performance of the new algorithm was measured by comparing the results of our method with that of an alternative approach that was based on fluorescence images from the same dataset. The comparison shows that the new algorithm performs very similarly to the fluorescence-based version. Consequently, the new algorithm is able to segment and characterize unlabeled cells, thus reducing the time and expense that would be spent on the fluorescent labeling in preparation for phagocytosis assays. By extending the proposed method to the label-free segmentation of fungal conidia, we will be able to reduce the need for fluorescence-based imaging even further. Our approach should thus help to minimize the possible side effects of fluorescence labeling on biological functions. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

  13. Quantitative assay and subcellular distribution of enzymes acting on dolichyl phosphate in rat liver

    PubMed Central

    Ravoet, A; Amar-Costesec, A; Godelaine, D; Beaufay, H

    1981-01-01

    To establish on a quantitative basis the subcellular distribution of the enzymes that glycosylate dolichyl phosphate in rat liver, preliminary kinetic studies on the transfer of mannose, glucose, and N-acetylglucosamine-1-phosphate from the respective (14)C- labeled nucleotide sugars to exogenous dolichyl phosphate were conducted in liver microsomes. Mannosyltransferase, glucosyltransferase, and, to a lesser extent, N- acetylglucosamine-phosphotransferase were found to be very unstable at 37 degrees C in the presence of Triton X-100, which was nevertheless required to disperse the membranes and the lipid acceptor in the aqueous reaction medium. The enzymes became fairly stable in the range of 10-17 degrees C and the reactions then proceeded at a constant velocity for at least 15 min. Conditions under which the reaction products are formed in amount proportional to that of microsomes added are described. For N- acetylglucosaminephosphotransferase it was necessary to supplement the incubation medium with microsomal lipids. Subsequently, liver homogenates were fractionated by differential centrifugation, and the microsome fraction, which contained the bulk of the enzymes glycosylating dolichyl phosphate, was analyzed by isopycnic centrifugation in a sucrose gradient without any previous treatment, or after addition of digitonin. The centrifugation behavior of these enzymes was compared to that of a number of reference enzymes for the endoplasmic reticulum, the golgi complex, the plasma membranes, and mitochondria. It was very simily to that of enzymes of the endoplasmic reticulum, especially glucose-6-phosphatase. Subcellular preparations enriched in golgi complex elements, plasma membranes, outer membranes of mitochondira, or mitoplasts showed for the transferases acting on dolichyl phosphate relative activities similar to that of glucose- 6-phosphatase. It is concluded that glycosylations of dolichyl phosphate into mannose, glucose, and N-acetylglucosamine-1

  14. Analyzing the Human Papillomavirus (HPV) Life Cycle in Primary Keratinocytes with a Quantitative Colony-Forming Assay.

    PubMed

    Lace, Michael J; Turek, Lubomir P; Anson, James R; Haugen, Thomas H

    2014-05-01

    Papillomavirus genomes replicate as extrachromosomal plasmids within infected keratinocytes, requiring the regulated expression of early viral gene products to initially amplify the viral genomes and subvert cell growth checkpoints as part of a complex path to immortalization. Building on contemporary keratinocyte transfection and culture systems, the methods described in this unit form a detailed approach to analyzing critical events in the human papillomavirus (HPV) life cycle, utilizing physiologic levels of viral gene products expressed from their native promoter(s) in the natural host cells for HPV infection. A quantitative colony-forming assay permits comparison of the capacities of various transfected HPV types and mutant HPV genomes to initially form colonies and immortalize human keratinocytes. In conjunction with additional methods, these protocols enable examination of genomic stability, viral and cellular gene expression, viral integration, and differentiation patterns influenced by HPV persistence in clonal human keratinocytes that effectively mimic early events in HPV infection. Copyright © 2014 John Wiley & Sons, Inc.

  15. Odorant Screening and Quantitation of Thiols in Carmenere Red Wine by Gas Chromatography-Olfactometry and Stable Isotope Dilution Assays.

    PubMed

    Pavez, Carolina; Agosin, Eduardo; Steinhaus, Martin

    2016-05-04

    The sensory impact of thiols in Vitis vinifera 'Carmenere' red wines was evaluated. For this purpose, aroma extract dilution analysis was applied to the thiols isolated from a Carmenere red wine by affinity chromatography with a mercurated agarose gel. Results revealed the presence of four odorants, identified as 2-furanylmethanethiol, 3-sulfanylhexyl acetate, 3-sulfanyl-1-hexanol, and 2-methyl-3-sulfanyl-1-butanol, with the latter being described here for the first time in Carmenere red wines. Quantitation of the four thiols in the Carmenere wine screened by aroma extract dilution analysis and in three additional Carmenere wines by stable isotope dilution assays resulted in concentrations above the respective orthonasal odor detection threshold values. Triangle tests applied to wine model solutions with and without the addition of the four thiols showed significant differences, thus suggesting that the compounds do have the potential to influence the overall aroma of red wine.

  16. Quantitative analysis of phosphinothricin-N-acetyltransferase in genetically modified herbicide tolerant pepper by an enzyme-linked immunosorbent assay.

    PubMed

    Shim, Youn-Young; Shin, Weon-Sun; Moon, Gi-Seong; Kim, Kyung-Hwan

    2007-04-01

    An immunoassay method was developed to quantitatively detect phosphinothricin-N-acetyltransferase (PAT) encoded by the Bialaphos resistance (bar) gene in genetically modified (GM) pepper. The histidine-tagged PAT was overexpressed in Escherichia coli M15 (pQE31-bar) and efficiently purified by Ni2+ affinity chromatography. A developed sandwich enzyme-linked immunosorbent assay (S-ELISA) method (detection limit: 0.01 microg/ml) was 100-fold more sensitive than a competitive indirect ELISA (CI-ELISA) method or Western blot analysis in detecting the recombinant PAT. In real sample tests, PAT in genetically modified herbicide-tolerant (GMHT) peppers was successfully quantified [4.9 +/- 0.4 microg/g of sample (n = 6)] by the S-ELISA method. The S-ELISA method developed here could be applied to other GMHT crops and vegetables producing PAT.

  17. Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis.

    PubMed

    Menard, J-P; Mazouni, C; Fenollar, F; Raoult, D; Boubli, L; Bretelle, F

    2010-12-01

    The purpose of this investigation was to determine the diagnostic accuracy of quantitative real-time polymerase chain reaction (PCR) assay in diagnosing bacterial vaginosis versus the standard methods, the Amsel criteria and the Nugent score. The Amsel criteria, the Nugent score, and results from the molecular tool were obtained independently from vaginal samples of 163 pregnant women who reported abnormal vaginal symptoms before 20 weeks gestation. To determine the performance of the molecular tool, we calculated the kappa value, sensitivity, specificity, and positive and negative predictive values. Either or both of the Amsel criteria (≥3 criteria) and the Nugent score (score ≥7) indicated that 25 women (15%) had bacterial vaginosis, and the remaining 138 women did not. DNA levels of Gardnerella vaginalis or Atopobium vaginae exceeded 10(9) copies/mL or 10(8) copies/mL, respectively, in 34 (21%) of the 163 samples. Complete agreement between both reference methods and high concentrations of G. vaginalis and A. vaginae was found in 94.5% of women (154/163 samples, kappa value = 0.81, 95% confidence interval 0.70-0.81). The nine samples with discordant results were categorized as intermediate flora by the Nugent score. The molecular tool predicted bacterial vaginosis with a sensitivity of 100%, a specificity of 93%, a positive predictive value of 73%, and a negative predictive value of 100%. The quantitative real-time PCR assay shows excellent agreement with the results of both reference methods for the diagnosis of bacterial vaginosis.

  18. Development of a sensitive E-screen assay for quantitative analysis of estrogenic activity in municipal sewage plant effluents.

    PubMed

    Körner, W; Hanf, V; Schuller, W; Kempter, C; Metzger, J; Hagenmaier, H

    1999-01-12

    A simplified proliferation test with human estrogen receptor-positive MCF-7 breast cancer cells (E-screen assay) was optimized and validated for the sensitive quantitative determination of total estrogenic activity in effluent samples from municipal sewage plants. After solid phase extraction of 1 l sewage on either 0.2 g polystyrene copolymer (ENV+) or 1 g RP-C18 material and removal of the solvent, analysis of the extracts in the E-screen assay could be performed without any clean-up step. This was even possible with untreated sewage. Parallel extraction of four sewage samples on both different solid phase materials gave comparable quantitative results in the E-screen. A blank sample did not induce cell proliferation. As additive behaviour of the estrogenic response of single compounds was proven for two different mixtures each containing three xenoestrogens, total estrogenic activity in the sewage samples, expressed as 17 beta-estradiol equivalent concentration (EEQ), could be calculated comparing the EC50 values of the samples with those of the positive control 17 beta-estradiol. The detection limit of the E-screen method was 0.05 pmol EEQ/l (0.014 ng EEQ/l), the limit of quantification 0.25-0.5 pmol EEQ/l (0.07-0.14 ng EEQ/l). In total, extracts of nine effluent and one influent sample from five different municipal sewage plants in South Germany were analyzed in the E-screen. All samples strongly induced cell proliferation in a dose-dependent manner which was completely inhibited by coincubation with 5 nM of the estrogen receptor-antagonist ICI 182,780. The proliferative effect relative to the positive control 17 beta-estradiol (RPE) was between 30 and 101%. 17 beta-Estradiol equivalent concentrations were between 2.5 and 25 ng/l indicating a significant input of estrogenic substances via sewage treatment plants into rivers.

  19. A quantitative assay for reductive metabolism of a pesticide in fish using electrochemistry coupled with liquid chromatography tandem mass spectrometry.

    PubMed

    Bussy, Ugo; Chung-Davidson, Yu-Wen; Li, Ke; Li, Weiming

    2015-04-07

    This is the first study to use electrochemistry to generate a nitro reduction metabolite as a standard for a liquid chromatography-mass spectrometry-based quantitative assay. This approach is further used to quantify 3-trifluoromethyl-4-nitrophenol (TFM) reductive metabolism. TFM is a widely used pesticide for the population control of sea lamprey (Petromyzon marinus), an invasive species of the Laurentian Great Lakes. Three animal models, sea lamprey, lake sturgeon (Acipenser fulvescens), and rainbow trout (Oncorhynchus mykiss), were selected to evaluate TFM reductive metabolism because they have been known to show differential susceptibilities to TFM toxicity. Amino-TFM (aTFM; 3-trifluoromethyl-4-aminophenol) was the only reductive metabolite identified through liquid chromatography-high-resolution mass spectrometry screening of liver extracts incubated with TFM and was targeted for electrochemical synthesis. After synthesis and purification, aTFM was used to develop a quantitative assay of the reductive metabolism of TFM through liquid chromatography and tandem mass spectrometry. The concentrations of aTFM were measured from TFM-treated cellular fractions, including cytosolic, nuclear, membrane, and mitochondrial protein extracts. Sea lamprey extracts produced the highest concentrations (500 ng/mL) of aTFM. In addition, sea lamprey and sturgeon cytosolic extracts showed concentrations of aTFM substantially higher than those of rainbow trout. However, other fractions of lake sturgeon extracts tend to show aTFM concentrations similar to those of rainbow trout but not with sea lamprey. These data suggest that the level of reductive metabolism of TFM may be associated with the sensitivities of the animals to this particular pesticide.

  20. Quantitative polymerase chain reaction (PCR) assays for a bacterial thiaminase I gene and the thiaminase-producing bacterium Paenibacillus thiaminolyticus.

    USGS Publications Warehouse

    Richter, C.A.; Wright-Osment, Maureen K.; Zajicek, J.L.; Honeyfield, D.C.; Tillitt, D.E.

    2009-01-01

    The thiaminase I enzyme produced by the gram-positive bacterium Paenibacillus thiaminolyticus isolated from the viscera of Lake Michigan alewives Alosa pseudoharengus is currently the only defined source of the thiaminase activity linked to thiamine (vitamin B1) deficiency in early mortality syndrome (EMS) in the larvae of Great Lakes salmonines. Diets of alewife or isolated strains of P. thiaminolyticus mixed in a semipurified diet and fed to lake trout Salvelinus namaycush have been shown to produce EMS in fry. We utilized quantitative polymerase chain reaction (Q-PCR) to aid in studies of the sources of P. thiaminolyticus and thiaminase I. Quantitative PCR assays were established to detect the thiaminase I gene of P. thiaminolyticus, the 16S rRNA gene from most species of bacteria, and the 16S rRNA gene specifically from P. thiaminolyticus and a few closely related taxa. The Q-PCR assays are linear over at least six orders of magnitude and can detect the thiaminase I gene of P. thiaminolyticus from as few as 1,000 P. thiaminolyticus cells/g of sample or the Paenibacillus 16S rRNA gene from as few as 100 P. thiaminolyticus cells/g of sample. The initial results from alewife viscera samples with high thiaminase activity yielded unexpectedly low densities of P. thiaminolyticus cells; Paenibacillus thiaminolyticus was detectable in 2 of 6 alewife viscera tested at densities on the order of 100 cells/g out of 100,000,000 total bacterial cells/g. The low numbers of P. thiaminolyticus detected suggest that alewives contain additional non-P. thiaminolyticus sources of thiaminase activity.

  1. PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities†

    PubMed Central

    Becker, Sven; Böger, Peter; Oehlmann, Ralfh; Ernst, Anneliese

    2000-01-01

    Succession of ecotypes, physiologically diverse strains with negligible rRNA sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (C. Postius and A. Ernst, Arch. Microbiol. 172:69–75, 1999). In order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of Taq nuclease assays (TNAs), PCR-based assays in which the amount of an amplicon is monitored by hydrolysis of a labeled oligonucleotide (TaqMan probe) when hybridized to the amplicon. High accuracy and a 7-order detection range made the real-time TNA superior to the corresponding end point technique. However, in samples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleotides and due to accumulation of amplicons that are not detected by the TaqMan probe. A decrease in reaction efficiency, which can be recognized by direct monitoring of amplification, provides experimental evidence for the presence of such a problem and emphasizes the need for real-time technology in quantitative PCR. Use of specific primers and probes and control of amplification efficiency allow correct quantification of target DNA in the presence of an up to 104-fold excess of phylogenetically similar DNA and of an up to 107-fold excess of dissimilar DNA. PMID:11055948

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

    PubMed

    André, Murielle; Reghin, Sylviane; Boussard, Estelle; Lempereur, Laurent; Maisonneuve, Stéphane

    2016-05-01

    Residual host cellular DNA (rcDNA) is one of the principal risk associated with continuous cell lines derived medicines such as viral vaccines. To assess rcDNA degradation, we suggest two quantitative real-time PCR assays designed to separately quantify target sequences shorter and longer than the 200 bp risk limit, the relative abundance of both targets reflecting the extent of rcDNA fragmentation. The conserved multicopy ribosomal 18S RNA gene was targeted to detect host cell templates from most mammalian cell substrates commonly used in the manufacture of human viral vaccines. The detection range of the method was assessed on purified DNA templates from different animal origins. The standard calibrator origin and structural conformation were shown crucial to achieve accurate quantification. Artificial mixtures of PCR products shorter and longer than 200 bp were used as a model to check the ability of the assay to estimate the fragment size distribution. The method was successfully applied to a panel of Vero cell derived vaccines and could be used as a universal method for determination of both content and size distribution of rcDNA in vaccines.

  3. Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria

    PubMed Central

    Gold, Ben; Lopez Quezada, Landys; Glasheen, Jou; Ballinger, Elaine; Somersan-Karakaya, Selin; Warrier, Thulasi; Nathan, Carl

    2016-01-01

    There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 °C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to ≥99% bacterial kill (MBC≥99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic. PMID:28060290

  4. Quantitative PCR assay for Mycobacterium pseudoshottsii and Mycobacterium shottsii and application to environmental samples and fishes from the Chesapeake Bay.

    PubMed

    Gauthier, D T; Reece, K S; Xiao, J; Rhodes, M W; Kator, H I; Latour, R J; Bonzek, C F; Hoenig, J M; Vogelbein, W K

    2010-09-01

    Striped bass (Morone saxatilis) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden (Brevoortia tyrannus) and bay anchovy (Anchoa mitchilli). Mycobacterium pseudoshottsii was found to be ubiquitous in water samples from the main stem of the Chesapeake Bay and was also present in water and sediments from the Rappahannock River, Virginia. M. pseudoshottsii was also detected in menhaden and anchovy tissues. In contrast, M. shottsii was not detected in water, sediment, or prey fish tissues. In conjunction with its nonpigmented phenotype, which is frequently found in obligately pathogenic mycobacteria of humans, this pattern of occurrence suggests that M. shottsii may be an obligate pathogen of striped bass.

  5. Quantitative PCR Assay for Mycobacterium pseudoshottsii and Mycobacterium shottsii and Application to Environmental Samples and Fishes from the Chesapeake Bay▿

    PubMed Central

    Gauthier, D. T.; Reece, K. S.; Xiao, J.; Rhodes, M. W.; Kator, H. I.; Latour, R. J.; Bonzek, C. F.; Hoenig, J. M.; Vogelbein, W. K.

    2010-01-01

    Striped bass (Morone saxatilis) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden (Brevoortia tyrannus) and bay anchovy (Anchoa mitchilli). Mycobacterium pseudoshottsii was found to be ubiquitous in water samples from the main stem of the Chesapeake Bay and was also present in water and sediments from the Rappahannock River, Virginia. M. pseudoshottsii was also detected in menhaden and anchovy tissues. In contrast, M. shottsii was not detected in water, sediment, or prey fish tissues. In conjunction with its nonpigmented phenotype, which is frequently found in obligately pathogenic mycobacteria of humans, this pattern of occurrence suggests that M. shottsii may be an obligate pathogen of striped bass. PMID:20656856

  6. Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria.

    PubMed

    Gold, Ben; Roberts, Julia; Ling, Yan; Lopez Quezada, Landys; Glasheen, Jou; Ballinger, Elaine; Somersan-Karakaya, Selin; Warrier, Thulasi; Nathan, Carl

    2016-12-14

    There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 °C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to ≥99% bacterial kill (MBC≥99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

  7. A Dual Electrochemical Sensor Based on a Test-strip Assay for the Quantitative Determination of Albumin and Creatinine.

    PubMed

    Yasukawa, Tomoyuki; Kiba, Yuya; Mizutani, Fumio

    2015-01-01

    A dual-electrochemical sensor based on a test-strip assay with immunochemistry and enzyme reactions has been developed for the determination of albumin and creatinine. Each nitrocellulose membrane with an immobilization area of an anti-albumin antibody or three enzymes was prepared in the device with three working electrodes for measuring albumin, creatinine, and ascorbic acid, as well as an Ag/AgCl electrode used as a counter/pseudo-reference electrode. The reactions of three enzymes were initiated by flowing a solution containing creatinine to detect an oxidation current of hydrogen peroxide. A sandwich-type immunocomplex was formed by albumin and antibody labeled with glucose oxidase (GOx). Captured GOx catalyzed the reduction of Fe(CN)6(3-) to Fe(CN)6(4-), which was oxidized electrochemically to determine the captured albumin. The responses for creatinine and albumin increased with the concentrations in millimolar order and over the range 18.75 - 150 μg mL(-1), respectively. The present sensor would be a distinct demonstration for producing quantitative dual-assays for various biomolecules used for clinical diagnoses.

  8. Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

    PubMed

    Xie, Xingmei; Liang, Qiaoyi

    2014-01-01

    Sex chromosome aneuploidies occur commonly in the general population, with an incidence of 1 in 400 newborns. However, no tests specifically targeting sex chromosomes have been carried out in prenatal diagnosis or newborn screening, resulting in late recognition of these diseases. In this study, a rapid diagnostic method for sex chromosome aneuploidies was established using Quantitative Fluorescent-PCR (QF-PCR). Ten markers were included in one multiplex QF-PCR assay, including two sex determination genes (AMXY and SRY), five X-linked short tandem repeats (STRs; DXS1053, DXS981, DXS6809, DXS1187, and DXS8377), one X/Y-common STR (X22), and two autosomal STRs (D13S305 and D21S11). Retrospective tests of 70 cases with known cytogenetic results indicated that the 10-plex QF-PCR assay could well determine sex chromosome copy numbers by both allelic peak numbers and a sex chromosome dosage calculation with the autosomal STRs as internal controls. Prospective comparison with cytogenetic karyotyping on 534 cases confirmed that the 10-plex QF-PCR assay could be well employed for sex chromosome aneuploidy diagnosis in at least the Chinese Han population. This is the first QF-PCR test for the diagnosis of sex chromosome aneuploidies in the Chinese population. This test is superior to previous designs by including up to 8 sex-linked markers covering different parts of sex chromosomes as well as employing internal controls for copy number dosage calculation in a single PCR reaction. Due to simple technique and data analysis, as well as easy implementation within routine clinical services, this method is of great clinical application value and could be widely applied.

  9. Real-time PCR-based assay for quantitative detection of Hematodinium sp. in the blue crab Callinectes sapidus.

    PubMed

    Nagle, L; Place, A R; Schott, E J; Jagus, R; Messick, G; Pitula, J S

    2009-03-09

    Hematodinium sp. is a parasitic dinoflagellate infecting the blue crab Callinectes sapidus and other crustaceans. PCR-based assays are currently being used to identify infections in crabs that would have been undetectable by traditional microscopic examination. We therefore sought to define the limits of quantitative PCR (qPCR) detection within the context of field collection protocols. We present a qPCR assay based on the Hematodinium sp. 18S rRNA gene that can detect 10 copies of the gene per reaction. Analysis of a cell dilution series vs. defined numbers of a cloned Hematodinium sp. 18S rRNA gene suggests a copy number of 10,000 per parasite and predicts a sensitivity of 0.001 cell equivalents. In practice, the assays are based on analysis of 1% of the DNA extracted from 200 microl of serum, yielding a theoretical detection limit of 5 cells ml(-1) hemolymph, assuming that 1 cell is present per sample. When applied to a limited field survey of blue crabs collected in Maryland coastal bays from May to August 2005, 24 of 128 crabs (18.8%) were identified as positive for Hematodinium sp. infection using qPCR. In comparison, only 6 of 128 crabs (4.7%) were identified as positive using traditional hemolymph microscopic examination. The qPCR method also detected the parasite in gill, muscle, heart and hepatopancreas tissues, with 17.2% of the crabs showing infection in at least one of these tissues. Importantly, it is now possible to enumerate parasites within defined quantities of crab tissue, which permits collection of more detailed information on the epizootiology of the pathogen.

  10. Molecular diagnostic toolkit for Rhizophagus irregularis isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome.

    PubMed

    Badri, Amine; Stefani, Franck O P; Lachance, Geneviève; Roy-Arcand, Line; Beaudet, Denis; Vialle, Agathe; Hijri, Mohamed

    2016-10-01

    Rhizophagus irregularis (previously named Glomus irregulare) is one of the most widespread and common arbuscular mycorrhizal fungal (AMF) species. It has been recovered worldwide in agricultural and natural soils, and the isolate DAOM-197198 has been utilized as a commercial inoculant for two decades. Despite the ecological and economical importance of this taxon, specific markers for quantification of propagules by quantitative real-time PCR (qPCR) are extremely limited and none have been rigorously validated for quality control of manufactured products such as biofertilizers. From the sequencing of 14 complete AMF mitochondrial (mt) genomes, a qPCR assay using a hydrolysis probe designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of R. irregularis isolate DAOM-197198. Specificity tests were performed using standard PCR and qPCR, and results clearly showed that the primers specifically amplified the isolate DAOM-197198, yielding a PCR product of 106 bp. According to the qPCR analyses on spores produced in vitro, the average copy number of mt genomes per spore was 3172 ± 304 SE (n = 6). Quantification assays were successfully undertaken on known and unknown samples in liquid suspensions and commercial dry formulations to show the accuracy, precision, robustness, and reproducibility of the qPCR assay. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate DAOM-197198. The approach of molecular toolkit used in our study could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.

  11. A novel real-time PCR assay for quantitative analysis of methylated alleles (QAMA): analysis of the retinoblastoma locus.

    PubMed

    Zeschnigk, Michael; Böhringer, Stefan; Price, Elizabeth Ann; Onadim, Zerrin; Masshöfer, Lars; Lohmann, Dietmar R

    2004-09-07

    Altered methylation patterns have been found to play a role in developmental disorders, cancer and aging. Increasingly, changes in DNA methylation are used as molecular markers of disease. Therefore, there is a need for reliable and easy to use techniques to detect and measure DNA methylation in research and routine diagnostics. We have established a novel quantitative analysis of methylated alleles (QAMA) which is essentially a major improvement over a previous method based on real-time PCR (MethyLight). This method is based on real-time PCR on bisulfite-treated DNA. A significant advantage over conventional MethyLight is gained by the use of TaqMan probes based on minor groove binder (MGB) technology. Their improved sequence specificity facilitates relative quantification of methylated and unmethylated alleles that are simultaneously amplified in single tube. This improvement allows precise measurement of the ratio of methylated versus unmethylated alleles and cuts down potential sources of inter-assay variation. Therefore, fewer control assays are required. We have used this novel technical approach to identify hypermethylation of the CpG island located in the promoter region of the retinoblastoma (RB1) gene and found that QAMA facilitates reliable and fast measurement of the relative quantity of methylated alleles and improves handling of diagnostic methylation analysis. Moreover, the simplified reaction setup and robustness inherent to the single tube assay facilitates high-throughput methylation analysis. Because the high sequence specificity inherent to the MGB technology is widely used to discriminate single nucleotide polymorphisms, QAMA potentially can be used to discriminate the methylation status of single CpG dinucleotides.

  12. Quantitation of Gingerols in Human Plasma by Newly Developed Stable Isotope Dilution Assays and Assessment of Their Immunomodulatory Potential.

    PubMed

    Schoenknecht, Carola; Andersen, Gaby; Schmidts, Ines; Schieberle, Peter

    2016-03-23

    In a pilot study with two volunteers, the main pungent and bioactive ginger (Zingiber officinale Roscoe) compounds, the gingerols, were quantitated in human plasma after ginger tea consumption using a newly established HPLC-MS/MS(ESI) method on the basis of stable isotope dilution assays. Limits of quantitation for [6]-, [8]-, and [10]-gingerols were determined as 7.6, 3.1, and 4.0 nmol/L, respectively. The highest plasma concentrations of [6]-, [8]-, and [10]-gingerols (42.0, 5.3, and 4.8 nmol/L, respectively) were reached 30-60 min after ginger tea intake. Incubation of activated human T lymphocytes with gingerols increased the intracellular Ca(2+) concentration as well as the IFN-γ secretion by about 20-30%. This gingerol-induced increase of IFN-γ secretion could be blocked by the specific TRPV1 antagonist SB-366791. The results of the present study point to an interaction of gingerols with TRPV1 in activated T lymphocytes leading to an augmentation of IFN-γ secretion.

  13. Quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions.

    PubMed

    Wilhelmsen, Kevin; Farrar, Katherine; Hellman, Judith

    2013-08-23

    The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

  14. A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4.

    PubMed

    Väisänen, E; Lahtinen, A; Eis-Hübinger, A M; Lappalainen, M; Hedman, K; Söderlund-Venermo, M

    2014-01-01

    Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes.

  15. Use of a chemiluminescent detector for quantitation of nitric oxide produced in assays of denitrifying enzymes. [Achromobacter cycloblastes; Pseudomonas perfectomarini

    SciTech Connect

    Pai, G.; Payne, W.J.; Le Gall, J.

    1987-05-01

    The authors developed a closed-flow system that continuously sweeps away gases evolved in enzyme assay mixtures into a commercially available oxides of nitrogen analyzer for the quantitation of any nitric oxide (NO) present in these gases. The system is particularly useful for the study of both the stoichiometry and kinetics of NO production from nitrite by cell-free extracts of denitrifying bacteria and by the purified denitrifying nitrite reductases. In addition to its much greater sensitivity when compared to standard gas chromatographic techniques, the method offers some unique advantages since it allows measurements of initial reaction velocities, without the problems of product inhibition, which often limit kinetic studies of denitrifying enzymes. The system also allows the immediate quantitation of any NO produced and so would be useful in detecting the transient presence of this very reactive free radical, if it is produced as a free intermediate in whole cell suspensions of denitrifying bacteria respiring nitrate. The apparatus has been calibrated using a purified copper nitrite reductase from Achromobacter cycloclastes and cell-free extracts from Pseudomonas Perfectomarini, and its utility in studies of the kinetics and stoichiometry of NO production from nitrite confirmed by comparison with results obtained using manometric and gas chromatographic techniques.

  16. Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays

    PubMed Central

    Kopp, Markus; Rychlik, Michael

    2015-01-01

    Because of minimal data available on folate analysis in dried matrix spots (DMSs), we combined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-methyltetrahydrofolic acid in dried blood spots (DBSs) and dried plasma spots (DPSs) as well as for the quantitation of whole blood 5-methyltetrahydrofolic acid in DBSs. We focused on two diagnostically conclusive parameters exhibited by the plasma and whole blood 5-methyltetrahydrofolic acid levels that reflect both temporary and long-term folate status. The method is performed using the [2H4]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only) and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/L whole blood 5-methyltetrahydrofolic acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-methyltetrahydrofolic acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-methyltetrahydrofolic acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and storage compared with conventional clinical sampling for the assessment of folate status. PMID:26605791

  17. Quantitative evaluation of pork adulteration in raw ground beef by radial immunodiffusion and enzyme-linked immunosorbent assay.

    PubMed

    Martin, D R; Chan, J; Chiu, J Y

    1998-12-01

    Quantitative estimates are important to establish whether pork adulteration in ground beef is accidental or intentional. A standard agar gel radial immunodiffusion (RID) test using forensic-grade antiserum to porcine albumin and an enzyme-linked immunosorbent assay (ELISA) using forensic-grade anti-porcine glycoprotein immunoglobulin were used to determine from 1 to 75% raw pork in raw ground beef. The RID test, which incorporated 1.5% anti-pork serum in 1% immunodiffusion agar, formed precipitin rings with pork albumin in agar wells. A linear standard curve was obtained by plotting the diffusion area against standard pork concentrations ranging from 0 to 80%. For the ELISA the endpoint optical density increased linearly versus log % pork between 0.0625% and 2% pork. In spiked samples, the RID test had a detection limit of 3 to 5%, a coefficient of variation (CV) of 22%, and a recovery of 105%. The ELISA had a detection limit of 1%, a CV of 18%, and a recovery of 114%. The mean recovery from the spiked samples by the ELISA and RID test was not significantly different (P > 0.05) from the known sample amounts. Quantitation by RID of 28 ground beef samples (27 of which were DTEK ELISA-positive for pork adulteration) revealed a wide range of pork content, with values as high as 48%.

  18. Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays.

    PubMed

    Kopp, Markus; Rychlik, Michael

    2015-01-01

    Because of minimal data available on folate analysis in dried matrix spots (DMSs), we combined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-methyltetrahydrofolic acid in dried blood spots (DBSs) and dried plasma spots (DPSs) as well as for the quantitation of whole blood 5-methyltetrahydrofolic acid in DBSs. We focused on two diagnostically conclusive parameters exhibited by the plasma and whole blood 5-methyltetrahydrofolic acid levels that reflect both temporary and long-term folate status. The method is performed using the [2H4]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only) and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/L whole blood 5-methyltetrahydrofolic acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-methyltetrahydrofolic acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-methyltetrahydrofolic acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and storage compared with conventional clinical sampling for the assessment of folate status.

  19. Detection limits of quantitative and digital PCR assays and their influence in presence-absence surveys of environmental DNA

    USGS Publications Warehouse

    Hunter, Margaret; Dorazio, Robert M.; Butterfield, John S.; Meigs-Friend, Gaia; Nico, Leo; Ferrante, Jason

    2017-01-01

    A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR-based analyses of low concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species’ presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty – indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis, and forensic and clinical diagnostics.

  20. Quantitative detection of methanotrophs in soil by novel pmoA-targeted real-time PCR assays.

    PubMed

    Kolb, Steffen; Knief, Claudia; Stubner, Stephan; Conrad, Ralf

    2003-05-01

    Methane oxidation in soils is mostly accomplished by methanotrophic bacteria. Little is known about the abundance of methanotrophs in soils, since quantification by cultivation and microscopic techniques is cumbersome. Comparison of 16S ribosomal DNA and pmoA (alpha subunit of the particulate methane monooxygenase) phylogenetic trees showed good correlation and revealed five distinct groups of methanotrophs within the alpha and gamma subclasses of Proteobacteria: the Methylococcus group, the Methylobacter/Methylosarcina group, the Methylosinus group, the Methylocapsa group, and the forest clones group (a cluster of pmoA sequences retrieved from forest soils). We developed quantitative real-time PCR assays with SybrGreen for each of these five groups and for all methanotrophic bacteria by targeting the pmoA gene. Detection limits were between 10(1) and 10(2) target molecules per reaction for all assays. Real-time PCR analysis of soil samples spiked with cells of Methylococcus capsulatus, Methylomicrobium album, and Methylosinus trichosporium recovered almost all the added bacteria. Only the Methylosinus-specific assay recovered only 20% of added cells, possibly due to a lower lysis efficiency of type II methanotrophs. Analysis of the methanotrophic community structure in a flooded rice field soil showed (5.0 +/- 1.4) x 10(6) pmoA molecules g(-1) for all methanotrophs. The Methylosinus group was predominant (2.7 x 10(6) +/- 1.1 x 10(6) target molecules g(-1)). In addition, bacteria of the Methylobacter/Methylosarcina group were abundant (2.0 x 10(6) +/- 0.9 x 10(6) target molecules g of soil(-1)). On the other hand, pmoA affiliated with the forest clones and the Methylocapsa group was below the detection limit of 1.9 x 10(4) target molecules g of soil(-1). Our results showed that pmoA-targeted real-time PCR allowed fast and sensitive quantification of the five major groups of methanotrophs in soil. This approach will thus be useful for quantitative analysis of the

  1. A Quantitative Toxicogenomics Assay Reveals the Evolution and Nature of Toxicity during the Transformation of Environmental Pollutants

    PubMed Central

    2015-01-01

    The incomplete mineralization of contaminants of emerging concern (CECs) during the advanced oxidation processes can generate transformation products that exhibit toxicity comparable to or greater than that of the original contaminant. In this study, we demonstrated the application of a novel, fast, and cost-effective quantitative toxicogenomics-based approach for the evaluation of the evolution and nature of toxicity along the electro-Fenton oxidative degradation of three representative CECs whose oxidative degradation pathways have been relatively well studied, bisphenol A, triclosan, and ibuprofen. The evolution of toxicity as a result of the transformation of parent chemicals and production of intermediates during the course of degradation are monitored, and the quantitative toxicogenomics assay results revealed the dynamic toxicity changes and mechanisms, as well as their association with identified intermediates during the electro-Fenton oxidation process of the selected CECs. Although for the three CECs, a majority (>75%) of the parent compounds disappeared at the 15 min reaction time, the nearly complete elimination of toxicity required a minimal 30 min reaction time, and they seem to correspond to the disappearance of identified aromatic intermediates. Bisphenol A led to a wide range of stress responses, and some identified transformation products containing phenolic or quinone group, such as 1,4-benzoquinone and hydroquinone, likely contributed to the transit toxicity exhibited as DNA stress (genotoxicity) and membrane stress during the degradation. Triclosan is known to cause severe oxidative stress, and although the oxidative damage potential decreased concomitantly with the disappearance of triclosan after a 15 min reaction, the sustained toxicity associated with both membrane and protein stress was likely attributed at least partially to the production of 2,4-dichlorophenol that is known to cause the production of abnormal proteins and affect the cell

  2. A Novel Stopped-Flow Assay for Quantitating Carbonic-Anhydrase Activity and Assessing Red-Blood-Cell Hemolysis.

    PubMed

    Zhao, Pan; Geyer, R Ryan; Boron, Walter F

    2017-01-01

    We report a novel carbonic-anhydrase (CA) assay and its use for quantitating red-blood-cell (RBC) lysis during stopped-flow (SF) experiments. We combine two saline solutions, one containing HEPES/pH 7.03 and the other, ~1% CO2/44 mM [Formula: see text]/pH 8.41, to generate an out-of-equilibrium CO2/[Formula: see text] solution containing ~0.5% CO2/22 [Formula: see text]/pH ~7.25 (10°C) in the SF reaction cell. CA catalyzes relaxation of extracellular pH to ~7.50: [Formula: see text] + H(+) → CO2 + H2O. Proof-of-concept studies (no intact RBCs) show that the pH-relaxation rate constant (kΔpH)-measured via pyranine fluorescence-rises linearly with increases in [bovine CAII] or [murine-RBC lysate]. The y-intercept (no CA) was kΔpH = 0.0183 s(-1). Combining increasing amounts of murine-RBC lysate with ostensibly intact RBCs (pre-SF hemolysis ≅0.4%)-fixing total [hemoglobin] at 2.5 μM in the reaction cell to simulate hemolysis from ostensibly 0 to 100%-causes kΔpH to increase linearly. This y-intercept (0% lysate/100% ostensibly intact RBCs) was kΔpH = 0.0820 s(-1), and the maximal kΔpH (100% lysate/0% intact RBCs) was 1.304 s(-1). Thus, mean percent hemolysis in the reaction cell was ~4.9%. Phenol-red absorbance assays yield indistinguishable results. The increase from 0.4 to 4.9% presumably reflects mechanical RBC disruption during rapid mixing. In all fluorescence studies, the CA blocker acetazolamide reduces kΔpH to near-uncatalyzed values, implying that all CA activity is extracellular. Our lysis assay is simple, sensitive, and precise, and will be valuable for correcting for effects of lysis in physiological SF experiments. The underlying CA assay, applied to blood plasma, tissue-culture media, and organ perfusates could assess lysis in a variety of applications.

  3. A Novel Stopped-Flow Assay for Quantitating Carbonic-Anhydrase Activity and Assessing Red-Blood-Cell Hemolysis

    PubMed Central

    Zhao, Pan; Geyer, R. Ryan; Boron, Walter F.

    2017-01-01

    We report a novel carbonic-anhydrase (CA) assay and its use for quantitating red-blood-cell (RBC) lysis during stopped-flow (SF) experiments. We combine two saline solutions, one containing HEPES/pH 7.03 and the other, ~1% CO2/44 mM HCO3-/pH 8.41, to generate an out-of-equilibrium CO2/HCO3- solution containing ~0.5% CO2/22 HCO3-/pH ~7.25 (10°C) in the SF reaction cell. CA catalyzes relaxation of extracellular pH to ~7.50: HCO3- + H+ → CO2 + H2O. Proof-of-concept studies (no intact RBCs) show that the pH-relaxation rate constant (kΔpH)—measured via pyranine fluorescence—rises linearly with increases in [bovine CAII] or [murine-RBC lysate]. The y-intercept (no CA) was kΔpH = 0.0183 s−1. Combining increasing amounts of murine-RBC lysate with ostensibly intact RBCs (pre-SF hemolysis ≅0.4%)—fixing total [hemoglobin] at 2.5 μM in the reaction cell to simulate hemolysis from ostensibly 0 to 100%—causes kΔpH to increase linearly. This y-intercept (0% lysate/100% ostensibly intact RBCs) was kΔpH = 0.0820 s−1, and the maximal kΔpH (100% lysate/0% intact RBCs) was 1.304 s−1. Thus, mean percent hemolysis in the reaction cell was ~4.9%. Phenol-red absorbance assays yield indistinguishable results. The increase from 0.4 to 4.9% presumably reflects mechanical RBC disruption during rapid mixing. In all fluorescence studies, the CA blocker acetazolamide reduces kΔpH to near-uncatalyzed values, implying that all CA activity is extracellular. Our lysis assay is simple, sensitive, and precise, and will be valuable for correcting for effects of lysis in physiological SF experiments. The underlying CA assay, applied to blood plasma, tissue-culture media, and organ perfusates could assess lysis in a variety of applications. PMID:28400735

  4. Quantitative analysis of the relative mutagenicity of five chemical constituents of tobacco smoke in the mouse lymphoma assay.

    PubMed

    Guo, Xiaoqing; Heflich, Robert H; Dial, Stacey L; Richter, Patricia A; Moore, Martha M; Mei, Nan

    2016-05-01

    Quantifying health-related biological effects, like genotoxicity, could provide a way of distinguishing between tobacco products. In order to develop tools for using genotoxicty data to quantitatively evaluate the risk of tobacco products, we tested five carcinogens found in cigarette smoke, 4-aminobiphenyl (4-ABP), benzo[a]pyrene (BaP), cadmium (in the form of CdCl2), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in the mouse lymphoma assay (MLA). The resulting mutagenicity dose responses were analyzed by various quantitative approaches and their strengths and weaknesses for distinguishing responses in the MLA were evaluated. L5178Y/Tk (+/-) 3.7.2C mouse lymphoma cells were treated with four to seven concentrations of each chemical for 4h. Only CdCl2 produced a positive response without metabolic activation (S9); all five chemicals produced dose-dependent increases in cytotoxicity and mutagenicity with S9. The lowest dose exceeding the global evaluation factor, the benchmark dose producing a 10%, 50%, 100% or 200% increase in the background frequency (BMD10, BMD50, BMD100 and BMD200), the no observed genotoxic effect level (NOGEL), the lowest observed genotoxic effect level (LOGEL) and the mutagenic potency expressed as a mutant frequency per micromole of chemical, were calculated for all the positive responses. All the quantitative metrics had similar rank orders for the agents' ability to induce mutation, from the most to least potent as CdCl2(-S9) > BaP(+S9) > CdCl2(+S9) > MeIQ(+S9) > 4-ABP(+S9) > NNK(+S9). However, the metric values for the different chemical responses (i.e. the ratio of the greatest value to the least value) for the different chemicals ranged from 16-fold (BMD10) to 572-fold (mutagenic potency). These results suggest that data from the MLA are capable of discriminating the mutagenicity of various constituents of cigarette smoke, and that quantitative analyses are available

  5. Quantitative PCR assay for detection and enumeration of ciguatera-causing dinoflagellate Gambierdiscus spp. (Gonyaulacales) in coastal areas of Japan.

    PubMed

    Nishimura, Tomohiro; Hariganeya, Naohito; Tawong, Wittaya; Sakanari, Hiroshi; Yamaguchi, Haruo; Adachi, Masao

    2016-02-01

    In Japan, ciguatera fish poisoning (CFP) has been increasingly reported not only in subtropical areas but also in temperate areas in recent years, causing a serious threat to human health. Ciguatera fish poisoning is caused by the consumption of fish that have accumulated toxins produced by an epiphytic/benthic dinoflagellate, genus Gambierdiscus. Previous studies revealed the existence of five Gambierdiscus species/phylotypes in Japan: Gambierdiscus australes, Gambierdiscus scabrosus, Gambierdiscus sp. type 2, Gambierdiscus sp. type 3, and Gambierdiscus (Fukuyoa) cf. yasumotoi. Among these, G. australes, G. scabrosus, and Gambierdiscus sp. type 3 strains exhibited toxicities in mice, whereas Gambierdiscus sp. type 2 strains did not show any toxicity. Therefore, it is important to monitor the cell abundance and dynamics of these species/phylotypes to identify and characterize CFP outbreaks in Japan. Because it is difficult to differentiate these species/phylotypes by observation under a light microscope, development of a rapid and reliable detection and enumeration method is needed. In this study, a quantitative PCR assay was developed using a TaqMan probe that targets unique SSU rDNA sequences of four Japanese Gambierdiscus species/phylotypes and incorporates normalization with DNA recovery efficiency. First, we constructed standard curves with high linearity (R(2)=1.00) and high amplification efficiency (≥1.98) using linearized plasmids that contained SSU rDNA of the target species/phylotypes. The detection limits for all primer and probe sets were approximately 10 gene copies. Further, the mean number of SSU rDNA copies per cell of each species/phylotype was determined from single cells in culture and from those in environmental samples using the qPCR assay. Next, the number of cells of each species/phylotype in the mixed samples, which were spiked with cultured cells of the four species/phylotypes, was calculated by division of the total number of rDNA copies

  6. Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control.

    PubMed

    Das, Amaresh; Deng, Ming Y; Babiuk, Shawn; McIntosh, Michael T

    2017-05-01

    Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

  7. Qualitative and quantitative results of interferon-γ release assays for monitoring the response to anti-tuberculosis treatment.

    PubMed

    Park, I-Nae; Shim, Tae Sun

    2017-03-01

    The usefulness of interferon-γ release assays (IGRAs) in monitoring to responses to anti-tuberculosis (TB) treatment is controversial. We compared the results of two IGRAs before and after anti-TB treatment in same patients with active TB. From a retrospective review, we selected patients with active TB who underwent repeated QuantiFERON-TB Gold (QFN-Gold, Cellestis Limited) and T-SPOT.TB (Oxford Immunotec) assays before and after anti-TB treatment with first-line drugs. Both tests were performed prior to the start of anti-TB treatment or within 1 week after the start of anti-TB treatment and after completion of treatment. A total of 33 active TB patients were included in the study. On the QFN-Gold test, at baseline, 23 cases (70%) were early secreted antigenic target 6-kDa protein 6 (ESAT-6) or culture filtrate protein 10 (CFP-10) positive. On the T-SPOT. TB test, at baseline, 31 cases (94%) were ESAT-6 or CFP-10 positive. Most of patients remained both test-positive after anti-TB treatment. Although changes in interferon-γ release responses over time were highly variable in both tests, there was a mean decline of 27 and 24 spot-forming counts for ESAT-6 and CFP-10, respectively on the T-SPOT.TB test (p < 0.05 for all). Although limited by the small number of patients and a short-term follow-up, there was significant decline in the quantitative result of the T-SPOT. TB test with treatment. However, both commercial IGRAs may not provide evidence regarding the cure of disease in Korea, a country where the prevalence of TB is within the intermediate range.

  8. Studies of plant colonisation by closely related Bacillus amyloliquefaciens biocontrol agents using strain specific quantitative PCR assays.

    PubMed

    Johansson, Anna H; Bejai, Sarosh; Niazi, Adnan; Manzoor, Shahid; Bongcam-Rudloff, Erik; Meijer, Johan

    2014-12-01

    Certain strains of Bacillus amyloliquefaciens can colonize plants and improve growth and stress management. In order to study these effects, bacterial growth dynamics on plants and in the rhizosphere are of interest calling for specific analytical tools. For that purpose, quantitative real-time PCR (qPCR) assays were developed in order to differentiate among three closely related B. amyloliquefaciens subsp. plantarum strains (UCMB5033, UCMB5036, UCMB5113) and to determine their levels with high accuracy. Oligonucleotide primers were designed for strain unique gene sequences and used for SYBR green based qPCR analysis. Standard curves covered a wide linear range (10(6)) of DNA amounts with the lowest detection level at 50 fg. Post-reaction melting curve analysis showed only a single product. Accurate threshold cycles were obtained, even in the presence of high excess of related Bacillus strains and total bacterial DNA from soil. Analysis of Bacillus colonisation after seed treatment of two oilseed rape cultivars (Oase and Ritz) grown on agar support showed a time dependent effect but that the bacteria mostly were found on root tissues and little on green tissues. The colonisation on plants grown in soil varied among the Bacillus strains where Oase seemed to house more bacteria than Ritz. Applied as a mixture, all three Bacillus strains co-existed on the roots of plants grown in soil. The qPCR assay in combination with other techniques will be a powerful tool to study plant interactions of these B. amyloliquefaciens biocontrol agents to further understand the requirements for successful interactions and improvement of plant properties.

  9. Detection of nonhemagglutinating influenza a(h3) viruses by enzyme-linked immunosorbent assay in quantitative influenza virus culture.

    PubMed

    van Baalen, C A; Els, C; Sprong, L; van Beek, R; van der Vries, E; Osterhaus, A D M E; Rimmelzwaan, G F

    2014-05-01

    To assess the efficacy of novel antiviral drugs against influenza virus in clinical trials, it is necessary to quantify infectious virus titers in respiratory tract samples from patients. Typically, this is achieved by inoculating virus-susceptible cells with serial dilutions of clinical specimens and detecting the production of progeny virus by hemagglutination, since influenza viruses generally have the capacity to bind and agglutinate erythrocytes of various species through their hemagglutinin (HA). This readout method is no longer adequate, since an increasing number of currently circulating influenza A virus H3 subtype (A[H3]) viruses display a reduced capacity to agglutinate erythrocytes. Here, we report the magnitude of this problem by analyzing the frequency of HA-deficient A(H3) viruses detected in The Netherlands from 1999 to 2012. Furthermore, we report the development and validation of an alternative method for monitoring the production of progeny influenza virus in quantitative virus cultures, which is independent of the capacity to agglutinate erythrocytes. This method is based on the detection of viral nucleoprotein (NP) in virus culture plates by enzyme-linked immunosorbent assay (ELISA), and it produced results similar to those of the hemagglutination assay using strains with good HA activity, including A/Brisbane/059/07 (H1N1), A/Victoria/210/09 (H3N2), other seasonal A(H1N1), A(H1N1)pdm09, and the majority of A(H3) virus strains isolated in 2009. In contrast, many A(H3) viruses that have circulated since 2010 failed to display HA activity, and infectious virus titers were determined only by detecting NP. The virus culture ELISA described here will enable efficacy testing of new antiviral compounds in clinical trials during seasons in which nonhemagglutinating influenza A viruses circulate.

  10. Sperm-macrophage interaction in the mouse: a quantitative assay in vitro using 111indium oxine-labeled sperm

    SciTech Connect

    Olive, D.L.; Weinberg, J.B.; Haney, A.F.

    1987-12-01

    The role of reproductive tract macrophages in contraception and reproductive failure has become widely recognized. However, in vitro analysis of sperm phagocytosis by macrophages has relied upon a semi-quantitative method of sperm counting that is of limited accuracy and reproducibility. We have developed an assay using murine sperm labeled with /sup 111/indium oxine, and results indicate the labeling to be rapid and efficient. Incorporation of /sup 111/indium into sperm increased the dose and sperm concentration and reached 90% maximal uptake after 15 min incubation, with maximal uptake occurring at 30 min. No decrease in sperm motility was noted with levels of oxine in excess of those required for significant labeling. Maximal labeling efficiency occurred in phosphate-buffered saline (PBS), with Dulbecco's modified Eagle's medium (DMEM) + 10% adult bovine serum (ABS) producing significantly less uptake. Label dissociation was detectable in PBS at room temperature, but at 37 degrees C in DMEM + 10% ABS, loss of label occurred at a rate of 23.5%/h. Addition of labeled sperm to murine macrophage monolayers under optimal conditions resulted in uptake of /sup 111/indium by macrophages, while free label was unincorporated. Results indicated assay specificity for macrophage-limited uptake, with insignificant label uptake by nonphagocytic murine fibroblasts and better sensitivity than sperm counting. Macrophages from Bacillus Calmette-Guerin (BCG)-infected mice resulted in a decrease in sperm uptake. Female macrophages showed greater capacity for sperm uptake than those of the male mouse. These initial studies demonstrated the utility of this model system in enhancing the understanding of sperm-macrophage interaction in the female reproductive tract.

  11. Qualitative and quantitative results of interferon-γ release assays for monitoring the response to anti-tuberculosis treatment

    PubMed Central

    Park, I-Nae; Shim, Tae Sun

    2017-01-01

    Background/Aims The usefulness of interferon-γ release assays (IGRAs) in monitoring to responses to anti-tuberculosis (TB) treatment is controversial. We compared the results of two IGRAs before and after anti-TB treatment in same patients with active TB. Methods From a retrospective review, we selected patients with active TB who underwent repeated QuantiFERON-TB Gold (QFN-Gold, Cellestis Limited) and T-SPOT.TB (Oxford Immunotec) assays before and after anti-TB treatment with first-line drugs. Both tests were performed prior to the start of anti-TB treatment or within 1 week after the start of anti-TB treatment and after completion of treatment. Results A total of 33 active TB patients were included in the study. On the QFN-Gold test, at baseline, 23 cases (70%) were early secreted antigenic target 6-kDa protein 6 (ESAT-6) or culture filtrate protein 10 (CFP-10) positive. On the T-SPOT. TB test, at baseline, 31 cases (94%) were ESAT-6 or CFP-10 positive. Most of patients remained both test-positive after anti-TB treatment. Although changes in interferon-γ release responses over time were highly variable in both tests, there was a mean decline of 27 and 24 spot-forming counts for ESAT-6 and CFP-10, respectively on the T-SPOT.TB test (p < 0.05 for all). Conclusions Although limited by the small number of patients and a short-term follow-up, there was significant decline in the quantitative result of the T-SPOT. TB test with treatment. However, both commercial IGRAs may not provide evidence regarding the cure of disease in Korea, a country where the prevalence of TB is within the intermediate range. PMID:27951621

  12. Immunochromatographic assay for quantitative and sensitive detection of hepatitis B virus surface antigen using highly luminescent quantum dot-beads.

    PubMed

    Shen, Jun; Zhou, Yaofeng; Fu, Fen; Xu, Hengyi; Lv, Jiaofeng; Xiong, Yonghua; Wang, Andrew

    2015-09-01

    Hepatitis B virus infection is one of the major causes of hepatitis, liver cirrhosis and liver cancer. In this study, we used highly luminescent quantum dot-beads (QBs) as signal amplification probes in the sandwich immunochromatographic assay (ICA) for ultrasensitive and quantitative detection of hepatitis B virus surface antigen (HBsAg) in human serum. Various parameters that influenced the sensitivity and stability of the QB-based ICA (QB-ICA) sensor were investigated. Two linear independent regression equations for detection of serum HBsAg were expressed with Y=0.3361X-0.0059 (R(2)=0.9983) for low HBsAg concentrations between 75 pg mL(-1) and 4.8 ng mL(-1), and Y=0.8404 X-2.9364 (R(2)=0.9939) for high HBsAg concentrations in the range from 4.8 ng mL(-1) to 75 ng mL(-1). The detection limit of the proposed ICA sensor achieved was 75 pg mL(-1), which is much higher than that of the routinely-used gold nanoparticle based ICA. The intra- and inter-assays recovery rates for spiked serum samples at HBsAg concentrations of 75 pg mL(-1), 3.75 ng mL(-1) and 18.75 ng mL(-1) ranged from 90.14% to 97.6%, and coefficients of variation were all below 7%, indicating that the QB-ICA sensor has an acceptable accuracy for HBsAg detection. Additionally, the quantitative method developed showed no false positive results in an analysis of 49 real HBsAg-negative serum samples, and exhibited excellent agreement (R(2)=0.9209) with a commercial chemiluminescence immunoassay kit in identifying 47 HBsAg-positive serum samples. In summary, due to its high fluorescence intensity, the sandwich QB-ICA sensor is a very promising point-of-care test for rapid, simple and ultrasensitive detection of HBsAg, as well as other disease-related protein biomarkers.

  13. A novel method for the quantitation of gingerol glucuronides in human plasma or urine based on stable isotope dilution assays.

    PubMed

    Schoenknecht, Carola; Andersen, Gaby; Schieberle, Peter

    2016-11-15

    The bio-active compounds of ginger (Zingiber officinale Roscoe), the gingerols, are gaining considerable attention due to their numerous beneficial health effects. In order to elucidate the physiological relevance of the ascribed effects their bioavailability has to be determined taking their metabolization into account. To quantitate in vivo generated [6]-, [8]- and [10]-gingerol glucuronides in human plasma and urine after ginger tea consumption, a simultaneous and direct liquid chromatography-tandem mass spectrometry method based on stable isotope dilution assays was established and validated. The respective references as well as the isotopically labeled substances were synthesized and characterized by mass spectrometry and NMR. Selective isolation of gingerol glucuronides from human plasma and urine by a mixed-phase anion-exchange SPE method led to recovery rates between 80.8 and 98.2%. LC-MS/MS analyses in selected reaction monitoring modus enabled a highly sensitive quantitation of gingerol glucuronides with LoQs between 3.9-9.8nmol/L in plasma and 39.3-161.1nmol/L in urine. The method precision in plasma and urine varied in the range±15%, whereas the intra-day accuracy in plasma and urine showed values between 78 and 122%. The developed method was then applied to a pilot study in which two volunteers consumed one liter ginger tea. Pharmacokinetic parameters like the maximum concentration (cmax), the time to reach cmax (tmax), area under the curve (AUC), elimination rate constant (kel) and elimination half-life (t1/2) were calculated from the concentration-time curve of each gingerol glucuronide. The obtained results will enable more detailed investigation of gingerol glucuronides as bioactives in their physiologically relevant concentrations.

  14. Detection limits of quantitative and digital PCR assays and their influence in presence-absence surveys of environmental DNA.

    PubMed

    Hunter, Margaret E; Dorazio, Robert M; Butterfield, John S S; Meigs-Friend, Gaia; Nico, Leo G; Ferrante, Jason A

    2017-03-01

    A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR-based analyses of low-concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species' presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty-indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis and forensic and clinical diagnostics. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  15. Development and validation of a real-time PCR assay for a novel HTLV-1 tax sequence detection and proviral load quantitation.

    PubMed

    Castro, Gonzalo M; Balangero, Marcos C; Maturano, Eduardo; Mangeaud, Arnaldo; Gallego, Sandra V

    2013-05-01

    A quantitative real-time PCR (qPCR) assay using SYBR Green dye was established in order to detect and quantify the proviral DNA of HTLV-1 in peripheral blood mononuclear cells (PBMCs). Primers were designed, and the assay was standardized to amplify a novel, conserved HTLV-1 tax region. Proviral load was normalized to the amount of cellular DNA by quantitation of the human albumin gene. Firstly, the qPCR was assessed determining the specificity, sensitivity, dynamic range and intra- and inter-assay reproducibility of the technique. The limit of detection as determined by PROBIT analysis using dilutions of the standard was 2.97 copies. The assay had an excellent dynamic range from 10⁵ to 10¹ copies per reaction and good intra- and inter-assay reproducibility, CVs less than 2%. Secondly, the performance of the qPCR was tested on 40 HTLV-1 seropositive individuals. Proviral load for HTLV-1 carriers ranged from 2.2×10² to more than 8.3×10⁴ copies/10⁶ PBMCs. The high sensitivity and wide dynamic range allowed the determination of a broad range of HTLV-1 proviral loads in infected individuals. This assay is a valuable alternative diagnostic tool when current available serological assays are insufficient. In addition, it will facilitate the study of the relationship between proviral load and pathogenesis. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Specificity of quantitative latex agglutination assay for D-dimer in exclusion of pulmonary embolism in the emergency department.

    PubMed

    Stein, Paul D; Matta, Fadi; Sabra, Michel J; Tana, Christopher; Gough, Andrew; Chabala, Steve; Kakish, Edward; Tworek, Joseph

    2014-11-01

    We assessed the prevalence of elevated quantitative latex agglutination assay for D-dimer in patients in the emergency department in whom pulmonary embolism (PE) was excluded. D-dimer was normal (<230 ng/mL) in 435 (83%) of the 522 patients. D-dimer was normal in 88% of the patients with musculoskeletal or related chest pain, 74% with pleurisy or pleuritic chest pain, and 85% with upper respiratory tract infection. D-dimer was 230 to 500 ng/mL in 65 (75%) of the 87 in whom D-dimer was elevated. Clinical probability was low in 31 (48%) of the 65 patients with D-dimer levels of 230 to 500 ng/mL. D-dimer was 230 to 500 ng/mL and clinical probability was low in 31 (36%) of the 87 patients who had computed tomographic (CT) angiograms because of elevated D-dimer. Negative likelihood ratio for PE is sufficiently low that PE can be excluded with reasonable certainty in such patients. Tailoring cutoff value to 500 ng/mL in patients with low clinical probability would have reduced CT angiograms by 36%.

  17. Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve

    PubMed Central

    Rueda-Martínez, Carmen; Fernández, M. Carmen; Soto-Navarrete, María Teresa; Jiménez-Navarro, Manuel; Durán, Ana Carmen; Fernández, Borja

    2016-01-01

    Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180–240 days old) and 56 old (300–440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta. PMID:27711171

  18. Evaluation of an enzyme-linked immunosorbent assay for quantitation of antibodies to Junin virus in human sera.

    PubMed

    García Franco, S; Ambrosio, A M; Feuillade, M R; Maiztegui, J I

    1988-01-01

    An enzyme-linked immunosorbent assay (ELISA) was evaluated for the quantitation of anti-Junin virus (JV) antibodies, in 83 selected cases of Argentine haemorrhagic fever (AHF). Serum samples were studied in two groups to facilitate comparative analysis; the first group was ELISA with indirect immunofluorescence (IF) test, in the second ELISA with plaque reduction neutralization test (PRINT). From the results obtained by using ELISA and IF on the same serum samples, a clear tendency of ELISA to demonstrate seroconversion for JV earlier and at higher frequency than IF test was noted. Simultaneous titration of specific antibodies by ELISA and PRNT tests rendered significantly correlated titers (r = 0.81), both methods being equivalently specific (100%). The demonstration of specific antibodies by ELISA in two cases that were undetected by the PRNT test resulted in a higher sensitivity index for ELISA than for PRNT (100% vs 97%). It is concluded that ELISA could efficiently replace IF and PRNT tests for the diagnosis of AHF.

  19. Generation of a recombinant classical swine fever virus stably expressing the firefly luciferase gene for quantitative antiviral assay.

    PubMed

    Shen, Liang; Li, Yongfeng; Chen, Jianing; Li, Chao; Huang, Junhua; Luo, Yuzi; Sun, Yuan; Li, Su; Qiu, Hua-Ji

    2014-09-01

    Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious swine disease leading to significant economic losses worldwide. Vaccines are widely used to control the disease, and no CSFV-specific antivirals are currently available. To facilitate anti-CSFV molecule discovery, we developed a reporter virus CSFV-N(pro)Fluc stably expressing the firefly luciferase (Fluc) gene in the N(pro) gene. The reporter virus enabled more sensitive and convenient detection of the N(pro) protein expression and the viral replication by luciferase reporter assay than by traditional methods. The CSFV N(pro) protein was detectable as early as 4.5h post-infection. As a proof-of-concept for its utility in rapid antiviral screening, this reporter virus was used to quantify anti-CSFV neutralizing antibodies of 50 swine sera and to assess 12 small interfering RNAs targeting different regions of the CSFV genome. The results were comparable to those obtained by traditional methods. Taken together, the reporter virus CSFV-N(pro)Fluc represents a useful tool for rapid and quantitative screening and evaluation of antivirals against CSFV.

  20. Real-Time Quantitative PCR Assay for Monitoring of Nervous Necrosis Virus Infection in Grouper Aquaculture▿†

    PubMed Central

    Kuo, Hsiao-Che; Wang, Ting-Yu; Chen, Peng-Peng; Chen, Young-Mao; Chuang, Hui-Ching; Chen, Tzong-Yueh

    2011-01-01

    Viral nervous necrosis caused by nervous necrosis virus (NNV) exacts a high mortality and results in huge economic losses in grouper aquaculture in Taiwan. The present study developed a real-time quantitative PCR (qPCR) method for NNV monitoring. The assay showed a strong linear correlation (r2 = 0.99) between threshold cycle (CT) and RNA quantities, which allowed identification of infected groupers by the CT value and could be exploited to warn of NNV infection prior to an outbreak in grouper fish farms. Real-time qPCR also confirmed the copious content of NNV in grouper fin, similar to that in primary tissues; the result was verified by using in situ reverse transcription-PCR (RT-PCR). This indicated that grouper fin was a suitable sample for NNV detection, in a manner that could be relatively benign to the fish. The rapid spread of NNV infection to the entire population of affected farms was evident. The developed real-time qPCR method is rapid, highly sensitive, and applicable to routine high-throughput detection of large numbers of samples and has potential as a suitable tool for diagnostic, epidemiological, and genetic studies of grouper aquaculture. PMID:21233077

  1. Quantitation of odor-active compounds in rye flour and rye sourdough using stable isotope dilution assays.

    PubMed

    Kirchhoff, Eva; Schieberle, Peter

    2002-09-11

    Application of the aroma extract dilution analysis on a flavor distillate prepared from freshly ground rye flour (type 1150) revealed 1-octen-3-one (mushroom-like), methional (cooked potato), and (E)-2-nonenal (fatty, green) with the highest flavor dilution (FD) factors among the 26 odor-active volatiles identified. Quantitative measurements performed by stable isotope dilution assays and a comparison to the odor thresholds of selected odorants in starch suggested methional, (E)-2-nonenal, and hexanal as contributors to the flour aroma, because their concentrations exceeded their odor thresholds by factors >100. Application of the same approach on a rye sourdough prepared from the same batch of flour revealed 3-methylbutanal, vanillin, 3-methylbutanoic acid, methional, (E,E)-2,4-decadienal, 2,3-butanedione, and acetic acid as important odorants; their concentrations exceeded their odor thresholds in water and starch by factors >100. A comparison of the concentrations of 20 odorants in rye flour and the sourdough made therefrom indicated that flour, besides the fermentation process, is an important source of aroma compounds in dough. However, 3-methylbutanol, acetic acid, and 2,3-butanedione were much increased during fermentation, whereas (E,E)-2,4-decadienal and 2-methylbutanal were decreased. Similar results were obtained for five different flours and sourdoughs, respectively, although the amounts of some odorants in the flour and the sourdough differed significantly within batches.

  2. DNA and RNA Extraction and Quantitative Real-Time PCR-Based Assays for Biogas Biocenoses in an Interlaboratory Comparison

    PubMed Central

    Lebuhn, Michael; Derenkó, Jaqueline; Rademacher, Antje; Helbig, Susanne; Munk, Bernhard; Pechtl, Alexander; Stolze, Yvonne; Prowe, Steffen; Schwarz, Wolfgang H.; Schlüter, Andreas; Liebl, Wolfgang; Klocke, Michael

    2016-01-01

    Five institutional partners participated in an interlaboratory comparison of nucleic acid extraction, RNA preservation and quantitative Real-Time PCR (qPCR) based assays for biogas biocenoses derived from different grass silage digesting laboratory and pilot scale fermenters. A kit format DNA extraction system based on physical and chemical lysis with excellent extraction efficiency yielded highly reproducible results among the partners and clearly outperformed a traditional CTAB/chloroform/isoamylalcohol based method. Analytical purpose, sample texture, consistency and upstream pretreatment steps determine the modifications that should be applied to achieve maximum efficiency in the trade-off between extract purity and nucleic acid recovery rate. RNA extraction was much more variable, and the destination of the extract determines the method to be used. RNA stabilization with quaternary ammonium salts was an as satisfactory approach as flash freezing in liquid N2. Due to co-eluted impurities, spectrophotometry proved to be of limited value for nucleic acid qualification and quantification in extracts obtained with the kit, and picoGreen® based quantification was more trustworthy. Absorbance at 230 nm can be extremely high in the presence of certain chaotropic guanidine salts, but guanidinium isothiocyanate does not affect (q)PCR. Absolute quantification by qPCR requires application of a reliable internal standard for which correct PCR efficiency and Y-intercept values are important and must be reported. PMID:28952569

  3. Development of a multiplex quantitative fluorescent PCR assay for identification of rearrangements in the AZFb and AZFc regions.

    PubMed

    Zhang, Jun; Li, Pei-qiong; Yu, Qi-hong; Chen, Hua-yun; Li, Juan; He, Yun-shao

    2008-06-01

    The azoospermia factor b (AZFb) and azoospermia factor c (AZFc) regions in the human Y chromosome consist of five palindromes constructed from six distinct families of amplicons and are prone to rearrangement. Partial deletion and duplication in the region can cause azoospermia or oligozoospermia and male infertility. The aim of the study was to establish a quantitative fluorescent PCR (QF-PCR) assay to classify AZFb and AZFc rearrangements. A single pair of fluorescent primers was designed to amplify simultaneously the amplicon in AZFc and the length-variant homologous sequences outside of the region as control. Since the copy number of the control sequences is fixed in the human genome, dosage of the target could be easily obtained through comparing the height of the fluorescent peaks between the target and the control after amplification with limited PCR cycles. Most types of rearrangements in AZFb and AZFc regions could be classified with QF-PCR containing four such primer pairs. Eleven types of rearrangement in AZFb and AZFc regions were well discriminated with QF-PCR. In conclusion, QF-PCR is a simple and reliable method to detect rearrangements in AZFb and AZFc.

  4. Identification of Reference Genes for Quantitative Real Time PCR Assays in Aortic Tissue of Syrian Hamsters with Bicuspid Aortic Valve.

    PubMed

    Rueda-Martínez, Carmen; Fernández, M Carmen; Soto-Navarrete, María Teresa; Jiménez-Navarro, Manuel; Durán, Ana Carmen; Fernández, Borja

    2016-01-01

    Bicuspid aortic valve (BAV) is the most frequent congenital cardiac malformation in humans, and appears frequently associated with dilatation of the ascending aorta. This association is likely the result of a common aetiology. Currently, a Syrian hamster strain with a relatively high (∼40%) incidence of BAV constitutes the only spontaneous animal model of BAV disease. The characterization of molecular alterations in the aorta of hamsters with BAV may serve to identify pathophysiological mechanisms and molecular markers of disease in humans. In this report, we evaluate the expression of ten candidate reference genes in aortic tissue of hamsters in order to identify housekeeping genes for normalization using quantitative real time PCR (RT-qPCR) assays. A total of 51 adult (180-240 days old) and 56 old (300-440 days old) animals were used. They belonged to a control strain of hamsters with normal, tricuspid aortic valve (TAV; n = 30), or to the affected strain of hamsters with TAV (n = 45) or BAV (n = 32). The expression stability of the candidate reference genes was determined by RT-qPCR using three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable reference genes for the three algorithms employed were Cdkn1β, G3pdh and Polr2a. We propose the use of Cdkn1β, or both Cdkn1β and G3pdh as reference genes for mRNA expression analyses in Syrian hamster aorta.

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

    PubMed

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

    2014-09-01

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

  6. Comparative evaluation of laboratory developed real-time PCR assays and RealStar(®) BKV PCR Kit for quantitative detection of BK polyomavirus.

    PubMed

    Hasan, Mohammad R; Tan, Rusung; Al-Rawahi, Ghada; Thomas, Eva; Tilley, Peter

    2016-08-01

    Quantitative, viral load monitoring for BK virus (BKV) by real-time PCR is an important tool in the management of polyomavirus associated nephropathy in renal transplant patients. However, variability in PCR results has been reported because of polymorphisms in viral genes among different subtypes of BKV, and lack of standardization of the PCR assays among different laboratories. In this study we have compared the performance of several laboratory developed PCR assays that target highly conserved regions of BKV genome with a commercially available, RealStar(®) BKV PCR Kit. Three real-time PCR assays (i) VP1 assay: selected from the literature that targets the major capsid protein (VP1) gene (ii) VP1MOD assay: VP1 assay with a modified probe, and (iii) BKLTA assay: newly designed assay that targets the large T antigen gene were assessed in parallel, using controls and clinical specimens that were previously tested using RealStar(®) BKV PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Nucleic acid from all samples were extracted using the QIA symphony virus/bacteria kit on an automated DNA extraction platform QIA symphony SP (Qiagen). Primer and probe concentration, and reaction conditions for laboratory developed assays were optimized and the limit of detection of different assays was determined. Positive control for laboratory developed BK assays was prepared through construction of a plasmid carrying respective amplicon sequences. The 95% detection limit of VP1, VP1MOD and BKLTA assays were 1.8×10(2), 3×10(3) and 3.5×10(2) genomic copies/ml, respectively, as determined by Probit regression analysis of data obtained by testing a dilution series of a titered patient specimen, using RealStar(®) BKV PCR Kit. The inter-assay and intra-assay, coefficient of variations of these assays using calibrated, plasmid standards were <1%. All assays, including the RealStar(®) BKV PCR assay, were highly specific when tested against a panel of external proficiency

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

    PubMed

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

    2017-08-01

    HHV-6 reactivation in immunocompromised patients is common and may be associated with serious morbidity and mortality; therefore, early detection and initiation of therapy might be of benefit. Real-time PCR assays allow for early identification of HHV-6 reactivation to assist in providing a timely response. Thus, we compared the performance of an in-house developed HHV-6 quantitative PCR assay with a commercially available kit, the RealStar(®) HHV-6 PCR Kit. The analytical sensitivity, analytical specificity, linearity, precision and accuracy of the in-house developed HHV-6 qPCR assay were evaluated. The diagnostic performance of the in-house HHV-6 qPCR assay was compared with the RealStar(®) HHV-6 PCR Kit, using 72 clinical specimens and 17 proficiency testing samples. Linear regression analysis of the quantitative results showed a dynamic range from 2 to 10 log10 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 log10 and 2 log10 copies/ml, respectively. The precision of the assay was highly reproducible among runs with coefficients of variance (CV) ranging from 0.27% to 4.37%. A comparison of 27 matched samples showed an excellent correlation between the quantitative viral loads measured by the in-house HHV-6 qPCR assay and the RealStar(®) HHV-6 PCR Kit (R(2)=0.926; P<0.0001), with an average bias of -0.24 log10 copies/ml. The in-house developed HHV-6 qPCR method is a sensitive and reliable assay with lower cost for the detection and quantification of HHV-6 DNA when compared to the RealStar(®) HHV-6 PCR Kit. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Development of a quantitative loop-mediated isothermal amplification (qLAMP) assay for the detection of Magnaporthe oryzae airborne inoculum in turf ecosystems

    USDA-ARS?s Scientific Manuscript database

    Grey Leaf Spot (GLS) is a detrimental disease of perennial ryegrass caused by a host-specialized form of Magnaporthe oryzae (Mot). In order to improve turf management, a quantitative loop-mediated isothermal amplification (LAMP) assay coupled with a simple spore trap is being developed to monitor GL...

  9. Factors That Contribute to Assay Variation in Quantitative Analysis of Sex Steroid Hormones Using Liquid and Gas Chromatography-Mass Spectrometry

    ERIC Educational Resources Information Center

    Xu, Xia; Veenstra, Timothy D.

    2012-01-01

    The list of physiological events in which sex steroids play a role continues to increase. To decipher the roles that sex steroids play in any condition requires high quality cohorts of samples and assays that provide highly accurate quantitative measures. Liquid and gas chromatography coupled with mass spectrometry (LC-MS and GC-MS) have…

  10. Development and Comparison of SYBR Green Quantitative Real-time PCR Assays for Detection and Enumeration of Sulfate-reducing Bacteria in Stored Swine Manure

    USDA-ARS?s Scientific Manuscript database

    A quantitative real-time polymerase chain reaction (PCR) assay for sulfate-reducing bacteria (SRB) was developed that targeted the dissimilatory sulfite reductase gene (dsrA). Degenerate primer sets were developed to detect three different groups of SRB in stored swine manure using a SYBR Green qua...

  11. Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status

    EPA Science Inventory

    Investigation of a redox-sensitive predictive model of mouse embryonic stem cell differentiation via quantitative nuclease protection assays and glutathione redox status Chandler KJ,Hansen JM, Knudsen T,and Hunter ES 1. U.S. Environmental Protection Agency, Research Triangl...

  12. Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status

    EPA Science Inventory

    Investigation of a redox-sensitive predictive model of mouse embryonic stem cell differentiation via quantitative nuclease protection assays and glutathione redox status Chandler KJ,Hansen JM, Knudsen T,and Hunter ES 1. U.S. Environmental Protection Agency, Research Triangl...

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  15. Factors That Contribute to Assay Variation in Quantitative Analysis of Sex Steroid Hormones Using Liquid and Gas Chromatography-Mass Spectrometry

    ERIC Educational Resources Information Center

    Xu, Xia; Veenstra, Timothy D.

    2012-01-01

    The list of physiological events in which sex steroids play a role continues to increase. To decipher the roles that sex steroids play in any condition requires high quality cohorts of samples and assays that provide highly accurate quantitative measures. Liquid and gas chromatography coupled with mass spectrometry (LC-MS and GC-MS) have…

  16. Quantitative estimation of diphtheria and tetanus toxoids. 5. Comparative assays in mice and in guinea-pigs of two diphtheria toxoid preparations.

    PubMed

    Lyng, J; Heron, I

    1991-10-01

    Two freeze-dried international reference diphtheria toxoids of different origin were compared in biological assays in guinea-pigs and mice under different adjuvant conditions. When the antigenic content in the two toxoids was used as denominator for determination of relative potency, that is to say quantitation of immunogenic power per unit amount of antigen, the design of the animal assay proved to have a major influence. Similar observations have been made previously also for tetanus vaccines. It is concluded that diphtheria vaccines as well as tetanus vaccines can hardly be quantitated unambiguously using the currently recommended potency assays in animals. A new scheme for control of toxoid vaccine production is suggested, with more emphasis on the control of the bulk purified toxoid, which would make the release of final products more simple and rapid.

  17. Development and evaluation of SYBR Green-I based quantitative PCR assays for herpes simplex virus type 1 whole transcriptome analysis.

    PubMed

    Garvey, Cathryn E; McGowin, Chris L; Foster, Timothy P

    2014-06-01

    There is an emerging need for viral gene specific quantitative PCR (qPCR) assays that validate and complement whole transcriptome level technologies, including microarray and next generation sequencing. Therefore, a compilation of qPCR assays that represented the breadth of the entire Herpes simplex virus type 1 (HSV-1) genome were developed and evaluated. SYBR Green-I-based quantitation of each of the 74 HSV-1 lytic genes enabled accurate and reproducible detection of viral genes using a minimal number of reaction conditions. The amplification specificity of these assays for HSV-1 target genes was confirmed by amplicon size and purity determination on agarose gels, melt temperature dissociation curve analysis, and direct DNA sequencing of amplified products. Analysis of representative target genes demonstrated that these assays accurately and reproducibly quantified target gene expression across a wide and linear range of detection. In addition, minimal intra- and inter-assay variability was observed with significant well-to-well and plate-to-plate/assay-to-assay precision. To evaluate the utility of the developed qPCR assay system, kinetic profiles of viral gene expression were determined for an array of representative genes from all HSV-1 transcriptional gene classes. Collectively, these data demonstrate that the compiled optimized qPCR assays is a scalable and cost-effective method to assess HSV-1 gene expression with broad application potential, including investigation of pathogenesis and antiviral therapies. In addition, they can be employed to validate and complement evolving technologies for genome-wide transcriptome analysis.

  18. Development and evaluation of SYBR Green-I based quantitative PCR assays for herpes simplex virus type 1 whole transcriptome analysis

    PubMed Central

    Garvey, Cathryn E.; McGowin, Chris L.; Foster, Timothy P.

    2014-01-01

    There is an emerging need for viral gene specific quantitative PCR (qPCR) assays that validate and complement whole transcriptome level technologies, including microarray and next generation sequencing. Therefore, a compilation of qPCR assays that represented the breadth of the entire Herpes simplex virus type 1 (HSV-1) genome were developed and evaluated. SYBR Green-I-based quantitation of each of the 74 HSV-1 lytic genes enabled accurate and reproducible detection of viral genes using a minimal number of reaction conditions. The amplification specificity of these assays for HSV-1 target genes was confirmed by amplicon size and purity determination on agarose gels, melt temperature dissociation curve analysis, and direct DNA sequencing of amplified products. Analysis of representative target genes demonstrated that these assays accurately and reproducibly quantified target gene expression across a wide and linear range of detection. In addition, minimal intra- and inter-assay variability was observed with significant well-to-well and plate-to-plate/assay-to-assay precision. To evaluate the utility of the developed qPCR assay system, kinetic profiles of viral gene expression were determined for an array of representative genes from all HSV-1 transcriptional gene classes. Collectively, these data demonstrate that the compiled optimized qPCR assays is a scalable and cost-effective method to assess HSV-1 gene expression with broad application potential, including investigation of pathogenesis and antiviral therapies. In addition, they can be employed to validate and complement evolving technologies for genome-wide transcriptome analysis. PMID:24607486

  19. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    SciTech Connect

    Fichorova, Raina N.; Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan; Chandra, Neelima; Doncel, Gustavo F.

    2015-06-15

    Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV + 2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P < 0.05), and decreased levels of TLR2 (P < 0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P < 0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product. - Highlights: • A transcriptome nuclease protection assay assessed microbicides for vaginal safety. • Biomarkers were

  20. Development of quantitative immunochromatographic assay for rapid and sensitive detection of carbohydrate antigen 19-9 (CA 19-9) in human plasma.

    PubMed

    Baryeh, Kwaku; Takalkar, Sunitha; Lund, Michelle; Liu, Guodong

    2017-09-05

    A quantitative immunochromatographic assay (QIA) was developed by using gold nanoparticle (GNP)-based lateral flow strip biosensor (LFSB) and a portable strip reader for rapid and sensitive quantitation of Carbohydrate Antigen 19-9 (CA 19-9) in human plasma. CA 19-9 is a biomarker that has been associated with cancers (such as pancreatic and colorectal cancers) and various non-cancerous diseases. The principle is based on sandwich-type immunoreactions between gold nanoparticle (GNP)-labelled detection antibody, anti-CA 19-9 capture antibody and CA 19-9to capture the GNPs on the test zone of LFSB. The accumulation of GNPs on the test zone gave a red line whose intensity was read with a portable strip reader to quantify the concentration of CA 19-9. Assay parameters including the membrane type, antibody concentration, amount of GNP-anti-CA 19-9 conjugates and the components of the running buffer were optimized to obtain the best sensitivity and reproducibility of the assay. The detection limit of the assay was determined to be 5UmL(-1) (S/N=3) with a linear range of 5UmL(-1)-100UmL(-1). CA 19-9 concentrations in healthy human and pancreatic cancer patient plasma samples were successfully evaluated using the developed quantitative immunochromatographic assay (QIA), and the results were in accordance with that obtained with enzyme linked immunosorbent assay (ELISA). The developed assay shows great promise for clinical application and biomedical diagnosis, particularly in limited resource settings. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.

    PubMed

    Fichorova, Raina N; Mendonca, Kevin; Yamamoto, Hidemi S; Murray, Ryan; Chandra, Neelima; Doncel, Gustavo F

    2015-06-15

    Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.

  2. Quantitation of viable Coxiella burnetii in milk using an integrated cell culture-polymerase chain reaction (ICC-PCR) assay.

    PubMed

    Stewart, Diana; Shieh, Y-Carol; Tortorello, Mary; Kukreja, Ankush; Shazer, Arlette; Schlesser, Joseph

    2015-11-01

    The obligate intracellular pathogen Coxiella burnetii has long been considered the most heat resistant pathogen in raw milk, making it the reference pathogen for determining pasteurisation conditions for milk products. New milk formulations and novel non-thermal processes require validation of effectiveness which requires a more practical method for analysis than using the currently used animal model for assessing Coxiella survival. Also, there is an interest in better characterising thermal inactivation of Coxiella in various milk formulations. To avoid the use of the guinea pig model for evaluating Coxiella survival, an Integrated Cell Culture-PCR (ICC-PCR) method was developed for determining Coxiella viability in milk. Vero cell cultures were directly infected from Coxiella-contaminated milk in duplicate 24-well plates. Viability of the Coxiella in milk was shown by a ≥ 0.5 log genome equivalent (ge)/ml increase in the quantity of IS111a gene from the baseline post-infection (day 0) level after 9-11 d propagation. Coxiella in skim, 2%, and whole milk, and half and half successfully infected Vero cells and increased in number by at least 2 logs using a 48-h infection period followed by 9-d propagation time. As few as 125 Coxiella ge/ml in whole milk was shown to infect and propagate at least 2 logs in the optimised ICC-PCR assay, though variable confirmation of propagation was shown for as low as 25 Coxiella ge/ml. Applicability of the ICC-PCR method was further proven in an MPN format to quantitate the number of viable Coxiella remaining in whole milk after 60 °C thermal treatment at 0, 20, 40, 60 and 90 min.

  3. Development of a quantitative enzyme-linked immunosorbent assay for vitellin in the mysid Neomysis integer (Crustacea: Mysidacea).

    PubMed

    Ghekiere, An; Fenske, Martina; Verslycke, Tim; Tyler, Charles; Janssen, Colin

    2005-09-01

    Mysid crustaceans have been put forward by several regulatory bodies as suitable test organisms to screen and test the potential effects of environmental endocrine disruptors. Despite the well-established use of mysid reproductive endpoints such as fecundity, egg development time, and time to first brood release in standard toxicity testing, little information exists on the hormonal regulation of these processes. Control of vitellogenesis is being studied intensively because yolk is an excellent model for studying mechanisms of hormonal control, and vitellogenesis can be chemically disrupted. Yolk protein or vitellin is a major source of nourishment during embryonic development of ovigorous egg-laying invertebrates. The accumulation of vitellin during oocyte development is vital for the production of viable offspring. In this context, we developed a competitive enzyme-linked immunosorbent assay (ELISA) for vitellin of the estuarine mysid Neomysis integer. Mysid vitellin was isolated using gel filtration, and the purified vitellin was used to raise polyclonal antibodies. The ELISA was sensitive within a working range of 4 to 500 ng vitellin/mL. Serial dilutions of whole body homogenates from female N. integer and the vitellin standard showed parallel binding curves, validating the specificity of the ELISA. The intra- and interassay coefficients of variation were 8.2% and 13.8%, respectively. Mysid vitellin concentrations were determined from ovigorous females and eggs at different developmental stages. The availability of a quantitative mysid vitellin ELISA should stimulate further studies on the basic biology of this process in mysids. Furthermore, it could provide a means to better understand and predict chemically induced reproductive effects in mysids.

  4. A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts

    PubMed Central

    Hostettler, Isabel; Müller, Joachim; Stephens, Chad E.; Haynes, Richard; Hemphill, Andrew

    2014-01-01

    Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48–72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development. PMID:25516828

  5. A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts.

    PubMed

    Hostettler, Isabel; Müller, Joachim; Stephens, Chad E; Haynes, Richard; Hemphill, Andrew

    2014-12-01

    Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48-72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development.

  6. Characterization of the rRNA Locus of Pfiesteria piscicida and Development of Standard and Quantitative PCR-Based Detection Assays Targeted to the Nontranscribed Spacer

    PubMed Central

    Saito, Keiko; Drgon, Tomás; Robledo, José A. F.; Krupatkina, Danara N.; Vasta, Gerardo R.

    2002-01-01

    Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 μg of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments. PMID:12406730

  7. Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the nontranscribed spacer.

    PubMed

    Saito, Keiko; Drgon, Tomás; Robledo, José A F; Krupatkina, Danara N; Vasta, Gerardo R

    2002-11-01

    Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 micro g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.

  8. Semi-quantitative Influenza A population averages from a multiplex respiratory viral panel (RVP): potential for reflecting target sequence changes affecting the assay.

    PubMed

    Rand, Kenneth H; Pieretti, Maura; Arcenas, Rodney; Beal, Stacy G; Houck, Herbert; Boslet, Emma; Lednicky, John A

    2017-07-14

    Yearly influenza virus mutations potentially affect the performance of molecular assays, if nucleic acid changes involve the sequences in the assay. Because individual patient viral loads depend on variables such as duration of illness, specimen type, age, and immunosuppression, we examined seasonal population averages of positive tests to smooth inherent variability. We studied the population seasonal averages of the semi-quantitative nAMPs for the influenza matrix and hemagglutinin genes in the GenMark (Carlsbad, CA) Respiratory Viral Panel assay between 3 institutions over 3 Influenza seasons. Population average nAMPs were strikingly consistent between separate institutions, but differed substantially between H3N2 and H1N1 seasons. In the 2012-2013 and 2014-2015 influenza seasons, matrix gene H3N2 nAMP averages were 50-70% less than those of the same assay in the 2013-2014 H1N1 season. Influenza strains representative of these seasons were grown in tissue culture and when the supernatant virus was adjusted to the same copy number using a TaqMan assay, the same relative differences were reproduced in the RVP assay. Because the sequences for the PCR and PCR product detection in the GenMark assay are proprietary, the manufacturer provided single stranded DNA matching the capture probe for the representative H3N2 (3 mismatches) and H1N1 strains (2 different mismatches). Equimolar concentrations of these synthetic DNA sequences gave average nAMP values that closely correlated with the average nAMPS of the representative strains and their respective seasonal averages. Seasonal averages of semi-quantitative data may provide a means to follow assay performance as a reflection of the effects of molecular drift.

  9. Development & validation of a quantitative anti-protective antigen IgG enzyme linked immunosorbent assay for serodiagnosis of cutaneous anthrax.

    PubMed

    Ghosh, N; Gunti, D; Lukka, H; Reddy, B R; Padmaja, Jyothi; Goel, A K

    2015-08-01

    Anthrax caused by Bacillus anthracis is primarily a disease of herbivorous animals, although several mammals are vulnerable to it. ELISA is the most widely accepted serodiagnostic assay for large scale surveillance of cutaneous anthrax. The aims of this study were to develop and evaluate a quantitative ELISA for determination of IgG antibodies against B. anthracis protective antigen (PA) in human cutaneous anthrax cases. Quantitative ELISA was developed using the recombinant PA for coating and standard reference serum AVR801 for quantification. A total of 116 human test and control serum samples were used in the study. The assay was evaluated for its precision, accuracy and linearity. The minimum detection limit and lower limit of quantification of the assay for anti-PA IgG were 3.2 and 4 µg/ml, respectively. The serum samples collected from the anthrax infected patients were found to have anti-PA IgG concentrations of 5.2 to 166.3 µg/ml. The intra-assay precision per cent CV within an assay and within an operator ranged from 0.99 to 7.4 per cent and 1.7 to 3.9 per cent, respectively. The accuracy of the assay was high with a per cent error of 6.5 - 24.1 per cent. The described assay was found to be linear between the range of 4 to 80 ng/ml (R [2] = 0.9982; slope = 0.9186; intercept = 0.1108). The results suggested that the developed assay could be a useful tool for quantification of anti-PA IgG response in human after anthrax infection or vaccination.

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

    PubMed

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

    2014-12-10

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-10-01

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

  12. A Quantitative Real-Time RT-PCR Assay for the Detection of Venezuelan equine encephalitis virus Utilizing a Universal Alphavirus Control RNA.

    PubMed

    Vina-Rodriguez, Ariel; Eiden, Martin; Keller, Markus; Hinrichs, Winfried; Groschup, Martin H

    2016-01-01

    Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV.

  13. A Quantitative Real-Time RT-PCR Assay for the Detection of Venezuelan equine encephalitis virus Utilizing a Universal Alphavirus Control RNA

    PubMed Central

    Vina-Rodriguez, Ariel; Eiden, Martin; Keller, Markus; Hinrichs, Winfried

    2016-01-01

    Venezuelan equine encephalitis virus (VEEV) is an Alphavirus from the family Togaviridae that causes epizootic outbreaks in equids and humans in Central and South America. So far, most studies use conventional reverse transcriptase PCR assays for the detection of the different VEEV subtypes. Here we describe the development of a TaqMan quantitative real-time reverse transcriptase PCR assay for the specific detection and quantitation of all VEEV subtypes which uses in parallel a universal equine encephalitis virus control RNA carrying target sequences of the three equine encephalitis viruses. The control RNA was used to generate standard curves for the calculation of copy numbers of viral genome of Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), and VEEV. The new assay provides a reliable high-throughput method for the detection and quantitation of VEEV RNA in clinical and field samples and allows a rapid differentiation from potentially cocirculating EEEV and WEEV strains. The capability to detect all known VEEV variants was experimentally demonstrated and makes this assay suitable especially for the surveillance of VEEV. PMID:28042576

  14. Quantitative Validation of the Presto Blue Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System.

    PubMed

    Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P; Schrooten, Jan Ir

    2015-06-01

    As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required.

  15. Quantitative Validation of the Presto Blue™ Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System

    PubMed Central

    Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P.

    2015-01-01

    As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue™, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required. PMID:25336207

  16. A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples

    PubMed Central

    2012-01-01

    Background CST6 promoter is highly methylated in cancer, and its detection can provide important prognostic information in breast cancer patients. The aim of our study was to develop a Methylation-Sensitive High Resolution Melting Analysis (MS-HRMA) assay for the investigation of CST6 promoter methylation. Methods We designed primers that amplify both methylated and unmethylated CST6 sequences after sodium bisulfate (SB) treatment and used spiked control samples of fully methylated to unmethylated SB converted genomic DNA to optimize the assay. We first evaluated the assay by analyzing 36 samples (pilot training group) and further analyzed 80 FFPES from operable breast cancer patients (independent group). MS-HRMA assay results for all 116 samples were compared with Methylation-Specific PCR (MSP) and the results were comparable. Results The developed assay is highly specific and sensitive since it can detect the presence of 1% methylated CST6 sequence and provides additionally a semi-quantitative estimation of CST6 promoter methylation. CST6 promoter was methylated in 39/80 (48.75%) of FFPEs with methylation levels being very different among samples. MS-HRMA and MSP gave comparable results when all samples were analyzed by both assays. Conclusions The developed MS-HRMA assay for CST6 promoter methylation is closed tube, highly sensitive, cost-effective, rapid and easy-to-perform. It gives comparable results to MSP in less time, while it offers the advantage of additionally providing an estimation of the level of methylation. PMID:23088560

  17. Development of low cost two-step reverse transcription-quantitative polymerase chain reaction assays for rotavirus detection in foul surface water drains.

    PubMed

    Chung, J W; Foppen, J W; Lens, P N L

    2013-06-01

    Commercial kits to determine RNA concentrations are expensive, and sometimes too expensive for laboratories working with tight budgets, especially those in developing countries. We developed, tested, and evaluated two home-made two-step reverse transcription-quantitative polymerase chain reaction assays aimed to detect rotavirus in surface water samples. A commercial one-step master kit was used for comparison. Our results indicated that the efficiency of the home-made assays was comparable to the commercial kit. Furthermore, the lowest detection limit of all assays corresponded to 10⁻⁰·² TCID₅₀ (50 % tissue Culture Infective Dose) per ml. The home-made assays were able to detect rotavirus concentrations in complex surface waters in a slum area in Kampala (Uganda) and their performance was comparable to the commercial kit. The total costs of the two home-made assays was 11 times less than the selected commercial kit. Although preparing home-made assays is more time consuming, the assays can be useful for cases in which consumable costs are more important than personnel costs.

  18. Optimization and validation of DNA extraction and real-time PCR assay for the quantitative measurement of residual host cell DNA in biopharmaceutical products.

    PubMed

    Hu, B; Sellers, J; Kupec, J; Ngo, W; Fenton, S; Yang, T-Y; Grebanier, A

    2014-01-01

    Host cell DNA contamination occurs during the production of biopharmaceuticals and must be controlled and monitored for the purity and safety of the drug products. A sodium iodide-based DNA extraction and a subsequent real time PCR assay were developed and validated for the quantitative measurement of residual host cell DNA impurity in monoclonal antibody therapeutic products. A sodium iodide-based commercial kit was optimized for the removal of interfering matrices. Several incubation steps from the kit protocol were combined and a neutralization buffer was introduced to protein digestion step to eliminate any precipitation from the detergent. The elimination of the two washing steps significantly reduced assay variability from loss of DNA pellets. The optimized DNA extraction procedure can recover DNA close to 100% for DNA concentrations from 10 to 100,000pg/mL. Of the published sequences of repetitive interspersed nuclear elements, we identified a nucleotide mismatch from the published CHO probe. Correction of this nucleotide increased DNA amplification by a thousand fold. The optimized assay was further validated for the quantitation of residual CHO DNA according to ICH guidelines with preset assay acceptance criteria. The method met all assay acceptance criteria and was found linear, accurate and precise for the quantitation of residual CHO in the linear range of 10-100,000pg DNA/mL. LOQ was measured at 10pg DNA/mL and LOD at 1pg DNA/mL. No matrix interference to our validated assay was detected from bioreactor harvest, Protein A eluate or eluate from ion exchange columns.

  19. Clinical Evaluation of the Immune Colloidal Gold Method for Rapid Qualitative and Quantitative Measurement of Thyroid-Stimulating Hormone as an Assay for Hypothyroidism.

    PubMed

    Wang, Tingting; Sheng, Shiwei; Ruan, Meifang; Yan, Jing; Gu, Jianying; Jiang, Yumin; Gao, Yunchao; Lu, Hankui

    2016-11-01

    The immune colloidal gold (ICG) method of measuring thyroid-stimulating hormone (TSH) is a rapid and easy-to-perform test, allowing off-site measurements. This study compared the clinical utility of the first ICG-based qualitative and quantitative TSH test methods in China with the third-generation serum TSH assay used worldwide. Fingertip and venous blood was collected within 30 min from 283 patients initially suspected of hypothyroidism. TSH was measured in fingertip blood using ICG-based qualitative quantitative tests. Serum TSH in venous blood was tested using the third-generation serum TSH assay. Correlations between systems were tested by kappa or Spearman correlation coefficients. Compared with the third-generation serum TSH assay, the ICG-qualitative TSH test kit had a kappa coefficient of 0.86, a sensitivity of 85.00%, and a specificity of 99.38% in screening for hypothyroidism. The percentages of false negatives and false positives among all subjects were 6.38% and 0.35% respectively; the total consistency rate of the two methods was 93.26%. When compared with the third-generation serum TSH assay, the ICG-quantitative TSH analysis system had a Spearman correlation coefficient of 0.91, a sensitivity of 88.43%, and a specificity of 98.77%. The percentages of false negatives and false positives among all subjects were 4.95% and 0.71%, respectively; the total consistency rate of the two methods was 94.35%. Both ICG-based assays are easier and faster to perform than the third-generation, laboratory-based serum TSH assay method. The ICG-based methods showed acceptable performance in the simplified screening for hypothyroidism. ClinicalTrials.gov identifier, NCT01921452. Merck Serono Co., Ltd.

  20. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

    PubMed

    Te, Shu Harn; Chen, Enid Yingru; Gin, Karina Yew-Hoong

    2015-08-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.

  1. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis

    PubMed Central

    Te, Shu Harn; Chen, Enid Yingru

    2015-01-01

    The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

  2. Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs.

    PubMed

    Decaro, Nicola; Martella, Vito; Ricci, Dominga; Elia, Gabriella; Desario, Costantina; Campolo, Marco; Cavaliere, Nicola; Di Trani, Livia; Tempesta, Maria; Buonavoglia, Canio

    2005-12-01

    Two genotype-specific fluorogenic RT-PCR assays were developed for the detection and quantitation of canine coronavirus (CCoV) type I and type II RNA in the faeces of dogs with diarrhoea. Both the fluorogenic assays showed high specificity, sensitivity and reproducibility, allowing a precise quantitation of CCoV type I and type II RNA over a linear range of about eight orders of magnitude (from 10(1) to 10(8) copies of standard RNA). Comparison with genotype-specific gel-based RT-PCR assays revealed that the fluorogenic assays were more sensitive and more rapid than conventional amplifications, with a large increase in throughput. The genotype-specific fluorogenic assays were then used to detect and measure viral loads in the faecal samples collected from dogs naturally or experimentally infected with type I, type II, or both genotypes. Of 174 samples collected from naturally infected dogs, 77 were positive for CCoV type I and 46 for CCoV type II. Thirty-eight dogs were found to be infected naturally by both genotypes, with viral RNA titres generally higher for type I in comparison to type II. At the same time, dogs infected experimentally shed type I RNA with higher titres with respect to type II.

  3. Imaging and quantitative analysis of tritium-labelled cells in lymphocyte proliferation assays using microchannel plate detectors originally developed for X-ray astronomy.

    PubMed

    Lees, J E; Hales, J M

    2001-01-01

    Microchannel plate detectors have been used in many astronomical X-ray telescopes. Recently we have begun to use similar detectors to image electron emission from radiolabelled biological assays. Here we show how a microchannel plate (MCP) detector can be used to image tritium uptake in T lymphocyte proliferation assays. Quantitative analysis using the MCP detector has the same sensitivity and speed as conventional liquid scintillation counter (LSC) analysis whilst obviating the need for scintillation fluid. In addition the system permits the imaging of whole plate harvests from a range of plate sizes. Here we present data obtained with 96-well plates and Terasaki plates.

  4. A quantitative real-time PCR assay for the identification and enumeration of Alexandrium cysts in marine sediments

    NASA Astrophysics Data System (ADS)

    Erdner, D. L.; Percy, L.; Keafer, B.; Lewis, J.; Anderson, D. M.

    2010-02-01

    Harmful algal blooms (HABs) are a global problem that affects both human and ecosystem health. One of the most serious and widespread HAB poisoning syndromes is paralytic shellfish poisoning, commonly caused by Alexandrium spp. dinoflagellates. Like many toxic dinoflagellates, Alexandrium produces resistant resting cysts as part of its life cycle. These cysts play a key role in bloom initiation and decline, as well as dispersal and colonization of new areas. Information on cyst numbers and identity is essential for understanding and predicting blooms, yet comprehensive cyst surveys are extremely time- and labor-intensive. Here we describe the development and validation of a quantitative real-time PCR (qPCR) technique for the enumeration of cysts of A. tamarense of the toxic North American/Group I ribotype. The method uses a cloned fragment of the large subunit ribosomal RNA gene as a standard for cyst quantification, with an experimentally determined conversion factor of 28,402±6152 LSU ribosomal gene copies per cyst. Tests of DNA extraction and PCR efficiency show that mechanical breakage is required for adequate cyst lysis, and that it was necessary to dilute our DNA extracts 50-fold in order to abolish PCR inhibition from compounds co-extracted from the sediment. The resulting assay shows a linear response over 6 orders of magnitude and can reliably quantify ≥10 cysts/cm 3 sediment. For method validation, 129 natural sediment samples were split and analyzed in parallel, using both the qPCR and primulin-staining techniques. Overall, there is a significant correlation ( p<0.001) between the cyst abundances determined by the two methods, although the qPCR counts tend to be lower than the primulin values. This underestimation is less pronounced in those samples collected from the top 1 cm of sediment, and more pronounced in those derived from the next 1-3 cm of the core. These differences may be due to the condition of the cysts in the different layers, as the

  5. Development and Evaluation of Up-Converting Phosphor Technology-Based Lateral Flow Assay for Quantitative Detection of NT-proBNP in Blood

    PubMed Central

    Hao, Qingfang; Zou, Deyong; Zhang, Xiaoli; Zhang, Liping; Li, Hongmei; Qiao, Yong; Zhao, Huansheng; Zhou, Lei

    2017-01-01

    A newly assay, up-converting phosphor technology-based lateral flow (UPT-LF) assay, was developed for rapid and quantitative detection of N-terminal fragment of B-type natriuretic peptide precursor (NT-proBNP), one of the most important serum molecular maker of heat failure, in plasma samples as a point of care testing (POCT) method for diagnosis of acute heart failure. Human plasma from 197 patients with acute heart failure and 200 healthy controls was assessed using the UPT-LF assay, in a comparison with a Roche Elecsys assay. The limit of detection of the UPT-LF assay, with a coefficient of variation (CV) of less than 15%, was 116 ng/L, which is lower than the clinical diagnosis cutoff (150 ng/mL). The linear range was 50–35,000 ng/L. The CVs were less than 10% for both UPT-LF and Roche Elecsys assays for plasma samples under different storages, demonstrating the good stability and reproducibility. There are certain linear correlations between the results of UPT-LF and Roche Elecsys assay for EDTA-K2 and heparin-anticoagulated plasma, as well as for serum samples. For UPT-LF assay, there is a significant correlation between the values derived from analysis of EDTA-K2 and heparin-anticoagulated plasma samples (R = 0.995). No statistically significant difference was found between serum and plasma samples for UPT-LF assay. Our results demonstrate that NT-proBNP levels in healthy adults are elevated with age and had a relationship with sex, and with the age increase the NT-proBNP levels of females are significantly higher than those of males (p<0.01). The UPT-LF assay has a high reproducibility, stability, sensitivity, specificity, and is consistent with Roche Elecsys assay, and therefore it could be used as a POCT method for the quantitative detection of NT-proBNP in blood for clinical diagnosis and research of acute heart failure. PMID:28151978

  6. A quantitative PCR assay for the detection and quantification of Shiga toxin-producing Escherichia coli (STEC) in minced beef and dairy products.

    PubMed

    Derzelle, S; Grine, A; Madic, J; de Garam, C Peytavin; Vingadassalon, N; Dilasser, F; Jamet, E; Auvray, F

    2011-11-15

    Shiga toxin (Stx)-producing Escherichia coli (STEC) are amongst major causes of food-borne infectious diseases and outbreaks. A new quantitative PCR (qPCR) assay was designed to detect all known stx gene subtypes in a single reaction, including the most distant variant stx2f. Performance of this assay was evaluated in combination with two different internal amplification controls (IAC), a competitive one specific for the assay and a universal IAC based on plasmid pUC19. The qPCR assay was 100% specific and showed analytical sensitivity of two STEC genome copies per reaction. The diagnostic approach proposed, combining enrichment, automated DNA extraction and qPCR detection, could reliably detect the presence of STEC in minced beef and cheese inoculated before enrichment at <4 CFU per 25 g. A comparative study performed on 240 minced beef and 113 raw milk cheese samples demonstrated that the method developed was as effective as two PCR screening assays used routinely in our laboratory to detect STEC. The new assay also proved to be appropriate for the direct quantification of STEC in milk and minced meat. It was found to be quantitative over a five log dynamic range, from 4 × 10⁶ to 40 CFU/mL for milk and from 10⁷ to 10² CFU/g for minced beef. In conclusion, the qPCR assay developed here represents a valuable tool for rapid detection and quantification of STEC in foods such as minced beef and dairy products as it ensures a high sensitivity and an optimal STEC diagnostic spectrum, taking into account the genetic stx variability observed in STEC population. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Detection of a Molecular Biomarker for Zygomycetes by Quantitative PCR Assays of Plasma, Bronchoalveolar Lavage, and Lung Tissue in a Rabbit Model of Experimental Pulmonary Zygomycosis▿

    PubMed Central

    Kasai, Miki; Harrington, Susan M.; Francesconi, Andrea; Petraitis, Vidmantas; Petraitiene, Ruta; Beveridge, Mara G.; Knudsen, Tena; Milanovich, Jeffery; Cotton, Margaret P.; Hughes, Johanna; Schaufele, Robert L.; Sein, Tin; Bacher, John; Murray, Patrick R.; Kontoyiannis, Dimitrios P.; Walsh, Thomas J.

    2008-01-01

    We developed two real-time quantitative PCR (qPCR) assays, targeting the 28S rRNA gene, for the diagnosis of zygomycosis caused by the most common, clinically significant Zygomycetes. The amplicons of the first qPCR assay (qPCR-1) from Rhizopus, Mucor, and Rhizomucor species were distinguished through melt curve analysis. The second qPCR assay (qPCR-2) detected Cunninghamella species using a different primer/probe set. For both assays, the analytic sensitivity for the detection of hyphal elements from germinating sporangiospores in bronchoalveolar lavage (BAL) fluid and lung tissue homogenates from rabbits was 1 to 10 sporangiospores/ml. Four unique and clinically applicable models of invasive pulmonary zygomycosis served as surrogates of human infections, facilitating the validation of these assays for potential diagnostic utility. For qPCR-1, 5 of 98 infarcted lung specimens were positive by qPCR and negative by quantitative culture (qCx). None were qCx positive only. Among 23 BAL fluid samples, all were positive by qPCR, while 22 were positive by qCx. qPCR-1 detected Rhizopus and Mucor DNA in 20 (39%) of 51 serial plasma samples as early as day 1 postinoculation. Similar properties were observed for qPCR-2, which showed greater sensitivity than qCx for BAL fluid (100% versus 67%; P = 0.04; n = 15). The assay detected Cunninghamella DNA in 18 (58%) of 31 serial plasma samples as early as day 1 postinoculation. These qPCR assays are sensitive and specific for the detection of Rhizopus, Mucor, Rhizomucor, and Cunninghamella species and can be used for the study and detection of infections caused by these life-threatening pathogens. PMID:18845827

  8. Assay of S for Quantitation of PEG and TNF Ligated Au Nanoparticles using ID-HR-MC-ICP-MS

    NASA Astrophysics Data System (ADS)

    Mann, J. L.; Vocke, R. D.; Newman, J. D.; Kelly, W. R.

    2009-12-01

    The use of nanomaterials in medicine has recently increased with the discovery that these materials can deliver drugs to specific sites within the body. An active area of nanomedicine involves the use of gold nanoparticles (AuNPs) as a delivery platform for anti-cancer agents such as tumor necrosis factor-α (TNF-α). TNF-α molecules attack the blood vessels in a tumor causing them to hemorrhage profusely. TNF-α is also highly toxic to normal cells, making targeted delivery to the tumor site crucial. AuNPs ensure the ligated TNF-α is delivered to its appropriate target, mitigating systemic toxicity. Additionally, coating AuNPs with the surface modifier polyethylene glycol (PEG) promotes bioavailability and biodistribution of TNF-α by preventing protein binding and bodily uptake thereby increasing the lifetime in the bloodstream. The FDA will likely mandate all nanotechnologies of this sort undergo quality control to ensure the nanoplatforms are modified with approximately the same number of drug molecules and other surface modifiers. Quality control methods, such as visible spectroscopy, can be used to qualitatively assess the total amounts of TNF-α and PEG on AuNPs, however, these methods need to be validated via calibration by an absolute technique. The sulfur content can be used as a proxy for both PEG and TNF-α concentrations because of the presence of at least one thiol in each of the ligands. The goal of this work was to provide a benchmark for future spectroscopic results by providing an accurate assessment of the TNF-α and PEG concentrations through the accurate quantitation of S. The sulfur concentration was assayed using isotope dilution (ID) combined with high resolution multi-collector inductively coupled plasma mass spectrometry (HR-MC-ICPMS). The measurement of S using ICPMS instrumentation is challenging because 1) molecular interferences (e.g. oxides, nitrides, and hydrides) exist on each of the S isotopes and 2) the need to perform the

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

    PubMed Central

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

    2013-01-01

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

  10. Development of quantitative PCR assays targeting the 16S rRNA genes of Enterococcus spp. and their application to the identification of enterococcus species in environmental samples.

    PubMed

    Ryu, Hodon; Henson, Michael; Elk, Michael; Toledo-Hernandez, Carlos; Griffith, John; Blackwood, Denene; Noble, Rachel; Gourmelon, Michèle; Glassmeyer, Susan; Santo Domingo, Jorge W

    2013-01-01

    The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium, although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water.

  11. Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

    PubMed Central

    Ryu, Hodon; Henson, Michael; Elk, Michael; Toledo-Hernandez, Carlos; Griffith, John; Blackwood, Denene; Noble, Rachel; Gourmelon, Michèle; Glassmeyer, Susan

    2013-01-01

    The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium, although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water. PMID:23087032

  12. Serum virus neutralization assay for detection and quantitation of serum neutralizing antibodies to influenza A virus in swine

    USDA-ARS?s Scientific Manuscript database

    The serum virus neutralization (SVN) assay is a serological test to detect the presence and magnitude of functional systemic antibodies that prevent infectivity of a virus. The SVN assay is a highly sensitive and specific test that may be applied to influenza A viruses (IAV) in swine to measure the ...

  13. Layer plate CAS assay for the quantitation of siderophore production and determination of exudation patterns for fungi.

    PubMed

    Andrews, Megan Y; Santelli, Cara M; Duckworth, Owen W

    2016-02-01

    The chrome azurol S (CAS) assay measures the chelating activity of siderophores, but its application (especially to fungi) is limited by toxicity issues. In this note, we describe a modified version of the CAS assay that is suitable for quantifying siderophore exudation for microorganisms, including fungi.

  14. Unsuitability of quantitative Bacteroidales 16S rRNA gene assays for discerning fecal contamination of drinking water.

    PubMed

    van der Wielen, Paul W J J; Medema, Gertjan

    2010-07-01

    Bacteroidales species were detected in (tap) water samples from treatment plants with three different PCR assays. 16S rRNA gene sequence analysis indicated that the sequences had an environmental rather than fecal origin. We conclude that assays for Bacteroidales 16S rRNA genes are not specific enough to discern fecal contamination of drinking water in the Netherlands.

  15. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

    PubMed

    Purcell, Maureen K; Pearman-Gillman, Schuyler; Thompson, Rachel L; Gregg, Jacob L; Hart, Lucas M; Winton, James R; Emmenegger, Eveline J; Hershberger, Paul K

    2016-07-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  16. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    USGS Publications Warehouse

    Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

    2016-01-01

    Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

  17. Evaluation of quantitative polymerase chain reaction assays targeting Mycobacterium avium, M. intracellulare, and M. avium subspecies paratuberculosis in drinking water biofilms.

    PubMed

    Chern, Eunice C; King, Dawn; Haugland, Richard; Pfaller, Stacy

    2015-03-01

    Mycobacterium avium (MA), Mycobacterium intracellulare (MI), and Mycobacterium avium subsp. paratuberculosis (MAP) are difficult to culture due to their slow growing nature. A quantitative polymerase chain reaction (qPCR) method for the rapid detection of MA, MI, and MAP can be used to provide data supporting drinking water biofilms as potential sources of human exposure. The aim of this study was to characterize two qPCR assays targeting partial 16S rRNA gene sequences of MA and MI and use these assays, along with two previously reported MAP qPCR assays (IS900 and Target 251), to investigate Mycobacterium occurrence in kitchen faucet biofilms. MA and MI qPCR assays demonstrated 100% specificity and sensitivity when evaluated against 18 non-MA complex, 76 MA, and 17 MI isolates. Both assays detected approximately 1,000 cells from a diluted cell stock inoculated on a sampling swab 100% of the time. DNA analysis by qPCR indicated that 35.3, 56.9 and 11.8% of the 51 kitchen faucet biofilm samples collected contained MA, MI, and MAP, respectively. This study introduces novel qPCR assays designed to specifically detect MA and MI in biofilm. Results support the use of qPCR as an alternative to culture for detection and enumeration of MA, MI, and MAP in microbiologically complex samples.

  18. A quantitative, facile, and high-throughput image-based cell migration method is a robust alternative to the scratch assay.

    PubMed

    Gough, Wendy; Hulkower, Keren I; Lynch, Renee; McGlynn, Patrick; Uhlik, Mark; Yan, Lei; Lee, Jonathan A

    2011-02-01

    Cell migration is a key phenotype for a number of therapeutically important biological responses, including angiogenesis. A commonly used method to assess cell migration is the scratch assay, which measures the movement of cells into a wound made by physically scoring a confluent cell monolayer to create an area devoid of cells. Although this method has been adequate for qualitative characterization of migration inhibitors, it does not provide the highly reproducible results required for quantitative compound structure-activity relationship evaluation because of the inconsistent size and placement of the wound area within the microplate well. The Oris™ Cell Migration Assay presents a superior alternative to the scratch assay, permitting formation of precisely placed and homogeneously sized cell-free areas into which migration can occur without releasing factors from wounded or dead cells or damaging the underlying extracellular matrix. Herein the authors compare results from the scratch and Oris™ cell migration assays using an endothelial progenitor cell line and the Src kinase inhibitor dasatinib. They find that using the Acumen™ Explorer laser microplate cytometer in combination with the Oris™ Cell Migration Assay plate provides a robust, efficient, and cost-effective cell migration assay exhibiting excellent signal to noise, plate uniformity, and statistical validation metrics.

  19. Histochemical technique: a general method for quantitative enzyme assays of single cell extracts with a time resolution of seconds and a reading precision of femtomoles

    SciTech Connect

    Outlaw, W.H. Jr.; Springer, S.A.; Tarczynski, M.C.

    1985-03-01

    Biochemists who study single cells have been constrained by the lack of a general methodology of high time resolution and high measurement sensitivity for quantitatively assaying enzyme activities using natural substrates in solution. The methods the authors describe will remove this limitation. In brief, nanogram tissue samples are dissected from frozen-dried tissue. The samples are extracted in microdroplets of assay cocktail. The enzyme activity, indicated fluorometrically by the oxidation/reduction of NAD(P), is followed in real time on a computer display. With these methods, assays of pyruvate orthophosphate dikinase on samples enriched in bundlesheath cells and mesophyll cells of Flaveria brownii yielded the predictable results. Assays of this enzyme in guard cells dissected from Vicia faba leaflets gave results like those recently reported by another laboratory for protoplasts derived from these cells. The results of assays by this method and by enzymic cycling for NAD(P) triose-P dehydrogenase were comparable. Phosphoenolpyruvate carboxylase, the most extensively studied enzyme activity, was present at high levels in guard cells, which has been demonstrated previously in other reports based on diverse assay approaches.

  20. Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma*

    PubMed Central

    Abbatiello, Susan E.; Schilling, Birgit; Mani, D. R.; Zimmerman, Lisa J.; Hall, Steven C.; MacLean, Brendan; Albertolle, Matthew; Allen, Simon; Burgess, Michael; Cusack, Michael P.; Gosh, Mousumi; Hedrick, Victoria; Held, Jason M.; Inerowicz, H. Dorota; Jackson, Angela; Keshishian, Hasmik; Kinsinger, Christopher R.; Lyssand, John; Makowski, Lee; Mesri, Mehdi; Rodriguez, Henry; Rudnick, Paul; Sadowski, Pawel; Sedransk, Nell; Shaddox, Kent; Skates, Stephen J.; Kuhn, Eric; Smith, Derek; Whiteaker, Jeffery R.; Whitwell, Corbin; Zhang, Shucha; Borchers, Christoph H.; Fisher, Susan J.; Gibson, Bradford W.; Liebler, Daniel C.; MacCoss, Michael J.; Neubert, Thomas A.; Paulovich, Amanda G.; Regnier, Fred E.; Tempst, Paul; Carr, Steven A.

    2015-01-01

    There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC

  1. A real-time RT-PCR assay for detection and absolute quantitation of Citrus tristeza virus in different plant tissues.

    PubMed

    Ruiz-Ruiz, Susana; Moreno, Pedro; Guerri, José; Ambrós, Silvia

    2007-11-01

    A real-time RT-PCR assay using SYBR Green was developed for specific and reliable quantitative detection of Citrus tristeza virus (CTV) in infected plants. A general primer set designed from conserved sequences in ORFs 1b and 2 enabled amplification of the genomic RNA (gRNA) while excluding most subgenomic and defective RNAs. Single RT-PCR products of 204 bp (isolate T36) or 186 bp (other isolates) were obtained with no primer-dimer or non-specific amplifications detected. Melting curve analysis revealed distinct melting temperature peaks (T(m)) for severe and mild isolates. External standard curves using RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) gRNA copies and with very low variation coefficient values. This protocol enabled reliable assessments of CTV accumulation in different tissues and from different citrus species, grown in the greenhouse or under field conditions, and infected with CTV isolates differing in their pathogenicity. CTV accumulation was higher in bark and fruits than in roots or leaves and showed minimal differences among several susceptible citrus species, but it was significantly lower in sour orange. This quantitative detection assay will be a valuable tool for diagnosis and molecular studies on CTV biology.

  2. Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples.

    PubMed

    Irenge, Léonid M; Durant, Jean-François; Tomaso, Herbert; Pilo, Paola; Olsen, Jaran S; Ramisse, Vincent; Mahillon, Jacques; Gala, Jean-Luc

    2010-11-01

    A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

  3. Development and evaluation of a new kinetic assay for the quantitation of fibronectin gelatin-binding activity.

    PubMed

    Gelder, F B; Brown, S T; Moore, C A

    1985-10-01

    A new rapid and sensitive kinetic assay that measures the gelatin-binding activity of fibronectin has been developed. This assay is based on the rate of fibronectin-mediated aggregation of covalently coupled latex-gelatin particles. The addition of human plasma and serum resulted in aggregation rates proportional to the concentration of fibronectin in the test sample. This assay was inhibited by the addition of gelatin, demonstrating substrate specificity. This new assay requires 50 microliter of sample and can be performed within 5 minutes. Particle aggregation rates were affected by pH, heparin, and coupled gelatin concentration per milligram of latex. Maximum aggregation rates were observed at pH 8.0. Heparin was not an absolute requirement for particle aggregation but enhanced rates up to 1 U heparin/ml with little additive effect at greater concentrations. Heparin had a relatively greater effect on assays performed in acidic buffers. The concentration of gelatin per milligram of latex was rate limiting up to approximately 50 micrograms gelatin/mg latex with little change in aggregation rates at higher concentrations. Good correlation between total antigenic fibronectin (electroimmunoassay) and fibronectin gelatin-binding activity (latex-gelatin kinetic aggregation assay) was demonstrated in plasma from normal blood donors. This new assay will allow further definition of the relationship between fibronectin gelatin-binding activity and antigenic fibronectin in normal and pathophysiologic states.

  4. A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences.

    PubMed

    Iraola, Gregorio; Pérez, Ruben; Betancor, Laura; Marandino, Ana; Morsella, Claudia; Méndez, Alejandra; Paolicchi, Fernando; Piccirillo, Alessandra; Tomás, Gonzalo; Velilla, Alejandra; Calleros, Lucía

    2016-12-15

    Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains. We developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 10(2) and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability. The possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay

  5. Quantitation of Acidothermus cellulolyticus E1 endoglucanase and Thermomonospora fusca E{sub 3} exoglucanase using enzyme-linked immunosorbent assay (ELISA)

    SciTech Connect

    Nieves, R.A.; Chou, Y.C.; Himmel, M.E.

    1995-12-31

    Two distinct quantitative indirect ELISAs were developed to determine the concentration of recombinant cellulose enzymes in culture filtrates. A monoclonal antibody (E1P7) was used as the primary antibody in developing an ELISA specific for Acidothermus cellulolyticus E1 endoglucanase. Likewise, a polyclonal rabbit serum (Ab684) was used to develop an ELISA specific for Thermomonospora fusca E{sub 3} exoglucanase. Dose-response curves indicated a dynamic range for both assays between 0.01 and 0.08 {mu}g/mL (1-8 ng/assay) when purified enzymes were used as standards. These assays have been used to estimate concentrations of secreted recombinant E1 and/or E{sub 3} in culture supernatants of Streptomyces lividans strain TK24 in which the corresponding genes have been cloned and expressed.

  6. Developing a quantitative in vivo tissue reconstitution assay to assess the relative potency of candidate populations of mouse oesophageal epithelial cells.

    PubMed

    Croagh, Daniel; Redvers, Rick; Phillips, Wayne A; Kaur, Pritinder

    2012-01-01

    Proliferation in mouse oesophageal epithelial cells is confined to the basal layer of the epithelium. Within this population, it is possible to discriminate different sub-populations using a combination of cell kinetic studies and functional assays. In particular, it is possible to distinguish basal epithelial cells, which are post-mitotic and destined to leave the basal layer and differentiate compared with those cells that remain in the cycling pool. Within the cycling basal population, there appears to be a hierarchy with respect to the rate of cell turnover which may reflect a hierarchy of "stemness", although it has not been possible to demonstrate functional differences between these populations using current in vivo tissue reconstitution assays. The aim of this chapter is to describe the development of a quantitative in vivo tissue reconstitution assay to assess the potency of candidate stem cell populations within the mouse oesophageal epithelium.

  7. Quantitation of IgE and carcinoembryonic antigen (CEA) by optical beam deflection (OBD) measurement of dot-immunobinding assay patterns visualized by an ELISA technique.

    PubMed

    Matsuzawa, S; Kimura, H; Tu, C Y; Kitamori, T; Sawada, T

    1993-05-05

    Dot-immunobinding assays of IgE and CEA were performed by a conventional dot-ELISA technique with diaminobenzidine staining, and the quantitative results were compared by densitometry and a new, spectroscopic, optical beam deflection (OBD) method using the same membrane. It was possible with the OBD method to detect quantities of these substances at least ten times smaller than with densitometry. Better intra-assay reproducibility for IgE and CEA measurements was obtained by the OBD method. The measurable ranges of the OBD method was broader than that of densitometry, because dark bands caused OBD in proportion to their color densities. When the dot-immunobinding assay with OBD measurement for CEA was also compared with a microtube ELISA using biotin-avidin conjugates, the sensitivities and reproducibilities of the two methods were found to be similar, with a correlation coefficient of 0.991.

  8. Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.

    PubMed

    Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

    2009-08-01

    An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.

  9. Evaluation and subsequent optimizations of the quantitative AmpliSens Florocenosis/Bacterial vaginosis-FRT multiplex real-time PCR assay for diagnosis of bacterial vaginosis.

    PubMed

    Rumyantseva, Tatiana; Shipitsyna, Elena; Guschin, Alexander; Unemo, Magnus

    2016-12-01

    Traditional microscopy-based methods for diagnosis of bacterial vaginosis (BV) are underutilized in many settings, and molecular techniques may provide opportunities for rapid, objective, and accurate BV diagnosis. This study evaluated the quantitative AmpliSens Florocenosis/Bacterial vaginosis-FRT multiplex real-time PCR (Florocenosis-BV) assay. Vaginal samples from a previous study including unselected female subjects (n = 163) and using Amsel criteria and 454 pyrosequencing for BV diagnosis were examined with the Florocenosis-BV test and additionally tested for the presence and quantity of Gardnerella vaginalis clades 3 and 4. The Florocenosis-BV assay demonstrated 100% and 98% sensitivity compared with the Amsel criteria and 454 pyrosequencing, respectively, with 91% specificity. The modified Florocenosis-BV assay (detecting also G. vaginalis clades 3 and 4) resulted in 100% sensitivity vs the Amsel criteria and 454 pyrosequencing with specificity of 86% and 88%, respectively. Further optimizations of thresholds for the quantitative parameters used in the kit resulted in 99-100% accuracy vs Amsel criteria and 454 pyrosequencing for selected parameters. The Florocenosis-BV assay is an objective, accurate, sensitive, and specific method for BV diagnosis; however, the performance of the test can be further improved with some minor optimizations. © 2016 APMIS. Published by John Wiley & Sons Ltd.

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

    PubMed

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

    2016-11-01

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

  11. Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load

    PubMed Central

    Armas Cayarga, Anny; Perea Hernández, Yenitse; González González, Yaimé J.; Dueñas Carrera, Santiago; González Pérez, Idania; Robaina Álvarez, René

    2011-01-01

    Human immunodeficiency virus type-1 (HIV-1) viral load is useful for monitoring disease progression in HIV-infected individuals. We generated RNA standards of HIV-1 and internal control (IC) by in vitro transcription and evaluated its performance in a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. HIV-1 and IC standards were obtained at high RNA concentrations, without DNA contamination. When these transcripts were included as standards in a qRT-PCR assay, it was obtained a good accuracy (±0.5 log10 unit of the expected results) in the quantification of the HIV-1 RNA international standard and controls. The lower limit detection achieved using these standards was 511.0 IU/mL. A high correlation (r = 0.925) was obtained between the in-house qRT-PCR assay and the NucliSens easyQ HIV-1 test (bioMerieux) for HIV-1 RNA quantitation with clinical samples (N = 14). HIV-1 and IC RNA transcripts, generated in this study, proved to be useful as standards in an in-house qRT-PCR assay for determination of HIV-1 viral load. PMID:21766036

  12. Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy.

    PubMed

    Bergallo, Massimiliano; Galliano, Ilaria; Montanari, Paola; Brusin, Martina Rosa; Finotti, Serena; Paderi, Giulia; Gabiano, Clara

    2017-04-01

    Gastroenteritis is a common disease in children. It is characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis, but it causes milder illness than do rotavirus and norovirus. There is high variability in the analytical performance of quantitative PCR-based assays among clinical laboratories. This study developed a reverse transcription real-time PCR method to detect SaV in fecal specimens collected from children under 5-years-old with acute gastroenteritis. Of 137 episodes of acute gastroenteritis, 15 (10.9%) were associated with SaV genomic detection, with a median viral load of 6.6(log10) ± 7.1(log10) genomes/mg fecal specimens. There was a significant difference in detection rate between males and females (9.48% (13/15) vs. 1.46% (2/15), p = 0.0232). Among the 15 SaV-positive cases, 6 were also positive for rotavirus. Viral RNA recovery rate ranged from 46% to 77% in the manual RNAzol protocol and from 31% to 90% in the automated Maxwell protocol. We also studied whether human genomic DNA influences the sensitivity of the assay: its presence caused a decrease in PCR sensitivity. The development of a laboratory-designed real-time PCR TaqMan assay for quantitative detection of SaV and the optimization and standardization of this assay, using stools of children with acute gastroenteritis, are described.

  13. Probe-based quantitative PCR assay for detecting constitutional and somatic deletions in the NF1 gene: application to genetic testing and tumor analysis.

    PubMed

    Terribas, Ernest; Garcia-Linares, Carles; Lázaro, Conxi; Serra, Eduard

    2013-06-01

    About 5% of patients with neurofibromatosis type 1 (NF1) bear constitutional microdeletions that encompass NF1 (neurofibromin 1) and neighboring genes. These patients are characterized by the development of a high number of dermal neurofibromas (dNFs), mental retardation, and an increased risk of developing a malignant peripheral nerve sheath tumor (MPNST). Additionally, 10% of somatic second hits identified in dNFs are caused by deletions involving the NF1 gene. To detect constitutional and somatic deletions, we developed a probe-based quantitative PCR (qPCR) assay for interrogating the copy number status of 11 loci distributed along a 2.8-Mb region around the NF1 gene. We developed the qPCR assay with Universal ProbeLibrary technology (Roche) and designed a Microsoft Excel spreadsheet to analyze qPCR data for copy number calculations. The assay fulfilled the essential aspects of the MIQE (minimum information for publication of quantitative real-time PCR experiments) guidelines and used the qBase relative quantification framework for calculations. The assay was validated with a set of DNA samples with known constitutional or somatic NF1 deletions. The assay showed high diagnostic sensitivity and specificity and distinguished between Type-1, Type-2, and atypical constitutional microdeletions in 14 different samples. It also identified 16 different somatic deletions in dNFs. These results were confirmed by multiplex ligation-dependent probe amplification. The qPCR assay provides a methodology for detecting constitutional NF1 microdeletions that could be incorporated as an additional technique in a genetic-testing setting. It also permits the identification of somatic NF1 deletions in tissues with a high percentage of cells bearing 2 copies of the NF1 gene. © 2013 American Association for Clinical Chemistry.

  14. A quantitative real-time reverse transcription PCR (qRT-PCR) assay to detect genome segment 9 of all 26 bluetongue virus serotypes.

    PubMed

    Maan, Narender S; Maan, Sushila; Belaganahalli, Manjunatha; Pullinger, Gillian; Montes, Antonio J Arenas; Gasparini, Marcela R; Guimera, Marc; Nomikou, Kyriaki; Mertens, Peter P C

    2015-03-01

    Bluetongue (BT) is an arboviral disease, which can often be fatal in naïve sheep and white tailed deer, but is usually less severe, or unapparent in other ruminants. Twenty-six bluetongue virus (BTV) serotypes have been recognised so far, two of which (BTV-25 and BTV-26) were recently identified by phylogenetic comparisons of genome-segment/outer-capsid protein VP2 (subsequently confirmed by serological 'virus-neutralisation' assays). Rapid, sensitive, reliable and quantitative diagnostic-assays for detection and identification of BTV represent important components of effective surveillance and control strategies. The BTV genome comprises 10 linear segments of dsRNA. We describe a 'TaqMan' fluorescence-probe based quantitative real-time RT-PCR assay, targeting the highly conserved genome-segment-9 (encoding the viral-helicase 'VP6' and NS4). The assay detected Seg-9 from isolates of all 26 BTV types, as well as from clinical samples derived from BTV-6w and BTV-8w outbreaks (in Europe), BTV-25 from Switzerland, BTV-26 from Kuwait, BTV-1w, BTV-4w and BTV-8w from Spain, BTV-4w, BTV-8, BTV-10 and BTV-16 from Brazil. Assay efficiency was evaluated with RNA derived from the reference strain of BTV-1w [RSArrrr/01] and was 99.6%, detecting down to 4 copies per reaction. Samples from uninfected insect or mammalian cell-cultures, hosts-species (uninfected sheep blood) or vector-insects, all gave negative results. The assay failed to detect RNA from heterologous but related Orbivirus species (including the nine African horse sickness virus [AHSV] and seven epizootic haemorrhagic disease virus [EHDV] serotypes). Copyright © 2014. Published by Elsevier B.V.

  15. Accurate, quantitative assays for the hydrolysis of soluble type I, II, and III /sup 3/H-acetylated collagens by bacterial and tissue collagenases

    SciTech Connect

    Mallya, S.K.; Mookhtiar, K.A.; Van Wart, H.E.

    1986-11-01

    Accurate and quantitative assays for the hydrolysis of soluble /sup 3/H-acetylated rat tendon type I, bovine cartilage type II, and human amnion type III collagens by both bacterial and tissue collagenases have been developed. The assays are carried out at any temperature in the 1-30/sup 0/C range in a single reaction tube and the progress of the reaction is monitored by withdrawing aliquots as a function of time, quenching with 1,10-phenanthroline, and quantitation of the concentration of hydrolysis fragments. The latter is achieved by selective denaturation of these fragments by incubation under conditions described in the previous paper of this issue. The assays give percentages of hydrolysis of all three collagen types by neutrophil collagenase that agree well with the results of gel electrophoresis experiments. The initial rates of hydrolysis of all three collagens are proportional to the concentration of both neutrophil or Clostridial collagenases over a 10-fold range of enzyme concentrations. All three assays can be carried out at collagen concentrations that range from 0.06 to 2 mg/ml and give linear double reciprocal plots for both tissue and bacterial collagenases that can be used to evaluate the kinetic parameters K/sub m/ and k/sub cat/ or V/sub max/. The assay developed for the hydrolysis of rat type I collagen by neutrophil collagenase is shown to be more sensitive by at least one order of magnitude than comparable assays that use rat type I collagen fibrils or gels as substrate.

  16. Detection of the pandemic H1N1/2009 influenza A virus by a highly sensitive quantitative real-time reverse-transcription polymerase chain reaction assay.

    PubMed

    Yang, Zhu; Mao, Guoliang; Liu, Yujun; Chen, Yuan-Chuan; Liu, Chengjing; Luo, Jun; Li, Xihan; Zen, Ke; Pang, Yanjun; Wu, Jianguo; Liu, Fenyong

    2013-02-01

    A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.

  17. Escherichia coli and Enterococcus spp. in rainwater tank samples: comparison of culture-based methods and 23S rRNA gene quantitative PCR assays.

    PubMed

    Ahmed, W; Richardson, K; Sidhu, J P S; Toze, S

    2012-10-16

    In this study, culture-based methods and quantitative PCR (qPCR) assays were compared with each other for the measurement of Escherichi