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Sample records for assaying quantitative

  1. A quantitative comet infection assay for influenza virus

    OpenAIRE

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

    2011-01-01

    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 in...

  2. Rapid quantitative assay for chloramphenicol acetyltransferase

    International Nuclear Information System (INIS)

    Measuring the expression of exogenous genetic material in mammalian cells is commonly done by fusing the DNA of interest to a gene encoding an easily-detected enzyme. Chloramphenicol acetyltransferase(CAT) is a convenient marker because it is not normally found in eukaryotes. CAT activity has usually been detected using a thin-layer chromatographic separation followed by autoradiography. An organic solvent extraction-based method for CAT detection has also been described, as well as a procedure utilizing HPLC analysis. Building on the extraction technique, they developed a rapid sensitive kinetic method for measuring CAT activity in cell homogenates. The method exploits the differential organic solubility of the substrate ([3H] or [14C]acetyl CoA) and the product (labeled acetylchloramphenicol). The assay is a simple one-vial, two-phase procedure and requires no tedious manipulations after the initial setup. Briefly, a 0.25 ml reaction with 100mM Tris-HCL, 1mM chloramphenicol, 0.1mM [14C]acetyl CoA and variable amounts of cell homogenate is pipetted into a miniscintillation vial, overlaid with 5 ml of a water-immiscible fluor, and incubated at 370C. At suitable intervals the vial is counted and the CAT level is quantitatively determined as the rate of increase in counts/min of the labeled product as it diffuses into the fluor phase, compared to a standard curve. When used to measure CAT in transfected Balb 3T3 cells the method correlated well with the other techniques

  3. A Quantitative Assay for Aggrecanase Activity

    OpenAIRE

    Will, Horst; Dettloff, Matthias; Bendzkô, Peter; Sveshnikov, Peter

    2005-01-01

    Aggrecanase activities of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteinases were measured with a recombinant aggrecan fragment and two monoclonal antibodies. Recombinant human aggrecan interglobular domain was first incubated in the presence of ADAMTS enzymes. The aggrecan peptide with the N-terminal sequence ARGSVIL released upon hydrolysis was then quantified in an enzyme-linked immunosorbent assay (ELISA) with an anti-neoepitope antibody specific for the N...

  4. Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    OpenAIRE

    Eriksson, Jonas; 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.

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

    Science.gov (United States)

    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.

  6. [Clinical evaluation of a novel HBsAg quantitative assay].

    Science.gov (United States)

    Takagi, Kazumi; Tanaka, Yasuhito; Naganuma, Hatsue; Hiramatsu, Kumiko; Iida, Takayasu; Takasaka, Yoshimitsu; Mizokami, Masashi

    2007-07-01

    The clinical implication of the hepatitis B surface antigen (HBsAg) concentrations in HBV-infected individuals remains unclear. The aim of this study was to evaluate a novel fully automated Chemiluminescence Enzyme Immunoassay (Sysmex HBsAg quantitative assay) by comparative measurements of the reference serum samples versus two independent commercial assays (Lumipulse f or Architect HBsAg QT). Furthermore, clinical usefulness was assessed for monitoring of the serum HBsAg levels during antiviral therapy. A dilution test using 5 reference-serum samples showed linear correlation curve in range from 0.03 to 2,360 IU/ml. The HBsAg was measured in total of 400 serum samples and 99.8% had consistent results between Sysmex and Lumipulse f. Additionally, a positive linear correlation was observed between Sysmex and Architect. To compare the Architect and Sysmex, both methods were applied to quantify the HBsAg in serum samples with different HBV genotypes/subgenotypes, as well as in serum contained HBV vaccine escape mutants (126S, 145R). Correlation between the methods was observed in results for escape mutants and common genotypes (A, B, C) in Japan. Observed during lamivudine therapy, an increase in HBsAg and HBV DNA concentrations preceded the aminotransferase (ALT) elevation associated with drug-resistant HBV variant emergence (breakthrough hepatitis). In conclusion, reliability of the Sysmex HBsAg quantitative assay was confirmed for all HBV genetic variants common in Japan. Monitoring of serum HBsAg concentrations in addition to HBV DNA quantification, is helpful in evaluation of the response to lamivudine treatment and diagnosis of the breakthrough hepatitis.

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

    OpenAIRE

    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 ...

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

    Science.gov (United States)

    Zhao, Yun; Xia, Qingyan; 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; Pvalue of ddPCR technology in the diagnosis of plant disease and quarantine applications. PMID:27427975

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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...

  11. Rapid Staphylococcus aureus agr Type Determination by a Novel Multiplex Real-Time Quantitative PCR Assay

    OpenAIRE

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-01-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information.

  12. Rapid Staphylococcus aureus agr type determination by a novel multiplex real-time quantitative PCR assay.

    Science.gov (United States)

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-05-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information. PMID:16672433

  13. Rapid Staphylococcus aureus agr Type Determination by a Novel Multiplex Real-Time Quantitative PCR Assay

    Science.gov (United States)

    Francois, Patrice; Koessler, Thibaud; Huyghe, Antoine; Harbarth, Stephan; Bento, Manuela; Lew, Daniel; Etienne, Jérôme; Pittet, Didier; Schrenzel, Jacques

    2006-01-01

    The accessory gene regulator (agr) is a crucial regulatory component of Staphylococcus aureus involved in the control of bacterial virulence factor expression. We developed a real-time multiplex quantitative PCR assay for the rapid determination of S. aureus agr type. This assay represents a rapid and affordable alternative to sequence-based strategies for assessing relevant epidemiological information. PMID:16672433

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. PMID:26356763

  16. Quantitative detection of RT activity by PERT assay: feasibility and limits to a standardized screening assay for human vaccines.

    Science.gov (United States)

    André, M; Morgeaux, S; Fuchs, F

    2000-06-01

    The detection of adventitious retroviruses has always been critical for assessing the safety concerns associated with viral vaccines. Assays for the enzymatic activity of reverse transcriptase (RT) are used as general methods for the detection of both known and unknown retroviruses. Several studies using newly-developed ultrasensitive PCR-based RT assays reported RT activity in viral vaccines grown in chicken cells. Here, we have assessed the performances of such a PCR-based RT assay--PERT assay--for the quantitative detection of RT activity in vaccines. Sensitivity, linearity and reproducibility of the method were studied on purified RT and viral vaccines treated to release RT from potentially contaminant retroviruses. The level of RT activity detected in chicken cell-derived vaccines was higher for live attenuated vaccines compared to inactivated ones. Contrary to other studies, RT activity was found in some mammalian cell-derived vaccines. AZT-TP sensitivity of RT activities detected in these vaccines and discrimination between retroviral and RT-like activities was further investigated. Feasibility and limits of PERT assay as a broad-spectrum retroviruses detection method in vaccines are discussed.

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

    Science.gov (United States)

    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.

  18. Imaging beads-retained prey assay for rapid and quantitative protein-protein interaction.

    Directory of Open Access Journals (Sweden)

    Yan Zhou

    Full Text Available Conventional Western blot based pull-down methods involve lengthy and laborious work and the results are generally not quantitative. Here, we report the imaging beads-retained prey (IBRP assay that is rapid and quantitative in studying protein-protein interactions. In this assay, the bait is immobilized onto beads and the prey is fused with a fluorescence protein. The assay takes advantage of the fluorescence of prey and directly quantifies the amount of prey binding to the immobilized bait under a microscope. We validated the assay using previously well studied interactions and found that the amount of prey retained on beads could have a relative linear relationship to both the inputs of bait and prey. IBRP assay provides a universal, fast, quantitative and economical method to study protein interactions and it could be developed to a medium- or high-throughput compatible method. With the availability of fluorescence tagged whole genome ORFs in several organisms, we predict IBRP assay should have wide applications.

  19. Quantitative comparison between microfluidic and microtiter plate formats for cell-based assays.

    Science.gov (United States)

    Yin, Huabing; Pattrick, Nicola; Zhang, Xunli; Klauke, Norbert; Cordingley, Hayley C; Haswell, Steven J; Cooper, Jonathan M

    2008-01-01

    In this paper, we compare a quantitative cell-based assay measuring the intracellular Ca2+ response to the agonist uridine 5'-triphosphate in Chinese hamster ovary cells, in both microfluidic and microtiter formats. The study demonstrates that, under appropriate hydrodynamic conditions, there is an excellent agreement between traditional well-plate assays and those obtained on-chip for both suspended immobilized cells and cultured adherent cells. We also demonstrate that the on-chip assay, using adherent cells, provides the possibility of faster screening protocols with the potential for resolving subcellular information about local Ca2+ flux.

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

    Science.gov (United States)

    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…

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

    Science.gov (United States)

    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.

  2. A DNA immunoprecipitation assay used in quantitative detection of in vitro DNA-protein complex binding.

    Science.gov (United States)

    Kim, Min Young; Chae, Ji Hyung; Oh, Chang-Ho; Kim, Chul Geun

    2013-10-15

    To begin gene transcription, several transcription factors must bind to specific DNA sequences to form a complex via DNA-protein interactions. We established an in vitro method for specific and sensitive analyses of DNA-protein interactions based on a DNA immunoprecipitation (DIP) method. We verified the accuracy and efficiency of the DIP assay in quantitatively measuring DNA-protein binding using transcription factor CP2c as a model. With our DIP assay, we could detect specific interactions within a DNA-CP2c complex, with reproducible and quantitative binding values. In addition, we were able to effectively measure the changes in DNA-CP2c binding by the addition of a small molecule, FQI1 (factor quinolinone inhibitor 1), previously identified as a specific inhibitor of this binding. To identify a new regulator of DNA-CP2c binding, we analyzed several CP2c binding peptides and found that only one class of peptide severely inhibits DNA-CP2c binding. These data show that our DIP assay is very useful in quantitatively detecting the binding dynamics of DNA-protein complex. Because DNA-protein interaction is very dynamic in different cellular environments, our assay can be applied to the detection of active transcription factors, including promoter occupancy in normal and disease conditions. Moreover, it may be used to develop a targeted regulator of specific DNA-protein interaction.

  3. Quantitative dynamic nuclear polarization‐NMR on blood plasma for assays of drug metabolism

    DEFF Research Database (Denmark)

    Lerche, Mathilde Hauge; Meier, Sebastian; Jensen, Pernille Rose;

    2011-01-01

    ‐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...... 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 13C‐carbamazepine and its pharmacologically active metabolite 13C‐carbamazepine‐10,11‐epoxide is 0.08 µg/mL in rabbit blood plasma analyzed by single......‐scan 13C 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...

  4. Quantitative immunobinding assay for calbindin D/sub 28k/ using nitrocellulose filters

    International Nuclear Information System (INIS)

    An immunobinding assay has been developed for the quantitative determination of vitamin D dependent calcium binding protein (calbindin D/sub 28k/) from rat kidney and cerebellum. The protein samples are applied to nitrocellulose membrane filters using a slot-blot apparatus. The remaining protein binding sites are blocked with bovine serum albumin and incubated sequentially with rat renal calbindin D (CaBP) antiserum (1:500 dilution) and 125I protein A (200,000 cpm/ml). After washing, the radioactivity bound to each sample is quantitated by cutting the nitrocellulose sheets and counting in a gamma counter. Different dilutions of purified rat renal CaBP result in standard curves which are linear over a 40 to 50 fold range. The sensitivity of the assay is such that 10 ng of calbindin D can be accurately quantitated. The assay is precise (intraassay variability 6%) and reproducible (interassay variability 11.9%). Using this assay, the concentration of rat renal CaBP was found to be 8.8 +/- 1.3 μg/mg protein and the concentration of rat cerebellar CaBP was 34.0 +/- 6.0 μg/mg protein. There was good agreement between the data in this assay and the data which the authors previously reported using radioimmunoassay. Some advantages of this assay are that it is fast, reproducible and large numbers of samples can be handled in parallel. The most considerable advantage is that it does not require iodination of the antigen

  5. Influence of hydrodynamic conditions on quantitative cellular assays in microfluidic systems.

    Science.gov (United States)

    Yin, Huabing; Zhang, Xunli; Pattrick, Nicola; Klauke, Norbert; Cordingley, Hayley C; Haswell, Stephen J; Cooper, Jonathan M

    2007-09-15

    This study demonstrates the importance of the hydrodynamic environment in microfluidic systems in quantitative cellular assays using live cells. Commonly applied flow conditions used in microfluidics were evaluated using the quantitative intracellular Ca2+ analysis of Chinese hamster ovary (CHO) cells as a model system. Above certain thresholds of shear stress, hydrodynamically induced intracellular Ca2+ fluxes were observed which mimic the responses induced by chemical stimuli, such as the agonist uridine 5'-triphosphate tris salt (UTP). This effect is of significance given the increasing application of microfluidic devices in high-throughput cellular analysis for biophysical applications and pharmacological screening.

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

    Science.gov (United States)

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-01

    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. PMID:27371921

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

    International Nuclear Information System (INIS)

    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

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

    Science.gov (United States)

    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. PMID:25904095

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

    Energy Technology Data Exchange (ETDEWEB)

    Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Gagnon, David [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Gjoerup, Ole [Molecular Oncology Research Institute, Tufts Medical Center, Boston, MA 02111 (United States); Archambault, Jacques [Institut de Recherches Cliniques de Montreal (IRCM), 110 Pine Avenue West, Montreal, Quebec, Canada H2W 1R7 (Canada); Department of Biochemistry and Molecular Medicine, Université de Montréal, Montréal, Quebec (Canada); Bullock, Peter A., E-mail: Peter.Bullock@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    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.

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

    OpenAIRE

    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 internalize...

  11. Multicenter Evaluation of the Elecsys Hepatitis B Surface Antigen Quantitative Assay

    Science.gov (United States)

    Zacher, B. J.; Moriconi, F.; Bowden, S.; Hammond, R.; Louisirirotchanakul, S.; Phisalprapa, P.; Tanwandee, T.; Wursthorn, K.; Brunetto, M. R.; Wedemeyer, H.; Bonino, F.

    2011-01-01

    The Elecsys hepatitis B surface antigen (HBsAg) II quantitative assay is a new quantitative electrochemiluminescence immunoassay which uses onboard dilution and a simple algorithm to determine HBsAg levels expressed in international units (IU)/ml (standardized against the World Health Organization [WHO] Second International Standard). This study evaluated its performance using routine serum samples from a wide range of HBsAg carriers and patients with chronic hepatitis B (CHB). HBsAg levels were measured in serum samples collected independently by five centers in Europe, Australia, and Asia. Serial dilution analyses were performed to assess the recommended dilution algorithm and determine the assay range free of hook effect. Assay precision was also established. Following assessment of serial dilutions (1:100 to 1:1,000,000) of the 611 samples analyzed, 70.0% and 85.6% of samples tested with analyzers incorporating 1:100 (Elecsys 2010 and cobas e 411) and 1:400 (Modular Analytics E170) onboard dilution, respectively, fell within the linear range of the assay, providing a final result on the first test. No high-dose hook effect was seen up to the maximum HBsAg serum level tested (870,000 IU/ml) using the dilution algorithm. HBsAg levels were reliably determined across all hepatitis B virus (HBV) genotypes, phases of HBV infection, and stages of disease tested. Precision was high across all analyzers (% coefficient of variation [CV], 1.4 to 9.6; HBsAg concentrations, 0.1 to 37,300 IU/ml). The Elecsys HBsAg II quantitative assay accurately and reliably quantifies HBsAg in routine clinical samples. Onboard dilution minimizes retesting and reduces the potential for error. PMID:21880853

  12. Multicenter evaluation of the Elecsys hepatitis B surface antigen quantitative assay.

    Science.gov (United States)

    Zacher, B J; Moriconi, F; Bowden, S; Hammond, R; Louisirirotchanakul, S; Phisalprapa, P; Tanwandee, T; Wursthorn, K; Brunetto, M R; Wedemeyer, H; Bonino, F

    2011-11-01

    The Elecsys hepatitis B surface antigen (HBsAg) II quantitative assay is a new quantitative electrochemiluminescence immunoassay which uses onboard dilution and a simple algorithm to determine HBsAg levels expressed in international units (IU)/ml (standardized against the World Health Organization [WHO] Second International Standard). This study evaluated its performance using routine serum samples from a wide range of HBsAg carriers and patients with chronic hepatitis B (CHB). HBsAg levels were measured in serum samples collected independently by five centers in Europe, Australia, and Asia. Serial dilution analyses were performed to assess the recommended dilution algorithm and determine the assay range free of hook effect. Assay precision was also established. Following assessment of serial dilutions (1:100 to 1:1,000,000) of the 611 samples analyzed, 70.0% and 85.6% of samples tested with analyzers incorporating 1:100 (Elecsys 2010 and cobas e 411) and 1:400 (Modular Analytics E170) onboard dilution, respectively, fell within the linear range of the assay, providing a final result on the first test. No high-dose hook effect was seen up to the maximum HBsAg serum level tested (870,000 IU/ml) using the dilution algorithm. HBsAg levels were reliably determined across all hepatitis B virus (HBV) genotypes, phases of HBV infection, and stages of disease tested. Precision was high across all analyzers (% coefficient of variation [CV], 1.4 to 9.6; HBsAg concentrations, 0.1 to 37,300 IU/ml). The Elecsys HBsAg II quantitative assay accurately and reliably quantifies HBsAg in routine clinical samples. Onboard dilution minimizes retesting and reduces the potential for error.

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

    Science.gov (United States)

    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.

  14. Use of coefficient of variation in assessing variability of quantitative assays.

    Science.gov (United States)

    Reed, George F; Lynn, Freyja; Meade, Bruce D

    2002-11-01

    We have derived the mathematical relationship between the coefficient of variation associated with repeated measurements from quantitative assays and the expected fraction of pairs of those measurements that differ by at least some given factor, i.e., the expected frequency of disparate results that are due to assay variability rather than true differences. Knowledge of this frequency helps determine what magnitudes of differences can be expected by chance alone when the particular coefficient of variation is in effect. This frequency is an operational index of variability in the sense that it indicates the probability of observing a particular disparity between two measurements under the assumption that they measure the same quantity. Thus the frequency or probability becomes the basis for assessing if an assay is sufficiently precise. This assessment also provides a standard for determining if two assay results for the same subject, separated by an intervention such as vaccination or infection, differ by more than expected from the variation of the assay, thus indicating an intervention effect. Data from an international collaborative study are used to illustrate the application of this proposed interpretation of the coefficient of variation, and they also provide support for the assumptions used in the mathematical derivation.

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

    Science.gov (United States)

    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. PMID:25348316

  16. Development of a quantitative bead capture assay for soluble IL-7 receptor alpha in human plasma.

    Directory of Open Access Journals (Sweden)

    Sylvie Faucher

    Full Text Available 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-7Ralpha (CD127 and common gamma (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.

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

    Directory of Open Access Journals (Sweden)

    Hanae Takatsuki

    Full Text Available 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 10(10/g brain (values varies 10(8.79-10.63/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.

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

    Science.gov (United States)

    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. PMID:26684676

  19. FungiQuant: A broad-coverage fungal quantitative real-time PCR assay

    Directory of Open Access Journals (Sweden)

    Liu Cindy M

    2012-11-01

    Full Text Available Abstract Background Fungal load quantification is a critical component of fungal community analyses. Limitation of current approaches for quantifying the fungal component in the human microbiome suggests the need for new broad-coverage techniques. Methods We analyzed 2,085 18S rRNA gene sequences from the SILVA database for assay design. We generated and quantified plasmid standards using a qPCR-based approach. We evaluated assay coverage against 4,968 sequences and performed assay validation following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE guidelines. Results We designed FungiQuant, a TaqMan® qPCR assay targeting a 351 bp region in the fungal 18S rRNA gene. Our in silico analysis showed that FungiQuant is a perfect sequence match to 90.0% of the 2,617 fungal species analyzed. We showed that FungiQuant’s is 100% sensitive and its amplification efficiencies ranged from 76.3% to 114.5%, with r2-values of >0.99 against the 69 fungal species tested. Additionally, FungiQuant inter- and intra-run coefficients of variance ranged from Conclusions FungiQuant has comprehensive coverage against diverse fungi and is a robust quantification and detection tool for delineating between true fungal detection and non-target human DNA.

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

    Science.gov (United States)

    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. PMID:27592286

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

    Science.gov (United States)

    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.

  2. Single-tube fluorescent product-enhanced reverse transcriptase assay with Ampliwax (STF-PERT) for retrovirus quantitation.

    Science.gov (United States)

    Sears, Johnna F; Khan, Arifa S

    2003-03-01

    A TaqMan fluorescent probe-based product enhanced reverse transcriptase (RT) assay is described in which the RT and polymerase chain reaction (PCR) steps are set-up in a single tube, in two compartments separated by Ampliwax (designated as single-tube fluorescent product-enhanced reverse transcriptase assay (STF-PERT)). This simplification of the two-step method resulted in increased assay reproducibility and handling efficiency while maintaining the sensitivity of the PERT assay (PERT assay can be used to quantitate low amounts of retrovirus in clinical and research materials and to evaluate retrovirus contamination in cell substrates and biological products in human use.

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

    Science.gov (United States)

    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...

  4. Quantitative Real-Time PCR Assay for QPX (Thraustochytriidae), a Parasite of the Hard Clam (Mercenaria mercenaria)▿

    OpenAIRE

    Liu, Qianqian; Allam, Bassem; Collier, Jackie L.

    2009-01-01

    We developed a real-time quantitative PCR (qPCR) assay targeting the rRNA internal transcribed spacer region of the hard clam pathogen QPX. The qPCR assay was more sensitive than was histology in detecting clams with light QPX infections. QPX was detected in 4 of 43 sediment samples but in none of 40 seawater samples.

  5. A lateral flow assay for quantitative detection of amplified HIV-1 RNA.

    Directory of Open Access Journals (Sweden)

    Brittany A Rohrman

    Full Text Available Although the accessibility of HIV treatment in developing nations has increased dramatically over the past decade, viral load testing to monitor the response of patients receiving therapy is often unavailable. Existing viral load technologies are often too expensive or resource-intensive for poor settings, and there is no appropriate HIV viral load test currently available at the point-of-care in low resource settings. Here, we present a lateral flow assay that employs gold nanoparticle probes and gold enhancement solution to detect amplified HIV RNA quantitatively. Preliminary results show that, when coupled with nucleic acid sequence based amplification (NASBA, this assay can detect concentrations of HIV RNA that match the clinically relevant range of viral loads found in HIV patients. The lateral flow test is inexpensive, simple and rapid to perform, and requires few resources. Our results suggest that the lateral flow assay may be integrated with amplification and sample preparation technologies to serve as an HIV viral load test for low-resource settings.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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. PMID:26087169

  9. Virological diagnosis of herpes simplex virus 1 esophagitis by quantitative real-time PCR assay.

    Science.gov (United States)

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

    2012-03-01

    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 × 10(6) ± 1.1 × 10(8)) than in histopathologically negative herpetic esophagitis (median = 3.1 × 10(3) ± 6.2 × 10(3)) (P = 0.0009). Moreover, a receiver operating characteristics analysis revealed that a viral load threshold greater than 2.5 × 10(4) 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

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

    Directory of Open Access Journals (Sweden)

    Zhang PF

    2015-09-01

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

  11. Performance evaluation of new automated hepatitis B viral markers in the clinical laboratory: two quantitative hepatitis B surface antigen assays and an HBV core-related antigen assay.

    Science.gov (United States)

    Park, Yongjung; Hong, Duck Jin; Shin, Saeam; Cho, Yonggeun; Kim, Hyon-Suk

    2012-05-01

    We evaluated quantitative hepatitis B surface antigen (qHBsAg) assays and a hepatitis B virus (HBV) core-related antigen (HBcrAg) assay. A total of 529 serum samples from patients with hepatitis B were tested. HBsAg levels were determined by using the Elecsys (Roche Diagnostics, Indianapolis, IN) and Architect (Abbott Laboratories, Abbott Park, IL) qHBsAg assays. HBcrAg was measured by using Lumipulse HBcrAg assay (Fujirebio, Tokyo, Japan). Serum aminotransferases and HBV DNA were respectively quantified by using the Hitachi 7600 analyzer (Hitachi High-Technologies, Tokyo, Japan) and the Cobas AmpliPrep/Cobas TaqMan test (Roche). Precision of the qHBsAg and HBcrAg assays was assessed, and linearity of the qHBsAg assays was verified. All assays showed good precision performance with coefficients of variation between 4.5% and 5.3% except for some levels. Both qHBsAg assays showed linearity from 0.1 to 12,000.0 IU/mL and correlated well (r = 0.9934). HBsAg levels correlated with HBV DNA (r = 0.3373) and with HBcrAg (r = 0.5164), and HBcrAg also correlated with HBV DNA (r = 0.5198; P HBcrAg assays.

  12. Quantitative immunobinding assay for vitamin D-dependent calcium-binding protein (calbindin-D28k) using nitrocellulose filters

    International Nuclear Information System (INIS)

    A sensitive dot immunobinding assay has been developed for the quantitative determination of vitamin D-dependent calcium-binding protein (calbindin-D28k; CaBP) in rat and human kidney and brain. Protein samples are spotted onto nitrocellulose sheets, fixed, and then rinsed with Tris-buffered saline. The remaining protein binding sites are blocked with bovine serum albumin, gelatin, or nonfat dry milk protein and the filters are then incubated sequentially with antiserum to calbindin-D28k (1:500 dilution) and 125I-protein A (200,000 cpm/ml). After washing, the radioactivity bound to each sample is quantitated by counting in a gamma counter. The sensitivity of the assay is such that 10 ng calbindin-D28k can be accurately quantitated. The highest levels of CaBP were detected in kidney (7.8 +/- 0.5 micrograms/mg protein) and cerebellum (22.1 +/- 1.4 micrograms/mg protein). Ten micrograms calmodulin, lactalbumin, or parvalbumin and 100 micrograms liver extract showed no reactivity in the assay. The assay is precise (intraassay variability, 4.0%) and reproducible (interassay variability, 8.8%). There was good agreement between the data in this assay and the data we obtained using radioimmunoassay (RIA). The assay has several advantages over the RIA. Iodination of pure antigen is not required and it is possible to detect membrane-bound and insoluble antigens using this assay. Also, the antiserum and 125I-protein A solutions can be saved and reused. This assay represents a major modification of the original immunobinding assays which used the less sensitive peroxidase stain. It is also an improvement over previous 125I immunobinding assays which were not quantitative but were used as antigen spot tests or which required iodination of the antibody

  13. Optimized semi-quantitative blot analysis in infection assays using the Stain-Free technology.

    Science.gov (United States)

    Zeitler, Anna F; Gerrer, Katrin H; Haas, Rainer; Jiménez-Soto, Luisa F

    2016-07-01

    Western blots are a commonly used method for protein detection and quantification in biological samples. Compensation of loading variations is achieved by housekeeping protein (HKP) normalization and/or total protein normalization (TPN). However, under infection conditions, HKP normalization, traditionally used in cell biology for quantification of western blots, can be problematic. Binding of microbes to target cells via specific receptors can induce signal transduction events resulting in drastic changes in the level of expression of HKPs. Additionally, samples collected after infection assays will include cellular and microbial proteins altering the analysis with TPN. Here we demonstrate under experimental infection conditions, how a reliable semi-quantitative analysis of proteins in western blots can be achieved using the Stain-Free technology. PMID:27150675

  14. LUCID: A Quantitative Assay of ESCRT-Mediated Cargo Sorting into Multivesicular Bodies.

    Science.gov (United States)

    Nickerson, Daniel P; Merz, Alexey J

    2015-12-01

    Endosomes are transportation nodes, mediating selective transport of soluble and transmembrane cargos to and from the Golgi apparatus, plasma membrane and lysosomes. As endosomes mature to become multivesicular bodies (MVBs), Endosomal Sorting Complexes Required for Transport (ESCRTs) selectively incorporate transmembrane cargos into vesicles that bud into the endosome lumen. Luminal vesicles and their cargoes are targeted for destruction when MVBs fuse with lysosomes. Common assays of endosomal luminal targeting, including fluorescence microscopy and monitoring of proteolytic cargo maturation, possess significant limitations. We present a quantitative assay system called LUCID (LUCiferase reporter of Intraluminal Deposition) that monitors exposure of chimeric luciferase-cargo reporters to cytosol. Luciferase-chimera signal increases when sorting to the endosome lumen is disrupted, and silencing of signal from the chimera depends upon luminal delivery of the reporter rather than proteolytic degradation. The system presents several advantages, including rapidity, microscale operation and a high degree of reproducibility that enables detection of subtle phenotypic differences. Luciferase reporters provide linear signal over an extremely broad dynamic range, allowing analysis of reporter traffic even at anemic levels of expression. Furthermore, LUCID reports transport kinetics when applied to inducible trafficking reporters.

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

    Science.gov (United States)

    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.

  16. A quantitative PCR (TaqMan assay for pathogenic Leptospira spp

    Directory of Open Access Journals (Sweden)

    Symonds Meegan L

    2002-07-01

    Full Text Available Abstract Background Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA and slide agglutination test (SAT, can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT. Methods The polymerase chain reaction (PCR has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples. Results and Conclusions The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.

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

    Science.gov (United States)

    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. PMID:25476288

  18. [Use of procalcitonine in intensive care units: comparison of semi quantitative PCT-Q Brahms assay with automated PCT-Kryptor assay].

    Science.gov (United States)

    Schuch, Géraldine; Duc-Marchand, Catherine; Venet, Cyrille; Mann, Hubert; Tixier, Anne; Bionda, Clara

    2011-01-01

    Procalcitonine (PCT) is recognized as a major and specific biomarker in diagnosis of bacterial infection. Used early in sepsis, it allows immediate administration of antibiotics and monitoring its effectiveness. Confronted on systemic inflammation response syndrom (SIRS), physicians must react quickly and effectively to evaluate bacterial infection and sepsis. The objective of this study was to compare analytical and clinical performances of semi-quantitative PCT-Q assay (Brahms) with quantitative and automated assay such on Kryptor (Brahms). Fifty blood samples of intensive care patients were compared. The analytical performance observed with PCT-Q assay is accurate: linear ratio kappa of 0.912 (95% CI 0.61, 0.97) and a good correlation between these techniques (p < 0.0001) (MedCalc software) were observed. Three discordances were observed and confirm the difficulties of reading for values close to 0.5 ng/mL. For these patients, PCT result showed its interest to discriminate local infection of a sepsis, to stop antibiotherapy with broad spectrum and to consolidate a therapeutic effectiveness in multi-visceral failure context. The semi-quantitative assay seems adapted for a fast and reliable evaluation of PCT in a general-purpose laboratory, not requiring neither dedicated analyzer, nor complex technicality but a control of the visual evaluation of results. It could be used for diagnosis of sepsis without monitoring precisely therapeutic follow-up.

  19. [Use of procalcitonine in intensive care units: comparison of semi quantitative PCT-Q Brahms assay with automated PCT-Kryptor assay].

    Science.gov (United States)

    Schuch, Géraldine; Duc-Marchand, Catherine; Venet, Cyrille; Mann, Hubert; Tixier, Anne; Bionda, Clara

    2011-01-01

    Procalcitonine (PCT) is recognized as a major and specific biomarker in diagnosis of bacterial infection. Used early in sepsis, it allows immediate administration of antibiotics and monitoring its effectiveness. Confronted on systemic inflammation response syndrom (SIRS), physicians must react quickly and effectively to evaluate bacterial infection and sepsis. The objective of this study was to compare analytical and clinical performances of semi-quantitative PCT-Q assay (Brahms) with quantitative and automated assay such on Kryptor (Brahms). Fifty blood samples of intensive care patients were compared. The analytical performance observed with PCT-Q assay is accurate: linear ratio kappa of 0.912 (95% CI 0.61, 0.97) and a good correlation between these techniques (p < 0.0001) (MedCalc software) were observed. Three discordances were observed and confirm the difficulties of reading for values close to 0.5 ng/mL. For these patients, PCT result showed its interest to discriminate local infection of a sepsis, to stop antibiotherapy with broad spectrum and to consolidate a therapeutic effectiveness in multi-visceral failure context. The semi-quantitative assay seems adapted for a fast and reliable evaluation of PCT in a general-purpose laboratory, not requiring neither dedicated analyzer, nor complex technicality but a control of the visual evaluation of results. It could be used for diagnosis of sepsis without monitoring precisely therapeutic follow-up. PMID:22123565

  20. A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden

    DEFF Research Database (Denmark)

    Kjær, L.; Riley, C.H.; Westman, M.;

    2012-01-01

    present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel...... tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well. © 2012...

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

    Science.gov (United States)

    Meitinger, Michael; Hartmann, Sandra; Schieberle, Peter

    2014-06-01

    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. PMID:24865106

  2. Quantitative Measurement of Serum Hepatitis B Surface Antigen Using an Immunoradiometric Assay in Chronic Hepatitis B

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Hyun Woo; Lee, Ho Young; Kim, Seog Gyun; Kim, Won; Jung, Wong Jin; Kang, Keon Wook; Chung, June Key; Lee, Myung Chul; Lee, Dong Soo [Seoul National Univ. Seoul (Korea, Republic of)

    2011-03-15

    Measurement of serum hepatitis B virus surface antigen (HBsAg) levels is important for the management of chronic hepatitis D 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 CMIA. Furthermors, serum HBsAg levels by IRMA were found to be highly correlated with those determined by CMIA (correlation coefficient R{sup 2=}0.838, P<0.001). Serum HBsAg level and serum HBV DNA copies were found to be linearly related by both methods (R{sup 2=}0.067, P=0.316 by IRMA, and R{sup 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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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...

  5. Bacteriophage P22 and Staphylococcus aureus Attenuation on Nonporous Fomites as Determined by Plate Assay and Quantitative PCR

    OpenAIRE

    Masago, Yoshifumi; Shibata, Tomoyuki; Rose, Joan B.

    2009-01-01

    Decay rates of bacteriophage P22 and Staphylococcus aureus on six types of common household inanimate surfaces were evaluated based on cultivation and quantitative PCR. A much higher level of inactivation was observed using the plate assay, suggesting that detection of the pathogen genome in samples from fomites does not necessarily imply a health risk to humans.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2013-09-01

    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.

  8. Validation of a primer optimisation matrix to improve the performance of reverse transcription – quantitative real-time PCR assays

    Directory of Open Access Journals (Sweden)

    Dobrovic Alexander

    2009-06-01

    Full Text Available Abstract Background The development of reverse transcription – quantitative real-time PCR (RT-qPCR platforms that can simultaneously measure the expression of multiple genes is dependent on robust assays that function under identical thermal cycling conditions. The use of a primer optimisation matrix to improve the performance of RT-qPCR assays is often recommended in technical bulletins and manuals. Despite this recommendation, a comprehensive introduction to and evaluation of this approach has been absent from the literature. Therefore, we investigated the impact of varying the primer concentration, leaving all the other reaction conditions unchanged, on a large number of RT-qPCR assays which in this case were designed to be monitored using hydrolysis probes from the Universal Probe Library (UPL library. Findings Optimal RT-qPCR conditions were determined for 60 newly designed assays. The calculated Cq (Quantification Cycle difference, non-specific amplification, and primer dimer formation for a given assay was often dependent on primer concentration. The chosen conditions were further optimised by testing two different probe concentrations. Varying the primer concentrations had a greater effect on the performance of a RT-qPCR assay than varying the probe concentrations. Conclusion Primer optimisation is important for improving the performance of RT-qPCR assays monitored by UPL probes. This approach would also be beneficial to the performance of other RT-qPCR assays such as those using other types of probes or fluorescent intercalating dyes.

  9. Real-time fluorescent quantitative PCR assay for measuring cytomegalovirus DNA load in patients after haematopoietic stem cell transplantation

    Institute of Scientific and Technical Information of China (English)

    FAN Jun; MA Wei-hang; YANG Mei-fang; XUE Han; GAO Hai-nü; LI Lan-juan

    2006-01-01

    @@ Cytomegalovirus (CMV) infection is a major and often deadly complication of haematopoietic stem cell (HSC) transplantation.1 Successful preemptive CMV therapy in transplant patients depends on the availability of sensitive, specific, and timely diagnostic tests for CMV infections.2 The pp65antigenemia assay has been used for this purposewith considerable success but has disadvantages of being time-consuming and labor-intensive.3 Recently,commercial quantitative polymerase chain reaction (PCR) methods based on TaqMan technique have become available and proven to be useful in the diagnosis of microbial infection as well as the determination of viral load.4 In this study, we developed a fluorescent-based quantitative real-time PCR (RT-FQ PCR) assay using TaqMan chemistry for rapid and quantitative detection of CMV DNA and assessed its clinic value for monitoring the reinfection of CMV in patients after HSC transplantion.

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

    Science.gov (United States)

    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. Development of a colorimetric assay for rapid quantitative measurement of clavulanic acid in microbial samples.

    Science.gov (United States)

    Dai, Xida; Xiang, Sihai; Li, Jia; Gao, Qiang; Yang, Keqian

    2012-02-01

    We developed a colorimetric assay to quantify clavulanic acid (CA) in culture broth of Streptomyces clavuligerus, to facilitate screening of a large number of S. clavuligerus mutants. The assay is based on a β-lactamase-catalyzed reaction, in which the yellow substrate nitrocefin (λ (max)=390 nm) is converted to a red product (λ (max)=486 nm). Since CA can irreversibly inhibit β-lactamase activity, the level of CA in a sample can be measured as a function of the A (390)/A (486) ratio in the assay mixture. The sensitivity and detection window of the assay were determined to be 50 μg L(-1) and 50 μg L(-1) to 10 mg L(-1), respectively. The reliability of the assay was confirmed by comparing assay results with those obtained by HPLC. The assay was used to screen a pool of 65 S. clavuligerus mutants and was reliable for identifying CA over-producing mutants. Therefore, the assay saves time and labor in large-scale mutant screening and evaluation tasks. The detection window and the reliability of this assay are markedly better than those of previously reported CA assays. This assay method is suitable for high throughput screening of microbial samples and allows direct visual observation of CA levels on agar plates.

  12. Novel wide-range quantitative nested real-time PCR assay for Mycobacterium tuberculosis DNA: development and methodology.

    Science.gov (United States)

    Takahashi, Teruyuki; Tamura, Masato; Asami, Yukihiro; Kitamura, Eiko; Saito, Kosuke; Suzuki, Tsukasa; Takahashi, Sachiko Nonaka; Matsumoto, Koichi; Sawada, Shigemasa; Yokoyama, Eise; Takasu, Toshiaki

    2008-05-01

    Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay for Mycobacterium tuberculosis DNA in order to rapidly diagnose tuberculous meningitis. This technique combined the high sensitivity of nested PCR with the accurate quantification of real-time PCR. In this study, we attempted to improve the original QNRT-PCR assay and newly developed the wide-range QNRT-PCR (WR-QNRT-PCR) assay, which is more accurate and has a wider detection range. For use as an internal-control "calibrator" to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. It had artificial random nucleotides in five regions annealing specific primers and probes. The NM-plasmid demonstrated statistically uniform amplifications (F = 1.086, P = 0.774) against a range (1 to 10(5)) of copy numbers of mimic M. tuberculosis DNA and was regarded as appropriate for use as a new internal control in the WR-QNRT-PSR assay. In addition, by the optimization of assay conditions in WR-QNRT-PCR, two-step amplification of target DNA was completely consistent with the standard curve of this assay. Due to the development of the NM-plasmid as the new internal control, significantly improved quantitative accuracy and a wider detection range were realized with the WR-QNRT-PCR assay. In the next study, we will try to use this novel assay method with actual clinical samples and examine its clinical usefulness.

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

    Science.gov (United States)

    Dysthe, Joseph C; 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.

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

    Science.gov (United States)

    Dysthe, Joseph C; 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

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

    Science.gov (United States)

    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

  16. A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia

    Institute of Scientific and Technical Information of China (English)

    Jason R. Schwartz; Purvaba J. Sarvaiya; Lily E. Leiva; Maria C. Velez; Tammuella C. Singleton; Lolie C. Yu; Wayne V. Vedeckis

    2012-01-01

    Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells.However,not all leukemia cells are sensitive to GC,and no assay to stratify patients is available.In the GC-sensitive T-cell ALL cell line CEM-C7,auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response.This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations.The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay.There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples,but not for a probe set that detects a specific,low abundance GR transcript (exon 1A3).Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and,with further development,may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients.

  17. Quantitative analysis of G-protein-coupled receptor internalization using DnaE intein-based assay.

    Science.gov (United States)

    Lu, Bin; Chen, Linjie; Zhang, Yaping; Shi, Ying; Zhou, Naiming

    2016-01-01

    G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, are involved in many physiological processes. They represent highly important therapeutic targets for drug discovery. Currently, there are numerous cell-based assays developed for the pharmacological profiling of GPCRs and the identification of novel agonists and antagonists. However, the development of new, faster, easier, and more cost-effective approaches to detect GPCR activity remains highly desirable. β-arrestin-dependent internalization has been demonstrated to be a common mechanism for most GPCRs. Here we describe a novel assay for quantitative analysis of GPCR internalization based on DnaE intein-mediated reconstitution of fragmented Renilla luciferase or Firefly luciferase when activated GPCRs interact with β-arrestin2 or Rab5. Further validation, using functionally divergent GPCRs, showed that EC50 values obtained for the known agonists and antagonists were in close agreement with the results of previous reports. This suggests that this assay is sensitive enough to permit quantification of GPCR internalization. Compared with conventional assays, this novel assay system is cost-effective, rapid, and easy to manipulate. These advantages may allow this assay to be used universally as a functional cell-based system for GPCR characterization and in the screening process of drug discovery. PMID:26928549

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

    Science.gov (United States)

    de Gier, Camilla; Pickering, Janessa L; Richmond, Peter C; Thornton, Ruth B; Kirkham, Lea-Ann S

    2016-09-01

    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).

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

    Directory of Open Access Journals (Sweden)

    Cameron R Turner

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  2. A new approach for quantitative analysis of L-phenylalanine using a novel semi-sandwich immunometric assay.

    Science.gov (United States)

    Kubota, Kazuyuki; Mizukoshi, Toshimi; Miyano, Hiroshi

    2013-10-01

    Here, we describe a novel method for L-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against L-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of L-phenylalanine were modified by "N-Fmoc-L-cysteine" (FC) residues and the derivative (FC-Phe) was used as a hapten. Immunization of mice with bovine serum albumin/FC-Phe conjugate successfully yielded specific monoclonal anti-FC-Phe antibodies. Second, a new derivatization reagent, "biotin linker conjugate of FC-Phe N-succinimidyl ester" (FC(Biotin)-NHS), was synthesized to convert L-phenylalanine to FC-(Biotin)-Phe as a hapten structure. The biotin moiety linked to the thiol group of cysteine formed a second binding site for streptavidin/horseradish peroxidase (HRP) conjugates for optical detection. Third, a new semi-sandwich-type immunometric assay was established using pre-derivatized L-phenylalanine, the monoclonal anti-FC-Phe antibody, and streptavidin/HRP conjugate (without second antibody). Using the new "semi-sandwich" immunometric assay system, a detection limit of 35 nM (60 amol per analysis) and a detection range of 0.1-20 μM were attained using a standard L-phenylalanine solution. Rat plasma samples were analyzed to test reliability. Intra-day assay precision was within 6% of the coefficient of variation; inter-day variation was 0.1%. The recovery rates were from 92.4 to 123.7%. This is the first report of the quantitative determination of L-phenylalanine using a reliable semi-sandwich immunometric assay approach and will be applicable to the quantitative determination of other amino acids.

  3. Quantitation of HIV-1 DNA with a sensitive TaqMan assay that has broad subtype specificity.

    Science.gov (United States)

    van der Sluis, Renée M; van Montfort, Thijs; Centlivre, Mireille; Schopman, Nick C T; Cornelissen, Marion; Sanders, Rogier W; Berkhout, Ben; Jeeninga, Rienk E; Paxton, William A; Pollakis, Georgios

    2013-01-01

    The increasing diversity of HIV-1 isolates makes virus quantitation challenging, especially when diverse isolates co-circulate in a geographical area. Measuring the HIV-1 DNA levels in cells has become a valuable practical tool for fundamental and clinical research. A quantitative HIV-1 DNA assay was developed based on TaqMan(®) technology. Primers that target the highly conserved LTR region were designed to detect a broad array of HIV-1 variants, including viral isolates from many subtypes, with high sensitivity. Introduction of a pre-amplification step prior to the TaqMan(®) reaction allowed the specific amplification of fully reverse transcribed viral DNA. Execution of the pre-amplification step with a second primer set enables for the exclusive quantitation of the 2-LTR circular HIV-1 DNA form. PMID:23059551

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

    Science.gov (United States)

    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.

  5. Colorimetric and fluorimetric assays to quantitate micromolar concentrations of transition metals.

    Science.gov (United States)

    McCall, K A; Fierke, C A

    2000-09-10

    Transition metal ions, although maintained at low concentrations, play diverse important roles in many biological processes. Two assays useful for the rapid quantification of a range of first-row transition metal ions have been developed. The colorimetric assay extends the 4-(2-pyridylazo)resorcinol assay of Hunt et al. (J. Biol. Chem. 255, 14793 (1984)) to measure nanomole quantities of Co(2+), Ni(2+), and Cu(2+) as well as Zn(2+). The fluorimetric assay takes advantage of the coordination of a number of metal ions (Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+)) by Fura-2 and can also be used to measure nanomole quantities of these ions. The assays developed here have the advantage of not requiring the extensive sample preparation necessary for other methodologies, such as atomic absorption spectroscopy and inductively coupled plasma emission spectroscopy (ICPES), while being comparable in accuracy to the detection limits of ICPES for the first-row transition metal ions. To demonstrate the effectiveness of these assays, we determined the affinity of carbonic anhydrase II (CA II), a prototypical zinc enzyme, for Ni(2+) and Cd(2+). These data indicate that CA II binds transition metals with high affinity and is much more selective for Zn(2+) over Ni(2+) or Cd(2+) than most small-molecule chelators or other metalloenzymes. PMID:10964414

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

    International Nuclear Information System (INIS)

    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

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

    Science.gov (United States)

    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

  8. The quantitation of human growth hormone by a radioreceptor assay using an established human cell line

    International Nuclear Information System (INIS)

    Membrane receptors on cultured human lymphocytes (IM-9) have been shown to bind human growth hormone (hGH) in a specific manner. The aim of the present study was to develop an in vitro assay of hGH based on this binding. The binding of [125I]hGH was studied as a function of time, temperature, cell density, tracer concentration and the concentration of unlabelled hGH and other related hormones. Also, the dissociation of bound hGH and the chemical stability of hGH in the incubation medium were studied. From these studies, the conditions for an appropriate radioreceptor assay were determined. Briefly, 1.5-3.0 x 107 cells ml-1 were incubated with 5-20 x 10-12 M [125I]hGH and three different concentrations of unlabelled hGH chosen from the linear part of the [125I]hGH displacement curve. The results were analyzed according to general pharmacopoeial principles. The mean values for growth hormone activity tested by radioreceptor assay were within the fiducial limits (P = 0.05) of the corresponding activity determined by the hypophysectomized rat body-weight gain assay. The in vitro assay was found to be more precise and less resource demanding than the in vivo bioassay of hGH. It is concluded that the in vitro bioassay described here is well suited as a screening method for potency determination of hGH preparations. (author)

  9. Rapid molecular assays for specific detection and quantitation of Loa loa microfilaremia.

    Directory of Open Access Journals (Sweden)

    Doran L Fink

    2011-08-01

    Full Text Available 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.

  10. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Rønne, E; Behrendt, N; Ploug, M;

    1994-01-01

    variant of uPAR, suPAR, has been constructed by recombinant technique and the protein content of a purified suPAR standard preparation was determined by amino acid composition analysis. The sensitivity of the assay (0.6 ng uPAR/ml) is strong enough to measure uPAR in extracts of cultured cells and cancer...... tissue. Recent studies have shown that a high uPA level in tumor extracts is in some cancers associated with poor prognosis. The present assay will now allow similar prognostic studies of uPAR levels....

  11. Quantitative radiological characterization of waste. Integration of gamma spectrometry and passive/active neutron assay

    Energy Technology Data Exchange (ETDEWEB)

    Simone, Gianluca; Mauro, Egidio; Gagliardi, Filippo; Gorello, Edoardo [Nucleco S.p.A., Rome (Italy)

    2016-06-15

    The radiological characterization of drums through Non-Destructive Assay (NDA) techniques commonly relies on gamma spectrometry. This paper introduces the procedure developed in Nucleco for the NDA radiological characterization of drums when the presence of Special Nuclear Material (SNM) is expected/observed. The procedure is based on the integration of a gamma spectrometry in SGS mode (Segmented Gamma Scanner) and a passive/active neutron assay. The application of this procedure is discussed on a real case of drums. The extension of the integration procedure to other gamma spectrometry systems is also discussed.

  12. Development of a quantitative real-time PCR assay for detection of Vibrio tubiashii targeting the metalloprotease gene.

    Science.gov (United States)

    Gharaibeh, Dima N; Hasegawa, Hiroaki; Häse, Claudia C

    2009-03-01

    Vibrio tubiashii has recently re-emerged as a pathogen of bivalve larvae, causing a marked increase in the mortality of these species within shellfish rearing facilities. This has resulted in substantial losses of seed production and thus created the need for specific as well as sensitive detection methods for this pathogen. In this project, quantitative PCR (qPCR) primers were developed and optimized based upon analysis of the V. tubiashii vtpA gene sequence, encoding a metalloprotease known to cause larval mortality. Standard curves were developed utilizing dilutions of known quantities of V. tubiashii cells that were compared to colony forming unit (CFU) plate counts. The assay was optimized for detection of vtpA with both lab-grown V. tubiashii samples and filter-captured environmental seawater samples seeded with V. tubiashii. In addition, the primers were confirmed to specifically detect only V. tubiashii when tested against a variety of non-target Vibrio species. Validation of the assay was completed by analyzing samples obtained from a shellfish hatchery. The development of this rapid and sensitive assay for quantitative detection of V. tubiashii will accurately determine levels of this bacterium in a variety of seawater samples, providing a useful tool for oyster hatcheries and a method to assess the presence of this bacterium in the current turbulent ocean environment.

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

    Science.gov (United States)

    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”. PMID:27335635

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

    Science.gov (United States)

    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".

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

    Science.gov (United States)

    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

  16. Quantitative comparison of DNA methylation assays for biomarker development and clinical applications

    DEFF Research Database (Denmark)

    Bock, Christoph; Halbritter, Florian; Carmona, Francisco J;

    2016-01-01

    Abstract DNA methylation patterns are altered in numerous diseases and often correlate with clinically relevant information such as disease subtypes, prognosis and drug response. With suitable assays and after validation in large cohorts, such associations can be exploited for clinical diagnostic...

  17. Quantitative predictivity of the transformation in vitro assay compared with the Ames test. [Hamsters

    Energy Technology Data Exchange (ETDEWEB)

    Parodi, S.; Taningher, M.; Russo, P.; Pala, M.; Vecchio, D.; Fassina, G.; Santi, L.

    For 59 chemical compounds, homogeneous data on transformation in vitro, mutagenicity in the Ames test, and carcinogenicity was reviewed. The potency in inducing transformation in vitro in hamster fibroblast cells was compared with the carcinogenic potency and a modest correlation coefficient was found between the two parameters. For these same 59 compounds it was also possible to compare mutagenic potency in the Ames test with carcinogenic potency. The correlation level was very similar. The predictivity of transformation in vitro increased significantly when only compounds for which some kind of dose-response relationship was available were utilized. This result stresses the importance of the quantitative aspect of the response in predictivity studies. The present study is compared with previous studies on the quantitative predictivity of different short-term tests. The work is not definitive, but gives an idea of the possible type of approach to the problem of comparing quantitative predictivities.

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-03-01

    Epizootic epitheliotropic disease virus (EEDV; salmonid herpesvirus [SalHV3]; family Alloherpesviridae) causes a systemic disease of juvenile and yearling Lake Trout Salvelinus namaycush. No cell lines are currently available for the culture and propagation of EEDV, so primary diagnosis is limited to PCR and electron microscopy. To better understand the pervasiveness of EEDV (carrier or latent state of infection) in domesticated and wild Lake Trout populations, we developed a sensitive TaqMan quantitative PCR (qPCR) assay to detect the presence of the EEDV terminase gene in Lake Trout tissues. This assay was able to detect a linear standard curve over nine logs of plasmid dilution and was sensitive enough to detect single-digit copies of EEDV. The efficiency of the PCR assay was 99.4 ± 0.06% (mean ± SD), with a 95% confidence limit of 0.0296 (R(2) = 0.994). Methods were successfully applied to collect preliminary data from a number of species and water bodies in the states of Pennsylvania, New York, and Vermont, indicating that EEDV is more common in wild fish than previously known. In addition, through the development of this qPCR assay, we detected EEDV in a new salmonid species, the Cisco Coregonus artedi. The qPCR assay was unexpectedly able to detect two additional herpesviruses, the Atlantic Salmon papillomatosis virus (ASPV; SalHV4) and the Namaycush herpesvirus (NamHV; SalHV5), which both share high sequence identity with the EEDV terminase gene. With these unexpected findings, we subsequently designed three primer sets to confirm initial TaqMan qPCR assay positives and to differentiate among EEDV, ASPV, and NamHV by detecting the glycoprotein genes via SYBR Green qPCR. Received April 20, 2015; accepted November 10, 2015. PMID:26980561

  20. Development of a Quantitative PCR Assay for Detection of Human Insulin-Like Growth Factor Receptor and Insulin Receptor Isoforms.

    Science.gov (United States)

    Flannery, Clare A; Rowzee, Anne M; Choe, Gina H; Saleh, Farrah L; Radford, Caitlin C; Taylor, Hugh S; Wood, Teresa L

    2016-04-01

    The biological activity of insulin and the insulin-like growth factor (IGF) ligands, IGF-I and IGF-II, is based in part on the relative abundance and distribution of their target receptors: the insulin receptor (IR) splice variants A (IR-A) and B (IR-B) and IGF 1 receptor (IGF-1R). However, the relative quantity of all three receptors in human tissues has never been measured together on the same scale. Due to the high homology between insulin receptor (IR)-A and IR-B proteins and lack of antibodies that discern the two IR splice variants, their mRNA sequence is the most reliable means of distinguishing between the receptors. Hence, highly specific primers for IR-A, IR-B, and IGF-1R mRNA were designed to accurately detect all three receptors by quantitative RT-PCR and enable direct quantification of relative receptor expression levels. A standard concentration curve of cDNA from each receptor was performed. Assay specificity was tested using competition assays and postamplification analysis by gel electrophoresis and cloning. Forward and reverse primer concentrations were optimized to ensure equal efficiencies across primer pairs. This assay enables a specific molecular signature of IGF/insulin signaling receptors to be assayed in different tissues, cell types, or cancers. PMID:26862994

  1. A protocol for the systematic and quantitative measurement of protein-lipid interactions using the liposome-microarray-based assay.

    Science.gov (United States)

    Saliba, Antoine-Emmanuel; Vonkova, Ivana; Deghou, Samy; Ceschia, Stefano; Tischer, Christian; Kugler, Karl G; Bork, Peer; Ellenberg, Jan; Gavin, Anne-Claude

    2016-06-01

    Lipids organize the activity of the cell's proteome through a complex network of interactions. The assembly of comprehensive atlases embracing all protein-lipid interactions is an important challenge that requires innovative methods. We recently developed a liposome-microarray-based assay (LiMA) that integrates liposomes, microfluidics and fluorescence microscopy and which is capable of measuring protein recruitment to membranes in a quantitative and high-throughput manner. Compared with previous assays that are labor-intensive and difficult to scale up, LiMA improves the protein-lipid interaction assay throughput by at least three orders of magnitude. Here we provide a step-by-step LiMA protocol that includes the following: (i) the serial and generic production of the liposome microarray; (ii) its integration into a microfluidic format; (iii) the measurement of fluorescently labeled protein (either purified proteins or from cell lysate) recruitment to liposomal membranes using high-throughput microscopy; (iv) automated image analysis pipelines to quantify protein-lipid interactions; and (v) data quality analysis. In addition, we discuss the experimental design, including the relevant quality controls. Overall, the protocol-including device preparation, assay and data analysis-takes 6-8 d. This protocol paves the way for protein-lipid interaction screens to be performed on the proteome and lipidome scales. PMID:27149326

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

    Directory of Open Access Journals (Sweden)

    Zsolt Czimmerer

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

  3. Establishment and Application of a TaqMan Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Rubella Virus RNA

    Institute of Scientific and Technical Information of China (English)

    Li-Hong ZHAO; Yu-Yan MA; Hong WANG; Shu-Ping ZHAO; Wei-Ming ZHAO; Hua LI; Lei-Yi WANG

    2006-01-01

    The aim of this study was to establish and apply a real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for rubella virus (RV) RNA. First, the primer and TaqMan probe concentrations, as well as reaction temperatures were optimized to establish an efficient real-time quantitative RT-PCR assay for RV RNA. Next, an RV-specific PCR amplicon was made as an external standard to estimate the linearity, amplification efficiency, analytical sensitivity and reproducibility of the real time quantitative assay. Finally, the assay was applied to quantify RVRNA in clinical samples for rubella diagnosis.The RV-specific PCR amplicon was prepared for evaluation of the assay at 503 bp, and its original concentration was 2.75×109 copies/μl. The real time quantitative assay was shown to have good linearity (R2=0.9920), high amplification efficiency (E=1.91), high sensitivity (275 copies/ml), and high reproducibility (variation coefficient range, from 1.25% to 3.58%). Compared with the gold standard, the specificity and sensitivity of the assay in clinical samples was 96.4% and 86.4%, respectively. Therefore, the established quantitative RT-PCR method is a simple, rapid, less-labored, quantitative, highly specific and sensitive assay for RV RNA.

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

    Science.gov (United States)

    de Gier, Camilla; Pickering, Janessa L; Richmond, Peter C; Thornton, Ruth B; Kirkham, Lea-Ann S

    2016-09-01

    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

  5. A Lateral Flow Assay for Quantitative Detection of Amplified HIV-1 RNA

    OpenAIRE

    Rohrman, Brittany A.; Veronica Leautaud; Elizabeth Molyneux; Richards-Kortum, Rebecca R.

    2012-01-01

    Although the accessibility of HIV treatment in developing nations has increased dramatically over the past decade, viral load testing to monitor the response of patients receiving therapy is often unavailable. Existing viral load technologies are often too expensive or resource-intensive for poor settings, and there is no appropriate HIV viral load test currently available at the point-of-care in low resource settings. Here, we present a lateral flow assay that employs gold nanoparticle probe...

  6. Viral Multiplex Quantitative PCR Assays for Tracking Sources of Fecal Contamination▿

    OpenAIRE

    Wolf, Sandro; Hewitt, Joanne; Greening, Gail E.

    2010-01-01

    Human and animal fecal pollution of the environment presents a risk to human health because of the presence of pathogenic viruses and bacteria. To distinguish between human and animal sources of pollution, we designed specific real-time reverse transcription (RT)-PCR assays for human and animal enteric viruses, including norovirus genogroups I, II, and III; porcine adenovirus types 3 and 5; ovine adenovirus; atadenovirus; and human adenovirus species C and F, which are excreted by infected hu...

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

    Science.gov (United States)

    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

    2015-07-01

    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

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

    Science.gov (United States)

    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.

  9. Slot immunoblot assay for detection and quantitation of periodontal disease-associated microorganisms in dental plaque.

    OpenAIRE

    van Poperin, N; Lopatin, D E

    1991-01-01

    A rapid method for qualitative and quantitative detection of specific oral microorganisms from subgingival dental plaque is described. Plaque samples were suspended in phosphate-buffered saline containing protease inhibitors and 0.5% formaldehyde, briefly sonicated to disperse bacterial aggregates, and applied to nitrocellulose membranes in a slot blot manifold. Subsequent incubations with species-specific rabbit antibody and anti-rabbit antibody-alkaline phosphatase conjugate and development...

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

    Science.gov (United States)

    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 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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Sónia Troeira Henriques

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    Science.gov (United States)

    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 2,000 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 1,000 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. PMID:6497105

  15. Evaluation of an infectivity standard for real-time quality control of human immunodeficiency virus type 1 quantitative micrococulture assays. Participating Laboratories of The AIDS Clinical Trials Group.

    OpenAIRE

    Scott, W A; Brambilla, D; Siwak, E; Beatty, C.; Bremer, J.; Coombs, R W; Farzadegan, H; Fiscus, S A; Hammer, S M; Hollinger, F. B.; Khan, N.; Rasheed, S; Reichelderfer, P S

    1996-01-01

    Quantitative microculture assays of cryopreserved human immunodeficiency virus type 1-infected cell suspensions and culture supernatants were compared among seven assays sites. There was no significant change in titer during 1 year of storage. The overall standard deviation for infected cell suspensions was approximately 0.8 log10 virus titer. A method for detecting deviant assay results was developed and was used to identify two donor cell preparations (n = 54) that gave consistently low tit...

  16. Development of a neutralization assay for influenza virus using an endpoint assessment based on quantitative reverse-transcription PCR.

    Directory of Open Access Journals (Sweden)

    Belete Teferedegne

    Full Text Available 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.

  17. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

    Science.gov (United States)

    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.

  18. PRUEBA CUANTITATIVA PARA PROTEASAS USANDO PELÍCULAS FOTOGRÁFICAS Quantitative Protease Assay Using Photographic Films

    Directory of Open Access Journals (Sweden)

    ANDREA PAREJA

    Full Text Available Se han desarrollado dos métodos para la medición cuantitativa de la actividad proteasa basados en la prueba de la película fotográfica. Una prueba discontinua puede ser implementada mediante la cuantificación de la cantidad de pigmento remanente en la película con cualquier programa de edición de imágenes. La medición continua de la actividad proteasa se puede obtener a través del cambio de absorbancia generado por la liberación de las sales de plata unidas al negativo fotográfico si se cuenta con un espectrofotómetro equipado con una celda con agitación.Two quantitative protease assays have been developed based on the classic photographic film test. A discontinuous assay can be performed by measuring the amount of pigment remaining in the film with any image editor software. A continuous assay can be implemented by the measuring the release of silver halide salts bonded in the film using a recording spectrophometer equipped with a Peltier Cell.

  19. Development of a quantitative, cell-based, high-content screening assay for epidermal growth factor receptor modulators

    Institute of Scientific and Technical Information of China (English)

    Jue WANG; Xin XIE

    2007-01-01

    Aim: To develop a robust, cell-based, high-content screening (HCS) assay based on receptor internalization for the identification of novel modulators of the epidermal growth factor receptor (EGFR). Methods: Agonist-induced receptor internalization is part of the signaling cascade of EGFR. Fluorescent-tagged epidermal growth factor (EGF) was used to visualize the internalized receptorligand complex. The fluorescent intracellular spots were detected and measured with an ArrayScan HCS reader. Compounds that can competitively bind to EGFR or interfere with EGFR internalization process would result in a reduced number and intensity of intracellular fluorescent spots. This assay was validated,optimized, and applied to a large-scale screening of a library containing 48 000 synthetic compounds. Results: The competition between fluorescent EGF and unlabeled EGF reveals the IC50 of unlabeled EGF is approximately 0.2 nmol/L,which is comparable with other published reports. Thirteen compounds with a relatively high degree of interference with EGFR internalization were identified.One of the compounds was proven to be agonist of the EGFR since it induced phosphorylation of the receptor and extracellular signal-regulated protein kinase (ERK). Conclusion: This automated, objective, and easy-to-use assay provided abundant information, quantitative results, and demonstrated the potential use of HCS methods in searching membrane receptor modulators.

  20. A Novel Qualitative and Quantitative Biofilm Assay Based on 3D Soft Tissue

    Directory of Open Access Journals (Sweden)

    Bodil Hakonen

    2014-01-01

    Full Text Available 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. Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors

    International Nuclear Information System (INIS)

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

  2. Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay.

    Directory of Open Access Journals (Sweden)

    Vasundhara Sharma

    Full Text Available The NF-κB transcription factor family plays a central role in innate immunity and inflammation processes and is frequently dysregulated in cancer. We developed an NF-κB functional assay in yeast to investigate the following issues: transactivation specificity of NF-κB proteins acting as homodimers or heterodimers; correlation between transactivation capacity and in vitro DNA binding measurements; impact of co-expressed interacting proteins or of small molecule inhibitors on NF-κB-dependent transactivation. Full-length p65 and p50 cDNAs were cloned into centromeric expression vectors under inducible GAL1 promoter in order to vary their expression levels. Since p50 lacks a transactivation domain (TAD, a chimeric construct containing the TAD derived from p65 was also generated (p50TAD to address its binding and transactivation potential. The p50TAD and p65 had distinct transactivation specificities towards seventeen different κB response elements (κB-REs where single nucleotide changes could greatly impact transactivation. For four κB-REs, results in yeast were predictive of transactivation potential measured in the human MCF7 cell lines treated with the NF-κB activator TNFα. Transactivation results in yeast correlated only partially with in vitro measured DNA binding affinities, suggesting that features other than strength of interaction with naked DNA affect transactivation, although factors such as chromatin context are kept constant in our isogenic yeast assay. The small molecules BAY11-7082 and ethyl-pyruvate as well as expressed IkBα protein acted as NF-κB inhibitors in yeast, more strongly towards p65. Thus, the yeast-based system can recapitulate NF-κB features found in human cells, thereby providing opportunities to address various NF-κB functions, interactions and chemical modulators.

  3. Potential of monitoring isotopologues by quantitative gas chromatography with time-of-flight mass spectrometry for metabolomic assay.

    Science.gov (United States)

    Wang, Yi; Hu, Haiyan; Su, Yue; Zhang, Fang; Guo, Yinlong

    2016-03-01

    Because of the extreme complexity of metabolomic samples, the effectiveness of quantitative gas chromatography with time-of-flight mass spectrometry depends substantially on the expansion of the linear dynamic range. Facing the existence of numerous saturated detector signals, a data processing method based on monitoring isotopologues has been developed. The monoisotopic ion kept the high mass spectrometry sensitivity, and the less abundant isotopologue ions extended the linear dynamic range. This alternative method was proved to extend the linear dynamic range to five orders of magnitude successfully and overcome the quantitative problems induced by the ion detector saturation. Finally, to validate the applicability, the method was applied to a metabolomic assay of Alzheimer's disease. Comparing with the traditional monoisotopic method, the use of monitoring isotopologues helped us to discover an additional eight metabolites with significant difference and to conduct a more reliable principal component analysis as well. The results demonstrated that monitoring isotopologues in quantitative gas chromatography with time-of-flight mass spectrometry could improve the authenticity of metabolomic analysis. PMID:26763370

  4. Multiplex time-reducing quantitative polymerase chain reaction assay for determination of telomere length in blood and tissue DNA.

    Science.gov (United States)

    Jiao, Jingjing; Kang, Jing X; Tan, Rui; Wang, Jingdong; Zhang, Yu

    2012-04-01

    In this paper we describe a multiplex time-reducing quantitative polymerase chain reaction (qPCR) method for determination of telomere length. This multiplex qPCR assay enables two pairs of primers to simultaneously amplify telomere and single copy gene (albumin) templates, thus reducing analysis time and labor compared with the previously established singleplex assay. The chemical composition of the master mix and primers for the telomere and albumin were systematically optimized. The thermal cycling program was designed to ensure complete separation of the melting processes of the telomere and albumin. Semi-log standard curves of DNA concentration versus cycle threshold (C (t)) were established, with a linear relationship over an 81-fold DNA concentration range. The well-performed intra-assay (RSD range 2.4-4.7%) and inter-assay (RSD range: 3.1-5.0%) reproducibility were demonstrated to ensure measurement stability. Using wild-type, Lewis lung carcinoma and H22 liver carcinoma C57BL/6 mouse models, significantly different telomere lengths among different DNA samples were not observed in wild-type mice. However, the relative telomere lengths of the tumor DNA in the two strains of tumor-bearing mice were significantly shorter than the lengths in the surrounding non-tumor DNA of tumor-bearing mice and the tissue DNA of wild-type mice. These results suggest that the shortening of telomere lengths may be regarded as an important indicator for cancer control and prevention. Quantification of telomere lengths was further confirmed by the traditional Southern blotting method. This method could be successfully used to reduce the time needed for rapid, precise measurement of telomere lengths in biological samples.

  5. Rapid agarose gel electrophoretic mobility shift assay for quantitating protein: RNA interactions.

    Science.gov (United States)

    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. PMID:27495142

  6. Quantitative assay for the colonization ability of heterogeneous bacteria on controlled nanopillar structures

    International Nuclear Information System (INIS)

    The colonization ability of bacteria on biomaterial surfaces is influenced by the morphology of the bacteria and the nanotopography of the biomaterial. However, interactions between the bacterial morphology and nanotopography of biomaterials have not yet been completely elucidated. In this article, we quantitatively characterized the bacterial morphology to illuminate the integrated effects of polyethylene terephthalate (PET) nanopillar arrays on the colonization of bacteria cells with different shapes. Our results demonstrated that the interaction between interpillar spacing and the diameter of the bacterial cells impacted the number of bacterial cells that adhered to different PET substrates. The interpillar spacing of nanopillar arrays promotes bacterial adhesion in a definite range (<50 nm). However, further increasing the interpillar spacing inhibited the adhesion of bacteria to the nanopillar arrays. Moreover, the interpillar spacing also influenced the morphologies of adherent bacterial cells on the PET nanopillar arrays, which consequently facilitated bacterial adhesion to the nanopillar arrays. Our findings enhance the understanding of interactions between controlled nanotopography and bacterial colonization and provide an appropriate parameter for the design of antibacterial materials with nanotopography. (paper)

  7. Assessment of Legionella pneumophila in recreational spring water with quantitative PCR (Taqman) assay

    Science.gov (United States)

    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. Magnetic particle separation technique: a reliable and simple tool for RIA/IRMA and quantitative PCR assay

    International Nuclear Information System (INIS)

    Five types of magnetic particles without or with aldehyde, amino and carboxyl functional groups, respectively were used to immobilize first or second antibody by three models, i. e. physical adsorption, chemical coupling and immuno-affinity, forming four types of magnetic particle antibodies. The second antibody immobilized on polyacrolein magnetic particles through aldehyde functional groups and the first antibodies immobilized on carboxylic polystyrene magnetic particles through carboxyl functional groups were recommended to apply to RIAs and/or IRMAs. Streptavidin immobilized on commercial magnetic particles through amino functional groups was successfully applied to separating specific PCR product for quantification of human cytomegalovirus. In the paper typical data on reliability of these magnetic particle ligands were reported and simplicity of the magnetic particle separation technique was discussed. The results showed that the technique was a reliable and simple tool for RIA/IRMA and quantitative PCR assay. (author)

  9. Odorant Screening and Quantitation of Thiols in Carmenere Red Wine by Gas Chromatography-Olfactometry and Stable Isotope Dilution Assays.

    Science.gov (United States)

    Pavez, Carolina; Agosin, Eduardo; Steinhaus, Martin

    2016-05-01

    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. PMID:27070203

  10. Quantitative polymerase chain reaction (PCR) assays for a bacterial thiaminase I gene and the thiaminase-producing bacterium Paenibacillus thiaminolyticus.

    Science.gov (United States)

    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.

  11. Development of real-time quantitative polymerase chain reaction assays to track treatment response in retinoid resistant acute promyelocytic leukemia

    Directory of Open Access Journals (Sweden)

    Jelena V Jovanovic

    2011-10-01

    Full Text Available Molecular detection of minimal residual disease (MRD has become established to assess remission status and guide therapy in patients with PML-RARA+ acute promyelocytic leukemia (APL. However, there are few data on tracking disease response in patients with rarer retinoid resistant subtypes of APL, characterized by PLZF-RARA and STAT5b-RARA. Despite their relative rarity (<1% of APL we identified 6 cases (PLZF-RARA, n=5; STAT5b-RARA, n=1, established the respective breakpoint junction regions and designed real-time quantitative polymerase chain reaction (RQ-PCR assays to detect leukemic transcripts. The relative level of fusion gene expression in diagnostic samples was comparable to that observed in t(15;17-associated APL, affording assay sensitivities of ~1 in 104-105. Serial samples were available from 2 PLZF-RARA APL patients. One showed persistent PCR positivity, predicting subsequent relapse, and remains in CR2, ~11 years post-autograft. The other, achieved molecular remission (CRm with combination chemotherapy, remaining in CR1 at 6 years. The STAT5b-RARA patient failed to achieve CRm following frontline combination chemotherapy and ultimately proceeded to allogeneic transplant on the basis of a steadily rising fusion transcript level. These data highlight the potential of RQ-PCR detection of MRD to facilitate development of more individualized approaches to the management of rarer molecularly-defined subsets of acute leukemia.

  12. Quantitative liquid chromatography/mass spectrometry/mass spectrometry warfarin assay for in vitro cytochrome P450 studies.

    Science.gov (United States)

    Zhang, Z Y; King, B M; Wong, Y N

    2001-11-01

    A sensitive assay using high-performance liquid chromatography tandem mass spectrometry (MS/MS) has been established for the quantitative analysis of cytochrome P450 form-specific activities using warfarin as a probe substrate. Four metabolites, 6-, 7-, 8-, and 10-hydroxywarfarin, were chromatographically resolved within 10 min using gradient mobile phases. The mass spectrometry was operated under negative ionization mode. The MS/MS product ion spectra of warfarin and the metabolites were generated using collision-activated dissociation and interpreted. The abundant product ions of the metabolites were selected for quantification applying multiple reaction monitoring. Quantification was based on a quadratic or power curve of the peak area ratio of the metabolite over the internal standard against the respective concentration of the metabolite. This assay has been validated from 2 to 1000 nM for 10-hydroxywarfarin and from 2 to 5000 nM for 6-, 7-, and 8-hydroxywarfarin and successfully applied to evaluate cytochrome P450-mediated drug-drug interactions in vitro using human hepatocytes and liver microsomal preparations. PMID:11673893

  13. Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

    Science.gov (United States)

    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.

  14. Establishment of a 10-Plex Quantitative Fluorescent-PCR Assay for rapid diagnosis of sex chromosome aneuploidies.

    Directory of Open Access Journals (Sweden)

    Xingmei Xie

    Full Text Available 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.

  15. Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays.

    Directory of Open Access Journals (Sweden)

    Markus Kopp

    Full Text Available 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.

  16. A two-step real-time PCR assay for quantitation and genotyping of human parvovirus 4.

    Science.gov (United States)

    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.

  17. Quantitation of Gingerols in Human Plasma by Newly Developed Stable Isotope Dilution Assays and Assessment of Their Immunomodulatory Potential.

    Science.gov (United States)

    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. PMID:26939769

  18. Development of plasmids for quantitative detection of integrated lentiviral vectors and evaluation of culture time to perform vector titer by real-time quantitative polymerase chain reaction assay

    Directory of Open Access Journals (Sweden)

    Elena Baiamonte

    2014-09-01

    Full Text Available The accurate assessment of provirus copy number per cell (VCN/cell is a fundamental issue in transgenesis as well as in gene therapy studies based on stably integrated vectors. To this end, real-time quantitative polymerase chain reaction (qPCR is a powerful method but it is sensible to differences in quality or concentration of the two-plasmid preparations used for the construction of the standard curves. In order to minimize technical errors we included genome specific sequences (mouse or human and vector specific sequences in the same plasmid. We evaluated the specificity and sensitivity of these bivalent plasmids by qPCR analysis on mouse and human genomic DNA containing a known number of a reporter lentiviral vector and we found that the system is reliable to measure up to 0.1 VCN/cell. Here we have applied this assay to measure vector titer of virus stock preparations and to determine the optimal cell passages at which viral titration effectively reflects the number of integrated vectors.

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

    OpenAIRE

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

    2007-01-01

    Evaluation of the fate and transport of biological warfare (BW) agents in landfills requires the development of specific and sensitive detection assays. The objective of the current study was to develop and validate SYBR green quantitative real-time PCR (Q-PCR) assays for the specific detection and quantification of surrogate BW agents in synthetic building debris (SBD) and leachate. Bacillus atrophaeus (vegetative cells and spores) and Serratia marcescens were used as surrogates for Bacillus...

  20. Immunochromatographic assay for quantitative and sensitive detection of hepatitis B virus surface antigen using highly luminescent quantum dot-beads.

    Science.gov (United States)

    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. PMID:26003704

  1. Implementation of a quantitative real-time PCR assay for the detection of Mycobacterium immunogenum in metalworking fluids.

    Science.gov (United States)

    Rhodes, Glenn; Fluri, Alexandra; Ruefenacht, Andrea; Gerber, Marco; Pickup, Roger

    2011-08-01

    The bacterium Mycobacterium immunogenum has been implicated in causing the lung condition hypersensitivity pneumonitis (HP) in factory workers exposed to colonized metalworking fluids (MWFs). M. immunogenum-specific, real-time quantitative PCR detection technique (MiRT-qPCR) was implemented on a large scale to 363 MWFs of varying types, originating from the United States and Europe, that had been in use for between 30 days and 1 year. In MWFs that contained between 10(3) and 10(9) culturable general heterotrophs mL(-1) the technique detected between 5 and 2 × 10(6) mL(-1) M. immunogenum cell equivalents (CE) in 12.2% (23 of 189) of U.S. samples and between 8 and 6 × 10(5) mL(-1) CE in 39.1% (68 of 174) of samples from Europe. In contrast, only three cultured presumptive mycobacterial isolates across all samples were confirmed as M. immunogenum. Implementation of the assay demonstrated its practicality and further emphasized the limitations of using cultivation alone. Interestingly, no M. immunogenum were detected in mineral oil-based Bio-Concept MWFs from the United States, while it was more commonly detected in used MWFs based on formaldehyde-releasing biocides than in MWFs free of formaldehyde-depot biocides. PMID:21756137

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

    Science.gov (United States)

    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 ...

  3. Investigation of a redox-sensitive predictive model of mouse embryonic stem cells differentiation using quantitative nuclease protection assays and glutathione redox status

    Science.gov (United States)

    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...

  4. Factors That Contribute to Assay Variation in Quantitative Analysis of Sex Steroid Hormones Using Liquid and Gas Chromatography-Mass Spectrometry

    Science.gov (United States)

    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…

  5. Serum Neutralization Assay Can Efficiently Replace Plaque Reduction Neutralization Test for Detection and Quantitation of West Nile Virus Antibodies in Human and Animal Serum Samples

    Science.gov (United States)

    Di Gennaro, Annapia; Casaccia, Claudia; Conte, Annamaria; Monaco, Federica; Savini, Giovanni

    2014-01-01

    A serum neutralization assay (SN) was compared with the official plaque reduction neutralization test for the quantitation of West Nile virus antibodies. A total of 1,348 samples from equid sera and 38 from human sera were tested by these two methods. Statistically significant differences were not observed, thus supporting the use of SN for routine purposes. PMID:25100824

  6. EVALUATION OF A PURIFICATION PROCEDURE FOR THE MUSCARINIC RECEPTOR FOR THE PURPOSE OF QUANTITATIVE RECEPTOR ASSAYS OF ANTICHOLINERGICS .B. THE SOLUBILIZED RECEPTOR

    NARCIS (Netherlands)

    SMISTEROVA, J; ENSING, K; DEBOER, J; DEZEEUW, RA

    1995-01-01

    For the purpose of quantitative receptor assays, a three-step solubilization procedure including three optimization sets for muscarinic receptor from calf striatum was developed. The first step includes the extraction of the P2-pellet with n-hexane and consequently with 2 M NaCl. By the latter, 39%

  7. A sensitive radioimmunoprecipitation assay for the detection and quantitation of antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1)

    International Nuclear Information System (INIS)

    A radioimmunoprecipitation (RIP) assay was developed to detect antibodies to the envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1). The assay, which utilized recombinant gp120 (rgp120), was quantitative, reproducible, and specific for antibodies to rgp120 or antibodies to native gp120 resulting from natural infection with HIV. Polyethylene glycol-8000 (PEG), used in the assay at a final concentration of 10% to precipitate immune complexes, was demonstrated to be effective in titering sera from different animal species. Provided samples were diluted at least 1:100, antibody titers could be determined either by the classical dilution method or by interpolation from a calibration curve prepared with a positive serum. The humoral response of animals immunized with rgp120 was monitored and a positive correlation was found between titers determined in the RIP assay and the ability of the sera to neutralize. In addition, RIP titers of HIV-positive human sera correlated very well with reactivity obtained in a commercial HIV immunoblot assay. The assay has the advantage of quantitation, fast turnaround time, and versatility

  8. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    Energy Technology Data Exchange (ETDEWEB)

    Fichorova, Raina N., E-mail: rfichorova@rics.bwh.harvard.edu [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Mendonca, Kevin; Yamamoto, Hidemi S.; Murray, Ryan [Laboratory of Genital Tract Biology, Department of Obstetrics, Gynecology and Reproductive Biology, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Chandra, Neelima; Doncel, Gustavo F. [CONRAD, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, VA (United States)

    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

  9. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation

    International Nuclear Information System (INIS)

    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

  10. Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number–qPCR Assay

    Science.gov (United States)

    Banting, Graham S.; Braithwaite, Shannon; Scott, Candis; Kim, Jinyong; Jeon, Byeonghwa; Ashbolt, Nicholas; Ruecker, Norma; Tymensen, Lisa; Charest, Jollin; Pintar, Katarina; Checkley, Sylvia

    2016-01-01

    ABSTRACT Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)–quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053–1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (Campylobacter-specific qPCR was used, with the most commonly detected species being C. jejuni, C. coli, and C. lari. Campylobacters in raw sewage were present at ∼102/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to

  11. A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts

    Directory of Open Access Journals (Sweden)

    Isabel Hostettler

    2014-12-01

    Full Text Available 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.

  12. A quantitative multiplex nuclease protection assay reveals immunotoxicity gene expression profiles in the rabbit model for vaginal drug safety evaluation.

    Science.gov (United States)

    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. PMID:25818602

  13. Quantitation of viable Coxiella burnetii in milk using an integrated cell culture-polymerase chain reaction (ICC-PCR) assay.

    Science.gov (United States)

    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.

  14. Development & validation of a quantitative anti-protective antigen IgG enzyme linked immunosorbent assay for serodiagnosis of cutaneous anthrax

    Directory of Open Access Journals (Sweden)

    N Ghosh

    2015-01-01

    Full Text Available Background & objectives: 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. Methods: 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. Results: 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. Interpretation & conclusions: 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.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Huang, S-H; Tsai, M-H; Lin, C-W [Department of Biotechnology, College of Health Science, Asia University, Wufeng, Taichung, Taiwan (China); Yang, T-C; Chuang, P-H [Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan (China); Tsai, I-S; Lu, H-C [Nanotechnology Research Center, Feng Chia University, Taichung, Taiwan (China); Wan Lei; Lin, Y-J [Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung, Taiwan (China); Lai, C-H [Department of Microbiology and Immunology, China Medical University, Taichung, Taiwan (China)], E-mail: cwlin@mail.cmu.edu.tw

    2008-10-08

    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.

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

    Science.gov (United States)

    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.

  18. Development and validation of a quantitative PCR assay using multiplexed hydrolysis probes for detection and quantification of Theileria orientalis isolates and differentiation of clinically relevant subtypes.

    Science.gov (United States)

    Bogema, D R; Deutscher, A T; Fell, S; Collins, D; Eamens, G J; Jenkins, C

    2015-03-01

    Theileria orientalis is an emerging pathogen of cattle in Asia, Australia, and New Zealand. This organism is a vector-borne hemoprotozoan that causes clinical disease characterized by anemia, abortion, and death, as well as persistent subclinical infections. Molecular methods of diagnosis are preferred due to their sensitivity and utility in differentiating between pathogenic and apathogenic genotypes. Conventional PCR (cPCR) assays for T. orientalis detection and typing are laborious and do not provide an estimate of parasite load. Current real-time PCR assays cannot differentiate between clinically relevant and benign genotypes or are only semiquantitative without a defined clinical threshold. Here, we developed and validated a hydrolysis probe quantitative PCR (qPCR) assay which universally detects and quantifies T. orientalis and identifies the clinically associated Ikeda and Chitose genotypes (UIC assay). Comparison of the UIC assay results with previously validated universal and genotype-specific cPCR results demonstrated that qPCR detects and differentiates T. orientalis with high sensitivity and specificiy. Comparison of quantitative results based on percent parasitemia, determined via blood film analysis and packed cell volume (PCV) revealed significant positive and negative correlations, respectively. One-way analysis of variance (ANOVA) indicated that blood samples from animals with clinical signs of disease contained statistically higher concentrations of T. orientalis DNA than animals with subclinical infections. We propose clinical thresholds to assist in classifying high-, moderate-, and low-level infections and describe how parasite load and the presence of the Ikeda and Chitose genotypes relate to disease.

  19. Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

    Science.gov (United States)

    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. PMID:26025892

  20. Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

    Energy Technology Data Exchange (ETDEWEB)

    Okita, Naoyuki, E-mail: nokita7@rs.noda.tus.ac.jp [Genome and Drug Research Center, Tokyo University of Science (Japan); Ashizawa, Daisuke; Ohta, Ryo [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022 (Japan); Abe, Hideaki [Genome and Drug Research Center, Tokyo University of Science (Japan); Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022 (Japan); Tanuma, Sei-ichi, E-mail: tanuma@rs.noda.tus.ac.jp [Genome and Drug Research Center, Tokyo University of Science (Japan); Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022 (Japan)

    2010-02-19

    Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V{sub max}) and the michaelis constant (k{sub m}) of PARG reaction were 4.46 {mu}M and 128.33 {mu}mol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 {mu}M. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.

  1. QUANTITATION OF DNA TOPOISOMERASE-II-ALPHA MESSENGER-RIBONUCLEIC-ACID LEVELS IN A SMALL-CELL LUNG-CANCER CELL-LINE AND 2 DRUG-RESISTANT SUBLINES USING A POLYMERASE CHAIN REACTION-AIDED TRANSCRIPT TITRATION ASSAY

    NARCIS (Netherlands)

    WITHOFF, S; SMIT, EF; MEERSMA, GJ; van den Berg, Anke; TIMMERBOSSCHA, H; KOK, K; POSTMUS, PE; MULDER, NH; DEVRIES, EGE; BUYS, CHCM

    1994-01-01

    BACKGROUND: We have modified a polymerase chain reaction (PCR)-aided transcript titration assay (1) in order to allow quantitation of low amounts of DNA topoisomerase II alpha mRNA in small RNA samples. EXPERIMENTAL DESIGN: The titration assay was used to quantitate the amount of DNA topoisomerase I

  2. 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.

    Science.gov (United States)

    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.

  3. Assay of S for Quantitation of PEG and TNF Ligated Au Nanoparticles using ID-HR-MC-ICP-MS

    Science.gov (United States)

    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

  4. Rapid Diagnosis of Mycobacterial Infections and Quantitation of Mycobacterium tuberculosis Load by Two Real-Time Calibrated PCR Assays

    Science.gov (United States)

    Broccolo, Francesco; Scarpellini, Paolo; Locatelli, Giuseppe; Zingale, Anna; Brambilla, Anna M.; Cichero, Paola; Sechi, Leonardo A.; Lazzarin, Adriano; Lusso, Paolo; Malnati, Mauro S.

    2003-01-01

    Sensitive and specific techniques to detect and identify Mycobacterium tuberculosis directly in clinical specimens are important for the diagnosis and management of patients with tuberculosis (TB). We developed two real-time PCR assays, based on the IS6110 multicopy element and on the senX3-regX3 intergenic region, which provide a rapid method for the diagnosis of mycobacterial infections. The sensitivity and specificity of both assays were established by using purified DNA from 71 clinical isolates and 121 clinical samples collected from 83 patients, 20 of whom were affected by TB. Both assays are accurate, sensitive, and specific, showing a complementary pattern of Mycobacterium recognition: broader for the IS6110-based assay and restricted to the M. tuberculosis complex for the senX3-regX3-based assay. Moreover, the addition of a synthetic DNA calibrator prior to DNA extraction allowed us to measure the efficiency of DNA recovery and to control for the presence of PCR inhibitors. The mycobacterial burden of the clinical samples, as assessed by direct microscopy, correlates with the M. tuberculosis DNA load measured by the senX3-regX3-based assay. In addition, reduced levels of M. tuberculosis DNA load are present in those patients subjected to successful therapy, suggesting a potential use of this assay for monitoring treatment efficacy. Therefore, these assays represent a fully controlled high-throughput system for the evaluation of mycobacterial burden in clinical specimens. PMID:14532183

  5. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA.

    Science.gov (United States)

    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. PMID:27154315

  6. Identification of the major capsid protein of erythrocytic necrosis virus (ENV) and development of quantitative real-time PCR assays for quantification of ENV DNA

    Science.gov (United States)

    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.

  7. 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

    Directory of Open Access Journals (Sweden)

    Anny Armas Cayarga

    2011-01-01

    Full Text Available 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 (=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 (=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.

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

    Science.gov (United States)

    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. PMID:27211626

  9. Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

    Institute of Scientific and Technical Information of China (English)

    Zhu Yang; Guoliang Mao; Yujun Liu; Yuan-Chuan Chen; Chengjing Liu; Jun Luo; Xihan Li

    2013-01-01

    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 H1N 1/2009 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.

  10. Quantitative identification of fecal water pollution sources by TaqMan real-time PCR assays using Bacteroidales 16S rRNA genetic markers.

    Science.gov (United States)

    Lee, Dae-Young; Weir, Susan C; Lee, Hung; Trevors, Jack T

    2010-12-01

    PCR-based analysis of Bacteroidales 16S rRNA genes has emerged as a promising tool to identify sources of fecal water pollution. In this study, three TaqMan real-time PCR assays (BacGeneral, BacHuman, and BacBovine) were developed and evaluated for their ability to quantitatively detect general (total), human-specific, and bovine-specific Bacteroidales 16S rRNA genetic markers. The detection sensitivity was determined to be 6.5 copies of 16S rRNA gene for the BacGeneral and BacHuman assays and 10 copies for the BacBovine assay. The assays were capable of detecting approximately one to two cells per PCR. When tested with 70 fecal samples from various sources (human, cattle, pig, deer, dog, cat, goose, gull, horse, and raccoon), the three assays positively identified the target markers in all samples without any false-negative results. The BacHuman and BacBovine assays exhibited false-positive reactions with non-target samples in a few cases. However, the level of the false-positive reactions was about 50 times smaller than that of the true-positive ones, and therefore, these cross-reactions were unlikely to cause misidentifications of the fecal pollution sources. Microbial source-tracking capability was tested at two freshwater streams of which water quality was influenced by human and cattle feces, respectively. The assays accurately detected the presence of the corresponding host-specific markers upon fecal pollution and the persistence of the markers in downstream areas. The assays are expected to reliably determine human and bovine fecal pollution sources in environmental water samples. PMID:20871990

  11. Detection of hemoplasma infection of goats by use of a quantitative polymerase chain reaction assay and risk factor analysis for infection.

    Science.gov (United States)

    Johnson, Kathy A; do Nascimento, Naíla C; Bauer, Amy E; Weng, Hsin-Yi; Hammac, G Kenitra; Messick, Joanne B

    2016-08-01

    OBJECTIVE To develop and validate a real-time quantitative PCR (qPCR) assay for the detection and quantification of Mycoplasma ovis in goats and investigate the prevalence and risk factors for hemoplasma infection of goats located in Indiana. ANIMALS 362 adult female goats on 61 farms. PROCEDURES Primers were designed for amplification of a fragment of the dnaK gene of M ovis by use of a qPCR assay. Blood samples were collected into EDTA-containing tubes for use in total DNA extraction, blood film evaluation, and determination of PCV. Limit of detection, intra-assay variability, interassay variability, and specificity of the assay were determined. RESULTS Reaction efficiency of the qPCR assay was 94.45% (R(2), 0.99; slope, -3.4623), and the assay consistently detected as few as 10 copies of plasmid/reaction. Prevalence of infection in goats on the basis of results for the qPCR assay was 18.0% (95% confidence interval, 14% to 22%), with infected goats ranging from 1 to 14 years old, whereby 61% (95% confidence interval, 47% to 73%) of the farms had at least 1 infected goat. Bacterial load in goats infected with M ovis ranged from 1.05 × 10(3) target copies/mL of blood to 1.85 × 10(5) target copies/mL of blood; however, no bacteria were observed on blood films. Production use of a goat was the only risk factor significantly associated with hemoplasma infection. CONCLUSIONS AND CLINICAL RELEVANCE The qPCR assay was more sensitive for detecting hemoplasma infection than was evaluation of a blood film, and production use of a goat was a risk factor for infection. PMID:27463552

  12. Quantitative digital image analysis of chromogenic assays for high throughput screening of alpha-amylase mutant libraries.

    Science.gov (United States)

    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%.

  13. Development of a novel HAC-based "gain of signal" quantitative assay for measuring chromosome instability (CIN) in cancer cells.

    Science.gov (United States)

    Kim, Jung-Hyun; Lee, Hee-Sheung; Lee, Nicholas C O; Goncharov, Nikolay V; Kumeiko, Vadim; Masumoto, Hiroshi; Earnshaw, William C; Kouprina, Natalay; Larionov, Vladimir

    2016-03-22

    Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene ("loss of signal" assay). Using this system, anticancer drugs can be easily ranked on by their effect on HAC loss. However, it is problematic to covert this "loss of signal" assay into a high-throughput screen to identify drugs and mutations that increase CIN levels. To address this point, we re-designed the HAC-based assay. In this new system, the HAC carries a constitutively expressed shRNA against the EGFP transgene integrated into human genome. Thus, cells that inherit the HAC display no green fluorescence, while cells lacking the HAC do. We verified the accuracy of this "gain of signal" assay by measuring the level of CIN induced by known antimitotic drugs and added to the list of previously ranked CIN inducing compounds, two newly characterized inhibitors of the centromere-associated protein CENP-E, PF-2771 and GSK923295 that exhibit the highest effect on chromosome instability measured to date. The "gain of signal" assay was also sensitive enough to detect increase of CIN after siRNA depletion of known genes controlling mitotic progression through distinct mechanisms. Hence this assay can be utilized in future experiments to uncover novel human CIN genes, which will provide novel insight into the pathogenesis of cancer. Also described is the possible conversion of this new assay into a high-throughput screen using a fluorescence microplate reader to characterize chemical libraries and identify new conditions that modulate CIN level. PMID:26943579

  14. Dose metric considerations in in vitro assays to improve quantitative in vitro-in vivo dose extrapolations.

    Science.gov (United States)

    Groothuis, Floris A; Heringa, Minne B; Nicol, Beate; Hermens, Joop L M; Blaauboer, Bas J; Kramer, Nynke I

    2015-06-01

    Challenges to improve toxicological risk assessment to meet the demands of the EU chemical's legislation, REACH, and the EU 7th Amendment of the Cosmetics Directive have accelerated the development of non-animal based methods. Unfortunately, uncertainties remain surrounding the power of alternative methods such as in vitro assays to predict in vivo dose-response relationships, which impedes their use in regulatory toxicology. One issue reviewed here, is the lack of a well-defined dose metric for use in concentration-effect relationships obtained from in vitro cell assays. Traditionally, the nominal concentration has been used to define in vitro concentration-effect relationships. However, chemicals may differentially and non-specifically bind to medium constituents, well plate plastic and cells. They may also evaporate, degrade or be metabolized over the exposure period at different rates. Studies have shown that these processes may reduce the bioavailable and biologically effective dose of test chemicals in in vitro assays to levels far below their nominal concentration. This subsequently hampers the interpretation of in vitro data to predict and compare the true toxic potency of test chemicals. Therefore, this review discusses a number of dose metrics and their dependency on in vitro assay setup. Recommendations are given on when to consider alternative dose metrics instead of nominal concentrations, in order to reduce effect concentration variability between in vitro assays and between in vitro and in vivo assays in toxicology. PMID:23978460

  15. Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters

    Science.gov (United States)

    Riedel, Timothy E.; Zimmer-Faust, Amity G.; Thulsiraj, Vanessa; Madi, Tania; Hanley, Kaitlyn T.; Ebentier, Darcy L.; Byappanahalli, Muruleedhara N.; Layton, Blythe; Raith, Meredith; Boehm, Alexandria B.; Griffith, John F.; Holden, Patricia A.; Shanks, Orin C.; Weisberg, Stephen B.; Jay, Jennifer A.

    2014-01-01

    Some molecular methods for tracking fecal pollution in environmental waters have both PCR and quantitative PCR (qPCR) assays available for use. To assist managers in deciding whether to implement newer qPCR techniques in routine monitoring programs, we compared detection limits (LODs) and costs of PCR and qPCR assays with identical targets that are relevant to beach water quality assessment. For human-associated assays targeting Bacteroidales HF183 genetic marker, qPCR LODs were 70 times lower and there was no effect of target matrix (artificial freshwater, environmental creek water, and environmental marine water) on PCR or qPCR LODs. The PCR startup and annual costs were the lowest, while the per reaction cost was 62% lower than the Taqman based qPCR and 180% higher than the SYBR based qPCR. For gull-associated assays, there was no significant difference between PCR and qPCR LODs, target matrix did not effect PCR or qPCR LODs, and PCR startup, annual, and per reaction costs were lower. Upgrading to qPCR involves greater startup and annual costs, but this increase may be justified in the case of the human-associated assays with lower detection limits and reduced cost per sample.

  16. Rapid and Quantitative Detection of Leifsonia xyli subsp. xyli in Sugarcane Stalk Juice Using a Real-Time Fluorescent (TaqMan) PCR Assay

    Science.gov (United States)

    Fu, Hua-Ying; Sun, Sheng-Ren; Wang, Jin-Da; Ahmad, Kashif; Wang, Heng-Bo; Chen, Ru-Kai

    2016-01-01

    Ratoon stunting disease (RSD) of sugarcane, one of the most important diseases seriously affecting the productivity of sugarcane crops, was caused by the bacterial agent Leifsonia xyli subsp. xyli (Lxx). A TaqMan probe-based real-time quantitative polymerase chain reaction (qPCR) assay was established in this study for the quantification of Lxx detection in sugarcane stalk juice. A pair of PCR primers (Pat1-QF/Pat1-QR) and a fluorogenic probe (Pat1-QP) targeting the Part1 gene of Lxx were used for the qPCR assay. The assay had a detection limit of 100 copies of plasmid DNA and 100 fg of Lxx genomic DNA, which was 100-fold more sensitive than the conventional PCR. Fifty (28.7%) of 174 stalk juice samples from two field trials were tested to be positive by qPCR assay, whereas, by conventional PCR, only 12.1% (21/174) were tested to be positive with a published primer pair CxxITSf#5/CxxITSr#5 and 15.5% (27/174) were tested to be positive with a newly designed primer pair Pat1-F2/Pat1-R2. The new qPCR assay can be used as an alternative to current diagnostic methods for Lxx, especially when dealing with certificating a large number of healthy cane seedlings and determining disease incidence accurately in commercial fields.

  17. A novel quantitative hemolytic assay coupled with restriction fragment length polymorphisms analysis enabled early diagnosis of atypical hemolytic uremic syndrome and identified unique predisposing mutations in Japan.

    Directory of Open Access Journals (Sweden)

    Yoko Yoshida

    Full Text Available For thrombotic microangiopathies (TMAs, the diagnosis of atypical hemolytic uremic syndrome (aHUS is made by ruling out Shiga toxin-producing Escherichia coli (STEC-associated HUS and ADAMTS13 activity-deficient thrombotic thrombocytopenic purpura (TTP, often using the exclusion criteria for secondary TMAs. Nowadays, assays for ADAMTS13 activity and evaluation for STEC infection can be performed within a few hours. However, a confident diagnosis of aHUS often requires comprehensive gene analysis of the alternative complement activation pathway, which usually takes at least several weeks. However, predisposing genetic abnormalities are only identified in approximately 70% of aHUS. To facilitate the diagnosis of complement-mediated aHUS, we describe a quantitative hemolytic assay using sheep red blood cells (RBCs and human citrated plasma, spiked with or without a novel inhibitory anti-complement factor H (CFH monoclonal antibody. Among 45 aHUS patients in Japan, 24% (11/45 had moderate-to-severe (≥50% hemolysis, whereas the remaining 76% (34/45 patients had mild or no hemolysis (<50%. The former group is largely attributed to CFH-related abnormalities, and the latter group has C3-p.I1157T mutations (16/34, which were identified by restriction fragment length polymorphism (RFLP analysis. Thus, a quantitative hemolytic assay coupled with RFLP analysis enabled the early diagnosis of complement-mediated aHUS in 60% (27/45 of patients in Japan within a week of presentation. We hypothesize that this novel quantitative hemolytic assay would be more useful in a Caucasian population, who may have a higher proportion of CFH mutations than Japanese patients.

  18. The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens.

    Directory of Open Access Journals (Sweden)

    Melina Messaoudi

    Full Text Available For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664 from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.

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

    Science.gov (United States)

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

    2016-09-01

    The Hologic Aptima(®) HIV-1 Quant Dx assay (Aptima HIV) is a real-time transcription-mediated amplification method CE-approved for use in diagnosis and monitoring of HIV-1 infection. The analytical performance of this new assay was compared to the FDA-approved Abbott RealTime HIV-1 (RealTime). The evaluation was performed using 220 clinical plasma samples, the WHO 3rd HIV-1 International Standard, and the QCMD HIV-1 RNA EQA. Concordance on qualitative results, correlation between quantitative results, accuracy, and reproducibility of viral load data were analyzed. The ability to measure HIV-1 subtypes was assessed on the second WHO International Reference Preparation Panel for HIV-1 Subtypes. With clinical samples, inter-assay agreement for qualitative results was high (91.8%) with Cohen's kappa statistic equal to 0.836. For samples with quantitative results in both assays (n = 93), Lin's concordance correlation coefficient was 0.980 (P R(2)  > 0.970) and showed higher sensitivity compared to RealTime being able to detect HIV-1 RNA in 10 out of 10 replicates containing down to 7 cp/ml (20 IU/ml). Reproducibility was very high, even at low HIV-1 RNA values. The Aptima HIV was able to detect and accurately quantify all the main HIV-1 subtypes in both reference panels and clinical samples. Besides excellent performance, Aptima HIV shows full automation, ease of use, and improved workflow compared to RealTime. J. Med. Virol. 88:1535-1544, 2016. © 2016 Wiley Periodicals, Inc. PMID:26864171

  20. Development of a quantitative PCR assay for rapid detection of Lactobacillus plantarum and Lactobacillus fermentum in cocoa bean fermentation.

    Science.gov (United States)

    Schwendimann, Livia; Kauf, Peter; Fieseler, Lars; Gantenbein-Demarchi, Corinne; Miescher Schwenninger, Susanne

    2015-08-01

    To monitor dominant species of lactic acid bacteria during cocoa bean fermentation, i.e. Lactobacillus plantarum and Lactobacillus fermentum, a fast and reliable culture-independent qPCR assay was developed. A modified DNA isolation procedure using a commercial kit followed by two species-specific qPCR assays resulted in 100% sensitivity for L. plantarum and L. fermentum. Kruskal-Wallis and post-hoc analyses of data obtained from experiments with cocoa beans that were artificially spiked with decimal concentrations of L. plantarum and L. fermentum strains allowed the calculation of a regression line suitable for the estimation of both species with a detection limit of 3 to 4 Log cells/g cocoa beans. This process was successfully tested for efficacy through the analyses of samples from laboratory-scale cocoa bean fermentations with both the qPCR assay and a culture-dependent method which resulted in comparable results.

  1. The utility of quantitative methylation assays at imprinted genes for the diagnosis of fetal and placental disorders.

    Science.gov (United States)

    Bourque, D K; Peñaherrera, M S; Yuen, R K C; Van Allen, M I; McFadden, D E; Robinson, W P

    2011-02-01

    An imbalance of imprinted gene expression within 11p15.5 is observed in Beckwith-Wiedemann syndrome (BWS), as well as in a variety of placental abnormalities including complete hydatidiform mole (CHM), placental mesenchymal dysplasia (PMD) and triploidy. To facilitate the diagnosis of epigenetic errors and chromosomal imbalance of 11p15.5, we validated a pyrosequencing assay to measure methylation at KvDMR1 using blood samples from 13 BWS cases, 8 of which showed reduced methylation as compared to control blood. An imbalance between maternal and paternal genomes as is found in triploidy, CHM or PMD was also associated with altered KvDMR1 methylation. A reciprocal pattern of methylation was obtained in the triploid cases by assaying the proximal 11p15.5 ICR associated with H19. To distinguish chromosome 11 specific alterations from whole genome imbalance, other imprinted differentially methylated regions (DMRs) can be utilized. Thus, pyrosequencing assays for DMRs associated with SGCE, SNRPN, and MEST were also compared for their utility in diagnosing parental imbalance in placental samples. While each of these assays could successfully distinguish parental origin of triploidy, SGCE showed the clearest separation between groups. The combined use of a chromosome 11p15.5 assay (e.g. KvDMR1 or H19-ICR) and non-chromosome 11 assay (e.g. SGCE) provides a potentially valuable diagnostic tool in the rapid screening of methylation errors in placental disorders. These results also show the maintenance of imprinting status at these loci in the human placenta, even in the presence of abnormal pathology.

  2. Establishment of Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

    Institute of Scientific and Technical Information of China (English)

    LIAN Hong-xia; LU De-xun; GAO Min

    2009-01-01

    Porcine lipoprotein lipase (LPL) cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP 10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR (FQ-PCR). The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 103 to 1010 copies. The standard curves showed high correlations (R2=0.9871). A series of standards for real-time PCR analysis have been constructed successfully, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.

  3. Validation of Bacteroidales quantitative PCR assays targeting human and animal fecal contamination in the public and domestic domains in India.

    Science.gov (United States)

    Odagiri, Mitsunori; Schriewer, Alexander; Hanley, Kaitlyn; Wuertz, Stefan; Misra, Pravas R; Panigrahi, Pinaki; Jenkins, Marion W

    2015-01-01

    We compared host-associated Bacteroidales qPCR assays developed in the continental United States and Europe for the purpose of measuring the effect of improved sanitation on human fecal exposure in rural Indian communities where both human and animal fecal loading are high. Ten candidate Bacteroidales qPCR assays were tested against fecal samples (human, sewage, cow, buffalo, goat, sheep, dog and chicken) from a test set of 30 individual human, 5 sewage, and 60 pooled animal samples collected in coastal Odisha, India. The two universal/general Bacteroidales assays tested (BacUni, GenBac3) performed equally well, achieving 100% sensitivity on the test set. Across the five human-associated assays tested (HF183 Taqman, BacHum, HumM2, BacH, HF183 SYBR), we found low sensitivity (17 to 49%) except for HF183 SYBR (89%), and moderate to high cross-reactivity with dog (20 to 80%) and chicken fecal samples (60 to 100%). BacHum had the highest accuracy (67%), amplified all sewage samples within the range of quantification (ROQ), and did not cross-react with any fecal samples from cows, the most populous livestock animal in India. Of the ruminant- and cattle-associated assays tested (BacCow, CowM2), BacCow was more sensitive in detecting the full range of common Indian livestock animal fecal sources, while CowM2 only detected cow sources with 50% sensitivity. Neither assay cross-reacted with human sources. BacCan, the dog-associated assay tested, showed no cross-reactivity with human sources, and high sensitivity (90%) for dog fecal samples. Overall, our results indicate BacUni, BacHum, HumM2, BacCan and BacCow would be the most suitable MST assays to distinguish and quantify relative amounts of human-associated and livestock/domestic animal-associated contributions to fecal contamination in Odisha, India.

  4. Establishment and Validation of a Non-Radioactive Method for In Vitro Transcription Assay Using Primer Extension and Quantitative Real Time PCR.

    Science.gov (United States)

    Wang, Juan; Zhao, Shasha; Zhou, Ying; Wei, Yun; Deng, Wensheng

    2015-01-01

    Primer extension-dependent in vitro transcription assay is one of the most important approaches in the research field of gene transcription. However, conventional in vitro transcription assays incorporates radioactive isotopes that cause environmental and health concerns and restricts its scope of application. Here we report a novel non-radioactive method for in vitro transcription analysis by combining primer extension with quantitative real time PCR (qPCR). We show that the DNA template within the transcription system can be effectively eliminated to a very low level by our specially designed approach, and that the primers uniquely designed for primer extension and qPCR can specifically recognize the RNA transcripts. Quantitative PCR data demonstrate that the novel method has successfully been applied to in vitro transcription analyses using the adenovirus E4 and major late promoters. Furthermore, we show that the TFIIB recognition element inhibits transcription of TATA-less promoters using both conventional and nonradioactive in vitro transcription assays. Our method will benefit the laboratories that need to perform in vitro transcription but either lack of or choose to avoid radioactive facilities.

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

    DEFF Research Database (Denmark)

    Larsen, Hans Henrik; Kovacs, Joseph A; Stock, Frida;

    2002-01-01

    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...

  6. Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses.

    Science.gov (United States)

    Simmons, Monika; Myers, Todd; Guevara, Carolina; Jungkind, Donald; Williams, Maya; Houng, Huo-Shu

    2016-07-01

    Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), and in vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping. PMID:27098955

  7. Real-time quantitative PCR assay with Taqman(®) probe for rapid detection of MCR-1 plasmid-mediated colistin resistance.

    Science.gov (United States)

    Chabou, S; Leangapichart, T; Okdah, L; Le Page, S; Hadjadj, L; Rolain, J-M

    2016-09-01

    Here we report the development of two rapid real-time quantitative PCR assays with TaqMan(®) probes to detect the MCR-1 plasmid-mediated colistin resistance gene from bacterial isolates and faecal samples from chickens. Specificity and sensitivity of the assay were 100% on bacterial isolates including 18 colistin-resistant isolates carrying the mcr-1 gene (six Klebsiella pneumoniae and 12 Escherichia coli) with a calibration curve that was linear from 10(1) to 10(8) DNA copies. Five out of 833 faecal samples from chickens from Algeria were positive, from which three E. coli strains were isolated and confirmed to harbour the mcr-1 gene by standard PCR and sequencing. PMID:27489722

  8. A High-Performance Liquid Chromatography-Mass Spectrometry Assay for Quantitation of the Tyrosine Kinase Inhibitor Nilotinib in Human Plasma and Serum

    OpenAIRE

    Parise, Robert A.; Egorin, Merrill J.; Christner, Susan M; Shah, Dhvani D; Zhou, Wei; Beumer, Jan H.

    2009-01-01

    Nilotinib (AMN-107, Tasigna™) is a small-molecule inhibitor of BCR/ABL, approved for chronic myelogenous leukemia. We developed and validated, according to FDA-guidelines, an LC-MS assay for sensitive, accurate and precise quantitation of nilotinib in 0.2 mL human plasma or serum. After acetonitrile protein precipitation, separation is achieved with a hydro-Synergi column and a 0.1% formic acid in methanol/water-gradient. Detection uses electrospray, positive-mode ionization mass spectrometry...

  9. Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

    OpenAIRE

    Pizeta Semighini, Camile; Marins, Mozart; Goldman, Maria Helena S.; Goldman, Gustavo Henrique

    2002-01-01

    The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated dr...

  10. Quantitation of sodium 35S-sulphate in nano mole levels by spectrophotometric assay using barium chloranilate and liquid scintillation counting

    International Nuclear Information System (INIS)

    In this assay, the absorbance of chloranilic acid released from buffered suspension of barium chloranilate, taken in excess, on reaction with solutions of known concentrations of authentic sodium sulphate (2-10 fg; 14-70 nanomoles) and that of sodium 35S-sulphate of known radioactivity (10-20 mci; 370-740 mBq) is measured spectrophotometrically and the quantitation done accordingly. The amount of chloranilic acid released has been found to be directly proportional to the amount of sulphate present in the original sample solution. (author). 8 refs., 1 tab

  11. Development of an isoform-specific tandem mass spectrometry assay for absolute quantitation of maize lipid transfer proteins.

    Science.gov (United States)

    Stevenson, Severin E; McClain, Scott; Thelen, Jay J

    2015-01-28

    Precise and accurate quantitation of maize grain allergens is important for seed and food industries. The major allergen in maize grain is Zea m 14, a lipid transfer protein (LTP). The B73 maize genome encodes for at least six LTPs sharing 15%-87% sequence identity to Zea m 14. Phylogenetic analysis of the maize LTP family revealed one gene that corresponds to Zea m 14 (denoted as LTPa) and two other genes sharing 43% (LTPc) and 74% (LTPb) identity with Zea m 14 that are putative homologues. Using stable isotope peptide mimics as internal standards for LTPs, we present a multiple reaction monitoring mass spectrometry approach for multiplexed, absolute quantitation of all three LTP proteins and alternative transcript models therein. To validate quantitative accuracy, a redundant peptide, simultaneously representing the two most abundant LTPs, was included. Analysis of 21 maize varieties revealed LTPa was most prominently expressed in maize grain, ranging from 9 to 32 μg LTP/mg protein. Proteins belonging to the LTPb and LTPc gene models were also expressed but at approximately 10- and 100-fold lower levels than LTPa, respectively. The quantitative results provided by the redundant peptide show around 95% agreement with the sum of the two unique peptides, thus providing support for the LTP gene models and validating the accuracy of this method. Though not all Zea m 14-related LTPs are abundant in grain, their high sequence homology and detectable expression in maize grain signify that LTPb and LTPc are putative allergens and should be accounted for in any quantitation strategy for maize LTP allergens. PMID:25540820

  12. Development of a novel HAC-based "gain of signal" quantitative assay for measuring chromosome instability (CIN) in cancer cells

    OpenAIRE

    Kim, Jung Hyun; Lee, Hee Sheung; Lee, Nicholas C.O.; Goncharov, Nikolay V.; Kumeiko, Vadim; Masumoto, Hiroshi; Earnshaw, William C.; Kouprina, Natalay; Larionov, Vladimir

    2016-01-01

    Accumulating data indicates that chromosome instability (CIN) common to cancer cells can be used as a target for cancer therapy. At present the rate of chromosome mis-segregation is quantified by laborious techniques such as coupling clonal cell analysis with karyotyping or fluorescence in situ hybridization (FISH). Recently, a novel assay was developed based on the loss of a non-essential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene ("loss of signal" a...

  13. A Fluorescence Immunochromatographic Assay Using Europium (III) Chelate Microparticles for Rapid, Quantitative and Sensitive Detection of Creatine Kinase MB.

    Science.gov (United States)

    Lai, Xiao-Hong; Liang, Rong-Liang; Liu, Tian-Cai; Dong, Zhi-Ning; Wu, Ying-Song; Li, Lin-Hai

    2016-05-01

    The isoenzyme creatine kinase MB is very important for diagnosis of acute myocardial infarction (AMI). Some CK-MB immunoassays are sensitive, accurate and available for clinical application, but they are expensive and time-consuming procedures. Furthermore, conventional fluorescence immunochromatographic assays (FL-ICAs) have suffered from background fluorescence interference and low analytical sensitivity. A rapid and simple FL-ICA with Eu (III) chelate polystyrene microparticles was developed to determine CK-MB in 50uL serum samples using a portable test strip reader by measuring the fluorescence peak heights of the test line (HT) and the control line (HC) in 12 min. The assay was reliable with a good correlation coefficient between HT/HC ratio and CK-MB concentration in samples. A linear range was 0.85-100.29 ng/mL for CK-MB, and the LOD was 0.029 ng/mL. The intra- and inter-assay coefficients of variation (CV) were both chelate microparticles-based FL-ICA is simple, fast, highly sensitive, reliable, and reproducible for point-of-care testing of CK-MB concentrations in serum. Graphical Abstract ᅟ.

  14. Identification and validation of suitable housekeeping genes for normalizing quantitative real-time PCR assays in injured peripheral nerves

    OpenAIRE

    Gambarotta, Giovanna; Ronchi, Giulia; Friard, Olivier; Galletta, Pantaleo; Perroteau, Isabelle; GEUNA, Stefano

    2014-01-01

    Injury to the peripheral nerve induces dramatic changes in terms of cellular composition that are reflected on RNA quality and quantity, making messenger RNA expression analysis very complex. Several commonly used housekeeping genes are regulated following peripheral nerve injury and are thus not suitable for quantitative real-time PCR normalization; moreover, the presence of pseudogenes in some of them impairs their use. To deal with this problem, we have developed a new method to identify n...

  15. Duplex quantitative real-time PCR assay for the detection and discrimination of the eggs of Toxocara canis and Toxocara cati (Nematoda, Ascaridoidea in soil and fecal samples

    Directory of Open Access Journals (Sweden)

    Durant Jean-Francois

    2012-12-01

    Full Text Available Abstract Background Toxocarosis is a zoonotic disease caused by Toxocara canis (T. canis and/or Toxocara cati (T. cati, two worldwide distributed roundworms which are parasites of canids and felids, respectively. Infections of humans occur through ingestion of embryonated eggs of T. canis or T. cati, when playing with soils contaminated with dogs or cats feces. Accordingly, the assessment of potential contamination of these areas with these roundworms eggs is paramount. Methods A duplex quantitative real-time PCR (2qPCR targeting the ribosomal RNA gene internal transcribed spacer (ITS2 has been developed and used for rapid and specific identification of T. canis and T. cati eggs in fecal and soil samples. The assay was set up on DNA samples extracted from 53 adult worms including T. canis, T. cati, T. leonina, Ascaris suum (A. suum and Parascaris equorum (P. equorum. The assay was used to assess the presence of T. cati eggs in several samples, including 12 clean soil samples spiked with eggs of either T. cati or A. suum, 10 actual soil samples randomly collected from playgrounds in Brussels, and fecal samples from cats, dogs, and other animals. 2qPCR results on dogs and cats fecal samples were compared with results from microscopic examination. Results 2qPCR assay allowed specific detection of T. canis and T. cati, whether adult worms, eggs spiked in soil or fecal samples. The 2qPCR limit of detection (LOD in spiked soil samples was 2 eggs per g of soil for a turnaround time of 3 hours. A perfect concordance was observed between 2qPCR assay and microscopic examination on dogs and cats feces. Conclusion The newly developed 2qPCR assay can be useful for high throughput prospective or retrospective detection of T.canis and/or T. cati eggs in fecal samples as well as in soil samples from playgrounds, parks and sandpits.

  16. Highly sensitive and broadly reactive quantitative reverse transcription-PCR assay for high-throughput detection of Rift Valley fever virus.

    Science.gov (United States)

    Bird, Brian H; Bawiec, Darcy A; Ksiazek, Thomas G; Shoemaker, Trevor R; Nichol, Stuart T

    2007-11-01

    Rift Valley fever (RVF) virus is a mosquito-borne virus associated with large-scale epizootics/epidemics throughout Africa and the Arabian peninsula. Virus infection can result in economically disastrous "abortion storms" and high newborn mortality in livestock. Human infections result in a flu-like illness, with 1 to 2% of patients developing severe complications, including encephalitis or hemorrhagic fever with high fatality rates. There is a critical need for a highly sensitive and specific molecular diagnostic assay capable of detecting the natural genetic spectrum of RVF viruses. We report here the establishment of a pan-RVF virus quantitative real-time reverse transcription-PCR assay with high analytical sensitivity (approximately 5 RNA copies of in vitro-transcribed RNA/reaction or approximately 0.1 PFU of infectious virus/reaction) and efficiency (standard curve slope = -3.66). Based on the alignments of the complete genome sequences of 40 ecologically and biologically diverse virus isolates collected over 56 years (1944 to 2000), the primer and probe annealing sites targeted in this assay are known to be located in highly conserved genomic regions. The performance of this assay relative to serologic assays is illustrated by testing of known RVF case materials obtained during the Saudi Arabia outbreak in 2000. Furthermore, analysis of acute-phase blood samples collected from human patients (25 nonfatal, 8 fatal) during that outbreak revealed that patient viremia at time of presentation at hospital may be a useful prognostic tool in determining patient outcome. PMID:17804663

  17. Interlaboratory comparison of three microbial source tracking quantitative polymerase chain reaction (qPCR) assays from fecal-source and environmental samples

    Science.gov (United States)

    Stelzer, Erin A.; Strickler, Kriston M.; Schill, William B.

    2012-01-01

    During summer and early fall 2010, 15 river samples and 6 fecal-source samples were collected in West Virginia. These samples were analyzed by three laboratories for three microbial source tracking (MST) markers: AllBac, a general fecal indicator; BacHum, a human-associated fecal indicator; and BoBac, a ruminant-associated fecal indicator. MST markers were analyzed by means of the quantitative polymerase chain reaction (qPCR) method. The aim was to assess interlaboratory precision when the three laboratories used the same MST marker and shared deoxyribonucleic acid (DNA) extracts of the samples, but different equipment, reagents, and analyst experience levels. The term assay refers to both the markers and the procedure differences listed above. Interlaboratory precision was best for all three MST assays when using the geometric mean absolute relative percent difference (ARPD) and Friedman's statistical test as a measure of interlaboratory precision. Adjustment factors (one for each MST assay) were calculated using results from fecal-source samples analyzed by all three laboratories and applied retrospectively to sample concentrations to account for differences in qPCR results among labs using different standards and procedures. Following the application of adjustment factors to qPCR results, ARPDs were lower; however, statistically significant differences between labs were still observed for the BacHum and BoBac assays. This was a small study and two of the MST assays had 52 percent of samples with concentrations at or below the limit of accurate quantification; hence, more testing could be done to determine if the adjustment factors would work better if the majority of sample concentrations were above the quantification limit.

  18. Aggregation of polyQ proteins is increased upon yeast aging and affected by Sir2 and Hsf1: novel quantitative biochemical and microscopic assays.

    Directory of Open Access Journals (Sweden)

    Aviv Cohen

    Full Text Available Aging-related neurodegenerative disorders, such as Parkinson's, Alzheimer's and Huntington's diseases, are characterized by accumulation of protein aggregates in distinct neuronal cells that eventually die. In Huntington's disease, the protein huntingtin forms aggregates, and the age of disease onset is inversely correlated to the length of the protein's poly-glutamine tract. Using quantitative assays to estimate microscopically and capture biochemically protein aggregates, here we study in Saccharomyces cerevisiae aging-related aggregation of GFP-tagged, huntingtin-derived proteins with different polyQ lengths. We find that the short 25Q protein never aggregates whereas the long 103Q version always aggregates. However, the mid-size 47Q protein is soluble in young logarithmically growing yeast but aggregates as the yeast cells enter the stationary phase and age, allowing us to plot an "aggregation timeline". This aging-dependent aggregation was associated with increased cytotoxicity. We also show that two aging-related genes, SIR2 and HSF1, affect aggregation of the polyQ proteins. In Δsir2 strain the aging-dependent aggregation of the 47Q protein is aggravated, while overexpression of the transcription factor Hsf1 attenuates aggregation. Thus, the mid-size 47Q protein and our quantitative aggregation assays provide valuable tools to unravel the roles of genes and environmental conditions that affect aging-related aggregation.

  19. Dried blood spots versus plasma for the quantitation of HIV-1 RNA using a real-Time PCR, m2000rt assay.

    Science.gov (United States)

    Vidya, Madhavan; Saravanan, Shanmugam; Rifkin, Samara; Solomon, Sunil S; Waldrop, Greer; Mayer, Kenneth H; Solomon, Suniti; Balakrishnan, Pachamuthu

    2012-05-01

    High costs and stringent requirements for storage and transport of plasma, often prohibit the availability of HIV viral load quantification in resource-limited settings. Dried blood spots (DBS) represent a better method of specimen collection that removes many of these logistical and technical limitations. The present study aimed to assess the performance of the Abbott m2000rt assay for quantitation of HIV-1 RNA in DBS specimens using plasma as a "gold standard" for comparison. One hundred paired DBS and plasma specimens were collected from patients infected with HIV, who were 18 years and older during routine visits to a private tertiary-care clinic in Chennai, India. HIV-1 RNA was extracted manually and then detected using the m2000rt assay. The mean plasma and DBS viral loads were 4.27 (95% CI: 2.65, 5.88) and 4.14 (95% CI: 1.96, 6.32) log copies/mL, respectively. The overall sensitivity of DBS reached 95%; with sensitivities of 62%, 88% and 100% when stratified by viral load ranges of ≤1000, 1000-3000 and >3000 copies/mL, respectively. An over quantitation of the viral load with DBS was observed in pairs with plasma viral loadfailure in resource-limited settings. PMID:22401801

  20. Development of monoclonal antibodies and quantitative sandwich enzyme linked immunosorbent assay for the characteristic sialoglycoprotein of edible bird's nest.

    Science.gov (United States)

    Zhang, Shiwei; Lai, Xintian; Liu, Xiaoqing; Li, Yun; Li, Bifang; Huang, Xiuli; Zhang, Qinlei; Chen, Wei; Lin, Lin; Yang, Guowu

    2013-01-01

    The article presents a sandwich enzyme linked immunosorbent assay (ELISA) for identification of edible bird's nest. The characteristic sialoglycoproteins were found by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and purified by liquid-phase isoelectric focusing (LIEF). According to the analysis, the molecular weight was 106-128 kDa and the isoelectric point was ≤pH 3.0. Two anti-characteristic sialoglycoprotein monoclonal antibodies were produced. The monoclonal antibodies were examined by western-blot assay. One of the monoclonal antibody was used as coating and the other as the enzyme-labeled antibody after being coupled to horseradish peroxidase (HRP). Based on the optimized ELISA condition, the method was established with IC(50) of 1.5 ng/mL, and low cross-reactivity with various fake materials (ELISA provided a suitable means for screening of a large number of samples. The coefficients of variation were between 2.9% and 5.8%.

  1. The development of a quantitative assay for the detection of anti-Ro/SS-A and anti-LA/SS-B autoantibodies using purified recombinant proteins.

    Science.gov (United States)

    Veldhoven, C H; Meilof, J F; Huisman, J G; Smeenk, R J

    1992-07-01

    A characteristic of patients with autoimmune diseases such as Sjögren's syndrome and systemic lupus erythematosus is the presence of anti-Ro/SS-A and anti-La/SS-B autoantibodies in their circulation. In order to investigate specific autoantibody levels in the sera of these patients quantitative assays for the detection of both anti-Ro/SS-A and anti-La/SS-B reactivity were developed. Ro/SS-A (60 kDa) and La-SS-B (50 kDa) cDNAs were cloned and expressed in E. coli as non-fusion proteins. These were purified to homogeneity using two different purification protocols. With these recombinant antigens, specific enzyme-linked immunosorbent assays (ELISAs) were developed. 40 sera positive for anti-Ro/SS-A autoantibodies in counterimmunoelectrophoresis (CIE) were tested in both the Ro/SS-A and La/SS-B ELISA. Activity values reproducibly ranged from 1536 to 120,000 U in the Ro/SS-A ELISA and from 763 to 2,500,000 U in the La/SS-B ELISA. The suitability of these ELISAs as screening assays was further investigated by testing 200 sera sent to our laboratory for routine detection of autoantibodies to extractable nuclear antigen (ENA: anti-Sm, anti-RNP, anti-Ro/SS-A and anti-La/SS-B). Both ELISAs showed a high sensitivity and specificity (Ro/SS-A ELISA 85% and 94%, La/SS-B ELISA 100% and 98% respectively), when compared to the standard assays, the RNA-precipitation assay and the HeLa immunoblotting test. From these data we conclude that a quantitative analysis of both anti-Ro/SS-A and anti-La/SS-B autoantibodies is now possible using purified recombinant non-fusion proteins. For screening purposes the La/SS-B ELISA showed a great improvement in sensitivity for the detection of anti-La/SS-B activity in comparison to the La/SS-B CIE, while the Ro/SS-A ELISA almost equalled the performance of the Ro/SS-A CIE.

  2. Allele Specific Locked Nucleic Acid Quantitative PCR (ASLNAqPCR): An Accurate and Cost-Effective Assay to Diagnose and Quantify KRAS and BRAF Mutation

    Science.gov (United States)

    Morandi, Luca; de Biase, Dario; Visani, Michela; Cesari, Valentina; De Maglio, Giovanna; Pizzolitto, Stefano; Pession, Annalisa; Tallini, Giovanni

    2012-01-01

    The use of tyrosine kinase inhibitors (TKIs) requires the testing for hot spot mutations of the molecular effectors downstream the membrane-bound tyrosine kinases since their wild type status is expected for response to TKI therapy. We report a novel assay that we have called Allele Specific Locked Nucleic Acid quantitative PCR (ASLNAqPCR). The assay uses LNA-modified allele specific primers and LNA-modified beacon probes to increase sensitivity, specificity and to accurately quantify mutations. We designed primers specific for codon 12/13 KRAS mutations and BRAF V600E, and validated the assay with 300 routine samples from a variety of sources, including cytology specimens. All were analyzed by ASLNAqPCR and Sanger sequencing. Discordant cases were pyrosequenced. ASLNAqPCR correctly identified BRAF and KRAS mutations in all discordant cases and all had a mutated/wild type DNA ratio below the analytical sensitivity of the Sanger method. ASLNAqPCR was 100% specific with greater accuracy, positive and negative predictive values compared with Sanger sequencing. The analytical sensitivity of ASLNAqPCR is 0.1%, allowing quantification of mutated DNA in small neoplastic cell clones. ASLNAqPCR can be performed in any laboratory with real-time PCR equipment, is very cost-effective and can easily be adapted to detect hot spot mutations in other oncogenes. PMID:22558339

  3. High Specificity of a Quantitative PCR Assay Targeting a Saxitoxin Gene for Monitoring Toxic Algae Associated with Paralytic Shellfish Toxins in the Yellow Sea.

    Science.gov (United States)

    Gao, Yan; Yu, Ren-Cheng; Murray, Shauna A; Chen, Jian-Hua; Kang, Zhen-Jun; Zhang, Qing-Chun; Kong, Fan-Zhou; Zhou, Ming-Jiang

    2015-10-01

    The identification of core genes involved in the biosynthesis of saxitoxin (STX) offers a great opportunity to detect toxic algae associated with paralytic shellfish toxins (PST). In the Yellow Sea (YS) in China, both toxic and nontoxic Alexandrium species are present, which makes it a difficult issue to specifically monitor PST-producing toxic algae. In this study, a quantitative PCR (qPCR) assay targeting sxtA4, a domain in the sxt gene cluster that encodes a unique enzyme involved in STX biosynthesis, was applied to analyze samples collected from the YS in spring of 2012. The abundance of two toxic species within the Alexandrium tamarense species complex, i.e., A. fundyense and A. pacificum, was also determined with TaqMan-based qPCR assays, and PSTs in net-concentrated phytoplankton samples were analyzed with high-performance liquid chromatography coupled with a fluorescence detector. It was found that the distribution of the sxtA4 gene in the YS was consistent with the toxic algae and PSTs, and the quantitation results of sxtA4 correlated well with the abundance of the two toxic species (r=0.857). These results suggested that the two toxic species were major PST producers during the sampling season and that sxtA-based qPCR is a promising method to detect toxic algae associated with PSTs in the YS. The correlation between PST levels and sxtA-based qPCR results, however, was less significant (r=0.552), implying that sxtA-based qPCR is not accurate enough to reflect the toxicity of PST-producing toxic algae. The combination of an sxtA-based qPCR assay and chemical means might be a promising method for monitoring toxic algal blooms. PMID:26231652

  4. A quantitative polymerase chain reaction assay for the enumeration of brown tide algae Aureococcusanophagefferens in coastal waters of Qinhuangdao

    Institute of Scientific and Technical Information of China (English)

    GUO Hao; LIU Yongjian; ZHANG Qi; YUAN Xiutang; ZHANG Weiwei; ZHANG Zhifeng

    2015-01-01

    Aureococcus anophagefferens, a small pelagophyte algae, has caused brown tide blooms in coastal waters of Qinhua-ngdao in recent years, presenting significant negative impacts on the shellfish mariculture industry. Under standard light microscopy, it is visually indistinguishable from other small algae in field samples due to its extremely small size. In this study, quantitative polymerase chain reaction (qPCR) based on 18S rDNA sequences was developed and used to detect and enumerate A. anophagefferens. A linear regression (R2=0.91) was generated based on cycle thr-esholds value (Ct) versus known concentrations of A. anophagefferens. Twenty-two field samples collected in coastal waters of Qinhuangdao were subjected to DNA extraction and then analyzed using qPCR. Results showed that A. anophagefferens had a wide distribution in coastal waters along Qinhuangdao. Elevated A. anophagefferens abun-dance, category 3 brown tide blooms (>200 000 cells/mL) occurred at Dongshan Beach and Tiger-stone Beach in August in 2013. In shellfish mariculture areas along coastal waters of Qinhuangdao, 4 stations had category 3 blooms, and 6 stations had category 2 blooms (35 000–200 000 cells/mL) in August and all stations had category 1 blooms (>0 to ≤35 000 cells/mL) in October. Quantitative PCR allows for detection of A. anophagefferens cells at low levels in filed samples, which is essential to effective management and prediction of brown tide blooms.

  5. Validation of a quantitative cerebrospinal fluid alpha-synuclein assay in a European-wide interlaboratory study

    DEFF Research Database (Denmark)

    Kruse, N.; Persson, S.; Alcolea, D.;

    2015-01-01

    Decreased levels of alpha-synuclein (aSyn) in cerebrospinal fluid (CSF) in Parkinson's disease and related synucleinopathies have been reported, however, not consistently in all cross-sectional studies. To test the performance of one recently released human-specific enzyme-linked immunosorbent...... assay (ELISA) for the quantification of aSyn in CSF, we carried out a round robin trial with 18 participating laboratories trained in CSF ELISA analyses within the BIOMARKAPD project in the EU Joint Program -Neurodegenerative Disease Research. CSF samples (homogeneous aliquots from pools) and ELISA kits...... (one lot) were provided centrally and data reported back to one laboratory for data analysis. Our study showed that although factors such as preanalytical sample handling and lot-to-lot variability were minimized by our study design, we identified high variation in absolute values of CSF aSyn even when...

  6. Comparative quantitation for the protein content of diphtheria and tetanus toxoids by DC protein assay and Kjeldahl method.

    Science.gov (United States)

    Doshi, J B; Ravetkar, S D; Ghole, V S; Rehani, K

    2003-09-01

    DPT, a combination vaccine against diphtheria, tetanus and pertussis is available since many years and still continued in the national immunisation schedule of many countries. Although highly potent, reactions to DPT vaccine are well known, mainly attributed to the factors like Pertussis component, aluminum adjuvant and lower purity of tetanus and diphtheria toxoids. The latter most important aspect has become a matter of concern, specially for the preparation of next generation combination vaccines with more number of antigens in combination with DPT. Purity of toxoid is expressed as Lf (Limes flocculation) per mg of protein nitrogen. The Kjeldahl method (KM) of protein nitrogen estimation suggested by WHO and British Pharmacopoeia is time consuming and less specific. Need has been felt to explore an alternative method which is quick and more specific for toxoid protein determination. DC (detergent compatible) protein assay, an improved Lowry's method, has been found to be much more advantageous than Kjeldahl method.

  7. Quantitation of minimal disease levels in chronic lymphocytic leukemia using a sensitive flow cytometric assay improves the prediction of outcome and can be used to optimize therapy.

    Science.gov (United States)

    Rawstron, A C; Kennedy, B; Evans, P A; Davies, F E; Richards, S J; Haynes, A P; Russell, N H; Hale, G; Morgan, G J; Jack, A S; Hillmen, P

    2001-07-01

    Previous studies have suggested that the level of residual disease at the end of therapy predicts outcome in chronic lymphocytic leukemia (CLL). However, available methods for detecting CLL cells are either insensitive or not routinely applicable. A flow cytometric assay was developed that can differentiate CLL cells from normal B cells on the basis of their CD19/CD5/CD20/CD79b expression. The assay is rapid and can detect one CLL cell in 10(4) to 10(5) leukocytes in all patients. We have compared this assay to conventional assessment in 104 patients treated with CAMPATH-1H and/or autologous transplant. During CAMPATH-1H therapy, circulating CLL cells were rapidly depleted in responding patients, but remained detectable in nonresponders. Patients with more than 0.01 x 10(9)/L circulating CLL cells always had significant (> 5%) marrow disease, and blood monitoring could be used to time marrow assessments. In 25 out of 104 patients achieving complete remission by National Cancer Institute (NCI) criteria, the detection of residual bone marrow disease at more than 0.05% of leukocytes in 6 out of 25 patients predicted significantly poorer event-free (P =.0001) and overall survival (P =.007). CLL cells are detectable at a median of 15.8 months (range, 5.5-41.8) posttreatment in 9 out of 18 evaluable patients with less than 0.05% CLL cells at end of treatment. All patients with detectable disease have progressively increasing disease levels on follow-up. The use of sensitive techniques, such as the flow assay described here, allow accurate quantitation of disease levels and provide an accurate method for guiding therapy and predicting outcome. These results suggest that the eradication of detectable disease may lead to improved survival and should be tested in future studies.

  8. Absolute quantitation of Met using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue.

    Directory of Open Access Journals (Sweden)

    Daniel V T Catenacci

    Full Text Available Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC of formalin-fixed paraffin-embedded (FFPE tissues is currently used to select for 'high Met' expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue.After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH.Proteomic mapping of recombinant Met identified 418TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD and quantitation (LOQ for this peptide were 150 and 200 amol/µg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/µg to 4669.5 amol/µg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898. IHC did not correlate well with SRM (n = 44; R2 = 0.537 nor FISH GCN (n = 31; R2 = 0.509. A Met SRM level of ≥1500 amol/µg was 100% sensitive (95% CI 0.69-1 and 100% specific (95% CI 0.92-1 for MET amplification.The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel

  9. Development of three stable isotope dilution assays for the quantitation of (E)-2-butenal (crotonaldehyde) in heat-processed edible fats and oils as well as in food.

    Science.gov (United States)

    Granvogl, Michael

    2014-02-12

    Three stable isotope dilution assays (SIDAs) were developed for the quantitation of (E)-2-butenal (crotonaldehyde) in heat-processed edible fats and oils as well as in food using synthesized [¹³C₄]-crotonaldehyde as internal standard. First, a direct headspace GC-MS method, followed by two indirect methods on the basis of derivatization with either pentafluorophenylhydrazine (GC-MS) or 2,4-dinitrophenylhydrazine (LC-MS/MS), was developed. All methods are also suitable for the quantitation of acrolein using the standard [¹³C₃]-acrolein. Applying these three methods on five different types of fats and oils varying in their fatty acid compositions revealed significantly varying crotonaldehyde concentrations for the different samples, but nearly identical quantitative data for all methods. Formed amounts of crotonaldehyde were dependent not only on the type of oil, e.g., 0.29-0.32 mg/kg of coconut oil or 33.9-34.4 mg/kg of linseed oil after heat-processing for 24 h at 180 °C, but also on the applied temperature and time. The results indicated that the concentration of formed crotonaldehyde seemed to be correlated with the amount of linolenic acid in the oils. Furthermore, the formation of crotonaldehyde was compared to that of its homologue acrolein, demonstrating that acrolein was always present in higher amounts in heat-processed oils, e.g., 12.3 mg of crotonaldehyde/kg of rapeseed oil in comparison to 23.4 mg of acrolein/kg after 24 h at 180 °C. Finally, crotonaldehyde was also quantitated in fried food, revealing concentrations from 12 to 25 μg/kg for potato chips and from 8 to 19 μg/kg for donuts, depending on the oil used.

  10. QUANTITATIVE ASSAY OF ALMOTRIPTAN MALATE IN PURE DRUG AND PHARMACEUTICAL PREPARATIONS USING SIMPLE AND CONVENIENT VISIBLE SPECTROPHOTOMETRIC METHODS

    Directory of Open Access Journals (Sweden)

    U. VIPLOVA PRASAD

    2012-05-01

    Full Text Available Two direct, simple and sensitive visible spectrophotometric methods (M1&M2 are described for the assay of almotripan malate in pure and solid dosage forms. The method M1 involves oxidative coupling of drug with brucine in presence of sodium meta periodate and purple red colored species is formed and exhibits absorption maxima at 520nm. The method M2 is based on the formation of yellowish brown colored species by the drug with Folin reagent and exhibits absorption maxima at 450nm. Regression analysis of Beer-Lambert plots showed good correlation in the concentration ranges (8.0-24 μg/ml for method M1, (16-48 μg/ml for method M2 respectively. The proposed methods are applied to commercial available tablets and the results are statistically compared with those obtained by the reported UV reference method and validated by recovery studies. The results are found satisfactory and reproducible. These methods are applied successfully for the estimation of the almotriptan malate in the presence of other ingredients that are usually present in dosageforms. These methods offer the advantages of rapidity, simplicity and sensitivity and normal cost and can be easily applied to resource-poor settings without the need for expensive instrumentation and reagents.

  11. Establishment of TaqMan Real-time Quantitative PCR Assay for Foreign Gene Copy Numbers in Transgenic Soybean

    Institute of Scientific and Technical Information of China (English)

    Qiu You-wen; Gao Xue-jun; Qi Bang-ruo; Li Lu; Zhen Zhen

    2012-01-01

    TaqMan quantitative PCR technique was used to detect the copies of exogenous CaMV35S flanks sequence in transgenic soybean. With soybean lectin as the endogenous reference gene, and gene complex DNA in non-GMO soybeans as the endogenous reference standard, the gradient dilution method was used to separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies, and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation. The standard curve equation of endogenous reference gene was y =–3.422x+35.201, R2=0.998; the standard curve equation of exogenous gene was y =–3.495x+35.303, R2=0.999. The sample copies was got by putting Ct value into the standard curve equation, and it was the ratio of exogenous gene and reference gene. We found that CaMV35S gene in transgenic soy was single copy.

  12. An in vitro system combined with an in-house quantitation assay for screening hepatitis C virus inhibitors.

    Science.gov (United States)

    Ho, Tin-Yun; Wu, Shih-Lu; Lai, I-Lu; Cheng, Ken-Sheng; Kao, Shung-Te; Hsiang, Chien-Yun

    2003-05-01

    Hepatitis C virus (HCV) infection is a serious global health problem. Interferon-alpha (IFN-alpha) and ribavirin have demonstrated efficacy in the treatment of HCV infection; however, these therapies display many side effects. To screen the anti-HCV compounds from plants, we established an in vitro model for inoculation of HCV by centrifugation-facilitated method. The HCV RNA molecules were then quantitated by nested competitive reverse transcription-polymerase chain reaction (cRT-PCR) using fluorescein-labeled primers, and analyzed by ABI Prism 310. The positive and negative strands of HCV RNA were detectable in Vero cells on Day 7 post-infection, suggesting that the HCV RNA was present in the cell model system. The cell culture system was further used to screen the anti-HCV activities of 4 Chinese herbal formulas and 15 formula components. IFN-alpha showed an antiviral effect. The formulas exhibited no anti-HCV activities, while Arnebia euchroma, Thlaspi arvense, and Poncirus trifoliata displayed anti-HCV activities. Therefore, these results pointed out the possibility by using the cell culture system established in this study to screen the herb extracts for their anti-HCV activities. PMID:12767467

  13. In-vitro Quantitative Assay of Interferon Gamma in Serum of Nigerian Indigenous and Exotic Breeds of Chickens

    Directory of Open Access Journals (Sweden)

    Esan Oluwaseun and Oladele Omolade

    2014-12-01

    Full Text Available The Nigerian Indigenous breeds of Chicken (NIC have thrived in harsh tropical environment with little veterinary care and poor nutrition compared with the introduced exotic breeds which performs sub-optimally in the tropics. However, they receive little attention for commercial production in spite of low input required. A comparative assessment of cellular immune response of the indigenous and exotic breeds was carried out to provide scientific explanation for their hardy nature and justify production for economic purposes. Fifteen chickens from each of three indigenous breeds i.e. Frizzled- feathered, Naked-neck and Smooth-feathered, and 8 Isa Brown pullets were 10 weeks old and reared in separate cages. The chickens were stabilized and administered Newcastle Disease Vaccine (NDV, LaSota strain. At 14 and 16 weeks old, all breeds were administered NDV Komarov strain in Freund’s adjuvant and in PBS intramuscularly as sensitizing and challenge inoculants, respectively. They were bled for serum 5 days later and concentrations of Interferon-gamma (IFN-gamma were determined using competitive Enzyme-linked immunosorbent assay. Results showed that the Frizzled-feathered chickens had the highest concentration of IFN-gamma (58±2.8 pg/ml which was significantly higher than 49±3.2 pg/ml and 44±2.5 pg/ml recorded for Smooth-feathered and Isa brown breeds respectively. Also, concentration in Naked-neck breed was 54±2.9 pg/ml, which was significantly higher than Isa Brown. Isa Brown had the significantly lowest concentration. It was concluded that the three NIC studied, have inherent capacity to mount higher levels of cellular immune response compared with the exotic Isa brown, when challenged.

  14. Quantitation of pulmonary surfactant protein SP-B in the absence or presence of phospholipids by enzyme-linked immunosorbent assay

    DEFF Research Database (Denmark)

    Oviedo, J M; Valiño, F; Plasencia, I;

    2001-01-01

    used to explore the effect of the presence of different phospholipids on the immunoreactivity of SP-B. Both polyclonal and monoclonal antibodies produced reproducible ELISA calibration curves for methanolic SP-B solutions with protein concentrations in the range of 20-1000 ng/mL. At these protein...... concentrations, neither dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, nor phosphatidylcholine or phosphatidylglycerol from egg yolk had significant effects on the binding of antibodies to SP-B up to protein-to-lipid weight ratios of 1:20. Coating of ELISA plates with SP-B concentrations higher......We have developed an enzyme-linked immunosorbent assay (ELISA) that uses polyclonal or monoclonal anti-surfactant protein SP-B antibodies to quantitate purified SP-B in chloroform/methanol and in chloroform/methanol extracts of whole pulmonary surfactant at nanogram levels. This method has been...

  15. A duplex real-time PCR assay for the quantitative detection of Naegleria fowleri in water samples.

    Science.gov (United States)

    Behets, Jonas; Declerck, Priscilla; Delaedt, Yasmine; Verelst, Lieve; Ollevier, Frans

    2007-01-01

    A fast and accurate duplex real-time PCR (qPCR) was developed to detect and quantify the human pathogenic amoeba Naegleria fowleri in water samples. In this study, primers and probe based on the Mp2Cl5 gene were designed to amplify and quantify N. fowleri DNA in a single duplex reaction. The qPCR detection limit (DL) corresponds to the minimum DNA quantity showing significant fluorescence with at least 90% of the positive controls in a duplex reaction. Using fluorescent Taqman technology the qPCR was found to be 100% specific for N. fowleri with a DL of 3 N. fowleri cell equivalents and a PCR efficiency of 99%. The quantification limit (QL) was 16 N. fowleri cell equivalents (corresponded with 320 N. fowleri cell equivalents l(-1) water sample) in a duplex qPCR reaction and corresponds to the lowest DNA quantity amplifiable with a coefficient of variation less than 25%. To detect inhibition an exogenous internal positive control (IPC) was included in each PCR reaction preventing false negative results. Comparison of qPCR and most probable number (MPN) culture results confirms that the developed qPCR is well suited for rapid and quantitative detection of this human pathogen in real water samples. Nevertheless 'low contamination levels' of water samples (fowleri cells l(-1)) still require culture method analyses. When other thermophilic Naegleria are very dominant, the MPN culture method could result in an underestimation in the real number of N. fowleri and some caution is necessary to interpret the data. The N. fowleri qPCR could be a useful tool to study further competitive phenomena between thermophilic Naegleria strains.

  16. Succession patterns of fungi associated to wound-induced agarwood in wild Aquilaria malaccensis revealed from quantitative PCR assay.

    Science.gov (United States)

    Mohamed, Rozi; Jong, Phai Lee; Nurul Irdayu, Ismail

    2014-09-01

    Aquilaria malaccensis produces agarwood in response to wounding and fungal attack. However, information is limited regarding Aquilaria's interaction with its diverse fungal community. In this study, time-related changes of three natural fungal colonizers in two wounded wild A. malaccensis were tracked, beginning a few hours after wounding up to 12 months. Using species-specific primers derived from their nrITS sequences in quantitative real-time PCR (qPCR), we quantified the amount of Cunninghamella bainieri, Fusarium solani and Lasiodiplodia theobromae. Because time is a major factor affecting agarwood quantity and quality, 14 wood samples were collected at different time points, i.e., 0-18 h, 2-13 days, 2-18 weeks, and 6-12 months after wounding. qPCR data revealed that the abundance of the three species decreased over time. The fungi were detected in high numbers during the first few hours and days after wounding (40- to 25,000-fold higher levels compared with initial counts) and in low numbers (<1- to 3,200-fold higher than initially) many months later. Consistent with its role in defense response, the accumulation of secondary metabolites at the wounding site could have caused the decline in fungal abundance. Succession patterns of the two trees were not identical, indicating that fungal populations may have been affected by tree environment and wound microclimate. Our results are important for understanding the diversity of microbial community in wild Aquilaria species and their association to wound-induced agarwood formation. Fungi could be secondary triggers to agarwood production in situations where trees are wounded in attempt to induce agarwood. PMID:24840100

  17. Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase α in Quantitative High-Throughput Screening (qHTS) Bioluminescent Format

    Science.gov (United States)

    Davis, Mindy I.; Sasaki, Atsuo T.; Simeonov, Anton

    2015-01-01

    Summary ipid kinases are important regulators of a variety of cellular processes and their dysregulation causes diseases such as cancer and metabolic diseases. Distinct lipid kinases regulate the seven different phosphorylated forms of phosphatidylinositol (PtdIns). Some lipid kinases utilize long-chain lipid substrates that have limited solubility in aqueous solutions, which can lead to difficulties in developing a robust and miniaturizable biochemical assay. The ability to prepare the lipid substrate and develop assays to identify modulators of lipid kinases is important and is the focus of this methods chapter. Herein, we describe a method to prepare a DMSO-based lipid mixture that enables the 1536-well screening of the lipid kinase phosphatidylinositol-5-phosphate 4-kinase α (PI5P4Kα) utilizing the D-myo-di16-PtIns(5)P substrate in quantitative high-throughput screening (qHTS) format using the ADP-Glo™ technology to couple the production of ADP to a bioluminescent readout. PMID:26552670

  18. Establishment of fluorescence quantitative PCR assay in detection of Mycobacterium tuberculosis%结核分枝杆菌荧光定量PCR检测方法建立

    Institute of Scientific and Technical Information of China (English)

    徐菊玲; 徐伯赢; 周洪昌; 张慧; 段劲; 薛利军; 邵圣文

    2011-01-01

    目的 探讨结核分枝杆菌实时定量检测方法并进行评价.方法针对结核杆菌(Mycobacterium tuberculosis,Mtb)16S rDNA基因设计引物,应用SYBR Green I建立荧光定量PCR( FQ-PCR)反应体系;提取Mtb基因组DNA,PCR扩增16S rDNA片段,构建重组质粒pMD-TB16S;检测11份肺结核患者痰标本;以甲型链球菌、大肠杆菌等基因组DNA作对照,检验方法特异性,对同一份Mtb DNA模板进行批内和批间检测,计算变异系数(CV).结果靶向Mtb 16S rDNA基因引物能够特异扩增Mtb 16S rDNA基因,对照组细菌基因未见扩增,灵敏度为(Mtb基因组DNA) 1.2 pg/μL,即(38.9 +3.54)拷贝/μL的16S rDNA基因,批内和批间Ct值变异系数分别为0.27%和1.26%.结论以16S rDNA为靶基因的FQ-PCR技术,能够对Mtb进行快速、敏感而特异的定量检测.%Objective To establish and assess a rapid real-time quantifiable method for the detection of Mycobacterium tuberculosis (Mtb). Methods The specific primers targetting 16S rDNA gene in Mtb were designed and the fluorescence quantitative PCR(FQ-PCR) assay was performed with SYBR Green I. After extraction of Mtb genomic DNA.the 16S rDNA fragment was amplified by conventional PCR and used to construct recombinant pMD-TB16S plasmid. Then, the serial dilutions of pMD-TB16S plasmid were subjected to the quantitation standard curve in FQ-PCR assay. The sputum samples from 11 patients with pulmonary tuberculosis were detected by the same FQ-PCR, with various genomic DNAs of Streptococcus viridans,Staphylococcus epidermidis,Staphylococcus aureus,and Escherichia coli used as the negative control to confirm specificity of FQ-PCR assay. The stabiity was analyzed with inter and intra FQ-PCR test by using the same Mtb genomic DNA,and the coefficient of variation(CV) values of threshold cycle(Ct ) were calculated. Results The primers targetting Mtb 16S rDNA gene were specific to the amplification of 16S rDNA gene of Mtb but not other bacteria in control

  19. Disruption of TgPHIL1 alters specific parameters of Toxoplasma gondii motility measured in a quantitative, three-dimensional live motility assay.

    Directory of Open Access Journals (Sweden)

    Jacqueline M Leung

    Full Text Available T. gondii uses substrate-dependent gliding motility to invade cells of its hosts, egress from these cells at the end of its lytic cycle and disseminate through the host organism during infection. The ability of the parasite to move is therefore critical for its virulence. T. gondii engages in three distinct types of gliding motility on coated two-dimensional surfaces: twirling, circular gliding and helical gliding. We show here that motility in a three-dimensional Matrigel-based environment is strikingly different, in that all parasites move in irregular corkscrew-like trajectories. Methods developed for quantitative analysis of motility parameters along the smoothed trajectories demonstrate a complex but periodic pattern of motility with mean and maximum velocities of 0.58 ± 0.07 µm/s and 2.01 ± 0.17 µm/s, respectively. To test how a change in the parasite's crescent shape might affect trajectory parameters, we compared the motility of Δphil1 parasites, which are shorter and wider than wild type, to the corresponding parental and complemented lines. Although comparable percentages of parasites were moving for all three lines, the Δphil1 mutant exhibited significantly decreased trajectory lengths and mean and maximum velocities compared to the parental parasite line. These effects were either partially or fully restored upon complementation of the Δphil1 mutant. These results show that alterations in morphology may have a significant impact on T. gondii motility in an extracellular matrix-like environment, provide a possible explanation for the decreased fitness of Δphil1 parasites in vivo, and demonstrate the utility of the quantitative three-dimensional assay for studying parasite motility.

  20. Disruption of TgPHIL1 alters specific parameters of Toxoplasma gondii motility measured in a quantitative, three-dimensional live motility assay.

    Science.gov (United States)

    Leung, Jacqueline M; Rould, Mark A; Konradt, Christoph; Hunter, Christopher A; Ward, Gary E

    2014-01-01

    T. gondii uses substrate-dependent gliding motility to invade cells of its hosts, egress from these cells at the end of its lytic cycle and disseminate through the host organism during infection. The ability of the parasite to move is therefore critical for its virulence. T. gondii engages in three distinct types of gliding motility on coated two-dimensional surfaces: twirling, circular gliding and helical gliding. We show here that motility in a three-dimensional Matrigel-based environment is strikingly different, in that all parasites move in irregular corkscrew-like trajectories. Methods developed for quantitative analysis of motility parameters along the smoothed trajectories demonstrate a complex but periodic pattern of motility with mean and maximum velocities of 0.58 ± 0.07 µm/s and 2.01 ± 0.17 µm/s, respectively. To test how a change in the parasite's crescent shape might affect trajectory parameters, we compared the motility of Δphil1 parasites, which are shorter and wider than wild type, to the corresponding parental and complemented lines. Although comparable percentages of parasites were moving for all three lines, the Δphil1 mutant exhibited significantly decreased trajectory lengths and mean and maximum velocities compared to the parental parasite line. These effects were either partially or fully restored upon complementation of the Δphil1 mutant. These results show that alterations in morphology may have a significant impact on T. gondii motility in an extracellular matrix-like environment, provide a possible explanation for the decreased fitness of Δphil1 parasites in vivo, and demonstrate the utility of the quantitative three-dimensional assay for studying parasite motility. PMID:24489670

  1. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia

    DEFF Research Database (Denmark)

    Jamal, Syed M.; Belsham, Graham

    2015-01-01

    . Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysableprobe-based real time reverse transcription quantitative polymerase chain reaction (RTqPCR) assays were developed for specific detection...... and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnosticassays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of OPan...... and detected the RNA from the targetviruses with cycle threshold (CT) values comparable with those obtained with the serotype independentpan-FMDV diagnostic assays. No cross-reactivity was observed in the seassays between the heterotypic viruses circulating in the region. The assays reported here have higher...

  2. Quantitative Analysis of the Relative Transcript Levels of ABC Transporter Atr Genes in Aspergillus nidulans by Real-Time Reverse Transcription-PCR Assay

    Science.gov (United States)

    Pizeta Semighini, Camile; Marins, Mozart; Goldman, Maria Helena S.; Goldman, Gustavo Henrique

    2002-01-01

    The development of assays for quantitative analysis of the relative transcript levels of ABC transporter genes by real-time reverse transcription-PCR (RT-PCR) might provide important information about multidrug resistance in filamentous fungi. Here, we evaluate the potential of real-time RT-PCR to quantify the relative transcript levels of ABC transporter Atr genes from Aspergillus nidulans. The AtrA to AtrD genes showed different and higher levels in the presence of structurally unrelated drugs, such as camptothecin, imazalil, itraconazole, hygromycin, and 4-nitroquinoline oxide. We also verified the relative transcript levels of the Atr genes in the A. nidulans imazalil-resistant mutants. These genes displayed a very complex pattern in different ima genetic backgrounds. The imaB mutant has higher basal transcript levels of AtrB and -D than those of the wild-type strain. The levels of these two genes are comparable when the imaB mutant is grown in the presence and absence of imazalil. The imaC, -D, and -H mutants have higher basal levels of AtrA than that of the wild type. The same behavior is observed for the relative transcript levels of AtrB in the imaG mutant background. PMID:11872487

  3. A Quick Assay for the Quantitation of Fumonisin B1 and B2 in Maize Samples by Liquid Chromatography and Mass Spectrometry.

    Science.gov (United States)

    Vega, Victor A

    2016-05-01

    A quick and effective extraction of fumonisin B1 (FB1) and B2 (FB2) in corn samples is described. The samples were extracted with an 80% acetonitrile-water solution, followed by a second extraction using an 80% methanol-water solution. The extract was then subjected to an immunoaffinity column for cleanup. Quantitation was then obtained by LC/MS. LC/MS parameters were optimized resulting in a 3 min run time. Corn certified reference materials (CRMs) at three different levels were analyzed. For the lowest level, a corn CRM sample containing 0.6 ppm of FB1 and 0.1 ppm of FB2 was analyzed. For the midlevel, analysis of a corn CRM sample containing 1.2 ppm FB1 and 0.3 ppm FB2 was conducted. A sample containing 3.6 ppm FB1 and 0.8 ppm FB2 was also analyzed. All recoveries were calculated using an external standard curve. Recoveries from all CRM samples ranged from 70 to 110%. Confirmation for both analytes in the sample extracts at each level was accomplished by comparing MS/MS spectra of the samples against those of the standards. This method provides a rapid, specific, robust, and easily controlled assay for the analysis of fumonisin in corn samples. PMID:27297840

  4. Pharmacological profile of brain-derived neurotrophic factor (BDNF) splice variant translation using a novel drug screening assay: a "quantitative code".

    Science.gov (United States)

    Vaghi, Valentina; Polacchini, Alessio; Baj, Gabriele; Pinheiro, Vera L M; Vicario, Annalisa; Tongiorgi, Enrico

    2014-10-01

    The neurotrophin brain-derived neurotrophic factor (BDNF) is a key regulator of neuronal development and plasticity. BDNF is a major pharmaceutical target in neurodevelopmental and psychiatric disorders. However, pharmacological modulation of this neurotrophin is challenging because BDNF is generated by multiple, alternatively spliced transcripts with different 5'- and 3'UTRs. Each BDNF mRNA variant is transcribed independently, but translation regulation is unknown. To evaluate the translatability of BDNF transcripts, we developed an in vitro luciferase assay in human neuroblastoma cells. In unstimulated cells, each BDNF 5'- and 3'UTR determined a different basal translation level of the luciferase reporter gene. However, constructs with either a 5'UTR or a 3'UTR alone showed poor translation modulation by BDNF, KCl, dihydroxyphenylglycine, AMPA, NMDA, dopamine, acetylcholine, norepinephrine, or serotonin. Constructs consisting of the luciferase reporter gene flanked by the 5'UTR of one of the most abundant BDNF transcripts in the brain (exons 1, 2c, 4, and 6) and the long 3'UTR responded selectively to stimulation with the different receptor agonists, and only transcripts 2c and 6 were increased by the antidepressants desipramine and mirtazapine. We propose that BDNF mRNA variants represent "a quantitative code" for regulated expression of the protein. Thus, to discriminate the efficacy of drugs in stimulating BDNF synthesis, it is appropriate to use variant-specific in vitro screening tests.

  5. TRI12 based quantitative real-time PCR assays reveal the distribution of trichothecene genotypes of F. graminearum and F. culmorum isolates in Danish small grain cereals.

    Science.gov (United States)

    Nielsen, L K; Jensen, J D; Rodríguez, A; Jørgensen, L N; Justesen, A F

    2012-07-16

    Quantitative real-time PCR assays, based on polymorphisms in the TRI12 gene of the trichothecene pathway, were developed to identify and quantify the trichothecene genotypes producing 3-acetyl-deoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) or nivalenol (NIV) in the Fusarium graminearum species complex, Fusarium culmorum, Fusarium cerealis and Fusarium pseudograminearum. These assays were applied on a total of 378 field samples of cereal grain of wheat, barley, triticale, rye and oats collected from 2003 to 2007 to study the trichothecene genotype composition in Danish cereals. The three genotypes, 3ADON, 15ADON and NIV were found in all five cereal species, great annual variation in the occurrence of the trichothecene genotypes was evident with considerable variation between the samples. 3ADON was the dominant genotype in barley, triticale, rye and oats while 15ADON was most dominant in wheat. The NIV genotype was found at low levels in most samples. Study of genotype composition within the Danish F. graminearum and F. culmorum population was based on principal component analysis (PCA). PCA revealed that the dominating genotype of F. graminearum in wheat is 15ADON. For barley, the PCA analysis indicated that the F. graminearum population consisted of all three genotypes, and in triticale, the F. graminearum population consisted mainly of 15ADON genotype. F. culmorum/F. cerealis showed correlation to the NIV genotype in wheat and triticale but not in barley. F. culmorum/F. cerealis also showed some correlation to 3ADON especially in wheat and triticale. Selected wheat and barley samples from 1957 to 2000 showed low amounts of F. graminearum and F. culmorum in general but with a dominance of the 3ADON genotype. 15ADON was not detected in these samples, except for very low amounts in the sample representing the years from 1997 to 2000. Detection of low amounts of the 15ADON genotype in these historical samples and the relatively high amounts of 15ADON

  6. Identification of novel anti-hepatitis C virus agents by a quantitative high throughput screen in a cell-based infection assay.

    Science.gov (United States)

    Hu, Zongyi; Hu, Xin; He, Shanshan; Yim, Hyung Joon; Xiao, Jingbo; Swaroop, Manju; Tanega, Cordelle; Zhang, Ya-qin; Yi, Guanghui; Kao, C Cheng; Marugan, Juan; Ferrer, Marc; Zheng, Wei; Southall, Noel; Liang, T Jake

    2015-12-01

    Hepatitis C virus (HCV) poses a major health threat to the world. The recent development of direct-acting antivirals (DAAs) against HCV has markedly improved the response rate of HCV and reduced the side effects in comparison to the interferon-based therapy. Despite this therapeutic advance, there is still a need to develop new inhibitors that target different stages of the HCV life cycle because of various limitations of the current regimens. In this study, we performed a quantitative high throughput screening of the Molecular Libraries Small Molecule Repository (MLSMR) of ∼350,000 chemicals for novel HCV inhibitors using our previously developed cell-based HCV infection assay. Following confirmation and structural clustering analysis, we narrowed down to 158 compounds from the initial ∼3000 molecules that showed inhibitory activity for further structural and functional analyses. We were able to assign the majority of these compounds to specific stage(s) in the HCV life cycle. Three of them are direct inhibitors of NS3/4A protease. Most of the compounds appear to act on novel targets in HCV life cycle. Four compounds with novel structure and excellent drug-like properties, three targeting HCV entry and one targeting HCV assembly/secretion, were advanced for further development as lead hits. These compounds represent diverse chemotypes that are potential lead compounds for further optimization and may offer promising candidates for the development of novel therapeutics against HCV infection. In addition, they represent novel molecular probes to explore the complex interactions between HCV and the cells. PMID:26515788

  7. Development of quantitative real-time PCR assays for fathead minnow (Pimephales promelas) gonadotropin ß subunit mRNAs to support endocrine disruptor research.

    Energy Technology Data Exchange (ETDEWEB)

    Villeneuve, Daniel L.; Miracle, Ann L.; Jensen, Kathleen M.; Degitz, Sigmund J.; Kahl, Michael D.; Korte, Joseph J.; Greene, Katie J.; Blake, Lindsey S.; Linnum, Ann; Ankley, Gerald T.

    2007-03-01

    Fathead minnows (Pimephales promelas) are one of the most widely-used small fish models for regulatory ecotoxicology testing and research related to endocrine disrupting chemicals (EDCs). In this study, we isolated and sequenced cDNAs for fathead minnow follicle-stimulating hormone-like and luteinizing hormone-like β (FSHβ and LHβ) and glycoprotein α (GPα) subunits. Quantitative real-time PCR assays for measuring gonadotropin (GtH) β subunit transcripts were developed and used to examine “baseline” transcript levels over a range of age classes and reproductive states encompassed in EDC testing. In females, FSHβ and LHβ transcripts were greater in 4-5 month old than in younger fish and were significantly correlated with one another across all age classes examined. In males, FSHβ transcripts were greatest in 2-3 month old fish and were inversely correlated with various measures of testis development including, gonadal-somatic index (GSI), and histological stage. Overall, the pattern of GtHβ expression over age classes associated with gonad development was similar to that reported for other asynchronous-spawning fish. Despite significant changes in female GSI, gonad stage, and plasma vitellogenin within 24 h of spawning, GtHβ transcript levels in fish that had spawned within the preceding 24 h were not significantly different from those in fish that were 2-3 days post-spawn and expected to spawn within the next 24 h based on spawning history. Results of this study provide insights related to the role of GtHs in fathead minnow reproductive development and function. Additionally they provide useful “baseline” data needed to design and interpret effective experiments for studying direct and indirect effects of EDCs on GtH subunit mRNA expression, which will facilitate a greater understanding of integrated system-wide responses of the fathead minnow brain-pituitary-gonadal axis to stressors including EDCs.

  8. Multicenter evaluation of the new Abbott Realtime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

    NARCIS (Netherlands)

    M. Schutten (Martin); D. Peters (D.); N. Back (Nicole); A.W. van den Beld (Annewieke); B. Beuselinck (B.); V. Foulongne (V.); A.M. Geretti (Anna Maria); L. Pandiani (L.); M. Tiemann; H.G.M. Niesters (Bert)

    2007-01-01

    textabstractThe analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche

  9. Multicenter evaluation of the new Abbott RealTime assays for quantitative detection of human immunodeficiency virus type 1 and hepatitis C virus RNA

    NARCIS (Netherlands)

    Schutten, M; Peters, D; Back, N K T; Beld, M; Beuselinck, K; Foulongne, V; Geretti, A-M; Pandiani, L; Tiemann, C; Niesters, H G M

    2007-01-01

    The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMa

  10. Quantitative and specific detection of the biocontrol agent, Serratia plymuthica, in plant extracts using a real-time TaqMan® assay

    NARCIS (Netherlands)

    Czajkowski, R.L.; Wolf, van der J.M.

    2012-01-01

    A Serratia plymuthica-specific TaqMan® assay was designed based on the consensus nucleotide sequence from the 3'- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to various s

  11. Bioluminescence Methods for Assaying Kinases in Quantitative High-Throughput Screening (qHTS) Format Applied to Yes1 Tyrosine Kinase, Glucokinase, and PI5P4Kα Lipid Kinase.

    Science.gov (United States)

    Davis, Mindy I; Auld, Douglas S; Inglese, James

    2016-01-01

    Assays in which the detection of a biological phenomenon is coupled to the production of bioluminescence by luciferase have gained widespread use. As firefly luciferases (FLuc) and kinases share a common substrate (ATP), coupling of a kinase to FLuc allows for the amount of ATP remaining following a kinase reaction to be assessed by quantitating the amount of luminescence produced. Alternatively, the amount of ADP produced by the kinase reaction can be coupled to FLuc through a two-step process. This chapter describes the bioluminescent assays that were developed for three classes of kinases (lipid, protein, and metabolic kinases) and miniaturized to 1536-well format, enabling their use for quantitative high-throughput (qHTS) of small-molecule libraries.

  12. Evaluation of Performance Characteristics of the Aptima HIV-1 Quant Dx Assay for Detection and Quantitation of Human Immunodeficiency Virus Type 1 in Plasma and Cervicovaginal Lavage Samples.

    Science.gov (United States)

    Sam, Soya S; Kurpewski, Jaclynn R; Cu-Uvin, Susan; Caliendo, Angela M

    2016-04-01

    Quantification of HIV-1 RNA has become the standard of care in the clinical management of HIV-1-infected individuals. The objective of this study was to evaluate performance characteristics and relative workflow of the Aptima HIV-1 Quant Dx assay in comparison with the Abbott RealTime HIV-1 assay using plasma and cervicovaginal lavage (CVL) specimens. Assay performance was evaluated by using an AcroMetrix HIV-1 panel, AcroMetrix positive controls, Qnostics and SeraCare HIV-1 evaluation panels, 208 clinical plasma samples, and 205 matched CVL specimens on the Panther and m2000 platforms. The Aptima assay demonstrated good linearity over the quantification range tested (2 to 5 log10copies/ml), and there was strong linear correlation between the assays (R(2)= 0.99), with a comparable coefficient of variance of laboratories demanding high-throughput sample processing. PMID:26842702

  13. Multicenter Evaluation of the New Abbott RealTime Assays for Quantitative Detection of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus RNA▿

    OpenAIRE

    Schutten, M.; Peters, D.; Back, N.K.T.; Beld, M; Beuselinck, K.; Foulongne, V.; Geretti, A.-M.; Pandiani, L.; Tiemann, C; Niesters, H.G.M.

    2007-01-01

    The analytical performances of the new Abbott RealTime hepatitis C virus (HCV) and human immunodeficiency virus type 1 viral load assays were compared at nine laboratories with different competitor assays. These included the Abbott LcX, Bayer Versant bDNA, Roche COBAS Amplicor, and Roche COBAS TaqMan assays. Two different protocols used during the testing period with and without a pre-m1000 RNA isolation spin were compared. The difference proved to be nonsignificant. A uracil-N-glycosylase (U...

  14. CPTAC Assay Portal: a repository of targeted proteomic assays

    Energy Technology Data Exchange (ETDEWEB)

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Rodriguez, Henry; Abbateillo, Susan E.; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Paulovich, Amanda G.

    2014-06-27

    To address these issues, the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as a public repository of well-characterized quantitative, MS-based, targeted proteomic assays. The purpose of the CPTAC Assay Portal is to facilitate widespread adoption of targeted MS assays by disseminating SOPs, reagents, and assay characterization data for highly characterized assays. A primary aim of the NCI-supported portal is to bring together clinicians or biologists and analytical chemists to answer hypothesis-driven questions using targeted, MS-based assays. Assay content is easily accessed through queries and filters, enabling investigators to find assays to proteins relevant to their areas of interest. Detailed characterization data are available for each assay, enabling researchers to evaluate assay performance prior to launching the assay in their own laboratory.

  15. Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

    Science.gov (United States)

    Yadav, Reena; Paria, Anutosh; Mankame, Smruti; Makesh, M; Chaudhari, Aparna; Rajendran, K V

    2015-12-01

    Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV.

  16. Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia.

    Directory of Open Access Journals (Sweden)

    Syed M Jamal

    Full Text Available Rapid and accurate diagnosis of foot-and-mouth disease (FMD and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08. In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsiaANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (CT values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4-100%] for serotype A positive samples and specificity (100% each for serotypes O, A and Asia-1 positive samples for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for

  17. Angiogenesis Assays.

    Science.gov (United States)

    Nambiar, Dhanya K; Kujur, Praveen K; Singh, Rana P

    2016-01-01

    Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis. PMID:26608294

  18. Binary Classification of a Large Collection of Environmental Chemicals from Estrogen Receptor Assays by Quantitative Structure-Activity Relationship and Machine Learning Methods

    Science.gov (United States)

    ABSTRACT: There are thousands of environmental chemicals subject to regulatory decisions for endocrine disrupting potential. A promising approach to manage this large universe of untested chemicals is to use a prioritization filter that combines in vitro assays with in silico QSA...

  19. Development of three quantitative real-time PCR assays for the detection of Rickettsia raoultii, Rickettsia slovaca, and Rickettsia aeschlimannii and their validation with ticks from the country of Georgia and the Republic of Azerbaijan.

    Science.gov (United States)

    Jiang, Ju; You, Brian J; Liu, Evan; Apte, Anisha; Yarina, Tamasin R; Myers, Todd E; Lee, John S; Francesconi, Stephen C; O'Guinn, Monica L; Tsertsvadze, Nikoloz; Vephkhvadze, Nino; Babuadze, Giorgi; Sidamonidze, Ketevan; Kokhreidze, Maka; Donduashvili, Marina; Onashvili, Tinatin; Ismayilov, Afrail; Agayev, Nigar; Aliyev, Mubariz; Muttalibov, Nizam; Richards, Allen L

    2012-12-01

    A previous surveillance study of human pathogens within ticks collected in the country of Georgia showed a relatively high infection rate for Rickettsia raoultii, R. slovaca, and R. aeschlimannii. These 3 spotted fever group rickettsiae are human pathogens: R. raoultii and R. slovaca cause tick-borne lymphadenopathy (TIBOLA), and R. aeschlimannii causes an infection characterized by fever and maculopapular rash. Three quantitative real-time polymerase chain reaction (qPCR) assays, Rraoul, Rslov, and Raesch were developed and optimized to detect R. raoultii, R. slovaca, and R. aeschlimannii, respectively, by targeting fragments of the outer membrane protein B gene (ompB) using species-specific molecular beacon or TaqMan probes. The 3 qPCR assays showed 100% specificity when tested against a rickettsiae DNA panel (n=20) and a bacteria DNA panel (n=12). The limit of detection was found to be at least 3 copies per reaction for all assays. Validation of the assays using previously investigated tick nucleic acid preparations, which included Rickettsia-free tick samples, tick samples that contain R. raoultii, R. slovaca, R. aeschlimannii, and other Rickettsia spp., gave 100% sensitivity for all 3 qPCR assays. In addition, a total of 65 tick nucleic acid preparations (representing 259 individual ticks) collected from the country of Georgia and the Republic of Azerbaijan in 2009 was tested using the 3 qPCR assays. R. raoultii, R. slovaca, and R. aeschlimannii were not detected in any ticks (n=31) from the Republic of Azerbaijan, but in the ticks from the country of Georgia (n=228) the minimal infection rate for R. raoultii and R. slovaca in Dermacentor marginatus was 10% and 4%, respectively, and for R. aeschlimannii in Haemaphysalis sulcata and Hyalomma spp. it was 1.9% and 20%, respectively.

  20. Detection limits and cost comparisons of human- and gull-associated conventional and quantitative PCR assays in artificial and environmental waters

    Science.gov (United States)

    Modern techniques for tracking fecal pollution in environmental waters require investing in DNA-based methods to determine the presence of specific fecal sources. To help water quality managers decide whether to employ routine polymerase chain reaction (PCR) or quantitative PC...

  1. Evaluation and comparison of SYBR Green I Real-Time PCR and TaqMan Real-Time PCR methods for quantitative assay of Listeria monocytogenes in nutrient broth and milk

    OpenAIRE

    Karatzas, Kimon Andreas G.

    2012-01-01

    Specific traditional plate count method and real-time PCR systems based on SYBR Green I and TaqMan technologies using a specific primer pair and probe for amplification of iap-gene were used for quantitative assay of Listeria monocytogenes in seven decimal serial dilution series of nutrient broth and milk samples containing 1.58 to 1.58×107 cfu /ml and the real-time PCR methods were compared with the plate count method with respect to accuracy and sensitivity. In this study, the plate count m...

  2. PROSPECTIVE STUDY OF MULTIPLE GENETIC TUMOR MARKER ASSAY BY QUANTITATIVE REAL-TIME PCR TO PREDICT RECURRENCE IN COLORECTAL CANCER PATIENTS

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Objective To describe correlation between multiple genetic tumor markers,carcinoembryonic antigen (CEA),cytokeratin 20 (CK20),and Survivin,and clinicopathological features of colorectal cancer (CRC) and to assess prognostic diagnosis value in cancer recurrence and metastasis.Methods A total of 92 patients with CRC,68 patients with precancerous lesions,and 29 control volunteers were collected for the detection of CEA,CK20,and Survivin expressions by using quantitative Real-Time PCR technology.Associations am...

  3. Molecular sensitivity threshold of wet mount and an immunochromatographic assay evaluated by quantitative real-time PCR for diagnosis of Trichomonas vaginalis infection in a low-risk population of childbearing women.

    Science.gov (United States)

    Leli, Christian; Castronari, Roberto; Levorato, Lucia; Luciano, Eugenio; Pistoni, Eleonora; Perito, Stefano; Bozza, Silvia; Mencacci, Antonella

    2016-06-01

    Vaginal trichomoniasis is a sexually transmitted infection caused by Trichomonas vaginalis, a flagellated protozoan. Diagnosis of T. vaginalis infection is mainly performed by wet mount microscopy, with a sensitivity ranging from 38% to 82%, compared to culture, still considered the gold standard. Commercial immunochromatographic tests for monoclonal-antibody-based detection have been introduced as alternative methods for diagnosis of T. vaginalis infection and have been reported in some studies to be more sensitive than wet mount. Real-time PCR methods have been recently developed, with optimal sensitivity and specificity. The aim of this study was to evaluate whether there is a molecular sensitivity threshold for both wet mount and imunochromatographic assays. To this aim, a total of 1487 low-risk childbearing women (median age 32 years, interquartile range 27-37) were included in the study, and underwent vaginal swab for T. vaginalis detection by means of a quantitative real-time PCR assay, wet mount and an immunochromatographic test. Upon comparing the results, prevalence values observed were 1.3% for real-time PCR, 0.5% for microscopic examination, and 0.8% for the immunochromatographic test. Compared to real-time PCR, wet mount sensitivity was 40% (95% confidence interval 19.1% to 63.9%) and specificity was 100% (95% CI 99.7% to 100%). The sensitivity and specificity of the immunochromatographic assay were 57.9% (95% CI 33.5% to 79.8%) and 99.9% (95% CI 99.6% to 100%), respectively. Evaluation of the wet mount results and those of immunochromatographic assay detection in relation to the number of T. vaginalis DNA copies detected in vaginal samples showed that the lower identification threshold for both wet mount (chi-square 6.1; P = 0.016) and the immunochromatographic assay (chi-square 10.7; P = 0.002) was ≥100 copies of T. vaginalis DNA/5 mcl of eluted DNA. PMID:27367320

  4. Development of a novel quantitative real-time RT-PCR assay for the simultaneous detection of all serotypes of Foot-and-mouth disease virus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; de Stricker, K.;

    2003-01-01

    Foot-and-mouth disease virus (FMDV) spreads extremely fast and the need for rapid and robust diagnostic virus detection systems was obvious during the recent European epidemic. Using a novel real-time RT-PCR system based on primer-probe energy transfer (PriProET) we present here an assay targeting...... to detect FMDV in materials from both cattle and buffalo. When compared to traditional virus cultivation the virus detection sensitivity was similar but the RT-PCR method can provide a laboratory result much faster than virus cultivation. The real-time PCR method confirms the identity of the amplicon...

  5. 盐酸克伦特罗的AlphaLISA检测方法的建立%Quantitative determination of clenbuterol using the amplified luminescent proximity homogeneous assay

    Institute of Scientific and Technical Information of China (English)

    赵芳; 岳振峰; 吕敬章; 洪丹漩; 张恒; 刘慧玲; 黄欣迪; 洪小柳

    2014-01-01

    目的:采用纳米均相时间分辨荧光免疫技术(amplified luminescent proximity homogeneous assay, Alpha-LISA)建立高灵敏检测盐酸克伦特罗(clenbuterol, CLB)残留的方法。方法在发光微粒上固定 CLB,与游离的CLB共同竞争固定在感光微粒上的CLB单克隆抗体,优化检测条件并进行方法学考核。结果该方法的灵敏度为0.04 ng/mL。猪肉样品回收率在83%~109%之间,牛肉样品回收率在92%~112%之间;猪肉样品检测的批内变异系数CV<10%、批间变异系数CV<15%。与特布他林、沙丁胺醇的交叉反应率分别为34.5%、25.7%,与莱克多巴胺、妥布特罗、齐帕特罗无交叉反应。结论 CLB-AlphaLISA 检测肉类中克伦特罗残留具有简单、快速、灵敏性高、特异性强、稳定等特点,具有广阔的应用前景。%Objective A direct competitive amplified luminescent proximity homogeneous assay (Alpha-LISA) for detecting clenbuterol(CLB) was established. Methods CLB was coated on emitting particles, and competed with sample CLB to anti-CLB which was coated on photosensitive particles. The analytical perfor-mance and optinal test conditions of the method were studied. Results The sensitivity of the assay was 0.04 ng/mL. The recoveries of the determination for CLB in pork and beer were respectively 83%~109% and 92%~112%. The CV of intra-and inter-assay were <10% and <15% respectively. The cross-reactivity of the CLB-AlphaLISA with ractopamine, tulobuterol and zilpaterol was negligible, while that with terbutaline and salbutamol was 34.5% and 25.7% respectively. Conclusion The CLB-AlphaLISA had the characteristics of excellent specificity and sensitivity, and good precision and economy. It could be widely used in rapid screen-ing for CLB contamination in meat or foods in the future.

  6. TRI12 based quantitative real-time PCR assays reveal the distribution of trichothecene genotypes of F. graminearum and F. culmorum isolates in Danish small grain cereals

    DEFF Research Database (Denmark)

    Nielsen, L.K.; Jensen, Jens Due; Rodriguez, A.;

    2012-01-01

    species complex, Fusarium culmorum, Fusarium cerealis and Fusarium pseudograminearum. These assays were applied on a total of 378 field samples of cereal grain of wheat, barley, triticale, rye and oats collected from 2003 to 2007 to study the trichothecene genotype composition in Danish cereals. The three...... genotypes, 3ADON, 15ADON and NIV were found in all five cereal species, great annual variation in the occurrence of the trichothecene genotypes was evident with considerable variation between the samples. 3ADON was the dominant genotype in barley, triticale, rye and oats while 15ADON was most dominant...

  7. Quantitative evaluation of viral fitness due to a single nucleotide polymorphism in the Marek's disease virus UL41 gene via an in vitro competition assay.

    Science.gov (United States)

    Mao, Weifeng; Niikura, Masahiro; Silva, Robert F; Cheng, Hans H

    2008-03-01

    Marek's disease, a T cell lymphoma, is an economically important disease of poultry caused by the Marek's disease virus (MDV), a highly cell-associated alphaherpesvirus. A greater understanding of viral gene function and the contribution of sequence variation to virulence should facilitate efforts to control Marek's disease in chickens. To characterize a naturally occurring single nucleotide polymorphism (SNP; AY510475:g.108,206C>T) in the MDV UL41 gene that results in a missense mutation (AAS01683:p.Arg377Cys), bacterial artificial chromosome (BAC)-derived MDVs that differed only in the UL41 SNP were evaluated using a head-to-head competition assay in vitro. Monitoring the frequency of each SNP by pyrosequencing during virus passage determined the ratio of each viral genome in a single monolayer, which is a very sensitive method to monitor viral fitness. MDV with the UL41*Cys allele showed enhanced fitness in vitro. To evaluate the mechanism of altered viral fitness caused by this SNP, the virion-associated host shutoff (vhs) activity of both UL41 alleles was determined. The UL41*Cys allele had no vhs activity, which suggests that enhanced fitness in vitro for MDV with inactive vhs was due to reduced degradation of viral transcripts. The in vitro competition assay should be applicable to other MDV genes and mutations.

  8. Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

    Science.gov (United States)

    Hou, Yan-Qiang; Liang, Dong-Yu; Lou, Xiao-Li; Zhang, Mei; Zhang, Zhen-huan; Zhang, Lu-rong

    2016-02-01

    Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P DNA-based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

  9. Study of quantitative assay and calculate method with RIA A dot-assay in titrating IU of anti-HBs%抗-HBs国际单位RIA一点法定量测定与计算方法的探讨

    Institute of Scientific and Technical Information of China (English)

    席三强; 虞现河

    2001-01-01

    在Radio-immuno Assay(RIA)试验测定抗-HBs国际单位的简易定量与计算中,采用RIA一点法测定,以分析表达式mIU/ml=SC(mIU/ml)[exp(0.69315S-NC/SC-NC)-1]进行结果计算.此式可以常用对数式表示为mIU/ml=SC(mIU/ml)[lg-1(0.30103S-NC/SC-NC)-1],当(S-NC)/(SC-NC)在0.3~1.1界线内时,其计算结果的相对误差小于6%,是目前误差最小的简易计算方法.同时还推算求出Holliger公式的阳性对照的最适含量为124.26mIU/ml,Richardson公式的阳性对照为125 mIU/ml,中国药品生物制品检定所公式的阳性对照为92.5 mIU/ml.

  10. Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

    Directory of Open Access Journals (Sweden)

    Hamel Christian

    2003-01-01

    Full Text Available Abstract Background X-linked ocular albinism type 1 (OA1 is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment. Results We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7–8, and a point mutation (310delG in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in OA1 gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA. Conclusion The identification of OA1 mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of OA1 exons with DMD exon as external standard performed on the LightCycler™ allows quick and accurate carrier-status assessment for at-risk females.

  11. Development and validation of a real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay for investigation of wild poliovirus type 1-South Asian (SOAS) strain reintroduced into Israel, 2013 to 2014.

    Science.gov (United States)

    Hindiyeh, M Y; Moran-Gilad, J; Manor, Y; Ram, D; Shulman, L M; Sofer, D; Mendelson, E

    2014-02-20

    In February 2013, wild poliovirus type 1 (WPV1) was reintroduced into southern Israel and resulted in continuous silent circulation in the highly immune population. As a part of the public health emergency response, a novel real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed, to allow for the sensitive and specific detection of the circulatingWPV1-South Asian (SOAS) strain. Specific primers and probes derived from the VP-1 region were designed, based on sequenced sewage isolates, and used to simultaneously amplify this WPV1-SOAS sequence together with bacteriophage MS-2 as internal control. High titre WPV1-SOAS stock virus was used for assay optimisation and 50 processed sewage samples collected from southern Israel and tested by reference culture based methods were used for analytical validation of the assay’s performance. The limit of detection of the multiplex qRT-PCR (SOAS/MS-2) assay was 0.1 plaque-forming unit (pfu)/reaction (20 pfu/mL) for WPV1-SOAS RNA with 100% sensitivity, specificity, positive and negative predictive values when compared to the culture based method. The turnaround time was rapid, providing results for environmental samples within 24 to 48 hours from completion of sewage processing, instead of five to seven days by culture-based analysis. Direct sewage testing by qRT-PCR assay proved to be a useful tool for rapid detection and environmental surveillance of WPV1-SOAS circulating strain during emergency response. Application of the approach for detection of WPV1-SOAS in stool samples obtained during acute flaccid paralysis (AFP) surveillance or field surveys should be further evaluated.

  12. 荧光微球免疫层析法定量检测牛乳中酪蛋白%Fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk

    Institute of Scientific and Technical Information of China (English)

    杨亚杰; 倪小琴; 王茜; 游琼; 张富生; 赖卫华

    2014-01-01

    Objective To develop a fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk. Methods Casein was used as the antigen to immunize the New Zealand rabbits to make the corresponding polyclonal antibody against the antigen, and EDC mediated method was used to couple the purified polyclonal antibody with fluorescent microspheres. The filter pad, sample pad, conjugate pad, NC membrane, and absorption pad were assembled to develop the fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk. Results The results could be obtained within 25 min without sophisticated equipment, and the limit of detection of the test strip was 100 ng/mL. The developed assay was highly specific to the casein protein as a result of that it had no cross-reactivity with other non-target proteins, such as BSA and OVA. The recovery rates of the intra-assay at casein concentrations of 100.0, 500.0, and 1000.0 ng/mL were (89.03±5.2)%, (93.47±6.9)% and (91.2±7.8)%, respectively, while the inter-assay were (87.69±6.2)%, (92.73±8.3)%and (89.82±8.5)%, respectively. Conclusion The fluorescent microsphere-based lateral flow assay for the quantitative detection of casein in milk with the advantages of rapid, convenient, and high sensitivity was established preliminarily.%目的:研究用荧光微球免疫层析法定量检测牛乳中的酪蛋白。方法本文以酪蛋白为抗原,免疫新西兰大白兔制备抗酪蛋白的多克隆抗体,将纯化后的多克隆抗体通过EDC介导法与荧光微球进行偶联,将滤纸、样品垫、结合垫、NC膜和吸水纸组装成试纸条,用此荧光微球免疫层析法定量检测牛乳中的酪蛋白。结果试纸条在25 min内就能判定结果,最低检测限为100 ng/mL,该方法与BSA、OVA均无交叉反应,具有很好的特异性。检测酪蛋白浓度为100.0、500.0、1000.0 ng/mL 的样品,试纸条的批内回收率分别为(89.03±5.2)%、(93.47±6

  13. Experimental Review of DNA-Based Methods for Wine Traceability and Development of a Single-Nucleotide Polymorphism (SNP) Genotyping Assay for Quantitative Varietal Authentication.

    Science.gov (United States)

    Catalano, Valentina; Moreno-Sanz, Paula; Lorenzi, Silvia; Grando, Maria Stella

    2016-09-21

    The genetic varietal authentication of wine was investigated according to DNA isolation procedures reported for enological matrices and also by testing 11 commercial extraction kits and various protocol modifications. Samples were collected at different stages of the winemaking process of renowned Italian wines Brunello di Montalcino, Lambruschi Modenesi, and Trento DOC. Results demonstrated not only that grape DNA loss is produced by the fermentation process but also that clarification and stabilization operations contribute to the reduction of double-stranded DNA content on wine. Despite the presence of inhibitors, downstream PCR genotyping yielded reliable nuclear and chloroplast SSR markers for must samples, whereas no amplification or inconsistent results were obtained at later stages of the vinification. In addition, a TaqMan genotyping assay based on cultivar-specific single-nucleotide polymorphisms (SNPs) was designed, which allowed assessment of grapevine DNA mixtures. Once the wine matrix limitations are overcome, this sensitive tool may be implemented for the relative quantification of cultivars used for blend wines or frauds. PMID:27573905

  14. Establishment of Double Real-Time Quantitative RT-PCR Assay for HCV and HIV%HCV及HIV二联病毒核酸荧光PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    容莹; 王文敬; 黎诚耀; 沈磁石; 郑欣

    2013-01-01

    目的 建立多联病毒核酸荧光PCR检测方法,为我国核酸检测技术的应用前景提供技术支持.方法 使用逆转录实时荧光PCR定量方法(QRT-PCR)检测HCV、HIV病毒,在成功扩增定量单一病毒的基础上,通过主要调整Mg2+离子、dNTPs、Taq的浓度等优化反应体系,建立同时检测两种病毒核酸的逆转录实时荧光PCR定量检测方法(一步法双检),并分析双检体系的扩增灵敏度.结果 通过优化筛选反应条件,使用QRT-PCR方法成功扩增双模板HCV/HIV RNA,且检测特异性好,灵敏度较高,能达到HCV RNA 20 IU/ml与HIV RNA 80IU/ml的检测灵敏度.结论 本试验成功建立能同时检测两种病毒核酸的QRT-PCR方法,为后续研究HBV、HCV、HIV多联荧光病毒核酸检测系统提供了理论依据与技术支持.%Objective To provide technical support and R&D background for nucleic acid technology in our country,a multiplex (HCV/HIV-1) real-time quantitative reverse transcription PCR assay (QRT-PCR)was investigated and established.Methods Based on the successful amplification and quantification of a single virus by QRT-PCR,we established a double real-time quantitative reverse transcription PCR assay for HCV/HIV RNA detection by optimizing the reaction system of adjusting the concentrations of Mg, dNTP and Taq. Furthermore, the sensitivity of detection the two viruses by QRT-PCR was analyzed. Results Double-templates of HCV and HIV RNA were amplified and quantified successfully with high specificity and sensitivity. The sensitivity limits were 20IU/ml and 80IU/ml for HCV RNA and HIV-1 RNA,respectively. Conclusion The establishment of QRT-PCR assay for HCV/HIV RNA detection will provide theoretical basis and technical support for developing multiplex QRT-PCR assay for several viruses (HBV/HCV/HIV-1) at the same time.

  15. Evaluation of Sensitivities and Specificities of SARS-CoV Detection by Real-time Quantitative Reverse Transcription-PCR Assays

    Institute of Scientific and Technical Information of China (English)

    Li-li XU; Zhi-hong HU; Hua-lin WANG; Xiao HAN; Fei DENG

    2009-01-01

    The etiological agent of severe acute respiratory syndrome (SARS) was identified as a new coronavirus, termed SARS-CoV. Establishment of an efficient and sensitive diagnostic system of SARS-CoV genetic materials is crucial for SARS control. In this study, we quantified SARS-CoV mRNAs in both infected cell culture lysate and in supernatant by using Real-time quantitative revere transcription-PCR based on EvaGreen鈩?dye and Taqman-MGB probes. For extensive evaluation of sensitivities and specificities, 13 pairs of primers and 4 probes were designed based on different genes of SARS-CoV. Glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was selected as the internal control gene. Results showed that S-gene-specific PCR was the most sensitive for detection, but because of its sequence variability in the different viral strains, primers and a probe based on the N gene were suitable substitutions. Meanwhile, we found the mRNA concentrations in cell culture lysates were much higher than in cell supernatant and facilited more sensitive detection of the SARS-CoV.

  16. In planta and soil quantification of Fusarium oxysporum f. sp. ciceris and evaluation of Fusarium wilt resistance in chickpea with a newly developed quantitative polymerase chain reaction assay.

    Science.gov (United States)

    Jiménez-Fernández, Daniel; Montes-Borrego, Miguel; Jiménez-Díaz, Rafael M; Navas-Cortés, Juan A; Landa, Blanca B

    2011-02-01

    Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris can be managed by risk assessment and use of resistant cultivars. A reliable method for the detection and quantification of F. oxysporum f. sp. ciceris in soil and chickpea tissues would contribute much to implementation of those disease management strategies. In this study, we developed a real-time quantitative polymerase chain reaction (q-PCR) protocol that allows quantifying F. oxysporum f. sp. ciceris DNA down to 1 pg in soil, as well as in the plant root and stem. Use of the q-PCR protocol allowed quantifying as low as 45 colony forming units of F. oxysporum f. sp. ciceris per gram of dry soil from a field plot infested with several races of the pathogen. Moreover, the q-PCR protocol clearly differentiated susceptible from resistant chickpea reactions to the pathogen at 15 days after sowing in artificially infested soil, as well as the degree of virulence between two F. oxysporum f. sp. ciceris races. Also, the protocol detected early asymptomatic root infections and distinguished significant differences in the level of resistance of 12 chickpea cultivars that grew in that same field plot infested with several races of the pathogen. Use of this protocol for fast, reliable, and cost-effective quantification of F. oxysporum f. sp. ciceris in asymptomatic chickpea tissues at early stages of the infection process can be of great value for chickpea breeders and for epidemiological studies in growth chambers, greenhouses and field-scale plots.

  17. Quantitative Real-Time Reverse Transcription-PCR Assay for the Expression of Tob mRNA in Human Colorectal Cancer

    Institute of Scientific and Technical Information of China (English)

    Dian-chao WU

    2010-01-01

    OBJECTIVE Tob is a member of Tob/BTG antiproliferative family. To date, Tob expression in human carcinoma using clinical specimens has not been studied in depth except for lung carcinoma and thyroid carcinoma. This study is the first to investigate the expression levels of Tob gene in human colorectal cancer tissues,and their corresponding para-cancerous tissues. The correlation of expression of the Tob gene with clinicopathological characteristics of colorectal cancer was also analyzed.METHODS Quantitative real time RT-PCR was used to detect the expression of Tob mRNA in 31 colorectal cancers.RESULTS Compared with normal tissues, up-regulation of Tob mRNA was observed in 31 colorectal cancer tissues (P = 0.020).The expression level of Tob at Dukes C + D phase was higher than Dukes A + B phase, and the difference was signifi cant (P < 0.05).However, in this study, it was found that the expression of Tob mRNA was not related with age, gender, and pathological type of colorectal cancer.CONCLUSION The up-regulation of Tob may be closely associated with tumorigenesis of colorectal carcinoma.

  18. Enabling comparative gene expression studies of thyroid hormone action through the development of a flexible real-time quantitative PCR assay for use across multiple anuran indicator and sentinel species.

    Science.gov (United States)

    Veldhoen, Nik; Propper, Catherine R; Helbing, Caren C

    2014-03-01

    Studies performed across diverse frog species have made substantial contributions to our understanding of basic vertebrate development and the natural or anthropogenic environmental factors impacting sensitive life stages. Because, anurans are developmental models, provide ecosystems services, and act as sentinels for the identification of environmental chemical contaminants that interfere with thyroid hormone (TH) action during postembryonic development, there is demand for flexible assessment techniques that can be applied to multiple species. As part of the "thyroid assays across indicator and sentinel species" (TAXISS) initiative, we have designed and validated a series of cross-species real time quantitative PCR (qPCR) primer sets that provide information on transcriptome components in evolutionarily distant anurans. Validation for fifteen gene transcripts involved a rigorous three-tiered quality control within tissue/development-specific contexts. Assay performance was confirmed on multiple tissues (tail fin, liver, brain, and intestine) of Rana catesbeiana and Xenopus laevis tadpoles enabling comparisons between tissues and generation of response profiles to exogenous TH. This revealed notable differences in TH-responsive gene transcripts including thra, thrb, thibz, klf9, col1a2, fn1, plp1, mmp2, timm50, otc, and dio2, suggesting differential regulation and susceptibility to contaminant effects. Evidence for the applicability of the TAXISS anuran qPCR assay across seven other species is also provided with five frog families represented and its utility in defining genome structure was demonstrated. This novel validated approach will enable meaningful comparative studies between frog species and aid in extending knowledge of developmental regulatory pathways and the impact of environmental factors on TH signaling in frog species for which little or no genetic information is currently available. PMID:24503578

  19. An Improved Quantitative Real-Time PCR Assay for the Enumeration of Heterosigma akashiwo (Raphidophyceae) Cysts Using a DNA Debris Removal Method and a Cyst-Based Standard Curve.

    Science.gov (United States)

    Kim, Joo-Hwan; Kim, Jin Ho; Wang, Pengbin; Park, Bum Soo; Han, Myung-Soo

    2016-01-01

    The identification and quantification of Heterosigma akashiwo cysts in sediments by light microscopy can be difficult due to the small size and morphology of the cysts, which are often indistinguishable from those of other types of algae. Quantitative real-time PCR (qPCR) based assays represent a potentially efficient method for quantifying the abundance of H. akashiwo cysts, although standard curves must be based on cyst DNA rather than on vegetative cell DNA due to differences in gene copy number and DNA extraction yield between these two cell types. Furthermore, qPCR on sediment samples can be complicated by the presence of extracellular DNA debris. To solve these problems, we constructed a cyst-based standard curve and developed a simple method for removing DNA debris from sediment samples. This cyst-based standard curve was compared with a standard curve based on vegetative cells, as vegetative cells may have twice the gene copy number of cysts. To remove DNA debris from the sediment, we developed a simple method involving dilution with distilled water and heating at 75°C. A total of 18 sediment samples were used to evaluate this method. Cyst abundance determined using the qPCR assay without DNA debris removal yielded results up to 51-fold greater than with direct counting. By contrast, a highly significant correlation was observed between cyst abundance determined by direct counting and the qPCR assay in conjunction with DNA debris removal (r2 = 0.72, slope = 1.07, p cysts in sediment samples.

  20. Development of a Quantitative TaqMan{trademark}-PCR Assay and Feasibility of Atmosphoric Collection for Coccidioides Immits for Ecological Studies

    Energy Technology Data Exchange (ETDEWEB)

    Daniels, J I; Wilson, W J; DeSantis, T Z; Gouveia, F J; Anderson, G L; Shinn, J H; Pletcher, R; Johnson, S M; Pappagianis, D

    2002-02-01

    identified as a select (biological) agent in the federal Anti-Terrorist and Effective Death Penalty Act. Successful demonstration of these tools in this study will place this multidisciplinary team in a credible position to proceed with additional research designed to determine the climatic signals and ecological triggers that would be associated with the presence of this microorganism environmentally and that would correlate with subsequent outbreaks of Valley Fever clinically. Results from such future research would then provide the information needed for environmental intervention of the disease occurrence, well before clinical cases appear. The technology and modeling developed for such a study also could be used for determining the ecology of other environmentally linked, medically important infectious diseases that occur naturally or that might be introduced deliberately into environmental media indoors or outside. The following approach was taken to achieve the technological objectives of this study. First, the protocols for the TaqMan{trademark}-PCR assay were enhanced to achieve the superior specificity and sensitivity required for quantifying, from a DNA signature, those C. immitis spores that are present in calibration samples (consisting of known quantities of pure culture inoculated onto air-filter concentrate, and then removed, and the DNA extracted) and those that are present within the calibration range on air filters obtained from the field and handled similarly. Second, the feasibility of using advanced nuclepore air-filter media to collect the spores from ambient air in C immitis endemic areas in the Central Valley of California was evaluated. These membranes permit the physics of high-volume air sampling to be used to filter larger amounts of air than previously possible for detecting airborne microorganisms. Thus, the higher volume of air flow improves the likelihood of capturing on the filter any C. immitis spores resuspended from nearby soil, where

  1. Development of a Quantitative TaqMan{trademark}-PCR Assay and Feasibility of Atmosphoric Collection for Coccidioides Immits for Ecological Studies

    Energy Technology Data Exchange (ETDEWEB)

    Daniels, J I; Wilson, W J; DeSantis, T Z; Gouveia, F J; Anderson, G L; Shinn, J H; Pletcher, R; Johnson, S M; Pappagianis, D

    2002-02-01

    identified as a select (biological) agent in the federal Anti-Terrorist and Effective Death Penalty Act. Successful demonstration of these tools in this study will place this multidisciplinary team in a credible position to proceed with additional research designed to determine the climatic signals and ecological triggers that would be associated with the presence of this microorganism environmentally and that would correlate with subsequent outbreaks of Valley Fever clinically. Results from such future research would then provide the information needed for environmental intervention of the disease occurrence, well before clinical cases appear. The technology and modeling developed for such a study also could be used for determining the ecology of other environmentally linked, medically important infectious diseases that occur naturally or that might be introduced deliberately into environmental media indoors or outside. The following approach was taken to achieve the technological objectives of this study. First, the protocols for the TaqMan{trademark}-PCR assay were enhanced to achieve the superior specificity and sensitivity required for quantifying, from a DNA signature, those C. immitis spores that are present in calibration samples (consisting of known quantities of pure culture inoculated onto air-filter concentrate, and then removed, and the DNA extracted) and those that are present within the calibration range on air filters obtained from the field and handled similarly. Second, the feasibility of using advanced nuclepore air-filter media to collect the spores from ambient air in C immitis endemic areas in the Central Valley of California was evaluated. These membranes permit the physics of high-volume air sampling to be used to filter larger amounts of air than previously possible for detecting airborne microorganisms. Thus, the higher volume of air flow improves the likelihood of capturing on the filter any C. immitis spores resuspended from nearby soil, where

  2. 牛副结核分枝杆菌实时荧光定量PCR检测方法的建立%Development of Real-time Fluorescence Quantitative PCR Assay for Detection of Bovine Mycobacterium paratuberculosis

    Institute of Scientific and Technical Information of China (English)

    王素华; 王忠才; 李孝军; 杜爱芳

    2012-01-01

    根据GenBank上公布的牛副结核分枝杆菌C-2染色体的ISMav2基因保守区域序列设计合成1对特异性引物,建立了一套SYBR Green Ⅰ荧光定量PCR检测牛副结核分枝杆菌(Mycobacterium paratuberculosis)的方法.以实验室构建的牛副结核分枝杆菌pMD-ISMav2阳性重组质粒为标准品,通过优化反应条件,建立了标准曲线,其相关系数为0.999.以构建的标准品为模板,进行了特异性和敏感性试验.结果显示,该方法检测布氏杆菌、大肠杆菌、沙门氏菌、链球菌DNA均为阴性;最低可检测到相当于每微升1.96×101拷贝数的标准品阳性质粒.本研究建立的实时荧光定量PCR具有特异、敏感和快速等优点,可用于牛副结核杆菌病的监测.%According to the published sequences of bovine Mycobacterium paratuberculosis in GenBank, a pair of primers were designed and synthesized for the C-2 chromosome(ISMav2)gene of Mycobacterium paratuberculosis, and a SYBR-Green I fluorescent Real-time quantitative PCR assay was developed. The positive standard plasmid pMD-ISMav2 were used as quantitative template to make the standard curves by optimizing the reaction conditions, the correlation coefficient (R2) was 0. 999. By using the positive standard as template, specificity and sensitivity were tested. According to the experiment, all negative controls such as Brucella, Escherichia coli, Salmonella and Streptococcus showed negative detection. By sensitivity analysis, the Real-time PCR indicated that a minimum of 1. 96×101 copies of plasmid DNA was detected. As a result of the specificity and sensitivity of the assay with a relatively rapid and simple procedure, the Real-time PCR can be used as a routine assay for the diagnosis of Mycobacterium paratuberculosis.

  3. Quantitation of N2-[1-(1-carboxy)ethyl]folic acid, a nonenzymatic glycation product of folic acid, in fortified foods and model cookies by a Stable isotope dilution assay.

    Science.gov (United States)

    Rychlik, Michael; Mayr, Anja

    2005-06-29

    A stable isotope dilution assay (SIDA) for the quantitation of N(2)-[1-(carboxy)ethyl]folic acid (CEF) has been developed by using [(2)H(4)]CEF as the internal standard. After sample cleanup by anion exchange chromatography, the three-dimensional specifity of liquid chromatography-tandem mass spectrometry enabled unequivocal determination of the nonenzymatic glycation product of folic acid (FA). When CEF was added to cornstarch, the detection limit for CEF was found to be 0.4 microg/100 g, and a recovery of 98.5% was determined. In analyses of cookies, the intra-assay coefficient of variation was 8.0% (n = 5). Application of the SIDA to commercial cookies produced from wheat flour fortified with FA revealed CEF contents of up to 7.1 microg/100 g, which accounted for approximately 10-20% of the cookies' FA content. In baby foods, multivitamin juices, and multivitamin sweets, however, CEF was not detectable. Further studies on CEF formation during baking of cookies made from fortified flour and different carbohydrates revealed that fructose was most effective in generating CEF followed by glucose, lactose, and sucrose with 12.5, 3.9, 2.5, and 2.5 microg/100 g of dry mass, respectively. During baking, approximately 50% of FA was retained for both monosaccharides fructose and glucose, and 77% as well as 85% of its initial content was retained for the disaccharides lactose and sucrose, respectively. Of the degraded amount of FA, CEF comprised 28% for fructose as well as 18, 12, and 8% for sucrose, lactose, and glucose, respectively. Therefore, CEF can be considered an important degradation product of FA in baked foods made from fructose. To retain a maximum amount of FA, products should rather be baked with sucrose than with reducing carbohydrates.

  4. Development of a quantitative assay to measure expression of transforming growth factor ß (TGF-ß) in Lost River sucker (Deltistes luxatus) and shortnose sucker (Chasmistes brevirostris) and evaluation of potential pitfalls in use with field-collected samples

    Science.gov (United States)

    Robertson, Laura S.; Ottinger, Christopher A.; Burdick, Summer M.; VanderKooi, Scott P.

    2012-01-01

    The Nature Conservancy is in the process of restoring the Williamson River Delta in an attempt to recreate important juvenile habitat for the endangered shortnose sucker Chasmistes brevirostris and the endangered Lost River sucker Deltistes luxatus. Measurement of TGF-β mRNA expression level was one of the indicators chosen to evaluate juvenile sucker health during the restoration process. TGF-β mRNA expression level has been correlated with disease status in several laboratory studies and TGF-β mRNA expression level has been used as a species-specific indicator of immune status in field-based fish health assessments. We describe here the identification of TGF-β and a possible splice variant from shortnose sucker and from Lost River sucker. The performance of a quantitative RT-PCR assay to measure TGF-β mRNA expression level was evaluated in field-collected spleen and kidney tissue samples. The quality of extracted RNA was higher in tissues harvested in September compared to July and higher in tissues harvested at lower temperature compared to higher temperature. In addition, the expression level of both TGF-β and 18S as assessed by qRT-PCR was higher in samples with higher quality RNA. TGF-β mRNA expression was lower in kidney than in spleen in both Lost River sucker and shortnose sucker.

  5. Development of a quantitative assay to measure expression of transforming growth factor β (TGF-β) in Lost River sucker (Deltistes luxatus) and shortnose sucker (Chasmistes brevirostris) and evaluation of potential pitfalls in use with field-collected samples.

    Science.gov (United States)

    Robertson, Laura S; Ottinger, Christopher A; Burdick, Summer M; VanderKooi, Scott P

    2012-05-01

    The Nature Conservancy is in the process of restoring the Williamson River Delta in an attempt to recreate important juvenile habitat for the endangered shortnose sucker Chasmistes brevirostris and the endangered Lost River sucker Deltistes luxatus. Measurement of TGF-β mRNA expression level was one of the indicators chosen to evaluate juvenile sucker health during the restoration process. TGF-β mRNA expression level has been correlated with disease status in several laboratory studies and TGF-β mRNA expression level has been used as a species-specific indicator of immune status in field-based fish health assessments. We describe here the identification of TGF-β and a possible splice variant from shortnose sucker and from Lost River sucker. The performance of a quantitative RT-PCR assay to measure TGF-β mRNA expression level was evaluated in field-collected spleen and kidney tissue samples. The quality of extracted RNA was higher in tissues harvested in September compared to July and higher in tissues harvested at lower temperature compared to higher temperature. In addition, the expression level of both TGF-β and 18S as assessed by qRT-PCR was higher in samples with higher quality RNA. TGF-β mRNA expression was lower in kidney than in spleen in both Lost River sucker and shortnose sucker. PMID:22366312

  6. Identification of recombinant baculovirus and determination of virus titer with fluorescence quantitative PCR assay%荧光定量PCR用于重组杆状病毒鉴定及病毒滴度检测的研究

    Institute of Scientific and Technical Information of China (English)

    童夏生; 孟哲峰

    2007-01-01

    AIM: To develop a real - time PCR assay based on TaqMan technology for the identification of recombinant baculovirus and determination of virus physical titers in Bac - to - Bac system. METHODS: The recombinant baculovirus containing human IL- 18 gene was produced using Bac -to- Bac system. A 10 -fold serially diluted primary viral stock was used for plaque assay and DNA extraction. Bacmid (baculovirus plasmid) was 10 -fold serially diluted and served as standards. Real - time PCR amplification of the IL - 18 gene was performed in triplicate for each diluted recombinant virus. At the same time, plaque assays were performed using overlay agarose method. RESULTS: The standard linear range (101 to 108 copies) for quantitation was achieved with the standard curve. We also find that the"vg/mL"titer value is generally about 10 times than"pfu/mL"titer of the same recombinant virus stock. CONCLUSION: A TaqMan real -time PCR method is established to identify the recombinant baculovirus and determine the"vg/mL"titer of virus. The method is rapid and quantitative over a wide range of virus titers.%目的:建立一种高效、简便的荧光实时定量PCR方法,用于重组杆状病毒鉴定及病毒滴度的检测.方法:利用Bac-to-Bac载体系统在昆虫细胞中构建含人IL-18基因的重组杆状病毒,收获的病毒母液以10倍梯度系列稀释后,提取病毒基因组DNA.以10倍梯度稀释的重组杆状病毒穿梭质粒(bacmid)作为标准模板,进行荧光定量PCR反应扩增IL-18基因片段并绘制标准曲线,然后以上述的重组杆状病毒基因组DNA作为模板,采用同样体系进行实时PCR反应检测,同时用琼脂糖空斑法测定病毒母液的滴度.结果:成功构建了重组杆状病毒并建立了病毒滴度的实时荧光PCR检测方法.运用标准模板进行的PCR反应显示该方法的线形范围为101-108拷贝,病毒母液的DNA拷贝浓度(vg/mL)值约为空斑检测的滴度pfu/mL值的10倍.结论:荧光定量PCR

  7. The haemolytic antibody isotope release (HAIR) assay; an efficient alternative technique to conventional plaque assays

    International Nuclear Information System (INIS)

    The haemolytic antibody isotope release (HAIR) assay quantitates antibody production by splenic antibody-producing cells by lysis of chromium-51-labelled sheep red blood cells. The amount of antibody quantitated by the HAIR assay directly correlates with the number of antibody-producing cells measured by a conventional plaque assay. The HAIR assay is an easy, sensitive, and reproducible technique that is especially useful when large numbers of animals are required for testing. (author)

  8. Development and Application of a Quantitative Competitive PCR Assay for Detecting Mycoplasma hyopneumoniae%猪肺炎支原体竞争定量 PCR检测方法的建立及初步应用

    Institute of Scientific and Technical Information of China (English)

    刘茂军; 李冬梅; 苏国东; 周勇岐; 白方方; 武昱孜; 刘蓓蓓; 邵国青

    2015-01-01

    [目的]本试验旨在探索一种对猪肺炎支原体(Mycoplasma hyopneumoniae)培养物中的支原体进行快速检测的竞争定量 PCR方法。[方法]设计一对Mhp特异性引物,检测Mhp培养物;又根据支原体种属保守基因序列设计一对引物,通过酶切缺失法构建竞争模板,其携带与目的片段相同的引物结合区。[结果]以一系列稀释的竞争模板的对数值为横坐标(X轴),竞争模板和目标模板扩增产物的光密度比值的校正值的对数值为纵坐标(Y轴)绘制标准曲线,根据 Y=0所对应的竞争模板的量推算求得支原体的拷贝数。以变色单位的对数值为 X轴,支原体的拷贝数为 Y轴,绘制出标准曲线,两者之间高度相关。[结论]成功建立了一种快速测定 Mhp培养物中支原体量的竞争PCR方法。%Objective] This study aimed to develop a quantitative competitive assay for detecting Mycoplasma hyopneumoniae in culture. [Method] One pair of Mhp-specific primers was designed for detecting Mhp in culture. Another pair of primers was designed based on the conserved gene sequences of Mycoplasma. The com-petitive template, which carried the same primer binding site with the target frag-ment, was constructed using enzyme digestion method. [Result] The logarithm of concentration of competitive template was treated as the abscissa (X-axis), and the logarithm of corrected optical density ratio between amplification products by compet-itive template and target template was treated as the ordinate (Y-axis). Thus the standard curve was drawn, and the regression equation was also obtained. When Y was assigned as 0, the concentration of the competitive plate was calculated, and then the concentration of Mhp was deduced. The logarithms of Color change unit (CCU) were treated as the abscissa (X-axis), and the copy numbers of Mhp were treated as the ordinate (Y-axis), so the standard curve was generated. It was found that the

  9. 荧光定量PCR技术检测结核杆菌临床应用分析%Analysis on the Clinical Application of Fluorescence Quantitative PCR Assay in Detection of Mycobacterium Tuberculosis

    Institute of Scientific and Technical Information of China (English)

    王雅宁

    2012-01-01

    Objective To investigate the clinical application value of fluorescence quantitative PCR (FQ — PCR) technigue in the detection of mycobacterium tuberculosis. Methods Sixty — eight patients clinically diagnosed as pulmonary tuberculosis in the First Hospital of Baoding, Hebei Province, were chosen as the research objects. Mycobacterium tuberculosis in peripheral blood and one portion of sputum was determined by FQ- PCR. Another portion of sputum was subject to sputum smear. Clinical diagnosis was used as the gold standard. The results were analyzed by χ2 test. Results The positive rate of mycobacterium tuberculosis detected by sputum smear, sputum FQ- PCR and peripheral blood FQ- PCR in 68 patients with pulmonary tuberculosis was 14.7%, 41.2% and 44.1%, respectively. The positive rates by FQ- PCR were significantly higher than that by sputum smear (P < 0.05). The positive rates by sputum FQ - PCR and peripheral blood FQ - PCR in 58 cases with negative detection results by sputum smear was 31.0% and 41.3% . The coincidence rate by FQ - PCR in sputum and in paired peripheral blood was 44. 1 % . Conclusions Fluorescence quantitative PCR assay is superior to conventional sputum smear in mycobacte rium tuber culosis detection. It can confirm and identify the positive results of sputum smear. To simultaneously conduct FQ-PCR assay in sputum and peripheral blood can have complementary effect and improve the detection accuracy.%目的 探讨荧光定量PCR技术在检测结核杆菌的临床应用价值. 方法 将本院临床诊断确诊为肺结核的68例患者为研究对象,采用荧光PCR技术测定两组痰液及外周血中的结核杆菌,以临床诊断结果为金标准,采用x2检验对结果进行分析比较. 结果 68例结核病患者中痰涂片、痰定量PCR、外周血定量PCR检测的阳性率为14.7%、41.2%、44.1%,定量PCR检测的阳性率显著高于痰涂片(P<0.05).痰涂片阴性的58例患者中痰及外周

  10. Label-Free Nanoplasmonic-Based Short Noncoding RNA Sensing at Attomolar Concentrations Allows for Quantitative and Highly Specific Assay of MicroRNA-10b in Biological Fluids and Circulating Exosomes.

    Science.gov (United States)

    Joshi, Gayatri K; Deitz-McElyea, Samantha; Liyanage, Thakshila; Lawrence, Katie; Mali, Sonali; Sardar, Rajesh; Korc, Murray

    2015-11-24

    MicroRNAs are short noncoding RNAs consisting of 18-25 nucleotides that target specific mRNA moieties for translational repression or degradation, thereby modulating numerous biological processes. Although microRNAs have the ability to behave like oncogenes or tumor suppressors in a cell-autonomous manner, their exact roles following release into the circulation are only now being unraveled and it is important to establish sensitive assays to measure their levels in different compartments in the circulation. Here, an ultrasensitive localized surface plasmon resonance (LSPR)-based microRNA sensor with single nucleotide specificity was developed using chemically synthesized gold nanoprisms attached onto a solid substrate with unprecedented long-term stability and reversibility. The sensor was used to specifically detect microRNA-10b at the attomolar (10(-18) M) concentration in pancreatic cancer cell lines, derived tissue culture media, human plasma, and media and plasma exosomes. In addition, for the first time, our label-free and nondestructive sensing technique was used to quantify microRNA-10b in highly purified exosomes isolated from patients with pancreatic cancer or chronic pancreatitis, and from normal controls. We show that microRNA-10b levels were significantly higher in plasma-derived exosomes from pancreatic ductal adenocarcinoma patients when compared with patients with chronic pancreatitis or normal controls. Our findings suggest that this unique technique can be used to design novel diagnostic strategies for pancreatic and other cancers based on the direct quantitative measurement of plasma and exosome microRNAs, and can be readily extended to other diseases with identifiable microRNA signatures.

  11. New Application of the Comet Assay: Chromosome–Comet Assay

    OpenAIRE

    Cortés-Gutiérrez, Elva I.; DÁVILA-RODRÍGUEZ, MARTHA I.; Fernández, José Luís; López-Fernández, Carmen; Gosálbez, Altea; Gosálvez, Jaime

    2011-01-01

    The comet assay is a well-established, simple, versatile, visual, rapid, and sensitive tool used extensively to assess DNA damage and DNA repair quantitatively and qualitatively in single cells. The comet assay is most frequently used to analyze white blood cells or lymphocytes in human biomonitoring studies, although other cell types have been examined, including buccal, nasal, epithelial, and placental cells and even spermatozoa. This study was conducted to design a protocol that can be use...

  12. A novel enzyme-linked immunosorbent assay for quantitative detection of anti-thyroid peroxidase antibodies in serum%血清抗甲状腺过氧化物酶抗体ELISA定量方法的建立

    Institute of Scientific and Technical Information of China (English)

    孙颖; 李会强; 陈寅; 仁杰; 李婵

    2011-01-01

    Objective To establish a novel enzyme-linked immunosorbent assay (ELISA) for quantitative detection of the concentra tion of anti-thyroid peroxidase antibody (TPOAb) in serum. Methods The microtiter plate was coated with biotinylated bovine serum albumin (BSA) and streptavidin. The biotinylated TPO antigen and standardized anti-TPOAb or test sera were successively added into the wells of plate. The HRP-anti-IgG was then added into the plate for colorization. The optimal concentrations of biotinylated TPO an tigen and HRP-anti-IgG were screened by chessboard titration, and the reaction conditions were optimized for the method evaluation. Results In the indirect-coating mode, the amount of coated antigen was 0. 083 μg/mL. The sensitivity of the assay was 0. 165 IU/mL. The coefficient of variations (CV) of inter-assay in high and low concentration of serum mixture were 9.2% and 9.0% , and the CV of intra-assay were 4.6% and 5.6% respectively. The recovery rate was between 96% and 104%. The coated ELISA plate re mained stable for 5 days at 37 ℃. The rate of cross-reaction with anti-thyroid globulin (TGAb) was 0. 22%. The reference range in serum was less than 65. 7 IU/mL. The correlation coefficient of the experimental results with those of Abbott kit was 0. 985 (P < 0.01). Conclusion In the developed ELISA of indirect-coated mode, the amount of needed purified antigen significantly reduced. The sensitivity and specificity of the assay were satisfied. The method is simple and cost-saving, so it should be very suitable for anti TPOAb detection in primary hospitals.%目的 建立一种ELISA方法用于定量分析血清抗甲状腺过氧化物酶(TPO)抗体(TPOAb).方法 用生物素化牛血清清蛋白(BSA)和链霉亲合素包被微孔板,同时加入生物素化TPO抗原和待检血清,再加入酶标记抗人IgG,建立间接包被模式酶联免疫法测定抗TPO抗体.经方阵滴定确定生物素化抗原和酶标抗体的最适浓度,优化反应条件,

  13. Staphylococcus aureus DNA in human venous blood detected by real-time quantitative PCR assay%实时定量PCR检测人全血中金黄色葡萄球菌DNA方法的建立

    Institute of Scientific and Technical Information of China (English)

    康军仁; 马恩陵; 房嘉宾; 崔希增

    2014-01-01

    目的 建立实时定量PCR (RQ-PCR)快速检测人全血中金黄色葡萄球菌DNA的方法,以便早期定量评估肠屏障损伤所致肠道内细菌移位引起或加重的全身感染.方法 对15份模拟全血标本及26份外科发热患者全血标本进行RQ-PCR检测.选择金黄色葡萄球菌高度保守的看家基因fem A基因作为靶基因设计引物和Taqman探针,建立20μl的RQ-PCR反应体系,采用含靶基因扩增片段的重组质粒建立标准曲线,提取血标本中的细菌基因组DNA.结果 引物和探针特异性好,检测限为100拷贝/μ1 (103 CFU/ml),灵敏度为99.7%,特异度为94.6%.标准曲线线性关系好,R2在0.9918 ~0.9997.不同浓度的金黄色葡萄球菌样本检测的平均准确性为(96.25±2.26)%,批内及批间重复性的平均变异系数分别为(8.06±0.07)%和(10.01±4.40)%.全血标本中金黄色葡萄球菌DNA平均回收率为(111.72±20.72)%.临床血标本RQ-PCR检测阳性率为15.4% (4/26),血培养结果皆为金黄色葡萄球菌阴性.结论 RQ-PCR可用于定量检测全血标本中的金黄色葡萄球菌DNA含量,具有快速、灵敏、特异性强、重复性好的优点.%Objective To establish a rapid real-time quantitative polymerase chain reaction (RQ-PCR)assay in quantifying and detecting Staphylococcus aureus DNA from human venous blood samples,so as to quantificationally evaluate the systemic infection caused or deteriorated by intestinal bacteria translocation.Methods Totally 26 clinical blood samples and 15 simulation blood samples were detected.The primers and TaqMan probe were designed targeting the highly conserved house-keeping femA gene of Staphylococcus aureus,and a 20 μl RQ-PCR amplification reaction system was established.The standard curve was built based on the recombinant plasmid DNA containing the amplicon of the target gene,and genomic DNA was extracted using QIAamp DNA Blood Mini Kit.Results The specificity of primers and probe was excellent

  14. Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infected bee tissues and in life stages of a symptomatic colony.

    OpenAIRE

    Blanchard, Philippe; Ribière, Magali; Celle, Olivier; Lallemand, Perrine; Schurr, Frank; Olivier, Violaine; Iscache, Anne Laure; Faucon, Jean Paul

    2007-01-01

    A two-step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify Chronic bee paralysis virus (CBPV) genome in bee samples. Standard curves obtained from a CBPV control RNA and from a plasmid containing a partial sequence of CBPV showed that this assay provided linear detection over a 7-log range (R(2)>0.99) with a limit of detection of 100 copies, and reliable inter-assay and intra-assay reproducibility. Standardisation including RN...

  15. 弯曲菌荧光定量PCR检测方法的建立及初步应用%Development and Application of Real-time PCR Assay for Quantitative Detection of Campylobacter spp

    Institute of Scientific and Technical Information of China (English)

    朱佳琪; 张小燕; 黄金林; 焦新安

    2014-01-01

    根据弯曲菌16S rRNA基因的靶序列设计特异性引物和探针,对反应条件和试剂浓度进行优化,建立了快速检测鸡肉中弯曲菌的荧光定量PCR方法,并对其特异性、敏感性和重复性进行评价。该方法除了对弯曲菌有扩增曲线外,对其他常见食源性病原菌均未有扩增曲线,具有较好的特异性;弯曲菌的最低检测限为10 CFU/mL;重复性实验获得标准曲线相关系数为R2=0.999,批内可重复性变异系数在0.12%-2.1%之间,批间可重复性变异系数为1.7%。应用该方法对68份鸡肉样品检测弯曲菌阳性率为95.6%(65/68),传统分离方法阳性率为89.5%(60/68),两种方法的阳性符合率为89.7%。本研究建立的荧光定量PCR方法灵敏度高、特异性好、操作简单,大大缩短检测周期,可作为检测鸡肉样品中弯曲菌的手段,为鸡肉样品弯曲菌的流行病学调查研究提供新的工具。%To establish a rapid assay for quantitative detection of Campylobacter spp.,a pair of primers and fluorescent probe were designed according to 16S rRNA sequence of Campylobacter spp.,and then a real-time PCR method was successfully established to detect Campylobacter spp. Results demonstrated that the method was speciifc for ampliifcation of Campylobacter spp.,but no ampliifcation for other related bacteria. The detection limit of the assay was 10CFU/mL and the correlation coefifcient of standard curve(R2)was 0.999. The detection of 68 chicken meat samples showed that the positive rate was 95.6%,while the positive rate was 89.5% by traditional culture method. The method was simple,rapid,speciifc and sensitive for rapid detection of Campylobacter spp. and a new tool for epidemiological investigation of Campylobacter spp. in chicken meats.

  16. Comparison of Three Enzyme-Linked Immunosorbent Assay Methods for Quantitative Determination of Ricin%3种酶联免疫分析法在蓖麻毒素定量测定中的比较

    Institute of Scientific and Technical Information of China (English)

    马小溪; 刘合珠; 唐吉军; 郭磊; 谢剑炜

    2011-01-01

    Three determination methods in enzyme-linked immunosorbent assay, namely optical absorption, fluorescent and chemiluminescent immunoassay, were established for quantitation of biotoxins protein ricin in various matrices. The detection conditions were systematically optimized.The results showed the best signal-to-noise (SNR) could be obtained for CLIA when dilution factor was 1:8000; and the chemiluminescence signal was stable at around 3 minutes after horseradish peroxidase reacted with its substrate. Whereafter, the three methods were compared for determination of ricin under individually optimized conditions. The comparative results indicated that chemiluminescent immunoassay could provide wider linear range (from 0.02 μg/L to 5.5 μg/L with correlation coefficient of 0.999), higher sensitivity (limit of detection was 0.005 μg/L), and could be adopted as a simple, rapid and robust approach. The CLIA method was applied to measure the spiked ricin in water, carbonated beverage, milk powder, coffee and human serum samples. The limits of detection and the recoveries were from 0.005 to 0.08 μg/L and from 89.6% to 108.8%, respectively.The method was quite suitable for quantitative determination of trace amounts of ricin in contaminated or poisoned samples.%建立了蓖麻毒素的3种酶联免疫定量分析法,即光吸收、荧光和化学发光免疫分析法,并系统优化了各项实验条件.结果显示:对于化学发光检测法,当酶标抗体HRP-4C13稀释倍数为1:8000时可获得最佳的信噪比,且酶与底物反应3 min后信号趋于稳定.随后在各自优化的条件下将3种方法用于毒素的检测.比较结果表明:化学发光酶联免疫分析法除具有线性范围宽(0.02~5.5 μg/L,r(2)=0.999)、灵敏度高(检出限为5 ng/L)的特点外,还具有简单、快速、体系稳定性好等优点.将本方法用于不同实际样品基质,如饮用水、碳酸饮料、奶粉、咖啡和血清中添加的蓖麻毒

  17. Assay Method for 235U in Low-Density Waste

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    <正>235U assay method will provide a semi-quantitative assay for any uranium lumps that might exist in low-density, low-Z material waste boxes within a short count time. These materials will consist of

  18. 检测伪狂犬病毒gB基因荧光定量PCR方法的建立%Development of fluorescence quantitative PCR assay to detect gB gene of pseudorabies virus

    Institute of Scientific and Technical Information of China (English)

    朱淑芬; 朱瑞良; 乔彩霞; 高志强; 张利峰; 张鹤晓; 吴亚琼; 王慧珊

    2012-01-01

    Pseudorabies virus(PRV) gB gene sequences including several genotypes strains available in GenBank were aligned with the DNAMAN software,the conserved region was then subjected to design specific primers and TaqMan probe.Then full-length PRV-gB gene amplified by PCR was cloned into pMD20-T vector and it was used as positive standard.A rapid fluorescence quantitative PCR detection method of pseudorabies virus was established by optimizing reaction conditions.The detection method had high specificity and reliability.The detection limit of FQ-PCR was 1.50×102 copies per reaction in analyzing pMD-gB standard.Compared with conventional PCR for diagnosis of PRV,the sensitivity of FQ-PCR method was 100 to 1 000-fold more than the conventional PCR and with good reproducibility.The detection results of 60 clinical specimens indicated that FQ-PCR not only checked out the positive samples which did the same with conventional PCR,but also checked out othed 2 positive samples which did not with conventional PCR.It confirmed that the method is sensitive.This assay can be used for early rapid detection of PRV,and for quarantine of import and export of meat products.%利用DNAMAN软件对GenBank登录的伪狂犬病病毒(PRV)各毒株gB基因序列进行比对分析,选择其保守区域设计合成特异性的引物和TaqMan探针,同时利用普通PCR技术扩增获得全长的伪狂犬病毒gB基因,并克隆到pMD20-T载体上作为阳性标准品。通过对荧光定量PCR反应条件的优化,建立了一种快速检测伪狂犬病病毒的荧光定量PCR技术。该检测技术具有较高的灵敏性、特异性和可靠性。对制备的pMD-gB阳性标准品的检测结果表明,所建立的伪狂犬病毒TaqMan荧光定量PCR最低检测极限可达1.50×102拷贝/反应;同时相比于普通PCR方法其灵敏度高100~1 000倍以上,并且重复性好。在对60份临床样品的检测中,荧光定量PCR不仅检出了普通PCR检测

  19. Two Types of Assays for Detecting Frog Sperm Chemoattraction

    OpenAIRE

    Burnett, Lindsey A.; Tholl, Nathan; Chandler, Douglas E.

    2011-01-01

    Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assay...

  20. An Evaluation of Quantitative PCR Assays (TaqMan® and SYBR Green for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates

    Directory of Open Access Journals (Sweden)

    Bing Zhang

    2016-09-01

    Full Text Available Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample.

  1. Is the Comet Assay a Sensitive Procedure for Detecting Genotoxicity?

    OpenAIRE

    Sasaki, Yu F.; Satomi Kawaguchi; Takanori Nakamura; Gisho Honda; Ayumi Yamamoto

    2010-01-01

    Although the Comet assay, a procedure for quantitating DNA damage in mammalian cells, is considered sensitive, it has never been ascertained that its sensitivity is higher than the sensitivity of other genotoxicity assays in mammalian cells. To determine whether the power of the Comet assay to detect a low level of genotoxic potential is superior to those of other genotoxicity assays in mammalian cells, we compared the results of Comet assay with those of micronucleus test (MN test). WTK1 hum...

  2. 实时荧光PCR技术在手足口病检测中的应用%Application of real-time fluorescent quantitative RT-PCR assay in the hand-foot-mouth disease detection

    Institute of Scientific and Technical Information of China (English)

    王敏; 周小蓟; 陈露

    2013-01-01

    Objective: To apply a method of real - time fluorescent quantitative RT - PCR for detecting samples from patients,and to examine its utility in diagnosis of hand - food - mouth disease( HFMD). Methods: A total of 257 samples from HFMD patients in Huaibei were collected in 2011 ,and they were detected by real - time fluorescent quantitative PCR method. The samples were subjected to screen in population of enterovirus with enterovinus 71 (EV71) and Coxasckievirus A 16 ( CoxA16) ,and the results were compared with those by conventional RT - PCR. Results: One hundred and thirty - eight samples were found intestinal virus nucleic acid positive in 257 specimens, the positive rate was 53.70% , of which, eighty - nine samples were EV71 positive, the positive rate was 34. 63% , thirty -four samples were CoxA16 positive, the positive rate was 13. 23% , while 15 samples were untyped, accounting for 5. 84% of the total cases. Conclusion: Real - time fluorescent quantitative PCR method has short time consumption, strong specificity, high sensitivity, can be used as a method for diagnosis of foot and mouth disease.%目的:对手足口病患者标本进行病毒核酸检测,探讨实时荧光定量RT-PCR方法在手足口病检测中的意义.方法:用实时荧光定量RT-PCR方法对2011年度淮北市采集的257份标本进行肠道病毒核酸检测,并对肠道病毒(EV71)、柯萨奇病毒A组16型(CoxA16)进行分型;同时,与常规RT-PCR的方法进行了比对.结果:257份标本中肠道病毒核酸阳性138份,阳性率为53.70%,其中,EV71阳性标本89份,阳性率34.63%,CoxA16型核酸阳性34份,阳性率13.23,未分型15份,占病例总数的5.84%.结论:实时荧光定量RT-PCR方法耗时短、特异性强、灵敏度高,可作为手足口病检测的诊断方法.

  3. Development of a multiplex Real-time fluorescent quantitative PCR assay for simultaneous detection of PRV,PPV and PCV2%3种猪繁殖障碍性病毒Real-time PCR快速检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    赵绪永; 马辉; 宁豫昌; 赵丽

    2012-01-01

    【目的】建立可同时检测猪伪狂犬病毒(PRV)、猪细小病毒(PPV)和猪圆环病毒Ⅱ型(PCV2)的多重实时荧光定量PCR方法。【方法】根据GenBank数据库中PRV、PPV和PCV2的核苷酸序列,设计3对特异性引物和探针,以10倍系列稀释的阳性质粒为模板,优化反应条件,建立检测PRV、PPV和PCV2的多重Real-time PCR方法,并对其敏感性、重复性和特异性进行检验;分别采用单项和多重Real-time PCR方法,对临床收集的42份疑似病料进行检测,比较2种方法的符合率。【结果】特异性和灵敏度试验表明,建立的多重Real-time PCR检测方法具有高度特异性,与其他病原无明显交叉反应;检测灵敏度高,可检出1.0×101拷贝/μL的阳性质粒或1TCID50/mL的病毒样品。用多重Real-time PCR对42份临床疑似病料进行检测,其检测结果与单重Real-time PCR结果完全一致,表明多重Real-time PCR方法是可行的。【结论】建立了可同时检测PRV、PPV和PCV2的多重Real-time PCR方法,该法具有快速、灵敏、特异和重复性好等优点。%【Objective】 The study developed a multiplex real-time fluorescent quantitative PCR which can simultaneously detect and discriminate porcine pseudorabies virus(PRV),porcine parvovirus(PPV) and porcine circovirus type 2(PCV2).【Method】 According to the nucleotide sequences of PRV,PPV and PCV2 from GenBank,3 pairs of specific primers and probes were designed.The positive plasmid diluted by 10 times was used as template to establish a multiplex Real-time PCR assay by optimizing the reaction conditions.Sensitivity,reproducibility and specificity assays were determined.42 clinical suspected disease materials were detected by the established multiplex Real-time PCR assay and compared with the result of singleplex assay.【Result】 The results of specificity and sensitivity assays showed that the specificity of the established multiplex Real-time PCR assay was high

  4. 联合应用实时荧光定量PCR和噬菌体生物扩增法在结核性脑膜炎诊断中的意义%The clinical significance of joint use of real-time fluorescence quantitative PCR and phage amplified biologically assay in the diagnosis of tuberculous meningitis

    Institute of Scientific and Technical Information of China (English)

    赵卫

    2012-01-01

    目的 分析联合应用实时荧光定量PCR和噬菌体生物扩增法在结核性脑膜炎诊断中的意义.方法 取该院于2007年4月至2011年4月收治的经临床确诊的结核性脑膜炎患者76例,随机分为观察组与对照组各38例.对照组患者使用涂片法进行结核分枝杆菌的检查,观察组联合使用实时荧光定量PCR和噬菌体生物扩增法,比较两者检测结果的真阳性率情况.结果 观察组阳性检出率为97.37%(37/38),对照组阳性检出率为57.89%(22/38),两组比较差异有统计学意义(P<0.05).结论 联合应用实时荧光定量PCR和噬菌体生物扩增法可以快速准确检测出早期结核性脑膜炎患者,改善其预后,值得推广使用.%Objective To explore the diagnostic value of joint use of real-time fluorescence quantitative PCR and phage amplified biologically assay in the diagnosis of tuberculous meningitis .Methods 76 patients with tuberculous meningitis in our hospital from April 2007 to April 2011,which were clinically diagnosed and randomly divided into the observation group and the control group with 38 cases in each group .Patients in the control group using the smear method for Mycobacterium tuberculosis screening,the observation group used a combination of real-time fluorescence quantitative PCR and phage amplified biologically assay,comparing detection results of true positive rate .Results The observation group positive rate was 97 .37/o (37/38) .The control groups positive rate was 57 .89% (22/38) .There was significant difference between two groups (P<0 .05) .Conclusion There were important values for early diagnosis of tuberculous meningitis in joint use of real-time fluorescence quantitative PCR and phage amplified biologically assay .

  5. Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

    KAUST Repository

    Abu Samra, Dina Bashir Kamil

    2015-06-29

    Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow onand off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Establishment and preliminary application of real time PCR assay for quantitative detection of CRLF2%CRL F2实时定量PCR检测方法的建立及初步应用

    Institute of Scientific and Technical Information of China (English)

    付晶晶; 李红; 易丽君; 乐萍; 黄慧

    2015-01-01

    Objective To establish a real‐time quantitative PCR method for the detection of cytokine receptor‐like factor 2 (CRLF2) expression .Methods Specific primers amplification target gene CRLF2 and housekeeping genes ABL were designed ,the purified PCR products were performed the TA cloning .After bacterial colony PCR screening and sequencing ,then the recombinant plasmids DNA was extracted and measured by using UV spectrophotometer and converted to copies/mL concentration .Finally it was diluted for preparing the plasmid standard substance ,then the standard curve was drawn for observing the sensitivity and linear rang ,meanwhile the stability of the plasmid DNA was evaluated .This method was initially applied to detect the CRLF2 level of bone marrow mononuclear cells in 10 cases of healthy children and 10 cases of newly diagnosed acute lymphoblastic leukemia (ALL) .Results CRLF2 PCR product had a single specific melting curve;the linear detection range of the standard substance was 103 - 108 copies /ml;the plasmid standard substance by freeze‐thawing for 3 times remained stable;the CRLF2 level of clinical sample was within the linear detection range of standard substance .Conclusion The real‐time quantitative PCR method for CRLF2 established by our laboratory has good specificity ,linearity range and stability ,which can be applied to the quantitative detection of CRLF2 gene in clinical ALL children .%目的:建立人细胞因子受体样因子2(CRLF2)实时定量 PCR检测方法。方法设计特异性引物扩增目的基因CRLF2及管家基因ABL ,将纯化的PCR产物进行TA克隆,经菌落PCR筛选并测序鉴定后,提取重组质粒DNA ,紫外分光光度计检测浓度换算成copies/mL浓度,稀释制备成质粒标准品,制作标准曲线观察灵敏度和线性范围,同时对质粒标准品的稳定性进行评估。初步应用该方法检测10例健康儿童和10例急性淋巴细胞白血病(ALL)初诊儿

  7. Comparison of the second-generation digene hybrid capture assay with the branched-DNA assay for measurement of hepatitis B virus DNA in serum

    OpenAIRE

    Ho, Stephen K. N.; Chan, Tak Mao; Cheng, Ignatius K. P.; Lai, Kar Neng

    1999-01-01

    The optimal hepatitis B virus (HBV) DNA quantitative assay for clinical use remains to be determined. We examined the sensitivity, linearity, and variability of a novel second-generation antibody capture solution hybridization assay, the Digene Hybrid Capture II assay (HCII), and compared it with another widely used solution hybridization assay, the branched-DNA (bDNA) assay (Quantiplex; Chiron Corp.). Our results showed similar and satisfactory assay linearity values, as well as interassay a...

  8. Preparation of monoclonal antibodies against ricin toxin and development of up-converting phosphor technology-based lateral flow assay for its quantitative detection%蓖麻毒素单抗制备及上转发光免疫层析定量检测方法研究

    Institute of Scientific and Technical Information of China (English)

    王晓晨; 周蕾; 孙崇云; 赵勇; 王鑫蕊; 张平平; 杨瑞馥; 马馨

    2016-01-01

    Objective To develop an up-converting phosphor technology based lateral flow assay ( UPT-LF) to detect ricin toxin ( RT) quickly, accurately and quantitatively.Methods Ricin-monoclonal antibodies were prepared and their affinity was evaluated before four types of monoclonal antibodies with the highest titer were applied to couple with the up-converting phosphor nano-particles ( UCP-NPs) as the bio-conjugate and disperse on the analysis membrane as the test line, respectively.Following systematic optimization to establish the RT-UPT-LF strip, the sensitivity, precision, quantita-tive ability and specificity of RT-UPT-LF were evaluated.Results The detection could be accomplished within 15 min and the detection limit of the RT-UPT-LF assay could reach 0.5 ng/ml within the quantitative detection range of 0.5-1000 ng/ml.Other non-specific toxins at a concentration of 1000 ng/ml did not cause any non-specific reactions.Conclusion The developed RT-UPT-LF strip provides a new means for on-site quantitative detection of ricin toxin.%目的:建立一种可快速、精确地对蓖麻毒素( RT)进行定量检测的上转发光免疫检测技术( UPT-LF),即RT-UPT-LF。方法采用杂交瘤细胞技术制备蓖麻毒素单克隆抗体并对效价进行评价;采用效价最高的4种单抗分别与上转发光纳米颗粒( UCP-NP)共价偶联,分别作为检测带,进而两两配伍,确定最优的配伍组合条件建立RT-UPT-LF方法;对RT-UPT-LF的敏感性、精密性、定量能力和特异性进行评价。结果与结论所建立的RT-UPT-LF方法可在15 min内完成对蓖麻毒素的检测,灵敏度可达0.5 ng/ml,定量范围为0.5~1000 ng/ml,与其他高浓度毒素无非特异反应,该法为蓖麻毒素检测提供了一种新手段。

  9. Development of real-time fluorescent quantitative PCR assay for detection of PRRSV based on TaqMan probe%基于TaqMan探针的猪繁殖与呼吸障碍综合症病毒实时荧光定量PCR方法的建立

    Institute of Scientific and Technical Information of China (English)

    祝秀梅; 马全英; 杜平; 王凡; 吕志慧; 牟克斌

    2012-01-01

    We establish a TaqMan real-time PCR assay for detection of PRRSV. The specific primers and probes were designed in the conserved region of the ORF7 gene for PRRSV, and the real-time fluorescent quantitative PCR was established by optimizing the probe concentration. Thirty clinical samples were detected by using the established quantitative RT-PCR assay, and the results were compared with that of conventional RT PCR and viral isolation tests. TaqMan fluorescent quantitative PCR for detection of PRRSV was established successfully with the optimal probe concentration 0. 4 μmol, and detection limit was as low as 3. 51 copies/μL The results by the TaqMan real-time PCR method were 100% consistent with the viral isolation tests. Sensitivity and positive rate (28/30) for clinical samples of TaqMan fluorescent quantitative PCR were relatively higher than conventional PCR (25/30). The results indicated this method has high specificity, sensitivity and reproducibility, and could be used for the diagnosis of PRRSV infection.%目的 建立一种能检测猪繁殖与呼吸障碍综合症病毒(PRRSV)的TaqMan探针荧光定量PCR方法.方法 根据PRRSV的ORF7基因保守区的核苷酸序列设计引物和TaqMan探针,通过探针浓度的优化,建立检测PRRSV的TaqMan探针荧光定量PCR方法.用该方法对30份临床疑似病料进行检测,并与常规RT-PCR方法和病毒分离方法进行比较.结果 TaqMan荧光PCR检测PRRSV的最佳探针浓度为0.4 μmol,检测灵敏度可达3.51拷贝/μL.检测的30份样品与病毒分离结果的符合率为100%,与普通PCR的检测结果(25/30)比较,本方法对临床样品的检出率(28/30)更高.结论 建立的方法特异性强、敏感性高、重复性好,可用于临床样品的检测.

  10. The skin-blanching assay.

    Science.gov (United States)

    Smit, P; Neumann, H A M; Thio, H B

    2012-10-01

    The skin-blanching assay is used for the determination and bioequivalence of dermatologic glucocorticoids (GCs). The exact mechanism of the production of blanching is not fully understood, but it is considered that local vasoconstriction of the skin microvasculature and the consequent blood-flow reduction cause this phenomenon. Several factors influence skin blanching, including drug concentration, duration of application, nature of vehicle, occlusion, posture and location. The intensity of vasoconstriction can be measured in several ways: visual or quantitative methods, such as reflectance spectroscopy, thermography, laser Doppler velocimetry and chromametry. In literature, contradicting results in the correlation of the skin-blanching assay with different tests to determine GC sensitivity have been reported, limiting its clinical usefulness.

  11. 赤拟谷盗HSP70基因克隆和竞争定量PCR检测%Cloning and quantitative competitive PCR assay of HSP70 gene in Tribolium castaneum ( Coleoptera: Tenebrionidae)

    Institute of Scientific and Technical Information of China (English)

    苏丽娟; 董晓慧; 尹新明

    2011-01-01

    To study the expression changes of heat shock protein 70 (a highly conserved protein) in Tribolium castaneum after exposure to heat stress, a fragment of 681 bp encoding for HSP70 was amplified and sequenced from T.castaneum.The fragment encoded 227 amino acid residues with the GenBank accession no.HM345948.The result of homology analysis showed that this fragment shared 97% identity with hsp70 from L.decemlineata (AF322911.1 ).Comparison of the deduced amino acid sequences of HSP70 in T.castaneum with those in Leptinotarsa decemlineata, Mamestra brassicae, Drosophila melanogaster and Liriomyza sativae indicated that they shared more than 94% identity.The internal competitor used in quantitative competitive PCR was obtained by RT-PCR.The detection system of hsp70 was constructed by PCR amplification using the same quantity of target cDNA and a series of diluted internal competitor as template.The linear equation of standard curve was Y= 1.032X- 1.618 (r2 =0.975).This study provides a very convenient method for the quantitation of the hsp70 expression changes in T.castaneum and offers the basic data for the prevention and control of pests using thermal control technology.%为研究赤拟谷盗Tribolium castaneum受到热胁迫后高度保守的热激蛋白70(heat shock protein 70,HSP70)基因的表达变化,本研究扩增了681 bp的赤拟谷盗hsp70片段,编码227个氨基酸残基,GenBank登录号为HM345948.同源性分析表明:赤拟谷盗hsp70核苷酸序列与马铃薯甲虫Leptinotarsa decemlineata的hsp70(GenBank登录号:AF322911.1)同源性最高,为97%;其推测的蛋白序列与马铃薯甲虫、甘蓝夜蛾Mamestra brassicae、黑腹果蝇Drosophila melanogaster和美洲斑潜蝇Liriomyza,sativae的HSP70蛋白均有94%以上的同源性.利用RT-PCR技术得到与赤拟谷盗hsp70进行竞争定量的内部竞争物,以等量的目标cDNA和一系列稀释的竞争模板进行竞争PCR扩增,构建了hsp70的竞争定量PCR检测体系,该

  12. Quantitative research.

    Science.gov (United States)

    Watson, Roger

    2015-04-01

    This article describes the basic tenets of quantitative research. The concepts of dependent and independent variables are addressed and the concept of measurement and its associated issues, such as error, reliability and validity, are explored. Experiments and surveys – the principal research designs in quantitative research – are described and key features explained. The importance of the double-blind randomised controlled trial is emphasised, alongside the importance of longitudinal surveys, as opposed to cross-sectional surveys. Essential features of data storage are covered, with an emphasis on safe, anonymous storage. Finally, the article explores the analysis of quantitative data, considering what may be analysed and the main uses of statistics in analysis.

  13. Computer-assisted assessment of the Human Epidermal Growth Factor Receptor 2 immunohistochemical assay in imaged histologic sections using a membrane isolation algorithm and quantitative analysis of positive controls

    International Nuclear Information System (INIS)

    Breast cancers that overexpress the human epidermal growth factor receptor 2 (HER2) are eligible for effective biologically targeted therapies, such as trastuzumab. However, accurately determining HER2 overexpression, especially in immunohistochemically equivocal cases, remains a challenge. Manual analysis of HER2 expression is dependent on the assessment of membrane staining as well as comparisons with positive controls. In spite of the strides that have been made to standardize the assessment process, intra- and inter-observer discrepancies in scoring is not uncommon. In this manuscript we describe a pathologist assisted, computer-based continuous scoring approach for increasing the precision and reproducibility of assessing imaged breast tissue specimens. Computer-assisted analysis on HER2 IHC is compared with manual scoring and fluorescence in situ hybridization results on a test set of 99 digitally imaged breast cancer cases enriched with equivocally scored (2+) cases. Image features are generated based on the staining profile of the positive control tissue and pixels delineated by a newly developed Membrane Isolation Algorithm. Evaluation of results was performed using Receiver Operator Characteristic (ROC) analysis. A computer-aided diagnostic approach has been developed using a membrane isolation algorithm and quantitative use of positive immunostaining controls. By incorporating internal positive controls into feature analysis a greater Area Under the Curve (AUC) in ROC analysis was achieved than feature analysis without positive controls. Evaluation of HER2 immunostaining that utilized membrane pixels, controls, and percent area stained showed significantly greater AUC than manual scoring, and significantly less false positive rate when used to evaluate immunohistochemically equivocal cases. It has been shown that by incorporating both a membrane isolation algorithm and analysis of known positive controls a computer-assisted diagnostic algorithm was

  14. Development of real-time fluorescent quantitative RT-PCR assay for detection of porcine reproductive and respiratory syndrome virus variant strains%猪繁殖与呼吸综合征病毒变异株实时荧光定量RT-PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    高诗敏; 王敏功; 邓瑞林; 姚晓辉; 王林川

    2011-01-01

    根据猪繁殖与呼吸综合征病毒(PRRSV)变异株和经典毒株的序列差异,分别设计了1对特异性鉴别引物和1条TagMan探针,建立了检测PRRSV变异株的荧光定量RT-PCR方法,并对该方法进行了特异性、敏感性、重复性试验.结果表明,建立的方法可鉴别诊断PRRSV变异株和经典毒株,具有较高的特异性;可以检测相当于1 TCID(50)的病毒模板,比常规RT-PCR方法的灵敏度高100倍;对同一样品进行4次重复试验,变异系数(CV)<10%,具有较好的重复性.应用建立的荧光定量RT-PCR和常规RT-PCR对13份PRRS疑似病例肺组织进行平行检测,结果荧光定量RT-PCR检出11份阳性,高于常规RT-PCR方法.本研究建立的荧光定量RT-PCR方法能检测高致病性PRRSV变异株,不仅特异性好、灵敏度高,而且快速简便,可用于变异PRRSV的临床诊断和流行病学调查.%To identify highly pathogenic porcine reproductive and respiratory syndrome virus(PRRSV) variants rapidly using real-time PCR technique, primers and TaqMan probe for specific PCR amplification were designed based on sequence differences between variant strains and classical strains.The fluorescent quantitative RT-PCR assay for detection of PRRSV variants was established.Its specificity,sensitivity and stability were examined.This assay had differentiate PRRSV variants.1 TCID50 of template RNA could be detected.The sensitivity of this assay was 100 times higher than the conventional RT-PCR.Content could be absolutely quantified of virus.The variation coefficients were less than 10% based on 4 replications of each sample.13 lung samples were examined by the developed fluorescent quantitative RT-PCR and the conventional PCR, respectively,and 11 were detected to be infected with PRRSV by the real-time PCR, but only 9 were confirmed to be infected with PRRSV by the conventional PCR.These results showed that the developed fluorescent quantitative RT-PCR assay was rapid, specific, sensitive

  15. 肝细胞生长因子mRNA实时荧光定量PCR检测体系的建立及其在淋巴瘤诊断中的价值%Establishment of real-time fluorescence quantitative polymerase chain reaction analysis assay quantification for hepatocyte growth flactor mRNA expression and its clinical relevance in lymphoma

    Institute of Scientific and Technical Information of China (English)

    岑东; 吕建新; 裴仁治; 涂植光; 余晓林; 文阳安

    2008-01-01

    Objective To construct quantitative standard for quantification of hepatocyte growth factor (HGF) mRNA and establish its real-time fluorescence quantitative(FQ)-PCR assay to estimate its clinical relevance in lymphoma.Methods Recombinant plasmid Was constructed with target cDNA obtained from isolated total RNA by RT-PCR After PCR products were identified and purified,recombined plasmids were quantitated and then acted as quantitative standard.A new real time FQ-PCR analysis system Was established with the second pair of primers and the probe after amplification condition and the concentrations of components were optimized.HGF mRNA expressions in 47 lymohoma cases[11 Hodgkin disease(HD) cases,36 non-Hodgkin lyphoma(NHL)cases.among these patients,36 patients in remission while 11 patients without remission ] were analyzed quantitatively,and its specificity and sensitivity for lymphoma diagnosis were evaluated by receiptor operation character(ROC)curve method.Results HGF mRNA quantitative standard was constructed successfully.and its real time FO.PCR analysis system Was established combined with hot.start PCR and down.touch PCR technique. According to slope of standard curve (-3.513)and correlation cofficient(0.999),amplification efficiency of the system was 92.6%.Coefficient variation of intra-assay,intra-day and inter-day-assay were 2.1%,4.0% and 6.8%,respectively.Sensitivity of FQ-PCR Was 2 eopies/μl.Expressions of HGF mRNA in lymphoma group Was higher than that in control group(6.425±2.172 and 0.317±0.192,respectively,t=15.883,P0.05).According to ROC analysis,its sensitivity and specificity were 93.6% and 100% when cutoff value for lymphoma clinical diagnosis Was 3.136.Conclusion HGF mRNA'8 quantitative standard and its real time F9-PCR analysis system have been successfully constructed,and it can be used for quantitative detection of its mRNA expression in lymphoma.%目的 构建肝细胞生长因子(HGF)mRNA定量标准,建立实时荧光定量(FQ)-PCR检测

  16. Microbead agglutination based assays

    KAUST Repository

    Kodzius, Rimantas

    2013-01-21

    We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

  17. Development of Real-time Fluorescence Quantitative PCR Assay for Detection of Bovine Viral Diarrhea Virus in Pigs%猪源牛病毒性腹泻病毒实时荧光定量PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    邓宇; 张荣; 丛雁方; 张建武; 袁世山; 文心田; 郑浩

    2011-01-01

    This study was aimed at developing a real-time fluorescence quantitative PCR assay for detecting and discriminating pig bovine viral diarrhea virus-1 (BVDVs) and classical swine fever virus (CSFV). According to the conserved gene sequence of 5 -UTR of BVDVs and CSFV, pairs of primer and specific TaqMan probe were designed. The positive standard plasmids were used as quantitative template to make the standard curves, through the method was optimized. Specificity, sensitivity, and conformity were tested. By sensitivity analysis, the FQ-PCR indicated that a minimum of 10 copies of plasmid DNA was detected. The assay exhibited reproducibility and specificity, and all negative controls such as CSFV and porcine reproductive and respiratory syndrome virus (PRRSV) showed negative detection. As a result of the sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time FQ-PCR can be used as a routine assay for the diagnosis of pig BVDV infection and CSFV vaccine polluted by BVDVs.%本研究旨在建立一种荧光定量PCR检测方法,用于猪源牛病毒性腹泻病毒(BVDVs)检测,同时能鉴别诊断猪瘟病毒(CSFV).根据BVDVs与CSFV 5 '-UTR保守序列,设计一套引物和特异TaqMan探针,以本实验室构建的猪源BVDVs 5′-UTR阳性重组质粒为标准品,通过优化反应条件,建立了标准曲线.以构建的标准品为模板进行了特异性、敏感性、重复性试验、并应用于临床检测.结果显示,该方法检测CSFV、猪繁殖与呼吸综合征病毒均为阴性;最低可检测到每个反应相当于10拷贝的标准品DNA;重复性试验的批内变异系数为0.20%~0.95%;应用所建立方法对临床样品进行检测,其结果与普通RT-PCR结果的符合率为86.7%.本研究建立的实时荧光定量PCR具有特异、敏感、快速、重复性好等优点,可用于猪感染BVDVs、猪瘟疫苗污染BVDVs的监测.

  18. Radioreceptor opioid assay

    International Nuclear Information System (INIS)

    A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

  19. Absolute nuclear material assay

    Science.gov (United States)

    Prasad, Manoj K.; Snyderman, Neal J.; Rowland, Mark S.

    2010-07-13

    A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

  20. Establishment and evaluation of a flow cytometric immune microsphere assay for quantitative detecting human serum insulin%流式免疫微球技术定量检测人血清胰岛素方法学研究和评价

    Institute of Scientific and Technical Information of China (English)

    王占科; 何丹; 祝仲珍; 胡晓璐; 潘小清; 常津

    2012-01-01

    We aim to establish a novel flow cytometric immune microsphere assay (FCIMA) for quantitative detecting human serum insulin by flow cytometric immune microsphere assay (FCIMA) and study its repeatability, linearity, and lowest detectable limit (LDL). Microsphere surface were labeled by rabbit against human insulin monoclonal antibody to prepare immune diagnosis microspheres, which could be combined with human insulin of serum, and appear fluorescent light by combining rat against human insulin monoclonal antibody labeled with FITC. The mean fluorescent intensity (MFI) of the surface of immune diagnosis microspheres were detected by flow cytometer (FC) and recorded. We found that the MFI of immune diagnosis microspheres were in direct proportion to the concentrations of human serum insulin. Moreover, both of the coefficient of variance (C V) of repeatability and the LDL of FCIMA were lower than those of ELISA, and the range of linearity of FCIMA was wider than that of ELISA. These results indicated that FCIMA can be used for quantitative determination of human serum insulin, which is better than ELISA in the repeatability, linearity range, and detection sensitivity.%目的 建立流式免疫微球定量检测人血清胰岛素的方法并对其重复性实验和线性实验以及最低检测限进行评价.方法 将兔抗人胰岛素单克隆抗体标记在微球表面制备免疫诊断微球,与血清中胰岛素结合后,加入荧光标记的鼠抗人胰岛素单克隆抗体,使微球表面呈现荧光,并通过流式细胞仪检测其平均荧光强度(mean fluorescent intensity,MFI)并记录.结果 微球表面MFI与人血清胰岛素浓度呈正比,流式免疫微球技术检测同一标本的重复性实验变异系数以及最低检测限均低于酶联免疫吸附试验,流式免疫微球技术线性范围大于酶联免疫吸附实验.结论 流式免疫诊断微球技术可用于人血清胰岛素定量检测,其测定重复性,线性范围和灵敏度

  1. Establishment of Single Tube Quantitative Detection Technology for Coliforms Assay Based on Time of Growth Spectrophotometer Method%基于生长时间光谱法的大肠菌群单管定量检测技术研究

    Institute of Scientific and Technical Information of China (English)

    蔡尽忠; 庄峙厦; 赵丽; 汤新华; 鄢庆枇

    2011-01-01

    通过研究大肠菌群初始接种浓度与达到0.2吸光度所需时间的关系,建立基于生长时间光谱法的大肠菌群单管定量检测技术。通过与多管发酵法比较发现单管定量检测技术的相对标准偏差小于传统的多管发酵法,而且反应所需的时间短,当初始菌浓为101.3cfu/mL时反应时间仅需9h,可以作为大肠菌群快速检测的方法,具有良好的应用前景。%By studying the initial inoculation concentration of coliforms and the time required to reach 0.2 absorbance,and establishment of single tube quantitative detection technology for coliforms assay based on time of growthspectrophotometer method.By comparison with the multi-tube fermentation method of quantitative detection of single-tube technology that the relative standard deviation less than the conventional multi-tube fermentation method,and the reaction time is short,when the initial bacterial concentration was 101.3cfu/mL,the reaction time was only 9h,can serve as a method of rapid detection of coliforms,has a good prospect.

  2. A quantitative assay for lysosomal acidification rates in human osteoclasts

    DEFF Research Database (Denmark)

    Jensen, Vicki Kaiser; Nosjean, Olivier; Dziegiel, Morten Hanefeld;

    2011-01-01

    The osteoclast initiates resorption by creating a resorption lacuna. The ruffled border surrounding the lacunae arises from exocytosis of lysosomes. To dissolve the inorganic phase of the bone, the vacuolar adenosine triphosphatase, located in the ruffled border, pumps protons into the resorption...... lacunae. The electroneutrality of the lacunae is maintained by chloride transport through the chloride-proton antiporter chloride channel 7. Inhibition of either proton or chloride transport prevents bone resorption. The aims of this study were to validate the human osteoclastic microsome- based influx......, the effect of valinomycin, inhibitor sensitivity, and the ion profile of the human osteoclast microsomes. The expression level of chloride channel 7 was increased in the human osteoclastic microsomes compared with whole osteoclasts. Acid influx was induced by 1.25 mM adenosine triphosphate. Further 1.1 μ...

  3. Comparison of Different Promoter Methylation Assays in Breast Cancer

    Directory of Open Access Journals (Sweden)

    Karijn P. M. Suijkerbuijk

    2010-01-01

    Full Text Available Background: Promoter hypermethylation has emerged as a promising cancer biomarker. Currently, a large variety of quantitative and non-quantitative techniques is used to measure methylation in clinical specimens. Here we directly compared three commonly used methylation assays and assessed the influence of tissue fixation, target sequence location and the amount of DNA on their performance.

  4. A quantitative ELISA for dystrophin.

    Science.gov (United States)

    Morris, G E; Ellis, J M; Nguyen, T M

    1993-05-01

    A novel approach to the quantitation of the muscular dystrophy protein, dystrophin, in muscle extracts is described. The two-site ELISA uses two monoclonal antibodies against dystrophin epitopes which lie close together in the rod domain of the dystrophin molecule in order to minimize the effects of dystrophin degradation. Dystrophin is assayed in its native form by extracting with non-ionic detergents and avoiding the use of SDS. PMID:8486926

  5. PCR-反向点杂交基因分型与实时荧光定量PCR检测人乳头瘤病毒的研究%Use of a PCR-based reverse blot hybridization assay for subtyping and real-time quantitative PCR to detect human papilloma virus

    Institute of Scientific and Technical Information of China (English)

    向华国; 曾锦婷; 何婉意; 黎国

    2012-01-01

    Objective To evaluate the significance of a PCR-based reverse blot hybridization (PCR-RDB) assay and realtime quantitative PCR for detecting human papilloma virus in female outpatients. Methods A total of 121 female outpatients were checked for 23 HFV DNA types by PCR-RDB and 13 high-risk HPV genotypes by real-time quantitative PCR. Results According to PCR-RDB, 28.10% of the women(34/121) tested positive while 16. 53%(20/121) tested positive according to real-time quantitative PCR. HPV was detected more often with PCR-RDB than with real-time quantitative PCR (P<0.05). The concordance rate for the two techniques was 93. 39%(113/121). Conclusion PCR-RDB can be used to screen for HPV infection while real-time quantitative PCR facilitates evaluation of the effectiveness of treatment and the prognosis for cervical carcinoma. Combining the two should increase the specificity and sensitivity of HPV detection.%目的 评价PCR-反向点杂交基因分型与实时荧光定量PCR在检测人乳头瘤病毒(HPV)的意义.方法 同时采用PCR-反向点杂交基因分型和实时荧光定量PCR对121例女性官颈脱离细胞标本进行HPV检测.其中PCR-反向点杂交基因分型能检测23种HPV亚型,实时荧光定量PCR定量检测常见的13种高危HPV亚型.结果 PCR-反向点杂交基因分型检测HPV的阳性率为28.10%(34/121),实时荧光定量PCR检测HPV的阳性率为16.53%(20/121),差异有统计学意义(P<0.05);二者检测的符合率为93.39%(113/121).结论 PCR-反向杂交基因分型适用于HPV感染的筛查,而实时荧光定量PCR适用于HPV感染相关疾病的疗效与预后的判断.PCR-反向杂交基因分型与实时荧光定量PCR联合检测可提高HPV检测的特异性和敏感度,对于生殖道HPV感染以及子宫颈癌的早期发现、预防和治疗具有重要意义.

  6. Cell Culture Assay for Human Noroviruses [response

    Energy Technology Data Exchange (ETDEWEB)

    Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

    2007-07-01

    We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

  7. Detection of Plasmodium falciparum by using magnetic nanoparticles separa-tion-based quantitative real-time PCR assay%纳米磁分离法结合实时荧光定量PCR检测恶性疟原虫的研究

    Institute of Scientific and Technical Information of China (English)

    王飞; 邓丛良; 刘翌; 田茵; 杨静; 孙福军; 孙宁; 刘必永; 田睿; 葛广路; 邹明强

    2014-01-01

    目的:建立恶性疟原虫纳米磁分离实时荧光定量PCR检测方法,快速准确地检测恶性疟原虫,为输入性恶性疟防控提供技术支持。方法根据恶性疟原虫基因组18S rRNA保守区序列,设计并合成引物和探针;构建质粒标准品,拟合标准曲线,采用纳米磁分离法提取核酸,建立恶性疟原虫纳米磁分离实时荧光定量PCR检测法,并进行该方法的灵敏度和特异性评价。结果与常规法(镜检法和快检法)相比,该方法检测恶性疟原虫更加灵敏,并具有良好的特异性,在(2.5×101~2.9×108)copies/ml拷贝数范围内循环阈值(Ct值)与质粒标准品拷贝数的对数值之间存在良好的线性关系(R2=0.999);应用该方法从非洲维和部队13名归国人员中检出1例低水平恶性疟原虫感染者,而常规法(镜检法和快检法)未检出。结论该方法能够快速准确地检测恶性疟原虫,在口岸低密度恶性疟原虫感染者的检测中具有很大的应用价值。%Objective 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 preven-tion of imported malaria. Methods 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 evaluat-ed. Results The relationship between the threshold cycle(Ct)and logarithm of initial templates copies was linear over a range of 2.5×101 to 2.5×108 copies/μl(R2=0.999). Among 13 subjects of entry frontier,a P. falciparum carrier with low load was de-tected by using the assay and none was detected with the conventional

  8. Transgenic Animal Mutation Assays

    Institute of Scientific and Technical Information of China (English)

    Tao Chen; Ph.D.D.A.B.T.

    2005-01-01

    @@ The novel transgenic mouse and rat mutation assays have provided a tool for analyzing in vivo mutation in any tissue, thus permitting the direct comparison of cancer incidence with mutant frequency.

  9. Lateral flow assays.

    Science.gov (United States)

    Koczula, Katarzyna M; Gallotta, Andrea

    2016-06-30

    Lateral flow assays (LFAs) are the technology behind low-cost, simple, rapid and portable detection devices popular in biomedicine, agriculture, food and environmental sciences. This review presents an overview of the principle of the method and the critical components of the assay, focusing on lateral flow immunoassays. This type of assay has recently attracted considerable interest because of its potential to provide instantaneous diagnosis directly to patients. The range and interpretation of results and parameters used for evaluation of the assay will also be discussed. The main advantages and disadvantages of LFAs will be summarized and relevant future improvements to testing devices and strategies will be proposed. Finally, the major recent advances and future diagnostic applications in the LFA field will be explored. PMID:27365041

  10. 沙门氏致病菌标准阳性模板的构建及实时荧光定量聚合酶链式反应检测%Construction of Standard Positive Template and Development of a Real-Time Fluorescence Quantitative Polymerase Chain Reaction (FQ-PCR) Assay for PathogenicSalmonella spp.

    Institute of Scientific and Technical Information of China (English)

    陈晨; 邵彪; 陈刚; 黄伟东

    2014-01-01

    Objective: To construct recombinant plasmids for use as standard positive template and establish a real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) assay for determining pathogenicSalmonellaspp. in foods. Methods: Primers and Taqman probe were designed and synthesized with the specific fragment of InvA gene as the target sequence. Recombinant plasmids were constructed by inserting the target gene into PGM-T carriers. Real-time fluorescent quantitative PCR method was established and its sensitivity, specificity, repeatability and accuracy were investigated. Results: The recombinant plasmids constructed using the specific sequence of pathogenicSalmonella spp. could be used as a standard positive template for fluorescent quantitative PCR. The standard curve wasY=-3.151 lgX+42.86 (R2=0.999), and the sensitivity of the method was 80 copies per reaction. It was specific to detect Salmonellaspp. with good repeatability (the inter-batch and intra-batch coefficients of variation were both less than 5%). Conclusion: The FQ-PCR method allows qualitative and quantitative detection of pathogenicSalmonellaspp.%目的:构建重组质粒作为标准阳性模板,建立食品中沙门氏致病菌实时荧光定量聚合酶链式反应检测方法。方法:以致病性沙门氏菌invA基因上特异性片段为目标,设计并合成引物和TaqMan探针,将目标片段连接到PGM-T载体上构建重组质粒,建立实时荧光定量检测体系,并考察方法的灵敏性、特异性、重复性和准确性。结果:构建出致病性沙门氏菌特异性基因片段的重组质粒,能够作为实时荧光定量聚合酶链式反应检测方法的标准阳性模板,标准曲线方程为Y=-3.151 lgX+42.86(R2=0.999),灵敏度80拷贝反应体系,能特异区分沙门氏菌与类型的细菌,同时,批内和批间的变异系数均小于5%,具有良好的重复性。结论:本方法能够实现对食品

  11. New Rapid Spore Assay

    Science.gov (United States)

    Kminek, Gerhard; Conley, Catharine

    2012-07-01

    The presentation will detail approved Planetary Protection specifications for the Rapid Spore Assay for spacecraft components and subsystems. Outlined will be the research and studies on which the specifications were based. The research, funded by ESA and NASA/JPL, was conducted over a period of two years and was followed by limited cleanroom studies to assess the feasibility of this assay during spacecraft assembly.

  12. Establishment of a Multiplex Real-Time Fluorescence Quantitative PCR Assay for Detection of Brucella and Mycobacterium tuberculosis%梅迪-维斯纳病毒和羊痘病毒多联实时定量PCR检测方法的建立及初步应用

    Institute of Scientific and Technical Information of China (English)

    徐军; 孙志华; 刘娟; 孟茹; 戴莉; 段晓东; 叶志辉

    2012-01-01

    To establish a method of multiplex real-time fluorescence quantitative PCR assay for fast diagnosis of Maedi-Visna virus (MVV) and Capripox virus (CPV). We designed and synthesized primers of MVV and CPV genes according to gene sequence published in GenBank,established multiplex RTFQ-PCR,then tested its stability,specificity and sensitivity,and detected the clinic and imitation samples with this method. The Tm of multiplex RT-PCR to amplify brucella and mycobacterium tuberculosis was 89~90 ℃ and 91~92 'C. But the results of the amplification of other bacteria were negative. The lowest detection limit for DNA of Brucella,MVV and CPV was 25 copies/μL,40 copies/μL,80 copies/μL,respectively. In conclusion,the assay could be used to detect CPV simultaneously.%利用多联实时荧光定量PCR技术建立了一种梅迪-维斯纳病毒和羊痘病毒快速鉴别诊断方法.分别设计并合成梅迪-维斯纳病毒和羊痘病毒基因的引物,建立多联实时定量PCR快速鉴别诊断方法;对所建立的方法进行稳定性、特异性和敏感性试验;并用所建立的方法对临床样品进行检测.结果显示:设计的引物敏感性和特异性较好,该多联实时荧光定量PCR方法中梅迪-维斯纳病毒Tm值为89~90℃,羊痘病毒Tm值为91~92℃,对其他供试的菌株则为阴性,并且该方法对梅迪-维斯纳病毒的DNA最低检出量为25拷贝/μL,羊痘病毒为40拷贝/μL,两病原都存在时为80拷贝/μL.研究结果表明本实验建立的方法可用于同时检测梅迪-维斯纳病毒和羊痘病毒,为动物检疫提供了一种有效的检测方法.

  13. Nuclear Resonance Fluorescence for Materials Assay

    International Nuclear Information System (INIS)

    This paper discusses the use of nuclear resonance fluorescence (NRF) techniques for the isotopic and quantitative assaying of radioactive material. Potential applications include age-dating of an unknown radioactive source, pre- and post-detonation nuclear forensics, and safeguards for nuclear fuel cycles Examples of age-dating a strong radioactive source and assaying a spent fuel pin are discussed. The modeling work has been performed with the Monte Carlo radiation transport computer code MCNPX, and the capability to simulate NRF has bee added to the code. Discussed are the limitations in MCNPX?s photon transport physics for accurately describing photon scattering processes that are important contributions to the background and impact the applicability of the NRF assay technique.

  14. Study and application of SYBR green I-based fluorescent quantitative PCR assay in type 2 porcine circovirus detection%荧光定量PCR检测猪圆环病毒2型方法的研究与应用

    Institute of Scientific and Technical Information of China (English)

    李志娟; 王永生; 陈陆; 杨霞; 赵军; 王川庆

    2011-01-01

    A SYBR Green Ⅰ-based real time quantitative PCR (Q-PCR) assay for the detection and quantitation of porcine circovirus type 2 (PCV2) was developed, a 276 bp conserved region of the ORF2 gene of PCV2 was amplified by conventional PCR and cloned into pMD-18 vector. The resulted pMD-18T-276 plasmid DNA was used as standard DNA template to optimize assay condition. The standard curve produced a good linear relationship between Ct value and initial amounts of total DNA at a range of 5.60× 101 -2. 74 × 109 copies per microlitre, and the correlation coefficient and slope were -1.00 and -3. 345 respectively. No cross-reactions were found in specimens containing PCV1, PRV, PRRSV,PPV and CSFV. The Q-PCR was used to detect the replication of PCV2 in infected cell. The result showed that the peak of virus replication was at 96 h after inoculation with PCV2 on PK15 cell. Four high titer PCV2 strains were screened from 11 isolates, which enriches the resources for PCV2 vaccine strains research.%利用常规PCR扩增出猪圆环病毒2型(PCV2)ORF2保守区域276 bp片段,将其克隆到pMD-18载体,以pMD-18T-276质粒DNA作为标准质粒模板优化反应条件,建立定量检测PCV2的荧光定量PCR方法.结果表明:Ct值与标准品模板在5.60×101~2.74×109拷贝·μL-1范围内呈良好的线性关系,相关系数为-1.00,斜率为-3.345.所建立的方法与猪1型圆环病毒、猪伪狂犬病毒、猪繁殖障碍与呼吸系统综合征病毒、猪细小病毒、猪瘟病毒均无交叉反应.应用该方法对PCV2感染细胞后的复制情况进行了研究,结果显示在感染细胞96 h时,PCV2复制达到最高点.此外,利用建立的方法对保存的11个分离株在PK15细胞上繁殖特性进行了研究,筛选到4株高滴度毒株,丰富了圆环病毒疫苗株的研究资源.

  15. Value of fluorescence quantitative PCR assay of bronchoalveolar lavage fluid samples in the diagnosis of Mycoplasma pneumoniae pneumonia in children%支气管肺泡灌洗液荧光定量PCR对儿童肺炎支原体肺炎诊断研究

    Institute of Scientific and Technical Information of China (English)

    钟礼立; 彭力; 黄寒; 梁沫; 周琼华; 张兵; 李云

    2011-01-01

    目的:探讨荧光实时定量PCR(FQ-PCR)检测肺泡灌洗液(BALF)中MP-DNA基因在肺炎支原体肺炎(MPP)早期病原诊断中的价值.方法:采用FQ-PCR检测61例患儿BALF中MP-DNA,并与目前常用血清MP抗体检测方法颗粒凝集法进行比较.结果:FQ-PCR检测MP-DNA敏感度94%,特异度100%;病程早期及难治性病例中FQ-PCR检测BALF中MP-DNA准确性优于单份血清MP抗体检测(P<0.01).难治性病例中MP-DNA拷贝数显著高于普通MPP组(P<0.05),且与CRP呈正相关(r=0.845,P<0.01).结论:FQ-PCR检测BALF中MP-DNA是早期MPP特别是难治性MPP病原诊断的可靠方法,优于血清MP抗体检测.MP-DNA的拷贝量可以作为评价难治性MPP的转归以及判断药物疗效的指标.%Objective To study the value of fluorescence quantitative polymerase chain reaction (FQ-PCR) for detecting Mycoplasma pneumoniae (MP)-DNA in bronchoalveolar lavage fluid (BALF) in the early diagnosis of Mycoplasma pneumoniae pneumonia (MPP). Methods FQ-PCR was used to detect MP-DNA in BALF obtained by fiberoptic bronchoscopy from 61 children with MPP, and the sensitivity and the specificity of FQ-PCR were compared with the traditional serological test. Results The sensitivity and the specificity of BALF FQ-PCR for detecting MP-DNA were 94% and 100% respectively. The accuracy of BALF FQ-PCR assay for detecting MP-DNA was higher than that of the serological test at the early stage of the disease (1-7 days) (P<0.01). In the children with refractory MPP, BALF FQPCR assay also showed higher accuracy for detecting MP-DNA than the serological test ( P < 0. 01 ). The copies of MPDNA in children with refractory MPP were significant higher than those in children with common MPP ( P <0.05). The copies of MP-DNA were positively correlated with CRP values ( r = 0. 845, P < 0.01 ). Conclusions FQ-PCR assay of BALF for detecting MP-DNA in BALF is superior to the serological test. It is a reliable method for the early diagnosis of MPP

  16. Assay method and compositions

    International Nuclear Information System (INIS)

    Methods are described for measuring catecholamine levels in human and animal body fluids and tissues using the catechol-O-methyl-transferase (COMT) radioassay. The assay involves incubating the biological sample with COMT and the tritiated methyl donor, S-adenosyl-L-methionine(3H)-methyl. The O-methylated (3H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin-3H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin-3H and raising the pH of the aqueous periodate phase from which O-methylated (3H) dopamine is extracted and counted. The assay may be modified depending on whether measurements of undifferentiated total endogenous catecholamine levels or differential analyses of the catecholamine levels are being performed. The sensitivity of the assay can be as low as 5 picograms for norepinephrine and epinephrine and 12 picograms for dopamine. The assemblance of the essential components of the assay into a kit for use in laboratories is also described. (U.K.)

  17. Rover waste assay system

    Energy Technology Data Exchange (ETDEWEB)

    Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

    1997-11-01

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

  18. Rover waste assay system

    International Nuclear Information System (INIS)

    The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for 137Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs

  19. Interpreting coagulation assays.

    Science.gov (United States)

    Green, David

    2010-09-01

    The interpretation of coagulation assays requires knowledge of the principal clotting pathways. The activated partial thromboplastin time is sensitive to all hemostatic factors except FVII, whereas the prothrombin time reflects levels of prothrombin and FV, FVII, and FX. Using the two tests in concert is helpful in identifying hemophilia, the coagulopathy of liver disease, and disseminated intravascular coagulation. In addition, the activated partial thromboplastin time and prothrombin time are used for monitoring anticoagulant therapy with heparin and warfarin, respectively. Measurement of D-dimer is informative in patients suspected of having thrombotic disorders and determining the risk of thrombosis recurrence. Mixing tests distinguish clotting factor deficiencies from circulating anticoagulants such as heparin, the lupus anticoagulant, and antibodies directed against specific clotting factors. The modified Bethesda assay detects and provides an indication of the strength of FVIII inhibitors. However, interpreting the results of coagulation assays is not always straightforward, and expert consultation is occasionally required to resolve difficult clinical situations. PMID:20855988

  20. Neutral Comet Assay

    OpenAIRE

    2013-01-01

    The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The ...

  1. Lateral flow strip assay

    Science.gov (United States)

    Miles, Robin R.; Benett, William J.; Coleman, Matthew A.; Pearson, Francesca S.; Nasarabadi, Shanavaz L.

    2011-03-08

    A lateral flow strip assay apparatus comprising a housing; a lateral flow strip in the housing, the lateral flow strip having a receiving portion; a sample collection unit; and a reagent reservoir. Saliva and/or buccal cells are collected from an individual using the sample collection unit. The sample collection unit is immersed in the reagent reservoir. The tip of the lateral flow strip is immersed in the reservoir and the reagent/sample mixture wicks up into the lateral flow strip to perform the assay.

  2. Automated phantom assay system

    International Nuclear Information System (INIS)

    This paper describes an automated phantom assay system developed for assaying phantoms spiked with minute quantities of radionuclides. The system includes a computer-controlled linear-translation table that positions the phantom at exact distances from a spectrometer. A multichannel analyzer (MCA) interfaces with a computer to collect gamma spectral data. Signals transmitted between the controller and MCA synchronize data collection and phantom positioning. Measured data are then stored on disk for subsequent analysis. The automated system allows continuous unattended operation and ensures reproducible results

  3. Multiplex cytological profiling assay to measure diverse cellular states.

    Directory of Open Access Journals (Sweden)

    Sigrun M Gustafsdottir

    Full Text Available Computational methods for image-based profiling are under active development, but their success hinges on assays that can capture a wide range of phenotypes. We have developed a multiplex cytological profiling assay that "paints the cell" with as many fluorescent markers as possible without compromising our ability to extract rich, quantitative profiles in high throughput. The assay detects seven major cellular components. In a pilot screen of bioactive compounds, the assay detected a range of cellular phenotypes and it clustered compounds with similar annotated protein targets or chemical structure based on cytological profiles. The results demonstrate that the assay captures subtle patterns in the combination of morphological labels, thereby detecting the effects of chemical compounds even though their targets are not stained directly. This image-based assay provides an unbiased approach to characterize compound- and disease-associated cell states to support future probe discovery.

  4. Lateral flow assays

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Amerongen, van A.

    2012-01-01

    A simple version of immunochemical-based methods is the Lateral Flow Assay (LFA). It is a dry chemistry technique (reagents are included); the fluid from the sample runs through a porous membrane (often nitrocellulose) by capillary force. Typically the membrane is cut as a strip of 0.5*5 cm. In most

  5. Hyaluronic Acid Assays

    DEFF Research Database (Denmark)

    Itenov, Theis Skovsgaard; Kirkby, Nikolai S; Bestle, Morten H;

    2015-01-01

    BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

  6. Instrument for assaying radiation

    Energy Technology Data Exchange (ETDEWEB)

    Coleman, Jody Rustyn; Farfan, Eduardo B.

    2016-03-22

    An instrument for assaying radiation includes a flat panel detector having a first side opposed to a second side. A collimated aperture covers at least a portion of the first side of the flat panel detector. At least one of a display screen or a radiation shield may cover at least a portion of the second side of the flat panel detector.

  7. Quantitative immunochemical evaluation of fish metallothionein upon exposure to cadmium

    DEFF Research Database (Denmark)

    Yudkovski, Yana; Rogowska-Wrzesinska, Adelina; Yankelevich, Irena;

    2008-01-01

    Efficient implementation of an environmental biomarker requires multi-annual comparability over a wide geographical range. The present study improved the comparability of a quantitative competitive metallothionein (MT) enzyme-linked-immuno-sorbent-assay (ELISA) in the sentinel fish Lithognathus...

  8. 鸡BF1和BF2基因实时荧光定量PCR检测方法的建立%Development of a real-time fluorescent quantitative PCR assay for BF1 and BF2 genes in chickens

    Institute of Scientific and Technical Information of China (English)

    金元昌; 李玉峰; 袁志栋

    2013-01-01

    目的 建立鸡主要组织相容性复合体(Major histocompatibility complex,MHC)BF1和BF2基因实时荧光定量PCR[Real-time fluorescent quantitative(FQ)-PCR]检测方法.方法 从鸡外周血淋巴白细胞(Peripheral blood leukocytes,PBLs)中提取细胞总RNA,以其为模板,根据BF1、BF2和GAPDH基因相对保守序列各设计一对引物,应用RT-PCR法扩增目的基因,分别克隆至pGM-T载体上,制备重组质粒标准品,经PCR、EcoR Ⅰ酶切及测序鉴定;优化反应体系及反应条件,绘制标准曲线,建立Real-time FQ-PCR检测方法,并验证其敏感性、特异性及重复性.结果 重组质粒标准品经PCR、酶切及测序鉴定构建正确;建立的定量标准曲线临界循环数(Threshold cycle,Ct)与BF1、BF2和GAPDH起始质粒DNA拷贝数的对数之间线性关系良好,相关系数分别为1.00、0.98、0.99,扩增效率分别为0.8、1.1、1.1;该方法的敏感性可达101 copies/μl;融解曲线分析表明扩增效率一致,特异性较好;质粒DNA标准品3次检测的CV值误差不超过0.5,均小于5%.结论 已成功建立了鸡BF1和BF2基因实时荧光定量PCR检测方法,为下一步应用该法检测BF1和BF2基因的转录水平奠定了基础.%Objective To develop a real-time fluorescent quantitative PCR assay for of BF1 and BF2 genes of major his— tocompatibility complex (MHC) in chickens. Methods Total RNA was extracted from chicken peripheral blood leukocytes (PBLs) and used as a template for amplification of BF1, BF2 and GAPDH genes by RT-PCR using the designed primers. The amplified genes were cloned into vector pGM-T respectively, and the constructed standard recombinant plasmids were identified by PCR, digestion with EcoR I and sequencing. The reaction system and condition were optimized, based on which a standard curve was plotted. The developed real-time FQ-PCR method was verified for sensitivity, specificity and reproducibility. Results PCR, restriction analysis and

  9. Quantitative multiplex detection of pathogen biomarkers

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-02-09

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

  10. 基于上转发光免疫层析技术的常见食源性致病菌快速检测方法研究与评价%Development of up-converting phosphor technology based lateral flow assay for quan-titative detection of foodborne pathogens

    Institute of Scientific and Technical Information of China (English)

    李春凤; 赵勇; 王晓英; 张平平; 刘晓; 孙崇云; 杨瑞馥; 王成彬; 周蕾

    2015-01-01

    Objective To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid detection of Salmonella paratyphi A, S.paratyphi B, Escherichia coli O157 ∶H7 and Vibrio parahaemolyticus. Methods With up-converting phosphor nano-particles ( UCP-NPs ) as the bio-marker, four double-antibody-sandwich mode based UPT-LF strips for detecting the above mentioned four pathogens were prepared respectively and their sensitivi-ty, accuracy, linearity and specificity were evaluated .Furthermore, the feasibility of detecting bacteria in food samples was evaluated by different food samples artificially contaminated with less than 10 CFU target pathogens .Results The sensitivi-ty of UPT-LF assays for four pathogens was 105 ~106 CFU/ml with excellent specificity .The four strips had a good linear response with the linear fitting coefficient of determination (r) for each target pathogen ranging from 0.985 to 0.996.The positive rate of detecting pathogens from samples was acceptable .Conclusion The four developed UPT-LF strips provide a new choice for rapid , specific and sensitive and quantitative detection of S.paratyphi A , S.paratyphi B, E.coli O157∶H7 and V.parahemolyticus.%目的:研制针对4种常见食源性致病菌:甲型副伤寒沙门菌( Salmonella paratyphi A)、乙型副伤寒沙门菌(Salmonella paratyphi B)、大肠埃希菌O157∶H7(Escherichia coli O157∶H7)、副溶血弧菌(Vibrio parahaemolyticus)的基于上转发光免疫层析技术( UPT-LF)的快速定量检测试纸,并对其检测性能进行评价。方法以上转发光纳米颗粒( UCP-NPs)作为示踪物,基于双抗体夹心检测模型,研制针对上述4种靶标菌的UPT-LF试纸;以4种靶标菌的系列浓度标准菌悬液作为标准样品,评价试纸的敏感性、特异性、线性和精密性,并进行模拟染菌食品的检测,评价模拟样品阳性检出率。结果针对4种靶标菌的UPT-LF试纸均具

  11. The comet assay – from toy to tool

    OpenAIRE

    Guenter Speit

    2015-01-01

    The comet assay is nowadays the most common method for measuring DNA damage and repair in single cells. It is based on the microelectrophoretic study published by Ostling and Johanson (1984) and was developed by Singh and coworkers (1988) to a versatile technique for quantitation of low levels of DNA damage in individual cells. This alkaline version still is the basis for the triumphant success of the comet assay in basic research into mechanisms of DNA damage and DNA repair, genotoxicity tes...

  12. 酶联免疫法检测血清乙型肝炎表面抗原的空白限检出限及定量限的建立与评价%The establishment and evaluation of limit of blank, limit of detection and limit of quantitation for detection of serum hepatitis B surface antigens by enzyme-linked immunosorbent assay

    Institute of Scientific and Technical Information of China (English)

    谭丽娜; 杨泽华; 赵克斌

    2013-01-01

    Objective To explore the question of serum hepatitis B surface antigens(HBsAg) results detected by enzyme-linked immunosorbent assay(ELISA) so as to prevent undetection of weakly positive samples, and to establish limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ). Methods Refering to CLSI EP17-A file, the HBsAg blank sample and a series of samples with low concentrations were performed ELJSA detection. According to the distribution, the results were analyzed using the relative statistical method. ELJSA was used to detect LOB, LOD and LOQ of HBsAg. Results The results were LOB=0.117 μg/L, LOD=0.317 μg/L and LOQ= 0.500 μg/L Conclusion The LOB established by referring to CLSI EP17-file was more scientific and reasonable than that established by conventional method and thus it might satisfy the clinical lab requirements better.%目的 探讨酶联免疫吸附试验(ELISA)检测血清乙型肝炎表面抗原(HBsAg)结果检测限的问题,防止漏检弱阳性标本,建立本实验室的空白限(LOB)、检出限(LOD)及定量限(LOQ).方法 参考美国临床实验室标准化委员会(CISI)发布的《确定检测低限和定量检测限的方案》(EP17-A)文件,将HBsAg的空白样本及一系列的低浓度样本进行ELISA检测,对得到的结果根据其分布状态,采用百分位数进行统计描述,建立ELISA法检测HBsAg的空白限、检出限及定量限.结果 空白限为0.117 μg/L,检出限为0.317 μg/L,定量限为0.500μg/L.结论 参考CLSI EP17-A文件建立的检测限比常规方法建立的检测限科学合理,更能满足临床实验室的要求.

  13. One-step quantitative cortisol dipstick with proportional reading

    NARCIS (Netherlands)

    Leung, Wingman; Chan, Puiyee; Bosgoed, Freddy; Lehmann, Karin; Renneberg, Ilka; Lehmann, Matthias; Renneberg, Reinhard

    2003-01-01

    Rapid quantitative immuno-detection of haptens by the lateral flow assay without “typical” competitive inhibition results is studied. In the present study, we describe an immuno-threshold-based assay for the quantification of cortisol. It gives a signal which is directly proportional to the cortisol

  14. Disagreement between Human Papillomavirus Assays

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte Møller;

    2014-01-01

    We aimed to determine the disagreement in primary cervical screening between four human papillomavirus assays: Hybrid Capture 2, cobas, CLART, and APTIMA. Material from 5,064 SurePath samples of women participating in routine cervical screening in Copenhagen, Denmark, was tested with the four...... assays. Positive agreement between the assays was measured as the conditional probability that the results of all compared assays were positive given that at least one assay returned a positive result. Of all 5,064 samples, 1,679 (33.2%) tested positive on at least one of the assays. Among these, 41......-65 years (n = 2,881), 23% tested positive on at least one assay, and 42 to 58% of these showed positive agreement on any compared pair of the assays. While 4% of primary screening samples showed abnormal cytology, 6 to 10% were discordant on any pair of assays. A literature review corroborated our findings...

  15. Lipase turbidimetric assay and acute pancreatitis.

    Science.gov (United States)

    Orda, R; Orda, S; Baron, J; Wiznitzer, T

    1984-04-01

    The simplified turbidimetric assay for lipase activity was used for the differential diagnosis of acute pancreatitis. Serum lipase levels were found to be increased in a group of 17 patients in whom acute pancreatitis was clinically suspected and confirmed by a high ACCR and decreased uptake of the radionuclide in the pancreas scan. The lipase levels were within normal limits in a control group of 14 patients suffering from diseases other than acute pancreatitis. The turbidimetric test was helpful for rapid quantitative determination of serum lipase and thus for the early and accurate diagnosis of acute pancreatitis. PMID:6200277

  16. Radon assay for SNO+

    International Nuclear Information System (INIS)

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+

  17. FLUIDICS DEVICE FOR ASSAY

    DEFF Research Database (Denmark)

    2007-01-01

    The present invention relates to a device for use in performing assays on standard laboratory solid supports whereon chemical entities are attached. The invention furthermore relates to the use of such a device and a kit comprising such a device. The device according to the present invention is a......, when operatively connected, one or more chambers (21) comprising the chemical entities (41), the inlet(s) (5) and outlet(s) (6) and chambers (21) being in fluid connection. The device further comprise means for providing differing chemical conditions in each chamber (21)....

  18. Growth cone collapse assay.

    Science.gov (United States)

    Cook, Geoffrey M W; Jareonsettasin, Prem; Keynes, Roger J

    2014-01-01

    The growth cone collapse assay has proved invaluable in detecting and purifying axonal repellents. Glycoproteins/proteins present in detergent extracts of biological tissues are incorporated into liposomes, added to growth cones in culture and changes in morphology are then assessed. Alternatively purified or recombinant molecules in aqueous solution may be added directly to the cultures. In both cases after a defined period of time (up to 1 h), the cultures are fixed and then assessed by inverted phase contrast microscopy for the percentage of growth cones showing a collapsed profile with loss of flattened morphology, filopodia, and lamellipodia.

  19. Radon assay for SNO+

    Energy Technology Data Exchange (ETDEWEB)

    Rumleskie, Janet [Laurentian University, Greater Sudbury, Ontario (Canada)

    2015-12-31

    The SNO+ experiment will study neutrinos while located 6,800 feet below the surface of the earth at SNOLAB. Though shielded from surface backgrounds, emanation of radon radioisotopes from the surrounding rock leads to back-grounds. The characteristic decay of radon and its daughters allows for an alpha detection technique to count the amount of Rn-222 atoms collected. Traps can collect Rn-222 from various positions and materials, including an assay skid that will collect Rn-222 from the organic liquid scintillator used to detect interactions within SNO+.

  20. One-step real-time fluorescent quantitative PCR assay for mumps virus in clinical saliva specimens%腮腺炎病毒临床口漱液样本的一步法实时荧光定量PCR检测

    Institute of Scientific and Technical Information of China (English)

    施海晶; 王晶晶; 陈俊英; 陈巍; 潘明; 赵钢; 孙强明

    2013-01-01

    Objective To develop an one-step real-time fluorescent quantitative PCR assay for mumps virus (MuV) in clinical saliva specimens and provide a basis for clinical diagnosis,prevention and control of mumps.Methods A total of 101 clinical saliva specimens collected from suspected patients with mumps in Yunnan and Sichuan Province as well as Guangxi Zhuang Autonomous Region,China were determined by cell culture-MuV specific nested PCR and real-time PCR with TaqMan probe respectively,based on which the sensitivities of the two methods were compared.Results Thirty-one MuV-positive specimens were found by cell culture-MuV specific nested PCR,all of which belonged to genotype F as proved by phylogenetic tree.However,41 MuV-positive specimens were found by real-time PCR with TaqMan probe,including the 31 positive specimens proved by cell culture-MuV specific nested PCR,indicating that the positive rate of Muv by real-time RT-PCR with TaqMan probe (40.59%) was higher than that by traditional cell culture-nested PCR (30.69%).Conclusion The sensitivity,specificity and simplicity of real-time PCR with TaqMan probe were higher than those of traditional cell culture-MuV specific nested PCR,which might be used for the clinical diagnosis,prevention and control of mumps.%目的 建立腮腺炎病毒(mumps virus,MuV)临床口漱液样本一步法荧光定量PCR检测方法,为腮腺炎的临床诊断及免疫防控提供依据.方法 对101份采集自中国南部3个省、自治区(云南、四川、广西)的临床疑似腮腺炎患者口漱液样本,分别采用细胞培养-MuV特异的巢式PCR法及TaqMan探针Real-time PCR法进行检测,比较两种方法检测MuV的灵敏性.结果 细胞培养-MuV特异的巢式PCR法共检出31份MuV阳性样本,系统进化分析表明,该31株MuV均属于F基因亚型;TaqMan探针Real-time PCR法共检出41份MuV阳性样本,其中包含了细胞培养-MuV特异的巢式PCR法检出的31份阳性样本,TaqMan探针Real-time RT

  1. Establishment of a Real-time Fluorescent Quantitative RT-PCR Assay for Detection of Porcine IFN-β and IRF-3 genes%猪IFN-β和IRF-3基因实时荧光定量RT-PCR检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    王蕊; 张改平; 乔松林; 刘永晖; 蒋志政; 万博; 鲍登克; 王爱萍

    2012-01-01

    为建立猪IFN-β及IRF-3基因实时荧光定量PCR检测方法,依据GenBank中IFN-β和IRF-3基因的保守序列,设计并合成各自特异性引物,并以β-actin为内参基因,采用SYBRGreen-Ⅰ为染料,建立实时荧光定量检测方法.提取猪肺泡巨噬细胞总RNA,经反转录得cDNA,用特异性引物经PCR扩增得到IFN-β和IRF-3基因,将其克隆至pMD-19T载体,经测序鉴定后得重组质粒,依次10倍稀释做为标准品,建立标准曲线及溶解曲线.结果表明,β3-actin基因、IFN-β基因和IRF-3基因标准曲线线性关系较好,R2≥0.997;溶解曲线为特异性单峰,无非特异性扩增,检测下限可达100个拷贝/μL.建立的猪IFN-β和IRF-3基因实时荧光定量RT-PCR检测方法,特异性强、重复性好,为从分子水平上研究猪的免疫应答奠定了基础.%To establish real-time fluorescent quantitative RT-PCR assays for detecting porcine IFN-β and IRF-3, sevral specific primer pairs were designed according to the porcine's IFN-β and IRF-3 gene sequences available in GenBank,and the porcine β-actin gene was used as an internal gene control. The total RNA was extracted from porcine alveolar macrophages. The reverse transcriptase PCR was used to obtain the first strand cDNA. Fragments for IFN-β and IRF-3 were amplified by PCR from the synthesized cDNA using the designed specific primers. The PCR products were purified and cloned into pMD-19T vector. The positive recombined plasmids were serially diluted and used as a standard. The standard and melting curve was analyzed. The results showed that the Ct value of β actin ,IFN-β and IRF-3 genes had a good linear relationship (R2≥ 0. 997) and the melting curve showed a single peak. The established real-time PCR methods can detect 100 copies of IFN-β and IRF-3 mRNA. The developed real-time PCR using SYBR Green I dye had high sensitivity,sepcifity and reproductivity.and could used as an effective tool for detection and quantification of IFN

  2. Development of real-time fluorescent quantitative PCR assays for detection of Serpina3g gene of Echinococcus multilocularis infected mouse%实时荧光定量PCR检测多房棘球蚴感染小鼠肝脏丝氨酸蛋白酶抑制基因方法的建立

    Institute of Scientific and Technical Information of China (English)

    王俊华; 刘涛; 王慧; 吕国栋; 卢晓梅; 王星; 姜涛; 温浩; 林仁勇

    2009-01-01

    目的 建立多房棘球蚴(泡球蚴)感染小鼠肝脏丝氨酸蛋白酶抑制基因(Serpina3g)实时荧光定量PCR(RT-qPCR)的检测方法及与常规RT-PCR检测方法的比较.方法 肉眼直视下肝脏穿刺注射100 μl泡球蚴组织匀浆液建立泡球蚴感染小鼠模型,3个月后将小鼠处死,Trizol法提取肝脏总RNA,反转录获得cDNA.依据Serpina3g的编码基因(NM_009251)保守序列,通过Primer 5软件设计定量引物,以β-actin作为内参.以健康小鼠肝脏获得的cDNA依次10倍梯度稀释制备标准品(1×10~2~1×10~6 pg),利用SYBR Green RT-qPCR方法检测Serpina3g基因,然后根据cDNA浓度与循环值(Ct)的线性关系,绘制标准曲线,确定RT-qPCR检测的最佳条件和重复性;并将RT-qPCR结果与常规RT-PCR琼脂糖凝胶电泳结果进行特异性、灵敏度和定量等方面的比较.结果 筛选出Serpina3g基因定量引物,最佳退火温度为56.4℃;RT-qPCR无非特异性扩增,标准品及样品熔解温度均在80.5℃,熔解峰单一;标准曲线斜率为-2.833(效率),相关系数r=0.996;5个不同梯度的样本(1×10~2-1×10~6pg)重复检测5次均为阳性,变异<5%;mRNA检出限为1×10~2 pg,总RNA在1×10~2~1×10~6 pg内均可以进行扩增;常规RT-PCR方法mRNA检出限仅为1×10~6 pg.结论 成功建立了小鼠源Serpina3g的PT-qPCR检测方法,在特异性、灵敏度、重复性和定量方面优于常规RT-PCR.%Objective To establish a real-time fluorescent quantitative PCR (RT-qPCR) for detection of Serpina3g of Echinococcus multilocularis infected mouse, and compare it with conventional RT-PCR. Methods To establish the mouse model, 100 μl homogenized metacestode was injected into the anterior liver lobe of the experimental mice. After 3 months, liver was sampled to extract the total RNA, and cDNA was obtained by reverse transcription. Primer was designed based on the nucleotide sequence of Serpina3g gene(NM_009251) by using soft of Primers 5. β-actin was used as a

  3. Quantitative lithofacies palaeogeography

    Institute of Scientific and Technical Information of China (English)

    Zeng-Zhao; Feng; Xiu-Juan; Zheng; Zhi-Dong; Bao; Zhen-Kui; Jin; Sheng-He; Wu; You-Bin; He; Yong-Min; Peng; Yu-Qing; Yang; Jia-Qiang; Zhang; Yong-Sheng; Zhang

    2014-01-01

    Quantitative lithofacies palaeogeography is an important discipline of palaeogeography.It is developed on the foundation of traditional lithofacies palaeogeography and palaeogeography,the core of which is the quantitative lithofacies palaeogeographic map.Quantity means that in the palaeogeographic map,the division and identification of each palaeogeographic unit are supported by quantitative data and quantitative fundamental maps.Our lithofacies palaeogeographic maps are quantitative or mainly quantitative.A great number of quantitative lithofacies palaeogeographic maps have been published,and articles and monographs of quantitative lithofacies palaeogeography have been published successively,thus the quantitative lithofacies palaeogeography was formed and established.It is an important development in lithofacies palaeogeography.In composing quantitative lithofacies palaeogeographic maps,the key measure is the single factor analysis and multifactor comprehensive mapping method—methodology of quantitative lithofacies palaeogeography.In this paper,the authors utilize two case studies,one from the Early Ordovician of South China and the other from the Early Ordovician of Ordos,North China,to explain how to use this methodology to compose the quantitative lithofacies palaeogeographic maps,and to discuss the palaeogeographic units in these maps.Finally,three characteristics,i.e.,quantification,multiple orders and multiple types,of quantitative lithofacies palaeogeographic maps are conclusively discussed.

  4. Quantitative investment analysis

    CERN Document Server

    DeFusco, Richard

    2007-01-01

    In the "Second Edition" of "Quantitative Investment Analysis," financial experts Richard DeFusco, Dennis McLeavey, Jerald Pinto, and David Runkle outline the tools and techniques needed to understand and apply quantitative methods to today's investment process.

  5. The Lumipulse G HBsAg-Quant assay for screening and quantification of the hepatitis B surface antigen.

    Science.gov (United States)

    Yang, Ruifeng; Song, Guangjun; Guan, Wenli; Wang, Qian; Liu, Yan; Wei, Lai

    2016-02-01

    Qualitative HBsAg assay is used to screen HBV infection for decades. The utility of quantitative assay is also rejuvenated recently. We aimed to evaluate and compare the performance of a novel ultra-sensitive and quantitative assay, the Lumipulse assay, with the Architect and Elecsys assays. As screening methods, specificity was compared using 2043 consecutive clinical routine samples. As quantitative assays, precision and accuracy were assessed. Sera from 112 treatment-naïve chronic hepatitis B patients, four patients undergoing antiviral therapy and one patient with acute infection were tested to compare the correlations. Samples with concurrent HBsAg/anti-HBs were also quantified. The Lumipulse assay precisely quantified ultra-low level of HBsAg (0.004 IU/mL). It identified additional 0.98% (20/2043) clinical samples with trance amount of HBsAg. Three assays displayed excellent linear correlations irrespective of genotypes and S-gene mutations (R(2)>0.95, PLumipulse assay did not yield higher HBsAg concentrations in samples with concomitant anti-HBs. Compared with other assays, the Lumipulse assay is sensitive and specific for detecting HBsAg. The interpretation of the extremely low-level results, however, is challenging. Quantitative HBsAg results by different assays are highly correlated, but they should be interpreted interchangeably only after conversion to eliminate the biases.

  6. Rigour in quantitative research.

    Science.gov (United States)

    Claydon, Leica Sarah

    2015-07-22

    This article which forms part of the research series addresses scientific rigour in quantitative research. It explores the basis and use of quantitative research and the nature of scientific rigour. It examines how the reader may determine whether quantitative research results are accurate, the questions that should be asked to determine accuracy and the checklists that may be used in this process. Quantitative research has advantages in nursing, since it can provide numerical data to help answer questions encountered in everyday practice.

  7. PathogenMip Assay: A Multiplex Pathogen Detection Assay

    OpenAIRE

    Akhras, Michael S.; Sreedevi Thiyagarajan; Villablanca, Andrea C.; Davis, Ronald W; Pål Nyrén; Nader Pourmand

    2007-01-01

    The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The ...

  8. 多重荧光定量PCR方法的建立及其在快速产前诊断中的应用%Establishment of multiple quantitative fluorescent polymerase chain reaction assay and its application in rapid prenatal diagnosis of common chromosome aneuploidies

    Institute of Scientific and Technical Information of China (English)

    徐爱群; 边旭明; 刘俊涛; 姚凤霞; 张为民; 郝娜; 周京

    2010-01-01

    术能成功用于常见非整倍体异常的快速产前诊断,检测结果准确,适合于规模较大的产前诊断中心进行大样本检测.%Objective To establish the multiple quantitative fluorescent polymerase chain reaction (QF-PCR)assay and evaluate its clinical application in prenatal diagnosis.Methods Totally 170 samples Were collected between May 2008 and July 2009 in prenatal center of Peking Union Medical College Hospital:123 of them were amniotic fluid,9 were chofionic villous samples,20 were fetal blood and 18 were villi from aborted fetuses.All samples were from women of Han nationality,with mean age of (34.1±4.6) years old,and with mean gestational age of(19.6±1.0)weeks.Cytogenetic cultures and karyotyping were made to every sample.Genomic DNA wag extracted from the samples.The sequences of twenty short tandem repeat (STR) markers were designed according to the GenBank and references,including 6 STR markers in chromosome 21.4 in chromosome 18.4 in chromosome 13,4 in chromosome X,1 in chromosome Y and 1 universal marker in both X and Y chromosome.Each sample was amplified by two sets of multiple QF-PCR,which included 4 STR markers in each of 21,18,13 and sex chromosomes. If the result was uninformative,the third set of anotherd 4 STR markers was added. Results ( 1 ) Karyotyping. Cytogenetic analysis were made for all the 170 samples, 151 (89%) of which were normal, and 19 (11% ) were abnormal (2)QF-PCR assay. 167(98% ) samples were detected by QF-PCR. The results were obtained within 2 -3 days after sampling. 134 samples were proved normal by QF-PCR, which was consistent with karyotyping. Among the 19 abnormal karyotype samples, 18 were detected as abnormal( eight were 21-trisomy, three were 18-trisomy)by QF-PCR. Among the 167 samples, 150(90% ) were detected using the first and second set of STR mixtures, and 3(2% ) were detected when the third set of STR was added. The remain 14(8% ) were uninformative. (3) The diagnostic efficiency of QF

  9. GC-MS DETERMINATION OF RATIOS OF STABLE-ISOTOPE LABELED TO NATURAL UREA USING [(CN2)-C-13-N-15]UREA FOR STUDYING UREA KINETICS IN SERUM AND AS A MEANS TO VALIDATE ROUTINE METHODS FOR THE QUANTITATIVE ASSAY OF UREA IN DIALYSATE

    NARCIS (Netherlands)

    WOLTHERS, BG; TEPPER, T; WITHAG, A; NAGEL, GT; DEHAAN, THY; VANLEEUWEN, JJ; STEGEMAN, CA; HUISMAN, RM

    1994-01-01

    A GC-MS determination of urea in serum or spent dialysate is described, using (CN2)-C-13-N-15-labelled urea and assaying the area ratio of labelled to natural urea by mass fragmentographic monitoring of fragments m/e 153 and 156, after its eventual conversion into the trimethylsilylether-derivative

  10. A luminescence-based assay of UDP-sugar producing pyrophosphorylases

    OpenAIRE

    Decker, Daniel; Lindberg, Stina; Eriksson, Jonas; Kleczkowski, Leszek A.

    2014-01-01

    A coupled luminescence assay was applied to monitor pyrophosphate (PPi) production by either purified barley UDP-glucose pyrophosphorylase (UGPase) or purified Leishmania UDP-sugar pyrophosphorylase (USPase). In the assay, the PPi produced by the pyrophosphorylases was converted to ATP by ATP-sulfurylase, and the ATP produced was linked to luminescent light formation through the action of firefly luciferase. The assay allowed for a quantitative measurement of UGPase and USPase activities, dow...

  11. Practical assay issues with the PERT/PBRT assay: a highly sensitive reverse transcriptase assay.

    Science.gov (United States)

    Chang, A; Dusing, S

    2006-01-01

    Product safety testing for retroviruses can be achieved by a panel of screening assays, including electron microscopy, viral gene specific PCRs, virus propagation, and detection of reverse transciptase activity. The application of PCR-based reverse transcriptase assays (PERT) that are approximately a million-fold more sensitive than conventional nucleotide incorporation assays in the testing of biologicals is described. Use of PERT assays can be applied to three areas: (i) screening for adventitious retrovirus contamination; (ii) detecting and quantifying endogenous viral particle load and (iii) monitoring levels of infectious retrovirus generation in cell lines that contain endogenous retroviruses.

  12. Quantitative analysis chemistry

    International Nuclear Information System (INIS)

    This book is about quantitative analysis chemistry. It is divided into ten chapters, which deal with the basic conception of material with the meaning of analysis chemistry and SI units, chemical equilibrium, basic preparation for quantitative analysis, introduction of volumetric analysis, acid-base titration of outline and experiment examples, chelate titration, oxidation-reduction titration with introduction, titration curve, and diazotization titration, precipitation titration, electrometric titration and quantitative analysis.

  13. On-line radiochemical assay for monoamine oxidase utilizing high-performance liquid chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Nissinen, E.; Linko-Loeppoenen SMae; Maennistoe P4

    1984-12-01

    A fast and sensitive assay for the determination of monoamine oxidase activity was developed. The method is based on the separation and quantitation of /sup 14/C-labeled assay products by high-performance liquid chromatography, which is interfaced directly into a flow-through radioactivity detector. This allows on-line quantitation of the radioactive compounds with picomole sensitivity. The method makes possible the complete separation and detection of the deaminated products of monoamine oxidase A and B substrates benzylamine and 5-hydroxytryptamine, respectively. This assay has been applied to the measurement of monoamine oxidase A and B activities in rat brain.

  14. From Antenna to Assay

    Science.gov (United States)

    Moore, Evan G.; Samuel, Amanda P. S.; Raymond, Kenneth N.

    2009-01-01

    Conspectus Ligand-sensitized, luminescent lanthanide(III) complexes are of considerable importance because their unique photophysical properties (microsecond to millisecond lifetimes, characteristic and narrow emission bands, and large Stokes shifts) make them well suited as labels in fluorescence-based bioassays. The long-lived emission of lanthanide(III) cations can be temporally resolved from scattered light and background fluorescence to vastly enhance measurement sensitivity. One challenge in this field is the design of sensitizing ligands that provide highly emissive complexes with sufficient stability and aqueous solubility for practical applications. In this Account, we give an overview of some of the general properties of the trivalent lanthanides and follow with a summary of advances made in our laboratory in the development of highly luminescent Tb(III) and Eu(III) complexes for applications in biotechnology. A focus of our research has been the optimization of these compounds as potential commercial agents for use in Homogeneous Time-Resolved Fluorescence (HTRF) technology. Our approach involves developing high-stability octadentate Tb(III) and Eu(III) complexes that rely on all-oxygen donor atoms and using multi-chromophore chelates to increase molar absorptivity; earlier examples utilized a single pendant chromophore (that is, a single “antenna”). Ligands based on 2-hydroxyisophthalamide (IAM) provide exceptionally emissive Tb(III) complexes with quantum yield values up to ∼60% that are stable at the nanomolar concentrations required for commercial assays. Through synthetic modification of the IAM chromophore and time-dependent density functional theory (TD-DFT) calculations, we have developed a method to predict absorption and emission properties of these chromophores as a tool to guide ligand design. Additionally, we have investigated chiral IAM ligands that yield Tb(III) complexes possessing both high quantum yield values and strong

  15. Comparison of chemosensitivity tests: clonogenic assay versus MTT assay.

    Directory of Open Access Journals (Sweden)

    Kawada K

    2002-06-01

    Full Text Available When the development of chemotherapeutic agents reaches the clinical trial stage, it is necessary to perform drug sensitivity tests quickly in order to select the most promising agents for the treatment of cancer. In order to assess the possibility of using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay as a substitute for the human tumor clonogenic assay (HTCA, we evaluated the correlation between the results obtained by these 2 assays in 5 human lung cancer cell lines. The correlation coefficient between the results of the HTCA and the MTT assay was 0.673, indicating a relatively good correlation. The correlation was most prominent in platinum analogues (r = 0.939 and good in anthracyclines/anthracenedione (r = 0.611. However, no significant correlation was observed in vinca alkaloids, etoposide, irinotecan, SN-38 (an active metabolite of irinotecan, and rhizoxin. The results of the MTT assay showed a high degree of correlation with those of the HTCA in predicting the sensitivity of cancer cell lines to platinum analogues, and anthracyclines/anthracenedione. These results suggest that the MTT assay may be more convenient and quickly performed than the HTCA and can replace HTCA in evaluating the effects of anticancer agents, especially the platinum analogues and anthracyclines/anthracenedione.

  16. Calorimetric assay of minor actinides

    Energy Technology Data Exchange (ETDEWEB)

    Rudy, C.; Bracken, D.; Cremers, T.; Foster, L.A.; Ensslin, N.

    1996-12-31

    This paper reviews the principles of calorimetric assay and evaluates its potential application to the minor actinides (U-232-4, Am-241, Am- 243, Cm-245, Np-237). We conclude that calorimetry and high- resolution gamma-ray isotopic analysis can be used for the assay of minor actinides by adapting existing methodologies for Pu/Am-241 mixtures. In some cases, mixtures of special nuclear materials and minor actinides may require the development of new methodologies that involve a combination of destructive and nondestructive assay techniques.

  17. Calorimetric assay of minor actinides

    International Nuclear Information System (INIS)

    This paper reviews the principles of calorimetric assay and evaluates its potential application to the minor actinides (U-232-4, Am-241, Am- 243, Cm-245, Np-237). We conclude that calorimetry and high- resolution gamma-ray isotopic analysis can be used for the assay of minor actinides by adapting existing methodologies for Pu/Am-241 mixtures. In some cases, mixtures of special nuclear materials and minor actinides may require the development of new methodologies that involve a combination of destructive and nondestructive assay techniques

  18. AFBI assay - Aptamer Fluorescence Binding and Internalization assay for cultured adherent cells.

    Science.gov (United States)

    Thiel, William H; Giangrande, Paloma H

    2016-07-01

    The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process allows for the enrichment of DNA or RNA aptamers from a complex nucleic acid library that are specific for a target molecule. The SELEX process has been adapted from identifying aptamers in vitro using recombinant target protein to cell-based methodologies (Cell-SELEX), where the targets are expressed on the surface of cells. One major advantage of Cell-SELEX is that the target molecules are maintained in a native confirmation. Additionally, Cell-SELEX may be used to discover novel therapeutic biomarkers by performing selections on diseased versus healthy cells. However, a caveat to Cell-SELEX is that testing of single aptamers identified in the selection is laborious, time-consuming, and expensive. The most frequently used methods to screen for aptamer binding and internalization on cells are flow cytometry and quantitative PCR (qPCR). While flow cytometry can directly assess binding of a fluorescently-labeled aptamer to a target, it requires significant starting material and is not easily scalable. qPCR-based approaches are highly sensitive but have non-negligible experiment-to-experiment variability due to the number of sample processing steps. Herein we describe a cell-based aptamer fluorescence binding and internalization (AFBI) assay. This assay requires minimal reagents and has few experimental steps/manipulations, thereby allowing for rapid screening of many aptamers and conditions simultaneously and direct quantitation of aptamer binding and internalization. PMID:26972784

  19. The Use of Chemical Reactivity Assays in Toxicity Prediction

    OpenAIRE

    ASTURIOL David; Worth, Andrew

    2011-01-01

    The use of so-called “in chemico” methodology - abiotic assays that measure chemical reactivity - is gaining ground as relevant and reliable means of toxicity prediction. In this report we explain the basis of the in chemico approach to toxicity prediction and we review the studies that have developed the concept and its practical application since the 1930s, with special attention being paid to studies aimed at the development of Quantitative Structure-Activity Relationship (QSAR) models and...

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

    International Nuclear Information System (INIS)

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

  1. Application of statistical process control to qualitative molecular diagnostic assays

    LENUS (Irish Health Repository)

    O'Brien, Cathal P.

    2014-11-01

    Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control (SPC). Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply SPC to an assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater sample numbers to mitigate a protracted time to detection. Modeled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of SPC to qualitative laboratory data.

  2. Development of robotic plasma radiochemical assays for positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Alexoff, D.L.; Shea, C.; Fowler, J.S.; Gatley, S.J.; Schlyer, D.J. [Brookhaven National Lab., Upton, NY (United States). Dept. of Chemistry

    1995-12-01

    A commercial laboratory robot system (Zymate PyTechnology II Laboratory Automation System; Zymark Corporation, Hopkinton, MA) was interfaced to standard and custom laboratory equipment and programmed to perform rapid radiochemical analyses for quantitative PET studies. A Zymark XP robot arm was used to carry out the determination of unchanged (parent) radiotracer in plasma using only solid phase extraction methods. Robotic throughput for the assay of parent radiotracer in plasma is 4--6 samples/hour depending on the radiotracer. Robotic assays of parent compound in plasma were validated for the radiotracers [{sup 11}C]Benztropine, [{sup 11}C]cocaine, [{sup 11}C]clorgyline, [{sup 11}C]deprenyl, [{sup 11}C]methadone, [{sup 11}C]methylphenidate, [{sup 11}C]raclorpride, and [{sup 11}C]SR46349B. A simple robot-assisted methods development strategy has been implemented to facilitate the automation of plasma assays of new radiotracers.

  3. Development of robotic plasma radiochemical assays for positron emission tomography

    International Nuclear Information System (INIS)

    A commercial laboratory robot system (Zymate PyTechnology II Laboratory Automation System; Zymark Corporation, Hopkinton, MA) was interfaced to standard and custom laboratory equipment and programmed to perform rapid radiochemical analyses for quantitative PET studies. A Zymark XP robot arm was used to carry out the determination of unchanged (parent) radiotracer in plasma using only solid phase extraction methods. Robotic throughput for the assay of parent radiotracer in plasma is 4--6 samples/hour depending on the radiotracer. Robotic assays of parent compound in plasma were validated for the radiotracers [11C]Benztropine, [11C]cocaine, [11C]clorgyline, [11C]deprenyl, [11C]methadone, [11C]methylphenidate, [11C]raclorpride, and [11C]SR46349B. A simple robot-assisted methods development strategy has been implemented to facilitate the automation of plasma assays of new radiotracers

  4. Development and Applification of TaqMan Fluorescence Quantitative PCR Assay to Detect Rabbit Bordetella Bronchiseptica%兔支气管败血波氏杆菌TaqMan荧光定量PCR检测方法的建立与应用

    Institute of Scientific and Technical Information of China (English)

    钱微; 刘燕; 肖琛闻; 韦强; 季权安; 鲍国连; 姚火春

    2013-01-01

    为了建立特异、敏感、快速检测兔支气管败血波氏杆菌(Bordetella bronchiseptica,Bb)的TaqMan荧光定量PCR方法,本研究以Bb的毒力因子CyaA为目的基因设计特异性引物和探针,并将PCR扩增产物克隆测序,测序结果与GenBank上Bb的CyaA的同源性达100%.以阳性克隆质粒作为定量检测标准品建立标准曲线,以提取的Bb基因组DNA为模板,进行特异性、灵敏度和重复性实验.该方法对波氏杆菌基因组DNA检测最低限为0.32 pg,灵敏度是普通PCR的25倍,与临床常见细菌无交叉反应.对45份疑似感染兔波氏杆菌病料的检测表明,TaqMan荧光定量PCR和普通PCR检测阳性率分别为75.6%和66.7%,两者符合率88.2%.结果表明,建立的TaqMan荧光定量Bb检测方法具有较好的特异性、敏感性和重复性.该方法的建立对Bb的临床高效诊断,Bb的防控提供了有效手段.%A specific, sensitive and rapid TaqMan fluorescence quantitative PCR was established for testing Rabbit Bordetella bronchiseptica. In present study, a pair of primers and probes were designed from target gene of virulence factors CyaA of Bordetella bronchiseptica. Amplified PCR product was cloned and sequenced, the results showed that the homology was 100% compared with the reference sequence published in GenBank. The positive recombinant plasmids were served as quantitative detection of standards to establish standard curve. The detectable quantity of Bordetella bronchiseptica genomic DNA was 0.32 pg, which was 25 times sensitivity compared with common PCR, there was no cross reaction with common clinical bacteria by TaqMan fluorescence quantitative PCR. The 45 suspected samples were detected by TaqMan fluorescence quantitative PCR or routine PCR. The positive detection rates were 75.5% and 66.7%, respectively, the coincidence rate was 88.2%. The results showed that the TaqMan fluorescent quantitative Rabbit Bordetella bronchiseptica detection method was

  5. Quantitative film radiography

    International Nuclear Information System (INIS)

    We have developed a system of quantitative radiography in order to produce quantitative images displaying homogeneity of parts. The materials that we characterize are synthetic composites and may contain important subtle density variations not discernible by examining a raw film x-radiograph. In order to quantitatively interpret film radiographs, it is necessary to digitize, interpret, and display the images. Our integrated system of quantitative radiography displays accurate, high-resolution pseudo-color images in units of density. We characterize approximately 10,000 parts per year in hundreds of different configurations and compositions with this system. This report discusses: the method; film processor monitoring and control; verifying film and processor performance; and correction of scatter effects

  6. On Quantitative Rorschach Scales.

    Science.gov (United States)

    Haggard, Ernest A.

    1978-01-01

    Two types of quantitative Rorschach scales are discussed: first, those based on the response categories of content, location, and the determinants, and second, global scales based on the subject's responses to all ten stimulus cards. (Author/JKS)

  7. SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries.

    Science.gov (United States)

    Wu, Jemma X; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P

    2016-07-01

    The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. PMID:27161445

  8. Comparison of an enzyme-linked immunoassay and a quantitative indirect fluorescent-antibody test with the conventional indirect fluorescent-antibody test for detecting antibodies to Toxoplasma gondii.

    OpenAIRE

    Violand, S A; Mitchell, T G; Kleeman, K T

    1982-01-01

    Two new methods for the detection of antibodies to Toxoplasma gondii, an enzyme-linked immunosorbent assay and a quantitative immunofluorescence assay, were evaluated and compared with the conventional indirect fluorescent-antibody slide test. Each of 100 human sera was assayed twice by the three procedures. Both the enzyme-linked immunosorbent assay and the quantitative immunofluorescence assay correlated well with serologically positive (indirect fluorescent-antibody titer greater than or e...

  9. Time-Resolved Fluorescence Assays.

    Science.gov (United States)

    Ma, Chen-Ting; Sergienko, Eduard A

    2016-01-01

    Fluorescence-based detection techniques are popular in high throughput screening due to sensitivity and cost-effectiveness. Four commonly used techniques exist, each with distinct characteristics. Fluorescence intensity assays are the simplest to run, but suffer the most from signal interference. Fluorescence polarization assays show less interference from the compounds or the instrument, but require a design that results in change of fluorophore-containing moiety size and usually have narrow assay signal window. Fluorescence resonance energy transfer (FRET) is commonly used for detecting protein-protein interactions and is constrained not by the sizes of binding partners, but rather by the distance between fluorophores. Time-resolved fluorescence resonance energy transfer (TR-FRET), an advanced modification of FRET approach utilizes special fluorophores with long-lived fluorescence and earns its place near the top of fluorescent techniques list by its performance and robustness, characterized by larger assay window and minimized compound spectral interference. TR-FRET technology can be applied in biochemical or cell-based in vitro assays with ease. It is commonly used to detect modulation of protein-protein interactions and in detection of products of biochemical reactions and cellular activities. PMID:27316992

  10. Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins.

    Science.gov (United States)

    Rosen, Osnat; Ozeri, Eyal; Barnea, Ada; David, Alon Ben; Zichel, Ran

    2016-09-24

    Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval.

  11. Barcoded microchips for biomolecular assays.

    Science.gov (United States)

    Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

    2015-01-20

    Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

  12. QUANTITATIVE PCR OF SELECTED ASPERGILLUS, PENICILLIUM AND PAECILOMYCES SPECIES

    Science.gov (United States)

    A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan®) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of Aspergillus, Penicillium and ...

  13. Assay for quantitative determination of CYP1A1 enzyme activity using 7-Ethoxyresorufin as standard substrate (EROD assay)

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Afshin Mohammadi-Bardbori ### Abstract The activity of the enzyme 7-ethoxy-resorufin-O-deethylase (EROD) has been extensively employed in biomonitoring studies of persistent organic pollutants (POPs) for more than a decade. Although the procedure is simple, convenient, sensitive and accurate. The cytochrome P450 monooxygenase 1A (CYP1A) is induced by several planar toxic compounds and endogenous chemicals, and the induction of this protein is often measured in terms of EROD a...

  14. H3亚型猪流感病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立%Development of a SYBR Green I real-time fluorescent quantitative PCR assay for detection of swine influenza virus H3 subtype

    Institute of Scientific and Technical Information of China (English)

    郭泗虎; 王文华; 张亮权; 齐海涛; 曹楠; 闫明菲; 黄昌力; 张超轶; 张桂红

    2012-01-01

    In this study, a SYBR Green I real-time PCR assay was developed for detection of swine influenza virus (SIV) subtype H3 with the specific primers designed according to the conserved sequence of HA genes of SIV H3 subtype in GenBank. The results showed that the standard curve established by recombinant plasmid showed a good linear relationship between template concentration and threshold cycle, the correlation coefficient (R2) was 0.994 and the coefficient of variation (CV) of intra-assay was between 0.17% and 1.41%. No cross-reaction was detected for other swine viruses and HI, H4, H6 and H9 subtypes of SFV. The results indicated that real-time PCR is more sensitive than routine PCR and virus isolation which was a promising application in diagnose of clinical suspected cases.%为建立一种快捷、可行的检测H3亚型猪流感病毒(SIV)的方法,本研究根据GenBank中登录的H3亚型SIV HA基因的保守序列,设计合成一对特异性引物,建立了一种检测H3亚型SIV的SYBR Green Ⅰ荧光定量检测方法,并进行灵敏度、稳定性和特异性试验,与普通PCR进行比较.研究结果表明,标准曲线的循环阈值与模板浓度呈现良好的线性关系,R2为0.994,CV在0.17%~1.41%之间,具有良好稳定性.除H3亚型SIV外,对H1、H4、H6、H9亚型SIV以及猪瘟病毒、猪繁殖与呼吸综合征病毒、口蹄疫病毒和猪圆环病毒2型的检测均为阴性,与普通PCR相比更灵敏.该方法特异性好,准确率高,适于临床分离鉴别H3亚型SIV.

  15. Parallel force assay for protein-protein interactions.

    Directory of Open Access Journals (Sweden)

    Daniela Aschenbrenner

    Full Text Available Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

  16. An improved europium release assay for complement-mediated cytolysis.

    Science.gov (United States)

    Cui, J; Bystryn, J C

    1992-02-14

    An improved assay for complement-mediated cytolysis is described. The target cells are labeled with europium complexed to diethylenetriaminopentaacetate (Eu-DTPA). Cytolysis caused by antibody plus complement leads to the release of the Eu-DTPA complex which is then formed into a highly fluorescent chelate by the addition of 2-naphthoyltrifluoroacetone (2-NTA). The amount of europium chelate formed--a measurement of cell death--is then quantified with a time-resolved fluorometer. The results of the assay are reproducible. Complement-mediated cytolysis when measured by europium release was five times more sensitive than when measured by conventional 51Cr release and three times than when measured by trypan blue exclusion. Because europium does not decay, target cells can be labelled in batches and stored frozen until use, which speeds and simplifies the assay. Thus, europium release assay is a simple and quantitative method to measure complement-mediated cytolysis which is sensitive and more rapid than conventional assays. PMID:1541836

  17. Uncertainty Quantification for Quantitative Imaging Holdup Measurements

    Energy Technology Data Exchange (ETDEWEB)

    Bevill, Aaron M [ORNL; Bledsoe, Keith C [ORNL

    2016-01-01

    In nuclear fuel cycle safeguards, special nuclear material "held up" in pipes, ducts, and glove boxes causes significant uncertainty in material-unaccounted-for estimates. Quantitative imaging is a proposed non-destructive assay technique with potential to estimate the holdup mass more accurately and reliably than current techniques. However, uncertainty analysis for quantitative imaging remains a significant challenge. In this work we demonstrate an analysis approach for data acquired with a fast-neutron coded aperture imager. The work includes a calibrated forward model of the imager. Cross-validation indicates that the forward model predicts the imager data typically within 23%; further improvements are forthcoming. A new algorithm based on the chi-squared goodness-of-fit metric then uses the forward model to calculate a holdup confidence interval. The new algorithm removes geometry approximations that previous methods require, making it a more reliable uncertainty estimator.

  18. Establishing Quantitative Standards for Residual Alkaline Phosphatase in Pasteurized Milk

    Science.gov (United States)

    Chon, Jung-Whan; Kim, Hyunsook; Kim, Kwang-Yup

    2016-01-01

    The alkaline phosphatase (ALP) assay is a rapid and convenient method for verifying milk pasteurization. Since colorimetric ALP assays rely on subjective visual assessments, their results are especially unreliable near the detection limits. In this study, we attempted to establish quantitative criteria for residual ALP in milk by using a more objective method based on spectrophotometric measurements. Raw milk was heat-treated for 0, 10, 20, 30, and 40 min and then subjected to ALP assays. The quantitative criteria for residual ALP in the milk was determined as 2 μg phenol/mL of milk, which is just above the ALP value of milk samples heat-treated for 30 min. These newly proposed methodology and criteria could facilitate the microbiological quality control of milk. PMID:27194927

  19. Establishment and application of SYBR Green Ⅰ real-time fluoresent quantitative RT-PCR assay for detection chicken interluekin-2 mRNA%鸡IL-2 mRNA SYBR Green Ⅰ实时荧光定量RT-PCR检测方法的建立与应用

    Institute of Scientific and Technical Information of China (English)

    王衡; 刘博奇; 宁章勇

    2013-01-01

    根据GenBank提供的ChIL-2参考序列设计了特异性引物,并以鸡3-磷酸甘油脱氢酶(GAPDH)基因为内参,以二者标准质粒为模板,应用SYBR Green Ⅰ实时荧光定量PCR技术,并采用双标准曲线相对定量方法建立了ChIL-2 mRNA荧光定量RT-PCR检测方法.结果显示,该方法该可以在3h左右对低拷贝量的模板(200 copy/μL)进行检测,同时该检测方法具有很好的特异性和重复性.雏鸡免疫试验IL-2 mRNA检测结果显示,该方法可有效应用于鸡体内IL-2在核酸水平的检测,并为今后IL-2相对定量的检测研究提供参考依据.%For establishing a method to detect chicken interleukin 2 (ChIL-2) mRNA expression,the primers were desigend for developing a SYBR Green Ⅰ real-time fluoresence quantitative RTPCR method,according to the chicken ChIL-2 gene and chicken glyceraldehyde-3-phosphate dehydrogenase (ChGAPDH) gene sequences available in GenBank of which plasmids were used as the templates.ChGAPDH was used as the internal control and "two standard curve relative quantitative" method was applied to compute the value.The results of this expriment revealed that this method could detect the template of low copies (200 copy/μL) in short time (about 3 hours),and also had a good specificity and repeatability.This method was used to detect the dynamic change of IL-2 mRNA in chicks which were inoculated with IBV S1 gene vaccine.The results of significant change between inoculated group and control group showed that this method could be effectively applied to detect the nucleic acid level of IL-2 in the body of chicken and provided a suitable reference approach for further research on relative quantification of ChIL-2.

  20. Chromosome aberration assays in Allium

    Energy Technology Data Exchange (ETDEWEB)

    Grant, W.F.

    1982-01-01

    The common onion (Allium cepa) is an excellent plant for the assay of chromosome aberrations after chemical treatment. Other species of Allium (A. cepa var. proliferum, A. carinatum, A. fistulosum and A. sativum) have also been used but to a much lesser extent. Protocols have been given for using root tips from either bulbs or seeds of Allium cepa to study the cytological end-points, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. It is considered that both mitotic and meiotic end-points should be used to a greater extent in assaying the cytogenetic effects of a chemical. From a literature survey, 148 chemicals are tabulated that have been assayed in 164 Allium tests for their clastogenic effect. Of the 164 assays which have been carried out, 75 are reported as giving a positive reaction, 49 positive and with a dose response, 1 positive and temperature-related, 9 borderline positive, and 30 negative; 76% of the chemicals gave a definite positive response. It is proposed that the Allium test be included among those tests routinely used for assessing chromosomal damage induced by chemicals.

  1. Bacterial mutagenicity assays: test methods.

    Science.gov (United States)

    Gatehouse, David

    2012-01-01

    The most widely used assays for detecting chemically induced gene mutations are those employing bacteria. The plate incorporation assay using various Salmonella typhimurium LT2 and E. coli WP2 strains is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances capable of causing DNA damage leading to gene mutations. The test is used worldwide as an initial screen to determine the mutagenic potential of new chemicals and drugs.The test uses several strains of S. typhimurium which carry different mutations in various genes of the histidine operon, and E. coli which carry the same AT base pair at the critical mutation site within the trpE gene. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When these auxotrophic bacterial strains are grown on a minimal media agar plates containing a trace of the required amino-acid (histidine or tryptophan), only those bacteria that revert to amino-acid independence (His(+) or Tryp(+)) will grow to form visible colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner.This chapter provides detailed procedures for performing the test in the presence and absence of a metabolic activation system (S9-mix), including advice on specific assay variations and any technical problems. PMID:22147566

  2. Assays for B lymphocyte function.

    Science.gov (United States)

    Bondada, Subbarao; Robertson, Darrell A

    2003-11-01

    This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. The first basic protocol is a generalized system for inducing in vitro antibody production and can accommodate various types of antigens under study. Secreted antibodies can then be measured with an enzyme-linked immunosorbent assay (ELISA) or other soluble-antibody detection systems. Alternatively, the number of antibody-producing cells can be quantified by plaque-forming cell (PFC) assays presented in this unit: the Cunningham-Szenberg and the Jerne-Nordin techniques. Both methods employ specially prepared slide chambers, described here, in which the antibody-producing B cells are mixed with complement and indicator sheep red blood cells (SRBC), or with trinitrophenol-modified SRBC (TNP-SRBC), with subsequent lysis and counting of plaques. Because IgM antibodies fix complement efficiently, whereas IgG and IgA antibodies do not, unmodified PFC assays measure only IgM antibodies. The assay can be modified, however, to measure all classes of antibodies or to enumerate total immunoglobulin-secreting B cells, as described in alternate protocols. Yet another method of measuring the number of antibody-producing B cells (in a class-specific fashion) is to use the ELISPOT technique described in UNIT 7.14. The resting B cells used in these procedures are prepared as described in the final support protocols for Percoll gradient centrifugation. PMID:18432909

  3. An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors

    DEFF Research Database (Denmark)

    Sørensen, A.H.; Ahring, B.K.

    1997-01-01

    An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens in ...

  4. 泡球蚴感染小鼠IL-17基因实时荧光定量RT-PCR方法的建立与检测%Development of real-time fluorescent quantitative PCR assays to detect IL-17 in mice infected with Echinococcus multilocularis

    Institute of Scientific and Technical Information of China (English)

    张志; 王俊华; 张传山; 吕国栋; 卢晓梅; 姜涛; 林仁勇; 温浩

    2011-01-01

    目的 建立检测泡球蚴感染小鼠IL-17的实时荧光定量RT-PCR方法.并用于泡球蚴病小鼠IL-17基因检测.方法 通过腹腔注射100 μl泡球蚴组织匀浆液建立泡球蚴感染小鼠模型,感染1个月后处死,提取肝脏总RNA,反转录得到cDNA;登陆GenBank,得到IL-17基因(U43088.1)全序列,选取保守序列,应用primer 5设计引物,β-actin作为内参,以10倍梯度稀释IL-17 PCR产物为标准品.进行实时荧光定量RT-PCR以确定方法的特异性和灵敏度;绘制标准曲线,确定IL-17基因检测的最佳条件. 结果 用建立的实时定量RT-PCR检测IL-17基因无非特异性扩增,各样品熔解温度均在81.0℃.熔解峰单一;mRNA检出限为1 × 102Pg,106~102pg总RNA均有扩增;泡球蚴感染1个月后,IL-17基因表达显著增加,感染组和对照组分别为(0.008728±0.000984)和(0.003823±0.00082).差异有统计学意义(P<0.05). 结论 成功建立了小鼠源IL-17基因实时定量RT-PCR检测体系.经检测,小鼠感染泡球蚴1个月后IL-17基因表达增加,表明IL-17在泡球蚴感染免疫调节中可能起重要作用.%Objective To establish a method of RT-q-PCR for detection of IL-17 in mice infected with Echinococcus multilocularis. Methods To establish the mouse model, 100 μl homogenized metacestodes were injected into the anterior lobe of the liver of laboratory mice. After 1 month, the animals were sacrificed and the liver was removed to extract total RNA. cDNA was obtained by reverse transcription. A primer was designed based on the nucleotide sequence of the IL-17 gene from GENE BANK(U43088. 1)by using primer 5. β-actin served as an internal control. cDNA of the liver was obtained to determine the sensitivity of RT-PCR and prepare a standard curve. Results The designed primer was specific for RT-q-PCR detection of IL-17 and the melting temperature for each sample was 81.0 ℃. The RT-q-PCR assay had a detection limit of 1× 102 pg mRNA, with a dynamic range of detection

  5. Quantitative analysis of cholinesterase from Daphnia magna by indirect and non-competitive enzyme-linked immunosorbent assay%大型溞胆碱酯酶间接非竞争酶联免疫吸附定量分析法的建立

    Institute of Scientific and Technical Information of China (English)

    刘洪翠; 杨艳霞; 李少南

    2012-01-01

    用达到电泳纯的大型溞(Daphnia magna)胆碱酯酶(cholinesterase,ChE)免疫BALB/c小鼠,得到效价达1∶8000的鼠抗大型溞ChE多克隆抗血清;通过优化抗原抗体反应条件,建立定量分析大型溞ChE含量的间接非竞争酶联免疫吸附法(indirect and non-competitive enzyme-linked immunosorbent assay).结果表明:此方法检测灵敏度为24 ng·mL-1;经免疫交叉反应鉴定,所获抗血清与5、10 μg·mL-1标准蛋白质和其他生物如花翅摇蚊、尖额溞、斑马鱼(脑、脑除外的整体)、家蚕、意大利蜜蜂、赤子爱胜蚯蚓、非洲爪蟾(蝌蚪)等来源的ChE交叉反应率分别为:<0.25%、<0.25%、2.13%、10.19%、3.88%、3.56%、2.81%、3.93%、5.00%和2.75%,具较高的特异性.说明用本试验得到的抗血清建立的间接非竞争酶联免疫吸附法可用于大型溞体内ChE含量的测定,以利于在农药暴露环境下大型溞体内ChE活性的精确检测.%Polyclonal anti-serum of cholinesteiase with dilution ratio of 1 :8 000 was obtained by immunizing BALB/c mice with purified cholinesterase (ChE) from Daphnia magna. An indirect and non-competitive enzyme-linked immunosorbent assay (ELISA), which used mice anti-Daphnia ChE as antibodies to react with the Daphnia ChE, was established to quantify immunoreactive content of ChE in D. magna. The minimal detection limit of the method was estimated to be 24 ng-mL"'. Specificity of anti-serum was high, with cross reaction of <0. 25%, <0. 25%, 2.13%, 10.19%, 3.88%, 3.56%, 2.81%, 3.93%, 5.00% and 2.75% towards non-ChE marker (5. 10 μg·mL-1), Chironomus kiinensis, Alona sp. , Brachydanio rerio (the brain, the whole body without brain), Bombyx mori, Apis mellifera L., Eisenia foetida , and tadpoles of Xenopus laevis, respectively. It is indicated that the established method can be used to quantify immunoreactive content of ChE in D. magna so as to measureme accurately in vivo activity of the ChE in pesticide

  6. A molecular assay for sensitive detection of pathogen-specific T-cells.

    Directory of Open Access Journals (Sweden)

    Victoria O Kasprowicz

    Full Text Available Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR for two reporters--monokine-induced by IFN-γ (MIG and the IFN-γ inducible protein-10 (IP10. We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001. Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings.

  7. Quantitative Decision Support Requires Quantitative User Guidance

    Science.gov (United States)

    Smith, L. A.

    2009-12-01

    Is it conceivable that models run on 2007 computer hardware could provide robust and credible probabilistic information for decision support and user guidance at the ZIP code level for sub-daily meteorological events in 2060? In 2090? Retrospectively, how informative would output from today’s models have proven in 2003? or the 1930’s? Consultancies in the United Kingdom, including the Met Office, are offering services to “future-proof” their customers from climate change. How is a US or European based user or policy maker to determine the extent to which exciting new Bayesian methods are relevant here? or when a commercial supplier is vastly overselling the insights of today’s climate science? How are policy makers and academic economists to make the closely related decisions facing them? How can we communicate deep uncertainty in the future at small length-scales without undermining the firm foundation established by climate science regarding global trends? Three distinct aspects of the communication of the uses of climate model output targeting users and policy makers, as well as other specialist adaptation scientists, are discussed. First, a brief scientific evaluation of the length and time scales at which climate model output is likely to become uninformative is provided, including a note on the applicability the latest Bayesian methodology to current state-of-the-art general circulation models output. Second, a critical evaluation of the language often employed in communication of climate model output, a language which accurately states that models are “better”, have “improved” and now “include” and “simulate” relevant meteorological processed, without clearly identifying where the current information is thought to be uninformative and misleads, both for the current climate and as a function of the state of the (each) climate simulation. And thirdly, a general approach for evaluating the relevance of quantitative climate model output

  8. Benefit of Hepatitis C Virus Core Antigen Assay in Prediction of Therapeutic Response to Interferon and Ribavirin Combination Therapy

    OpenAIRE

    Takahashi, Masahiko; Saito, Hidetsugu; Higashimoto, Makiko; Atsukawa, Kazuhiro; Ishii, Hiromasa

    2005-01-01

    A highly sensitive second-generation hepatitis C virus (HCV) core antigen assay has recently been developed. We compared viral disappearance and first-phase kinetics between commercially available core antigen (Ag) assays, Lumipulse Ortho HCV Ag (Lumipulse-Ag), and a quantitative HCV RNA PCR assay, Cobas Amplicor HCV Monitor test, version 2 (Amplicor M), to estimate the predictive benefit of a sustained viral response (SVR) and non-SVR in 44 genotype 1b patients treated with interferon (IFN) ...

  9. Screening for pulmonary embolism with a D-dimer assay: do we still need to assess clinical probability as well?

    OpenAIRE

    Hammond, Christopher J; Hassan, Tajek B.

    2005-01-01

    Clinical risk stratification and D-dimer assay can be of use in excluding pulmonary embolism in patients presenting to emergency departments but many D-dimer assays exist and their accuracy varies. We used clinical risk stratification combined with a quantitative latex-agglutination D-dimer assay to screen patients before arranging further imaging if required. Retrospective analysis of a sequential series of 376 patients revealed that no patient with a D-dimer of

  10. Establishment and preliminary application of a real-time fluorescent quantitative PCR assay for detection of feline herpesvirus 1%猫疱疹病毒Ⅰ型实时荧光定量 PCR方法的建立及初步应用

    Institute of Scientific and Technical Information of China (English)

    王吉; 卫礼; 付瑞; 李晓波; 王淑菁; 巩薇; 岳秉飞; 贺争鸣

    2014-01-01

    Objective To establish a real-time fluorescent quantitative PCR ( Q-PCR) method for detection of feline herpesvirus 1 ( FHV-1 ) in experiment cats and clinical sick cats.Methods Primers and TaqMan probes were designed and synthesized according to the published FHV-1 specific sequences of TK gene.FHV DNA standards were prepared using molecular biological techniques.The linearity, specificity, sensitivity, stability of the established Q-PCR method were tested.The method was used to detect 48 samples of cats.Results The linear range was 102 copies/μL to 109 copies/μL.The developed Q-PCR method showed no cross reaction with herpes virus type 1 ( HSV-1 ) , canine herpesvirus (CHV), pig pseudo rabies virus (PRV) and cat parvovirus (FPV).The sensitivity was 10 copies/μL.The coefficient of variation ( CV ) was less than 5%.There were 33 positive cases detected in the 48 samples of cats. Conclusions The developed Q-PCR method is good in linearity, specificity, sensitivity, stability, and may be used for rapid quantitative detection of FHV-1 in cats.%目的:建立猫疱疹病毒I型( FHV-1)实时荧光定量PCR检测方法,应用于实验用猫及临床患猫FHV-1的快速检测。方法根据已发表的FHV-1 TK基因序列设计特异引物和TaqMan探针,使用分子生物学方法制备质粒标准品,建立FHV-1实时荧光定量PCR方法,并对方法的线性、特异性、敏感性、及稳定性等进行测定。用建立的方法对48份猫临床样品进行检测。结果成功建立FHV-1实时荧光定量PCR方法;该方法的线性范围为(1×102~1×109)copies/μL,与单纯疱疹病毒Ⅰ型(HSV-1)、犬疱疹病毒(CHV)、猪伪狂犬病毒(PRV)、猫细小病毒( FPV)均无交叉反应,检测灵敏度可达到10 copies/μL。批内变异系数均小于5%。应用该方法从48份猫临床样本中检测出33份FHV-1核酸阳性。结论建立的FHV-1PCR检测方法具有特异、敏感及稳定

  11. Establishment and Application of Real-time TaqMan-quantitative PCR Assay for Detection of Mycoplasma suis (Eperythrozoon suis)%猪附红细胞体荧光定量PCR检测方法的建立及应用

    Institute of Scientific and Technical Information of China (English)

    闫若潜; 赵明军; 张志凌; 张健; 盛敏; 刘光辉; 吴志明

    2008-01-01

    根据GenBank中猪附红细胞体(Mycoplasma suis)推测的功能性基因ORF2序列设计引物和TaqMan荧光探针,经优化反应条件,建立了M.suis的TaqMan荧光定量PCR检测方法(fluorescent quantitative real-time PCR,FQ-PCR);进行了FQ-PCR检测方法的敏感性、特异性和重复性实验,并对疑似M.suis感染临床抗凝全血样品进行了应用检测,同时与普通PCR方法进行了对比实验.结果成功地建立了M.suis的FQ-PCR方法和定量标准曲线,标准曲线的曲线循环阈值与模板浓度有良好的线性关系,相关系数为0.998;FQ-PCR方法检测灵敏度可达10个拷贝/霯,比普通PCR灵敏度高100倍;FQ-PCR方法特异性高,对pGEX-T/M.suis重组质粒扩增呈现阳性反应曲线,而对8个对照细菌、病毒和寄生虫DNA扩增曲线均呈现阴性反应;不同浓度的pGEX-T/M.suis重组质粒分别重复扩增2次,重复结果良好;对24份临床疑似M.suis感染样品进行了应用检测,阳性检出率为83.3%,较普通PCR方法阳性检出率高.

  12. Quantitative Compositional Reasoning

    NARCIS (Netherlands)

    Chatterjee, K.; Alfaro, de L.; Faella, M.; Henzinger, T.A.; Majumdar, R.; Stoelinga, M.I.A.

    2006-01-01

    We present a compositional theory of system verifica tion, where specifications assign real-numbered costs to systems. These costs can express a wide variety of quantitative system properties, such as resource consumption, price or a measure of how well a system satisfies its specification. The theo

  13. Development of a real-time PCR assay for quantification of Citrobacter rodentium.

    Science.gov (United States)

    Sagaidak, Sofia; Taibi, Amel; Wen, Bijun; Comelli, Elena M

    2016-07-01

    Molecular tools to quantify Citrobacter rodentium are not available. We developed a quantitative PCR assay targeting the espB gene. This assay is specific, has a linearity range of about 6.7×10(1) to 6.7×10(6)cells/PCR reaction (92% efficiency) and a detection limit of about 10(4)cells/g wet feces.

  14. RNase protection assays and RNA gel blots: a direct comparison of sensitivity.

    Science.gov (United States)

    Higgs, D C; Colbert, J T

    1992-01-01

    RNase protection assays are commonly thought to be a more sensitive means of detecting and quantitating specific mRNAs than are RNA gel blots (Northern blots). We have directly compared the sensitivity of these two approaches by assaying for known amounts of in vitro synthesized beta-glucuronidase mRNA. With the probes and protocols employed here, the ability to detect a specific mRNA was similar whether RNase protection or RNA gel blot analyses were performed.

  15. A new assay for lignin-type peroxidases employing the dye azure B.

    OpenAIRE

    Archibald, F S

    1992-01-01

    The discovery in 1983 of fungal "ligninases" capable of catalyzing the peroxidation of nonphenolic aromatic lignin components has been seen as a major advance in understanding how certain basidiomycete fungi can completely degrade lignin. The ability of these lignin-type peroxidases to covert millimolar concentrations of veratryl alcohol to veratraldehyde, indicated by a change in the A310 of veratraldehyde, has become the standard assay for routine quantitation of LP activity. A new assay ba...

  16. Estudos sobre a dosagem microbiologica das vitaminas do complexo B - II. Niacina Microbiological assay for niacine

    Directory of Open Access Journals (Sweden)

    Gilberto G. Villela

    1945-02-01

    Full Text Available The microbiological assay method of Snell and Wright for niacine was studied and some modifications of the basal medium were proposed. A maximal growth of the "Lactobacillus arabinosus" was obtained by the addition to the basal medium of 25 mg % asparagine and increasing the percentages of glucose and sodium acetate. Liver and yeast extracts were assayed satisfactory and the niacine added was recovered quantitatively.

  17. Designs, formats and applications of lateral flow assay: A literature review

    OpenAIRE

    Muhammad Sajid; Abdel-Nasser Kawde; Muhammad Daud

    2015-01-01

    This manuscript provides a brief overview of latest research involving the use of lateral flow assay for qualitative and quantitative analysis in different areas. The excellent features and versatility of detection formats make these strips an ideal choice for point of care applications. We outline and critically discuss detection formats, molecular recognition probes, labels, and detection systems used in lateral flow assay. Applications in different fields along with selected examples from ...

  18. New competition assay for the solubilized hepatic galactosyl receptor

    Energy Technology Data Exchange (ETDEWEB)

    Ray, D.A.; Weigel, P.H.

    1985-02-15

    The present method of quantitating soluble asialoglycoprotein (galactosyl) receptor activity relies on the selective precipitation of receptor-ligand complexes to allow separation from free ligand. To provide an alternative to selective precipitation procedures, a simple and rapid method to assay for detergent-solubilized galactosyl receptor activity has been developed which uses permeabilized, fixed cells as a source of immobilized solid-phase receptors. Isolated rat hepatocytes were treated with digitonin to make available the internal as well as the external receptors. The permeable cells were also treated with glutaraldehyde to prevent further protein loss during subsequent exposure to detergents such as Triton X-100. The permeable/fixed cells, which retained about 70% of their total /sup 125/I-asialo-orosomucoid (/sup 125/I-ASOR)-binding activity, with 89% specific binding, were insoluble even in 0.5% Triton X-100 and were easily pelleted. The permeable/fixed cells can be prepared in advance and stored frozen for months. A detergent extract of receptor is mixed with a constant amount of both /sup 125/I-ASOR and permeable/fixed cells. Soluble active receptors compete with immobilized receptors on the treated cell for binding of the /sup 125/I-ASOR. The assay is reproducible, linear over a broad range of soluble receptor concentration, and can quantitate receptor activity from as few as 10(5) hepatocytes. A modified purification procedure for the rat hepatic galactosyl receptor using this competition assay is also described.

  19. Critical Quantitative Inquiry in Context

    Science.gov (United States)

    Stage, Frances K.; Wells, Ryan S.

    2014-01-01

    This chapter briefly traces the development of the concept of critical quantitative inquiry, provides an expanded conceptualization of the tasks of critical quantitative research, offers theoretical explanation and justification for critical research using quantitative methods, and previews the work of quantitative criticalists presented in this…

  20. Controlling variation in the comet assay

    OpenAIRE

    Collins, Andrew R; El Yamani, Naouale; Lorenzo, Yolanda; Shaposhnikov, Sergey; Brunborg, Gunnar; Azqueta, Amaya

    2014-01-01

    Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of ...

  1. Co-incubation with IL-18 potentiates antigen-specific IFN-γ response in a whole-blood stimulation assay for measurement of cell-mediated immune responses in pigs experimentally infected with Lawsonia intracellularis

    DEFF Research Database (Denmark)

    Riber, Ulla; Boesen, Henriette Toft; Jakobsen, Jeanne Toft;

    2011-01-01

    The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in-vitro assay for a direct read out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult to evalu......The whole-blood interferon-gamma (IFN-γ) assay is a quantitative in-vitro assay for a direct read out of Ag-specific cell-mediated immune (CMI) responses to infectious diseases. The IFN-γ assay is robust in severe intracellular infections like Brucella or mycobacteria, but more difficult...

  2. Comet Assay in Cancer Chemoprevention.

    Science.gov (United States)

    Santoro, Raffaela; Ferraiuolo, Maria; Morgano, Gian Paolo; Muti, Paola; Strano, Sabrina

    2016-01-01

    The comet assay can be useful in monitoring DNA damage in single cells caused by exposure to genotoxic agents, such as those causing air, water, and soil pollution (e.g., pesticides, dioxins, electromagnetic fields) and chemo- and radiotherapy in cancer patients, or in the assessment of genoprotective effects of chemopreventive molecules. Therefore, it has particular importance in the fields of pharmacology and toxicology, and in both environmental and human biomonitoring. It allows the detection of single strand breaks as well as double-strand breaks and can be used in both normal and cancer cells. Here we describe the alkali method for comet assay, which allows to detect both single- and double-strand DNA breaks. PMID:26608293

  3. Nondestructive assay of sale materials

    International Nuclear Information System (INIS)

    This paper covers three primary areas: (1) reasons for performing nondestructive assay on SALE materials; (2) techniques used; and (3) discussion of investigators' revised results. The study shows that nondestructive calorimetric assay of plutonium offers a viable alternative to traditional wet chemical techniques. For these samples, the precision ranged from 0.4 to 0.6% with biases less than 0.2%. Thus, for those materials where sampling errors are the predominant source of uncertainty, this technique can provide improved accuracy and precision while saving time and money as well as reducing the amount of liquid wastes to be handled. In addition, high resolution gamma-ray spectroscopy measurements of solids can provide isotopic analysis data in a cost effective and timely manner. The timeliness of the method can be especially useful to the plant operator for production control and quality control measurements

  4. Protein binding assay for hyaluronate

    Energy Technology Data Exchange (ETDEWEB)

    Lacy, B.E.; Underhill, C.B.

    1986-11-01

    A relatively quick and simple assay for hyaluronate was developed using the specific binding protein, hyaluronectin. The hyaluronectin was obtained by homogenizing the brains of Sprague-Dawley rats, and then centrifuging the homogenate. The resulting supernatant was used as a source of crude hyaluronectin. In the binding assay, the hyaluronectin was mixed with (/sup 3/H)hyaluronate, followed by an equal volume of saturated (NH/sub 4/)/sub 2/SO/sub 4/, which precipitated the hyaluronectin and any (/sup 3/H)hyaluronate associated with it, but left free (/sup 3/H)hyaluronate in solution. The mixture was then centrifuged, and the amount of bound (/sup 3/H)hyaluronate in the precipitate was determined. Using this assay, the authors found that hyaluronectin specifically bound hyaluronate, since other glycosaminoglycans failed to compete for the binding protein. In addition, the interaction between hyaluronectin and hyaluronate was of relatively high affinity, and the size of the hyaluronate did not appear to substantially alter the amount of binding. To determine the amount of hyaluronate in an unknown sample, they used a competition assay in which the binding of a set amount of (/sup 3/H)hyaluronate was blocked by the addition of unlabeled hyaluronate. By comparing the degree of competition of the unknown samples with that of known amounts of hyaluronate, it was possible to determine the amount of hyaluronate in the unknowns. They have found that this method is sensitive to 1 ..mu..g or less of hyaluronate, and is unaffected by the presence of proteins.

  5. Establishment of indirect immunofluorescence assay for rotavirus.

    Science.gov (United States)

    Tao, J; Zhang, J; Liu, X; Jin, H; Jiang, C; Yin, Y

    2016-03-01

    Rotavirus infection is the most frequent cause of infantile gastroenteritis worldwide and a significant cause of death in infants and young children, following severe diarrhea and dehydration. Rotavirus vaccines are considered the most effective way to prevent rotavirus infections. In the process of developing rotavirus vaccines, it is crucial to establish a reliable and standardized method to determine vaccine titer. In this study, we developed an indirect immunofluorescence assay (IFA) to determine the infectious titer of Lanzhou lamb rotavirus (LLR) vaccine grown in MA104 cells. The activating concentration of trypsin was 1 µg/ml for healthy monolayers of MA104 cells at 100% confluence. After incubation for 18 hr, a rabbit anti-SA11 polyclonal antibody, diluted at 1:800 in PBS, was added to all wells, followed by an Alexa-488-conjugated secondary antibody diluted at 1:500 in PBS. Cells were examined with a fluorescence microscope. Our results show that IFA was more reproducible, more sensitive, simpler, and more rapid than the log 50% cell culture infectious dose-ELISA (lgCCID50-ELISA) in measuring the rotavirus vaccines. IFA provided a reliable basis for the qualitative and quantitative analysis of rotavirus, and the certification of rotavirus vaccine production.

  6. Fluorescence Polarization Assays in Small Molecule Screening

    Science.gov (United States)

    Lea, Wendy A.; Simeonov, Anton

    2011-01-01

    Importance of the field Fluorescence polarization (FP) is a homogeneous method that allows rapid and quantitative analysis of diverse molecular interactions and enzyme activities. This technique has been widely utilized in clinical and biomedical settings, including the diagnosis of certain diseases and monitoring therapeutic drug levels in body fluids. Recent developments in the field has been symbolized by the facile adoption of FP in high-throughput screening (HTS) and small molecule drug discovery of an increasing range of target classes. Areas covered in this review The article provides a brief overview on the theoretical foundation of FP, followed by updates on recent advancements in its application for various drug target classes, including G-protein coupled receptors (GPCRs), enzymes and protein-protein interactions (PPIs). The strengths and weaknesses of this method, practical considerations in assay design, novel applications, and future directions are also discussed. What the reader will gain The reader will be informed of the most recent advancements and future directions of FP application to small molecule screening. Take home message In addition to its continued utilization in high-throughput screening, FP has expanded into new disease and target areas and has been marked by increased use of labeled small molecule ligands for receptor binding studies. PMID:22328899

  7. Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey

    OpenAIRE

    Posthuma-Trumpie, G. A.; Korf, J.; Amerongen, van, H.

    2009-01-01

    Lateral flow (immuno)assays are currently used for qualitative, semiquantitative and to some extent quantitative monitoring in resource-poor or non-laboratory environments. Applications include tests on pathogens, drugs, hormones and metabolites in biomedical, phytosanitary, veterinary, feed/food and environmental settings. We describe principles of current formats, applications, limitations and perspectives for quantitative monitoring. We illustrate the potentials and limitations of analysis...

  8. A high-throughput chemically induced inflammation assay in zebrafish

    Directory of Open Access Journals (Sweden)

    Liebel Urban

    2010-12-01

    Full Text Available Abstract Background Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immunomodulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high-content screening. Results Here we show that specific, noninvasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sublethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immunomodulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction. Conclusions This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds toward new immunomodulatory therapies. We have called this method the chemically induced inflammation assay, or ChIn assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.

  9. Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.

    Directory of Open Access Journals (Sweden)

    Ahmed Abd El Wahed

    Full Text Available Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF. Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR are the standard method for molecular detection of the dengue virus (DENV. Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA assays were developed to detect DENV1-4.Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4 to 241 (DENV1-3 RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal and in Bangkok (Thailand. In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31 and 100% (n=23, respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90 and 100%(n=41, respectively.During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

  10. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    Directory of Open Access Journals (Sweden)

    Carina Ladeira

    2015-06-01

    The use of the CBMN assay in in vitro genetic toxicology testing is well established and in fact it has become an accepted standard method to assess the genotoxic hazard of chemicals which led to the development of a special guideline by the Organization for Economic Cooperation and Development (OECD, the OECD 487 guideline (Kirsch-Volders et al., 2014. The CBMN assay is an effective tool for the study of cellular and nuclear dysfunction caused by in vitro or in vivo aging, micronutrient deficiency or excess, genotoxins exposure and genetic defects in genome maintenance. It is also fruitful in the emerging fields of nutrigenomics and toxicogenomics and their combinations, as it becomes increasingly clear that nutrient status also impacts on sensitivity to exogenous genotoxins (Fenech, 2005, 2007. Many results obtained by this assay indicate the potential predictive value of the CBMN assay with respect to cancer risk and validate its use as a test for detecting nutritional, environmental and genetic factors that are potentially carcinogenic. Also it is used by pharmaceutical industry, human biomonitoring of genotoxic exposures and its increasing application in preventive medicine and nutrition and the increased investment in the automation of the CBMN assay are indicative of the increasing importance of this test (Fenech, 2007. The comet assay or single-cell gel electrophoresis (SCGE is a simple, sensitive method for detecting DNA-strand breaks. Cells embedded in agarose on a microscope slide are lysed with detergent and 2.5 M NaCl and fresh Triton X-100 to remove membranes and soluble cell constituents, including most histones, leaving the DNA, still supercoiled and attached to a nuclear matrix, as a nucleoid. A break in one strand of a DNA loop is enough to release the supercoiling, and during electrophoresis the relaxed loops are able to extend towards the anode (Fairbairn et al., 1995; Collins et al., 1997; Moller et al., 2000; Azqueta et al., 2009; Collins

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

    Science.gov (United States)

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

    2012-03-01

    Chronic bee paralysis virus (CBPV) is responsible for chronic bee paralysis, an infectious and contagious disease in adult honey bees (Apis mellifera L.). A real-time RT-PCR assay to quantitate the CBPV load is now available. To propose this assay as a reference method, it was characterised further in an intra-laboratory study during which the reliability and the repeatability of results and the performance of the assay were confirmed. The qPCR assay alone and the whole quantitation method (from sample RNA extraction to analysis) were both assessed following the ISO/IEC 17025 standard and the recent XP U47-600 standard issued by the French Standards Institute. The performance of the qPCR assay and of the overall CBPV quantitation method were validated over a 6 log range from 10(2) to 10(8) with a detection limit of 50 and 100 CBPV RNA copies, respectively, and the protocol of the real-time RT-qPCR assay for CBPV quantitation was approved by the French Accreditation Committee. PMID:22207079

  12. Cp*Rh-based indicator-displacement assays for the identification of amino sugars and aminoglycosides.

    Science.gov (United States)

    Zaubitzer, Friederike; Buryak, Andrey; Severin, Kay

    2006-05-01

    Indicator-displacement assays based on the organometallic complex [{Cp*RhCl2}2] (Cp*=pentamethylcyclopentadienyl) and the dye gallocyanine were used to sense amino sugars and aminoglycosides in buffered aqueous solution by conducting UV-visible spectroscopy. The data of three assays at pH 7.0, 8.0, and 9.0 were sufficient to distinguish between the amino sugars galactosamine, glucosamine, mannosamine and the aminoglycosides kanamycin A, kanamycin B, amikacin, apramycin, paromomycin, and streptomycin. Furthermore, the assays were used to characterize mixtures of aminoglycosides and obtain quantitative information about the respective analytes. PMID:16521137

  13. 检测家蝇抗菌肽基因diptericin时间表达模式的实时荧光定量方法的建立%Establishing Real-time Quantitative PCR Assay for Detecting Expression Pattern of The Antimicrobial Peptide gene Diptericin from the Musca domestica

    Institute of Scientific and Technical Information of China (English)

    杨小蓉; 金小宝; 朱家勇; 丁彩屏

    2012-01-01

    OBJECTIVE To establish a fluorescent dye (SYBR Green I) real-time quantitative PCR (real-timePCR) to detect antimicrobial peptide gene diptericin expression patterns method from Musca domestica. METHODS Based on the cloned diptericin gene, software Prmier 5.0 was used for designing primers for non-induced, different developmental stages and different times (2h, 4h, 8h, 12h, 24h, 36h and 48h) of Musca domestica were induced by real-time PCR. 10 times of serial dilution of plasmid with target gene was used. Real-time PCR was used for standard curve. RESULTS The gene expression level that Uninduced third-instar was low compared with that induced. The greatest level of mRNA for all nine infection time was in 12 hours. After induced 2 hours, a significantly transcript level 107 was reflected. After 36 hours, mRNA levels were significantly descend. In different stages of development, the greatest level of mRNA for all six stages was in the 3rd- instars and adult. The lowest level of transcript detected was in the eggs and 1st- instars. A 102 -fold increased in mRNA was revealed between the 3rd- and eggs, 1st-, 2nd- instar and pupae. Almost no change was found between the 3rd- instar and adult. CONCLUSION Real-time PCR detection method housefly gene diptericin specificity, high sensitivity, in order to further explore the biological activity of antimicrobial peptides housefly to the study of new antibiotics to lay the initial foundation.%目的 建立荧光染料(SYBR Green Ⅰ)实时荧光定量PCR(real-timePCR)方法检测的家蝇抗菌肽基因diptericin 时间表达模式方法.方法 基于已克隆出的diptericin基因,利用软件Prmier5.0设计上下游引物,对未诱导,诱导后2h、4h、8h、12 h、24 h、36 h、48 h及不同发育阶段的家蝇三龄幼虫进行real-time PCR设计引物,然后利用10倍系列稀释含有目的基因的质粒,进行实时荧光定量PCR反应,制作标准曲线.结果 未刺激的三龄幼虫diptericin基因

  14. Difference of Fluorescent Quantitative PCR Assay for Detection of Tuberculosis in Clinical Samples%不同临床标本实时荧光定量 PCR诊断结核分枝杆菌的检出率差异研究

    Institute of Scientific and Technical Information of China (English)

    李光友; 苏贵灵; 高杰; 查争; 黄亚银; 王亮; 冯岚

    2013-01-01

    Objective To observe the difference in detection rate of Tuberculosis in different types of samples. Methods Sam pies were taken from 1 343 suspected patients with Tuberculosis to detect TB DNA by real time fluorescence quantitative PCR (FQ PCR) ,and meaaody 393 suspected patients to detect TB Ab in serum by Tuberculosis antibody colloidal gold diag nostic kit. Results The positive rate of bronchial fluid,bronchial brush biopsy,urine,sputum,pleural fluid and ascites in dif ferent types of samples from 1 343 suspected patients were 57. 14% ,21. 21 % ,13. 33% ,11. 23%,9. 09% and 3. 85% respec tively. The positive rate of sputum was lower than that of bronchial fluid and bronchial brush biopsy (X2 =14. 32 ,5. 87, P< 0. 05). 393 suspected tuberculosis patients,different types of samples were detected by FQ PCR and TB Ab colloidal gold di agnostic kit and the positive detection rates were 24. 43% and 47. 58% , respectively. There was significant difference be tween two methods (X2= 44. 73, P<0. 01) ,and the coincidence rate between two methods was 61. 57%. Conclusion The detection rate was highest in the bronchial fluid,and the detection rate was the lowest in ascites sample.%目的 讨论不同临床标本中结核分枝杆菌的检出率有何差异.方法 对1 343例疑似结核病患者的呼吸道和体液标本进行实时荧光定量PCR检测,同时对其中393例进行血清结核抗体检测.结果 1 343例疑似结核病患者不同临床标本经实时荧光定量PCR检测显示,支气管灌洗液检出率最高为57.14%,其次为支气管刷检物21.21%,尿液13.33%,痰液11.23%,胸腔积液9.09%,最低为腹腔积液3.85%.痰液与支气管灌洗液、支气管刷检物之间检出率差异具有统计学意义(χ2=14.32,5.87,P<0.05);393例不同临床标本同时进行实时荧光定量PCR和血清结核抗体检测,实时荧光定量PCR检出率为24.43%,血清结核抗体检出率为47.58%,两种方法的阳性率差异有统计学意义(χ2=44.73,P<0

  15. Quantitative social science

    Science.gov (United States)

    Weidlich, W.

    1987-03-01

    General concepts for the quantitative description of the dynamics of social processes are introduced. They allow for embedding social science into the conceptual framework of synergetics. Equations of motion for the socioconfiguration are derived on the stochastic and quasideterministic level. As an application the migration of interacting human populations is treated. The solutions of the nonlinear migratory equations include limit cycles and strange attractors. The empiric evaluation of interregional migratory dynamics is exemplified in the case of Germany.

  16. Quantitative FDG in depression

    International Nuclear Information System (INIS)

    Full text: Studies of regional cerebral glucose metabolism (rCMRGlu) using positron emission tomography (PET) in patients with affective disorders have consistently demonstrated reduced metabolism in the frontal regions. Different quantitative and semi-quantitative rCMRGlu regions of interest (ROI) comparisons, e.g. absolute metabolic rates, ratios of dorsolateral prefrontal cortex (DLPFC) to ipsilateral hemisphere cortex, have been reported. These studies suffered from the use of a standard brain atlas to define ROls, whereas in this case study, the individual''s magnetic resonance imaging (MRI) scan was registered with the PET scan to enable accurate neuroanatomical ROI definition for the subject. The patient is a 36-year-old female with a six-week history of major depression (HAM-D = 34, MMSE = 28). A quantitative FDG PET study and an MRI scan were performed. Six MRI-guided ROls (DLPFC, PFC, whole hemisphere) were defined. The average rCMRGlu in the DLPFC (left = 28.8 + 5.8 mol/100g/min; right = 25.6 7.0 mol/100g/min) were slightly reduced compared to the ipsilateral hemispherical rate (left = 30.4 6.8 mol/100g/min; right = 29.5 7.2 mol/100g/min). The ratios of DLPFC to ipsilateral hemispheric rate were close to unity (left = 0.95 0.29; right 0.87 0.32). The right to left DLPFC ratio did not show any significant asymmetry (0.91 0.30). These results do not correlate with earlier published results reporting decreased left DLPFC rates compared to right DLPFC, although our results will need to be replicated with a group of depressed patients. Registration of PET and MRI studies is necessary in ROI-based quantitative FDG PET studies to allow for the normal anatomical variation among individuals, and thus is essential for accurate comparison of rCMRGlu between individuals

  17. Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

    OpenAIRE

    Carina Ladeira; Susana Viegas; Gomes, Manuel C.

    2015-01-01

    The cytokinesis-block micronucleus cytome (CBMN) assay is a comprehensive system for measuring DNA damage; cytostasis and cytotoxicity-DNA damage events are scored specifically in once-divided binucleated cells. The endpoints possible to be measured are micronuclei (MN), a biomarker of chromosome breakage and/or whole chromosome loss, nucleoplasmic bridges (NPB), a biomarker of DNA misrepair and/or telomere end-fusions, and nuclear buds (NBUD), a biomarker of elimination of amplified DNA and/...

  18. The Genomic Grade Assay Compared With Ki67 to Determine Risk of Distant Breast Cancer Recurrence

    DEFF Research Database (Denmark)

    Ignatiadis, Michail; Azim, Hatem A; Desmedt, Christine;

    2016-01-01

    Importance: The Genomic Grade Index (GGI) was previously developed, evaluated on frozen tissue, and shown to be prognostic in early breast cancer. To test the GGI in formalin-fixed, paraffin-embedded breast cancer tumors, a quantitative reverse transcriptase polymerase chain reaction assay...... was developed and named the Genomic Grade (GG). The GG assay has the potential to increase the clinical application of the GGI, but robust demonstration of the clinical validity of the GG assay is required. Objective: To evaluate the prognostic ability of the GG assay to detect breast cancer recurrence compared...... cancer. Patients included in this study had available formalin-fixed, paraffin-embedded samples of their primary tumors and were randomized to either a 5-year tamoxifen monotherapy arm or a 5-year letrozole monotherapy arm. Associations between either GG assay results or log2-transformed Ki67 data...

  19. The Physcomitrella patens System for Transient Gene Expression Assays.

    Science.gov (United States)

    Thévenin, Johanne; Xu, Wenjia; Vaisman, Louise; Lepiniec, Loïc; Dubreucq, Bertrand; Dubos, Christian

    2016-01-01

    Transient expression assays are valuable techniques to study in vivo the transcriptional regulation of gene expression. These methods allow to assess the transcriptional properties of a given transcription factor (TF) or a complex of regulatory proteins against specific DNA motifs, called cis-regulatory elements. Here, we describe a fast, efficient, and reliable method based on the use of Physcomitrella patens protoplasts that allows the study of gene expression in a qualitative and quantitative manner by combining the advantage of GFP (green fluorescent protein) as a marker of promoter activity with flow cytometry for accurate measurement of fluorescence in individual cells. PMID:27557766

  20. Verification of uranium 238 quantity calculated using waste assay systems

    International Nuclear Information System (INIS)

    The amount of 238U in uranium-contaminated waste drums generated in the decommissioning of nuclear facilities is evaluated from γ-ray measurement. We used the γ-ray measurement system made from CANBERRA(Qualitative and Quantitative (Q2) Low Level Waste Assay Systems) and measured the waste drums. This system assumes uniform distribution of uranium. But, homogeneity can not be checked with real waste drums. Authors developed the new analysis technique which calculates the amount of uranium by correcting the influence of uneven distribution of the uranium. As a result of evaluating using the new analysis technique, the error which influences quantitative value of 238U has been evaluated. (author)

  1. Electrophoretic assay of specific estrogen receptors: a contribution to methodology.

    Science.gov (United States)

    van Netten, J P; Algard, F T; Montessori, G; Weare, B

    1977-11-01

    Experimental evidence is presented that supports the use of the cold agar-gel electrophoretic method for the clinical quantitation of specific estrogen-binding protein present in some human mammary carcinomas. It is necessary to dilute tumor extracts to avoid interference by serum-borne, non-relevant hormone-binding proteins such as albumin, which migrates to the same anodal region as does the binding protein. Dilution to 2.5 mg or less of total protein per milliliter circumvents such interference while still permitting reliable quantitation of the binding protein. Seventy-two mammary carcinomas were compared for binding-protein content by both the cold agar-gel electrophoresis and a single-point dextran-coated charcoal assay. The correlation coefficient (0.96) indicated excellent agreement between results by the two methods. In addition results are presented which indicate that the preparation of tumor extracts for electrophoresis does not require the use of an ultracentrifuge. PMID:912871

  2. Comparative evaluation of two radioenzymatic procedures designed to determine noradrenaline in the plasma (COMT assay and PNMT assay)

    International Nuclear Information System (INIS)

    A comparative evaluation of two radioenzymatic procedures to determine the concentration of noradrenaline in the plasma - with linearity, sensitivity, specifity and accuracy serving as test criteria - led to the following results: In view of a probability of error in the order of 2% both methods were judged to show a satisfactory sensitivity. The specific of the COMT assay, by contrast with that of the PNMT assay, was found to be wanting, as the noradrenaline measurements in the presence of other biogenic amines were biassed in such a way that the values determined were higher than the actual concentrations. During antihypertensive treatment even minimal changes in the noradrenaline concentration can be ascertained on a quantitative basis. If suitable hardware is available, the COMT assay permits up to 25 single determinations to be carried out per day, while the number of double determinations is restricted to 7 per day. One advantage, however, lies in the fact that several catecholamines in the plasma can be detected simultaneously, if required. In cases where the noradrenaline concentration alone is to be determined for clinical purposes, preference should be given to the PNMT assay, as both tests showed equal linearity and sensitivity. (TRV)

  3. A gold nanoparticle-based semi-quantitative and quantitative ultrasensitive paper sensor for the detection of twenty mycotoxins

    Science.gov (United States)

    Kong, Dezhao; Liu, Liqiang; Song, Shanshan; Suryoprabowo, Steven; Li, Aike; Kuang, Hua; Wang, Libing; Xu, Chuanlai

    2016-02-01

    A semi-quantitative and quantitative multi-immunochromatographic (ICA) strip detection assay was developed for the simultaneous detection of twenty types of mycotoxins from five classes, including zearalenones (ZEAs), deoxynivalenols (DONs), T-2 toxins (T-2s), aflatoxins (AFs), and fumonisins (FBs), in cereal food samples. Sensitive and specific monoclonal antibodies were selected for this assay. The semi-quantitative results were obtained within 20 min by the naked eye, with visual limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.1-0.5, 2.5-250, 0.5-1, 0.25-1 and 2.5-10 μg kg-1, and cut-off values of 0.25-1, 5-500, 1-10, 0.5-2.5 and 5-25 μg kg-1, respectively. The quantitative results were obtained using a hand-held strip scan reader, with the calculated limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.04-0.17, 0.06-49, 0.15-0.22, 0.056-0.49 and 0.53-1.05 μg kg-1, respectively. The analytical results of spiked samples were in accordance with the accurate content in the simultaneous detection analysis. This newly developed ICA strip assay is suitable for the on-site detection and rapid initial screening of mycotoxins in cereal samples, facilitating both semi-quantitative and quantitative determination.A semi-quantitative and quantitative multi-immunochromatographic (ICA) strip detection assay was developed for the simultaneous detection of twenty types of mycotoxins from five classes, including zearalenones (ZEAs), deoxynivalenols (DONs), T-2 toxins (T-2s), aflatoxins (AFs), and fumonisins (FBs), in cereal food samples. Sensitive and specific monoclonal antibodies were selected for this assay. The semi-quantitative results were obtained within 20 min by the naked eye, with visual limits of detection for ZEAs, DONs, T-2s, AFs and FBs of 0.1-0.5, 2.5-250, 0.5-1, 0.25-1 and 2.5-10 μg kg-1, and cut-off values of 0.25-1, 5-500, 1-10, 0.5-2.5 and 5-25 μg kg-1, respectively. The quantitative results were obtained using a hand-held strip scan

  4. Quantitative SPECT techniques.

    Science.gov (United States)

    Watson, D D

    1999-07-01

    Quantitative imaging involves first, a set of measurements that characterize an image. There are several variations of technique, but the basic measurements that are used for single photon emission computed tomography (SPECT) perfusion images are reasonably standardized. Quantification currently provides only relative tracer activity within the myocardial regions defined by an individual SPECT acquisition. Absolute quantification is still a work in progress. Quantitative comparison of absolute changes in tracer uptake comparing a stress and rest study or preintervention and postintervention study would be useful and could be done, but most commercial systems do not maintain the data normalization that is necessary for this. Measurements of regional and global function are now possible with electrocardiography (ECG) gating, and this provides clinically useful adjunctive data. Techniques for measuring ventricular function are evolving and promise to provide clinically useful accuracy. The computer can classify images as normal or abnormal by comparison with a normal database. The criteria for this classification involve more than just checking the normal limits. The images should be analyzed to measure how far they deviate from normal, and this information can be used in conjunction with pretest likelihood to indicate the level of statistical certainty that an individual patient has a true positive or true negative test. The interface between the computer and the clinician interpreter is an important part of the process. Especially when both perfusion and function are being determined, the ability of the interpreter to correctly assimilate the data is essential to the use of the quantitative process. As we become more facile with performing and recording objective measurements, the significance of the measurements in terms of risk evaluation, viability assessment, and outcome should be continually enhanced. PMID:10433336

  5. Serum indices: managing assay interference.

    Science.gov (United States)

    Farrell, Christopher-John L; Carter, Andrew C

    2016-09-01

    Clinical laboratories frequently encounter samples showing significant haemolysis, icterus or lipaemia. Technical advances, utilizing spectrophotometric measurements on automated chemistry analysers, allow rapid and accurate identification of such samples. However, accurate quantification of haemolysis, icterus and lipaemia interference is of limited value if laboratories do not set rational alert limits, based on sound interference testing experiments. Furthermore, in the context of increasing consolidation of laboratories and the formation of laboratory networks, there is an increasing requirement for harmonization of the handling of haemolysis, icterus and lipaemia-affected samples across different analytical platforms. Harmonization may be best achieved by considering both the analytical aspects of index measurement and the possible variations in the effects of haemolysis, icterus and lipaemia interferences on assays from different manufacturers. Initial verification studies, followed up with ongoing quality control testing, can help a laboratory ensure the accuracy of haemolysis, icterus and lipaemia index results, as well as assist in managing any biases in index results from analysers from different manufacturers. Similarities, and variations, in the effect of haemolysis, icterus and lipaemia interference in assays from different manufacturers can often be predicted from the mechanism of interference. Nevertheless, interference testing is required to confirm expected similarities or to quantify differences. It is important that laboratories are familiar with a number of interference testing protocols and the particular strengths and weaknesses of each. A rigorous approach to all aspects of haemolysis, icterus and lipaemia interference testing allows the analytical progress in index measurement to be translated into improved patient care. PMID:27147624

  6. SNO+ Scintillator Purification and Assay

    Science.gov (United States)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.

    2011-04-01

    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  7. Proteasome Assay in Cell Lysates

    Science.gov (United States)

    Maher, Pamela

    2016-01-01

    The ubiquitin-proteasome system (UPS) mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes including tumorigenesis, inflammation and cell death (Ciechanover, 2005; Kisselev and Goldberg, 2001). A number of recent studies have shown that proteasome activity is decreased in a variety of neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke as well as during normal aging (Chung et al., 2001; Ciechanover and Brundin, 2003; Betarbet et al., 2005). This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Protein clearance by the UPS involves two sequential reactions. The first is the tagging of protein lysine residues with ubiquitin (Ub) and the second is the subsequent degradation of the tagged proteins by the proteasome. We herein describe an assay for the second of these two reactions (Valera et al., 2013). This assay uses fluorogenic substrates for each of the three activities of the proteasome: chymotrypsin-like activity, trypsin-like activity and caspase-like activity. Cleavage of the fluorophore from the substrate by the proteasome results in fluorescence that can be detected with a fluorescent plate reader.

  8. The chemistry behind antioxidant capacity assays.

    Science.gov (United States)

    Huang, Dejian; Ou, Boxin; Prior, Ronald L

    2005-03-23

    This review summarizes the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addition, other assays intended to measure a sample's scavenging capacity of biologically relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this analysis, it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addition to these two commonly accepted assays. PMID:15769103

  9. F# for quantitative finance

    CERN Document Server

    Astborg, Johan

    2013-01-01

    To develop your confidence in F#, this tutorial will first introduce you to simpler tasks such as curve fitting. You will then advance to more complex tasks such as implementing algorithms for trading semi-automation in a practical scenario-based format.If you are a data analyst or a practitioner in quantitative finance, economics, or mathematics and wish to learn how to use F# as a functional programming language, this book is for you. You should have a basic conceptual understanding of financial concepts and models. Elementary knowledge of the .NET framework would also be helpful.

  10. 21 CFR 864.7525 - Heparin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and... HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A heparin assay is a device used to determine the level of the anticoagulant heparin in the...

  11. Multicentre comparison of a diagnostic assay

    DEFF Research Database (Denmark)

    Waters, Patrick; Reindl, Markus; Saiz, Albert;

    2016-01-01

    ) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4...

  12. 21 CFR 864.7490 - Sulfhemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Sulfhemoglobin assay. 864.7490 Section 864.7490 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... assay. (a) Identification. A sulfhemoglobin assay is a device consisting of the reagents,...

  13. 21 CFR 864.7250 - Erythropoietin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythropoietin assay. 864.7250 Section 864.7250... assay. (a) Identification. A erythropoietin assay is a device that measures the concentration of erythropoietin (an enzyme that regulates the production of red blood cells) in serum or urine. This...

  14. 21 CFR 864.7425 - Carboxyhemoglobin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Carboxyhemoglobin assay. 864.7425 Section 864.7425 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED... assay. (a) Identification. A carboxyhemoglobin assay is a device used to determine the...

  15. 21 CFR 866.3210 - Endotoxin assay.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Endotoxin assay. 866.3210 Section 866.3210 Food... DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3210 Endotoxin assay. (a) Identification. An endotoxin assay is a device that uses serological techniques in whole blood. The device...

  16. Isotopes——Development of a lmmunoradiometric Assay for Quantifying NSE in Human Serum

    Institute of Scientific and Technical Information of China (English)

    FANGJun-jian; YANQiang-feng

    2003-01-01

    Neuron-specific enolase(NSE) is a tumor marker for neuroblastoma and also one of the most sensitive and specific marker for small-cell lung cancer(SCLC) which has neuroendocrine properties with over-expressing of NSE. Quantitative assay for circulating NSE has clinic significance in monitoring of therapy and prognosis for the cancer.

  17. A multiplex real-time PCR assay for routine diagnosis of bacterial vaginosis

    NARCIS (Netherlands)

    Kusters, J. G.; Reuland, E. A.; Bouter, S.; Koenig, P.; Dorigo-Zetsma, J. W.

    2015-01-01

    A semi-quantitative multiplex PCR assay for the diagnosis of bacterial vaginosis (BV) was evaluated in a prospective study in a population of Dutch women with complaints of abnormal vaginal discharge. The PCR targets Gardnerella vaginalis, Atopobium vaginae, Megasphaera phylotype 1, Lactobacillus cr

  18. Development of an immunochromatographic assay based on carbon nanoparticles for the determination of the phytoregulator forchlorfenuron

    NARCIS (Netherlands)

    Suaréz-Pantaleón, C.; Wichers, J.H.; Abad-Somovilla, A.; Amerongen, van A.

    2013-01-01

    Rapid analytical methods enabling the determination of diverse targets are essential in a number of research areas, from clinical diagnostics to feed and food quality and safety. Herein, the development of a quantitative immunochromatographic assay for the detection of the synthetic phytoregulator f

  19. An enzyme-linked immunosorbent assay for autoantibodies against the nuclear protein Scl-70

    DEFF Research Database (Denmark)

    Geisler, C; Høier-Madsen, M

    1985-01-01

    This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of autoantibodies against the nuclear protein Scl-70. The isolation of Scl-70 from rat livers and the conditions for the ELISA are described. Compared with the already established...

  20. Lateral flow (immuno) assay : its strengths, weaknesses, opportunities and threats. A literature survey

    NARCIS (Netherlands)

    Posthuma-Trumpie, Geertruida A.; Korf, Jakob; van Amerongen, Aart

    2009-01-01

    Lateral flow (immuno) assays are currently used for qualitative, semiquantitative and to some extent quantitative monitoring in resource-poor or non-laboratory environments. Applications include tests on pathogens, drugs, hormones and metabolites in biomedical, phytosanitary, veterinary, feed/food a

  1. Lateral flow (immuno)assay: its strengths, weaknesses, opportunities and threats. A literature survey

    NARCIS (Netherlands)

    Posthuma-Trumpie, G.A.; Korf, J.; Amerongen, van A.

    2009-01-01

    Lateral flow (immuno)assays are currently used for qualitative, semiquantitative and to some extent quantitative monitoring in resource-poor or non-laboratory environments. Applications include tests on pathogens, drugs, hormones and metabolites in biomedical, phytosanitary, veterinary, feed/food an

  2. Making transuranic assay measurements using modern controllers

    International Nuclear Information System (INIS)

    This paper describes methodology and computer-controlled instrumentation developed at the Los Alamos National Laboratory that accurately performs nondestructive assays of large containers bearing transuranic wastes and nonradioactive matrix materials. These assay systems can measure fissile isotopes with 1-mg sensitivity and spontaneous neutron-emitting isotopes at a 10-mg sensitivity. The assays are performed by neutron interrogation, detection, and counting in a custom assay chamber. An International Business Machines Personal Computer (IBM-PC) is used to control the CAMAC-based instrumentation system that acquires the assay data. 6 refs., 7 figs

  3. Establishment of Immunoradiometric Assay for Carcinoembryonic Antigen

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Two anti-CEA monoclonal antibodies are used, one is coated on the microtiter plate, the other is labeled to make 125I-CEAMcAb. The one-step assay is established based on immunoradiometric assay(IRMA). The sensitivity of the assay is 0.5 μ g/L. The intra-assay CVs and the inter-assay CVs are lower than 10.0% and 15.0%, respectively. The analytical recoveries are ranged from 97.4% to 107.8%. The reference cut-out value of 35 normal serum is lower than

  4. Predictive Assay For Cancer Targets

    Energy Technology Data Exchange (ETDEWEB)

    Suess, A; Nguyen, C; Sorensen, K; Montgomery, J; Souza, B; Kulp, K; Dugan, L; Christian, A

    2005-09-19

    Early detection of cancer is a key element in successful treatment of the disease. Understanding the particular type of cancer involved, its origins and probable course, is also important. PhIP (2-amino-1-methyl-6 phenylimidazo [4,5-b]pyridine), a heterocyclic amine produced during the cooking of meat at elevated temperatures, has been shown to induce mammary cancer in female, Sprague-Dawley rats. Tumors induced by PhIP have been shown to contain discreet cytogenetic signature patterns of gains and losses using comparative genomic hybridization (CGH). To determine if a protein signature exists for these tumors, we are analyzing expression levels of the protein products of the above-mentioned tumors in combination with a new bulk protein subtractive assay. This assay produces a panel of antibodies against proteins that are either on or off in the tumor. Hybridization of the antibody panel onto a 2-D gel of tumor or control protein will allow for identification of a distinct protein signature in the tumor. Analysis of several gene databases has identified a number of rat homologs of human cancer genes located in these regions of gain and loss. These genes include the oncogenes c-MYK, ERBB2/NEU, THRA and tumor suppressor genes EGR1 and HDAC3. The listed genes have been shown to be estrogen-responsive, suggesting a possible link between delivery of bio-activated PhIP to the cell nucleus via estrogen receptors and gene-specific PhIP-induced DNA damage, leading to cell transformation. All three tumors showed similar silver staining patterns compared to each other, while they all were different than the control tissue. Subsequent screening of these genes against those from tumors know to be caused by other agents may produce a protein signature unique to PhIP, which can be used as a diagnostic to augment optical and radiation-based detection schemes.

  5. Activity assay of membrane transport proteins

    Institute of Scientific and Technical Information of China (English)

    Hao Xie

    2008-01-01

    Membrane transport proteins are integral membrane proteins and considered as potential drug targets. Activity assay of transport proteins is essential for developing drugs to target these proteins. Major issues related to activity assessment of transport proteins include availability of transporters,transport activity of transporters, and interactions between ligands and transporters. Researchers need to consider the physiological status of proteins (bound in lipid membranes or purified), availability and specificity of substrates, and the purpose of the activity assay (screening, identifying, or comparing substrates and inhibitors) before choosing appropriate assay strategies and techniques. Transport proteins bound in vesicular membranes can be assayed for transporting substrate across membranes by means of uptake assay or entrance counterflow assay. Alternatively, transport proteins can be assayed for interactions with ligands by using techniques such as isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, or surface plasmon resonance. Other methods and techniques such as fluorometry, scintillation proximity assay, electrophysiological assay, or stopped-flow assay could also be used for activity assay of transport proteins. In this paper the major strategies and techniques for activity assessment of membrane transport proteins are reviewed.

  6. Quantitative gamma scintillometry and spectrometry in uranium exploration

    International Nuclear Information System (INIS)

    This article discusses aspects of quantitative gamma scintillometry and spectrometry in uranium exploration with particular emphasis on field instrument capabilities and the calibration of surface radiometric field instruments. Factors that influence the accuracy of in-situ (surface) radiometric assaying, for example, disequilibrium, geometry, the physico-chemical characteristics of the calibration source, the presence of thorium and potassium and the non-uniform distribution of the source material are also discussed

  7. Development of a heavy metals enzymatic-based assay using papain

    Energy Technology Data Exchange (ETDEWEB)

    Shukor, Yunus [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia)]. E-mail: yunus@biotech.upm.edu.my; Baharom, Nor Azlan [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Rahman, Fadhil Abd. [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Abdullah, Mohd. Puad [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Shamaan, Nor Aripin [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia); Syed, Mohd. Arif [Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia, 43400 Serdang, Selangor (Malaysia)

    2006-05-04

    A heavy metals enzymatic-based assay using papain was developed. Papain was assayed using the Casein-coomassie-dye-binding assay. The assay is sensitive to several heavy metals. The IC{sub 50} (concentration of toxicant giving 50% inhibition) of Hg{sup 2+}, Ag{sup 2+}, Pb{sup 2}, Zn{sup 2+} is 0.39, 0.40, 2.16, 2.11 mg l{sup -1}, respectively. For Cu{sup 2+} and Cd{sup 2+} the LOQ (limits of quantitation) is 0.004 and 0.1 mg l{sup -1}, respectively. The IC{sub 50} and LOQ values were found to be generally comparable to several other enzymatic and bioassays tests such as: immobilized urease, 15-min Microtox{sup TM}, 48 h Daphnia magna, and 96 h Rainbow trout. The papain assay is xenobiotics tolerant, has a wide pH for optimum activity, is temperature stable, and has a relatively quick assay time. The papain assay was used to identify polluted water samples from industrial sources in Penang, Malaysia. We found one site where the assay gave a positive toxic response. The toxicity of the site was confirmed using Atomic Emission Spectrometry analysis.

  8. A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes.

    Science.gov (United States)

    Das, Debasis; Walvoort, Marthe T C; Lukose, Vinita; Imperiali, Barbara

    2016-01-01

    Phosphoglycosyltransferases (PGTs) are families of integral membrane proteins with intriguingly diverse architectures. These enzymes function to initiate many important biosynthetic pathways including those leading to peptidoglycan, N-linked glycoproteins and lipopolysaccharide O-antigen. In spite of tremendous efforts, characterization of these enzymes remains a challenge not only due to the inherent difficulties associated with the purification of integral membrane proteins but also due to the limited availability of convenient assays. Current PGT assays include radioactivity-based methods, which rely on liquid-liquid or solid-liquid extractions, multienzyme systems linked to lactate dehydrogenase and NAD(+) generation, and HPLC-based approaches, all of which may suffer from low sensitivity and low throughput. Herein, we present the validation of a new luminescence-based assay (UMP-Glo) for measuring activities of PGT enzymes. This assay measures UMP, the by-product of PGT reactions, in a sensitive and quantitative manner by measuring the luminescence output in a discontinuous coupled assay system. The assay is rapid and robust in nature, and also compatible with microtiter plate formats. Activity and kinetic parameters of PglC, a PGT from Campylobacter jejuni, were quickly established using this assay. The efficacy of the assay was further corroborated using two different PGTs; PglC from Helicobacter pullorum and WecA from Thermatoga maritima. PMID:27624811

  9. ColonyArea: an ImageJ plugin to automatically quantify colony formation in clonogenic assays.

    Directory of Open Access Journals (Sweden)

    Camilo Guzmán

    Full Text Available The clonogenic or colony formation assay is a widely used method to study the number and size of cancer cell colonies that remain after irradiation or cytotoxic agent administration and serves as a measure for the anti-proliferative effect of these treatments. Alternatively, this assay is used to quantitate the transforming potential of cancer associated genes and chemical agents. Therefore, there is a need for a simplified and standardized analysis of colony formation assays for both routine laboratory use and for parallelized automated analysis. Here we describe the freely available ImageJ-plugin "ColonyArea", which is optimized for rapid and quantitative analysis of focus formation assays conducted in 6- to 24-well dishes. ColonyArea processes image data of multi-well dishes, by separating, concentrically cropping and background correcting well images individually, before colony formation is quantitated. Instead of counting the number of colonies, ColonyArea determines the percentage of area covered by crystal violet stained cell colonies, also taking the intensity of the staining and therefore cell density into account. We demonstrate that these parameters alone or in combination allow for robust quantification of IC50 values of the cytotoxic effect of two staurosporines, UCN-01 and staurosporine (STS on human glioblastoma cells (T98G. The relation between the potencies of the two compounds compared very well with that obtained from an absorbance based method to quantify colony growth and to published data. The ColonyArea ImageJ plugin provides a simple and efficient analysis routine to quantitate assay data of one of the most commonly used cellular assays. The bundle is freely available for download as supporting information. We expect that ColonyArea will be of broad utility for cancer biologists, as well as clinical radiation scientists.

  10. Quantitative velocity modulation spectroscopy

    Science.gov (United States)

    Hodges, James N.; McCall, Benjamin J.

    2016-05-01

    Velocity Modulation Spectroscopy (VMS) is arguably the most important development in the 20th century for spectroscopic study of molecular ions. For decades, interpretation of VMS lineshapes has presented challenges due to the intrinsic covariance of fit parameters including velocity modulation amplitude, linewidth, and intensity. This limitation has stifled the growth of this technique into the quantitative realm. In this work, we show that subtle changes in the lineshape can be used to help address this complexity. This allows for determination of the linewidth, intensity relative to other transitions, velocity modulation amplitude, and electric field strength in the positive column of a glow discharge. Additionally, we explain the large homogeneous component of the linewidth that has been previously described. Using this component, the ion mobility can be determined.

  11. Quantitative Concept Analysis

    CERN Document Server

    Pavlovic, Dusko

    2012-01-01

    Formal Concept Analysis (FCA) begins from a context, given as a binary relation between some objects and some attributes, and derives a lattice of concepts, where each concept is given as a set of objects and a set of attributes, such that the first set consists of all objects that satisfy all attributes in the second, and vice versa. Many applications, though, provide contexts with quantitative information, telling not just whether an object satisfies an attribute, but also quantifying this satisfaction. Contexts in this form arise as rating matrices in recommender systems, as occurrence matrices in text analysis, as pixel intensity matrices in digital image processing, etc. Such applications have attracted a lot of attention, and several numeric extensions of FCA have been proposed. We propose the framework of proximity sets (proxets), which subsume partially ordered sets (posets) as well as metric spaces. One feature of this approach is that it extracts from quantified contexts quantified concepts, and thu...

  12. Quantitative metamaterial property extraction

    CERN Document Server

    Schurig, David

    2015-01-01

    We examine an extraction model for metamaterials, not previously reported, that gives precise, quantitative and causal representation of S parameter data over a broad frequency range, up to frequencies where the free space wavelength is only a modest factor larger than the unit cell dimension. The model is comprised of superposed, slab shaped response regions of finite thickness, one for each observed resonance. The resonance dispersion is Lorentzian and thus strictly causal. This new model is compared with previous models for correctness likelihood, including an appropriate Occam's factor for each fit parameter. We find that this new model is by far the most likely to be correct in a Bayesian analysis of model fits to S parameter simulation data for several classic metamaterial unit cells.

  13. Environmental Technology Verification Report for Abraxis Ecologenia® 17β-Estradiol (E2) Microplate Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits

    Science.gov (United States)

    This verification test was conducted according to procedures specifiedin the Test/QA Planfor Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kis for the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples. Deviations to the...

  14. Detection of PCV2 DNA by SYBR Green I-based quantitative PCR

    Institute of Scientific and Technical Information of China (English)

    YANG Zong-zhao; HABIB Mudasser; SHUAI Jiang-bing; FANG Wei-huan

    2007-01-01

    We developed an assay for the detection and quantitation ofporcine circovirus type 2 (PCV2) with the SYBR Green I-based real-time PCR. The real-time PCR provides a broad dynamic range, detecting from 103 to 1011 copies of DNA per reaction.No cross-reactions were found in specimens containing PCV1. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, real-time PCR can be used as a routine assay for the clinical diagnosis of PCV2 infection. In this study we applied real-time PCR assay to 80 clinical samples, collected from 40 pigs with postweaning multisystemic wasting syndrome (PMWS) and 40 healthy pigs in comparison with conventional PCR assay. In 56 of 80 samples, PCV2 DNA was detected by conventional PCR assay. All samples positive for PCV2 DNA in conventional PCR assay were also positive in real-time assay, and 12 of 24 samples that tested negative for PCV2 DNA in the conventional assay were tested positive in real-time PCR assay. Real-time PCR assay increased the number of samples in which PCV2 was detected by 15%. It is, therefore, considered to be a useful tool for the detection of PCV2.

  15. Comet assay as a predictive assay for radiosensitivity of two human brain tumor cell lines

    International Nuclear Information System (INIS)

    Micronucleus assay and comet assay were compared as a predictive assay for radiosensitivity of tumors. Two human brain tumor cell lines, Becker (derived from astrocytoma) and ONS76 (derived from medulloblastoma) were used. Colony methods as the gold standard showed ONS76 as radiosensitive and Becker as radioresistant cell lines. Micronucleus assay revealed no different radiosensitivity between them. With comet assay, Becker cells received irradiation showed less damage to the DNA and faster repair of the damage than ONS76 cells did. The results correlate with those from colony methods. Comet assay is simple and rapid method for clinical use and it has an advantage not to establish the primary culture. Moreover, the results of comet assay showed not only DNA damage but also repair from the damage. It is concluded that comet assay is a superior method than micronucleus assay and has a potent candidate for clinical predictive assay. (author)

  16. Application of statistical process control to qualitative molecular diagnostic assays.

    Directory of Open Access Journals (Sweden)

    Cathal P O'brien

    2014-11-01

    Full Text Available Modern pathology laboratories and in particular high throughput laboratories such as clinical chemistry have developed a reliable system for statistical process control. Such a system is absent from the majority of molecular laboratories and where present is confined to quantitative assays. As the inability to apply statistical process control to assay is an obvious disadvantage this study aimed to solve this problem by using a frequency estimate coupled with a confidence interval calculation to detect deviations from an expected mutation frequency. The results of this study demonstrate the strengths and weaknesses of this approach and highlight minimum sample number requirements. Notably, assays with low mutation frequencies and detection of small deviations from an expected value require greater samples with a resultant protracted time to detection. Modelled laboratory data was also used to highlight how this approach might be applied in a routine molecular laboratory. This article is the first to describe the application of statistical process control to qualitative laboratory data.

  17. Optimization of a colorimetric assay for glycosylated human serum albumin

    Energy Technology Data Exchange (ETDEWEB)

    Bohney, J.P.; Feldhoff, R.C.

    1986-05-01

    The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100/sup 0/C. A NaBH/sub 4/ reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with (/sup 3/H)glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation.

  18. Expert system for transuranic waste assay

    International Nuclear Information System (INIS)

    Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs

  19. Highly sensitive, quantitative cell-based assay for prions adsorbed to solid surfaces

    OpenAIRE

    Edgeworth, J. A.; Jackson, G. S.; Clarke, A R; Weissmann, C; Collinge, J.

    2009-01-01

    Prions are comprised principally of aggregates of a misfolded host protein and cause fatal transmissible neurodegenerative disorders of humans and animals, such as variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Prions pose significant public health concerns, including contamination of blood products and surgical instruments; require laborious and often insensitive animal bioassay to detect; and resist conventional hospital sterilization methods. A major experimental a...

  20. Shedding light on microbial predator-prey population dynamics using a quantitative bioluminescence assay.

    Science.gov (United States)

    Im, Hansol; Kim, Dasol; Ghim, Cheol-Min; Mitchell, Robert J

    2014-01-01

    This study assessed the dynamics of predation by Bdellovibrio bacteriovorus HD 100. Predation tests with two different bioluminescent strains of Escherichia coli, one expressing a heat-labile bacterial luciferase and the other a heat-stable form, showed near identical losses from both, indicating that protein expression and stability are not responsible for the "shutting-off" of the prey bioluminescence (BL). Furthermore, it was found that the loss in the prey BL was not proportional with the predator-to-prey ratio (PPR), with significantly greater losses seen as this value was increased. This suggests that other factors also play a role in lowering the prey BL. The loss in BL, however, was very consistent within nine independent experiments to the point that we were able to reliably estimate the predator numbers within only 1 h when present at a PPR of 6 or higher, Using a fluorescent prey, we found that premature lysis of the prey occurs at a significant level and was more prominent as the PPR ratio increased. Based upon the supernatant fluorescent signal, even a relatively low PPR of 10-20 led to approximately 5% of the prey population being prematurely lysed within 1 h, while a PPR of 90 led to nearly 15% lysis. Consequently, we developed a modified Lotka-Volterra predator-prey model that accounted for this lysis and is able to reliably estimate the prey and bdelloplast populations for a wide range of PPRs. PMID:24272279

  1. Detecting cervical cancer by quantitative promoter hypermethylation assay on cervical scrapings : A feasibility study

    NARCIS (Netherlands)

    Reesink-Peters, N; Wisman, G.B.A.; Jeronimo, C; Tokumaru, CY; Cohen, Y; Dong, SM; Klip, HG; Buikema, HJ; Suurmeijer, AJH; Hollema, H; Boezen, HM; Sidransky, D; van der Zee, AGJ

    2004-01-01

    Current morphology-based cervical cancer screening is associated with significant false-positive and false-negative results. Tumor suppressor gene hypermethylation is frequently present in cervical cancer. It is unknown whether a cervical scraping reflects the methylation status of the underlying ep

  2. Quantitative Assay and Evaluation of Uranium Levels in Water Resources of Egypt

    International Nuclear Information System (INIS)

    Quality of water resources are one of the vital components for industrial, agricultural and economical development as well as for security issue. The aim of this study is to evaluate the levels of uranium in the Egyptian water resources used for drinking purposes. Fifty-seven water samples representing Egyptian water resources for drinking water (unpurified, purified, tap and ground) were collected. Sensitive and rapid destructive laser fluorimetry technique was used for uranium determination. The results showed that the level of uranium in most of the collected samples was below than the safe limit recommended by the international organizations. The average annual equivalent dose due to the consumption of water ranged from 0.1 μSv/y to 2.0 μSv/y which is lower than the international recommendation

  3. Identification of Different States of Hepatitis B Virus Infection with a Quantitative PCR Assay

    OpenAIRE

    Kessler, Harald H.; Preininger, Sabine; Stelzl, Evelyn; Daghofer, Elisabeth; Santner, Brigitte I.; Marth, Egon; Lackner, Herwig; Stauber, Rudolf E

    2000-01-01

    The level of hepatitis B virus (HBV) DNA in serum reflects the replicative activity of HBV. To compare serum HBV DNA levels in different states of hepatitis B, 47 sera of patients with HBeAg-positive chronic hepatitis B, 4 sera of patients with HBeAg-negative chronic hepatitis B, 40 samples of patients after HBeAg seroconversion during alpha interferon treatment, 57 sera of inactive HBsAg carriers, and 42 sera of patients who had recovered from chronic hepatitis B more than 12 months prior to...

  4. Development of a Real-Time Quantitative PCR Assay to Detect Cryptosporidium parvum Oocysts in Soil

    Science.gov (United States)

    The risk of Cryptosporidium parvum (C. parvum) contamination is a serious issue with respect to drinking water, as evidenced by the cryptosporidiosis outbreak in Milwaukee WI, in 1993, which involved over 400,000 infections and at least 54 deaths. Ground-water contamination by C...

  5. Quantitative assay of element mass inventories in single cell biological systems with micro-PIXE

    International Nuclear Information System (INIS)

    Elemental concentrations in micro-PIXE (Particle Induced X-ray Emission) maps of elements in biological tissue slices have been determined using auxiliary information on the sample matrix composition from EBS (Elastic Backscattering Spectroscopy) and STIM (Scanning Transmission Ion Microscopy). The thin sample approximation may be used for evaluating micro-PIXE data in cases, where X-ray absorption in the sample can be neglected and the mass of elements in a selected area can be estimated. The resulting sensitivity amounts to an impressive 10−12 g of the selected elements. Two cases are presented as examples. In the first, we determined the total mass of gold nanoparticles internalized by human monocyte-derived dendritic cells (MDDC). In the second, an inventory of the mass of elements in the micro-particulate material adsorbed at the wall of the lorica of the microzooplankton species Tintinnopsis radix has been created

  6. Quantitative microtiter fibronectin fibrillogenesis assay: use in high throughput screening for identification of inhibitor compounds

    OpenAIRE

    Tomasini-Johansson, Bianca R.; Johnson, Ian A.; Hoffmann, F. Michael; Mosher, Deane F.

    2012-01-01

    Fibronectin (FN) is a plasma glycoprotein that circulates in the near micromolar concentration range and is deposited along with locally produced FN in the extracellular matrices of many tissues. Control of FN deposition is tightly controlled by cells. Agents that modulate FN assembly may be useful therapeutically in conditions characterized by excessive FN deposition, such as fibrosis, inflammatory diseases, and malignancies. To identify such agents by high throughput screening (HTS), we dev...

  7. Quantitative assay of element mass inventories in single cell biological systems with micro-PIXE

    Energy Technology Data Exchange (ETDEWEB)

    Ogrinc, Nina [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); LOTRIČ Metrology, Selca 163, SI-4227 Selca (Slovenia); Pelicon, Primož, E-mail: primoz.pelicon@ijs.si [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); Vavpetič, Primož; Kelemen, Mitja; Grlj, Nataša; Jeromel, Luka [Jožef Stefan Institute, Jamova 39, SI-1000 Ljubljana (Slovenia); Tomić, Sergej [Medical Faculty of the Military Medical Academy, University of Defense, Crnotravska 17, Belgrade (Serbia); Čolić, Miodrag [Medical Faculty of the Military Medical Academy, University of Defense, Crnotravska 17, Belgrade (Serbia); Medical Faculty, University of Niš, Boulevard of Dr. Zoran Djindjić 81, 18000 Niš (Serbia); Beran, Alfred [Dipartimento di Oceanografia Biologica, Istituto Nazionale di Oceanografia e Geofisica Sperimentale, Via Auguste Piccard 54, 34151 Trieste (Italy)

    2013-07-01

    Elemental concentrations in micro-PIXE (Particle Induced X-ray Emission) maps of elements in biological tissue slices have been determined using auxiliary information on the sample matrix composition from EBS (Elastic Backscattering Spectroscopy) and STIM (Scanning Transmission Ion Microscopy). The thin sample approximation may be used for evaluating micro-PIXE data in cases, where X-ray absorption in the sample can be neglected and the mass of elements in a selected area can be estimated. The resulting sensitivity amounts to an impressive 10{sup −12} g of the selected elements. Two cases are presented as examples. In the first, we determined the total mass of gold nanoparticles internalized by human monocyte-derived dendritic cells (MDDC). In the second, an inventory of the mass of elements in the micro-particulate material adsorbed at the wall of the lorica of the microzooplankton species Tintinnopsis radix has been created.

  8. A comparison of quantitative and qualitative superoxide dismutase assays for application to low temperature microalgae

    NARCIS (Netherlands)

    Janknegt, P.J.; Rijstenbil, J.W.; van de Poll, W.H.; Gechev, T.S.; Buma, A.G.J.

    2007-01-01

    Antioxidant enzymes such as superoxide dismutase (SOD) play a key role in the removal of reactive oxygen species produced during visible and ultraviolet irradiance stress in microalgae and plants. However, little is known about the enzymatic antioxidative stress responses in ecologically important A

  9. Quantitative Evaluation of Sensitivity and Selectivity of Multiplex NanoSPR Biosensor Assays

    OpenAIRE

    Yu, Chenxu; Irudayaraj, Joseph

    2007-01-01

    A new functionalization procedure was developed to replace cyltrimethylammoniumbromide coating on gold nanorods (GNRs) fabricated through seed-mediated growth with chemically active alkanethiols; antibodies were then attached to the GNRs to yield gold nanorod molecular probes (GNrMPs). The functionalization procedure was shown to minimize nonspecific binding. Multiplex sensing was demonstrated for three targets (goat anti-human IgG, goat anti-rabbit IgG, and goat anti-mouse IgG) through the d...

  10. Fluorometric polyethyleneglycol-peptide hybrid substrates for quantitative assay of protein disulfide isomerase

    DEFF Research Database (Denmark)

    Christiansen, Camilla; St Hilaire, Phaedria M; Winther, Jakob R.

    2004-01-01

    of substrate for PDI where two cysteine-containing oligopeptides are connected by an onameric ethylene glycol linker. We term such hybrid compounds PEGtides. The oligopeptides are each marked with a fluorescent aminobenzoic acid and a quenching nitrotyrosine group, respectively. The reversible formation...

  11. The quantitative real-time PCR applications in the monitoring of marine harmful algal bloom (HAB) species

    OpenAIRE

    Antonella, Penna; Luca, Galluzzi

    2012-01-01

    In the last decade, various molecular methods (e.g., fluorescent hybridization assay, sandwich hybridization assay, automatized biosensor detection, real-time PCR assay) have been developed and implemented for accurate and specific identification and estimation of marine toxic microalgal species. This review focuses on the recent quantitative real-time PCR (qrt-PCR) technology developed for the control and monitoring of the most important taxonomic phytoplankton groups producing biotoxins wit...

  12. Vitellogenin assay by enzyme-linked immunosorbant assay as a biomarker of endocrine disruptor chemicals pollution

    Directory of Open Access Journals (Sweden)

    Ebrahimi M

    2005-01-01

    Full Text Available There is increasing evidence that many xenobiotic chemicals (called as endocrine disruptor chemicals, EDCs through interfering with the endocrine system, have the capability to induce developmental and reproductive abnormalities in humans and animals. The yolk protein precursor vitellogenin (Vtg has proved to be a simple and sensitive biomarker for assessing exposure of fish to EDCs, especially the estrogenic compounds. Work is ongoing to develop screening and testing programmes for endocrine disrupting effects of new chemicals, and in the focus of this development are the fish test species common carp ( Cyprinus carpio . In this study, we have developed quantitative enzyme-linked immunosorbent assays (ELISA for VTS in common carp. The working range of the ELISA was 11.25 - 2000 ng/ml (75 - 25% specific binding/maximum antibody binding [B/B0] with a 50% B/B0 intra and interassay variation of 3.9% ( n = 10 and 12.5% ( n = 30, respectively. This ELISA is capable of detecting Vtg as low as 6 ng/ml, and can accurately detect Vtg in even 10 ml of plasma. The ELISA was applied to measurement of Vtg production by male carp ( C. carpio , Cyprinidae fish exposure to ethynylestradiol. The results showed that the amount of Vtg produced in plasma of exposed fish increased in logarithmic order comparing to the control group and the ELISA described here could be used as an indicator of water pollution to estrogenic pollutants.

  13. Electrokinetically-controlled RNA-DNA hybridization assay for foodborne pathogens

    International Nuclear Information System (INIS)

    We have developed a microfluidic chip for use in an RNA-DNA hybridization assay for foodborne pathogens. Automatic sequential reagent dispensing and washing was realized with a programmable DC voltage sequencer. Signal detection was achieved with a miniaturized optical detection module. Salmonella and Listeria monocytogenes bacteria in different concentrations were quantitatively determined by this RNA-DNA hybridization assay in the microfluidic chip. The detection limit for the Salmonella and Listeria monocytogenes bacteria is 103 to 104 CFU mL-1. The method excels by a significant reduction in the consumption of sample and reagent, and a short assay time. This automatic-operating microfluidic RNA-DNA hybridization assay is promising for on-site pathogen detection. (author)

  14. Comparison of Three Nucleic Acid Amplification Assays of Cerebrospinal Fluid for Diagnosis of Cytomegalovirus Encephalitis

    Science.gov (United States)

    Bestetti, Arabella; Pierotti, Chiara; Terreni, Mariarosa; Zappa, Alessandra; Vago, Luca; Lazzarin, Adriano; Cinque, Paola

    2001-01-01

    The diagnostic reliabilities of three cytomegalovirus (CMV) nucleic acid amplification assays of cerebrospinal fluid (CSF) were compared by using CSF samples from human immunodeficiency virus-infected patients with a postmortem histopathological diagnosis of CMV encephalitis (n = 15) or other central nervous system conditions (n = 16). By using a nested PCR assay, the quantitative COBAS AMPLICOR CMV MONITOR PCR, and the NucliSens CMV pp67 nucleic acid sequence-based amplification assay, sensitivities were 93.3, 86.6, and 93.3%, respectively, and specificities were 93.7, 93.7, and 87.5%, respectively. The COBAS AMPLICOR assay revealed significantly higher CMV DNA levels in patients with diffuse ventriculoencephalitis than in patients with focal periventricular lesions. PMID:11230445

  15. Development and evaluation of real-time PCR assays for bloodmeal identification in Culicoides midges.

    Science.gov (United States)

    VAN DER Saag, M R; Gu, X; Ward, M P; Kirkland, P D

    2016-06-01

    Culicoides (Diptera: Ceratopogonidae) midges are the biological vectors of a number of arboviruses of veterinary importance. However, knowledge relating to the basic biology of some species, including their host-feeding preferences, is limited. Identification of host-feeding preferences in haematophagous insects can help to elucidate the transmission dynamics of the arboviruses they may transmit. In this study, a series of semi-quantitative real-time polymerase chain reaction (qPCR) assays to identify the vertebrate host sources of bloodmeals of Culicoides midges was developed. Two pan-reactive species group and seven species-specific qPCR assays were developed and evaluated. The assays are quick to perform and less expensive than nucleic acid sequencing of bloodmeals. Using these assays, it was possible to rapidly test nearly 700 blood-fed midges of various species from several geographic locations in Australia. PMID:26854008

  16. Programmable Quantitative DNA Nanothermometers.

    Science.gov (United States)

    Gareau, David; Desrosiers, Arnaud; Vallée-Bélisle, Alexis

    2016-07-13

    Developing molecules, switches, probes or nanomaterials that are able to respond to specific temperature changes should prove of utility for several applications in nanotechnology. Here, we describe bioinspired strategies to design DNA thermoswitches with programmable linear response ranges that can provide either a precise ultrasensitive response over a desired, small temperature interval (±0.05 °C) or an extended linear response over a wide temperature range (e.g., from 25 to 90 °C). Using structural modifications or inexpensive DNA stabilizers, we show that we can tune the transition midpoints of DNA thermometers from 30 to 85 °C. Using multimeric switch architectures, we are able to create ultrasensitive thermometers that display large quantitative fluorescence gains within small temperature variation (e.g., > 700% over 10 °C). Using a combination of thermoswitches of different stabilities or a mix of stabilizers of various strengths, we can create extended thermometers that respond linearly up to 50 °C in temperature range. Here, we demonstrate the reversibility, robustness, and efficiency of these programmable DNA thermometers by monitoring temperature change inside individual wells during polymerase chain reactions. We discuss the potential applications of these programmable DNA thermoswitches in various nanotechnology fields including cell imaging, nanofluidics, nanomedecine, nanoelectronics, nanomaterial, and synthetic biology. PMID:27058370

  17. Quantitative Electron Nanodiffraction.

    Energy Technology Data Exchange (ETDEWEB)

    Spence, John [Arizona State Univ., Mesa, AZ (United States)

    2015-01-30

    This Final report summarizes progress under this award for the final reporting period 2002 - 2013 in our development of quantitive electron nanodiffraction to materials problems, especially devoted to atomistic processes in semiconductors and electronic oxides such as the new artificial oxide multilayers, where our microdiffraction is complemented with energy-loss spectroscopy (ELNES) and aberration-corrected STEM imaging (9). The method has also been used to map out the chemical bonds in the important GaN semiconductor (1) used for solid state lighting, and to understand the effects of stacking sequence variations and interfaces in digital oxide superlattices (8). Other projects include the development of a laser-beam Zernike phase plate for cryo-electron microscopy (5) (based on the Kapitza-Dirac effect), work on reconstruction of molecular images using the scattering from many identical molecules lying in random orientations (4), a review article on space-group determination for the International Tables on Crystallography (10), the observation of energy-loss spectra with millivolt energy resolution and sub-nanometer spatial resolution from individual point defects in an alkali halide, a review article for the Centenary of X-ray Diffration (17) and the development of a new method of electron-beam lithography (12). We briefly summarize here the work on GaN, on oxide superlattice ELNES, and on lithography by STEM.

  18. Quantitive DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.

    2008-01-28

    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  19. A lateral electrophoretic flow diagnostic assay

    OpenAIRE

    Lin, R.; Skandarajah, A.; Gerver, RE; Neira, HD; Fletcher, DA; Herr, AE

    2015-01-01

    © 2015 The Royal Society of Chemistry. Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to ...

  20. Validation of the Filovirus Plaque Assay for Use in Preclinical Studies.

    Science.gov (United States)

    Shurtleff, Amy C; Bloomfield, Holly A; Mort, Shannon; Orr, Steven A; Audet, Brian; Whitaker, Thomas; Richards, Michelle J; Bavari, Sina

    2016-04-01

    A plaque assay for quantitating filoviruses in virus stocks, prepared viral challenge inocula and samples from research animals has recently been fully characterized and standardized for use across multiple institutions performing Biosafety Level 4 (BSL-4) studies. After standardization studies were completed, Good Laboratory Practices (GLP)-compliant plaque assay method validation studies to demonstrate suitability for reliable and reproducible measurement of the Marburg Virus Angola (MARV) variant and Ebola Virus Kikwit (EBOV) variant commenced at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). The validation parameters tested included accuracy, precision, linearity, robustness, stability of the virus stocks and system suitability. The MARV and EBOV assays were confirmed to be accurate to ±0.5 log10 PFU/mL. Repeatability precision, intermediate precision and reproducibility precision were sufficient to return viral titers with a coefficient of variation (%CV) of ≤30%, deemed acceptable variation for a cell-based bioassay. Intraclass correlation statistical techniques for the evaluation of the assay's precision when the same plaques were quantitated by two analysts returned values passing the acceptance criteria, indicating high agreement between analysts. The assay was shown to be accurate and specific when run on Nonhuman Primates (NHP) serum and plasma samples diluted in plaque assay medium, with negligible matrix effects. Virus stocks demonstrated stability for freeze-thaw cycles typical of normal usage during assay retests. The results demonstrated that the EBOV and MARV plaque assays are accurate, precise and robust for filovirus titration in samples associated with the performance of GLP animal model studies.

  1. Quantitation of latex allergens.

    Science.gov (United States)

    Palosuo, Timo; Alenius, Harri; Turjanmaa, Kristiina

    2002-05-01

    Minimizing allergen concentration in latex goods to prevent sensitization to natural rubber latex (NRL) and thereby the development of clinical allergy is acknowledged as of mutual interest for rubber manufacturers and regulatory health authorities. However, measuring total protein, the principal currently available method, cannot be deemed a satisfactory regulatory measure to control allergen content. Specific methods based on human IgE-containing reagents, such as radioallergosorbent test (RAST) inhibition, have been available in certain laboratories for demonstrating NRL allergens in rubber products but the methods lack standardization. Currently, one commercial test has become available for measuring individual NRL allergens by capture ELISA-based assays using monoclonal antibodies and purified or recombinant allergens. Such methods are specific, they can be properly standardized, and they are of sufficient sensitivity and reproducibility. Results from medical gloves collected in two national market surveys in Finland in 1995 and 1999, respectively, show that Hev b 6.02 and Hev b 5, the two major allergens for NRL-allergic adults, are the most abundant allergens regularly detectable in high- and moderate-allergen gloves. In addition, Hev b 3 and Hev b 1, the two major allergens for children with spina bifida, are also commonly found. In general, when the sum of the four allergens exceeded 1 microg/g, most NRL-allergic patients showed positive skin prick test reactions against them. Using these new methods assessment of threshold levels that could in due course become guidelines for the rubber industry and regulatory health authorities is becoming possible. Eventually, this progress is expected to lead to a declining incidence of latex allergy. PMID:12079417

  2. LT-HSC Methylcellulose Assay

    Science.gov (United States)

    Kerenyi, Marc A.

    2016-01-01

    Hematopoietic differentiation is a highly complex process originating from an extraordinary population of cells called long-term repopulating hematopoietic stem cells (LT-HSCs). The unique feature of all stem cells, including HSCs, is their exceptional ability to divide asymmetrically giving rise to two different kinds of offspring. One daughter cell becomes an LT-HSC itself (self-renews) to maintain the LT-HSC pool, whereas the second daughter cell pursues a differentiation fate to ultimately give rise to terminally differentiated mature blood cells (Orkin and Zon, 2008). Quantification of phenotypic LT-HSCs can be performed by multi-color flow cytometry and the gold standard for assessment of LT-HSC self-renewal and function is competitive bone marrow transplantation (Miller et al., 2008). Although these methods are irreplaceable to determine LT-HSC abundance and functionality, they have their disadvantages and limitations. For example, competitive bone marrow transplantation is typically monitored as a function of peripheral blood donor contribution over 12–16 weeks. While reduced peripheral blood donor contribution by itself signifies impairment in the stem/progenitor cells compartment, it cannot unambiguously discriminate between reduced LT-HSC self-renewal, impaired LT-HSC differentiation or compromised progenitor cell differentiation. Here we describe an LT-HSCs methylcellulose colony-forming assay, as a fast complementary in vitro method to directly assess LT-HSC differentiation capacity. As described in Kerenyi et al. (2013), this technique acts as a powerful tool to differentiate between LT-HSC or progenitor cell differentiation defects.

  3. Universal and specific quantitative detection of botulinum neurotoxin genes

    Directory of Open Access Journals (Sweden)

    Arnon Stephen S

    2010-10-01

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

  4. Quantitative Literacy: Geosciences and Beyond

    Science.gov (United States)

    Richardson, R. M.; McCallum, W. G.

    2002-12-01

    Quantitative literacy seems like such a natural for the geosciences, right? The field has gone from its origin as a largely descriptive discipline to one where it is hard to imagine failing to bring a full range of mathematical tools to the solution of geological problems. Although there are many definitions of quantitative literacy, we have proposed one that is analogous to the UNESCO definition of conventional literacy: "A quantitatively literate person is one who, with understanding, can both read and represent quantitative information arising in his or her everyday life." Central to this definition is the concept that a curriculum for quantitative literacy must go beyond the basic ability to "read and write" mathematics and develop conceptual understanding. It is also critical that a curriculum for quantitative literacy be engaged with a context, be it everyday life, humanities, geoscience or other sciences, business, engineering, or technology. Thus, our definition works both within and outside the sciences. What role do geoscience faculty have in helping students become quantitatively literate? Is it our role, or that of the mathematicians? How does quantitative literacy vary between different scientific and engineering fields? Or between science and nonscience fields? We will argue that successful quantitative literacy curricula must be an across-the-curriculum responsibility. We will share examples of how quantitative literacy can be developed within a geoscience curriculum, beginning with introductory classes for nonmajors (using the Mauna Loa CO2 data set) through graduate courses in inverse theory (using singular value decomposition). We will highlight six approaches to across-the curriculum efforts from national models: collaboration between mathematics and other faculty; gateway testing; intensive instructional support; workshops for nonmathematics faculty; quantitative reasoning requirement; and individual initiative by nonmathematics faculty.

  5. Deterministic quantitative risk assessment development

    Energy Technology Data Exchange (ETDEWEB)

    Dawson, Jane; Colquhoun, Iain [PII Pipeline Solutions Business of GE Oil and Gas, Cramlington Northumberland (United Kingdom)

    2009-07-01

    Current risk assessment practice in pipeline integrity management is to use a semi-quantitative index-based or model based methodology. This approach has been found to be very flexible and provide useful results for identifying high risk areas and for prioritizing physical integrity assessments. However, as pipeline operators progressively adopt an operating strategy of continual risk reduction with a view to minimizing total expenditures within safety, environmental, and reliability constraints, the need for quantitative assessments of risk levels is becoming evident. Whereas reliability based quantitative risk assessments can be and are routinely carried out on a site-specific basis, they require significant amounts of quantitative data for the results to be meaningful. This need for detailed and reliable data tends to make these methods unwieldy for system-wide risk k assessment applications. This paper describes methods for estimating risk quantitatively through the calibration of semi-quantitative estimates to failure rates for peer pipeline systems. The methods involve the analysis of the failure rate distribution, and techniques for mapping the rate to the distribution of likelihoods available from currently available semi-quantitative programs. By applying point value probabilities to the failure rates, deterministic quantitative risk assessment (QRA) provides greater rigor and objectivity than can usually be achieved through the implementation of semi-quantitative risk assessment results. The method permits a fully quantitative approach or a mixture of QRA and semi-QRA to suit the operator's data availability and quality, and analysis needs. For example, consequence analysis can be quantitative or can address qualitative ranges for consequence categories. Likewise, failure likelihoods can be output as classical probabilities or as expected failure frequencies as required. (author)

  6. Quantitative Luminescence Imaging System

    International Nuclear Information System (INIS)

    The goal of the MEASUREMENT OF CHEMILUMINESCENCE project is to develop and deliver a suite of imaging radiometric instruments for measuring spatial distributions of chemiluminescence. Envisioned deliverables include instruments working at the microscopic, macroscopic, and life-sized scales. Both laboratory and field portable instruments are envisioned. The project also includes development of phantoms as enclosures for the diazoluminomelanin (DALM) chemiluminescent chemistry. A suite of either phantoms in a variety of typical poses, or phantoms that could be adjusted to a variety of poses, is envisioned. These are to include small mammals (rats), mid-sized mammals (monkeys), and human body parts. A complete human phantom that can be posed is a long-term goal of the development. Taken together, the chemistry and instrumentation provide a means for imaging rf dosimetry based on chemiluminescence induced by the heat resulting from rf energy absorption. The first delivered instrument, the Quantitative Luminescence Imaging System (QLIS), resulted in a patent, and an R ampersand D Magazine 1991 R ampersand D 100 award, recognizing it as one of the 100 most significant technological developments of 1991. The current status of the project is that three systems have been delivered, several related studies have been conducted, two preliminary human hand phantoms have been delivered, system upgrades have been implemented, and calibrations have been maintained. Current development includes sensitivity improvements to the microscope-based system; extension of the large-scale (potentially life-sized targets) system to field portable applications; extension of the 2-D large-scale system to 3-D measurement; imminent delivery of a more refined human hand phantom and a rat phantom; rf, thermal and imaging subsystem integration; and continued calibration and upgrade support

  7. Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution▿ †

    OpenAIRE

    Shanks, Orin C.; Atikovic, Emina; Blackwood, A. Denene; Lu, Jingrang; Noble, Rachel T.; Domingo, Jorge Santo; Seifring, Shawn; Sivaganesan, Mano; Haugland, Richard A.

    2007-01-01

    Accurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 × 106 copies of target DNA, with a coeff...

  8. Comparison of radioimmunoassay and fluorescent polarization immunoassay for quantitative determination of vancomycin concentrations in serum.

    OpenAIRE

    Ackerman, B H; Berg, H G; Strate, R G; Rotschafer, J C

    1983-01-01

    A new fluorescent polarization immunoassay (Abbott Laboratories, Diagnostics Division, North Chicago, Ill.) was compared with a standard radioimmunoassay (American Diagnostics Corp., Newport Beach, Calif.) in 34 patients being treated with vancomycin. A total of 123 serum samples were divided and quantitatively analyzed for vancomycin by both assay methods. The results obtained indicated that the two assay methods are comparable (y = 1.01x - 0.81; r = 0.99).

  9. Quantitative analysis of plasma interleiukin-6 by immunoassay on microchip

    Science.gov (United States)

    Abe, K.; Hashimoto, Y.; Yatsushiro, S.; Yamamura, S.; Tanaka, M.; Ooie, T.; Baba, Y.; Kataoka, M.

    2012-03-01

    Sandwich enzyme-linked immunoassay (ELISA) is one of the most frequently employed assays for clinical diagnosis, since this enables the investigator to identify specific protein biomarkers. However, the conventional assay using a 96-well microtitration plate is time- and sample-consuming, and therefore is not suitable for rapid diagnosis. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. We employed the piezoelectric inkjet printing for deposition and fixation of 1st antibody on the microchannnel surface (300 μm width and 100 μm depth). Model analyte was interleukin-6 (IL-6) which was one of the inflammatory cytokine. After blocking the microchannel, antigen, biotin-labeled 2nd antibody, and avidin-labeled peroxidase were infused into the microchannel and incubated for 20 min, 10 min, and 5 min, respectively. This assay could detect 2 pg/ml and quantitatively measure the range of 0-32 pg/ml. Liner regression analysis of plasma IL-6 concentration obtained by microchip and conventional methods exhibited a significant relationship (R2 = 0.9964). This assay reduced the time for the antigen-antibody reaction to 1/6, and the consumption of samples and reagents to 1/50 compared with the conventional method. This assay enables us to determine plasma IL-6 with accuracy, high sensitivity, time saving ability, and low consumption of sample and reagents, and thus will be applicable to clinic diagnosis.

  10. A Method for Quantitative Determination of Biofilm Viability

    Directory of Open Access Journals (Sweden)

    Maria Strømme

    2012-06-01

    Full Text Available In this study we present a scheme for quantitative determination of biofilm viability offering significant improvement over existing methods with metabolic assays. Existing metabolic assays for quantifying viable bacteria in biofilms usually utilize calibration curves derived from planktonic bacteria, which can introduce large errors due to significant differences in the metabolic and/or growth rates of biofilm bacteria in the assay media compared to their planktonic counterparts. In the presented method we derive the specific growth rate of Streptococcus mutans bacteria biofilm from a series of metabolic assays using the pH indicator phenol red, and show that this information could be used to more accurately quantify the relative number of viable bacteria in a biofilm. We found that the specific growth rate of S. mutans in biofilm mode of growth was 0.70 h−1, compared to 1.09 h−1 in planktonic growth. This method should be applicable to other bacteria types, as well as other metabolic assays, and, for example, to quantify the effect of antibacterial treatments or the performance of bactericidal implant surfaces.

  11. Characterization of a New Sensitive PCR Assay for Quantification of Viral DNA Isolated from Patients with Hepatitis B Virus Infections▿

    OpenAIRE

    Thibault, Vincent; Pichoud, Christian; Mullen, Carolyn; Rhoads, James; Smith, Jane B.; Bitbol, Alain; Thamm, Sven; Zoulim, Fabien

    2007-01-01

    Sensitive and accurate quantification of hepatitis B virus (HBV) DNA is necessary for monitoring patients with chronic hepatitis receiving antiviral therapy in order to determine treatment response and to adapt therapy in case of inadequate virologic control. The development of quantitative PCR assays has been crucial in meeting these needs. The objective of this study was to compare the performance of a new real-time PCR assay (Abbott RealTime) for HBV DNA with that of three other commercial...

  12. Quantitative Nuclear Medicine. Chapter 17

    International Nuclear Information System (INIS)

    Planar imaging is still used in clinical practice although tomographic imaging (single photon emission computed tomography (SPECT) and positron emission tomography (PET)) is becoming more established. In this chapter, quantitative methods for both imaging techniques are presented. Planar imaging is limited to single photon. For both SPECT and PET, the focus is on the quantitative methods that can be applied to reconstructed images

  13. Mastering R for quantitative finance

    CERN Document Server

    Berlinger, Edina; Badics, Milán; Banai, Ádám; Daróczi, Gergely; Dömötör, Barbara; Gabler, Gergely; Havran, Dániel; Juhász, Péter; Margitai, István; Márkus, Balázs; Medvegyev, Péter; Molnár, Julia; Szucs, Balázs Árpád; Tuza, Ágnes; Vadász, Tamás; Váradi, Kata; Vidovics-Dancs, Ágnes

    2015-01-01

    This book is intended for those who want to learn how to use R's capabilities to build models in quantitative finance at a more advanced level. If you wish to perfectly take up the rhythm of the chapters, you need to be at an intermediate level in quantitative finance and you also need to have a reasonable knowledge of R.

  14. Assessing sediment contamination using six toxicity assays

    Directory of Open Access Journals (Sweden)

    Allen G. BURTON Jr.

    2001-08-01

    Full Text Available An evaluation of sediment toxicity at Lake Orta, Italy was conducted to compare a toxicity test battery of 6 assays and to evaluate the extent of sediment contamination at various sediment depths. Lake Orta received excessive loadings of copper and ammonia during the 1900’s until a large remediation effort was conducted in 1989-90 using lime addition. Since that time, the lake has shown signs of a steady recovery of biological communities. The study results showed acute toxicity still exists in sediments at a depth of 5 cm and greater. Assays that detected the highest levels of toxicity were two whole sediment exposures (7 d using Hyalella azteca and Ceriodaphnia dubia. The MicrotoxR assay using pore water was the third most sensitive assay. The Thamnotox, Rototox, Microtox solid phase, and Seed Germination-Root Elongation (pore and solid phase assays showed occasional to no toxicity. Based on similarity of responses and assay sensitivity, the two most useful assays were the C. dubia (or H. azteca and Microtox pore water. These assays were effective at describing sediment toxicity in a weight-of-evidence approach.

  15. A bioluminescent assay for measuring glucose uptake.

    Science.gov (United States)

    Valley, Michael P; Karassina, Natasha; Aoyama, Natsuyo; Carlson, Coby; Cali, James J; Vidugiriene, Jolanta

    2016-07-15

    Identifying activators and inhibitors of glucose uptake is critical for both diabetes management and anticancer therapy. To facilitate such studies, easy-to-use nonradioactive assays are desired. Here we describe a bioluminescent glucose uptake assay for measuring glucose transport in cells. The assay is based on the uptake of 2-deoxyglucose and the enzymatic detection of the 2-deoxyglucose-6-phosphate that accumulates. Uptake can be measured from a variety of cell types, it can be inhibited by known glucose transporter inhibitors, and the bioluminescent assay yields similar results when compared with the radioactive method. With HCT 116 cells, glucose uptake can be detected in as little as 5000 cells and remains linear up to 50,000 cells with signal-to-background values ranging from 5 to 45. The assay can be used to screen for glucose transporter inhibitors, or by multiplexing with viability readouts, changes in glucose uptake can be differentiated from overall effects on cell health. The assay also can provide a relevant end point for measuring insulin sensitivity. With adipocytes and myotubes, insulin-dependent increases in glucose uptake have been measured with 10- and 2-fold assay windows, respectively. Significant assay signals of 2-fold or more have also been measured with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and skeletal myoblasts. PMID:27130501

  16. Development of an upconverting chelate assay

    Science.gov (United States)

    Xiao, Xudong; Haushalter, Jeanne P.; Kotz, Kenneth T.; Faris, Gregory W.

    2005-04-01

    We report progress on performing a cell-based assay for the detection of EGFR on cell surfaces by using upconverting chelates. An upconversion microscope has been developed for performing assays and testing optical response. A431 cells are labeled with europium DOTA and imaged using this upconverting microscope.

  17. Radioreceptor assay: theory and applications to pharmacology

    Energy Technology Data Exchange (ETDEWEB)

    Perret, G. (U.E.R. de Medecine, Sante et Biologie Humaine, 93 - Bobigny (France)); Simon, P. (Faculte de Medecine Pitie-Salpetriere, 75 - Paris (France))

    The aim of the first part of this work is to present the theory of the radioreceptor assay and to compare it to the other techniques of radioanalysis (radioimmunoassay, competitive protein binding assays). The technology of the radioreceptor assay is then presented and its components (preparation of the receptors, radioligand, incubation medium) are described. The analytical characteristics of the radioreceptor assay (specificity, sensitivity, reproductibility, accuracy) and the pharmacological significance of the results are discussed. The second part is devoted to the description of the radioreceptor assays of some pharmacological classes (neuroleptics, tricyclic antidepressants, benzodiazepines, ..beta..-blockers, anticholinergic drugs) and to their use in therapeutic drug monitoring. In conclusion, by their nature, radioreceptor assays are highly sensitive, reliable, precise, accurate and simple to perform. Their chief disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand. Paradoxically in some cases, this lack of specificity may be advantageous in that it allows for the detection of not only the apparent compound but of active metabolites and endogenous receptor agonists as well and in that radioreceptors assays can be devised for a whole pharmacological class and not only for one drug as it is the case for classical physico-chemical techniques. For all these reasons future of radioreceptor assay in pharmacology appears promising.

  18. Acellular comet assay: A tool for assessing variables influencing the alkaline comet assay

    International Nuclear Information System (INIS)

    In this study, an acellular modification to the alkaline comet assay to further evaluate key variables within the assay that may influence the outcome of genotoxicity studies is described. This acellular comet assay can detect differences of 0.2 Gy of 60Co gamma-ray radiation between 0 and 1 Gy and differences of 1 Gy between 0 and 8 Gy; thus, this assay is applicable for a wide range of DNA damage levels. It is also shown that DNA damage from different radiation energies was not significantly different from 60Co gamma-ray. This assay displayed a statistical increase in DNA damage due to uncontrolled exposure to natural light; however, the slope of the dose-response curve for light-exposed samples was similar to that for samples protected from light. A comparison of the alkaline comet assay with the acellular comet assay allowed for the intrinsic repair capacity of the alkaline comet assay to be quantified. (authors)

  19. Capillary Electrophoresis Analysis of Conventional Splicing Assays

    DEFF Research Database (Denmark)

    de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida;

    2014-01-01

    Rare sequence variants in "high-risk" disease genes, often referred as unclassified variants (UVs), pose a serious challenge to genetic testing. However, UVs resulting in splicing alterations can be readily assessed by in vitro assays. Unfortunately, analytical and clinical interpretation...... of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...... International Agency for Research on Cancer guidelines), we performed qPCR and/or minigene assays. The latter were performed with a new splicing vector (pSAD) developed by authors of the present manuscript (patent #P201231427 CSIC). We have identified three clinically relevant Class-5 variants (c.682-2A>G, c...

  20. Probabilistic interpretation of radioactive waste assay

    International Nuclear Information System (INIS)

    The non-destructive assay of radioactive wastes from nuclear power plants and fuel cycle facilities is required for assessing the disposal risks. Such assay is influenced by two distinct facts: the statistical uncertainty of the measurement and the spatial uncertainty due to the random or at least unknown spatial distribution of the assayed material in a waste container. In this paper a probabilistic interpretation procedure is presented for a single-detector assay system of nuclear waste by one-shot passive gamma technique. The key parameter for this study, the average escape probability for photons, has been obtained by a specific-purpose Monte Carlo code, MCRW. The code has been developed to simulate the entire pulse height spectral responses for point sources located within the compartment which is the basic idea describing the degree of spatial homogeneity of the nuclear and matrix materials. The methodology presented here can be extended to actual radioactive waste assay