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

PubMed

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

2017-10-23

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

A quantitative assay measuring the function of lipase maturation factor 1

PubMed Central

Yin, Fen; Doolittle, Mark H.; Péterfy, Miklós

2009-01-01

Newly synthesized lipoprotein lipase (LPL) and related members of the lipase gene family require an endoplasmic reticulum maturation factor for attainment of enzyme activity. This factor has been identified as lipase maturation factor 1 (Lmf1), and mutations affecting its function and/or expression result in combined lipase deficiency (cld) and hypertriglyceridemia. To assess the functional impact of Lmf1 sequence variations, both naturally occurring and induced, we report the development of a cell-based assay using LPL activity as a quantitative reporter of Lmf1 function. The assay uses a cell line homozygous for the cld mutation, which renders endogenous Lmf1 nonfunctional. LPL transfected into the mutant cld cell line fails to attain activity; however, cotransfection of LPL with wild-type Lmf1 restores its ability to support normal lipase maturation. In this report, we describe optimized conditions that ensure the detection of a complete range of Lmf1 function (full, partial, or complete loss of function) using LPL activity as the quantitative reporter. To illustrate the dynamic range of the assay, we tested several novel mutations in mouse Lmf1. Our results demonstrate the ability of the assay to detect and analyze Lmf1 mutations having a wide range of effects on Lmf1 function and protein expression. PMID:19471043

Multiplexed MRM-based assays for the quantitation of proteins in mouse plasma and heart tissue.

PubMed

Percy, Andrew J; Michaud, Sarah A; Jardim, Armando; Sinclair, Nicholas J; Zhang, Suping; Mohammed, Yassene; Palmer, Andrea L; Hardie, Darryl B; Yang, Juncong; LeBlanc, Andre M; Borchers, Christoph H

2017-04-01

The mouse is the most commonly used laboratory animal, with more than 14 million mice being used for research each year in North America alone. The number and diversity of mouse models is increasing rapidly through genetic engineering strategies, but detailed characterization of these models is still challenging because most phenotypic information is derived from time-consuming histological and biochemical analyses. To expand the biochemists' toolkit, we generated a set of targeted proteomic assays for mouse plasma and heart tissue, utilizing bottom-up LC/MRM-MS with isotope-labeled peptides as internal standards. Protein quantitation was performed using reverse standard curves, with LC-MS platform and curve performance evaluated by quality control standards. The assays comprising the final panel (101 peptides for 81 proteins in plasma; 227 peptides for 159 proteins in heart tissue) have been rigorously developed under a fit-for-purpose approach and utilize stable-isotope labeled peptides for every analyte to provide high-quality, precise relative quantitation. In addition, the peptides have been tested to be interference-free and the assay is highly multiplexed, with reproducibly determined protein concentrations spanning >4 orders of magnitude. The developed assays have been used in a small pilot study to demonstrate their application to molecular phenotyping or biomarker discovery/verification studies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA Meter: Energy Tunable, Quantitative Hybridization Assay

PubMed Central

Braunlin, William; Völker, Jens; Plum, G. Eric; Breslauer, Kenneth J.

2015-01-01

We describe a novel hybridization assay that employs a unique class of energy tunable, bulge loop-containing competitor strands (C*) that hybridize to a probe strand (P). Such initial “pre-binding” of a probe strand modulates its effective “availability” for hybridizing to a target site (T). More generally, the assay described here is based on competitive binding equilibria for a common probe strand (P) between such tunable competitor strands (C*) and a target strand (T). We demonstrate that loop variable, energy tunable families of C*P complexes exhibit enhanced discrimination between targets and mismatched targets, thereby reducing false positives/negatives. We refer to a C*P complex between a C* competitor single strand and the probe strand as a “tuning fork,” since the C* strand exhibits branch points (forks) at the duplex-bulge interfaces within the complex. By varying the loop to create families of such “tuning forks,” one can construct C*P “energy ladders” capable of resolving small differences within the target that may be of biological/functional consequence. The methodology further allows quantification of target strand concentrations, a determination heretofore not readily available by conventional hybridization assays. The dual ability of this tunable assay to discriminate and quantitate targets provides the basis for developing a technology we refer to as a “DNA Meter.” Here we present data that establish proof-of-principle for an in solution version of such a DNA Meter. We envision future applications of this tunable assay that incorporate surface bound/spatially resolved DNA arrays to yield enhanced discrimination and sensitivity. PMID:23529692

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

PubMed

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

2017-01-01

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

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

PubMed Central

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

2013-01-01

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

A quantitative assay for mitochondrial fusion using Renilla luciferase complementation

PubMed Central

Huang, Huiyan; Choi, Seok-Yong; Frohman, Michael A.

2010-01-01

Mitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Large mitochondria produce energy more efficiently, whereas small mitochondria translocate better to subcellular sites where local production of ATP is acutely required. Mitochondrial fusion is currently assayed by fusing together cells expressing GFP or RFP in their mitochondria and then scoring the frequency of cells with yellow mitochondria (representing fused green and red mitochondria). However, this assay is labor-intensive and only semi-quantitative. We describe here a reporter system consisting of split fragments of Renilla luciferase and YFP fused to mitochondrial matrix-targeting sequences and to leucine zippers to trigger dimerization. The assay enables fusion to be quantitated both visually for individual cells and on a population level using chemiluminescence, laying the foundation for high throughput small molecule and RNAi screens for modulators of mitochondrial fusion. We use the assay to examine cytoskeletal roles in fusion progression. PMID:20488258

A quantitative assay for mitochondrial fusion using Renilla luciferase complementation.

PubMed

Huang, Huiyan; Choi, Seok-Yong; Frohman, Michael A

2010-08-01

Mitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Large mitochondria produce energy more efficiently, whereas small mitochondria translocate better to subcellular sites where local production of ATP is acutely required. Mitochondrial fusion is currently assayed by fusing together cells expressing GFP or RFP in their mitochondria and then scoring the frequency of cells with yellow mitochondria (representing fused green and red mitochondria). However, this assay is labor-intensive and only semi-quantitative. We describe here a reporter system consisting of split fragments of Renilla luciferase and YFP fused to mitochondrial matrix-targeting sequences and to leucine zippers to trigger dimerization. The assay enables fusion to be quantitated both visually for individual cells and on a population level using chemiluminescence, laying the foundation for high throughput small molecule and RNAi screens for modulators of mitochondrial fusion. We use the assay to examine cytoskeletal roles in fusion progression. (c) 2010 Mitochondria Research Society. Published by Elsevier B.V. All rights reserved.

A multiplex branched DNA assay for parallel quantitative gene expression profiling.

PubMed

Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

2006-05-01

We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

Modified telomeric repeat amplification protocol: a quantitative radioactive assay for telomerase without using electrophoresis.

PubMed

Szatmari, I; Tókés, S; Dunn, C B; Bardos, T J; Aradi, J

2000-06-15

A polymerase chain reaction (PCR)-based radioactive telomerase assay was developed in our laboratory which is quantitative and does not require electrophoretic evaluation (designated as TP-TRAP; it utilizes two reverse primers). The main steps of the assay include (1) extension of a 20-mer oligonucleotide substrate (MTS) by telomerase, (2) amplification of the telomerase products in the presence of [(3)H]dTTP using the substrate oligonucleotide and two reverse primers (RPC3, 38 mer; RP, 20 mer), (3) isolation of the amplified radioactive dsDNA by precipitation and filtration, (4) determination of the radioactivity of the acid-insoluble DNA. The length of the telomerase products does not increase on amplification. This valuable feature of the assay is achieved by utilization of the two reverse primers and a highly specific PCR protocol. The assay is linear, accurate, and suitable for cell-biological studies where slight quantitative differences in telomerase activity must be detected. The assay is also suitable for screening and characterization of telomerase inhibitors, as shown with a chemically modified oligonucleotide reverse transcriptase inhibitor [(s(4)dU)(35)]. Copyright 2000 Academic Press.

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-01-01

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

Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

Cancer.gov

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

Proteomics: from hypothesis to quantitative assay on a single platform. Guidelines for developing MRM assays using ion trap mass spectrometers.

PubMed

Han, Bomie; Higgs, Richard E

2008-09-01

High-throughput HPLC-mass spectrometry (HPLC-MS) is routinely used to profile biological samples for potential protein markers of disease, drug efficacy and toxicity. The discovery technology has advanced to the point where translating hypotheses from proteomic profiling studies into clinical use is the bottleneck to realizing the full potential of these approaches. The first step in this translation is the development and analytical validation of a higher throughput assay with improved sensitivity and selectivity relative to typical profiling assays. Multiple reaction monitoring (MRM) assays are an attractive approach for this stage of biomarker development given their improved sensitivity and specificity, the speed at which the assays can be developed and the quantitative nature of the assay. While the profiling assays are performed with ion trap mass spectrometers, MRM assays are traditionally developed in quadrupole-based mass spectrometers. Development of MRM assays from the same instrument used in the profiling analysis enables a seamless and rapid transition from hypothesis generation to validation. This report provides guidelines for rapidly developing an MRM assay using the same mass spectrometry platform used for profiling experiments (typically ion traps) and reviews methodological and analytical validation considerations. The analytical validation guidelines presented are drawn from existing practices on immunological assays and are applicable to any mass spectrometry platform technology.

Analytical validation of quantitative immunohistochemical assays of tumor infiltrating lymphocyte biomarkers.

PubMed

Singh, U; Cui, Y; Dimaano, N; Mehta, S; Pruitt, S K; Yearley, J; Laterza, O F; Juco, J W; Dogdas, B

2018-06-04

Tumor infiltrating lymphocytes (TIL), especially T-cells, have both prognostic and therapeutic applications. The presence of CD8+ effector T-cells and the ratio of CD8+ cells to FOXP3+ regulatory T-cells have been used as biomarkers of disease prognosis to predict response to various immunotherapies. Blocking the interaction between inhibitory receptors on T-cells and their ligands with therapeutic antibodies including atezolizumab, nivolumab, pembrolizumab and tremelimumab increases the immune response against cancer cells and has shown significant improvement in clinical benefits and survival in several different tumor types. The improved clinical outcome is presumed to be associated with a higher tumor infiltration; therefore, it is thought that more accurate methods for measuring the amount of TIL could assist prognosis and predict treatment response. We have developed and validated quantitative immunohistochemistry (IHC) assays for CD3, CD8 and FOXP3 for immunophenotyping T-lymphocytes in tumor tissue. Various types of formalin fixed, paraffin embedded (FFPE) tumor tissues were immunolabeled with anti-CD3, anti-CD8 and anti-FOXP3 antibodies using an IHC autostainer. The tumor area of stained tissues, including the invasive margin of the tumor, was scored by a pathologist (visual scoring) and by computer-based quantitative image analysis. Two image analysis scores were obtained for the staining of each biomarker: the percent positive cells in the tumor area and positive cells/mm 2 tumor area. Comparison of visual vs. image analysis scoring methods using regression analysis showed high correlation and indicated that quantitative image analysis can be used to score the number of positive cells in IHC stained slides. To demonstrate that the IHC assays produce consistent results in normal daily testing, we evaluated the specificity, sensitivity and reproducibility of the IHC assays using both visual and image analysis scoring methods. We found that CD3, CD8 and

Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR.

PubMed

Dalla Valle, L; Toffolo, V; Lamprecht, M; Maltese, C; Bovo, G; Belvedere, P; Colombo, L

2005-10-31

The aim of the present work was to develop two new independent SYBR Green I-based real-time PCR assays for both detection and quantification of betanodavirus, an RNA virus that infects several species of marine teleost fish causing massive mortalities in larvae and juveniles. The assays utilized two pairs of primers targeting highly conserved regions of both the RNA molecules forming the betanodavirus genome: RNA1 encoding the RNA-dependent RNA polymerase (RdRP) and RNA2 encoding the coat protein (CP). The specificity of amplifications was monitored by the melting analysis and agarose gel electrophoresis of the amplified products. The applicability of these assays was confirmed with 21 betanodavirus strains, covering all the four main clades. In addition, a BLAST (NCBI) search with the primer sequences showed no genomic cross-reactivity with other viruses. The new assays were able to quantify concentrations of betanodavirus genes ranging from 10(1) to 10(8) copies per reaction. The intra-assay coefficients of variation (CV) of threshold cycle (Ct) values of the assays were 1.5% and 1.4% for CP and RdRP RNAs, respectively. The inter-assay CVs of Ct values were 2.3% and 2.4% for CP and RdRP RNAs, respectively. Moreover, regression analysis showed a significant correlation (R2>0.97) between genome number, as determined by real-time PCR assays and the corresponding virus titer expressed as TCID50/ml of two different betanodavirus strains propagated in cell culture. The two assays were compared with a previously established one-step RT-PCR assay and with the classical virus isolation test and found to be more sensitive. In conclusion, the developed real-time RT-PCR assays are a reliable, specific and sensitive tool for the quantitative diagnosis of betanodavirus.

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

PubMed Central

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

2015-01-01

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

Quantitative Assay of Pyrazofurin a New Antiviral, Antitumor Antibiotic1

PubMed Central

Westhead, J. E.; Price, H. D.

1974-01-01

Pyrazofurin, a carbon-linked nucleoside, has been previously reported to possess antiviral and antitumor activity. The antagonistic effect of pyrazofurin against Neurospora crassa has been utilized to develop a quantitative assay for the compound. PMID:4275616

A comparison of sperm agglutination and immobilization assays with a quantitative ELISA for anti-sperm antibody in serum.

PubMed

Lynch, D M; Leali, B A; Howe, S E

1986-08-01

An enzyme-linked immunosorbent assay (ELISA) that quantitates antisperm antibody in serum was compared with standard sperm agglutination and immobilization assays with the use of sera from 40 normal and 292 subfertile individuals. Quantitation of the assay was accomplished by standardizing assay parameters, including the incorporation of a standard reference curve, the number of whole target sperm, the optimal dilution of serum, the selection of microtiter plate, and the time and temperatures involved in the adsorption and incubation phases. With this method, the level of antisperm antibody binding to target sperm in 40 normal fertile individuals was found to be 2.3 (+/- 1.1 standard deviation [SD]) fg immunoglobulin (Ig)/sperm. An increased mean level of 7.4 +/- 3.7 fg Ig/sperm was determined in 84 infertile patients with positive agglutination and/or immobilization tests. In 208 individuals with negative agglutination and immobilization tests the mean concentration of antisperm antibody was 2.5 +/- 1.3 fg Ig/sperm. Postvasectomy patients assayed by this method had a mean Ig binding value of 7.1 +/- 2.4 fg Ig/sperm. The infertile group with positive agglutination and/or immobilization tests had a significantly higher mean antisperm antibody level than the normal fertile group, according to the Student's t-test for independent samples (P less than 0.001). This indirect serum-based assay reproducibly quantitates antisperm antibody binding to whole target sperm, suggests the normal and abnormal levels of antisperm antibody, and correlates with standard functional assays.

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

PubMed

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

2017-05-01

Identifying the most efficient oxidation process to achieve maximum removal of a target pollutant compound forms the subject of much research. There exists a need to develop rapid screening tools to support research in this area. In this work we report on the development of a quantitative assay as a means for identifying catalysts capable of decolourising methylene blue through the generation of oxidising species from hydrogen peroxide. Here, a previously described methylene blue test strip method was repurposed as a quantitative, aqueous-based spectrophotometric assay. From amongst a selection of metal salts and metallophthalocyanine complexes, monitoring of the decolourisation of the cationic dye methylene blue (via Fenton-like and non-Fenton oxidation reactions) by the assay identified the following to be suitable oxidation catalysts: CuSO 4 (a Fenton-like catalyst), iron(II)phthalocyanine (a non-Fenton oxidation catalyst), as well as manganese(II) phthalocyanine. The applicability of the method was examined for the removal of bisphenol A (BPA), as measured by HPLC, during parallel oxidation experiments. The order of catalytic activity was identified as FePc > MnPc > CuSO 4 for both BPA and MB. The quantitative MB decolourisation assay may offer a rapid method for screening a wide range of potential catalysts for oxidation processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Rapid 2,2'-bicinchoninic-based xylanase assay compatible with high throughput screening

Treesearch

William R. Kenealy; Thomas W. Jeffries

2003-01-01

High-throughput screening requires simple assays that give reliable quantitative results. A microplate assay was developed for reducing sugar analysis that uses a 2,2'-bicinchoninic-based protein reagent. Endo-1,4-â-D-xylanase activity against oat spelt xylan was detected at activities of 0.002 to 0.011 IU ml−1. The assay is linear for sugar...

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

NASA Technical Reports Server (NTRS)

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

2002-01-01

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

Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator.

PubMed

Thiessen, Lindsey D; Neill, Tara M; Mahaffee, Walter F

2018-01-01

Plant pathogen detection systems have been useful tools to monitor inoculum presence and initiate management schedules. More recently, a loop-mediated isothermal amplification (LAMP) assay was successfully designed for field use in the grape powdery mildew pathosystem; however, false negatives or false positives were prevalent in grower-conducted assays due to the difficulty in perceiving the magnesium pyrophosphate precipitate at low DNA concentrations. A quantitative LAMP (qLAMP) assay using a fluorescence resonance energy transfer-based probe was assessed by grape growers in the Willamette Valley of Oregon. Custom impaction spore samplers were placed at a research vineyard and six commercial vineyard locations, and were tested bi-weekly by the lab and by growers. Grower-conducted qLAMP assays used a beta-version of the Smart-DART handheld LAMP reaction devices (Diagenetix, Inc., Honolulu, HI, USA), connected to Android 4.4 enabled, Bluetooth-capable Nexus 7 tablets for output. Quantification by a quantitative PCR assay was assumed correct to compare the lab and grower qLAMP assay quantification. Growers were able to conduct and interpret qLAMP results; however, the Erysiphe necator inoculum quantification was unreliable using the beta-Smart-DART devices. The qLAMP assay developed was sensitive to one spore in early testing of the assay, but decreased to >20 spores by the end of the trial. The qLAMP assay is not likely a suitable management tool for grape powdery mildew due to losses in sensitivity and decreasing costs and portability for other, more reliable molecular tools.

A Dual Electrochemical Sensor Based on a Test-strip Assay for the Quantitative Determination of Albumin and Creatinine.

PubMed

Yasukawa, Tomoyuki; Kiba, Yuya; Mizutani, Fumio

2015-01-01

A dual-electrochemical sensor based on a test-strip assay with immunochemistry and enzyme reactions has been developed for the determination of albumin and creatinine. Each nitrocellulose membrane with an immobilization area of an anti-albumin antibody or three enzymes was prepared in the device with three working electrodes for measuring albumin, creatinine, and ascorbic acid, as well as an Ag/AgCl electrode used as a counter/pseudo-reference electrode. The reactions of three enzymes were initiated by flowing a solution containing creatinine to detect an oxidation current of hydrogen peroxide. A sandwich-type immunocomplex was formed by albumin and antibody labeled with glucose oxidase (GOx). Captured GOx catalyzed the reduction of Fe(CN)6(3-) to Fe(CN)6(4-), which was oxidized electrochemically to determine the captured albumin. The responses for creatinine and albumin increased with the concentrations in millimolar order and over the range 18.75 - 150 μg mL(-1), respectively. The present sensor would be a distinct demonstration for producing quantitative dual-assays for various biomolecules used for clinical diagnoses.

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

PubMed

Yang, Yang; Wong, Gary; Ye, Baoguo; Li, Shihua; Li, Shanqin; Zheng, Haixia; Wang, Qiang; Liang, Mifang; Gao, George F; Liu, Lei; Liu, Yingxia; Bi, Yuhai

2017-06-01

The Zika virus (ZIKV) is an arbovirus that has spread rapidly worldwide within recent times. There is accumulating evidence that associates ZIKV infections with Guillain-Barré Syndrome (GBS) and microcephaly in humans. The ZIKV is genetically diverse and can be separated into Asian and African lineages. A rapid, sensitive, and specific assay is needed for the detection of ZIKV across various pandemic regions. So far, the available primers and probes do not cover the genetic diversity and geographic distribution of all ZIKV strains. To this end, we have developed a one-step quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based on conserved sequences in the ZIKV envelope (E) gene. The detection limit of the assay was determined to be five RNA transcript copies and 2.94 × 10 -3 50% tissue culture infectious doses (TCID 50 ) of live ZIKV per reaction. The assay was highly specific and able to detect five different ZIKV strains covering the Asian and African lineages without nonspecific amplification, when tested against other flaviviruses. The assay was also successful in testing for ZIKV in clinical samples. Our assay represents an improvement over the current methods available for the detection ZIKV and would be valuable as a diagnostic tool in various pandemic regions.

Quantitative Validation of the Presto Blue Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System.

PubMed

Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P; Schrooten, Jan Ir

2015-06-01

As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required.

Quantitative Validation of the Presto Blue™ Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System

PubMed Central

Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P.

2015-01-01

As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue™, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required. PMID:25336207

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

PubMed

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

2005-07-01

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

Optimization of a resazurin-based microplate assay for large-scale compound screenings against Klebsiella pneumoniae.

PubMed

Kim, Hyung Jun; Jang, Soojin

2018-01-01

A new resazurin-based assay was evaluated and optimized using a microplate (384-well) format for high-throughput screening of antibacterial molecules against Klebsiella pneumoniae . Growth of the bacteria in 384-well plates was more effectively measured and had a > sixfold higher signal-to-background ratio using the resazurin-based assay compared with absorbance measurements at 600 nm. Determination of minimum inhibitory concentrations of the antibiotics revealed that the optimized assay quantitatively measured antibacterial activity of various antibiotics. An edge effect observed in the initial assay was significantly reduced using a 1-h incubation of the bacteria-containing plates at room temperature. There was an approximately 10% decrease in signal variability between the edge and the middle wells along with improvement in the assay robustness ( Z ' = 0.99). This optimized resazurin-based assay is an efficient, inexpensive, and robust assay that can quantitatively measure antibacterial activity using a high-throughput screening system to assess a large number of compounds for discovery of new antibiotics against K. pneumoniae .

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

PubMed

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

1984-10-01

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

Antigen detection based on background fluorescence quenching immunochromatographic assay.

PubMed

Chen, Xiangjun; Xu, Yangyang; Yu, Jinsheng; Li, Jiutong; Zhou, Xuelei; Wu, Chuanyong; Ji, Qiuliang; Ren, Yuan; Wang, Liqun; Huang, Zhengyi; Zhuang, Hanling; Piao, Long; Head, Richard; Wang, Yajie; Lou, Jiatao

2014-09-02

Gold immunochromatographic assay (GICA) has been around for quite a while, but it is qualitative in the vast majority of applications. A fast, simple and quantitative GICA is in call for better medicine. In the current study, we have established a novel, quantitative GICA based on fluorescence quenching and nitrocellulose membrane background signals, called background fluorescence quenching immunochromatographic assay (bFQICA). Using model analyte alpha-fetoprotein (AFP), the present study assessed the performance of bFQICA in numerous assay aspects. With serial dilutions of the international AFP standard, standard curves for the calculation of AFP concentration were successfully established. At 10 and 100ngmL(-1) of the international AFP standard, the assay variability was defined with a coefficient of variance at 10.4% and 15.2%, respectively. For samples with extended range of AFP levels, bFQICA was able to detect AFP at as low as 1ngmL(-1). Fluorescence in bFQICA strips stayed constant over months. A good correlation between the results from bFQICA and from a well-established Roche electrochemiluminescence immunoassay was observed in 27 serum samples (r=0.98, p<0.001). In conclusion, our study has demonstrated distinctive features of bFQICA over conventional GICA, including utilization of a unique fluorescence ratio between nitrocellulose membrane background and specific signals (F1/F2) to ensure accurate measurements, combined qualitative and quantitative capabilities, and exceptionally high sensitivity for detection of very low levels of antigens. All of these features could make bFQICA attractive as a model for antigen-antibody complex based GICA, and could promote bFQICA to a broad range of applications for investigation of a variety of diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

STANDARDIZATION OF A FLUORESCENT-BASED QUANTITATIVE ADHESION ASSAY TO STUDY ATTACHMENT OF Taenia solium ONCOSPHERE TO EPITHELIAL CELLS In Vitro

PubMed Central

Chile, Nancy; Evangelista, Julio; Gilman, Robert H.; Arana, Yanina; Palma, Sandra; Sterling, Charles R; Garcia, Hector H.; Gonzalez, Armando; Verastegui, Manuela

2012-01-01

To fully understand the preliminary stages of Taenia solium oncosphere attachment in the gut, adequate tools and assays are necessary to observe and quantify this event that leads to infection. A fluorescent-based quantitative adhesion assay, using biotinylated activated-oncospheres and monolayers of Chinese hamster ovary cells (CHO-K1) or human intestinal monolayer cells (INT-407, HCT-8 or HT-29), was developed to study initial events during the infection of target cells and to rapidly quantify the in vitro adhesion of T. solium oncospheres. Fluorescein streptavidin was used to identify biotinylated activated-oncospheres adhered to cells. This adherence was quantified using an automated fluorescence plate reader, and the results were expressed as fluorescence intensity values. A series of three assays were performed. The first was to identify the optimum number of biotinylated activated-oncospheres to be used in the adhesion assay. The goal of the second assay was to validate this novel method with the established oncosphere-binding system using the immunofluorescent-antibody assay (IFA) method to quantify oncosphere adhesion. A total of 10,000 biotinylated activated-oncospheres were utilized to assess the role of sera and laminin (LM) in oncosphere adherence to a CHO-K1 cell monolayer. The findings that sera and LM increase the adhesion of oncospheres to monolayer cells were similar to results that were previously obtained using the IFA method. The third assay compared the adherence of biotinylated activated-oncospheres to different types of human intestinal monolayer cells. In this case, the fluorescence intensity was greatest when using the INT-407 cell monolayer. We believe this new method of quantification offers the potential for rapid, large-scale screening to study and elucidate specific molecules and mechanisms involved in oncosphere-host cell attachment. PMID:22178422

Quantitative genotoxicity assays for analysis of medicinal plants: A systematic review.

PubMed

Sponchiado, Graziela; Adam, Mônica Lucia; Silva, Caroline Dadalt; Soley, Bruna Silva; de Mello-Sampayo, Cristina; Cabrini, Daniela Almeida; Correr, Cassyano Januário; Otuki, Michel Fleith

2016-02-03

Medicinal plants are known to contain numerous biologically active compounds, and although they have proven pharmacological properties, they can cause harm, including DNA damage. Review the literature to evaluate the genotoxicity risk of medicinal plants, explore the genotoxicity assays most used and compare these to the current legal requirements. A quantitative systematic review of the literature, using the keywords "medicinal plants", "genotoxicity" and "mutagenicity", was undertakenQ to identify the types of assays most used to assess genotoxicity, and to evaluate the genotoxicity potential of medicinal plant extracts. The database searches retrieved 2289 records, 458 of which met the inclusion criteria. Evaluation of the selected articles showed a total of 24 different assays used for an assessment of medicinal plant extract genotoxicity. More than a quarter of those studies (28.4%) reported positive results for genotoxicity. This review demonstrates that a range of genotoxicity assay methods are used to evaluate the genotoxicity potential of medicinal plant extracts. The most used methods are those recommended by regulatory agencies. However, based on the current findings, in order to conduct a thorough study concerning the possible genotoxic effects of a medicinal plant, we indicate that it is important always to include bacterial and mammalian tests, with at least one in vivo assay. Also, these tests should be capable of detecting outcomes that include mutation induction, clastogenic and aneugenic effects, and structural chromosome abnormalities. In addition, the considerable rate of positive results detected in this analysis further supports the relevance of assessing the genotoxicity potential of medicinal plants. Copyright © 2016. Published by Elsevier Ireland Ltd.

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

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

Shin, Jong; Phelan, Paul J.; Chhum, Panharith

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 JCVmore » 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.« less

Smartphone based visual and quantitative assays on upconversional paper sensor.

PubMed

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

2016-01-15

The integration of smartphone with paper sensors recently has been gain increasing attentions because of the achievement of quantitative and rapid analysis. However, smartphone based upconversional paper sensors have been restricted by the lack of effective methods to acquire luminescence signals on test paper. Herein, by the virtue of 3D printing technology, we exploited an auxiliary reusable device, which orderly assembled a 980nm mini-laser, optical filter and mini-cavity together, for digitally imaging the luminescence variations on test paper and quantitative analyzing pesticide thiram by smartphone. In detail, copper ions decorated NaYF4:Yb/Tm upconversion nanoparticles were fixed onto filter paper to form test paper, and the blue luminescence on it would be quenched after additions of thiram through luminescence resonance energy transfer mechanism. These variations could be monitored by the smartphone camera, and then the blue channel intensities of obtained colored images were calculated to quantify amounts of thiram through a self-written Android program installed on the smartphone, offering a reliable and accurate detection limit of 0.1μM for the system. This work provides an initial demonstration of integrating upconversion nanosensors with smartphone digital imaging for point-of-care analysis on a paper-based platform. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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

2015-01-01

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

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

PubMed Central

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 (<2 MPN/300 ml) when this 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

[Clinical evaluation of a novel HBsAg quantitative assay].

PubMed

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.

Quantitation of HBV DNA in human serum using a branched DNA (bDNA) signal amplification assay.

PubMed

Hendricks, D A; Stowe, B J; Hoo, B S; Kolberg, J; Irvine, B D; Neuwald, P D; Urdea, M S; Perrillo, R P

1995-11-01

The aim of this study was to establish the performance characteristics of a nonradioisotopic branched DNA (bDNA) signal amplification assay for quantitation of hepatitis B virus (HBV) DNA in human serum. Quantitation was determined from a standard curve and expressed as HBV DNA equivalents/mL (Eq/mL; 285,000 Eq = 1 pg of double stranded HBV DNA). The bDNA assay exhibited a nearly four log dynamic range of quantitation and an analytical detection limit of approximately 100,000 Eq/mL. To ensure a specificity of 99.7%, the quantitation limit was set at 700,000 Eq/mL. The interassay percent coefficient of variance for quantification values ranged from 10% to 15% when performed by novice users with different sets of reagents. Using the bDNA assay, HBV DNA was detected in 94% to 100% of hepatitis B e antigen-positive specimens and 27% to 31% of hepatitis B e antigen-negative specimens from chronic HBV-infected patients. The bDNA assay may be useful as a prognostic and therapy monitoring tool for the management of HBV-infected patients undergoing antiviral treatment.

A rapid fluorescence assay for danofloxacin in beef muscle: effect of muscle type on limit of quantitation.

PubMed

Schneider, Marilyn J

2008-08-01

A simple, rapid fluorescence screening assay was applied to the analysis of beef muscle for danofloxacin at the U.S. tolerance level of 200 ng/g. Muscle samples were homogenized in acetic acid-acetonitrile, the resultant mixture centrifuged, and fluorescence of the supernatants was then measured. The significant difference between the fluorescence of control muscle sample extracts and extracts of samples fortified at 200 ng/g allowed for successful discrimination between the samples. Setting a threshold level at the average 200 ng/g fortified sample extract fluorescence -3sigma allowed for identification of potentially violative samples. Successful analysis of a group of blind fortified samples over a range of concentrations was accomplished in this manner, without any false-negative results. The limits of quantitation for danofloxacin, as well as enrofloxacin, using this assay were determined in three types of beef muscle (hanging tenderloin, neck, and eye round steak), as well as in serum. Significant differences in limits of quantitation were found among the three different muscle types examined, with hanging tenderloin muscle providing the lowest value. This work not only shows the potential for use of the fluorescence screening assay as an alternative to currently used microbial or antibody-based assays for the analysis of danofloxacin in beef muscle, but also suggests that assays using beef muscle may vary in performance depending on the specific muscle selected for analysis.

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

PubMed

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

2015-08-21

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

Printing 2-dimentional droplet array for single-cell reverse transcription quantitative PCR assay with a microfluidic robot.

PubMed

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-04-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis.

Printing 2-Dimentional Droplet Array for Single-Cell Reverse Transcription Quantitative PCR Assay with a Microfluidic Robot

PubMed Central

Zhu, Ying; Zhang, Yun-Xia; Liu, Wen-Wen; Ma, Yan; Fang, Qun; Yao, Bo

2015-01-01

This paper describes a nanoliter droplet array-based single-cell reverse transcription quantitative PCR (RT-qPCR) assay method for quantifying gene expression in individual cells. By sequentially printing nanoliter-scale droplets on microchip using a microfluidic robot, all liquid-handling operations including cell encapsulation, lysis, reverse transcription, and quantitative PCR with real-time fluorescence detection, can be automatically achieved. The inhibition effect of cell suspension buffer on RT-PCR assay was comprehensively studied to achieve high-sensitivity gene quantification. The present system was applied in the quantitative measurement of expression level of mir-122 in single Huh-7 cells. A wide distribution of mir-122 expression in single cells from 3061 copies/cell to 79998 copies/cell was observed, showing a high level of cell heterogeneity. With the advantages of full-automation in liquid-handling, simple system structure, and flexibility in achieving multi-step operations, the present method provides a novel liquid-handling mode for single cell gene expression analysis, and has significant potentials in transcriptional identification and rare cell analysis. PMID:25828383

CPTAC Assay Portal: a repository of targeted proteomic assays

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

Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.

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 investigatorsmore » 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.« less

Effects of Holding Time, Storage, and the Preservation of Samples on Sample Integrity for the Detection of Fecal Indicator Bacteria by Quantitative Polymerase Chain Reaction (qPCR)-based assays.

EPA Science Inventory

The purpose of this project was to answer questions related to storage of samples to be analyzed by the quantitative polymerase chain reaction (qPCR)-based assays for fecal indicator bacteria. The project was divided into two parts. The first part was to determine if filters th...

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

USDA-ARS?s Scientific Manuscript database

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

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

PubMed

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

2007-12-25

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

Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolites.

PubMed

Sakamoto, Seiichi; Putalun, Waraporn; Vimolmangkang, Sornkanok; Phoolcharoen, Waranyoo; Shoyama, Yukihiro; Tanaka, Hiroyuki; Morimoto, Satoshi

2018-01-01

Immunoassays are antibody-based analytical methods for quantitative/qualitative analysis. Since the principle of immunoassays is based on specific antigen-antibody reaction, the assays have been utilized worldwide for diagnosis, pharmacokinetic studies by drug monitoring, and the quality control of commercially available products. Berson and Yalow were the first to develop an immunoassay, known as radioimmunoassay (RIA), for detecting endogenous plasma insulin [1], a development for which Yalow was awarded the Nobel Prize in Physiology or Medicine in 1977. Even today, after half a century, immunoassays are widely utilized with some modifications from the originally proposed system, e.g., radioisotopes have been replaced with enzymes because of safety concerns regarding the use of radioactivity, which is referred to as enzyme immunoassay/enzyme-linked immunosorbent assay (ELISA). In addition, progress has been made in ELISA with the recent advances in recombinant DNA technology, leading to increase in the range of antibodies, probes, and even systems. This review article describes ELISA and its applications for the detection of plant secondary metabolites.

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

EPA Science Inventory

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

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

PubMed Central

Lin, Yunfeng

2015-01-01

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

Impedance-based cellular assays for regenerative medicine.

PubMed

Gamal, W; Wu, H; Underwood, I; Jia, J; Smith, S; Bagnaninchi, P O

2018-07-05

Therapies based on regenerative techniques have the potential to radically improve healthcare in the coming years. As a result, there is an emerging need for non-destructive and label-free technologies to assess the quality of engineered tissues and cell-based products prior to their use in the clinic. In parallel, the emerging regenerative medicine industry that aims to produce stem cells and their progeny on a large scale will benefit from moving away from existing destructive biochemical assays towards data-driven automation and control at the industrial scale. Impedance-based cellular assays (IBCA) have emerged as an alternative approach to study stem-cell properties and cumulative studies, reviewed here, have shown their potential to monitor stem-cell renewal, differentiation and maturation. They offer a novel method to non-destructively assess and quality-control stem-cell cultures. In addition, when combined with in vitro disease models they provide complementary insights as label-free phenotypic assays. IBCA provide quantitative and very sensitive results that can easily be automated and up-scaled in multi-well format. When facing the emerging challenge of real-time monitoring of three-dimensional cell culture dielectric spectroscopy and electrical impedance tomography represent viable alternatives to two-dimensional impedance sensing.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'. © 2018 The Author(s).

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

PubMed

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

2016-02-01

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

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

EPA Science Inventory

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

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

PubMed Central

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

2017-01-01

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

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

PubMed Central

Higa, Futoshi; Kusano, Nobuchika; Tateyama, Masao; Shinzato, Takashi; Arakaki, Noriko; Kawakami, Kazuyoshi; Saito, Atsushi

1998-01-01

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples. PMID:9574712

Quantitative CrAssphage PCR Assays for Human Fecal ...

EPA Pesticide Factsheets

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

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

PubMed

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

2017-12-27

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

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

PubMed

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

2013-03-01

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

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

PubMed

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

2008-07-01

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

An Alu-based, MGB Eclipse real-time PCR method for quantitation of human DNA in forensic samples.

PubMed

Nicklas, Janice A; Buel, Eric

2005-09-01

The forensic community needs quick, reliable methods to quantitate human DNA in crime scene samples to replace the laborious and imprecise slot blot method. A real-time PCR based method has the possibility of allowing development of a faster and more quantitative assay. Alu sequences are primate-specific and are found in many copies in the human genome, making these sequences an excellent target or marker for human DNA. This paper describes the development of a real-time Alu sequence-based assay using MGB Eclipse primers and probes. The advantages of this assay are simplicity, speed, less hands-on-time and automated quantitation, as well as a large dynamic range (128 ng/microL to 0.5 pg/microL).

Quantitative relationship between the local lymph node assay and human skin sensitization assays.

PubMed

Schneider, K; Akkan, Z

2004-06-01

The local lymph node assay (LLNA) is a new test method which allows for the quantitative assessment of sensitizing potency in the mouse. Here, we investigate the quantitative correlation between results from the LLNA and two human sensitization tests--specifically, human repeat insult patch tests (HRIPTs) and human maximization tests (HMTs). Data for 57 substances were evaluated, of which 46 showed skin sensitizing properties in human tests, whereas 11 yielded negative results in humans. For better comparability data from mouse and human tests were transformed to applied doses per skin area, which ranged over four orders of magnitude for the substances considered. Regression analysis for the 46 human sensitizing substances revealed a significant positive correlation between the LLNA and human tests. The correlation was better between LLNA and HRIPT data (n=23; r=0.77) than between LLNA and HMT data (n=38; r=0.65). The observed scattering of data points is related to various uncertainties, in part associated with insufficiencies of data from older HMT studies. Predominantly negative results in the LLNA for another 11 substances which showed no skin sensitizing activity in human maximization tests further corroborate the correspondence between LLNA and human tests. Based on this analysis, the LLNA can be considered a reliable basis for relative potency assessments for skin sensitizers. Proposals are made for the regulatory exploitation of the LLNA: four potency groups can be established, and assignment of substances to these groups according to the outcome of the LLNA can be used to characterize skin sensitizing potency in substance-specific assessments. Moreover, based on these potency groups, a more adequate consideration of sensitizing substances in preparations becomes possible. It is proposed to replace the current single concentration limit for skin sensitizers in preparations, which leads to an all or nothing classification of a preparation as sensitizing to

Evaluation of the Abbott RealTime HCV assay for quantitative detection of hepatitis C virus RNA.

PubMed

Michelin, Birgit D A; Muller, Zsofia; Stelzl, Evelyn; Marth, Egon; Kessler, Harald H

2007-02-01

The Abbott RealTime HCV assay for quantitative detection of HCV RNA has recently been introduced. In this study, the performance of the Abbott RealTime HCV assay was evaluated and compared to the COBAS AmpliPrep/COBAS TaqMan HCV test. Accuracy, linearity, interassay and intra-assay variations were determined, and a total of 243 routine clinical samples were investigated. When accuracy of the new assay was tested, the majority of results were found to be within +/-0.5 log(10) unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve up to 1.0 x 10(6)IU/ml. The interassay variation ranged from 15% to 32%, and the intra-assay variation ranged from 5% to 8%. When clinical samples were tested by the Abbott RealTime HCV assay and the results were compared with those obtained by the COBAS AmpliPrep/COBAS TaqMan HCV test, the results for 93% of all samples with positive results by both tests were found to be within +/-1.0 log(10) unit. The viral loads for all patients measured by the Abbott and Roche assays showed a high correlation (R(2)=0.93); quantitative results obtained by the Abbott assay were found to be lower than those obtained by the Roche assay. The Abbott RealTime HCV assay proved to be suitable for use in the routine diagnostic laboratory. The time to results was similar for both of the assays.

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

PubMed Central

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

2015-01-01

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 AC50 (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

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

PubMed

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

2015-08-01

A main goal of the U.S. Tox21 program is to profile a 10K-compound library for activity against a panel of stress-related and nuclear receptor signaling pathway assays using a quantitative high-throughput screening (qHTS) approach. However, assay artifacts, including nonreproducible signals and assay interference (e.g., autofluorescence), complicate compound activity interpretation. To address these issues, we have developed a data analysis pipeline that includes an updated signal noise-filtering/curation protocol and an assay interference flagging system. To better characterize various types of signals, we adopted a weighted version of the area under the curve (wAUC) to quantify the amount of activity across the tested concentration range in combination with the assay-dependent point-of-departure (POD) concentration. Based on the 32 Tox21 qHTS assays analyzed, we demonstrate that signal profiling using wAUC affords the best reproducibility (Pearson's r = 0.91) in comparison with the POD (0.82) only or the AC(50) (i.e., half-maximal activity concentration, 0.81). Among the activity artifacts characterized, cytotoxicity is the major confounding factor; on average, about 8% of Tox21 compounds are affected, whereas autofluorescence affects less than 0.5%. To facilitate data evaluation, we implemented two graphical user interface applications, allowing users to rapidly evaluate the in vitro activity of Tox21 compounds. © 2015 Society for Laboratory Automation and Screening.

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

PubMed

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

2012-12-04

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

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

PubMed

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

2017-06-01

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

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

EPA Science Inventory

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

Qualitative and quantitative evaluation of a local lymph node assay based on ex vivo interleukin-2 production.

PubMed

Azam, Philippe; Peiffer, Jean-Luc; Ourlin, Jean-Claude; Bonnet, Pierre-Antoine; Tissier, Marie-Hélène; Vian, Laurence; Fabre, Isabelle

2005-01-15

The local lymph node assay (LLNA) is a regular method for the detection of sensitizing chemicals in mice which measures the incorporation of tritiated thymidine in lymph node cells. We have evaluated an alternative to this method based on the interleukin-2 (IL-2) production of lymph node cells. At the mRNA level, no change in the IL-2 gene expression level was detected by real-time PCR analysis. At the protein level, various experimental conditions were checked in order to improve the irritant versus sensitizer discrimination with a restricted set of prototypic compounds. In particular, the use of phytohemagglutinin A (PHA) in an ex vivo cell culture step showed an improvement of both signal and discrimination. In these optimised conditions, a panel of irritants and potency-graded sensitizers was used to assess the performance of the modified method. IFN-gamma production was used as a positive control. For each compound, a dose-response was performed and stimulation indexes (SI) were determined. Effective concentrations (EC) for each sensitizers were then extracted and compared to the literature data of the regular LLNA. The IL-2-based LLNA showed similar performances at both qualitative and quantitative levels compared to regular LLNA.

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

PubMed Central

2014-01-01

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

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

PubMed

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

2013-01-01

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

Comparative evaluation of hepatitis C virus RNA quantitation by branched DNA, NASBA, and monitor assays.

PubMed

Lunel, F; Cresta, P; Vitour, D; Payan, C; Dumont, B; Frangeul, L; Reboul, D; Brault, C; Piette, J C; Huraux, J M

1999-02-01

Several studies have shown a relationship between pretreatment hepatitis C virus (HCV) viral load and the response to interferon (IFN) therapy, creating a need for quantitative HCV-RNA assays. Here, we compared three commercial methods: nucleic acid sequence-based amplification NASBA (Organon), branched DNA 2.0 (bDNA) (Chiron), and Monitor (Roche), with reverse-transcription polymerase chain reaction (RT-PCR) as the reference. We assessed sensitivity and reproducibility on a well-characterized panel of sera (EUROHEP), a Chimp Rodney plasma pool, and samples from IFN-treated and -untreated patients with chronic hepatitis C caused by different HCV genotypes. The reproducibility of the NASBA and bDNA methods was slightly better than that of Monitor, especially for genotypes 2 and 4. NASBA had the highest sensitivity (99% vs. 94% and 88% with Monitor and bDNA, respectively), especially for the follow-up of patients on IFN. NASBA gave the highest HCV-RNA concentrations, which were approximately 10-fold more than with the bDNA assay and 100-fold more than with the Monitor kit. The linearity, tested on the chimp Rodney plasma pool, was better with bDNA for high viral load than with NASBA and Monitor, although for low concentration of HCV RNA, bDNA was negative. Pretreatment viral load was lower in patients who had a sustained virological response to IFN, although the bDNA method was not sensitive enough to quantify all pretreatment samples. This study indicates that gene amplification methods (NASBA or Monitor) have better sensitivity than bDNA assays for quantification of HCV RNA in patients with chronic HCV infection, although the bDNA and NASBA methods are more likely to quantify all genotypes. Prospective studies are needed to demonstrate the usefulness of quantitative assays for the follow-up of patients with chronic hepatitis C.

Evaluation of the Aptima HIV-1 Quant Dx Assay for HIV-1 RNA Quantitation in Different Biological Specimen Types

PubMed Central

Yek, Christina; Massanella, Marta; Peling, Tashi; Lednovich, Kristen; Nair, Sangeetha V.; Worlock, Andrew; Vargas, Milenka; Gianella, Sara; Ellis, Ronald J.; Strain, Matthew C.; Busch, Michael P.; Nugent, C. Thomas

2017-01-01

ABSTRACT The search for a cure for HIV infection has highlighted the need for increasingly sensitive and precise assays to measure viral burden in various tissues and body fluids. We describe the application of a standardized assay for HIV-1 RNA in multiple specimen types. The fully automated Aptima HIV-1 Quant Dx assay (Aptima assay) is FDA cleared for blood plasma HIV-1 RNA quantitation. In this study, the Aptima assay was applied for the quantitation of HIV RNA in peripheral blood mononuclear cells (PBMCs; n = 72), seminal plasma (n = 20), cerebrospinal fluid (CSF; n = 36), dried blood spots (DBS; n = 104), and dried plasma spots (DPS; n = 104). The Aptima assay was equivalent to or better than commercial assays or validated in-house assays for the quantitation of HIV RNA in CSF and seminal plasma. For PBMC specimens, the sensitivity of the Aptima assay in the detection of HIV RNA decayed as background uninfected PBMC counts increased; proteinase K treatment demonstrated some benefit in restoring signal at higher levels of background PBMCs. Finally, the Aptima assay yielded 100% detection rates of DBS in participants with plasma HIV RNA levels of ≥35 copies/ml and 100% detection rates of DPS in participants with plasma HIV RNA levels of ≥394 copies/ml. The Aptima assay can be applied to a variety of specimens from HIV-infected subjects to measure HIV RNA for studies of viral persistence and cure strategies. It can also detect HIV in dried blood and plasma specimens, which may be of benefit in resource-limited settings. PMID:28592548

Evaluation of the Aptima HIV-1 Quant Dx Assay for HIV-1 RNA Quantitation in Different Biological Specimen Types.

PubMed

Yek, Christina; Massanella, Marta; Peling, Tashi; Lednovich, Kristen; Nair, Sangeetha V; Worlock, Andrew; Vargas, Milenka; Gianella, Sara; Ellis, Ronald J; Strain, Matthew C; Busch, Michael P; Nugent, C Thomas; Richman, Douglas D

2017-08-01

The search for a cure for HIV infection has highlighted the need for increasingly sensitive and precise assays to measure viral burden in various tissues and body fluids. We describe the application of a standardized assay for HIV-1 RNA in multiple specimen types. The fully automated Aptima HIV-1 Quant Dx assay (Aptima assay) is FDA cleared for blood plasma HIV-1 RNA quantitation. In this study, the Aptima assay was applied for the quantitation of HIV RNA in peripheral blood mononuclear cells (PBMCs; n = 72), seminal plasma ( n = 20), cerebrospinal fluid (CSF; n = 36), dried blood spots (DBS; n = 104), and dried plasma spots (DPS; n = 104). The Aptima assay was equivalent to or better than commercial assays or validated in-house assays for the quantitation of HIV RNA in CSF and seminal plasma. For PBMC specimens, the sensitivity of the Aptima assay in the detection of HIV RNA decayed as background uninfected PBMC counts increased; proteinase K treatment demonstrated some benefit in restoring signal at higher levels of background PBMCs. Finally, the Aptima assay yielded 100% detection rates of DBS in participants with plasma HIV RNA levels of ≥35 copies/ml and 100% detection rates of DPS in participants with plasma HIV RNA levels of ≥394 copies/ml. The Aptima assay can be applied to a variety of specimens from HIV-infected subjects to measure HIV RNA for studies of viral persistence and cure strategies. It can also detect HIV in dried blood and plasma specimens, which may be of benefit in resource-limited settings.

Microplate-based filter paper assay to measure total cellulase activity.

PubMed

Xiao, Zhizhuang; Storms, Reginald; Tsang, Adrian

2004-12-30

The standard filter paper assay (FPA) published by the International Union of Pure and Applied Chemistry (IUPAC) is widely used to determine total cellulase activity. However, the IUPAC method is not suitable for the parallel analyses of large sample numbers. We describe here a microplate-based method for assaying large sample numbers. To achieve this, we reduced the enzymatic reaction volume to 60 microl from the 1.5 ml used in the IUPAC method. The modified 60-microl format FPA can be carried out in 96-well assay plates. Statistical analyses showed that the cellulase activities of commercial cellulases from Trichoderma reesei and Aspergillus species determined with our 60-microl format FPA were not significantly different from the activities measured with the standard FPA. Our results also indicate that the 60-microl format FPA is quantitative and highly reproducible. Moreover, the addition of excess beta-glucosidase increased the sensitivity of the assay by up to 60%. 2004 Wiley Periodicals, Inc.

A Flow Cytometry-Based Assay for Quantifying Non-Plaque Forming Strains of Yellow Fever Virus

PubMed Central

Hammarlund, Erika; Amanna, Ian J.; Dubois, Melissa E.; Barron, Alex; Engelmann, Flora; Messaoudi, Ilhem; Slifka, Mark K.

2012-01-01

Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses. PMID:23028428

A flow cytometry-based assay for quantifying non-plaque forming strains of yellow fever virus.

PubMed

Hammarlund, Erika; Amanna, Ian J; Dubois, Melissa E; Barron, Alex; Engelmann, Flora; Messaoudi, Ilhem; Slifka, Mark K

2012-01-01

Primary clinical isolates of yellow fever virus can be difficult to quantitate by standard in vitro methods because they may not form discernable plaques or induce a measurable cytopathic effect (CPE) on cell monolayers. In our hands, the Dakar strain of yellow fever virus (YFV-Dakar) could not be measured by plaque assay (PA), focus-forming assay (FFA), or by measurement of CPE. For these reasons, we developed a YFV-specific monoclonal antibody (3A8.B6) and used it to optimize a highly sensitive flow cytometry-based tissue culture limiting dilution assay (TC-LDA) to measure levels of infectious virus. The TC-LDA was performed by incubating serial dilutions of virus in replicate wells of C6/36 cells and stained intracellularly for virus with MAb 3A8.B6. Using this approach, we could reproducibly quantitate YFV-Dakar in tissue culture supernatants as well as from the serum of viremic rhesus macaques experimentally infected with YFV-Dakar. Moreover, the TC-LDA approach was >10-fold more sensitive than standard plaque assay for quantitating typical plaque-forming strains of YFV including YFV-17D and YFV-FNV (French neurotropic vaccine). Together, these results indicate that the TC-LDA technique is effective for quantitating both plaque-forming and non-plaque-forming strains of yellow fever virus, and this methodology may be readily adapted for the study and quantitation of other non-plaque-forming viruses.

A sensitive and high throughput TaqMan-based reverse transcription quantitative polymerase chain reaction assay efficiently discriminates ALK rearrangement from overexpression for lung cancer FFPE specimens.

PubMed

Lung, Jrhau; Lin, Yu-Ching; Hung, Ming-Szu; Jiang, Yuan Yuan; Lee, Kuan-Der; Lin, Paul Yann; Tsai, Ying Huang

2016-04-01

ALK fusion gene is an oncogenic driver in lung cancer with low prevalence, which can be ameliorated by crizotinib. Currently, ALK fusion gene can be diagnosed by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), but inconstistnt results between the two methods are encountered regularly. To make the ALK fusion gene screening more efficient and to provide a simple solution to clarify the discrepancy between FISH and IHC results, a sensitive TaqMan-based reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay was established. The 3-plex TaqMan-based RT-qPCR assay was established and performed on 102 archived formalin-fixed, paraffin-embedded (FFPE) NSCLC samples to detect ALK rearrangement and overexpression. Break-apart FISH and automatic immunohistochemistry based ALK assays were performed side by side using tissue microarray. The RT-qPCR was performed successfully for 80 samples and 10 of them showed positive signals. Three out of the 10 qPCR positive cases were further confirmed by FISH and IHC test. Two others were IHC positive and FISH negative, and expressed full-length ALK transcript. The rest were neither FISH nor IHC positive and their ALK expression level was significantly lower than those FISH or IHC positive cases. Our RT-qPCR assay demonstrates that the capability and reliability of ALK detection is comparable to FISH and IHC, but it is more effective at discriminating ALK rearrangement from overexpression. The RT-qPCR assay easily clarifies the discrepancy between FISH and IHC, and can be incorporated into routine ALK screening for lung cancer. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Quantitative analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay and the COBAS AMPLICOR CMV MONITOR PCR test after blood and marrow allogeneic transplantation.

PubMed

Boivin, G; Bélanger, R; Delage, R; Béliveau, C; Demers, C; Goyette, N; Roy, J

2000-12-01

The performance of a commercially available qualitative PCR test for plasma (AMPLICOR CMV Test; Roche Diagnostics) and a quantitative PCR test for plasma and leukocytes (COBAS AMPLICOR CMV MONITOR Test; Roche Diagnostics) was evaluated with samples from 50 blood or marrow allogeneic transplant recipients who received short courses of sequential ganciclovir therapy (2 weeks intravenously followed by 2 weeks orally) based on a positive cytomegalovirus (CMV) pp65 antigenemia (AG) assay. The number of persons with a positive CMV test was significantly higher for leukocyte-based assays (AG, 67.5%; PCR, 62.5%) compared to both quantitative and qualitative PCR tests of plasma (42.5 and 35%, respectively). One person developed CMV disease during the study despite a negative AG assay; in this particular case, all PCR assays were found to be positive 10 days before his death. There was a trend for earlier positivity after transplantation and more rapid negativity after initiation of ganciclovir for the tests performed on leukocytes. The mean number of CMV copies as assessed by PCR was significantly higher in leukocytes than in plasma (P = 0.02). Overall, excellent agreement (kappa coefficient, >0.75) was found only between the two PCR assays (qualitative and quantitative) based on plasma. These results suggest that either the pp65 AG assay or the COBAS AMPLICOR CMV MONITOR Test using leukocytes could be used to safely monitor CMV viremia in related allogeneic blood or marrow transplant recipients. Such a strategy will result in preemptive treatment for about two-thirds of the persons with a relatively low rate (<33%) of secondary viremic episodes following short courses of ganciclovir therapy.

Validation and application of quantitative PCR assays using host-specific Bacteroidales genetic markers for swine fecal pollution tracking.

PubMed

Fan, Lihua; Shuai, Jiangbing; Zeng, Ruoxue; Mo, Hongfei; Wang, Suhua; Zhang, Xiaofeng; He, Yongqiang

2017-12-01

Genome fragment enrichment (GFE) method was applied to identify host-specific bacterial genetic markers that differ among different fecal metagenomes. To enrich for swine-specific DNA fragments, swine fecal DNA composite (n = 34) was challenged against a DNA composite consisting of cow, human, goat, sheep, chicken, duck and goose fecal DNA extracts (n = 83). Bioinformatic analyses of 384 non-redundant swine enriched metagenomic sequences indicated a preponderance of Bacteroidales-like regions predicted to encode metabolism-associated, cellular processes and information storage and processing. After challenged against fecal DNA extracted from different animal sources, four sequences from the clone libraries targeting two Bacteroidales- (genes 1-38 and 3-53), a Clostridia- (gene 2-109) as well as a Bacilli-like sequence (gene 2-95), respectively, showed high specificity to swine feces based on PCR analysis. Host-specificity and host-sensitivity analysis confirmed that oligonucleotide primers and probes capable of annealing to select Bacteroidales-like sequences (1-38 and 3-53) exhibited high specificity (>90%) in quantitative PCR assays with 71 fecal DNAs from non-target animal sources. The two assays also demonstrated broad distributions of corresponding genetic markers (>94% positive) among 72 swine feces. After evaluation with environmental water samples from different areas, swine-targeted assays based on two Bacteroidales-like GFE sequences appear to be suitable quantitative tracing tools for swine fecal pollution. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

Treml, Diana; Venturelli, Gustavo L; Brod, Fábio C A; Faria, Josias C; Arisi, Ana C M

2014-12-10

A genetically modified (GM) common bean event, namely Embrapa 5.1, resistant to the bean golden mosaic virus (BGMV), was approved for commercialization in Brazil. Brazilian regulation for genetically modified organism (GMO) labeling requires that any food containing more than 1% GMO be labeled. The event-specific polymerase chain reaction (PCR) method has been the primary trend for GMO identification and quantitation because of its high specificity based on the flanking sequence. This work reports the development of an event-specific assay, named FGM, for Embrapa 5.1 detection and quantitation by use of SYBR Green or hydrolysis probe. The FGM assay specificity was tested for Embrapa 2.3 event (a noncommercial GM common bean also resistant to BGMV), 46 non-GM common bean varieties, and other crop species including maize, GM maize, soybean, and GM soybean. The FGM assay showed high specificity to detect the Embrapa 5.1 event. Standard curves for the FGM assay presented a mean efficiency of 95% and a limit of detection (LOD) of 100 genome copies in the presence of background DNA. The primers and probe developed are suitable for the detection and quantitation of Embrapa 5.1.

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

PubMed

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

2017-02-24

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

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

PubMed

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

2008-05-15

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

A simple and widely applicable hit validation strategy for protein-protein interaction inhibitors based on a quantitative ligand displacement assay.

PubMed

Sameshima, Tomoya; Miyahisa, Ikuo; Homma, Misaki; Aikawa, Katsuji; Hixon, Mark S; Matsui, Junji

2014-12-15

Identification of inhibitors for protein-protein interactions (PPIs) from high-throughput screening (HTS) is challenging due to the weak affinity of primary hits. We present a hit validation strategy of PPI inhibitors using quantitative ligand displacement assay. From an HTS for Bcl-xL/Mcl-1 inhibitors, we obtained a hit candidate, I1, which potentially forms a reactive Michael acceptor, I2, inhibiting Bcl-xL/Mcl-1 through covalent modification. We confirmed rapid reversible and competitive binding of I1 with a probe peptide, suggesting non-covalent binding. The advantages of our approach over biophysical assays include; simplicity, higher throughput, low protein consumption and universal application to PPIs including insoluble membrane proteins. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

PubMed

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

2016-01-01

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

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

PubMed

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

2015-03-01

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

A gold nanoparticle-based semi-quantitative and quantitative ultrasensitive paper sensor for the detection of twenty mycotoxins

NASA Astrophysics Data System (ADS)

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

Quantitation of hepatitis B virus DNA in plasma using a sensitive cost-effective "in-house" real-time PCR assay.

PubMed

Daniel, Hubert Darius J; Fletcher, John G; Chandy, George M; Abraham, Priya

2009-01-01

Sensitive nucleic acid testing for the detection and accurate quantitation of hepatitis B virus (HBV) is necessary to reduce transmission through blood and blood products and for monitoring patients on antiviral therapy. The aim of this study is to standardize an "in-house" real-time HBV polymerase chain reaction (PCR) for accurate quantitation and screening of HBV. The "in-house" real-time assay was compared with a commercial assay using 30 chronically infected individuals and 70 blood donors who are negative for hepatitis B surface antigen, hepatitis C virus (HCV) antibody and human immunodeficiency virus (HIV) antibody. Further, 30 HBV-genotyped samples were tested to evaluate the "in-house" assay's capacity to detect genotypes prevalent among individuals attending this tertiary care hospital. The lower limit of detection of this "in-house" HBV real-time PCR was assessed against the WHO international standard and found to be 50 IU/mL. The interassay and intra-assay coefficient of variation (CV) of this "in-house" assay ranged from 1.4% to 9.4% and 0.0% to 2.3%, respectively. Virus loads as estimated with this "in-house" HBV real-time assay correlated well with the commercial artus HBV RG PCR assay ( r = 0.95, P < 0.0001). This assay can be used for the detection and accurate quantitation of HBV viral loads in plasma samples. This assay can be employed for the screening of blood donations and can potentially be adapted to a multiplex format for simultaneous detection of HBV, HIV and HCV to reduce the cost of testing in blood banks.

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

PubMed Central

Richards-Kortum, Rebecca

2015-01-01

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

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

PubMed

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

2015-03-30

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

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

PubMed

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

2015-11-17

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

Hessian-based quantitative image analysis of host-pathogen confrontation assays.

PubMed

Cseresnyes, Zoltan; Kraibooj, Kaswara; Figge, Marc Thilo

2018-03-01

Host-fungus interactions have gained a lot of interest in the past few decades, mainly due to an increasing number of fungal infections that are often associated with a high mortality rate in the absence of effective therapies. These interactions can be studied at the genetic level or at the functional level via imaging. Here, we introduce a new image processing method that quantifies the interaction between host cells and fungal invaders, for example, alveolar macrophages and the conidia of Aspergillus fumigatus. The new technique relies on the information content of transmitted light bright field microscopy images, utilizing the Hessian matrix eigenvalues to distinguish between unstained macrophages and the background, as well as between macrophages and fungal conidia. The performance of the new algorithm was measured by comparing the results of our method with that of an alternative approach that was based on fluorescence images from the same dataset. The comparison shows that the new algorithm performs very similarly to the fluorescence-based version. Consequently, the new algorithm is able to segment and characterize unlabeled cells, thus reducing the time and expense that would be spent on the fluorescent labeling in preparation for phagocytosis assays. By extending the proposed method to the label-free segmentation of fungal conidia, we will be able to reduce the need for fluorescence-based imaging even further. Our approach should thus help to minimize the possible side effects of fluorescence labeling on biological functions. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

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

PubMed Central

Te, Shu Harn; Chen, Enid Yingru

2015-01-01

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. PMID:26025892

A Rapid and Quantitative Recombinase Activity Assay

USDA-ARS?s Scientific Manuscript database

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

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

PubMed Central

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

2018-01-01

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

Development of a fluorescent quantitative real-time polymerase chain reaction assay for the detection of Goose parvovirus in vivo

PubMed Central

Yang, Jin-Long; Cheng, An-Chun; Wang, Ming-Shu; Pan, Kang-Cheng; Li, Min; Guo, Yu-Fei; Li, Chuan-Feng; Zhu, De-Kang; Chen, Xiao-Yue

2009-01-01

Background Goose parvovirus (GPV) is a Dependovirus associated with latent infection and mortality in geese. Currently, it severely affects geese production worldwide. The objective of this study was to develop a fluorescent quantitative real-time polymerase chain reaction (PCR) (FQ-PCR) assay for fast and accurate quantification of GPV DNA in infected goslings, which can aid in the understanding of the regular distribution pattern and the nosogenesis of GPV in vivo. Results The detection limit of the assay was 2.8 × 101 standard DNA copies, with a sensitivity of 3 logs higher than that of the conventional gel-based PCR assay targeting the same gene. The real-time PCR was reproducible, as shown by satisfactory low intraassay and interassay coefficients of variation. Conclusion The high sensitivity, specificity, simplicity, and reproducibility of the GPV fluorogenic PCR assay, combined with a high throughput, make this method suitable for a broad spectrum of GPV etiology-related applications. PMID:19754946

Assay Portal | Office of Cancer Clinical Proteomics Research

Cancer.gov

The CPTAC Assay Portal serves as a centralized public repository of "fit-for-purpose," multiplexed quantitative mass spectrometry-based proteomic targeted assays. Targeted proteomic assays eliminate issues that are commonly observed using conventional protein detection systems.

Real-time quantitative reverse transcription-PCR assay for renal cell carcinoma-associated antigen G250.

PubMed

Chuanzhong, Ye; Ming, Guan; Fanglin, Zhang; Haijiao, Chen; Zhen, Lin; Shiping, Chen; YongKang, Zhang

2002-04-01

Gene amplification/expression of G250 is a major event in human renal tumorigenesis. G250-based therapeutic agents and G250-specific gene therapy are under development. These new perspectives call for a sensitive and accurate method to screen G250 alterations in renal cell cancer (RCC) patients and investigate the relationship between G250 mRNA expression and RCC. We developed a quantitative RT-PCR assay for the measurement of G250 mRNA expression using a real-time procedure based on the use of fluorogenic probes and the ABI PRISM 7700 Sequence Detector System. The method has been applied to the measurement of quantitative mRNA level of G250 in 31 cases RCC and 6 normal renal tissues. The dynamic range was 10(3)-10(8). The relationship between Ct and log starting concentration was linear (r=0.99). G250 expression was present in all RCCs with G250 amplification but was absent in normal ones. G250 mRNA expression ranged from 2.9 x 10(3) to 6.5 x 10(7) copy/microg RNA, with a mean value of 3.5 x 10(6) copy/microg RNA. The expression of G250 revealed an inverse correlation to tumor grade. G250 mRNA level did not correlate with the cell types and clinical stages (P>0.05). G250 has the potential to be used as a marker of diagnosis and increasing proliferation in RCC. This new simple, rapid, semi-automated assay was a major alternative to competitive PCR and Northern blot analysis for gene alteration analysis in human tumors and might be a powerful tool for large randomized, prospective cooperative group trials and supporting future G250-based biological and gene therapy approaches.

Comparison of central HER2 testing with quantitative total HER2 expression and HER2 homodimer measurements using a novel proximity-based assay.

PubMed

Huang, Weidong; Reinholz, Monica; Weidler, Jodi; Yolanda, Lie; Paquet, Agnes; Whitcomb, Jeannette; Lingle, Wilma; Jenkins, Robert B; Chen, Beiyun; Larson, Jeffrey S; Tan, Yuping; Sherwood, Thomas; Bates, Michael; Perez, Edith A

2010-08-01

The accuracy and reliability of immunohistochemical analysis and in situ hybridization for the assessment of HER2 status remains a subject of debate. We developed a novel assay (HERmark Breast Cancer Assay, Monogram Biosciences, South San Francisco, CA) that provides precise quantification of total HER2 protein expression (H2T) and HER2 homodimers (H2D) in formalin-fixed, paraffin-embedded tissue specimens. H2T and H2D results of 237 breast cancers were compared with those of immunohistochemical studies and fluorescence in situ hybridization (FISH) centrally performed at the Mayo Clinic, Rochester, MN. H2T described a continuum across a wide dynamic range ( approximately 2.5 log). Excluding the equivocal cases, HERmark showed 98% concordance with immunohistochemical studies for positive and negative assay values. For the 94 immunohistochemically equivocal cases, 67% and 39% concordance values were observed between HERmark and FISH for positive and negative assay values, respectively. Polysomy 17 in the absence of HER2 gene amplification did not result in HER2 overexpression as evaluated quantitatively using the HERmark assay.

A simple competitive RT-PCR assay for quantitation of HIV-1 subtype B and non-B RNA in plasma.

PubMed

Hamatake, Makiko; Nishizawa, Masako; Yamamoto, Naoki; Kato, Shingo; Sugiura, Wataru

2007-06-01

An easy, inexpensive competitive RT-PCR assay for HIV-1 RNA quantitation was constructed. A 138-bp sequence in the HIV-1 gag p24 region was selected as the target and co-amplified with competitor RNA containing an internal 44-bp deletion. Quantitation of serial dilutions of control RNA samples prepared from the LAI isolate demonstrated a good linearity (R(2)=0.991) within the range between 10 and 250 copies/sample. The detection limit of the assay was determined to be 3.8 copies/sample by Probit analysis and corresponded to 110 copies/ml in plasma. The intra-assay CV value was 9.1%, and the inter-assay value was 25.9%. Both were comparable to those obtained with commercially available HIV-1 RNA quantitation kits. The correlation efficient for the results obtained in 47 plasma samples from HIV-1-infected individuals (subtype A in 1, subtype B in 25, subtype C in 4, subtype F in 1, and CRF01 AE in 16) with the competitive RT-PCR and Cobas Amplicor HIV-1 Monitor test v1.5 was 0.956 for subtype B and 0.947 for subtype non-B. The assay devised is a good alternative for monitoring antiretroviral therapy in resource-poor countries.

Comparative cytotoxic and genotoxic potential of 13 drinking water disinfection by-products using a microplate-based cytotoxicity assay and a developed SOS/umu assay.

PubMed

Zhang, Shao-Hui; Miao, Dong-Yue; Tan, Li; Liu, Ai-Lin; Lu, Wen-Qing

2016-01-01

The implications of disinfection by-products (DBPs) present in drinking water are of public health concern because of their potential mutagenic, carcinogenic and other toxic effects on humans. In this study, we selected 13 main DBPs found in drinking water to quantitatively analyse their cytotoxicity and genotoxicity using a microplate-based cytotoxicity assay and a developed SOS/umu assay in Salmonella typhimurium TA1535/pSK1002. With the developed SOS/umu test, eight DBPs: 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-fura3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H]-furanone (MX), dibromoacetonitrile (DBN), iodoacetic acid (IA), bromochloroacetonitrile (BCN), bromoacetic acid (BA), trichloroacetonitrile (TCN), dibromoacetic acid (DBA) and dichloroacetic acid (DCA) were significantly genotoxic to S. typhimurium. Three DBPs: chloroacetic acid (CA), trichloroacetic acid (TCA) and dichloroacetonitrile (DCN) were weakly genotoxic, whereas the remaining DBPs: chloroacetonitrile (CN) and chloral hydrate (CH) were negative. The rank order in decreasing genotoxicity was as follows: MX > DBN > IA > BCN > BA > TCN > DBA > DCA > CA, TCA, DCN > CN, CH. MX was approximately 370 000 times more genotoxic than DCA. In the microplate-based cytotoxicity assay, cytotoxic potencies of the 13 DBPs were compared and ranked in decreasing order as follows: MX > IA > DBN > BCN > BA > TCN > DCN > CA > DCA > DBA > CN > TCA > CH. MX was approximately 19 200 times more cytotoxic than CH. A statistically significant correlation was found between cytotoxicity and genotoxicity of the 13 DBPs in S. typhimurium. Results suggest that microplate-based cytotoxicity assay and the developed SOS/umu assay are feasible tools for analysing the cytotoxicity and genotoxicity of DBPs, particularly for comparing their toxic intensities quantitatively. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e

Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica.

PubMed

Wilbur, Ami E; Ford, Susan E; Gauthier, Julie D; Gomez-Chiarri, Marta

2012-12-27

The continuing challenges to the management of both wild and cultured eastern oyster Crassostrea virginica populations resulting from protozoan parasites has stimulated interest in the development of molecular assays for their detection and quantification. For Haplosporidium nelsoni, the causative agent of multinucleated sphere unknown (MSX) disease, diagnostic evaluations depend extensively on traditional but laborious histological approaches and more recently on rapid and sensitive (but not quantitative) end-point polymerase chain reaction (PCR) assays. Here, we describe the development and application of a quantitative PCR (qPCR) assay for H. nelsoni using an Applied Biosystems TaqMan® assay designed with minor groove binder (MGB) probes. The assay was highly sensitive, detecting as few as 20 copies of cloned target DNA. Histologically evaluated parasite density was significantly correlated with the quantification cycle (Cq), regardless of whether quantification was categorical (r2 = 0.696, p < 0.0001) or quantitative (r2 = 0.797, p < 0.0001). Application in field studies conducted in North Carolina, USA (7 locations), revealed widespread occurrence of the parasite with moderate to high intensities noted in some locations. In Rhode Island, USA, application of the assay on oysters from 2 locations resulted in no positives.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2015-07-01

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

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

PubMed

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

2018-05-02

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

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

PubMed

Te, Shu Harn; Chen, Enid Yingru; Gin, Karina Yew-Hoong

2015-08-01

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

PubMed

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

2014-04-01

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

USGS Publications Warehouse

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.

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

PubMed Central

2013-01-01

Background A rapid, non-invasive, and inexpensive point-of-care (POC) diagnostic for malaria followed by therapeutic intervention would improve the ability to control infection in endemic areas. Methods A semi-nested PCR amplification protocol is described for quantitative detection of Plasmodium falciparum and is compared to a traditional nested PCR. The approach uses primers that target the P. falciparum dihydrofolate reductase gene. Results This study demonstrates that it is possible to perform an uninterrupted, asymmetric, semi-nested PCR assay with reduced assay time to detect P. falciparum without compromising the sensitivity and specificity of the assay using saliva as a testing matrix. Conclusions The development of this PCR allows nucleic acid amplification without the need to transfer amplicon from the first PCR step to a second reaction tube with nested primers, thus reducing both the chance of contamination and the time for analysis to < two hours. Analysis of the PCR amplicon yield was adapted to lateral flow detection using the quantitative up-converting phosphor (UCP) reporter technology. This approach provides a basis for migration of the assay to a POC microfluidic format. In addition the assay was successfully evaluated with oral samples. Oral fluid collection provides a simple non-invasive method to collect clinical samples. PMID:23433252

Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study

USGS Publications Warehouse

Layton, Blythe A.; Cao, Yiping; Ebentier, Darcy L.; Hanley, Kaitlyn; Ballesté, Elisenda; Brandão, João; Byappanahalli, Muruleedhara N.; Converse, Reagan; Farnleitner, Andreas H.; Gentry-Shields, Jennifer; Gourmelon, Michèle; Lee, Chang Soo; Lee, Jiyoung; Lozach, Solen; Madi, Tania; Meijer, Wim G.; Noble, Rachel; Peed, Lindsay; Reischer, Georg H.; Rodrigues, Raquel; Rose, Joan B.; Schriewer, Alexander; Sinigalliano, Chris; Srinivasan, Sangeetha; Stewart, Jill; ,; Laurie, C.; Wang, Dan; Whitman, Richard; Wuertz, Stefan; Jay, Jenny; Holden, Patricia A.; Boehm, Alexandria B.; Shanks, Orin; Griffith, John F.

2013-01-01

A number of PCR-based methods for detecting human fecal material in environmental waters have been developed over the past decade, but these methods have rarely received independent comparative testing in large multi-laboratory studies. Here, we evaluated ten of these methods (BacH, BacHum-UCD, Bacteroides thetaiotaomicron (BtH), BsteriF1, gyrB, HF183 endpoint, HF183 SYBR, HF183 Taqman®, HumM2, and Methanobrevibacter smithii nifH (Mnif)) using 64 blind samples prepared in one laboratory. The blind samples contained either one or two fecal sources from human, wastewater or non-human sources. The assay results were assessed for presence/absence of the human markers and also quantitatively while varying the following: 1) classification of samples that were detected but not quantifiable (DNQ) as positive or negative; 2) reference fecal sample concentration unit of measure (such as culturable indicator bacteria, wet mass, total DNA, etc); and 3) human fecal source type (stool, sewage or septage). Assay performance using presence/absence metrics was found to depend on the classification of DNQ samples. The assays that performed best quantitatively varied based on the fecal concentration unit of measure and laboratory protocol. All methods were consistently more sensitive to human stools compared to sewage or septage in both the presence/absence and quantitative analysis. Overall, HF183 Taqman® was found to be the most effective marker of human fecal contamination in this California-based study.

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

PubMed Central

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

2013-01-01

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

Discordances with HIV-1 RNA quantitative determinations by three commercial assays in Pointe Noire, Republic of Congo.

PubMed

Bruzzone, Bianca; Bisio, Francesca; Caligiuri, Patrizia; Mboungou, Franc A Mayinda; Nigro, Nicola; Sticchi, Laura; Ventura, Agostina; Saladini, Francesco; Zazzi, Maurizio; Icardi, Giancarlo; Viscoli, Claudio

2014-07-01

Accurate HIV-1 RNA quantitation is required to support the scale up of antiretroviral therapy in African countries. Extreme HIV-1 genetic variability in Africa may affect the ability of commercially available assays to detect and quantify HIV-1 RNA accurately. The aim of this study was to compare three real-time PCR assays for quantitation of plasma HIV-1 RNA levels in patients from the Republic of Congo, an area with highly diversified HIV-1 subtypes and recombinants. The Abbott RealTime HIV-1, BioMérieux HIV-1 EasyQ test 1.2 and Cobas AmpliPrep/Cobas TaqMan HIV-1 1.0 were compared for quantitation of HIV-1 RNA in 37 HIV-1 seropositive pregnant women enrolled in the Kento-Mwana project for prevention of mother-to-child transmission in Pointe-Noire, Republic of Congo. The sample panel included a variety of HIV-1 subtypes with as many as 21 (56.8%) putative unique recombinant forms. Qualitative detection of HIV-1 RNA was concordant by all three assays in 33/37 (89.2%) samples. Of the remaining 4 (10.8%) samples, all were positive by Roche, three by Abbott and none by BioMérieux. Differences exceeding 1Log in positive samples were found in 4/31 (12.9%), 10/31 (32.3%) and 5/31 (16.1%) cases between Abbott and BioMérieux, Roche and BioMérieux, and Abbott and Roche, respectively. In this sample panel representative of highly polymorphic HIV-1 in Congo, the agreement among the three assays was moderate in terms of HIV-1 RNA detectability and rather inconsistent in terms of quantitation. Copyright © 2014. Published by Elsevier B.V.

Quantitative Analysis of Energy Metabolic Pathways in MCF-7 Breast Cancer Cells by Selected Reaction Monitoring Assay*

PubMed Central

Drabovich, Andrei P.; Pavlou, Maria P.; Dimitromanolakis, Apostolos; Diamandis, Eleftherios P.

2012-01-01

To investigate the quantitative response of energy metabolic pathways in human MCF-7 breast cancer cells to hypoxia, glucose deprivation, and estradiol stimulation, we developed a targeted proteomics assay for accurate quantification of protein expression in glycolysis/gluconeogenesis, TCA cycle, and pentose phosphate pathways. Cell growth conditions were selected to roughly mimic the exposure of cells in the cancer tissue to the intermittent hypoxia, glucose deprivation, and hormonal stimulation. Targeted proteomics assay allowed for reproducible quantification of 76 proteins in four different growth conditions after 24 and 48 h of perturbation. Differential expression of a number of control and metabolic pathway proteins in response to the change of growth conditions was found. Elevated expression of the majority of glycolytic enzymes was observed in hypoxia. Cancer cells, as opposed to near-normal MCF-10A cells, exhibited significantly increased expression of key energy metabolic pathway enzymes (FBP1, IDH2, and G6PD) that are known to redirect cellular metabolism and increase carbon flux through the pentose phosphate pathway. Our quantitative proteomic protocol is based on a mass spectrometry-compatible acid-labile detergent and is described in detail. Optimized parameters of a multiplex selected reaction monitoring (SRM) assay for 76 proteins, 134 proteotypic peptides, and 401 transitions are included and can be downloaded and used with any SRM-compatible mass spectrometer. The presented workflow is an integrated tool for hypothesis-driven studies of mammalian cells as well as functional studies of proteins, and can greatly complement experimental methods in systems biology, metabolic engineering, and metabolic transformation of cancer cells. PMID:22535206

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

NASA Astrophysics Data System (ADS)

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

2011-05-01

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

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

PubMed

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

2002-03-15

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

Mass Spectrometry-based Assay for High Throughput and High Sensitivity Biomarker Verification

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

Guo, Xuejiang; Tang, Keqi

Searching for disease specific biomarkers has become a major undertaking in the biomedical research field as the effective diagnosis, prognosis and treatment of many complex human diseases are largely determined by the availability and the quality of the biomarkers. A successful biomarker as an indicator to a specific biological or pathological process is usually selected from a large group of candidates by a strict verification and validation process. To be clinically useful, the validated biomarkers must be detectable and quantifiable by the selected testing techniques in their related tissues or body fluids. Due to its easy accessibility, protein biomarkers wouldmore » ideally be identified in blood plasma or serum. However, most disease related protein biomarkers in blood exist at very low concentrations (<1ng/mL) and are “masked” by many none significant species at orders of magnitude higher concentrations. The extreme requirements of measurement sensitivity, dynamic range and specificity make the method development extremely challenging. The current clinical protein biomarker measurement primarily relies on antibody based immunoassays, such as ELISA. Although the technique is sensitive and highly specific, the development of high quality protein antibody is both expensive and time consuming. The limited capability of assay multiplexing also makes the measurement an extremely low throughput one rendering it impractical when hundreds to thousands potential biomarkers need to be quantitatively measured across multiple samples. Mass spectrometry (MS)-based assays have recently shown to be a viable alternative for high throughput and quantitative candidate protein biomarker verification. Among them, the triple quadrupole MS based assay is the most promising one. When it is coupled with liquid chromatography (LC) separation and electrospray ionization (ESI) source, a triple quadrupole mass spectrometer operating in a special selected reaction monitoring (SRM

Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual determinations of ergot contamination.

PubMed

Comte, Alexia; Gräfenhan, Tom; Links, Matthew G; Hemmingsen, Sean M; Dumonceaux, Tim J

2017-01-01

We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes.

Quantitative molecular diagnostic assays of grain washes for Claviceps purpurea are correlated with visual determinations of ergot contamination

PubMed Central

Comte, Alexia; Gräfenhan, Tom; Links, Matthew G.; Hemmingsen, Sean M.

2017-01-01

We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (cpn60). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined cpn60 sequences from field-collected and reference strains of the ergot fungus, Claviceps purpurea. These data allowed us to identify this fungal sequence as deriving from C. purpurea, and suggested that C. purpurea DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of C. purpurea DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of Claviceps DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a cpn60-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes. PMID:28257512

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

PubMed Central

Jamal, Syed M.; Belsham, Graham J.

2015-01-01

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 the poor

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.

PubMed

Jamal, Syed M; Belsham, Graham J

2015-01-01

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 the poor

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.

PubMed

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 < 0.001). Therefore, this improved qPCR method should be a powerful tool for the accurate quantification of H. akashiwo cysts in sediment samples.

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

ERIC Educational Resources Information Center

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

2012-01-01

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

A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

PubMed Central

Wilson, Peter M.; LaBonte, Melissa J.; Russell, Jared; Louie, Stan; Ghobrial, Andrew A.; Ladner, Robert D.

2011-01-01

Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5–3′ exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R2 > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC–MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation. PMID:21576234

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

PubMed Central

Ogrean, Christy; Jackson, Ben; Covino, James

2010-01-01

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

Performance Assessment PCR-Based Assays Targeting Bacteroidales Genetic Markers of Bovine Fecal Pollution▿

PubMed Central

Shanks, Orin C.; White, Karen; Kelty, Catherine A.; Hayes, Sam; Sivaganesan, Mano; Jenkins, Michael; Varma, Manju; Haugland, Richard A.

2010-01-01

There are numerous PCR-based assays available to characterize bovine fecal pollution in ambient waters. The determination of which approaches are most suitable for field applications can be difficult because each assay targets a different gene, in many cases from different microorganisms, leading to variation in assay performance. We describe a performance evaluation of seven end-point PCR and real-time quantitative PCR (qPCR) assays reported to be associated with either ruminant or bovine feces. Each assay was tested against a reference collection of DNA extracts from 247 individual bovine fecal samples representing 11 different populations and 175 fecal DNA extracts from 24 different animal species. Bovine-associated genetic markers were broadly distributed among individual bovine samples ranging from 39 to 93%. Specificity levels of the assays spanned 47.4% to 100%. End-point PCR sensitivity also varied between assays and among different bovine populations. For qPCR assays, the abundance of each host-associated genetic marker was measured within each bovine population and compared to results of a qPCR assay targeting 16S rRNA gene sequences from Bacteroidales. Experiments indicate large discrepancies in the performance of bovine-associated assays across different bovine populations. Variability in assay performance between host populations suggests that the use of bovine microbial source-tracking applications will require a priori characterization at each watershed of interest. PMID:20061457

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

PubMed

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.

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

PubMed

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

2011-12-01

Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples. Copyright © 2011 Elsevier B.V. All rights reserved.

High-throughput quantitative luminescence assay of the growth in planta of Pseudomonas syringae chromosomally tagged with Photorhabdus luminescens luxCDABE.

PubMed

Fan, Jun; Crooks, Casey; Lamb, Chris

2008-01-01

Bioluminescent strains of the Arabidopsis thaliana pathogens Pseudomonas syringae pathovar (pv.) tomato and pv. maculicola were made by insertion of the luxCDABE operon from Photorhabdus luminescens into the P. syringae chromosome under the control of a constitutive promoter. Stable integration of luxCDABE did not affect bacterial fitness, growth in planta or disease outcome. Luminescence accurately and reliably reported bacterial growth in infected Arabidopsis leaves both with a fixed inoculum followed over time and with varying inocula assayed at a single time point. Furthermore, the bioluminescence assay could detect a small (1.3-fold) difference in bacterial growth between different plant genotypes with a precision comparable to that of the standard plate assay. Luminescence of luxCDABE-tagged P. syringae allows rapid and convenient quantification of bacterial growth without the tissue extraction, serial dilution, plating and manual scoring involved in standard assays of bacterial growth by colony formation in plate culture of samples from infected tissue. The utility of the bioluminescence assay was illustrated by surveying the 500-fold variation in growth of the universally virulent P. syringae pv. maculicola ES4326 among more than 100 Arabidopsis ecotypes and identification of two quantitative trait loci accounting for 48% and 16%, respectively, of the variance of basal resistance to P. syringae pv. tomato DC3000 in the Col-0 x Fl-1 F(2) population. Luminescence assay of bacteria chromosomally tagged with luxCDABE should greatly facilitate the genetic dissection of quantitative differences in gene-for-gene, basal and acquired disease resistance and other aspects of plant interactions with bacterial pathogens requiring high-throughput assays or large-scale quantitative screens.

Evaluation of New Quantitative Assays for Diagnosis and Monitoring of Cytomegalovirus Disease in Human Immunodeficiency Virus-Positive Patients

PubMed Central

Pellegrin, Isabelle; Garrigue, Isabelle; Binquet, Christine; Chene, Genevieve; Neau, Didier; Bonot, Pascal; Bonnet, Fabrice; Fleury, Herve; Pellegrin, Jean-Luc

1999-01-01

Cobas Amplicor CMV Monitor (CMM) and Quantiplex CMV bDNA 2.0 (CMV bDNA 2.0), two new standardized and quantitative assays for the detection of cytomegalovirus (CMV) DNA in plasma and peripheral blood leukocytes (PBLs), respectively, were compared to the CMV viremia assay, pp65 antigenemia assay, and the Amplicor CMV test (P-AMP). The CMV loads were measured in 384 samples from 58 human immunodeficiency virus (HIV) type 1-infected, CMV-seropositive subjects, including 13 with symptomatic CMV disease. The assays were highly concordant (agreement, 0.88 to 0.97) except when the CMV load was low. Quantitative results for plasma and PBLs were significantly correlated (Spearman ρ = 0.92). For PBLs, positive results were obtained 125 days before symptomatic CMV disease by CMV bDNA 2.0 and 124 days by pp65 antigenemia assay, whereas they were obtained 46 days before symptomatic CMV disease by CMM and P-AMP. At the time of CMV disease diagnosis, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 97.8, 92.3, and 97.8%, respectively, whereas they were 92.3, 93.3, 80, and 97.8%, respectively, for the pp65 antigenemia assay; 84.6, 100, 100, and 95.7%, respectively, for CMM; and 76.9, 100, 100, and 93.8%, respectively, for P-AMP. Considering the entire follow-up, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 73.3, 52.1, and 97.1%, respectively, whereas they were 100, 55.5, 39.4, and 100%, respectively, for the pp65 antigenemia assay; 92.3, 86.7, 66.7, and 97.5%, respectively, for CMM; and 84.6, 91.1, 73.3, and 95.3%, respectively, for P-AMP. Detection of CMV in plasma is technically easy and, despite its later positivity (i.e., later than in PBLs), can provide enough information sufficiently early so that HIV-infected patients can be effectively treated. In addition, these standardized quantitative assays accurately monitor the efficacy of anti-CMV treatment. PMID:10488165

High-throughput multiplexed T-cell-receptor excision circle quantitative PCR assay with internal controls for detection of severe combined immunodeficiency in population-based newborn screening.

PubMed

Gerstel-Thompson, Jacalyn L; Wilkey, Jonathan F; Baptiste, Jennifer C; Navas, Jennifer S; Pai, Sung-Yun; Pass, Kenneth A; Eaton, Roger B; Comeau, Anne Marie

2010-09-01

Real-time quantitative PCR (qPCR) targeting a specific marker of functional T cells, the T-cell-receptor excision circle (TREC), detects the absence of functional T cells and has a demonstrated clinical validity for detecting severe combined immunodeficiency (SCID) in infants. There is need for a qPCR TREC assay with an internal control to monitor DNA quality and the relative cellular content of the particular dried blood spot punch sampled in each reaction. The utility of the qPCR TREC assay would also be far improved if more tests could be performed on the same newborn screening sample. We approached the multiplexing of qPCR for TREC by attenuating the reaction for the reference gene, with focus on maintaining tight quality assurance for reproducible slopes and for prevention of sample-to-sample cross contamination. Statewide newborn screening for SCID using the multiplexed assay was implemented, and quality-assurance data were recorded. The multiplex qPCR TREC assay showed nearly 100% amplification efficiency for each of the TREC and reference sequences, clinical validity for multiple forms of SCID, and an analytic limit of detection consistent with prevention of contamination. The eluate and residual ghost from a 3.2-mm dried blood spot could be used as source material for multiplexed immunoassays and multiplexed DNA tests (Multiplex Plus), with no disruption to the multiplex TREC qPCR. Population-based SCID newborn screening programs should consider multiplexing for quality assurance purposes. Potential benefits of using Multiplex Plus include the ability to perform multianalyte profiling.

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

PubMed

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

2006-04-28

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

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

PubMed

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.

Precise Quantitation of MicroRNA in a Single Cell with Droplet Digital PCR Based on Ligation Reaction.

PubMed

Tian, Hui; Sun, Yuanyuan; Liu, Chenghui; Duan, Xinrui; Tang, Wei; Li, Zhengping

2016-12-06

MicroRNA (miRNA) analysis in a single cell is extremely important because it allows deep understanding of the exact correlation between the miRNAs and cell functions. Herein, we wish to report a highly sensitive and precisely quantitative assay for miRNA detection based on ligation-based droplet digital polymerase chain reaction (ddPCR), which permits the quantitation of miRNA in a single cell. In this ligation-based ddPCR assay, two target-specific oligonucleotide probes can be simply designed to be complementary to the half-sequence of the target miRNA, respectively, which avoids the sophisticated design of reverse transcription and provides high specificity to discriminate a single-base difference among miRNAs with simple operations. After the miRNA-templated ligation, the ddPCR partitions individual ligated products into a water-in-oil droplet and digitally counts the fluorescence-positive and negative droplets after PCR amplification for quantification of the target molecules, which possesses the power of precise quantitation and robustness to variation in PCR efficiency. By integrating the advantages of the precise quantification of ddPCR and the simplicity of the ligation-based PCR, the proposed method can sensitively measure let-7a miRNA with a detection limit of 20 aM (12 copies per microliter), and even a single-base difference can be discriminated in let-7 family members. More importantly, due to its high selectivity and sensitivity, the proposed method can achieve precise quantitation of miRNAs in single-cell lysate. Therefore, the ligation-based ddPCR assay may serve as a useful tool to exactly reveal the miRNAs' actions in a single cell, which is of great importance for the study of miRNAs' biofunction as well as for the related biomedical studies.

A high-throughput assay for DNA topoisomerases and other enzymes, based on DNA triplex formation.

PubMed

Burrell, Matthew R; Burton, Nicolas P; Maxwell, Anthony

2010-01-01

We have developed a rapid, high-throughput assay for measuring the catalytic activity (DNA supercoiling or relaxation) of topoisomerase enzymes that is also capable of monitoring the activity of other enzymes that alter the topology of DNA. The assay utilises intermolecular triplex formation to resolve supercoiled and relaxed forms of DNA, the principle being the greater efficiency of a negatively supercoiled plasmid to form an intermolecular triplex with an immobilised oligonucleotide than the relaxed form. The assay provides a number of advantages over the standard gel-based methods, including greater speed of analysis, reduced sample handling, better quantitation and improved reliability and accuracy of output data. The assay is performed in microtitre plates and can be adapted to high-throughput screening of libraries of potential inhibitors of topoisomerases including bacterial DNA gyrase.

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

EPA Science Inventory

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

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

PubMed Central

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

2006-01-01

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

Quantitative non-destructive assay of PuBe neutron sources

NASA Astrophysics Data System (ADS)

Lakosi, László; Bagi, János; Nguyen, Cong Tam

2006-02-01

PuBe neutron sources were assayed, using a combination of high resolution γ-spectrometry (HRGS) and neutron correlation technique. In a previous publication [J. Bagi, C. Tam Nguyen, L. Lakosi, Nucl. Instr. and Meth. B 222 (2004) 242] a passive neutron well-counter was reported with 3He tubes embedded in a polyamide (TERRAMID) moderator (lined inside with Cd) surrounding the sources to be measured. Gross and coincidence neutron counting was performed, and the Pu content of the sources was found out from isotope analysis and by adopting specific (α, n) reaction yields of the Pu isotopes and 241Am in Be, based on supplier's information and literature data. The method was further developed and refined. Evaluation algorithm was more precisely worked out. The contribution of secondary (correlated) neutrons to the total neutron output was derived from the coincidence (doubles) count rate and taken into account in assessing the Pu content. A new evaluation of former results was performed. Assay was extended to other PuBe sources, and new results were added. In order to attain higher detection efficiency, a more efficient moderator was also applied, with and without Cd shielding around the assay chamber. Calibration seems possible using neutron measurements only (without γ-spectrometry), based on a correlation between the Pu amount and the coincidence-to-total ratio. It is expected that the method could be used for Pu accountancy and safeguards verification as well as identification and assay of seized, found, or not documented PuBe neutron sources.

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

PubMed

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

2013-12-27

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

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

EPA Science Inventory

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

Distance-Based Tear Lactoferrin Assay on Microfluidic Paper Device Using Interfacial Interactions on Surface-Modified Cellulose.

PubMed

Yamada, Kentaro; Henares, Terence G; Suzuki, Koji; Citterio, Daniel

2015-11-11

"Distance-based" detection motifs on microfluidic paper-based analytical devices (μPADs) allow quantitative analysis without using signal readout instruments in a similar manner to classical analogue thermometers. To realize a cost-effective and calibration-free distance-based assay of lactoferrin in human tear fluid on a μPAD not relying on antibodies or enzymes, we investigated the fluidic mobilities of the target protein and Tb(3+) cations used as the fluorescent detection reagent on surface-modified cellulosic filter papers. Chromatographic elution experiments in a tear-like sample matrix containing electrolytes and proteins revealed a collapse of attractive electrostatic interactions between lactoferrin or Tb(3+) and the cellulosic substrate, which was overcome by the modification of the paper surface with the sulfated polysaccharide ι-carrageenan. The resulting μPAD based on the fluorescence emission distance successfully analyzed 0-4 mg mL(-1) of lactoferrin in complex human tear matrix with a lower limit of detection of 0.1 mg mL(-1) by simple visual inspection. Assay results of 18 human tear samples including ocular disease patients and healthy volunteers showed good correlation to the reference ELISA method with a slope of 0.997 and a regression coefficient of 0.948. The distance-based quantitative signal and the good batch-to-batch fabrication reproducibility relying on printing methods enable quantitative analysis by simply reading out "concentration scale marks" printed on the μPAD without performing any calibration and using any signal readout instrument.

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

PubMed Central

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

2013-01-01

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

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

PubMed

Ghosh, N; Gunti, D; Lukka, H; Reddy, B R; Padmaja, Jyothi; Goel, A K

2015-08-01

Anthrax caused by Bacillus anthracis is primarily a disease of herbivorous animals, although several mammals are vulnerable to it. ELISA is the most widely accepted serodiagnostic assay for large scale surveillance of cutaneous anthrax. The aims of this study were to develop and evaluate a quantitative ELISA for determination of IgG antibodies against B. anthracis protective antigen (PA) in human cutaneous anthrax cases. Quantitative ELISA was developed using the recombinant PA for coating and standard reference serum AVR801 for quantification. A total of 116 human test and control serum samples were used in the study. The assay was evaluated for its precision, accuracy and linearity. The minimum detection limit and lower limit of quantification of the assay for anti-PA IgG were 3.2 and 4 µg/ml, respectively. The serum samples collected from the anthrax infected patients were found to have anti-PA IgG concentrations of 5.2 to 166.3 µg/ml. The intra-assay precision per cent CV within an assay and within an operator ranged from 0.99 to 7.4 per cent and 1.7 to 3.9 per cent, respectively. The accuracy of the assay was high with a per cent error of 6.5 - 24.1 per cent. The described assay was found to be linear between the range of 4 to 80 ng/ml (R [2] = 0.9982; slope = 0.9186; intercept = 0.1108). The results suggested that the developed assay could be a useful tool for quantification of anti-PA IgG response in human after anthrax infection or vaccination.

Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

PubMed

Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen

2015-02-06

Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

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

PubMed

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

2012-06-01

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

Development and validation of a liquid chromatography tandem mass spectrometry assay for the quantitation of a protein therapeutic in cynomolgus monkey serum.

PubMed

Zhao, Yue; Liu, Guowen; Angeles, Aida; Hamuro, Lora L; Trouba, Kevin J; Wang, Bonnie; Pillutla, Renuka C; DeSilva, Binodh S; Arnold, Mark E; Shen, Jim X

2015-04-15

We have developed and fully validated a fast and simple LC-MS/MS assay to quantitate a therapeutic protein BMS-A in cynomolgus monkey serum. Prior to trypsin digestion, a recently reported sample pretreatment method was applied to remove more than 95% of the total serum albumin and denature the proteins in the serum sample. The pretreatment procedure simplified the biological sample prior to digestion, improved digestion efficiency and reproducibility, and did not require reduction and alkylation. The denatured proteins were then digested with trypsin at 60 °C for 30 min and the tryptic peptides were chromatographically separated on an Acquity CSH column (2.1 mm × 50 mm, 1.7 μm) using gradient elution. One surrogate peptide was used for quantitation and another surrogate peptide was selected for confirmation. Two corresponding stable isotope labeled peptides were used to compensate variations during LC-MS detection. The linear analytical range of the assay was 0.50-500 μg/mL. The accuracy (%Dev) was within ± 5.4% and the total assay variation (%CV) was less than 12.0% for sample analysis. The validated method demonstrated good accuracy and precision and the application of the innovative albumin removal sample pretreatment method improved both assay sensitivity and robustness. The assay has been applied to a cynomolgus monkey toxicology study and the serum sample concentration data were in good agreement with data generated using a quantitative ligand-binding assay (LBA). The use of a confirmatory peptide, in addition to the quantitation peptide, ensured the integrity of the drug concentrations measured by the method. Copyright © 2015 Elsevier B.V. All rights reserved.

MRM assay for quantitation of complement components in human blood plasma - a feasibility study on multiple sclerosis.

PubMed

Rezeli, Melinda; Végvári, Akos; Ottervald, Jan; Olsson, Tomas; Laurell, Thomas; Marko-Varga, György

2011-12-10

As a proof-of-principle study, a multiple reaction monitoring (MRM) assay was developed for quantitation of proteotypic peptides, representing seven plasma proteins associated with inflammation (complement components and C-reactive protein). The assay development and the sample analysis were performed on a linear ion trap mass spectrometer. We were able to quantify 5 of the 7 target proteins in depleted plasma digests with reasonable reproducibility over a 2 orders of magnitude linear range (RSD≤25%). The assay panel was utilized for the analysis of a small multiple sclerosis sample cohort with 10 diseased and 8 control patients. Copyright © 2011 Elsevier B.V. All rights reserved.

Quantitative determination of polysulfide in albumins, plasma proteins and biological fluid samples using a novel combined assays approach.

PubMed

Ikeda, Mayumi; Ishima, Yu; Shibata, Akitomo; Chuang, Victor T G; Sawa, Tomohiro; Ihara, Hideshi; Watanabe, Hiroshi; Xian, Ming; Ouchi, Yuya; Shimizu, Taro; Ando, Hidenori; Ukawa, Masami; Ishida, Tatsuhiro; Akaike, Takaaki; Otagiri, Masaki; Maruyama, Toru

2017-05-29

Hydrogen sulfide (H 2 S) signaling involves polysulfide (RSS n SR') formation on various proteins. However, the current lack of sensitive polysulfide detection assays poses methodological challenges for understanding sulfane sulfur homeostasis and signaling. We developed a novel combined assay by modifying Sulfide Antioxidant Buffer (SAOB) to produce an "Elimination Method of Sulfide from Polysulfide" (EMSP) treatment solution that liberates sulfide, followed with methylene blue (MB) sulfide detection assay. The combined EMSP-MB sulfide detection assay performed on low molecular weight sulfur species showed that sulfide was produced from trisulfide compounds such as glutathione trisulfide and diallyl trisulfide, but not from the thiol compounds such as cysteine, cystine and glutathione. In the case of plasma proteins, this novel combined detection assay revealed that approximately 14.7, 1.7, 3.9, 3.7 sulfide mol/mol released from human serum albumin, α 1 -anti-trypsin, α 1 -acid glycoprotein and ovalbumin, respectively, suggesting that serum albumin is a major pool of polysulfide in human blood circulation. Taken together with the results of albumins of different species, the liberated sulfide has a good correlation with cysteine instead of methionine, indicating the site of incorporation of polysulfide is cysteine. With this novel sulfide detention assay, approximately 8,000, 120 and 1100 μM of polysulfide concentrations was quantitated in human healthy plasma, saliva and tear, respectively. Our promising polysulfide specific detection assay can be a very important tool because quantitative determination of polysulfide sheds light on the functional consequence of protein-bound cysteine polysulfide and expands the research area of reactive oxygen to reactive polysulfide species. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel, multiplexed, probe-based quantitative PCR assay for the soybean root- and stem-rot pathogen, Phytophthora sojae, utilizes its transposable element

PubMed Central

Haudenshield, James S.; Song, Jeong Y.; Hartman, Glen L.

2017-01-01

Phytophthora root rot of soybean [Glycine max (L.) Merr.] is caused by the oomycete Phytophthora sojae (Kaufm. & Gerd.). P. sojae has a narrow host range, consisting primarily of soybean, and it is a serious pathogen worldwide. It exists in root and stem tissues as mycelium, wherein it can form oospores which subsequently germinate to release motile, infectious zoospores. Molecular assays detecting DNA of P. sojae are useful in disease diagnostics, and for determining the presence of the organism in host tissues, soils, and runoff or ponded water from potentially infested fields. Such assays as published have utilized ITS sequences from the nuclear ribosomal RNA genes in conventional PCR or dye-binding quantitative PCR (Q-PCR) but are not amenable to multiplexing, and some of these assays did not utilize control strategies for type I or type II errors. In this study, we describe primers and a bifunctional probe with specificity to a gypsy-like retroelement in the P. sojae genome to create a fluorogenic 5’-exonuclease linear hydrolysis assay, with a multiplexed internal control reaction detecting an exogenous target to validate negative calls, and with uracil-deglycosylase-mediated protection against carryover contamination. The assay specifically detected 13 different P. sojae isolates, and excluded 17 other Phytophthora species along with 20 non-Phytophthora fungal and oomycete species pathogenic on soybean. A diagnostic limit of detection of 34 fg total P. sojae DNA was observed in serial dilutions, equivalent to 0.3 genome, and a practical detection sensitivity of four zoospores per sample was achieved, despite losses during DNA extraction. PMID:28441441

Comparison of the COBAS TAQMAN HIV-1 HPS with VERSANT HIV-1 RNA 3.0 assay (bDNA) for plasma RNA quantitation in different HIV-1 subtypes.

PubMed

Gomes, Perpétua; Palma, Ana Carolina; Cabanas, Joaquim; Abecasis, Ana; Carvalho, Ana Patrícia; Ziermann, Rainer; Diogo, Isabel; Gonçalves, Fátima; Lobo, Céu Sousa; Camacho, Ricardo

2006-08-01

Quantitation of HIV-1 RNA levels in plasma has an undisputed prognostic value and is extremely important for evaluating response to antiretroviral therapy. The purpose of this study was to evaluate the performance of the real-time PCR COBAS TaqMan 48 analyser, comparing it to the existing VERSANT 3.0 (bDNA) for HIV-1 RNA quantitation in plasma of individuals infected with different HIV-1 subtypes (104 blood samples). A positive linear correlation between the two tests (r2 = 0.88) was found. Quantitation by the COBAS TaqMan assay was approximately 0.32log10 higher than by bDNA. The relationship between the two assays was similar within all subtypes with a Deming regression of <1 and <0 for the Bland-Altman plots. Overall, no significant differences were found in plasma viral load quantitation in different HIV-1 subtypes between both assays; therefore these assays are suitable for viral load quantitation of highly genetically diverse HIV-1 plasma samples.

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures

PubMed Central

Wiklander, Oscar P. B.; Bostancioglu, R. Beklem; Welsh, Joshua A.; Zickler, Antje M.; Murke, Florian; Corso, Giulia; Felldin, Ulrika; Hagey, Daniel W.; Evertsson, Björn; Liang, Xiu-Ming; Gustafsson, Manuela O.; Mohammad, Dara K.; Wiek, Constanze; Hanenberg, Helmut; Bremer, Michel; Gupta, Dhanu; Björnstedt, Mikael; Giebel, Bernd; Nordin, Joel Z.; Jones, Jennifer C.; EL Andaloussi, Samir; Görgens, André

2018-01-01

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell’s activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived

Systematic Methodological Evaluation of a Multiplex Bead-Based Flow Cytometry Assay for Detection of Extracellular Vesicle Surface Signatures.

PubMed

Wiklander, Oscar P B; Bostancioglu, R Beklem; Welsh, Joshua A; Zickler, Antje M; Murke, Florian; Corso, Giulia; Felldin, Ulrika; Hagey, Daniel W; Evertsson, Björn; Liang, Xiu-Ming; Gustafsson, Manuela O; Mohammad, Dara K; Wiek, Constanze; Hanenberg, Helmut; Bremer, Michel; Gupta, Dhanu; Björnstedt, Mikael; Giebel, Bernd; Nordin, Joel Z; Jones, Jennifer C; El Andaloussi, Samir; Görgens, André

2018-01-01

Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell's activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived

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

PubMed

Väisänen, E; Lahtinen, A; Eis-Hübinger, A M; Lappalainen, M; Hedman, K; Söderlund-Venermo, M

2014-01-01

Human parvovirus 4 (PARV4) of the family Parvoviridae was discovered in a plasma sample of a patient with an undiagnosed acute infection in 2005. Currently, three PARV4 genotypes have been identified, however, with an unknown clinical significance. Interestingly, these genotypes seem to differ in epidemiology. In Northern Europe, USA and Asia, genotypes 1 and 2 have been found to occur mainly in persons with a history of injecting drug use or other parenteral exposure. In contrast, genotype 3 appears to be endemic in sub-Saharan Africa, where it infects children and adults without such risk behaviour. In this study, a novel straightforward and cost-efficient molecular assay for both quantitation and genotyping of PARV4 DNA was developed. The two-step method first applies a single-probe pan-PARV4 qPCR for screening and quantitation of this relatively rare virus, and subsequently, only the positive samples undergo a real-time PCR-based multi-probe genotyping. The new qPCR-GT method is highly sensitive and specific regardless of the genotype, and thus being suitable for studying the clinical impact and occurrence of the different PARV4 genotypes. Copyright © 2013 Elsevier B.V. All rights reserved.

Recommendations for the generation, quantification, storage and handling of peptides used for mass spectrometry-based assays

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

Hoofnagle, Andrew N.; Whiteaker, Jeffrey R.; Carr, Steven A.

2015-12-30

The Clinical Proteomic Tumor Analysis Consortium (1) (CPTAC) of the National Cancer Institute (NCI) is a comprehensive and coordinated effort to accelerate the understanding of the molecular basis of cancer through the application of robust technologies and workflows for the quantitative measurements of proteins. The Assay Development Working Group of the CPTAC Program aims to foster broad uptake of targeted mass spectrometry-based assays employing isotopically labeled peptides for confident assignment and quantification, including multiple reaction monitoring (MRM; also referred to as Selected Reaction Monitoring), parallel reaction monitoring (PRM), and other targeted methods.

Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay

PubMed Central

Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

2016-01-01

The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 104 CFU mL−1 or 105 CFU mL−1 for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R2) of 0.916–0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water. PMID:26884128

Rapid multiplex detection of 10 foodborne pathogens with an up-converting phosphor technology-based 10-channel lateral flow assay.

PubMed

Zhao, Yong; Wang, Haoran; Zhang, Pingping; Sun, Chongyun; Wang, Xiaochen; Wang, Xinrui; Yang, Ruifu; Wang, Chengbin; Zhou, Lei

2016-02-17

The rapid high-throughput detection of foodborne pathogens is essential in controlling food safety. In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was established for the rapid and simultaneous detection of 10 epidemic foodborne pathogens. Ten different single-target UPT-LF strips were developed and integrated into one TC-UPT-LF disc with optimization. Without enrichment the TC-UPT-LF assay had a detection sensitivity of 10(4) CFU mL(-1) or 10(5) CFU mL(-1) for each pathogen, and after sample enrichment it was 10 CFU/0.6 mg. The assay also showed good linearity, allowing quantitative detection, with a linear fitting coefficient of determination (R(2)) of 0.916-0.998. The 10 detection channels did not cross-react, so multiple targets could be specifically detected. When 279 real food samples were tested, the assay was highly consistent (100%) with culture-based methods. The results for 110 food samples artificially contaminated with single or multiple targets showed a high detection rate (≥ 80%) for most target bacteria. Overall, the TC-UPT-LF assay allows the rapid, quantitative, and simultaneous detection of 10 kinds of foodborne pathogens within 20 min, and is especially suitable for the rapid detection and surveillance of foodborne pathogens in food and water.

GFP-based fluorescence assay for CAG repeat instability in cultured human cells.

PubMed

Santillan, Beatriz A; Moye, Christopher; Mittelman, David; Wilson, John H

2014-01-01

Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries.

Quantitatively and Kinetically Identifying Binding Motifs of Amelogenin Proteins to Mineral Crystals Through Biochemical and Spectroscopic Assays

PubMed Central

Zhu, Li; Hwang, Peter; Witkowska, H. Ewa; Liu, Haichuan; Li, Wu

2014-01-01

Tooth enamel is the hardest tissue in vertebrate animals. Consisting of millions of carbonated hydroxyapatite crystals, this highly mineralized tissue develops from a protein matrix in which amelogenin is the predominant component. The enamel matrix proteins are eventually and completely degraded and removed by proteinases to form mineral-enriched tooth enamel. Identification of the apatite-binding motifs in amelogenin is critical for understanding the amelogenin–crystal interactions and amelogenin–proteinases interactions during tooth enamel biomineralization. A stepwise strategy is introduced to kinetically and quantitatively identify the crystal-binding motifs in amelogenin, including a peptide screening assay, a competitive adsorption assay, and a kinetic-binding assay using amelogenin and gene-engineered amelogenin mutants. A modified enzyme-linked immunosorbent assay on crystal surfaces is also applied to compare binding amounts of amelogenin and its mutants on different planes of apatite crystals. We describe the detailed protocols for these assays and provide the considerations for these experiments in this chapter. PMID:24188774

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

EPA Science Inventory

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

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

DOE PAGES

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

2017-07-19

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

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

PubMed Central

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

2014-01-01

ABSTRACT Background Both real-time-polymerase chain reaction (PCR) and hybrid capture 2 (HC2) assay can detect and quantify hepatitis B virus (HBV) DNA. However, real-time-PCR can detect a wide range of HBV DNA, while HC2 assay could not detect lower levels of viremia. The present study was designed to detect and quantify HBV DNA by real-time-PCR and HC2 assay and compare the quantitative data of these two assays. Materials and methods A cross-sectional study was conducted in between July 2010 and June 2011. A total of 66 serologically diagnosed chronic hepatitis B (CHB) patients were selected for the study. Real-time-PCR and HC2 assay was done to detect HBV DNA. Data were analyzed by statistical Package for the social sciences (SPSS). Results Among 66 serologically diagnosed chronic hepatitis B patients 40 (60.61%) patients had detectable and 26 (39.39%) had undetectable HBV DNA by HC2 assay. Concordant results were obtained for 40 (60.61%) out of these 66 patients by real-time-PCR and HC2 assay with mean viral load of 7.06 ± 1.13 log10 copies/ml and 6.95 ± 1.08 log10 copies/ml, respectively. In the remaining 26 patients, HBV DNA was detectable by real-time-PCR in 20 patients (mean HBV DNA level was 3.67 ± 0.72 log10 copies/ml. However, HBV DNA could not be detectable in six cases by the both assays. The study showed strong correlation (r = 0.915) between real-time-PCR and HC2 assay for the detection and quantification of HBV DNA. Conclusion HC2 assay may be used as an alternative to real-time-PCR for CHB patients. How to cite this article: Majid F, Jahan M, Moben AL, Tabassum S. Comparison of Hybrid Capture 2 Assay with Real-time-PCR for Detection and Quantitation of Hepatitis B Virus DNA. Euroasian J Hepato-Gastroenterol 2014;4(1):31-35. PMID:29264316

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

PubMed

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

2014-01-01

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

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

PubMed

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

2016-01-01

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

Detection of the downy mildew pathogens of spinach (Peronospora effusa) and beet (P. schachtii) using spore traps and quantitative PCR assays

USDA-ARS?s Scientific Manuscript database

Downy mildew of spinach, caused by Peronospora effusa, is a disease constraint on spinach production worldwide. The aim of this study was to develop a real-time quantitative PCR assay for detection of airborne inoculum of P. effusa in California. This type of assay may, in combination with disease-...

Array-based comparative genomic hybridization-guided identification of reference genes for normalization of real-time quantitative polymerase chain reaction assay data for lymphomas, histiocytic sarcomas, and osteosarcomas of dogs.

PubMed

Tsai, Pei-Chien; Breen, Matthew

2012-09-01

To identify suitable reference genes for normalization of real-time quantitative PCR (RT-qPCR) assay data for common tumors of dogs. Malignant lymph node (n = 8), appendicular osteosarcoma (9), and histiocytic sarcoma (12) samples and control samples of various nonneoplastic canine tissues. Array-based comparative genomic hybridization (aCGH) data were used to guide selection of 9 candidate reference genes. Expression stability of candidate reference genes and 4 commonly used reference genes was determined for tumor samples with RT-qPCR assays and 3 software programs. LOC611555 was the candidate reference gene with the highest expression stability among the 3 tumor types. Of the commonly used reference genes, expression stability of HPRT was high in histiocytic sarcoma samples, and expression stability of Ubi and RPL32 was high in osteosarcoma samples. Some of the candidate reference genes had higher expression stability than did the commonly used reference genes. Data for constitutively expressed genes with high expression stability are required for normalization of RT-qPCR assay results. Without such data, accurate quantification of gene expression in tumor tissue samples is difficult. Results of the present study indicated LOC611555 may be a useful RT-qPCR assay reference gene for multiple tissue types. Some commonly used reference genes may be suitable for normalization of gene expression data for tumors of dogs, such as lymphomas, osteosarcomas, or histiocytic sarcomas.

Development of a F actin-based live-cell fluorimetric microplate assay for diarrhetic shellfish toxins.

PubMed

Leira, F; Alvarez, C; Cabado, A G; Vieites, J M; Vieytes, M R; Botana, L M

2003-06-15

A new cytotoxicity assay for detection and quantitation of diarrhetic shellfish toxins (DSP) is presented. This assay is based upon fluorimetric determination of F-actin depolymerization induced by okadaic acid (OA)-class compounds in the BE(2)-M17 neuroblastoma cell line. No interferences were observed with other marine toxins such as saxitoxin, domoic acid, or yessotoxin, thus indicating a good specificity of the assay as expected by the direct relationship between protein phosphatase inhibition and cytoskeletal changes. The proposed method is rapid (<2h) and shows a linear response in the range of 50-300 nM OA. The detection limit of the assay for crude methanolic extracts of bivalves lies between 0.2 and 1.0 microg OA per gram of digestive glands, depending on the type of samples (fresh or canned), thus being similar to that of the mouse bioassay. The performance of this assay has been evaluated by comparative analysis of 32 toxic mussel samples by the F-actin assay, mouse bioassay, HPLC and PP2A inhibition assay. Results obtained by the F-actin method showed no differences with HPLC and significant correlation with PP2A inhibition assay (r(2)=0.71). No false negative results were obtained with this new cell assay, which also showed optimum reproducibility.

Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays.

PubMed

Mattiuzzo, Giada; Ashall, James; Doris, Kathryn S; MacLellan-Gibson, Kirsty; Nicolson, Carolyn; Wilkinson, Dianna E; Harvey, Ruth; Almond, Neil; Anderson, Robert; Efstathiou, Stacey; Minor, Philip D; Page, Mark

2015-01-01

The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA.

Analytical characteristics and comparative evaluation of Aptima HCV quant Dx assay with the Abbott RealTime HCV assay and Roche COBAS AmpliPrep/COBAS TaqMan HCV quantitative test v2.0.

PubMed

Worlock, A; Blair, D; Hunsicker, M; Le-Nguyen, T; Motta, C; Nguyen, C; Papachristou, E; Pham, J; Williams, A; Vi, M; Vinluan, B; Hatzakis, A

2017-04-04

The Aptima HCV Quant Dx assay (Aptima assay) is a fully automated quantitative assay on the Panther® system. This assay is intended for confirmation of diagnosis and monitoring of HCV RNA in plasma and serum specimens. The purpose of the testing described in this paper was to evaluate the performance of the Aptima assay. The analytical sensitivity, analytical specificity, precision, and linearity of the Aptima assay were assessed. The performance of the Aptima assay was compared to two commercially available HCV assays; the Abbott RealTime HCV assay (Abbott assay, Abbott Labs Illinois, USA) and the Roche COBAS Ampliprep/COBAS Taqman HCV Quantitative Test v2.0 (Roche Assay, Roche Molecular Systems, Pleasanton CA, USA). The 95% Lower Limit of Detection (LoD) of the assay was determined from dilutions of the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) and HCV positive clinical specimens in HCV negative human plasma and serum. Probit analysis was performed to generate the 95% predicted detection limits. The Lower Limit of Quantitation (LLoQ) was established for each genotype by diluting clinical specimens and the 2nd HCV WHO International Standard (NIBSC 96/798 genotype 1) in HCV negative human plasma and serum. Specificity was determined using 200 fresh and 536 frozen HCV RNA negative clinical specimens including 370 plasma specimens and 366 serum specimens. Linearity for genotypes 1 to 6 was established by diluting armored RNA or HCV positive clinical specimens in HCV negative serum or plasma from 8.08 log IU/mL to below 1 log IU/mL. Precision was tested using a 10 member panel made by diluting HCV positive clinical specimens or spiking armored RNA into HCV negative plasma and serum. A method comparison was conducted against the Abbott assay using 1058 clinical specimens and against the Roche assay using 608 clinical specimens from HCV infected patients. In addition, agreement between the Roche assay and the Aptima assay using specimens with low

Performance of the New Bayer VERSANT HCV RNA 3.0 assay for quantitation of hepatitis C virus RNA in plasma and serum: conversion to international units and comparison with the Roche COBAS Amplicor HCV Monitor, Version 2.0, assay.

PubMed

Beld, Marcel; Sentjens, Roel; Rebers, Sjoerd; Weegink, Christine; Weel, Jan; Sol, Cees; Boom, René

2002-03-01

We have evaluated the VERSANT HCV RNA 3.0. Assay (HCV 3.0 bDNA assay) (Bayer Diagnostics, Berkeley, Calif.), which is an improved signal amplification procedure for the HCV 2.0 bDNA assay for the quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The HCV 3.0 bDNA assay has a linear dynamic range of 2.5 x 10(3) to 4.0 x 10(7) HCV RNA copies per ml (c/ml). The performance of the HCV 3.0 bDNA assay was evaluated using three different test panels. An overall specificity of 96.8% relative to the detection limit of the HCV 3.0 bDNA assay was found. The intra- and interrun reproducibilities for both the dilution panel and the NAP (AcroMetrix, Benicia, Calif.) panel were consistent with coefficients of variation of less than 9%. Quantitation with the HCV 3.0 bDNA assay was linear over the entire range of both panels (ranges of 4.4 x 10(3) to 3.5 x 10(6) c/ml and 5 x 10(3) to 2 x 10(6) IU/ml, respectively), with correlation coefficients of 0.999, slopes close to one, and intercepts close to zero. The regression equation indicated that 1 IU corresponded to about 4.8 copies of HCV RNA. A correlation coefficient of 0.941 was found for HCV RNA values (in international units per milliliter) obtained from the HCV 3.0 bDNA assay and the HCV Monitor version 2.0 assay (HCV Monitor 2.0 assay) (Roche Diagnostic Systems, Branchburg, N.J.). Quantitative results obtained close to the lower limit of the HCV 3.0 bDNA assay might imply that its lower limit should be reconsidered and raised, if necessary. It appeared that quantitation values obtained from the HCV Monitor 2.0 assay of between 5 x 10(2) and 10(5) IU/ml were in general higher than those obtained from the HCV 3.0 bDNA assay, whereas values obtained from the HCV Monitor 2.0 assay were underestimated for samples with HCV RNA levels above 10(5) IU/ml.

Performance of the New Bayer VERSANT HCV RNA 3.0 Assay for Quantitation of Hepatitis C Virus RNA in Plasma and Serum: Conversion to International Units and Comparison with the Roche COBAS Amplicor HCV Monitor, Version 2.0, Assay

PubMed Central

Beld, Marcel; Sentjens, Roel; Rebers, Sjoerd; Weegink, Christine; Weel, Jan; Sol, Cees; Boom, René

2002-01-01

We have evaluated the VERSANT HCV RNA 3.0. Assay (HCV 3.0 bDNA assay) (Bayer Diagnostics, Berkeley, Calif.), which is an improved signal amplification procedure for the HCV 2.0 bDNA assay for the quantitation of hepatitis C virus (HCV) RNA in serum or plasma of HCV-infected individuals. The HCV 3.0 bDNA assay has a linear dynamic range of 2.5 × 103 to 4.0 × 107 HCV RNA copies per ml (c/ml). The performance of the HCV 3.0 bDNA assay was evaluated using three different test panels. An overall specificity of 96.8% relative to the detection limit of the HCV 3.0 bDNA assay was found. The intra- and interrun reproducibilities for both the dilution panel and the NAP (AcroMetrix, Benicia, Calif.) panel were consistent with coefficients of variation of less than 9%. Quantitation with the HCV 3.0 bDNA assay was linear over the entire range of both panels (ranges of 4.4 × 103 to 3.5 × 106 c/ml and 5 × 103 to 2 × 106 IU/ml, respectively), with correlation coefficients of 0.999, slopes close to one, and intercepts close to zero. The regression equation indicated that 1 IU corresponded to about 4.8 copies of HCV RNA. A correlation coefficient of 0.941 was found for HCV RNA values (in international units per milliliter) obtained from the HCV 3.0 bDNA assay and the HCV Monitor version 2.0 assay (HCV Monitor 2.0 assay) (Roche Diagnostic Systems, Branchburg, N.J.). Quantitative results obtained close to the lower limit of the HCV 3.0 bDNA assay might imply that its lower limit should be reconsidered and raised, if necessary. It appeared that quantitation values obtained from the HCV Monitor 2.0 assay of between 5 × 102 and 105 IU/ml were in general higher than those obtained from the HCV 3.0 bDNA assay, whereas values obtained from the HCV Monitor 2.0 assay were underestimated for samples with HCV RNA levels above 105 IU/ml. PMID:11880394

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

PubMed Central

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” 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

[Simultaneous quantitative analysis of five alkaloids in Sophora flavescens by multi-components assay by single marker].

PubMed

Chen, Jing; Wang, Shu-Mei; Meng, Jiang; Sun, Fei; Liang, Sheng-Wang

2013-05-01

To establish a new method for quality evaluation and validate its feasibilities by simultaneous quantitative assay of five alkaloids in Sophora flavescens. The new quality evaluation method, quantitative analysis of multi-components by single marker (QAMS), was established and validated with S. flavescens. Five main alkaloids, oxymatrine, sophocarpine, matrine, oxysophocarpine and sophoridine, were selected as analytes to evaluate the quality of rhizome of S. flavescens, and the relative correction factor has good repeatibility. Their contents in 21 batches of samples, collected from different areas, were determined by both external standard method and QAMS. The method was evaluated by comparison of the quantitative results between external standard method and QAMS. No significant differences were found in the quantitative results of five alkaloids in 21 batches of S. flavescens determined by external standard method and QAMS. It is feasible and suitable to evaluate the quality of rhizome of S. flavescens by QAMS.

Performance of NucliSens HIV-1 EasyQ Version 2.0 compared with six commercially available quantitative nucleic acid assays for detection of HIV-1 in China.

PubMed

Xu, Sihong; Song, Aijing; Nie, Jianhui; Li, Xiuhua; Wang, Youchun

2010-10-01

Six HIV-1 viral load assays have been widely used in China. These include the Cobas Amplicor HIV-1 Monitor Version 1.5 ('Amplicor'), Cobas AmpliPrep/Cobas TaqMan HIV-1 test Version 1.0 ('CAP/CTM'), Versant HIV-1 RNA Version 3.0 (branched DNA [bDNA]-based assay; 'Versant bDNA'), Abbott RealTime HIV-1 assay ('Abbott RealTime'), NucliSens HIV-1 QT (nucleic acid sequence-based amplification assay; 'NucliSens NASBA'), and NucliSens EasyQ HIV-1 Version 1.1 ('EasyQ V1.1'). Recently, an updated version of EasyQ V1.1, NucliSens EasyQ HIV-1 Version 2.0 ('EasyQ V2.0') was introduced into China. It is important to evaluate the impact of HIV-1 genotypes on the updated assay compared with the other commercial available assays in China. A total of 175 plasma samples with different HIV-1 clades prevalent in China were collected from treatment-naïve patients. The viral loads of those samples were determined with the seven HIV-1 viral load assays, and the quantitative differences between them were evaluated. Overall, EasyQ V2.0 exhibited a significant correlation (R = 0.769-0.850, p ≤ 0.001) and high agreement (94.77-97.13%, using the Bland-Altman model) with the other six assays. Although no significant differences between EasyQ V2.0 and the other six assays were observed when quantifying clade B' samples, there were statistically significant differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays when quantifying clade BC samples, and between EasyQ V2.0 and the Versant bDNA and Abbott RealTime assays when quantifying clade AE samples. For clade BC samples, the quantitative differences between EasyQ V2.0 and the Amplicor, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log(10) IU/mL in approximately 50% of samples and exceeded 1 log(10) IU/mL in approximately 15% of samples. For clade AE samples, the quantitative differences between EasyQ V2.0 and the CAP/CTM, Versant bDNA, and Abbott RealTime assays exceeded 0.5 log(10) IU/mL in

Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy.

PubMed

Bergallo, Massimiliano; Galliano, Ilaria; Montanari, Paola; Brusin, Martina Rosa; Finotti, Serena; Paderi, Giulia; Gabiano, Clara

2017-04-01

Gastroenteritis is a common disease in children. It is characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis, but it causes milder illness than do rotavirus and norovirus. There is high variability in the analytical performance of quantitative PCR-based assays among clinical laboratories. This study developed a reverse transcription real-time PCR method to detect SaV in fecal specimens collected from children under 5-years-old with acute gastroenteritis. Of 137 episodes of acute gastroenteritis, 15 (10.9%) were associated with SaV genomic detection, with a median viral load of 6.6(log 10 ) ± 7.1(log 10 ) genomes/mg fecal specimens. There was a significant difference in detection rate between males and females (9.48% (13/15) vs. 1.46% (2/15), p = 0.0232). Among the 15 SaV-positive cases, 6 were also positive for rotavirus. Viral RNA recovery rate ranged from 46% to 77% in the manual RNAzol protocol and from 31% to 90% in the automated Maxwell protocol. We also studied whether human genomic DNA influences the sensitivity of the assay: its presence caused a decrease in PCR sensitivity. The development of a laboratory-designed real-time PCR TaqMan assay for quantitative detection of SaV and the optimization and standardization of this assay, using stools of children with acute gastroenteritis, are described.

A multisite assessment of the quantitative capabilities of the Xpert MTB/RIF assay.

PubMed

Blakemore, Robert; Nabeta, Pamela; Davidow, Amy L; Vadwai, Viral; Tahirli, Rasim; Munsamy, Vanisha; Nicol, Mark; Jones, Martin; Persing, David H; Hillemann, Doris; Ruesch-Gerdes, Sabine; Leisegang, Felicity; Zamudio, Carlos; Rodrigues, Camilla; Boehme, Catharina C; Perkins, Mark D; Alland, David

2011-11-01

The Xpert MTB/RIF is an automated molecular test for Mycobacterium tuberculosis that estimates bacterial burden by measuring the threshold-cycle (Ct) of its M. tuberculosis-specific real-time polymerase chain reaction. Bacterial burden is an important biomarker for disease severity, infection control risk, and response to therapy. Evaluate bacterial load quantitation by Xpert MTB/RIF compared with conventional quantitative methods. Xpert MTB/RIF results were compared with smear-microscopy, semiquantiative solid culture, and time-to-detection in liquid culture for 741 patients and 2,008 samples tested in a multisite clinical trial. An internal control real-time polymerase chain reaction was evaluated for its ability to identify inaccurate quantitative Xpert MTB/RIF results. Assays with an internal control Ct greater than 34 were likely to be inaccurately quantitated; this represented 15% of M. tuberculosis-positive tests. Excluding these, decreasing M. tuberculosis Ct was associated with increasing smear microscopy grade for smears of concentrated sputum pellets (r(s) = -0.77) and directly from sputum (r(s) =-0.71). A Ct cutoff of approximately 27.7 best predicted smear-positive status. The association between M. tuberculosis Ct and time-to-detection in liquid culture (r(s) = 0.68) and semiquantitative colony counts (r(s) = -0.56) was weaker than smear. Tests of paired same-patient sputum showed that high viscosity sputum samples contained ×32 more M. tuberculosis than nonviscous samples. Comparisons between the grade of the acid-fast bacilli smear and Xpert MTB/RIF quantitative data across study sites enabled us to identify a site outlier in microscopy. Xpert MTB/RIF quantitation offers a new, standardized approach to measuring bacterial burden in the sputum of patients with tuberculosis.

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.

PubMed

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 < .0001). This observation could provide impetus for further research to elucidate the clinical usefulness of the qHBsAg and HBcrAg assays.

A whole-cell bioreporter assay for quantitative genotoxicity evaluation of environmental samples.

PubMed

Jiang, Bo; Li, Guanghe; Xing, Yi; Zhang, Dayi; Jia, Jianli; Cui, Zhisong; Luan, Xiao; Tang, Hui

2017-10-01

Whole-cell bioreporters have emerged as promising tools for genotoxicity evaluation, due to their rapidity, cost-effectiveness, sensitivity and selectivity. In this study, a method for detecting genotoxicity in environmental samples was developed using the bioluminescent whole-cell bioreporter Escherichia coli recA::luxCDABE. To further test its performance in a real world scenario, the E. coli bioreporter was applied in two cases: i) soil samples collected from chromium(VI) contaminated sites; ii) crude oil contaminated seawater collected after the Jiaozhou Bay oil spill which occurred in 2013. The chromium(VI) contaminated soils were pretreated by water extraction, and directly exposed to the bioreporter in two phases: aqueous soil extraction (water phase) and soil supernatant (solid phase). The results indicated that both extractable and soil particle fixed chromium(VI) were bioavailable to the bioreporter, and the solid-phase contact bioreporter assay provided a more precise evaluation of soil genotoxicity. For crude oil contaminated seawater, the response of the bioreporter clearly illustrated the spatial and time change in genotoxicity surrounding the spill site, suggesting that the crude oil degradation process decreased the genotoxic risk to ecosystem. In addition, the performance of the bioreporter was simulated by a modified cross-regulation gene expression model, which quantitatively described the DNA damage response of the E. coli bioreporter. Accordingly, the bioluminescent response of the bioreporter was calculated as the mitomycin C equivalent, enabling quantitative comparison of genotoxicities between different environmental samples. This bioreporter assay provides a rapid and sensitive screening tool for direct genotoxicity assessment of environmental samples. Copyright © 2017. Published by Elsevier Ltd.

A novel method for the quantitation of gingerol glucuronides in human plasma or urine based on stable isotope dilution assays.

PubMed

Schoenknecht, Carola; Andersen, Gaby; Schieberle, Peter

2016-11-15

The bio-active compounds of ginger (Zingiber officinale Roscoe), the gingerols, are gaining considerable attention due to their numerous beneficial health effects. In order to elucidate the physiological relevance of the ascribed effects their bioavailability has to be determined taking their metabolization into account. To quantitate in vivo generated [6]-, [8]- and [10]-gingerol glucuronides in human plasma and urine after ginger tea consumption, a simultaneous and direct liquid chromatography-tandem mass spectrometry method based on stable isotope dilution assays was established and validated. The respective references as well as the isotopically labeled substances were synthesized and characterized by mass spectrometry and NMR. Selective isolation of gingerol glucuronides from human plasma and urine by a mixed-phase anion-exchange SPE method led to recovery rates between 80.8 and 98.2%. LC-MS/MS analyses in selected reaction monitoring modus enabled a highly sensitive quantitation of gingerol glucuronides with LoQs between 3.9-9.8nmol/L in plasma and 39.3-161.1nmol/L in urine. The method precision in plasma and urine varied in the range±15%, whereas the intra-day accuracy in plasma and urine showed values between 78 and 122%. The developed method was then applied to a pilot study in which two volunteers consumed one liter ginger tea. Pharmacokinetic parameters like the maximum concentration (c max ), the time to reach c max (t max ), area under the curve (AUC), elimination rate constant (k el ) and elimination half-life (t 1/2 ) were calculated from the concentration-time curve of each gingerol glucuronide. The obtained results will enable more detailed investigation of gingerol glucuronides as bioactives in their physiologically relevant concentrations. Copyright © 2016 Elsevier B.V. All rights reserved.

Multicolor Bioluminescence Boosts Malaria Research: Quantitative Dual-Color Assay and Single-Cell Imaging in Plasmodium falciparum Parasites

PubMed Central

2015-01-01

New reliable and cost-effective antimalarial drug screening assays are urgently needed to identify drugs acting on different stages of the parasite Plasmodium falciparum, and particularly those responsible for human-to-mosquito transmission, that is, the P. falciparum gametocytes. Low Z′ factors, narrow dynamic ranges, and/or extended assay times are commonly reported in current gametocyte assays measuring gametocyte-expressed fluorescent or luciferase reporters, endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent dye fluorescence. We hereby report on a dual-luciferase gametocyte assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases from Pyrophorus plagiophthalamus under the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial drugs and allowed to quantitatively and simultaneously measure stage-specific drug effects on parasites at different developmental stages. The optimized assay, requiring only 48 h incubation with drugs and using a cost-effective luminogenic substrate, significantly reduces assay cost and time in comparison to state-of-the-art analogous assays. The assay had a Z′ factor of 0.71 ± 0.03, and it is suitable for implementation in 96- and 384-well microplate formats. Moreover, the use of a nonlysing d-luciferin substrate significantly improved the reliability of the assay and allowed one to perform, for the first time, P. falciparum bioluminescence imaging at single-cell level. PMID:25102353

Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients.

PubMed

Caliendo, A M; St George, K; Kao, S Y; Allega, J; Tan, B H; LaFontaine, R; Bui, L; Rinaldo, C R

2000-06-01

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.

Comparison of Quantitative Cytomegalovirus (CMV) PCR in Plasma and CMV Antigenemia Assay: Clinical Utility of the Prototype AMPLICOR CMV MONITOR Test in Transplant Recipients

PubMed Central

Caliendo, Angela M.; St. George, Kirsten; Kao, Shaw-Yi; Allega, Jessica; Tan, Ban-Hock; LaFontaine, Robert; Bui, Larry; Rinaldo, Charles R.

2000-01-01

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients. PMID:10834964

Evaluation of a newly developed quantitative heart-type fatty acid binding protein assay based on fluorescence immunochromatography using specific monoclonal antibodies.

PubMed

Kang, Keren; Wu, Peidian; Li, Wenmei; Tang, Shixing; Wang, Jihua; Luo, Xiaochun; Xie, Mingquan

2015-01-01

To develop a rapid, sensitive and specific assay for quantification of serum heart-type fatty acid binding protein (H-FABP) based on immunofluorescence of specific monoclonal antibodies. We generated novel H-FABP-directed monoclonal antibodies by cloning of spleen cells of mice immunized with H-FABP. Epitopes were mapped and antigen affinity was assessed by surface plasmon resonance (SPR). The H-FABP specific monoclonal antibodies were coupled to fluorescent beads and sprayed onto a nitrocellulose membrane facilitating quantification of H-FABP by immunofluorescence. Reagent cross-reactivity, interference resistance, accuracy and sensitivity were examined. A total of 103 clinical samples were used to compare the sensitivity and specificity of the new assay to a commercially available Randox kit. This new assay could be finished within 15 min, with sensitivity reaching 1 ng/ml. In a trial of 103 clinical serum samples, the new testing kit results were highly correlated with those from the Randox kit (R(2) = 0.9707). Using the Randox kit as the reference kit, the sensitivity of the new assay was 98.25%, and specificity was 100%. An immunofluorescence-based H-FABP assay employing novel monoclonal antibodies could rapidly, specifically and sensitively detect H-FABP in serum samples, providing an effective method for rapid clinical assessment of H-FABP index in the clinic.

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

PubMed

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

2012-03-01

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

A quantitative PCR assay for aerobic, vinyl chloride- and ethene-assimilating microorganisms in groundwater.

PubMed

Jin, Yang Oh; Mattes, Timothy E

2010-12-01

Vinyl chloride (VC) is a known human carcinogen that is primarily formed in groundwater via incomplete anaerobic dechlorination of chloroethenes. Aerobic, ethene-degrading bacteria (etheneotrophs), which are capable of both fortuitous and growth-linked VC oxidation, could be important in natural attenuation of VC plumes that escape anaerobic treatment. In this work, we developed a quantitative, real-time PCR (qPCR) assay for etheneotrophs in groundwater. We designed and tested degenerate qPCR primers for two functional genes involved in aerobic, growth-coupled VC- and ethene-oxidation (etnC and etnE). Primer specificity to these target genes was tested by comparison to nucleotide sequence databases, PCR analysis of template DNA extracted from isolates and environmental samples, and sequencing of qPCR products obtained from VC-contaminated groundwater. The assay was made quantitative by constructing standard curves (threshold cycle vs log gene copy number) with DNA amplified from Mycobacterium strain JS60, an etheneotrophic isolate. Analysis of groundwater samples from three different VC-contaminated sites revealed that etnC abundance ranged from 1.6 × 10(3) - 1.0 × 10(5) copies/L groundwater while etnE abundance ranged from 4.3 × 10(3) - 6.3 × 10(5) copies/L groundwater. Our data suggest this novel environmental measurement method will be useful for supporting VC bioremediation strategies, assisting in site closure, and conducting microbial ecology studies involving etheneotrophs.

Battery operated preconcentration-assisted lateral flow assay.

PubMed

Kim, Cheonjung; Yoo, Yong Kyoung; Han, Sung Il; Lee, Junwoo; Lee, Dohwan; Lee, Kyungjae; Hwang, Kyo Seon; Lee, Kyu Hyoung; Chung, Seok; Lee, Jeong Hoon

2017-07-11

Paper-based analytical devices (e.g. lateral flow assays) are highly advantageous as portable diagnostic systems owing to their low costs and ease of use. Because of their low sensitivity and detection limits for biomolecules, these devices have several limitations in applications for real-field diagnosis. Here, we demonstrate a paper-based preconcentration enhanced lateral flow assay using a commercial β-hCG-based test. Utilizing a simple 9 V battery operation with a low power consumption of approximately 81 μW, we acquire a 25-fold preconcentration factor, demonstrating a clear sensitivity enhancement in the colorimetric lateral flow assay; consequently, clear colors are observed in a rapid kit test line, which cannot be monitored without preconcentration. This device can also facilitate a semi-quantitative platform using the saturation value and/or color intensity in both paper-based colorimetric assays and smartphone-based diagnostics.

GFP-Based Fluorescence Assay for CAG Repeat Instability in Cultured Human Cells

PubMed Central

Santillan, Beatriz A.; Moye, Christopher; Mittelman, David; Wilson, John H.

2014-01-01

Trinucleotide repeats can be highly unstable, mutating far more frequently than point mutations. Repeats typically mutate by addition or loss of units of the repeat. CAG repeat expansions in humans trigger neurological diseases that include myotonic dystrophy, Huntington disease, and several spinocerebellar ataxias. In human cells, diverse mechanisms promote CAG repeat instability, and in mice, the mechanisms of instability are varied and tissue-dependent. Dissection of mechanistic complexity and discovery of potential therapeutics necessitates quantitative and scalable screens for repeat mutation. We describe a GFP-based assay for screening modifiers of CAG repeat instability in human cells. The assay exploits an engineered intronic CAG repeat tract that interferes with expression of an inducible GFP minigene. Like the phenotypes of many trinucleotide repeat disorders, we find that GFP function is impaired by repeat expansion, in a length-dependent manner. The intensity of fluorescence varies inversely with repeat length, allowing estimates of repeat tract changes in live cells. We validate the assay using transcription through the repeat and engineered CAG-specific nucleases, which have previously been reported to induce CAG repeat instability. The assay is relatively fast and should be adaptable to large-scale screens of chemical and shRNA libraries. PMID:25423602

Gold nanoparticle-based enzyme-linked antibody-aptamer sandwich assay for detection of Salmonella Typhimurium.

PubMed

Wu, Wenhe; Li, Jun; Pan, Dun; Li, Jiang; Song, Shiping; Rong, Mingge; Li, Zixi; Gao, Jimin; Lu, Jianxin

2014-10-08

Enzyme-linked immunosorbent assay (ELISA) provides a convenient means for the detection of Salmonella enterica serovar Typhimurium (STM), which is important for rapid diagnosis of foodborne pathogens. However, conventional ELISA is limited by antibody-antigen immunoreactions and suffers from poor sensitivity and tedious sample pretreatment. Therefore, development of novel ELISA remains challenging. Herein, we designed a comprehensive strategy for rapid, sensitive, and quantitative detection of STM with high specificity by gold nanoparticle-based enzyme-linked antibody-aptamer sandwich (nano-ELAAS) method. STM was captured and preconcentrated from samples with aptamer-modified magnetic particles, followed by binding with detector antibodies. Then nanoprobes carrying a large amount of reporter antibodies and horseradish peroxidase molecules were used for colorimetric signal amplification. Under the optimized reaction conditions, the nano-ELAAS assay had a quantitative detection range from 1 × 10(3) to 1 × 10(8) CFU mL(-1), a limit of detection of 1 × 10(3) CFU mL(-1), and a selectivity of >10-fold for STM in samples containing other bacteria at higher concentration with an assay time less than 3 h. In addition, the developed nanoprobes were improved in terms of detection range and/or sensitivity when compared with two commercial enzyme-labeled antibody signal reporters. Finally, the nano-ELAAS method was demonstrated to work well in milk samples, a common source of STM contamination.

A monoclonal antibody based elisa for quantitation of human leukaemia inhibitory factor.

PubMed

Taupin, J L; Gualde, N; Moreau, J F

1997-02-01

The authors report on the development of a new sandwich enzyme-linked immunoabsorbent assay (ELISA) for the quantitation of the human cytokine leukaemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) with high accuracy and sensitivity (23 pg/ml), in less than 5 h and in various biological fluids. The antibodies used in this assay were raised against recombinant glycosylated LIF expressed in vivo following inoculation of recombinant vaccinia viruses, and screened with the biologically active cytokine in a flow cytometry assay using cells expressing a membrane-bound form of LIF. Furthermore, this home-made assay was compared with two commercially available ELISA kits. The results led to the conclusion that these three assays are far from being equivalent between each other, in terms of sensitivity towards non-glycosylated vs glycosylated LIF. Two major parameters must be incriminated: the glycosylation status of the LIF molecule used as the calibrator, and the binding characteristics of the monoclonal antibodies used to set up these assays toward LIF derived from Escherichia coli or from eukaryotic cells. This points out the importance of these parameters for the design of ELISAs meant for the quantitation of glycosylated cytokines in biological fluids.

A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

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

Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain

2016-09-15

The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc.more » The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.« less

Multiplexed and Microparticle-based Analyses: Quantitative Tools for the Large-Scale Analysis of Biological Systems

PubMed Central

Nolan, John P.; Mandy, Francis

2008-01-01

While the term flow cytometry refers to the measurement of cells, the approach of making sensitive multiparameter optical measurements in a flowing sample stream is a very general analytical approach. The past few years have seen an explosion in the application of flow cytometry technology for molecular analysis and measurements using micro-particles as solid supports. While microsphere-based molecular analyses using flow cytometry date back three decades, the need for highly parallel quantitative molecular measurements that has arisen from various genomic and proteomic advances has driven the development in particle encoding technology to enable highly multiplexed assays. Multiplexed particle-based immunoassays are now common place, and new assays to study genes, protein function, and molecular assembly. Numerous efforts are underway to extend the multiplexing capabilities of microparticle-based assays through new approaches to particle encoding and analyte reporting. The impact of these developments will be seen in the basic research and clinical laboratories, as well as in drug development. PMID:16604537

Determination of Interference During In Vitro Pyrogen Detection: Development and Characterization of a Cell-Based Assay.

PubMed

Palma, Linda; Rossetti, Francesca; Dominici, Sabrina; Buondelmonte, Costantina; Rocchi, Marco B L; Rizzardi, Gian P; Vallanti, Giuliana; Magnani, Mauro

Contamination of pharmaceutical products and medical devices with pyrogens such as endotoxins is the most common cause of systemic inflammation and, in worst cases, of septic shock. Thus, quantification of pyrogens is crucial. The limulus amebocyte lysate (LAL)-based assays are the reference tests for in vitro endotoxin detection, in association with the in vivo rabbit pyrogen test (RPT), according to European Pharmacopoeia (EP 2.6.14), and U.S. Pharmacopoeia (USP <85>). However, several substances interfere with LAL assay, while RPT is not accurate, not quantitative, and raises ethical limits. Biological assays, as monocyte activation tests, have been developed and included in European Pharmacopoeia (EP 7.0; 04/2010:20630) guidelines as an alternative to RPT and proved relevant to the febrile reaction in vivo. Because this reaction is carried out by endogenous mediators under the transcriptional control of nuclear factor-kappaB (NF-kappaB), we sought to determine whether a NF-kappaB reporter-gene assay, based on MonoMac-6 (MM6) cells, could reconcile the basic mechanism of innate immune response with the relevance of monocytoid cell lines to the organism reaction to endotoxins. This article describes both optimization and characterization of the reporter cells-based assay, which overall proved the linearity, accuracy, and precision of the test, and demonstrated the sensitivity of the assay to 0.24 EU/mL endotoxin, close to the pyrogenic threshold in humans. Moreover, the assay was experimentally compared to the LAL test in the evaluation of selected interfering samples. The good performance of the MM6 reporter test demonstrates the suitability of this assay to evaluate interfering or false-positive samples.

Laboratory assessment of Activated Protein C Resistance/Factor V-Leiden and performance characteristics of a new quantitative assay.

PubMed

Amiral, Jean; Vissac, Anne Marie; Seghatchian, Jerard

2017-12-01

Activated Protein C Resistance is mainly associated to a factor V mutation (RQ506), which induces a deficient inactivation of activated factor V by activated protein C, and is associated to an increased risk of venous and arterial thrombosis in affected individuals, caused by the prolonged activated factor V survival. Its prevalence is mainly in Caucasians (about 5%), and this mutation is absent in Africans and Asians. Presence of Factor V-Leiden is usually evidenced with clotting methods, using a two-step APTT assay performed without or with APC: prolongation of blood coagulation time is decreased if this factor is present. The R506Q Factor V-Leiden mutation is now usually characterized using molecular biology, and this technique tends to become the first intention assay for characterization of patients. Both techniques are qualitative, and allow classifying tested individuals as heterozygotes or homozygotes for the mutation, when present. A new quantitative assay for Factor V-Leiden, using a one-step clotting method, has been developed, and designed with highly purified human coagulation proteins. Clotting is triggered with human Factor Xa, in presence of calcium and phospholipids (mixture which favours APC action over clotting process). Diluted tested plasma, is supplemented with a clotting mixture containing human fibrinogen, prothrombin, and protein S at a constant concentration. APC is added, and clotting is initiated with calcium. Calibration is performed with a pool of plasmas from patients carrying the R506Q Factor V mutation, and its mixtures with normal plasma. Homozygous patients have clotting times of about <40sec; heterozygous patients have clotting times of about 40-60sec and normal individuals yield clotting times >70sec. Factor V-Leiden concentration is usually >75% in homozygous patients, 30-60% in heterozygous patients and below 5% in normal. The assay is insensitive to clotting factor deficiencies (II, VII, VIII: C, IX, X), dicoumarol or heparin

A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods.

PubMed

Nixon, Gavin J; Svenstrup, Helle F; Donald, Carol E; Carder, Caroline; Stephenson, Judith M; Morris-Jones, Stephen; Huggett, Jim F; Foy, Carole A

2014-12-01

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These 'isothermal' methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

Comparative Analysis of Predictive Models for Liver Toxicity Using ToxCast Assays and Quantitative Structure-Activity Relationships (MCBIOS)

EPA Science Inventory

Comparative Analysis of Predictive Models for Liver Toxicity Using ToxCast Assays and Quantitative Structure-Activity Relationships Jie Liu1,2, Richard Judson1, Matthew T. Martin1, Huixiao Hong3, Imran Shah1 1National Center for Computational Toxicology (NCCT), US EPA, RTP, NC...

A prospective study of a quantitative PCR ELISA assay for the diagnosis of CMV pneumonia in lung and heart-transplant recipients.

PubMed

Barber, L; Egan, J J; Lomax, J; Haider, Y; Yonan, N; Woodcock, A A; Turner, A J; Fox, A J

2000-08-01

Qualitative polymerase chain reaction (PCR) for the identification of cytomegalovirus (CMV) infection has a low predictive value for the identification of CMV pneumonia. This study prospectively evaluated the application of a quantitative PCR Enzyme-Linked Immuno-Sorbent Assay (ELISA) assay in 9 lung- and 18 heart-transplant recipients who did not receive ganciclovir prophylaxis. DNA was collected from peripheral blood polymorphonuclear leucocytes (PMNL) posttransplantation. Oligonucleotide primers for the glycoprotein B gene (149 bp) were used in a PCR ELISA assay using an internal standard for quantitation. CMV disease was defined as histological evidence of end organ damage. The median level CMV genome equivalents in patients with CMV disease was 2665/2 x 10(5) PMNL (range 1,200 to 61,606) compared to 100 x 10(5) PMNL (range 20 to 855) with infection but no CMV disease (p = 0.036). All patients with CMV disease had genome equivalents levels of >1200/2 x 10(5) PMNL. A cut-off level of 1,200 PMNL had a positive predictive value for CMV disease of 100% and a negative predictive value of 100%. The first detection of levels of CMV genome equivalents above a level of 1200/2 x 10(5) PMNL was at a median of 58 days (range 47 to 147) posttransplant. Quantitative PCR assays for the diagnosis of CMV infection may predict patients at risk of CMV disease and thereby direct preemptive treatment to high-risk patients.

Pentobarbital quantitation using EMIT serum barbiturate assay reagents: application to monitoring of high-dose pentobarbital therapy.

PubMed

Pape, B E; Cary, P L; Clay, L C; Godolphin, W

1983-01-01

Pentobarbital serum concentrations associated with a high-dose therapeutic regimen were determined using EMIT immunoassay reagents. Replicate analyses of serum controls resulted in a within-assay coefficient of variation of 5.0% and a between-assay coefficient of variation of 10%. Regression analysis of 44 serum samples analyzed by this technique (y) and a reference procedure (x) were y = 0.98x + 3.6 (r = 0.98; x = ultraviolet spectroscopy) and y = 1.04x + 2.4 (r = 0.96; x = high-performance liquid chromatography). Clinical evaluation of the results indicates the immunoassay is sufficiently sensitive and selective for pentobarbital to allow accurate quantitation within the therapeutic range associated with high-dose therapy.

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

PubMed

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

2012-10-01

Human enteroviruses are the most frequent cause of aseptic meningitis and are involved in other neurological infections. Qualitative detection of enterovirus genomes in cerebrospinal fluid is a prerequisite in diagnosing neurological diseases. The pathogenesis of these infections is not well understood and research in this domain would benefit from the availability of a quantitative technique to determine viral load in clinical specimens. This study describes the development of a real-time RT-qPCR assay using hydrolysis TaqMan probe and a competitive RNA internal control. The assay has high specificity and can be used for a large sample of distinct enterovirus strains and serotypes. The reproducible limit of detection was estimated at 1875 copies/ml of quantitative standards composed of RNA transcripts obtained from a cloned echovirus 30 genome. Technical performance was unaffected by the introduction of a competitive RNA internal control before RNA extraction. The mean enterovirus RNA concentration in an evaluation series of 15 archived cerebrospinal fluid specimens was determined at 4.78 log(10)copies/ml for the overall sample. The sensitivity and reproducibility of the real time RT-qPCR assay used in combination with the internal control to monitor the overall specimen process make it a valuable tool with applied research into enterovirus infections. Copyright © 2012 Elsevier B.V. All rights reserved.

Heterogeneous Antibody-Based Activity Assay for Lysine Specific Demethylase 1 (LSD1) on a Histone Peptide Substrate.

PubMed

Schmitt, Martin L; Ladwein, Kathrin I; Carlino, Luca; Schulz-Fincke, Johannes; Willmann, Dominica; Metzger, Eric; Schilcher, Pierre; Imhof, Axel; Schüle, Roland; Sippl, Wolfgang; Jung, Manfred

2014-07-01

Posttranslational modifications of histone tails are very important for epigenetic gene regulation. The lysine-specific demethylase LSD1 (KDM1A/AOF2) demethylates in vitro predominantly mono- and dimethylated lysine 4 on histone 3 (H3K4) and is a promising target for drug discovery. We report a heterogeneous antibody-based assay, using dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) for the detection of LSD1 activity. We used a biotinylated histone 3 peptide (amino acids 1-21) with monomethylated lysine 4 (H3K4me) as the substrate for the detection of LSD1 activity with antibody-mediated quantitation of the demethylated product. We have successfully used the assay to measure the potency of reference inhibitors. The advantage of the heterogeneous format is shown with cumarin-based LSD1 inhibitor candidates that we have identified using virtual screening. They had shown good potency in an established LSD1 screening assay. The new heterogeneous assay identified them as false positives, which was verified using mass spectrometry. © 2014 Society for Laboratory Automation and Screening.

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

PubMed

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

2016-01-01

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

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

PubMed

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

2015-01-01

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

Optical assays based on colloidal inorganic nanoparticles.

PubMed

Ghasemi, Amir; Rabiee, Navid; Ahmadi, Sepideh; Hashemzadeh, Shabnam; Lolasi, Farshad; Bozorgomid, Mahnaz; Kalbasi, Alireza; Nasseri, Behzad; Shiralizadeh Dezfuli, Amin; Aref, Amir Reza; Karimi, Mahdi; Hamblin, Michael R

2018-06-20

Colloidal inorganic nanoparticles have wide applications in the detection of analytes and in biological assays. A large number of these assays rely on the ability of gold nanoparticles (AuNPs, in the 20 nm diameter size range) to undergo a color change from red to blue upon aggregation. AuNP assays can be based on cross-linking, non-cross linking or unmodified charge-based aggregation. Nucleic acid-based probes, monoclonal antibodies, and molecular-affinity agents can be attached by covalent or non-covalent means. Surface plasmon resonance and SERS techniques can be utilized. Silver NPs also have attractive optical properties (higher extinction coefficient). Combinations of AuNPs and AgNPs in nanocomposites can have additional advantages. Magnetic NPs and ZnO, TiO2 and ZnS as well as insulator NPs including SiO2 can be employed in colorimetric assays, and some can act as peroxidase mimics in catalytic applications. This review covers the synthesis and stabilization of inorganic NPs and their diverse applications in colorimetric and optical assays for analytes related to environmental contamination (metal ions and pesticides), and for early diagnosis and monitoring of diseases, using medically important biomarkers.

Comparison of the CMV brite turbo assay and the digene hybrid capture CMV DNA (Version 2.0) assay for quantitation of cytomegalovirus in renal transplant recipients.

PubMed

Ho, S K; Li, F K; Lai, K N; Chan, T M

2000-10-01

We compared the CMV Brite Turbo Kit (BT) and the Digene Hybrid Capture CMV DNA (version 2.0) assay (HC2) in the quantitation of pp65 antigenemia and cytomegalovirus (CMV) DNA levels in immunosuppressed renal transplant recipients. Of 123 blood specimens collected from 24 renal transplant recipients, BT and HC2 assays detected 35 and 39 positive samples, respectively. The overall concordance rate between the two assays was 90%. Discordant results were observed at low levels of viremia, so that 8 samples were HC2 positive but BT negative and another 4 were BT positive but HC2 negative. There was good correlation (R(2) = 0.766; P<0.01) between the levels of CMV DNA and pp65 antigenemia in the 31 concordant positive samples. Correlation between results obtained with the two assays was confirmed by longitudinal studies for a patient who developed clinical CMV disease. HC2 may be more sensitive at low viremia levels and allow earlier detection of impending CMV disease. The BT assay offered the advantage of a rapid (2-h) turnaround time. We conclude that BT and HC2 assays have similar sensitivity and efficacy in the diagnosis and monitoring of CMV infection and disease in renal transplant recipients. While the HC2 assay would be appropriate for centers that handle a large number of samples, the BT test may be more suitable for small sample numbers or when results are needed urgently.

Impedance-based cellular assay technologies: recent advances, future promise.

PubMed

McGuinness, Ryan

2007-10-01

Cell-based assays are continuing to grow in importance in the drug discovery workflow. Their early introduction holds the promise of limiting attrition in the later, more costly phases of the process. This article reviews recent advances in the development of impedance technologies for label-free cell-based assays. These systems are capable of monitoring endogenous receptor activation, and thus generate more physiologically relevant measures of pharmacological endpoints. Primary cells can be investigated as well, thus producing disease relevant information. Label-free assays significantly decrease assay development efforts and avoid many complications inherent in recombinant readout systems. Impedance-based systems have great potential to advance the utility of cell-based assays as they are applied to drug discovery and pharmacology.

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

PubMed Central

Li, Wenli; Drake, Mary Anne

2001-01-01

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

A novel pyrogallol red-based assay to assess catalase activity: Optimization by response surface methodology.

PubMed

Abderrahim, Mohamed; Arribas, Silvia M; Condezo-Hoyos, Luis

2017-05-01

Pyrogallol red (PGR) was identified as a novel optical probe for the detection of hydrogen peroxide (H 2 O 2 ) based on horseradish peroxidase (HRP)-catalyzed oxidation. Response surface methodology (RSM) was applied as a tool to optimize the concentrations of PGR (100µmolL -1 ), HRP (1UmL -1 ) and H 2 O 2 (250µmolL -1 ) and used to develop a sensitive PGR-based catalase (CAT) activity assay (PGR-CAT assay). N-ethylmaleimide -NEM- (102mmolL -1 ) was used to avoid interference produced by thiol groups while protecting CAT activity. Incubation time (30min) for samples or CAT used as standard and H 2 O 2 as well as signal stability (stable between 5 and 60min) were also evaluated. PGR-CAT assay was linear within the range of 0-4UmL -1 (R 2 =0.993) and very sensitive with limits of detection (LOD) of 0.005UmL -1 and quantitation (LOQ) of 0.01UmL -1 . PGR-CAT assay showed an adequate intra-day RSD=0.6-9.5% and inter-day RSD=2.4-8.9%. Bland-Altman analysis and Passing-Bablok and Pearson correlation analysis showed good agreement between CAT activity as measured by the PRG-CAT assay and the Amplex Red assay. The PGR-CAT assay is more sensitive than all the other colorimetric assays reported, particularly the Amplex Red assay, and the cost of PGR is a small fraction (about 1/1000) of that of an Amplex Red probe, so it can be expected to find wide use among scientists studying CAT activity in biological samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Multiplexed profiling of GPCR activities by combining split TEV assays and EXT-based barcoded readouts.

PubMed

Galinski, Sabrina; Wichert, Sven P; Rossner, Moritz J; Wehr, Michael C

2018-05-25

G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and are implicated in the physiological regulation of many biological processes. The high diversity of GPCRs and their physiological functions make them primary targets for therapeutic drugs. For the generation of novel compounds, however, selectivity towards a given target is a critical issue in drug development as structural similarities between members of GPCR subfamilies exist. Therefore, the activities of multiple GPCRs that are both closely and distantly related to assess compound selectivity need to be tested simultaneously. Here, we present a cell-based multiplexed GPCR activity assay, termed GPCRprofiler, which uses a β-arrestin recruitment strategy and combines split TEV protein-protein interaction and EXT-based barcode technologies. This approach enables simultaneous measurements of receptor activities of multiple GPCR-ligand combinations by applying massively parallelized reporter assays. In proof-of-principle experiments covering 19 different GPCRs, both the specificity of endogenous agonists and the polypharmacological effects of two known antipsychotics on GPCR activities were demonstrated. Technically, normalization of barcode reporters across individual assays allows quantitative pharmacological assays in a parallelized manner. In summary, the GPCRprofiler technique constitutes a flexible and scalable approach, which enables simultaneous profiling of compound actions on multiple receptor activities in living cells.

SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes.

PubMed

Tien, Wei-Ping; Lim, Gareth; Yeo, Gladys; Chiang, Suzanna Nicole; Chong, Chee-Seng; Ng, Lee-Ching; Hapuarachchi, Hapuarachchige Chanditha

2017-09-19

The monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures. We developed a rapid, sensitive and specific real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the detection of ZIKV in field-caught mosquitoes. The primers and PCR cycling conditions were optimized to minimize non-specific amplification due to cross-reactivity with the genomic material of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex tritaeniorhynchus, Culex sitiens and Anopheles sinensis, as well as accompanying microbiota. The performance of the assay was further evaluated with a panel of flaviviruses and alphaviruses as well as in field-caught Ae. aegypti mosquitoes confirmed to be positive for ZIKV. As compared to a probe-based assay, the newly developed assay demonstrated 100% specificity and comparable detection sensitivity for ZIKV in mosquitoes. Being a SYBR Green-based method, the newly-developed assay is cost-effective and easy to adapt, thus is applicable to large-scale vector surveillance activities in endemic countries, including those with limited resources and expertise. The amplicon size (119 bp) also allows sequencing to confirm the virus type. The primers flank relatively conserved regions of ZIKV genome, so that, the assay is able to detect genetically diverse ZIKV strains. Our findings, therefore, testify the potential use of the newly-developed assay in vector surveillance programmes for ZIKV in endemic regions.

Colorimetric micro-assay for accelerated screening of mould inhibitors

Treesearch

Carol A. Clausen; Vina W. Yang

2013-01-01

Since current standard laboratory methods are time-consuming macro-assays that rely on subjective visual ratings of mould growth, rapid and quantitative laboratory methods are needed to screen potential mould inhibitors for use in and on cellulose-based products. A colorimetric micro-assay has been developed that uses XTT tetrazolium salt to enzymatically assess...

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

EPA Science Inventory

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

NCI Launches Proteomics Assay Portal | Office of Cancer Clinical Proteomics Research

Cancer.gov

In a paper recently published by the journal Nature Methods, Investigators from the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (NCI-CPTAC) announced the launch of a proteomics Assay Portal for multiple reaction monitoring-mass spectrometry (MRM-MS) assays.  This community web-based repository for well-characterized quantitative proteomic assays currently consists of 456 unique peptide assays to 282 unique proteins and ser

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

PubMed Central

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

2015-01-01

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. PMID:25588653

An MRM-Based Cytokeratin Marker Assay as a Tool for Cancer Studies: Application to Lung Cancer Pleural Effusions.

PubMed

Perzanowska, Anna; Fatalska, Agnieszka; Wojtas, Grzegorz; Lewandowicz, Andrzej; Michalak, Agata; Krasowski, Grzegorz; Borchers, Christoph H; Dadlez, Michal; Domanski, Dominik

2018-03-01

The goal of this work was to develop an LC-MRM assay for the quantitative analysis of a set of established and diagnostically important cytokeratin (CK) markers used in cancer diagnosis, prognosis, and therapy monitoring. Second, the potential of this assay in lung cancer diagnosis through pleural effusion (PE) analysis was examined. A multiplexed MRM assay was developed for 17 CKs and their select caspase-cleaved fragments. Isotope-labeled standard peptides were used for high assay specificity and absolute peptide quantitation; with robust standard-flow LC coupled to a latest-generation triple-quadrupole instrument for high sensitivity. The potential clinical applicability was demonstrated by the analysis of 118 PE samples. The MRM assay was evaluated for endogenous detection, linearity, precision, upper and lower limits of quantification, selectivity, reproducibility and peptide stability, and is generally applicable to any epithelial cancer study. A set of 118 patients with known pathologies allowed us to define the range of CK levels in clinical PE samples. Specific CKs were able to differentiate cancer-related PEs from those caused by benign ailments. In addition, they allowed to differentiate between PEs from subjects with small cell lung cancer versus non-small cell lung carcinoma, and to further differentiate the latter into its two subtypes, adenocarcinoma and squamous cell carcinoma. An MRM-based CK assay for carcinoma studies can differentiate between the three lung cancer histological types using less-invasive PE sampling providing potential therapy-guiding information on patients that are inoperable. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Luciferase Functional Quantitative Assay for Measuring NF-ĸB Promoter Transactivation Mediated by HTLV-1 and HTLV-2 Tax Proteins.

PubMed

Bergamo, Elisa; Diani, Erica; Bertazzoni, Umberto; Romanelli, Maria Grazia

2017-01-01

HTLV-1 and HTLV-2 viruses express Tax transactivator proteins required for viral genome transcription and capable of transforming cells in vivo and in vitro. Although Tax oncogenic potential needs to be further elucidated, it is well established that Tax proteins activate, among others, transcription factors of the NF-ĸB family, which are involved in immune and inflammatory responses, cell growth, apoptosis, stress responses and oncogenesis. Here, we describe a reporter gene assay applied for quantitative analysis of Tax-dependent NF-ĸB activation. The procedure is based on co-transfection of two individual vectors containing the cDNA for firefly and Renilla luciferase enzymes and vectors expressing Tax proteins. The luciferase expression is driven by cis-NF-ĸB promoter regulatory elements responsive to Tax transactivating factor. This assay is particularly useful to investigate Tax influence on NF-ĸB activation mediated by viral or host factors.

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

USDA-ARS?s Scientific Manuscript database

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

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

NASA Technical Reports Server (NTRS)

Bush, V. N.

1974-01-01

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

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

USGS Publications Warehouse

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

2013-01-01

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

A novel robust quantitative Förster resonance energy transfer assay for protease SENP2 kinetics determination against its all natural substrates.

PubMed

Liu, Yan; Shen, Yali; Zheng, Shasha; Liao, Jiayu

2015-12-01

SUMOylation (the process of adding the SUMO [small ubiquitin-like modifier] to substrates) is an important post-translational modification of critical proteins in multiple processes. Sentrin/SUMO-specific proteases (SENPs) act as endopeptidases to process the pre-SUMO or as isopeptidases to deconjugate the SUMO from its substrate. Determining the kinetics of SENPs is important for understanding their activities. Förster resonance energy transfer (FRET) technology has been widely used in biomedical research and is a powerful tool for elucidating protein interactions. In this paper we report a novel quantitative FRET-based protease assay for SENP2 endopeptidase activity that accounts for the self-fluorescent emissions of the donor (CyPet) and the acceptor (YPet). The kinetic parameters, k(cat), K(M), and catalytic efficiency (k(cat)/K(M)) of catalytic domain SENP2 toward pre-SUMO1/2/3, were obtained by this novel design. Although we use SENP2 to demonstrate our method, the general principles of this quantitative FRET-based protease kinetic determination can be readily applied to other proteases.

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

PubMed

Berzofsky, Ronald N.

1995-01-01

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

Development of in vitro and in vivo neutralization assays based on the pseudotyped H7N9 virus.

PubMed

Tian, Yabin; Zhao, Hui; Liu, Qiang; Zhang, Chuntao; Nie, Jianhui; Huang, Weijing; Li, Changgui; Li, Xuguang; Wang, Youchun

2018-05-31

H7N9 viral infections pose a great threat to both animal and human health. This avian virus cannot be handled in level 2 biocontainment laboratories, substantially hindering evaluation of prophylactic vaccines and therapeutic agents. Here, we report a high-titer pseudoviral system with a bioluminescent reporter gene, enabling us to visually and quantitatively conduct analyses of virus replications in both tissue cultures and animals. For evaluation of immunogenicity of H7N9 vaccines, we developed an in vitro assay for neutralizing antibody measurement based on the pseudoviral system; results generated by the in vitro assay were found to be strongly correlated with those by either hemagglutination inhibition (HI) or micro-neutralization (MN) assay. Furthermore, we injected the viruses into Balb/c mice and observed dynamic distributions of the viruses in the animals, which provides an ideal imaging model for quantitative analyses of prophylactic and therapeutic monoclonal antibodies. Taken together, the pseudoviral systems reported here could be of great value for both in vitro and in vivo evaluations of vaccines and antiviral agents without the need of wild type H7N9 virus.

Gold nanoparticles-based protease assay

PubMed Central

Guarise, Cristian; Pasquato, Lucia; De Filippis, Vincenzo; Scrimin, Paolo

2006-01-01

We describe here a simple assay that allows the visual detection of a protease. The method takes advantage of the high molar absorptivity of the plasmon band of gold colloids and is based on the color change of their solution when treated with dithiols. We used C- and N-terminal cysteinyl derivatives of a peptide substrate exploiting its selective recognition and cleavage by a specific protease. Contrary to the native ones, cleaved peptides are unable to induce nanoparticles aggregation; hence, the color of the solution does not change. The detection of two proteases is reported: thrombin (involved in blood coagulation and thrombosis) and lethal factor (an enzyme component of the toxin produced by Bacillus anthracis). The sensitivity of this nanoparticle-based assay is in the low nanomolar range. PMID:16537471

Gold nanoparticles-based protease assay.

PubMed

Guarise, Cristian; Pasquato, Lucia; De Filippis, Vincenzo; Scrimin, Paolo

2006-03-14

We describe here a simple assay that allows the visual detection of a protease. The method takes advantage of the high molar absorptivity of the plasmon band of gold colloids and is based on the color change of their solution when treated with dithiols. We used C- and N-terminal cysteinyl derivatives of a peptide substrate exploiting its selective recognition and cleavage by a specific protease. Contrary to the native ones, cleaved peptides are unable to induce nanoparticles aggregation; hence, the color of the solution does not change. The detection of two proteases is reported: thrombin (involved in blood coagulation and thrombosis) and lethal factor (an enzyme component of the toxin produced by Bacillus anthracis). The sensitivity of this nanoparticle-based assay is in the low nanomolar range.

Detection and quantification of serum or plasma HCV RNA: mini review of commercially available assays.

PubMed

Le Guillou-Guillemette, Helene; Lunel-Fabiani, Francoise

2009-01-01

The treatment schedule (combination of compounds, doses, and duration) and the virological follow-up for management of antiviral treatment in patients chronically infected by HCV is now well standardized, but to ensure good monitoring of the treated patients, physicians need rapid, reproducible, and sensitive molecular virological tools with a wide range of detection and quantification of HCV RNA in blood samples. Several assays for detection and/or quantification of HCV RNA are currently commercially available. Here, all these assays are detailed, and a brief description of each step of the assay is provided. They are divided into two categories by method: those based on signal amplification and those based on target amplification. These two categories are then divided into qualitative, quantitative, and quantitative detection assays. The real-time reverse-transcription polymerase chain reaction (RT-PCR)-based assays are the most promising strategy in the HCV virological area.

Analysis of Gold Ores by Fire Assay

ERIC Educational Resources Information Center

Blyth, Kristy M.; Phillips, David N.; van Bronswijk, Wilhelm

2004-01-01

Students of an Applied Chemistry degree course carried out a fire-assay exercise. The analysis showed that the technique was a worthwhile quantitative analytical technique and covered interesting theory including acid-base and redox chemistry and other concepts such as inquarting and cupelling.

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

PubMed

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

2016-11-01

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

A real-time RT-PCR assay for molecular identification and quantitation of feline morbillivirus RNA from biological specimens.

PubMed

De Luca, Eliana; Crisi, Paolo Emidio; Di Domenico, Marco; Malatesta, Daniela; Vincifori, Giacomo; Di Tommaso, Morena; Di Guardo, Giovanni; Di Francesco, Gabriella; Petrini, Antonio; Savini, Giovanni; Boari, Andrea; Lorusso, Alessio

2018-05-03

The aim of this study was to develop a real-time RT-PCR to detect and quantitate feline morbillivirus (FeMV) RNA in biological samples. Primers and probe were targeted on a conserved region of FeMV P/V/C gene. To validate the assay with field samples, a total number of specimens of cats have been recruited including 264 urine and blood samples and compared with a generic RT-PCR targeting the L protein encoding gene of morbilliviruses. In addition, 385 tissue samples from 35 carcasses of cats have been also employed. RNA titres were low in all tested samples. Results also indicated the absence of cross-reaction with related morbilliviruses and existing pathogens of cats. In tissues with low levels of FeMV RNA, the presence of viral antigen was also evidenced by immunohistochemistry targeting the N viral protein. This newly described assay allows for a rapid, accurate and reliable quantitative detection of FeMV RNA that can be applied for diagnostics and research studies. Copyright © 2018 Elsevier B.V. All rights reserved.

Parallel force assay for protein-protein interactions.

PubMed

Aschenbrenner, Daniela; Pippig, Diana A; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E

2014-01-01

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.

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

PubMed Central

Sharma, Vasundhara; Jordan, Jennifer J.; Ciribilli, Yari; Resnick, Michael A.; Bisio, Alessandra; Inga, Alberto

2015-01-01

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. PMID:26147604

DEVELOPMENT OF CRASSPHAGE-BASED QPCR ASSAYS ...

EPA Pesticide Factsheets

A newly discovered bacteriophage, “crAssphage”, is predicted to be both highlyabundant and predominantly human-associated, both ideal characteristics for a human-specific fecal indicator. A total of 384 end-point PCR primers were designed along the length of the crAssphage genome, eliminating regions suspected to be hypervariable or react with other animal sources. The primer pairs were rigorously tested in three rounds of screening for specificity, geographic variability, limit of detection, and environmental water performance. The two best performing assays, crAss056 and crAss064, were adapted to a qPCR platform and exhibited a specificity of 98.0% and 98.9%, respectively. The markers’ abundance was compared with two bacterial based assays and were found at concentrations at or above the bacterial based assays in wastewater influent and impacted environmental waters. This poster will present the methodology of the novel marker development and the potential uses for this technology in maintaining sustainable waterways in the future. To inform the public.

Photon upconversion in homogeneous fluorescence-based bioanalytical assays.

PubMed

Soukka, Tero; Rantanen, Terhi; Kuningas, Katri

2008-01-01

Upconverting phosphors (UCPs) are very attractive reporters for fluorescence resonance energy transfer (FRET)-based bioanalytical assays. The large anti-Stokes shift and capability to convert near-infrared to visible light via sequential absorption of multiple photons enable complete elimination of autofluorescence, which commonly impairs the performance of fluorescence-based assays. UCPs are ideal donors for FRET, because their very narrow-banded emission allows measurement of the sensitized acceptor emission, in principle, without any crosstalk from the donor emission at a wavelength just tens of nanometers from the emission peak of the donor. In addition, acceptor dyes emitting at visible wavelengths are essentially not excited by near-infrared, which further emphasizes the unique potential of upconversion FRET (UC-FRET). These characteristics result in favorable assay performance using detection instrumentation based on epifluorometer configuration and laser diode excitation. Although UC-FRET is a recently emerged technology, it has already been applied in both immunoassays and nucleic acid hybridization assays. The technology is also compatible with optically difficult biological samples, such as whole blood. Significant advances in assay performance are expected using upconverting lanthanide-doped nanocrystals, which are currently under extensive research. UC-FRET, similarly to other fluorescence techniques based on resonance energy transfer, is strongly distance dependent and may have limited applicability, for example in sandwich-type assays for large biomolecules, such as viruses. In this article, we summarize the essentials of UC-FRET, describe its current applications, and outline the expectations for its future potential.

Evaluation of a real-time PCR assay based on the single-copy SAG1 gene for the detection of Toxoplasma gondii.

PubMed

Yu, Haijie; Huang, Bin; Zhuo, Xunhui; Chen, Xueqiu; Du, Aifang

2013-11-08

Real-time PCR-based detection of Toxoplasma gondii is very sensitive and convenient for diagnosing toxoplasmosis. However, the performance of the PCR assays could be influenced by the target gene chosen. Here we evaluate a real-time PCR assay using double-stranded DNA dyes (SYBR(®) Green I assay) with a new set of primers targeting the SAG1 gene for the fast and specific detection of T. gondii. The assay showed higher sensitivity than conventional PCR protocols using T. gondii DNA as template. The detection limit of the developed real-time PCR assay was in the order of 1 tachyzoite. The assay was also assessed by experimentally infected mice and showed positive results for blood (25%), spleen (50%) and lung (50%) as early as 1 dpi. The specificity of the assay was confirmed by using DNA from Neospora caninum, Escherichia coli, Babesia bovis, Trypanosoma brucei, Cryptosporidium parvum, and Toxocara canis. Assay applicability was successfully tested in blood samples collected from slaughtered pigs. These results indicate that, based on SYBR(®) green I, the quantitative SAG1 assay may also be useful in the study of the pathogenicity, immunoprophylaxis, and treatment of T. gondii. Copyright © 2013 Elsevier B.V. All rights reserved.

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

PubMed

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

2010-12-01

A growing concern over endocrine disruption in aquatic species has prompted the development of molecular assays to monitor environmental impacts. This study describes the development of quantitative polymerase chain reaction (qPCR) assays to characterize the expression of two vitellogenin (Vtg) genes in the invasive round goby (Neogobius melanostomus). Fragments from the 18SrRNA (housekeeping gene), Vtg II, and Vtg III genes were cloned and sequenced. The qPCR assays were developed to detect hepatic Vtg expression in goby. The assays detected induction of both Vtg genes in nonreproductive males following a two-week laboratory exposure to 17β-estradiol (≥1 mg/kg i.p. injection). The assays were applied to goby from Hamilton Harbour, Lake Ontario (Canada), including those from sites where feminization and intersex of goby has been documented. Both Vtg genes had significantly higher expression in females compared to males. Male reproductive goby adopt either parental or sneaker tactics; Vtg II expression was higher in sneaker than in parental males but parental and nonreproductive males did not differ from each other. The Vtg III expression was significantly higher in sneaker males followed by parental males and nonreproductive males, respectively. The Vtg II and III expression in nonreproductive males was elevated in the contaminated site with documented intersex. This assay provides an important tool for the use of an invasive species in monitoring endocrine disruption in the Great Lakes region. Copyright © 2010 SETAC.

Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

PubMed

Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

2015-02-21

This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays.

PubMed

Blagojević Zagorac, Gordana; Mahmutefendić, Hana; Maćešić, Senka; Karleuša, Ljerka; Lučin, Pero

2017-03-01

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 232: 463-476, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Pathway Based Toxicology and Fit-for-Purpose Assays.

PubMed

Clewell, Rebecca A; McMullen, Patrick D; Adeleye, Yeyejide; Carmichael, Paul L; Andersen, Melvin E

The field of toxicity testing for non-pharmaceutical chemicals is in flux with multiple initiatives in North America and the EU to move away from animal testing to mode-of-action based in vitro assays. In this arena, there are still obstacles to overcome, such as developing appropriate cellular assays, creating pathway-based dose-response models and refining in vitro-in vivo extrapolation (IVIVE) tools. Overall, it is necessary to provide assurances that these new approaches are adequately protective of human and ecological health. Another major challenge for individual scientists and regulatory agencies is developing a cultural willingness to shed old biases developed around animal tests and become more comfortable with mode-of-action based assays in human cells. At present, most initiatives focus on developing in vitro alternatives and assessing how well these alternative methods reproduce past results related to predicting organism level toxicity in intact animals. The path forward requires looking beyond benchmarking against high dose animal studies. We need to develop targeted cellular assays, new cell biology-based extrapolation models for assessing regions of safety for chemical exposures in human populations, and mode-of-action-based approaches which are constructed on an understanding of human biology. Furthermore, it is essential that assay developers have the flexibility to 'validate' against the most appropriate mode-of-action data rather than against apical endpoints in high dose animal studies. This chapter demonstrates the principles of fit-for-purpose assay development using pathway-targeted case studies. The projects include p53-mdm2-mediated DNA-repair, estrogen receptor-mediated cell proliferation and PPARα receptor-mediated liver responses.

A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina.

PubMed

Buling, A; Criado-Fornelio, A; Asenzo, G; Benitez, D; Barba-Carretero, J C; Florin-Christensen, M

2007-06-20

The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome b gene, this method shows both high sensitivity and specificity, and allows quantification of parasite DNA. In tests, reproducible quantitative results were obtained over the range of 0.1 ng to 0.1 fg of parasite DNA. Melting curve analysis differentiated between B. bovis and B. bigemina. To assess the performance of the new qPCR procedure it was used to screen for babesiosis in 40 cows and 80 horses. B. bigemina was detected in five cows (three of these were also found to be positive by standard PCR techniques targeting the 18S rRNA gene). In addition, B. bovis was detected in one horse and B. bigemina in two horses using the proposed method, while none was found positive by ribosomal standard PCR. The sequences of the B. bigemina cytochrome b and 18S rRNA genes were completely conserved in isolates from Spain and Argentina, while those of B. bovis showed moderate polymorphism.

Multiplexed MRM-based quantitation of candidate cancer biomarker proteins in undepleted and non-enriched human plasma.

PubMed

Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Borchers, Christoph H

2013-07-01

An emerging approach for multiplexed targeted proteomics involves bottom-up LC-MRM-MS, with stable isotope-labeled internal standard peptides, to accurately quantitate panels of putative disease biomarkers in biofluids. In this paper, we used this approach to quantitate 27 candidate cancer-biomarker proteins in human plasma that had not been treated by immunoaffinity depletion or enrichment techniques. These proteins have been reported as biomarkers for a variety of human cancers, from laryngeal to ovarian, with breast cancer having the highest correlation. We implemented measures to minimize the analytical variability, improve the quantitative accuracy, and increase the feasibility and applicability of this MRM-based method. We have demonstrated excellent retention time reproducibility (median interday CV: 0.08%) and signal stability (median interday CV: 4.5% for the analytical platform and 6.1% for the bottom-up workflow) for the 27 biomarker proteins (represented by 57 interference-free peptides). The linear dynamic range for the MRM assays spanned four orders-of-magnitude, with 25 assays covering a 10(3) -10(4) range in protein concentration. The lowest abundance quantifiable protein in our biomarker panel was insulin-like growth factor 1 (calculated concentration: 127 ng/mL). Overall, the analytical performance of this assay demonstrates high robustness and sensitivity, and provides the necessary throughput and multiplexing capabilities required to verify and validate cancer-associated protein biomarker panels in human plasma, prior to clinical use. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Optical assay for biotechnology and clinical diagnosis.

PubMed

Moczko, Ewa; Cauchi, Michael; Turner, Claire; Meglinski, Igor; Piletsky, Sergey

2011-08-01

In this paper, we present an optical diagnostic assay consisting of a mixture of environmental-sensitive fluorescent dyes combined with multivariate data analysis for quantitative and qualitative examination of biological and clinical samples. The performance of the assay is based on the analysis of spectrum of the selected fluorescent dyes with the operational principle similar to electronic nose and electronic tongue systems. This approach has been successfully applied for monitoring of growing cell cultures and identification of gastrointestinal diseases in humans.

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

PubMed

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

2017-08-01

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

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

PubMed

Kruse, Niels; Persson, Staffan; Alcolea, Daniel; Bahl, Justyna M C; Baldeiras, Ines; Capello, Elisabetta; Chiasserini, Davide; Bocchio Chiavetto, Luisella; Emersic, Andreja; Engelborghs, Sebastiaan; Eren, Erden; Fladby, Tormod; Frisoni, Giovanni; García-Ayllón, María-Salud; Genc, Sermin; Gkatzima, Olymbia; Heegaard, Niels H H; Janeiro, André M; Kováčech, Branislav; Kuiperij, H Bea; Leitão, Maria J; Lleó, Alberto; Martins, Madalena; Matos, Mafalda; Mollergard, Hanne M; Nobili, Flavio; Öhrfelt, Annika; Parnetti, Lucilla; de Oliveira, Catarina Resende; Rot, Uros; Sáez-Valero, Javier; Struyfs, Hanne; Tanassi, Julia T; Taylor, Peggy; Tsolaki, Magda; Vanmechelen, Eugeen; Verbeek, Marcel M; Zilka, Norbert; Blennow, Kaj; Zetterberg, Henrik; Mollenhauer, Brit

2015-09-01

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 the same samples and same lots of assays were applied. We further demonstrate that although absolute concentrations differ between laboratories the quantitative results are comparable. With further standardization this assay may become an attractive tool for comparing aSyn measurements in diverse settings. Recommendations for further validation experiments and improvement of the interlaboratory results obtained are given. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed Central

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

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. PMID:22739263

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

PubMed

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

2018-01-01

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

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

PubMed Central

Hajeri, Subhas; Chen, Jianchi; McCollum, Thomas Greg

2018-01-01

Huanglongbing (HLB, citrus greening) is a devastating citrus disease affecting citrus production worldwide. It is associated with the bacterium “Candidatus Liberibacter asiaticus” (CLas) and is vectored by the Asian citrus psyllid (ACP). Currently, diagnosis of CLas in regulatory samples is based on real-time quantitative polymerase chain reaction (qPCR) using 16S rRNA gene specific primers/probe. The detection of CLas using qPCR is challenging due to low pathogen titer and uneven distribution in infected plants and exacerbated by sampling issues and presence of inhibitors. This study evaluated a duplex droplet digital polymerase chain reaction (ddPCR) using multi-copy gene targets, 16S and RNR, to simultaneously detect CLas DNA targets in the same sample for unambiguous detection of the HLB pathogen in DNA extracts from citrus leaves and ACP. Standard curve analyses on tenfold dilution series with plasmid, citrus leaf and ACP DNA showed that both ddPCR and qPCR exhibited good linearity and efficiency in the duplex assay. CLas-infected low titer samples were used to validate the duplex ddPCR and qPCR performance and demonstrated that detection rate is higher when both 16S and RNR primers were used in duplex assay. However, the receiver operating characteristic analysis indicated that area under the curve for RNR primer was significantly broader, compared to 16S primers for CLas detection at low target titer. The absolute quantification of CLas at variable titers was reproducible and repeatable for both primer sets and the ddPCR showed higher resilience to PCR inhibitors with citrus leaf and ACP extracts. Hence, the resultant duplex ddPCR assay resulted in a significantly improved detection platform for diagnosis of CLas in samples with low pathogen titer. PMID:29772016

Development of an LC-MS based enzyme activity assay for MurC: application to evaluation of inhibitors and kinetic analysis.

PubMed

Deng, Gejing; Gu, Rong-Fang; Marmor, Stephen; Fisher, Stewart L; Jahic, Haris; Sanyal, Gautam

2004-06-29

An enzyme activity assay, based on mass spectrometric (MS) detection of specific reaction product following HPLC separation, has been developed to evaluate pharmaceutical hits identified from primary high throughput screening (HTS) against target enzyme Escherichia coli UDP-N-acetyl-muramyl-L-alanine ligase (MurC), an essential enzyme in the bacterial peptidoglycan biosynthetic pathway, and to study the kinetics of the enzyme. A comparative analysis of this new liquid chromatographic-MS (LC-MS) based assay with a conventional spectrophotometric Malachite Green (MG) assay, which detects phosphate produced in the reaction, was performed. The results demonstrated that the LC-MS assay, which determines specific ligase activity of MurC, offers several advantages including a lower background (0.2% versus 26%), higher sensitivity (> or = 10 fold), lower limit of quantitation (LOQ) (0.02 microM versus 1 microM) and wider linear dynamic range (> or = 4 fold) than the MG assay. Good precision for the LC-MS assay was demonstrated by the low intraday and interday coefficient of variation (CV) values (3 and 6%, respectively). The LC-MS assay, free of the artifacts often seen in the Malachite Green assay, offers a valuable secondary assay for hit evaluation in which the false positives from the primary high throughput screening can be eliminated. In addition, the applicability of this assay to the study of enzyme kinetics has also been demonstrated. Copyright 2004 Elsevier B.V.

SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries*

PubMed Central

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

2016-01-01

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

Large-Scale Interlaboratory Study to Develop, Analytically Validate and Apply Highly Multiplexed, Quantitative Peptide Assays to Measure Cancer-Relevant Proteins in Plasma*

PubMed Central

Abbatiello, Susan E.; Schilling, Birgit; Mani, D. R.; Zimmerman, Lisa J.; Hall, Steven C.; MacLean, Brendan; Albertolle, Matthew; Allen, Simon; Burgess, Michael; Cusack, Michael P.; Gosh, Mousumi; Hedrick, Victoria; Held, Jason M.; Inerowicz, H. Dorota; Jackson, Angela; Keshishian, Hasmik; Kinsinger, Christopher R.; Lyssand, John; Makowski, Lee; Mesri, Mehdi; Rodriguez, Henry; Rudnick, Paul; Sadowski, Pawel; Sedransk, Nell; Shaddox, Kent; Skates, Stephen J.; Kuhn, Eric; Smith, Derek; Whiteaker, Jeffery R.; Whitwell, Corbin; Zhang, Shucha; Borchers, Christoph H.; Fisher, Susan J.; Gibson, Bradford W.; Liebler, Daniel C.; MacCoss, Michael J.; Neubert, Thomas A.; Paulovich, Amanda G.; Regnier, Fred E.; Tempst, Paul; Carr, Steven A.

2015-01-01

There is an increasing need in biology and clinical medicine to robustly and reliably measure tens to hundreds of peptides and proteins in clinical and biological samples with high sensitivity, specificity, reproducibility, and repeatability. Previously, we demonstrated that LC-MRM-MS with isotope dilution has suitable performance for quantitative measurements of small numbers of relatively abundant proteins in human plasma and that the resulting assays can be transferred across laboratories while maintaining high reproducibility and quantitative precision. Here, we significantly extend that earlier work, demonstrating that 11 laboratories using 14 LC-MS systems can develop, determine analytical figures of merit, and apply highly multiplexed MRM-MS assays targeting 125 peptides derived from 27 cancer-relevant proteins and seven control proteins to precisely and reproducibly measure the analytes in human plasma. To ensure consistent generation of high quality data, we incorporated a system suitability protocol (SSP) into our experimental design. The SSP enabled real-time monitoring of LC-MRM-MS performance during assay development and implementation, facilitating early detection and correction of chromatographic and instrumental problems. Low to subnanogram/ml sensitivity for proteins in plasma was achieved by one-step immunoaffinity depletion of 14 abundant plasma proteins prior to analysis. Median intra- and interlaboratory reproducibility was <20%, sufficient for most biological studies and candidate protein biomarker verification. Digestion recovery of peptides was assessed and quantitative accuracy improved using heavy-isotope-labeled versions of the proteins as internal standards. Using the highly multiplexed assay, participating laboratories were able to precisely and reproducibly determine the levels of a series of analytes in blinded samples used to simulate an interlaboratory clinical study of patient samples. Our study further establishes that LC

Modification of two capripoxvirus quantitative real-time PCR assays to improve diagnostic sensitivity and include beta-actin as an internal positive control.

PubMed

Das, Amaresh; Deng, Ming Y; Babiuk, Shawn; McIntosh, Michael T

2017-05-01

Capripoxviruses (CaPVs), consisting of Sheeppox virus (SPV), Goatpox virus (GPV), and Lumpy skin disease virus (LSDV) species, cause economically significant diseases in sheep, goats, and cattle, respectively. Quantitative real-time polymerase chain reaction (qPCR) assays are routinely used for rapid detection of CaPVs in surveillance and outbreak management programs. We further modified and optimized 2 previously published CaPV qPCR assays, referred to as the Balinsky and Bowden assays, by changing commercial PCR reagents used in the tests. The modified assays displayed 100% analytical specificity and showed no apparent changes in analytical sensitivities for detection of CaPVs compared with the original assays. Diagnostic sensitivities, assessed using 50 clinical reference samples from experimentally infected sheep, goats, and cattle, improved from 82% to 92% for the modified Balinsky assay and from 58% to 82% for the modified Bowden assay. The modified qPCR assays were multiplexed for detection of beta-actin as an indicator for potential false-negative results. The multiplex modified qPCR assays exhibited the same diagnostic sensitivities as the singleplex assays suggesting their utility in the detection of CaPVs.

Molecular diagnostic toolkit for Rhizophagus irregularis isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome.

PubMed

Badri, Amine; Stefani, Franck O P; Lachance, Geneviève; Roy-Arcand, Line; Beaudet, Denis; Vialle, Agathe; Hijri, Mohamed

2016-10-01

Rhizophagus irregularis (previously named Glomus irregulare) is one of the most widespread and common arbuscular mycorrhizal fungal (AMF) species. It has been recovered worldwide in agricultural and natural soils, and the isolate DAOM-197198 has been utilized as a commercial inoculant for two decades. Despite the ecological and economical importance of this taxon, specific markers for quantification of propagules by quantitative real-time PCR (qPCR) are extremely limited and none have been rigorously validated for quality control of manufactured products such as biofertilizers. From the sequencing of 14 complete AMF mitochondrial (mt) genomes, a qPCR assay using a hydrolysis probe designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of R. irregularis isolate DAOM-197198. Specificity tests were performed using standard PCR and qPCR, and results clearly showed that the primers specifically amplified the isolate DAOM-197198, yielding a PCR product of 106 bp. According to the qPCR analyses on spores produced in vitro, the average copy number of mt genomes per spore was 3172 ± 304 SE (n = 6). Quantification assays were successfully undertaken on known and unknown samples in liquid suspensions and commercial dry formulations to show the accuracy, precision, robustness, and reproducibility of the qPCR assay. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate DAOM-197198. The approach of molecular toolkit used in our study could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.

Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples.

PubMed

Pulford, D J; Gias, E; Bueno, I M; McFadden, Amj

2016-01-01

To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis. Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay. The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R(2) = 0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6 × 10(4) and 3.3 × 10(6) genomes per

Development of a Quantitative Sandwich Enzyme-Linked Immunosorbent Assay for Detecting the MPT64 Antigen of Mycobacterium tuberculosis

PubMed Central

Ji, Mijung; Cho, Byungki; Cho, Young Shik; Park, Song-Yong; Cho, Sang-Nae

2014-01-01

Purpose Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. Materials and Methods The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. Results The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×104 CFU/mL and 2.0×106 CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). Conclusion The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis. PMID:24719143

Development of a quantitative sandwich enzyme-linked immunosorbent assay for detecting the MPT64 antigen of Mycobacterium tuberculosis.

PubMed

Ji, Mijung; Cho, Byungki; Cho, Young Shik; Park, Song-Yong; Cho, Sang-Nae; Jeon, Bo-Young; Yoon, Byoung-Su

2014-05-01

Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

Clinical comparison of branched DNA and reverse transcriptase-PCR and nucleic acid sequence-based amplification assay for the quantitation of circulating recombinant form_BC HIV-1 RNA in plasma.

PubMed

Pan, Pinliang; Tao, Xiaoxia; Zhang, Qi; Xing, Wenge; Sun, Xianguang; Pei, Lijian; Jiang, Yan

2007-12-01

To investigate the correlation between three viral load assays for circulating recombinant form (CRF)_BC. Recent studies in HIV-1 molecular epidemiology, reveals that CRF_BC is the dominant subtype of HIV-1 virus in mainland China, representing over 45% of the HIV-1 infected population. The performances of nucleic acid sequence-based amplification (NASBA), branched DNA (bDNA) and reverse transcriptase polymerase chain reaction (RT-PCR) were compared for the HIV-1 viral load detection and quantitation of CRF_BC in China. Sixteen HIV-1 positive and three HIV-1 negative samples were collected. Sequencing of the positive samples in the gp41 region was conducted. The HIV-1 viral load values were determined using bDNA, RT-PCR and NASBA assays. Deming regression analysis with SPSS 12.0 (SPS Inc., Chicago, Illinois, USA) was performed for data analysis. Sequencing and phylogenetic analysis of env gene (gp41) region of the 16 HIV-1 positive clinical specimens from Guizhou Province in southwest China revealed the dominance of the subtype CRF_BC in that region. A good correlation of their viral load values was observed among three assays. Pearson's correlation between RT-PCR and bDNA is 0.969, Lg(VL)RT-PCR = 0.969 * Lg(VL)bDNA + 0.55; Pearson's correlation between RT-PCR and NASBA is 0.968, Lg(VL)RT-PCR = 0.968 * Lg(VL)NASBA + 0.937; Pearson's correlation between NASBA and bDNA is 0.980, Lg(VL)NASBA = 0.980 * Lg(VL)bDNA - 0.318. When testing with 3 different assays, RT-PCR, bDNA and NASBA, the group of 16 HIV-1 positive samples showed the viral load value was highest for RT-PCR, followed by bDNA then NASBA, which is consistent with the former results in subtype B. The three viral load assays are highly correlative for CRF_BC in China.

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

PubMed Central

Albrich, Werner C.; van der Linden, Mark P. G.; Bénet, Thomas; Chou, Monidarin; Sylla, Mariam; Barreto Costa, Patricia; Richard, Nathalie; Klugman, Keith P.; Endtz, Hubert P.; Paranhos-Baccalà, Gláucia; Telles, Jean-Noël

2016-01-01

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. PMID:26986831

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

PubMed Central

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

2012-01-01

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

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

PubMed

Bassler, H A; Flood, S J; Livak, K J; Marmaro, J; Knorr, R; Batt, C A

1995-10-01

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.

Parallel Force Assay for Protein-Protein Interactions

PubMed Central

Aschenbrenner, Daniela; Pippig, Diana A.; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E.

2014-01-01

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. PMID:25546146

Assays for the determination of the activity of DNA nucleases based on the fluorometric properties of the YOYO dye.

PubMed

Fernández-Sierra, Mónica; Quiñones, Edwin

2015-03-15

Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA-protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA-YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect. Copyright © 2015 Elsevier Inc. All rights reserved.

Development of an SRM method for absolute quantitation of MYDGF/C19orf10 protein.

PubMed

Dwivedi, Ravi C; Krokhin, Oleg V; El-Gabalawy, Hani S; Wilkins, John A

2016-06-01

To develop a MS-based selected reaction monitoring (SRM) assay for quantitation of myeloid-derived growth factor (MYDGF) formerly chromosome 19 open reading frame (C19orf10). Candidate reporter peptides were identified in digests of recombinant MYDGF. Isotopically labeled forms of these reporter peptides were employed as internal standards for assay development. Two reference peptides were selected SYLYFQTFFK and GAEIEYAMAYSK with respective LOQ of 42 and 380 attomole per injection. Application of the assay to human serum and synovial fluid determined that the assay sensitivity was reduced and quantitation was not achievable. However, the partial depletion of albumin and immunoglobulin from synovial fluids provided estimates of 300-650 femtomoles per injection (0.7-1.6 nanomolar (nM) fluid concentrations) in three of the six samples analyzed. A validated sensitive assay for the quantitation of MYDGF in biological fluids was developed. However, the endogenous levels of MYDGF in such fluids are at or below the current levels of quantitation. The levels of MYDGF are lower than those previously reported using an ELISA. The current results suggest that additional steps may be required to remove high abundance proteins or to enrich MYDGF for SRM-based quantitation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

Purcell, Maureen K; Pearman-Gillman, Schuyler; Thompson, Rachel L; Gregg, Jacob L; Hart, Lucas M; Winton, James R; Emmenegger, Eveline J; Hershberger, Paul K

2016-07-01

Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea. © 2016 The Author(s).

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

USGS Publications Warehouse

Purcell, Maureen K.; Pearman-Gillman, Schuyler; Thompson, Rachel L.; Gregg, Jacob L.; Hart, Lucas M.; Winton, James R.; Emmenegger, Eveline J.; Hershberger, Paul K.

2016-01-01

Viral erythrocytic necrosis (VEN) is a disease of marine and anadromous fish that is caused by the erythrocytic necrosis virus (ENV), which was recently identified as a novel member of family Iridoviridae by next-generation sequencing. Phylogenetic analysis of the ENV DNA polymerase grouped ENV with other erythrocytic iridoviruses from snakes and lizards. In the present study, we identified the gene encoding the ENV major capsid protein (MCP) and developed a quantitative real-time PCR (qPCR) assay targeting this gene. Phylogenetic analysis of the MCP gene sequence supported the conclusion that ENV does not group with any of the currently described iridovirus genera. Because there is no information regarding genetic variation of the MCP gene across the reported host and geographic range for ENV, we also developed a second qPCR assay for a more conserved ATPase-like gene region. The MCP and ATPase qPCR assays demonstrated good analytical and diagnostic sensitivity and specificity based on samples from laboratory challenges of Pacific herring Clupea pallasii. The qPCR assays had similar diagnostic sensitivity and specificity as light microscopy of stained blood smears for the presence of intraerythrocytic inclusion bodies. However, the qPCR assays may detect viral DNA early in infection prior to the formation of inclusion bodies. Both qPCR assays appear suitable for viral surveillance or as a confirmatory test for ENV in Pacific herring from the Salish Sea.

Hepatitis C Virus RNA Real-Time Quantitative RT-PCR Method Based on a New Primer Design Strategy.

PubMed

Chen, Lida; Li, Wenli; Zhang, Kuo; Zhang, Rui; Lu, Tian; Hao, Mingju; Jia, Tingting; Sun, Yu; Lin, Guigao; Wang, Lunan; Li, Jinming

2016-01-01

Viral nucleic acids are unstable when improperly collected, handled, and stored, resulting in decreased sensitivity of currently available commercial quantitative nucleic acid testing kits. Using known unstable hepatitis C virus RNA, we developed a quantitative RT-PCR method based on a new primer design strategy to reduce the impact of nucleic acid instability on nucleic acid testing. The performance of the method was evaluated for linearity, limit of detection, precision, specificity, and agreement with commercial hepatitis C virus assays. Its clinical application was compared to that of two commercial kits--Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) and Kehua. The quantitative RT-PCR method delivered a good performance, with a linearity of R(2) = 0.99, a total limit of detection (genotypes 1 to 6) of 42.6 IU/mL (95% CI, 32.84 to 67.76 IU/mL), a CV of 1.06% to 3.34%, a specificity of 100%, and a high concordance with the CAP/CTM assay (R(2) = 0.97), with a means ± SD value of -0.06 ± 1.96 log IU/mL (range, -0.38 to 0.25 log IU/mL). The method was superior to commercial assays in detecting unstable hepatitis C virus RNA (P < 0.05). This quantitative RT-PCR method can effectively eliminate the influence of RNA instability on nucleic acid testing. The principle of primer design strategy may be applied to the detection of other RNA or DNA viruses. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

New design, development, and optimization of an in-house quantitative TaqMan Real-time PCR assay for HIV-1 viral load measurement.

PubMed

Noorbazargan, Hassan; Nadji, Seyed Alireza; Samiee, Siamak Mirab; Paryan, Mahdi; Mohammadi-Yeganeh, Samira

2018-04-01

Background Viral load measurement is commonly applicable to monitor HIV infection in patients to determine the number of HIV-RNA in serum samples of individuals. The aim of the present study was to set up a highly specific, sensitive, and reproducible home-brewed Real-time PCR assay based on TaqMan chemistry to quantify HIV-1 RNA genome. Methods In this study, three sets of primer pairs and a TaqMan probe were designed for HIV subtypes conserved sequences. An internal control was included in this assay to evaluate the presence of inhibition. Standard curve and threshold cycle values were determined using in vitro transcribed RNA from int region of HIV-1. A serial dilution of RNA standards was generated by in vitro transcription, from 10 to 10 9 copies/ml to find the sensitivity and the limit of detection (LOD) of the assay and to evaluate its performance in a quantitative RT-PCR assay. Results The assay has a low LOD equivalent to 33.13 copies/ml of HIV-1 RNA and a linear range of detection from 10 to 10 9 copies/ml. The coefficient of variation (CV) for Inter and Intra-assay precision of this in-house HIV Real-time RT-PCR ranged from 0.28 to 2.49% and 0.72 to 4.47%, respectively. The analytical and clinical specificity was 100%. Conclusions The results indicate that the developed method has a suitable specificity and sensitivity and is highly reproducible and cost-benefit. Therefore, it will be useful to monitor HIV infection in plasma samples of individuals.

Development of a BK virus real-time quantitative assay using the bioMérieux analyte-specific reagents in plasma specimens.

PubMed

Rennert, Hanna; Fernandes, Helen; Gilani, Zahid; Sipley, John

2015-12-01

Viral load testing for BK virus (BKV) has become the standard of care for diagnosing BKV infection and monitoring therapy in kidney transplant patients. However, there are currently no US Food and Drug Administration-approved assays and no standardization among available tests. This study evaluated the performance of the analyte-specific reagent (ASR) BKV primers r-gene and probe r-gene reagents (bioMérieux, Marcy l'Étoile, France) soon to become available on the US market for accuracy, linearity, precision, analytical sensitivity, specificity, and correlation with the Qiagen (Germantown, MD) BKV ASR test using commercial material and patient plasma samples. The assay was linear from 204 to 3.92 million (2.31-6.6 log10) DNA copies/mL (coefficient of determination: R(2) =0.999). A dilution series demonstrated limits of detection and quantitation of 2.14 log10 and 2.30 log10 copies/mL (95% hit rate detection), respectively. Interrun precision was highly reproducible, with coefficients of variance ranging from 2.2% to 6.0%. A comparison of 34 matched samples showed a good agreement (R(2) = 0.87) between the bioMérieux BKV laboratory test and the Qiagen BKV ASR assay results, with an average negative bias (-0.28 log10 copies/mL). The laboratory-developed test with bioMérieux BKV reagents is a reliable and sensitive assay for BKV DNA quantitation compared with the Qiagen ASR test. Copyright© by the American Society for Clinical Pathology.

A low cost mobile phone dark-field microscope for nanoparticle-based quantitative studies.

PubMed

Sun, Dali; Hu, Tony Y

2018-01-15

Dark-field microscope (DFM) analysis of nanoparticle binding signal is highly useful for a variety of research and biomedical applications, but current applications for nanoparticle quantification rely on expensive DFM systems. The cost, size, limited robustness of these DFMs limits their utility for non-laboratory settings. Most nanoparticle analyses use high-magnification DFM images, which are labor intensive to acquire and subject to operator bias. Low-magnification DFM image capture is faster, but is subject to background from surface artifacts and debris, although image processing can partially compensate for background signal. We thus mated an LED light source, a dark-field condenser and a 20× objective lens with a mobile phone camera to create an inexpensive, portable and robust DFM system suitable for use in non-laboratory conditions. This proof-of-concept mobile DFM device weighs less than 400g and costs less than $2000, but analysis of images captured with this device reveal similar nanoparticle quantitation results to those acquired with a much larger and more expensive desktop DFMM system. Our results suggest that similar devices may be useful for quantification of stable, nanoparticle-based activity and quantitation assays in resource-limited areas where conventional assay approaches are not practical. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of Quantitative PCR Assays Targeting the 16S rRNA Genes of Enterococcus spp. and Their Application to the Identification of Enterococcus Species in Environmental Samples

PubMed Central

Ryu, Hodon; Henson, Michael; Elk, Michael; Toledo-Hernandez, Carlos; Griffith, John; Blackwood, Denene; Noble, Rachel; Gourmelon, Michèle; Glassmeyer, Susan

2013-01-01

The detection of environmental enterococci has been determined primarily by using culture-based techniques that might exclude some enterococcal species as well as those that are nonculturable. To address this, the relative abundances of enterococci were examined by challenging fecal and water samples against a currently available genus-specific assay (Entero1). To determine the diversity of enterococcal species, 16S rRNA gene-based group-specific quantitative PCR (qPCR) assays were developed and evaluated against eight of the most common environmental enterococcal species. Partial 16S rRNA gene sequences of 439 presumptive environmental enterococcal strains were analyzed to study further the diversity of enterococci and to confirm the specificities of group-specific assays. The group-specific qPCR assays showed relatively high amplification rates with targeted species (>98%), although some assays cross-amplified with nontargeted species (1.3 to 6.5%). The results with the group-specific assays also showed that different enterococcal species co-occurred in most fecal samples. The most abundant enterococci in water and fecal samples were Enterococcus faecalis and Enterococcus faecium, although we identified more water isolates as Enterococcus casseliflavus than as any of the other species. The prevalence of the Entero1 marker was in agreement with the combined number of positive signals determined by the group-specific assays in most fecal samples, except in gull feces. On the other hand, the number of group-specific assay signals was lower in all water samples tested, suggesting that other enterococcal species are present in these samples. While the results highlight the value of genus- and group-specific assays for detecting the major enterococcal groups in environmental water samples, additional studies are needed to determine further the diversity, distributions, and relative abundances of all enterococcal species found in water. PMID:23087032

Development of fluorogenic probe-based PCR assays for the detection and quantification of bovine piroplasmids.

PubMed

Criado-Fornelio, A; Buling, A; Asenzo, G; Benitez, D; Florin-Christensen, M; Gonzalez-Oliva, A; Henriques, G; Silva, M; Alongi, A; Agnone, A; Torina, A; Madruga, C R

2009-06-10

This paper reports two new quantitative PCR (qPCR) assays, developed in an attempt to improve the detection of bovine piroplasmids. The first of these techniques is a duplex TaqMan assay for the simultaneous diagnosis of Babesia bovis and B. bigemina. This technique is ideal for use in South America where bovids harbour no theilerids. The second technique, which is suitable for the diagnosis of both babesiosis and theileriosis worldwide, involves fluorescence resonance energy transfer (FRET) probes. In FRET assays, Babesia bovis, B. divergens, Babesia sp. (B. major or B. bigemina), Theileria annae and Theileria sp. were all identifiable based on the melting temperatures of their amplified fragments. Both techniques provided linear calibration curves over the 0.1fg/microl to 0.01ng/microl DNA range. The assays showed good sensitivity and specificity. To assess their performance, both procedures were compared in two separate studies: the first was intended to monitor the experimental infection of calves with B. bovis and the second was a survey where 200 bovid/equine DNA samples from different countries were screened for piroplasmids. Comparative studies showed that duplex TaqMan qPCR was more sensitive than FRET qPCR in the detection of babesids.

Identification of compounds that modulate retinol signaling using a cell-based qHTS assay

PubMed Central

Chen, Yanling; Sakamuru, Srilatha; Huang, Ruili; Reese, David H.; Xia, Menghang

2016-01-01

In vertebrates, the retinol (vitamin A) signaling pathway (RSP) controls the biosynthesis and catabolism of all-trans retinoic acid (atRA), which regulates transcription of genes essential for embryonic development. Chemicals that interfere with the RSP to cause abnormal intracellular levels of atRA are potential developmental toxicants. To assess chemicals for the ability to interfere with retinol signaling, we have developed a cell-based RARE (Retinoic Acid Response Element) reporter gene assay to identify RSP disruptors. To validate this assay in a quantitative high-throughput screening (qHTS) platform, we screened the Library of Pharmacologically Active Compounds (LOPAC) in both agonist and antagonist modes. The screens detected known RSP agonists, demonstrating assay reliability, and also identified novel RSP agonists including kenpaullone, niclosamide, PD98059 and SU4312, and RSP antagonists including Bay 11-7085, LY294002, 3,4-Methylenedioxy-β-nitrostyrene, and topoisomerase inhibitors (camptothecin, topotecan, amsacrine hydrochloride, and idarubicin). When evaluated in the P19 pluripotent cell, these compounds were found to affect the expression of the Hoxa1 gene that is essential for embryo body patterning. These results show that the RARE assay is an effective qHTS approach for screening large compound libraries to identify chemicals that have the potential to adversely affect embryonic development through interference with retinol signaling. PMID:26820057

A minor groove binder probe real-time PCR assay for discrimination between type 2-based vaccines and field strains of canine parvovirus.

PubMed

Decaro, Nicola; Elia, Gabriella; Desario, Costantina; Roperto, Sante; Martella, Vito; Campolo, Marco; Lorusso, Alessio; Cavalli, Alessandra; Buonavoglia, Canio

2006-09-01

A minor groove binder (MGB) probe assay was developed to discriminate between type 2-based vaccines and field strains of canine parvovirus (CPV). Considering that most of the CPV vaccines contain the old type 2, no longer circulating in canine population, two MGB probes specific for CPV-2 and the antigenic variants (types 2a, 2b and 2c), respectively, were labeled with different fluorophores. The MGB probe assay was able to discriminate correctly between the old type and the variants, with a detection limit of 10(1) DNA copies and a good reproducibility. Quantitation of the viral DNA loads was accurate, as demonstrated by comparing the CPV DNA titres to those calculated by means of the TaqMan assay recognising all CPV types. This assay will ensure resolution of most diagnostic problems in dogs showing CPV disease shortly after CPV vaccination, although it does not discriminate between field strains and type 2b-based vaccines, recently licensed to market in some countries.

Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.

PubMed Central

Ackerman, S B; Kelley, E A

1983-01-01

The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units. Images PMID:6341399

Probe colorimeter for quantitating enzyme-linked immunosorbent assays and other colorimetric assays performed with microplates.

PubMed

Ackerman, S B; Kelley, E A

1983-03-01

The performance of a fiberoptic probe colorimeter (model PC800; Brinkmann Instruments, Inc., Westbury, N.Y.) for quantitating enzymatic or colorimetric assays in 96-well microtiter plates was compared with the performances of a spectrophotometer (model 240; Gilford Instrument Laboratories, Inc., Oberlin, Ohio) and a commercially available enzyme immunoassay reader (model MR590; Dynatech Laboratories, Inc., Alexandria, Va.). Alkaline phosphatase-p-nitrophenyl phosphate in 3 M NaOH was used as the chromophore source. Six types of plates were evaluated for use with the probe colorimeter; they generated reproducibility values (100% coefficient of variation) ranging from 91 to 98% when one individual made 24 independent measurements on the same dilution of chromophore on each plate. Eleven individuals each performed 24 measurements with the colorimeter on either a visually light (absorbance of 0.10 at 420 nm) or a dark (absorbance of 0.80 at 420 nm) dilution of chromophore; reproducibilities averaged 87% for the light dilution and 97% for the dark dilution. When one individual measured the same chromophore sample at least 20 times in the colorimeter, in the spectrophotometer or in the enzyme immunoassay reader, reproducibility for each instrument was greater than 99%. Measurements of a dilution series of chromophore in a fixed volume indicated that the optical responses of each instrument were linear in a range of 0.05 to 1.10 absorbance units.

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

PubMed Central

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

2016-01-01

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

An improved 96-well turbidity assay for T4 lysozyme activity.

PubMed

Toro, Tasha B; Nguyen, Thao P; Watt, Terry J

2015-01-01

T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: •Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays;•Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and•Incorporates a simplified expression and purification protocol for T4 lysozyme.

An improved 96-well turbidity assay for T4 lysozyme activity

PubMed Central

Toro, Tasha B.; Nguyen, Thao P.; Watt, Terry J.

2015-01-01

T4 lysozyme (T4L) is an important model system for investigating the relationship between protein structure and function. Despite being extensively studied, a reliable, quantitative activity assay for T4L has not been developed. Here, we present an improved T4L turbidity assay as well as an affinity-based T4L expression and purification protocol. This assay is designed for 96-well format and utilizes conditions amenable for both T4L and other lysozymes. This protocol enables easy, efficient, and quantitative characterization of T4L variants and allows comparison between different lysozymes. Our method: • Is applicable for all lysozymes, with enhanced sensitivity for T4 lysozyme compared to other 96-well plate turbidity assays; • Utilizes standardized conditions for comparing T4 lysozyme variants and other lysozymes; and • Incorporates a simplified expression and purification protocol for T4 lysozyme. PMID:26150996

Real-time PCR assays using internal controls for quantitation of HPV-16 and beta-globin DNA in cervicovaginal lavages.

PubMed

Lefevre, Jonas; Hankins, Catherine; Pourreaux, Karina; Voyer, Hélène; Coutlée, François

2003-12-01

High-risk human papillomavirus 16 (HPV-16) DNA viral load has been measured with real-time PCR assays by amplifying HPV-16 and a human gene. However, these assays have not used internal controls (ICs) to screen for the presence of inhibitors contained in samples. To quantitate HPV-16 DNA and cell content with real-time PCR, ICs for HPV-16 DNA and beta-globin were synthesised and used to control for inhibition. The assays were sensitive and linear over 5 logs. Good reproducibility was achieved with inter-run coefficients of variation of 23% (10(2) HPV-16 copies), 12% (10(4) HPV-16 copies), 17% (274 beta-globin DNA copies) and 7% (27,400 beta-globin DNA copies). Samples containing 56,800,000, 306,000, 18,000, and 4,070 HPV-16 copies/microg of cellular DNA were tested blindly and estimated to contain 48,800,000, 479,000, 20,300, and 6,620 HPV-16 copies/microg of DNA (mean ratio of measured to expected viral load of 1.27+/-0.32). Inhibition of amplification of HPV-16 and beta-globin ICs by six samples known to contain PCR inhibitors was variable: four inhibited both ICs while two inhibited only the HPV-16 IC. The use of internal controls with real-time PCR for HPV-16 quantitation allows to screen for the presence of inhibitors that do not affect equally primer-driven genomic amplification.

Quantitation of 47 human tear proteins using high resolution multiple reaction monitoring (HR-MRM) based-mass spectrometry.

PubMed

Tong, Louis; Zhou, Xi Yuan; Jylha, Antti; Aapola, Ulla; Liu, Dan Ning; Koh, Siew Kwan; Tian, Dechao; Quah, Joanne; Uusitalo, Hannu; Beuerman, Roger W; Zhou, Lei

2015-02-06

Tear proteins are intimately related to the pathophysiology of the ocular surface. Many recent studies have demonstrated that the tear is an accessible fluid for studying eye diseases and biomarker discovery. This study describes a high resolution multiple reaction monitoring (HR-MRM) approach for developing assays for quantification of biologically important tear proteins. Human tear samples were collected from 1000 subjects with no eye complaints (411 male, 589 female, average age: 55.5±14.5years) after obtaining informed consent. Tear samples were collected using Schirmer's strips and pooled into a single global control sample. Quantification of proteins was carried out by selecting "signature" peptides derived by trypsin digestion. A 1-h nanoLC-MS/MS run was used to quantify the tear proteins in HR-MRM mode. Good reproducibility of signal intensity (using peak areas) was demonstrated for all 47 HR-MRM assays with an average coefficient of variation (CV%) of 4.82% (range: 1.52-10.30%). All assays showed consistent retention time with a CV of less than 0.80% (average: 0.57%). HR-MRM absolute quantitation of eight tear proteins was demonstrated using stable isotope-labeled peptides. In this study, we demonstrated for the first time the technique to quantify 47 human tear proteins in HR-MRM mode using approximately 1μl of human tear sample. These multiplexed HR-MRM-based assays show great promise of further development for biomarker validation in human tear samples. Both discovery-based and targeted quantitative proteomics can be achieved in a single quadrupole time-of-flight mass spectrometer platform (TripleTOF 5600 system). Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

EPA Science Inventory

Investigation of a redox-sensitive predictive model of mouse embryonic stem cell differentiation via quantitative nuclease protection assays and glutathione redox status Chandler KJ,Hansen JM, Knudsen T,and Hunter ES 1. U.S. Environmental Protection Agency, Research Triangl...

Quantitative Assessment of Commutability for Clinical Viral Load Testing Using a Digital PCR-Based Reference Standard

PubMed Central

Tang, L.; Sun, Y.; Buelow, D.; Gu, Z.; Caliendo, A. M.; Pounds, S.

2016-01-01

Given recent advances in the development of quantitative standards, particularly WHO international standards, efforts to better understand the commutability of reference materials have been made. Existing approaches in evaluating commutability include prediction intervals and correspondence analysis; however, the results obtained from existing approaches may be ambiguous. We have developed a “deviation-from-ideal” (DFI) approach to evaluate commutability of standards and applied it to the assessment of Epstein-Bar virus (EBV) load testing in four quantitative PCR assays, treating digital PCR as a reference assay. We then discuss advantages and limitations of the DFI approach as well as experimental design to best evaluate the commutability of an assay in practice. PMID:27076654

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

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

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

2009-01-01

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

Comparative evaluation of laboratory developed real-time PCR assays and RealStar(®) BKV PCR Kit for quantitative detection of BK polyomavirus.

PubMed

Hasan, Mohammad R; Tan, Rusung; Al-Rawahi, Ghada; Thomas, Eva; Tilley, Peter

2016-08-01

Quantitative, viral load monitoring for BK virus (BKV) by real-time PCR is an important tool in the management of polyomavirus associated nephropathy in renal transplant patients. However, variability in PCR results has been reported because of polymorphisms in viral genes among different subtypes of BKV, and lack of standardization of the PCR assays among different laboratories. In this study we have compared the performance of several laboratory developed PCR assays that target highly conserved regions of BKV genome with a commercially available, RealStar(®) BKV PCR Kit. Three real-time PCR assays (i) VP1 assay: selected from the literature that targets the major capsid protein (VP1) gene (ii) VP1MOD assay: VP1 assay with a modified probe, and (iii) BKLTA assay: newly designed assay that targets the large T antigen gene were assessed in parallel, using controls and clinical specimens that were previously tested using RealStar(®) BKV PCR Kit (Altona Diagnostics GmbH, Hamburg, Germany). Nucleic acid from all samples were extracted using the QIA symphony virus/bacteria kit on an automated DNA extraction platform QIA symphony SP (Qiagen). Primer and probe concentration, and reaction conditions for laboratory developed assays were optimized and the limit of detection of different assays was determined. Positive control for laboratory developed BK assays was prepared through construction of a plasmid carrying respective amplicon sequences. The 95% detection limit of VP1, VP1MOD and BKLTA assays were 1.8×10(2), 3×10(3) and 3.5×10(2) genomic copies/ml, respectively, as determined by Probit regression analysis of data obtained by testing a dilution series of a titered patient specimen, using RealStar(®) BKV PCR Kit. The inter-assay and intra-assay, coefficient of variations of these assays using calibrated, plasmid standards were <1%. All assays, including the RealStar(®) BKV PCR assay, were highly specific when tested against a panel of external proficiency

Interference of magnesium corrosion with tetrazolium-based cytotoxicity assays.

PubMed

Fischer, Janine; Prosenc, Marc H; Wolff, Martin; Hort, Norbert; Willumeit, Regine; Feyerabend, Frank

2010-05-01

Magnesium (Mg) alloys are promising materials for the development of biodegradable implants. However, the current in vitro test procedures for cytotoxicity, cell viability and proliferation are not always suitable for this class of materials. In this paper we show that tetrazolium-salt-based assays, which are widely used in practice, are influenced by the corrosion products of Mg-based alloys. Corroded Mg converts tetrazolium salts to formazan, leading to a higher background and falsifying the results of cell viability. Tetrazolium-based assays are therefore not a useful tool for testing the cytotoxicity of Mg in static in vitro assays. Copyright (c) 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration.

PubMed

LeBlanc, André; Michaud, Sarah A; Percy, Andrew J; Hardie, Darryl B; Yang, Juncong; Sinclair, Nicholas J; Proudfoot, Jillaine I; Pistawka, Adam; Smith, Derek S; Borchers, Christoph H

2017-07-07

When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.

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

ERIC Educational Resources Information Center

Xu, Xia; Veenstra, Timothy D.

2012-01-01

The list of physiological events in which sex steroids play a role continues to increase. To decipher the roles that sex steroids play in any condition requires high quality cohorts of samples and assays that provide highly accurate quantitative measures. Liquid and gas chromatography coupled with mass spectrometry (LC-MS and GC-MS) have…

Measurement of filter paper activities of cellulase with microplate-based assay.

PubMed

Yu, Xiaoxiao; Liu, Yan; Cui, Yuxiao; Cheng, Qiyue; Zhang, Zaixiao; Lu, Jia Hui; Meng, Qingfan; Teng, Lirong; Ren, Xiaodong

2016-01-01

It is always a challenge to determine the total cellulase activity efficiently without reducing accuracy. The most common total cellulase activity assay is the filter paper assay (FPA) established by the International Union of Pure and Applied Chemistry (IUPAC). A new procedure to measure the FPA with microplate-based assay was studied in this work, which followed the main idea of IUPAC to dilute cellulase preparation to get fixed glucose release. FPAs of six cellulase preparations were determined with the microplate-based assay. It is shown that FPAs of cellulase Youtell, RCconc, R-10, Lerkam, Yishui and Sinopharm were 67.9, 46.0, 46.1, 27.4, 7.6 and 8.0 IU/ml respectively. There was no significant difference at the 95% confidence level between the FPA determined with IUPAC and the microplate-based assay. It could be concluded that the FPA could be determined by the microplate-based assay with the same accuracy and much more efficiency compared with that by IUPAC.

Measurement of filter paper activities of cellulase with microplate-based assay

PubMed Central

Yu, Xiaoxiao; Liu, Yan; Cui, Yuxiao; Cheng, Qiyue; Zhang, Zaixiao; Lu, Jia Hui; Meng, Qingfan; Teng, Lirong; Ren, Xiaodong

2015-01-01

It is always a challenge to determine the total cellulase activity efficiently without reducing accuracy. The most common total cellulase activity assay is the filter paper assay (FPA) established by the International Union of Pure and Applied Chemistry (IUPAC). A new procedure to measure the FPA with microplate-based assay was studied in this work, which followed the main idea of IUPAC to dilute cellulase preparation to get fixed glucose release. FPAs of six cellulase preparations were determined with the microplate-based assay. It is shown that FPAs of cellulase Youtell, RCconc, R-10, Lerkam, Yishui and Sinopharm were 67.9, 46.0, 46.1, 27.4, 7.6 and 8.0 IU/ml respectively. There was no significant difference at the 95% confidence level between the FPA determined with IUPAC and the microplate-based assay. It could be concluded that the FPA could be determined by the microplate-based assay with the same accuracy and much more efficiency compared with that by IUPAC. PMID:26858572

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

USDA-ARS?s Scientific Manuscript database

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

A microsphere-based assay for mutation analysis of the biotinidase gene using dried blood spots

PubMed Central

Lindau-Shepard, Barbara; Janik, David K.; Pass, Kenneth A.

2012-01-01

Biotinidase deficiency is an autosomal recessive syndrome caused by defects in the biotinidase gene, the product of which affects biotin metabolism. Newborn screening (NBS) for biotinidase deficiency can identify affected infants prior to onset of symptoms; biotin supplementation can resolve or prevent the clinical features. In NBS, dry blood spots (DBS) are usually tested for biotinidase enzyme activity by colorimetric analysis. By taking advantage of the multiplexing capabilities of the Luminex platform, we have developed a microsphere-based array genotyping method for the simultaneous detection of six disease causing mutations in the biotinidase gene, thereby permitting a second tier of molecular analysis. Genomic DNA was extracted from 3.2 mm DBS. Biotinidase gene sequences, containing the mutations of interest, were amplified by multiplexed polymerase chain reaction, followed by multiplexed allele-specific primer extension using universally tagged genotyping primers. The products were then hybridized to anti-tag carrying xTAG microspheres and detected on the Luminex platform. Genotypes were verified by sequencing. Genotyping results of 22 known biotinidase deficient samples by our xTAG biotinidase assay was in concordance with the results obtained from DNA sequencing, for all 6 mutations used in our panel. These results indicate that genotyping by an xTAG microsphere-based array is accurate, flexible, and can be adapted for high-throughput. Since NBS for biotinidase deficiency is by enzymatic assay, less than optimal quality of the DBS itself can compromise enzyme activity, while the DNA from these samples mostly remains unaffected. This assay warrants evaluation as a viable complement to the biotinidase semi-quantitative colorimetric assay. PMID:27625817

Development of a liquid chromatography-mass spectrometry based enzyme activity assay for phosphatidylcholine-specific phospholipase C.

PubMed

Murakami, Chiaki; Mizuno, Satoru; Kado, Sayaka; Sakane, Fumio

2017-06-01

Phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) hydrolyzes PC to generate the second messenger 1,2-diacylglycerol (DG) and phosphocholine. PC-PLC plays pivotal roles in inflammation, carcinogenesis, tumor progression, atherogenesis, and subarachnoid hemorrhage. Although the activity of PC-PLC in mammalian tissues was discovered approximately 40 years ago, neither the protein nor its gene has been identified. In the present study, we developed a non-radioactive enzyme activity assay for PC-PLC based on mass spectrometric detection of DG following HPLC separation. This new liquid chromatography-mass spectrometry (LC-MS) assay directly determines a specific reaction product, 1-palmitoyl-2-oleoyl-DG, that is generated from 1-palmitoyl-2-oleoyl-PC by purified Bacillus cereus PC-PLC. The LC-MS assay offers several advantages including a lower background (0.02% versus 91%), higher signal background ratio (4242 versus 1.06)/signal noise ratio (7494 versus 4.4), higher sensitivity (≥32-fold), and lower limit of quantitation (0.04 pmol versus 0.69 pmol of PC-PLC), than a conventional fluorometric assay, which indirectly detects phosphocholine produced in the reaction. In addition to Bacillus cereus PC-PLC, the LC-MS assay was applicable to the measurement of mammalian PC-PLC prepared from the mouse brain. The radioisotope-free, highly sensitive and precise LC-MS assay for PC-PLC would be useful for the purification and identification of PC-PLC protein. Copyright © 2017 Elsevier Inc. All rights reserved.

Clinical Usefulness of a One-Tube Nested Reverse Transcription Quantitative Polymerase Chain Reaction Assay for Evaluating Human Epidermal Growth Factor Receptor 2 mRNA Overexpression in Formalin-Fixed and Paraffin-Embedded Breast Cancer Tissue Samples.

PubMed

Wang, Hye-Young; Ahn, Sungwoo; Park, Sunyoung; Kim, SeungIl; Lee, Hyeyoung

2017-01-01

Currently, the two main methods used to analyze human epidermal growth factor receptor 2 (HER2) amplification or overexpression have a limited accuracy and high costs. These limitations can be overcome by the development of complementary quantitative methods. In this study, we analyzed HER2 mRNA expression in clinical formalin-fixed and paraffin-embedded (FFPE) samples using a one-tube nested reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay. We measured expression relative to 3 reference genes and compared the results to those obtained by conventional immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) assays with 226 FFPE breast cancer tissue samples. The one-tube nested RT-qPCR assay proved to be highly sensitive and specific based on comparisons with IHC (96.9 and 97.7%, respectively) and FISH (92.4 and 92.9%, respectively) obtained with the validation set. Comparisons with clinicopathological data revealed significant associations between HER2 overexpression and TNM stage (p < 0.01), histological type (p < 0.01), ER status (p < 0.001), PR status (p < 0.05), HER2 status (p < 0.001), and molecular subtypes (p < 0.001). Based on these findings, our one-tube nested RT-qPCR assay is a potentially useful and complementary screening tool for the detection of HER2 mRNA overexpression. © 2016 S. Karger AG, Basel.

Analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay, the amplicor CMV test, and a semi-quantitative polymerase chain reaction test after allogeneic marrow transplantation.

PubMed

Ksouri, H; Eljed, H; Greco, A; Lakhal, A; Torjman, L; Abdelkefi, A; Ben Othmen, T; Ladeb, S; Slim, A; Zouari, B; Abdeladhim, A; Ben Hassen, A

2007-03-01

A pp65 antigenemia assay for polymorphonuclear leukocytes (PMNLs) (CINAkit Rapid Antigenemia), and a qualitative polymerase chain reaction (PCR) test for plasma 'PCR-P qual' (Amplicor cytomegalovirus [CMV] test) were performed for 126 samples (blood and plasma) obtained from 18 bone marrow transplant patients, over a 9-month surveillance period. Among those samples, 92 were assayed with a semi-quantitative PCR test for PMNLs 'PCR-L quant.' The number of samples with a positive CMV test for antigenemia and PCR-P qual assays was 20.63% and 12.7%, respectively, whereas the PCR-L quant assay was positive in 48 of the 92 samples assayed (52.17%). The rates of concordance of the results of PCR-P qual and antigenemia, PCR-P qual and PCR-L quant, antigenemia and PCR-L quant were 92%, 65.2% and 66.8%, respectively. The analysis of the results for the 92 specimens tested by all 3 methods showed a rate of concordance of 63% among all methods. Good agreement (kappa=0.72) was found only between pp65 Ag and PCR-P qual assays. Clinical disease correlates with an antigenemia high viral load. Three patients had CMV disease despite preemptive therapy, and all of them had graft-versus-host-disease (GVHD). PMNLs-based assays are more efficient in monitoring CMV reactivation, but for high-risk patients with GVHD, more sensitive assays (real-time PCR) must be done.

Cyquant cell proliferation assay as a fluorescence-based method for in vitro screening of antimalarial activity.

PubMed

Sriwilaijaroen, Nongluk; Kelly, Jane Xu; Riscoe, Michael; Wilairat, Prapon

2004-12-01

The appearance of drug resistant parasites and the absence of an effective vaccine have resulted in the need for new effective antimalarial drugs. Consequently, a convenient method for in vitro screening of large numbers of antimalarial drug candidates has become apparent. The CyQUANT cell proliferation assay is a highly sensitive fluorescence-based method for quantitation of cell number by measuring the strong fluorescence produced when green GR dye binds to nucleic acids. We have applied the CyQUANT assay method to evaluate the growth of Plasmodium falciparum D6 strain in culture. The GR-nucleic acid fluorescence linearly correlated with percent parasitemia at both 0.75 or 1 percent hematocrit with the same correlation coefficient of r2 = 0.99. The sensitivity of P. falciparum D6 strain to chloroquine and to 3,6-bis-omega-diethylaminoamyloxyxanthone, a novel antimalarial, determined by the CyQUANT assay were comparable to those obtained by the traditional [3H]-ethanolamine assay: IC50 value of chloroquine was 54 nM and 51 nM by the CyQUANT and [3H]-ethanolamine assay, respectively; IC50 value for 3,6-bis-omega-diethylaminoamyloxyxanthone was 254 nM and 223 nM by the CyQUANT and [3H]-ethanolamine assay, respectively. This procedure requires no radioisotope, uses simple equipment, and is an easy and convenient procedure, with no washing and harvesting steps. Moreover, all procedures can be set up continuously and thus, the CyQUANT assay is suitable in automatic high through-put drug screening of antimalarial drugs.

A multiparametric assay for quantitative nerve regeneration evaluation.

PubMed

Weyn, B; van Remoortere, M; Nuydens, R; Meert, T; van de Wouwer, G

2005-08-01

We introduce an assay for the semi-automated quantification of nerve regeneration by image analysis. Digital images of histological sections of regenerated nerves are recorded using an automated inverted microscope and merged into high-resolution mosaic images representing the entire nerve. These are analysed by a dedicated image-processing package that computes nerve-specific features (e.g. nerve area, fibre count, myelinated area) and fibre-specific features (area, perimeter, myelin sheet thickness). The assay's performance and correlation of the automatically computed data with visually obtained data are determined on a set of 140 semithin sections from the distal part of a rat tibial nerve from four different experimental treatment groups (control, sham, sutured, cut) taken at seven different time points after surgery. Results show a high correlation between the manually and automatically derived data, and a high discriminative power towards treatment. Extra value is added by the large feature set. In conclusion, the assay is fast and offers data that currently can be obtained only by a combination of laborious and time-consuming tests.

Quantification of equine immunoglobulin A in serum and secretions by a fluorescent bead-based assay.

PubMed

Schnabel, Christiane L; Babasyan, Susanna; Freer, Heather; Wagner, Bettina

2017-06-01

Only few quantitative reports exist about the concentrations and induction of immunoglobulin A (IgA) in mucosal secretions of horses. Despite this, it is widely assumed that IgA is the predominant immunoglobulin on mucosal surfaces in the horse. Here, two new monoclonal antibodies (mAbs) against equine IgA, clones 84-1 and 161-1, were developed and characterized in detail. Both IgA mAbs specifically bound monomeric and dimeric equine IgA in different applications, such as Western blots and fluorescent bead-based assays. Cross-reactivity with other equine immunoglobulin isotypes was not observed. The new IgA mAb 84-1 was used in combination with the previously characterized anti-equine IgA mAb BVS2 for the development and validation of a fluorescent bead-based assay to quantify total IgA in equine serum and various secretions. The IgA assay's linear detection ranged from 64pg/ml to 1000ng/ml. For the quantification of IgA in serum or in secretions an IgA standard was purified from serum or nasal wash fluid (secretory IgA), respectively. The different standards were needed for accurate IgA quantification in the respective samples taking the different signal intensities of monomeric and dimeric IgA on the florescent bead-based assay into account. IgA was quantified by the bead-based assay established here in different equine samples of healthy adult individuals. In serum the median total IgA was 0.45mg/ml for Thoroughbred horses (TB, n=10) and 1.16mg/ml in Icelandic horses (ICH, n=12). In nasopharyngeal secretions of TB (n=7) 0.13mg/ml median total IgA was measured, and 0.25mg/ml for ICH (n=12). Saliva of ICH (n=6) contained a median of 0.15mg/ml, colostrum of Warmbloods (n=8) a median of 1.89mg/ml IgA. Compared to IgG1 and IgG4/7 quantified in the same samples, IgA appeared as the major immunoglobulin isotype in nasopharyngeal secretions and saliva while it is a minor isotype in serum and colostrum. The newly developed monoclonal antibodies against equine IgA and the

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

PubMed

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

2012-04-27

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

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

PubMed Central

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

2012-01-01

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

Quantitative detection of crystalline lysine supplementation in poultry feeds using a rapid bacterial bioluminescence assay.

PubMed

Zabala Díaz, I B; Ricke, S C

2003-08-01

Lysine is an essential amino acid for both humans and animals; and it is usually the first or second limiting amino acid in most formulated diets. In order to estimate the lysine content in feeds and feed sources, rapid amino acid bioassays have been developed. The objective of this work is to assess a rapid assay for lysine supplementation in chicken feeds, using a luminescent Escherichia coli lysine-auxotrophic strain, to avoid prior thermal sterilization. An E. coli lysine auxotroph carrying a plasmid with lux genes was used as the test organism. The lysine assay was conducted using depleted auxotrophic cells in lysine samples. Luminescence was measured with a Dynex MLX luminometer after addition of the aldehyde substrate. Growth response (monitored as optical density at 600 nm) and light emission response of the assay E. coli strain were monitored to generate standard curves. Bioluminescent analysis of feed samples indicated that the method works well in the presence of a complex feed matrix. Comparison of both optical density and luminescent-based methods indicated that, when the assay takes place under optimal conditions, both methodologies correlated well ( r(2)=0.99). Except for the 0.64% lysine-supplemented feed, estimates for lysine based on the bacterial assay were over 80% (82-97%) of the theoretical values. Animal data showed that the bacterial bioluminescent method correlated well with the chick bioassay when diets with different levels of lysine supplementation were assayed for lysine bioavailability ( r(2)=0.97). Luminescent methodology coupled with a bacterial growth assay is a promising technique to assess lysine availability in supplemented animal feeds.

Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics

PubMed Central

2012-01-01

Multiple reaction monitoring mass spectrometry (MRM-MS) with stable isotope dilution (SID) is increasingly becoming a widely accepted assay for the quantification of proteins and peptides. These assays have shown great promise in relatively high throughput verification of candidate biomarkers. While the use of MRM-MS assays is well established in the small molecule realm, their introduction and use in proteomics is relatively recent. As such, statistical and computational methods for the analysis of MRM-MS data from proteins and peptides are still being developed. Based on our extensive experience with analyzing a wide range of SID-MRM-MS data, we set forth a methodology for analysis that encompasses significant aspects ranging from data quality assessment, assay characterization including calibration curves, limits of detection (LOD) and quantification (LOQ), and measurement of intra- and interlaboratory precision. We draw upon publicly available seminal datasets to illustrate our methods and algorithms. PMID:23176545

Statistical characterization of multiple-reaction monitoring mass spectrometry (MRM-MS) assays for quantitative proteomics.

PubMed

Mani, D R; Abbatiello, Susan E; Carr, Steven A

2012-01-01

Multiple reaction monitoring mass spectrometry (MRM-MS) with stable isotope dilution (SID) is increasingly becoming a widely accepted assay for the quantification of proteins and peptides. These assays have shown great promise in relatively high throughput verification of candidate biomarkers. While the use of MRM-MS assays is well established in the small molecule realm, their introduction and use in proteomics is relatively recent. As such, statistical and computational methods for the analysis of MRM-MS data from proteins and peptides are still being developed. Based on our extensive experience with analyzing a wide range of SID-MRM-MS data, we set forth a methodology for analysis that encompasses significant aspects ranging from data quality assessment, assay characterization including calibration curves, limits of detection (LOD) and quantification (LOQ), and measurement of intra- and interlaboratory precision. We draw upon publicly available seminal datasets to illustrate our methods and algorithms.

Capillary electrophoresis-based assay of phosphofructokinase-1.

PubMed

Malina, Andrew; Bryant, Sherrisse K; Chang, Simon H; Waldrop, Grover L; Gilman, S Douglass

2014-02-15

An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg-ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg-ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by ultraviolet (UV) absorbance at 260 nm of Mg-ATP and Mg-ADP. The separation was enhanced by the addition of Mg²⁺ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC₅₀ value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors.

Capillary Electrophoresis-Based Assay of Phosphofructokinase-1

PubMed Central

Malina, Andrew; Bryant, Sherrisse K.; Chang, Simon H.; Waldrop, Grover L.; Gilman, S. Douglass

2013-01-01

An assay was developed for phosphofructokinase-1 (PFK-1) using capillary electrophoresis (CE). In the glycolytic pathway, this enzyme catalyzes the rate-limiting step from fructose-6-phosphate and magnesium-bound adenosine triphosphate (Mg-ATP) to fructose-1,6-bisphosphate and magnesium-bound adenosine diphosphate (Mg-ADP). This enzyme has recently become a research target because of the importance of glycolysis in cancer and obesity. The CE assay for PFK-1 is based on the separation and detection by UV absorbance at 260 nm of Mg-ATP and Mg-ADP. The separation was enhanced by addition of Mg2+ to the separation buffer. Inhibition studies of PFK-1 by aurintricarboxylic acid and palmitoyl coenzyme A were also performed. An IC50 value was determined for aurintricarboxylic acid, and this value matched values in the literature obtained using coupled spectrophotometric assays. This assay for PFK-1 directly monitors the enzyme-catalyzed reaction, and the CE separation reduces the potential of spectral interference by inhibitors. PMID:24444856

A Dual-Color Reporter Assay of Cohesin-Mediated Gene Regulation in Budding Yeast Meiosis.

PubMed

Fan, Jinbo; Jin, Hui; Yu, Hong-Guo

2017-01-01

In this chapter, we describe a quantitative fluorescence-based assay of gene expression using the ratio of the reporter green fluorescence protein (GFP) to the internal red fluorescence protein (RFP) control. With this dual-color heterologous reporter assay, we have revealed cohesin-regulated genes and discovered a cis-acting DNA element, the Ty1-LTR, which interacts with cohesin and regulates gene expression during yeast meiosis. The method described here provides an effective cytological approach for quantitative analysis of global gene expression in budding yeast meiosis.

Detection limits of quantitative and digital PCR assays and their influence in presence-absence surveys of environmental DNA

USGS Publications Warehouse

Hunter, Margaret; Dorazio, Robert M.; Butterfield, John S.; Meigs-Friend, Gaia; Nico, Leo; Ferrante, Jason A.

2017-01-01

A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR-based analyses of low concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species’ presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty – indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis, and forensic and clinical diagnostics.

ORGANOPHOSPHORUS HYDROLASE-BASED ASSAY FOR ORGANOPHOSPHATE PESTICIDES

EPA Science Inventory

We report a rapid and versatile Organophosphorus hydrolase (OPH)-based method for measurement of organophosphates. This assay is based on a substrate-dependent change in pH at the local vicinity of the enzyme. The pH change is monitored using fluorescein isothiocyanate (FITC), ...

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

PubMed

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

2012-03-01

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

Targeting Anti-Cancer Active Compounds: Affinity-Based Chromatographic Assays

PubMed Central

de Moraes, Marcela Cristina; Cardoso, Carmen Lucia; Seidl, Claudia; Moaddel, Ruin; Cass, Quezia Bezerra

2016-01-01

Affinity-based chromatography assays encompass the use of solid supports containing immobilized biological targets to monitor binding events in the isolation , identification and/or characterization of bioactive compounds. This powerful bioanalytical technique allows the screening of potential binders through fast analyses that can be directly performed using isolated substances or complex matrices. An overview of the recent researches in frontal and zonal affinity-based chromatography screening assays, which has been used as a tool in the identification and characterization of new anti-cancer agents, is discussed. In addition, a critical evaluation of the recently emerged ligands fishing assays in complex mixtures is also discussed. PMID:27306095

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

PubMed

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

2014-01-01

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

Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths.

PubMed

Ditommaso, Savina; Giacomuzzi, Monica; Ricciardi, Elisa; Zotti, Carla M

2015-08-01

Two different real-time quantitative PCR (PMA-qPCR) assays were applied for quantification of Legionella spp. by targeting a long amplicon (approx 400 bp) of 16S rRNA gene and a short amplicon (approx. 100 bp) of 5S rRNA gene. Purified DNA extracts from pure cultures of Legionella spp. and from environmental water samples were quantified. Application of the two assays to quantify Legionella in artificially contaminated water achieved that both assays were able to detect Legionella over a linear range of 10 to 10(5) cells ml(-1). A statistical analysis of the standard curves showed that both assays were linear with a good correlation coefficient (R(2) = 0.99) between the Ct and the copy number. Amplification with the reference assay was the most effective for detecting low copy numbers (1 bacterium per PCR mixture). Using selective quantification of viable Legionella by the PMA-qPCR method we obtained a greater inhibition of the amplification of the 400-bp 16S gene fragment (Δlog(10) = 3.74 ± 0.39 log(10) GU ml(-1)). A complete inhibition of the PCR signal was obtained when heat-killed cells in a concentration below 1 × 10(5) cells ml(-1) were pretreated with PMA. Analysing short amplicon sizes led to only 2.08 log reductions in the Legionella dead-cell signal. When we tested environmental water samples, the two qPCR assays were in good agreement according to the kappa index (0.741). Applying qPCR combined with PMA treatment, we also obtained a good agreement (kappa index 0.615). The comparison of quantitative results shows that both assays yielded the same quantification sensitivity (mean log = 4.59 vs mean log = 4.31). Copyright © 2015 Elsevier Ltd. All rights reserved.

Two types of nanoparticle-based bio-barcode amplification assays to detect HIV-1 p24 antigen.

PubMed

Dong, Huahuang; Liu, Jianli; Zhu, Hong; Ou, Chin-Yih; Xing, Wenge; Qiu, Maofeng; Zhang, Guiyun; Xiao, Yao; Yao, Jun; Pan, Pinliang; Jiang, Yan

2012-08-31

HIV-1 p24 antigen is a major viral component of human immunodeficiency virus type 1 (HIV-1) which can be used to identify persons in the early stage of infection and transmission of HIV-1 from infected mothers to infants. The detection of p24 is usually accomplished by using an enzyme-linked immunosorbent assay (ELISA) with low detection sensitivity. Here we report the use of two bio-barcode amplification (BCA) assays combined with polymerase chain reaction (PCR) and gel electrophoresis to quantify HIV-1 p24 antigen. A pair of anti-p24 monoclonal antibodies (mAbs) were used in BCA assays to capture HIV-1 p24 antigen in a sandwich format and allowed for the quantitative measurement of captured p24 using PCR and gel electrophoresis. The first 1 G12 mAb was coated on microplate wells or magnetic microparticles (MMPs) to capture free p24 antigens. Captured p24 in turn captured 1D4 mAb coated gold nanoparticle probes (GNPs) containing double-stranded DNA oligonucleotides. One strand of the oligonucleotides was covalently immobilized whereas the unbound complimentary bio-barcode DNA strand could be released upon heating. The released bio-barcode DNA was amplified by PCR, electrophoresed in agarose gel and quantified. The in-house ELISA assay was found to quantify p24 antigen with a limit of detection (LOD) of 1,000 pg/ml and a linear range between 3,000 and 100,000 pg/ml. In contrast, the BCA-based microplate method yielded an LOD of 1 pg/ml and a linear detection range from 1 to 10,000 pg/ml. The BCA-based MMP method yielded an LOD of 0.1 pg/ml and a linear detection range from 0.1 to 1,000 pg/ml. When combined with PCR and simple gel electrophoresis, BCA-based microplate and MMPs assays can be used to quantify HIV-1 p24 antigen. These methods are 3-4 orders of magnitude more sensitive than our in-house ELISA-based assay and may provide a useful approach to detect p24 in patients newly infected with HIV.

Development of a quantitative loop-mediated isothermal amplification (qLAMP) assay for the detection of Magnaporthe oryzae airborne inoculum in turf ecosystems

USDA-ARS?s Scientific Manuscript database

Grey Leaf Spot (GLS) is a detrimental disease of perennial ryegrass caused by a host-specialized form of Magnaporthe oryzae (Mot). In order to improve turf management, a quantitative loop-mediated isothermal amplification (LAMP) assay coupled with a simple spore trap is being developed to monitor GL...

A simple, specific high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin in cell culture medium.

PubMed

Li, Ye; Cassone, Vincent M

2015-09-01

A simple, specific, high-throughput enzyme-linked immunosorbent assay (ELISA) for quantitative determination of melatonin was developed for directly measuring melatonin in cell culture medium with 10% FBS. This assay adopts a commercial monoclonal melatonin antibody and melatonin-HRP conjugate, so it can be applied in multiple labs rapidly with low cost compared with commercial RIA and ELISA kits. In addition, the procedure is much simpler with only four steps: 1) sample/conjugate incubation, 2) plate washing, 3) TMB color reaction and 4) reading of results. The standards of the assay cover a wide working range from 100 pg/mL to 10 ng/mL. The sensitivity was 68 pg/mL in cell culture medium with 10% FBS and 26 pg/mL in PBS with as little as 25 μL sample volume. The recovery of melatonin from cell culture medium was 101.0%. The principal cross-reacting compound was 5-methoxytryptophol (0.1%). The variation coefficients of the assay, within and between runs, ranged between 6.68% and 15.76% in cell culture medium. The mean linearity of a series diluted cell culture medium sample was 105% (CV=5%), ranging between 98% and 111%, y=5.5263x+0.0646, R(2)=0.99. The assay enables small research and teaching labs to reliably measure this important neurohormone. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2016-01-01

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

Performance of PCR-based and Bioluminescent assays for mycoplasma detection.

PubMed

Falagan-Lotsch, Priscila; Lopes, Talíria Silva; Ferreira, Nívea; Balthazar, Nathália; Monteiro, Antônio M; Borojevic, Radovan; Granjeiro, José Mauro

2015-11-01

Contaminated eukaryotic cell cultures are frequently responsible for unreliable results. Regulatory entities request that cell cultures must be mycoplasma-free. Mycoplasma contamination remains a significant problem for cell cultures and may have an impact on biological analysis since they affect many cell parameters. The gold standard microbiological assay for mycoplasma detection involves laborious and time-consuming protocols. PCR-based and Bioluminescent assays have been considered for routine cell culture screening in research laboratories since they are fast, easy and sensitive. Thus, the aim of this work is to compare the performance of two popular commercial assays, PCR-based and Bioluminescent assays, by assessing the level of mycoplasma contamination in cell cultures from Rio de Janeiro Cell Bank (RJCB) and also from customers' laboratories. The results obtained by both performed assays were confirmed by scanning electron microscopy. In addition, we evaluated the limit of detection of the PCR kit under our laboratory conditions and the storage effects on mycoplasma detection in frozen cell culture supernatants. The performance of both assays for mycoplasma detection was not significantly different and they showed very good agreement. The Bioluminescent assay for mycoplasma detection was slightly more dependable than PCR-based due to the lack of inconclusive results produced by the first technique, especially considering the ability to detect mycoplasma contamination in frozen cell culture supernatants. However, cell lines should be precultured for four days or more without antibiotics to obtain safe results. On the other hand, a false negative result was obtained by using this biochemical approach. The implementation of fast and reliable mycoplasma testing methods is an important technical and regulatory issue and PCR-based and Bioluminescent assays may be good candidates. However, validation studies are needed. Copyright © 2015 Elsevier B.V. All rights

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

PubMed

Togashi, Katsuhiro; Sasaki, Shiho; Sato, Wataru

2015-08-01

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

Optimized protocol for quantitative multiple reaction monitoring-based proteomic analysis of formalin-fixed, paraffin embedded tissues

PubMed Central

Kennedy, Jacob J.; Whiteaker, Jeffrey R.; Schoenherr, Regine M.; Yan, Ping; Allison, Kimberly; Shipley, Melissa; Lerch, Melissa; Hoofnagle, Andrew N.; Baird, Geoffrey Stuart; Paulovich, Amanda G.

2016-01-01

Despite a clinical, economic, and regulatory imperative to develop companion diagnostics, precious few new biomarkers have been successfully translated into clinical use, due in part to inadequate protein assay technologies to support large-scale testing of hundreds of candidate biomarkers in formalin-fixed paraffin embedded (FFPE) tissues. While the feasibility of using targeted, multiple reaction monitoring-mass spectrometry (MRM-MS) for quantitative analyses of FFPE tissues has been demonstrated, protocols have not been systematically optimized for robust quantification across a large number of analytes, nor has the performance of peptide immuno-MRM been evaluated. To address this gap, we used a test battery approach coupled to MRM-MS with the addition of stable isotope labeled standard peptides (targeting 512 analytes) to quantitatively evaluate the performance of three extraction protocols in combination with three trypsin digestion protocols (i.e. 9 processes). A process based on RapiGest buffer extraction and urea-based digestion was identified to enable similar quantitation results from FFPE and frozen tissues. Using the optimized protocols for MRM-based analysis of FFPE tissues, median precision was 11.4% (across 249 analytes). There was excellent correlation between measurements made on matched FFPE and frozen tissues, both for direct MRM analysis (R2 = 0.94) and immuno-MRM (R2 = 0.89). The optimized process enables highly reproducible, multiplex, standardizable, quantitative MRM in archival tissue specimens. PMID:27462933

An assay for lateral line regeneration in adult zebrafish.

PubMed

Pisano, Gina C; Mason, Samantha M; Dhliwayo, Nyembezi; Intine, Robert V; Sarras, Michael P

2014-04-08

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Overproduction and characterization of a lytic polysaccharide monooxygenase in Bacillus subtilis using an assay based on ascorbate consumption.

PubMed

Yu, Mi-Ji; Yoon, Sun-Hee; Kim, Young-Wan

2016-11-01

Lytic polysaccharide monooxygenases (LPMOs) are copper ion-containing enzymes that degrade crystalline polysaccharides, such as cellulose or chitin, through an oxidative mechanism. To the best of our knowledge, there are no assay methods for the direct characterization of LPMOs that degrade substrates without coupled enzymes. As such, in this study, a coupled enzyme-free assay method for LPMOs was developed, which is based on measuring the consumption of ascorbic acid used as an external electron donor for LPMOs. To establish this new assay method, a chitin-active LPMO from Bacillus atrophaeus (BatLPMO10) was cloned as a model enzyme. An expression system using B. subtilis as the host cell yielded a simple purification process without complicated periplasmic fractionation, as well as improved productivity by 3.7-fold higher than that of Escherichia coli BL21(DE3). At the optimum pH determined using a newly developed assay, BatLPMO10 showed the highest activity in terms of promoting chitin degradation by a chitinase. In addition, the assay method indicated that BatLPMO10 was inhibited by sodium ions, and BatLPMO10 and a chitinase mutually enhanced each other's activities upon degrading chitin as the substrate. In conclusion, this hydrolase-free ascorbate assay allows quantitative analysis of BatLPMO10 without a coupled enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

Coupling spore traps and quantitative PCR assays for detection of the downy mildew pathogens of spinach (Peronospora effusa) and beet (Peronospora schachtii)

USDA-ARS?s Scientific Manuscript database

Downy mildew of spinach (Spinacia oleracea L.), caused by Peronospora effusa, is a disease constraint on production worldwide, including in California where the majority of United States spinach is grown. The aim of this study was to develop a real-time quantitative PCR (qPCR) assay for detection o...

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

PubMed

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

2014-01-01

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

Carbon quantum dots-based recyclable real-time fluorescence assay for alkaline phosphatase with adenosine triphosphate as substrate.

PubMed

Qian, Zhaosheng; Chai, Lujing; Tang, Cong; Huang, Yuanyuan; Chen, Jianrong; Feng, Hui

2015-03-03

A convenient, reliable, and highly sensitive real-time assay for alkaline phosphatase (ALP) activity in the continuous and recyclable way is established on the basis of aggregation and disaggregation of carbon quantum dots (CQDs) through the competitive assay approach. CQDs and adenosine triphosphate (ATP) were used as the fluorescent indicator and substrate for ALP activity assessment, respectively. Richness of carboxyl groups on the surface of CQDs enables their severe aggregation triggered by cerium ions, which results in effective fluorescence quenching. Under the catalytic hydrolysis of ALP, ATP can be rapidly transformed to phosphate ions. Stronger affinity of phosphate ions to cerium ions than carboxyl groups is taken advantage of to achieve fluorescence recovery induced by redispersion of CQDs in the presence of ALP and ATP. Quantitative evaluation of ALP activity in a broad range from 4.6 to 383.3 U/L with the detection limit of 1.4 U/L can be realized in this way, which endows the assay with high enough sensitivity for practical detection in human serum. The assay can be used in a recyclable way for more than three times since the generated product CePO4 as a precipitate can be easily removed from the standard assay system. This strategy broadens the sensing application of fluorescent CQDs with excellent biocompatibility and provides an example based on disaggregation in optical probe development.

The absolute counting of red cell-derived microparticles with red cell bead by flow rate based assay.

PubMed

Nantakomol, Duangdao; Imwong, Malika; Soontarawirat, Ingfar; Kotjanya, Duangporn; Khakhai, Chulalak; Ohashi, Jun; Nuchnoi, Pornlada

2009-05-01

Activation of red blood cell is associated with the formation of red cell-derived microparticles (RMPs). Analysis of circulating RMPs is becoming more refined and clinically useful. A quantitative Trucount tube method is the conventional method uses for quantitating RMPs. In this study, we validated a quantitative method called "flow rate based assay using red cell bead (FCB)" to measure circulating RMPs in the peripheral blood of healthy subjects. Citrated blood samples collected from 30 cases of healthy subjects were determined the RMPs count by using double labeling of annexin V-FITC and anti-glycophorin A-PE. The absolute RMPs numbers were measured by FCB, and the results were compared with the Trucount or with flow rate based calibration (FR). Statistical correlation and agreement were analyzed using linear regression and Bland-Altman analysis. There was no significant difference in the absolute number of RMPs quantitated by FCB when compared with those two reference methods including the Trucount tube and FR method. The absolute RMPs count obtained from FCB method was highly correlated with those obtained from Trucount tube (r(2) = 0.98, mean bias 4 cell/microl, limit of agreement [LOA] -20.3 to 28.3 cell/microl), and FR method (r(2) = 1, mean bias 10.3 cell/microl, and LOA -5.5 to 26.2 cell/microl). This study demonstrates that FCB is suitable and more affordable for RMPs quantitation in the clinical samples. This method is a low cost and interchangeable to latex bead-based method for generating the absolute counts in the resource-limited areas. (c) 2008 Clinical Cytometry Society.

Domain-based assays of individual antibody concentrations in an oligoclonal combination targeting a single protein.

PubMed

Meng, Q; Li, M; Silberg, M A; Conrad, F; Bettencourt, J; To, R; Huang, C; Ma, J; Meyer, K; Shimizu, R; Cao, L; Tomic, M T; Marks, J D

2012-02-15

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development, including pharmacokinetic (PK), toxicology, stability, and biochemical characterization studies of such drugs. We have developed an antitoxin, XOMA 3AB, consisting of three recombinant mAbs that potently neutralize the known subtypes of type A botulinum neurotoxin (BoNT/A). The three mAbs bind nonoverlapping BoNT/A epitopes with high affinity. XOMA 3AB is being developed as a treatment for botulism resulting from BoNT/A. To develop antibody-specific assays, we cloned, expressed, and purified BoNT/A domains from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. mAb-specific domains were used to develop an enzyme-linked immunosorbent assay (ELISA) for characterization of the integrity and binding activity of the three mAbs in the drug product. An electrochemiluminescence bridging assay that is robust to interference from components in serum was also developed, and we demonstrate that it can be used for PK assays. This type of antigen engineering to generate mAb-specific domains is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that binds the same protein and is superior to anti-idiotype approaches. Copyright © 2011 Elsevier Inc. All rights reserved.

Japanese Reference Panel of Blood Specimens for Evaluation of Hepatitis C Virus RNA and Core Antigen Quantitative Assays

PubMed Central

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

2012-01-01

An accurate and reliable quantitative assay for hepatitis C virus (HCV) is essential for measuring viral propagation and the efficacy of antiviral therapy. There is a growing need for domestic reference panels for evaluation of clinical assay kits because the performance of these kits may vary with region-specific genotypes or polymorphisms. In this study, we established a reference panel by selecting 80 donated blood specimens in Japan that tested positive for HCV. Using this panel, we quantified HCV viral loads using two HCV RNA kits and five core antigen (Ag) kits currently available in Japan. The data from the two HCV RNA assay kits showed excellent correlation. All RNA titers were distributed evenly across a range from 3 to 7 log IU/ml. Although the data from the five core Ag kits also correlated with RNA titers, the sensitivities of individual kits were not sufficient to quantify viral load in all samples. As calculated by the correlation with RNA titers, the theoretical lower limits of detection by these core Ag assays were higher than those for the detection of RNA. Moreover, in several samples in our panel, core Ag levels were underestimated compared to RNA titers. Sequence analysis in the HCV core region suggested that polymorphisms at amino acids 47 to 49 of the core Ag were responsible for this underestimation. The panel established in this study will be useful for estimating the quality of currently available and upcoming HCV assay kits; such quality control is essential for clinical usage of these kits. PMID:22495557

Studies of plant colonisation by closely related Bacillus amyloliquefaciens biocontrol agents using strain specific quantitative PCR assays.

PubMed

Johansson, Anna H; Bejai, Sarosh; Niazi, Adnan; Manzoor, Shahid; Bongcam-Rudloff, Erik; Meijer, Johan

2014-12-01

Certain strains of Bacillus amyloliquefaciens can colonize plants and improve growth and stress management. In order to study these effects, bacterial growth dynamics on plants and in the rhizosphere are of interest calling for specific analytical tools. For that purpose, quantitative real-time PCR (qPCR) assays were developed in order to differentiate among three closely related B. amyloliquefaciens subsp. plantarum strains (UCMB5033, UCMB5036, UCMB5113) and to determine their levels with high accuracy. Oligonucleotide primers were designed for strain unique gene sequences and used for SYBR green based qPCR analysis. Standard curves covered a wide linear range (10(6)) of DNA amounts with the lowest detection level at 50 fg. Post-reaction melting curve analysis showed only a single product. Accurate threshold cycles were obtained, even in the presence of high excess of related Bacillus strains and total bacterial DNA from soil. Analysis of Bacillus colonisation after seed treatment of two oilseed rape cultivars (Oase and Ritz) grown on agar support showed a time dependent effect but that the bacteria mostly were found on root tissues and little on green tissues. The colonisation on plants grown in soil varied among the Bacillus strains where Oase seemed to house more bacteria than Ritz. Applied as a mixture, all three Bacillus strains co-existed on the roots of plants grown in soil. The qPCR assay in combination with other techniques will be a powerful tool to study plant interactions of these B. amyloliquefaciens biocontrol agents to further understand the requirements for successful interactions and improvement of plant properties.

An automated smartphone-based diagnostic assay for point-of-care semen analysis

PubMed Central

Kanakasabapathy, Manoj Kumar; Sadasivam, Magesh; Singh, Anupriya; Preston, Collin; Thirumalaraju, Prudhvi; Venkataraman, Maanasa; Bormann, Charles L.; Draz, Mohamed Shehata; Petrozza, John C.; Shafiee, Hadi

2017-01-01

Male infertility affects up to 12% of the world’s male population and is linked to various environmental and medical conditions. Manual microscope-based testing and computer-assisted semen analysis (CASA) are the current standard methods to diagnose male infertility; however, these methods are labor-intensive, expensive, and laboratory-based. Cultural and socially dominated stigma against male infertility testing hinders a large number of men from getting tested for infertility, especially in resource-limited African countries. We describe the development and clinical testing of an automated smartphone-based semen analyzer designed for quantitative measurement of sperm concentration and motility for point-of-care male infertility screening. Using a total of 350 clinical semen specimens at a fertility clinic, we have shown that our assay can analyze an unwashed, unprocessed liquefied semen sample with <5-s mean processing time and provide the user a semen quality evaluation based on the World Health Organization (WHO) guidelines with ~98% accuracy. The work suggests that the integration of microfluidics, optical sensing accessories, and advances in consumer electronics, particularly smartphone capabilities, can make remote semen quality testing accessible to people in both developed and developing countries who have access to smartphones. PMID:28330865

Comparative study of β-glucan induced respiratory burst measured by nitroblue tetrazolium assay and real-time luminol-enhanced chemiluminescence assay in common carp (Cyprinus carpio L.).

PubMed

Vera-Jimenez, N I; Pietretti, D; Wiegertjes, G F; Nielsen, M E

2013-05-01

The respiratory burst is an important feature of the immune system. The increase in cellular oxygen uptake that marks the initiation of the respiratory burst is followed by the production of reactive oxygen species (ROS) such as superoxide anion and hydrogen peroxide which plays a role in the clearance of pathogens and tissue regeneration processes. Therefore, the respiratory burst and associated ROS constitute important indicators of fish health status. This paper compares two methods for quantitation of ROS produced during the respiratory burst in common carp: the widely used, single-point measurement based on the intracellular reduction of nitroblue tetrazolium (NBT) and a real-time luminol-enhanced assay based on the detection of native chemiluminescence. Both assays allowed for detection of dose-dependent changes in magnitude of the respiratory burst response induced by β-glucans in head kidney cells of carp. However, whereas the NBT assay was shown to detect the production of only superoxide anions, the real-time luminol-enhanced assay could detect the production of both superoxide anions and hydrogen peroxide. Only the chemiluminescence assay could reliably record the production of ROS on a real-time scale at frequent and continual time intervals for time course experiments, providing more detailed information on the respiratory burst response. The real-time chemiluminescence assay was used to measure respiratory burst activity in macrophage and neutrophilic granulocyte-enriched head kidney cell fractions and total head kidney cell suspensions and proved to be a fast, reliable, automated multiwell microplate assay to quantitate fish health status modulated by β-glucans. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Quantitative Point-of-Need Assay for the Assessment of Vitamin D3 Deficiency.

PubMed

Vemulapati, S; Rey, E; O'Dell, D; Mehta, S; Erickson, D

2017-10-26

Vitamin D is necessary for the healthy growth and development of bone and muscle. Vitamin D deficiency, which is present in 42% of the US population, is often undiagnosed as symptoms may not manifest for several years and long-term deficiency has been linked to osteoporosis, diabetes and cancer. Currently the majority of vitamin D testing is performed in large-scale commercial laboratories which have high operational costs and long times-to-result. Development of a low-cost point-of-need assay could be transformative to deficiency analysis in limited-resource settings. The best biomarker of vitamin D status, 25hydroxyvitamin D 3 (25(OH)D 3 ), however, is particularly challenging to measure in such a format due to complexities involved in sample preparation, including the need to separate the marker from its binding protein. Here we present a rapid diagnostic test for the accurate, quantitative assessment of 25(OH)D 3 in finger-stick blood. The assay is accompanied by a smartphone-assisted portable imaging device that can autonomously perform the necessary image processing. To achieve accurate quantification of 25(OH)D 3 , we also demonstrate a novel elution buffer that separates 25(OH)D 3 from its binding protein in situ, eliminating the need for sample preparation. In human trials, the accuracy of our platform is 90.5%.

Quantitative serology assays for determination of antibody responses to Ebola virus glycoprotein and matrix protein in nonhuman primates and humans.

PubMed

Vu, Hong; Shulenin, Sergey; Grolla, Allen; Audet, Jonathan; He, Shihua; Kobinger, Gary; Unfer, Robert C; Warfield, Kelly L; Aman, M Javad; Holtsberg, Frederick W

2016-02-01

The West Africa Ebola virus disease (EVD) outbreak has reached unprecedented magnitude and caused worldwide concerns for the spread of this deadly virus. Recent findings in nonhuman primates (NHPs) demonstrate that antibodies can be protective against EVD. However, the role of antibody response in vaccine-mediated protection is not fully understood. To address these questions quantitative serology assays are needed for measurement of the antibody response to key Ebola virus (EBOV) proteins. Serology enzyme-linked immunosorbent assays (ELISA's), using a reference detection antibody, were developed in order to standardize the quantitation of antibody levels in vaccinated NHPs or in humans exposed to EBOV or immunized with an EBOV vaccine. Critical reagents were generated to support the development of the serology ELISAs. Recombinant EBOV matrix protein (VP40) was expressed in Escherichia coli and purified. Two variants of the glycoprotein (GP), the ectodomain lacking the transmembrane domain (GPΔTM), and an engineered GP lacking the mucin-like domain (GPΔmuc) were expressed and purified from mammalian cell systems. Using these proteins, three ELISA methods were developed and optimized for reproducibility and robustness, including stability testing of critical reagents. The assay was used to determine the antibody response against VP40, GPΔTM, and GPΔmuc in a NHP vaccine study using EBOV virus-like particles (VLP) vaccine expressing GP, VP40 and the nucleoprotein. Additionally, these ELISAs were used to successfully detect antibody responses to VP40, GPΔTM and GPΔmuc in human sera from EBOV infected individuals. Copyright © 2015 Elsevier B.V. All rights reserved.

Clinical Comparison of an Enhanced-Sensitivity Branched-DNA Assay and Reverse Transcription-PCR for Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

PubMed Central

Nolte, Frederick S.; Boysza, Jodi; Thurmond, Cathy; Clark, W. Scott; Lennox, Jeffrey L.

1998-01-01

The performance characteristics of an enhanced-sensitivity branched-DNA assay (bDNA) (Quantiplex HIV-1 version 2.0; Chiron Corp., Emeryville, Calif.) and a reverse transcription (RT)-PCR assay (AMPLICOR HIV-1 Monitor; Roche Diagnostic Systems, Inc., Branchburg, N.J.) were compared in a molecular diagnostic laboratory. Samples used in this evaluation included linearity and reproducibility panels made by dilution of a human immunodeficiency virus type 1 (HIV-1) stock culture of known virus particle count in HIV-1-negative plasma, a subtype panel consisting of HIV-1 subtypes A through F at a standardized level, and 64 baseline plasma specimens from HIV-1-infected individuals. Plots of log10 HIV RNA copies per milliliter versus log10 nominal virus particles per milliliter demonstrated that both assays were linear over the stated dynamic ranges (bDNA, r = 0.98; RT-PCR, r = 0.99), but comparison of the slopes of the regression lines (bDNA, m = 0.96; RT-PCR, m = 0.83) suggested that RT-PCR had greater proportional systematic error. The between-run coefficients of variation for bDNA and RT-PCR were 24.3 and 34.3%, respectively, for a sample containing 1,650 nominal virus particles/ml and 44.0 and 42.7%, respectively, for a sample containing 165 nominal virus particles/ml. Subtypes B, C, and D were quantitated with similar efficiencies by bDNA and RT-PCR; however, RT-PCR was less efficient in quantitating subtypes A, E, and F. One non-B subtype was recognized in our clinical specimens based on the ratio of values obtained with the two methods. HIV-1 RNA was quantitated in 53 (83%) baseline plasma specimens by bDNA and in 55 (86%) specimens by RT-PCR. RT-PCR values were consistently greater than bDNA values, with population means of 142,419 and 67,580 copies/ml, respectively (P < 0.01). The results were highly correlated (r = 0.91), but the agreement was poor (mean difference in log10 copies per milliliter ± 2 standard deviations, 0.45 ± 0.61) for the 50 clinical specimens

Development of two stable isotope dilution assays for the quantitation of acrolein in heat-processed fats.

PubMed

Ewert, Alice; Granvogl, Michael; Schieberle, Peter

2011-04-27

Two stable isotope dilution assays were developed for the quantitation of acrolein in fats and oils using [(13)C(3)]-acrolein as the internal standard. First, a direct GC-MS headspace method, followed by an indirect GC-MS method using derivatization with pentafluorophenyl hydrazine, was established. Analysis of six different types of oils varying in their pattern of fatty acids showed significant differences in the amounts of acrolein formed after heating at various temperatures and for various times. For example, after 24 h at 140 °C, coconut oil contained 6.7 mg/kg, whereas linseed oil was highest with 242.3 mg/kg. A comparison of the results showed that the extent of acrolein formation seemed to be correlated with the amount of linolenic acid in the oils. Although the acrolein concentrations were lowered in all six oils after frying of potato crisps, linseed and rapeseed oil still contained the highest amounts of acrolein after frying. By applying both methods on different thermally treated fats and oils, nearly identical quantitative data were obtained.

Development of an Escherichia coli K12-specific quantitative polymerase chain reaction assay and DNA isolation suited to biofilms associated with iron drinking water pipe corrosion products.

PubMed

Lu, Jingrang; Gerke, Tammie L; Buse, Helen Y; Ashbolt, Nicholas J

2014-12-01

A quantitative polymerase chain reaction assay (115 bp amplicon) specific to Escherichia coli K12 with an ABI(TM) internal control was developed based on sequence data encoding the rfb gene cluster. Assay specificity was evaluated using three E. coli K12 strains (ATCC W3110, MG1655 & DH1), 24 non-K12 E. coli and 23 bacterial genera. The biofilm detection limit was 10(3) colony-forming units (CFU) E. coli K12 mL(-1), but required a modified protocol, which included a bio-blocker Pseudomonas aeruginosa with ethylenediaminetetraacetic acid buffered to pH 5 prior to cell lysis/DNA extraction. The novel protocol yielded the same sensitivity for drinking water biofilms associated with Fe3O4 (magnetite)-coated SiO2 (quartz) grains and biofilm-surface iron corrosion products from a drinking water distribution system. The novel DNA extraction protocol and specific E. coli K12 assay are sensitive and robust enough for detection and quantification within iron drinking water pipe biofilms, and are particularly well suited for studying enteric bacterial interactions within biofilms.

Rapid Real-Time PCR Assay for Detection and Quantitation of Mycobacterium avium subsp. paratuberculosis DNA in Artificially Contaminated Milk

PubMed Central

O'Mahony, Jim; Hill, Colin

2004-01-01

Using fluorescence resonance energy transfer technology and Lightcycler analysis, we developed a real-time PCR assay with primers and probes designed by using IS900 which allowed rapid detection of Mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Initially, the PCR parameters (including primer and probe levels, assay volume, Mg2+ concentration, and annealing temperature) were optimized. Subsequently, the quantitative ability of the assay was tested and was found to be accurate over a broad linear range (3 × 106 to 3 × 101 copies). The assay sensitivity when purified DNA was used was determined to be as low as five copies, with excellent reproducibility. A range of DNA isolation strategies was developed for isolating M. avium subsp. paratuberculosis DNA from spiked milk, the most effective of which involved the use of 50 mM Tris HCl, 10 mM EDTA, 2% Triton X-100, 4 M guanidinium isothiocyante, and 0.3 M sodium acetate combined with boiling, physical grinding, and nucleic acid spin columns. When this technique was used in conjunction with the real-time PCR assay, it was possible to consistently detect <100 organisms per ml of milk (equivalent to 2,000 organisms per 25 ml). Furthermore, the entire procedure (extraction and PCR) was performed in less than 3 h and was successfully adapted to quantify M. avium subsp. paratuberculosis in spiked milk from heavily and mildly contaminated samples. PMID:15294786

Detection limits of quantitative and digital PCR assays and their influence in presence-absence surveys of environmental DNA.

PubMed

Hunter, Margaret E; Dorazio, Robert M; Butterfield, John S S; Meigs-Friend, Gaia; Nico, Leo G; Ferrante, Jason A

2017-03-01

A set of universal guidelines is needed to determine the limit of detection (LOD) in PCR-based analyses of low-concentration DNA. In particular, environmental DNA (eDNA) studies require sensitive and reliable methods to detect rare and cryptic species through shed genetic material in environmental samples. Current strategies for assessing detection limits of eDNA are either too stringent or subjective, possibly resulting in biased estimates of species' presence. Here, a conservative LOD analysis grounded in analytical chemistry is proposed to correct for overestimated DNA concentrations predominantly caused by the concentration plateau, a nonlinear relationship between expected and measured DNA concentrations. We have used statistical criteria to establish formal mathematical models for both quantitative and droplet digital PCR. To assess the method, a new Grass Carp (Ctenopharyngodon idella) TaqMan assay was developed and tested on both PCR platforms using eDNA in water samples. The LOD adjustment reduced Grass Carp occupancy and detection estimates while increasing uncertainty-indicating that caution needs to be applied to eDNA data without LOD correction. Compared to quantitative PCR, digital PCR had higher occurrence estimates due to increased sensitivity and dilution of inhibitors at low concentrations. Without accurate LOD correction, species occurrence and detection probabilities based on eDNA estimates are prone to a source of bias that cannot be reduced by an increase in sample size or PCR replicates. Other applications also could benefit from a standardized LOD such as GMO food analysis and forensic and clinical diagnostics. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Alchemy: A Web 2.0 Real-time Quality Assurance Platform for Human Immunodeficiency Virus, Hepatitis C Virus, and BK Virus Quantitation Assays.

PubMed

Agosto-Arroyo, Emmanuel; Coshatt, Gina M; Winokur, Thomas S; Harada, Shuko; Park, Seung L

2017-01-01

The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45-60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician-supervised resident/fellow programming projects as learning

Alchemy: A Web 2.0 Real-time Quality Assurance Platform for Human Immunodeficiency Virus, Hepatitis C Virus, and BK Virus Quantitation Assays

PubMed Central

Agosto-Arroyo, Emmanuel; Coshatt, Gina M.; Winokur, Thomas S.; Harada, Shuko; Park, Seung L.

2017-01-01

Background: The molecular diagnostics laboratory faces the challenge of improving test turnaround time (TAT). Low and consistent TATs are of great clinical and regulatory importance, especially for molecular virology tests. Laboratory information systems (LISs) contain all the data elements necessary to do accurate quality assurance (QA) reporting of TAT and other measures, but these reports are in most cases still performed manually: a time-consuming and error-prone task. The aim of this study was to develop a web-based real-time QA platform that would automate QA reporting in the molecular diagnostics laboratory at our institution, and minimize the time expended in preparing these reports. Methods: Using a standard Linux, Nginx, MariaDB, PHP stack virtual machine running atop a Dell Precision 5810, we designed and built a web-based QA platform, code-named Alchemy. Data files pulled periodically from the LIS in comma-separated value format were used to autogenerate QA reports for the human immunodeficiency virus (HIV) quantitation, hepatitis C virus (HCV) quantitation, and BK virus (BKV) quantitation. Alchemy allowed the user to select a specific timeframe to be analyzed and calculated key QA statistics in real-time, including the average TAT in days, tests falling outside the expected TAT ranges, and test result ranges. Results: Before implementing Alchemy, reporting QA for the HIV, HCV, and BKV quantitation assays took 45–60 min of personnel time per test every month. With Alchemy, that time has decreased to 15 min total per month. Alchemy allowed the user to select specific periods of time and analyzed the TAT data in-depth without the need of extensive manual calculations. Conclusions: Alchemy has significantly decreased the time and the human error associated with QA report generation in our molecular diagnostics laboratory. Other tests will be added to this web-based platform in future updates. This effort shows the utility of informatician

Diagnostic accuracy of quantitative real-time PCR assay versus clinical and Gram stain identification of bacterial vaginosis.

PubMed

Menard, J-P; Mazouni, C; Fenollar, F; Raoult, D; Boubli, L; Bretelle, F

2010-12-01

The purpose of this investigation was to determine the diagnostic accuracy of quantitative real-time polymerase chain reaction (PCR) assay in diagnosing bacterial vaginosis versus the standard methods, the Amsel criteria and the Nugent score. The Amsel criteria, the Nugent score, and results from the molecular tool were obtained independently from vaginal samples of 163 pregnant women who reported abnormal vaginal symptoms before 20 weeks gestation. To determine the performance of the molecular tool, we calculated the kappa value, sensitivity, specificity, and positive and negative predictive values. Either or both of the Amsel criteria (≥3 criteria) and the Nugent score (score ≥7) indicated that 25 women (15%) had bacterial vaginosis, and the remaining 138 women did not. DNA levels of Gardnerella vaginalis or Atopobium vaginae exceeded 10(9) copies/mL or 10(8) copies/mL, respectively, in 34 (21%) of the 163 samples. Complete agreement between both reference methods and high concentrations of G. vaginalis and A. vaginae was found in 94.5% of women (154/163 samples, kappa value = 0.81, 95% confidence interval 0.70-0.81). The nine samples with discordant results were categorized as intermediate flora by the Nugent score. The molecular tool predicted bacterial vaginosis with a sensitivity of 100%, a specificity of 93%, a positive predictive value of 73%, and a negative predictive value of 100%. The quantitative real-time PCR assay shows excellent agreement with the results of both reference methods for the diagnosis of bacterial vaginosis.

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

PubMed

Shankar, Manoharan; Priyadharshini, Ramachandran; Gunasekaran, Paramasamy

2009-08-01

An image analysis-based method for high throughput screening of an alpha-amylase mutant library using chromogenic assays was developed. Assays were performed in microplates and high resolution images of the assay plates were read using the Virtual Microplate Reader (VMR) script to quantify the concentration of the chromogen. This method is fast and sensitive in quantifying 0.025-0.3 mg starch/ml as well as 0.05-0.75 mg glucose/ml. It was also an effective screening method for improved alpha-amylase activity with a coefficient of variance of 18%.

Piezo- and solenoid valve-based liquid dispensing for miniaturized assays.

PubMed

Niles, Walter D; Coassin, Peter J

2005-04-01

Miniaturization of biological assays requires dispensing liquids in the submicroliter range of volumes. Accuracy and reproducibility of dispensing this range depend on both the dispenser and the receptacle in which the assay is constructed. Miniaturization technologies developed by Aurora Discovery, Inc. (San Diego, CA) include high-density multiwell plates for assay samples and reagent storage, as well as piezo-based and solenoid valve-based liquid dispensers. Some basic principles of small-volume dispensing by jetting are described to provide context for dispenser design and function. Performance of the latest instruments incorporating these dispensing devices is presented.

Quantitative enzyme-linked immunosorbent assay for determination of polychlorinated biphenyls in environmental soil and sediment samples.

PubMed

Johnson, J C; Van Emon, J M

1996-01-01

An enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of Aroclors 1242, 1248, 1254, and 1260 in soil and sediments was developed and its performance compared with that of gas chromatography (GC). The detection limits for Aroclors 1242 and 1248 in soil are 10.5 and 9 ng/g, respectively. The assay linear dynamic range is 50-1333 ng/g. Cross-reactivity of the assay with 37 structurally related potential cocontaminants in environmental soil samples was examined; none of the chlorinated anisoles, benzenes, or phenols exhibited >3% cross-reactivity, with <0.1% cross-reactivity being the norm. Soil spike recoveries of 107% and 104% were obtained for Aroclors 1242 and 1248, respectively, for a spike level of 5 mg/kg, with corresponding relative standard deviations of 14% and 17%. One hundred forty-eight environmental soil, sediment, and paper pulp samples, obtained from two EPA listed Superfund sites, were analyzed by ELISA and standard GC methods. Samples were extracted for ELISA analysis by shaking with methanol. Additional extractions of the same samples were performed either with supercritical carbon dioxide or by Soxhlet extraction with methanol. ELISA results for both the supercritical fluid and the Soxhlet extracts were in close agreement with the GC results, while the ELISA results for the methanol shake extracts were not. The data for the environmental samples demonstrated the capability of the ELISA to provide accurate results and reinforced the dependence of any detection method, including ELISA, on appropriate extraction procedures.

Detection of influenza antigenic variants directly from clinical samples using polyclonal antibody based proximity ligation assays

PubMed Central

Martin, Brigitte E.; Jia, Kun; Sun, Hailiang; Ye, Jianqiang; Hall, Crystal; Ware, Daphne; Wan, Xiu-Feng

2016-01-01

Identification of antigenic variants is the key to a successful influenza vaccination program. The empirical serological methods to determine influenza antigenic properties require viral propagation. Here a novel quantitative PCR-based antigenic characterization method using polyclonal antibody and proximity ligation assays, or so-called polyPLA, was developed and validated. This method can detect a viral titer that is less than 1000 TCID50/mL. Not only can this method differentiate between different HA subtypes of influenza viruses but also effectively identify antigenic drift events within the same HA subtype of influenza viruses. Applications in H3N2 seasonal influenza data showed that the results from this novel method are consistent with those from the conventional serological assays. This method is not limited to the detection of antigenic variants in influenza but also other pathogens. It has the potential to be applied through a large-scale platform in disease surveillance requiring minimal biosafety and directly using clinical samples. PMID:25546251

Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens.

PubMed

Larson, Jeffrey S; Goodman, Laurie J; Tan, Yuping; Defazio-Eli, Lisa; Paquet, Agnes C; Cook, Jennifer W; Rivera, Amber; Frankson, Kristi; Bose, Jolly; Chen, Lili; Cheung, Judy; Shi, Yining; Irwin, Sarah; Kiss, Linda D B; Huang, Weidong; Utter, Shannon; Sherwood, Thomas; Bates, Michael; Weidler, Jodi; Parry, Gordon; Winslow, John; Petropoulos, Christos J; Whitcomb, Jeannette M

2010-06-28

We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7-10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH).

Evaluation of quantitative polymerase chain reaction assays targeting Mycobacterium avium, M. intracellulare, and M. avium subspecies paratuberculosis in drinking water biofilms.

PubMed

Chern, Eunice C; King, Dawn; Haugland, Richard; Pfaller, Stacy

2015-03-01

Mycobacterium avium (MA), Mycobacterium intracellulare (MI), and Mycobacterium avium subsp. paratuberculosis (MAP) are difficult to culture due to their slow growing nature. A quantitative polymerase chain reaction (qPCR) method for the rapid detection of MA, MI, and MAP can be used to provide data supporting drinking water biofilms as potential sources of human exposure. The aim of this study was to characterize two qPCR assays targeting partial 16S rRNA gene sequences of MA and MI and use these assays, along with two previously reported MAP qPCR assays (IS900 and Target 251), to investigate Mycobacterium occurrence in kitchen faucet biofilms. MA and MI qPCR assays demonstrated 100% specificity and sensitivity when evaluated against 18 non-MA complex, 76 MA, and 17 MI isolates. Both assays detected approximately 1,000 cells from a diluted cell stock inoculated on a sampling swab 100% of the time. DNA analysis by qPCR indicated that 35.3, 56.9 and 11.8% of the 51 kitchen faucet biofilm samples collected contained MA, MI, and MAP, respectively. This study introduces novel qPCR assays designed to specifically detect MA and MI in biofilm. Results support the use of qPCR as an alternative to culture for detection and enumeration of MA, MI, and MAP in microbiologically complex samples.

Relative Chemical Binding Affinities for Trout and Human Estrogen Receptor Using Different Competitive Binding Assays

EPA Science Inventory

Rainbow trout-based assays for estrogenicity are currently being used for development of predictive models based upon quantitative structure activity relationships. A predictive model based on a single species raises the question of whether this information is valid for other spe...

Development of a high-throughput assay for rapid screening of butanologenic strains.

PubMed

Agu, Chidozie Victor; Lai, Stella M; Ujor, Victor; Biswas, Pradip K; Jones, Andy; Gopalan, Venkat; Ezeji, Thaddeus Chukwuemeka

2018-02-21

We report a Thermotoga hypogea (Th) alcohol dehydrogenase (ADH)-dependent spectrophotometric assay for quantifying the amount of butanol in growth media, an advance that will facilitate rapid high-throughput screening of hypo- and hyper-butanol-producing strains of solventogenic Clostridium species. While a colorimetric nitroblue tetrazolium chloride-based assay for quantitating butanol in acetone-butanol-ethanol (ABE) fermentation broth has been described previously, we determined that Saccharomyces cerevisiae (Sc) ADH used in this earlier study exhibits approximately 13-fold lower catalytic efficiency towards butanol than ethanol. Any Sc ADH-dependent assay for primary quantitation of butanol in an ethanol-butanol mixture is therefore subject to "ethanol interference". To circumvent this limitation and better facilitate identification of hyper-butanol-producing Clostridia, we searched the literature for native ADHs that preferentially utilize butanol over ethanol and identified Th ADH as a candidate. Indeed, recombinant Th ADH exhibited a 6-fold higher catalytic efficiency with butanol than ethanol, as measured using the reduction of NADP + to NADPH that accompanies alcohol oxidation. Moreover, the assay sensitivity was not affected by the presence of acetone, acetic acid or butyric acid (typical ABE fermentation products). We broadened the utility of our assay by adapting it to a high-throughput microtiter plate-based format, and piloted it successfully in an ongoing metabolic engineering initiative.

New generation QuIC assays for prion seeding activity.

PubMed

Orrù, Christina D; Wilham, Jason M; Vascellari, Sarah; Hughson, Andrew G; Caughey, Byron

2012-01-01

The ability of abnormal TSE-associated forms of PrP to seed the formation of amyloid fibrils from recombinant PrP(Sen) has served as the basis for several relatively rapid and highly sensitive tests for prion diseases. These tests include rPrP-PMCA (rPMCA), standard quaking-induced conversion (S-QuIC), amyloid seeding assay (ASA), real-time QuIC (RT-QuIC) and enhanced QuIC (eQuIC). Here, we summarize recent improvements in the RT-QuIC-based assays that enhance the practicality, sensitivity and quantitative attributes of assays QuIC and promote the detection of prion seeding activity in dilute, inhibitor-laden fluids such as blood plasma.

A TaqMan real-time PCR-based assay for the identification of Fasciola spp.

PubMed

Alasaad, Samer; Soriguer, Ramón C; Abu-Madi, Marawan; El Behairy, Ahmed; Jowers, Michael J; Baños, Pablo Díez; Píriz, Ana; Fickel, Joerns; Zhu, Xing-Quan

2011-06-30

Real time quantitative PCR (qPCR) is one of the key technologies of the post-genome era, with clear advantages compared to normal end-point PCR. In this paper, we report the first qPCR-based assay for the identification of Fasciola spp. Based on sequences of the second internal transcribed spacers (ITS-2) of the ribosomal rRNA gene, we used a set of genus-specific primers for Fasciola ITS-2 amplification, and we designed species-specific internal TaqMan probes to identify F. hepatica and F. gigantica, as well as the hybrid 'intermediate'Fasciola. These primers and probes were used for the highly specific, sensitive, and simple identification of Fasciola species collected from different animal host from China, Spain, Niger and Egypt. The novel qPCR-based technique for the identification of Fasciola spp. may provide a useful tool for the epidemiological investigation of Fasciola infection, including their intermediate snail hosts. Copyright © 2011 Elsevier B.V. All rights reserved.

MTS dye based colorimetric CTLL-2 cell proliferation assay for product release and stability monitoring of interleukin-15: assay qualification, standardization and statistical analysis.

PubMed

Soman, Gopalan; Yang, Xiaoyi; Jiang, Hengguang; Giardina, Steve; Vyas, Vinay; Mitra, George; Yovandich, Jason; Creekmore, Stephen P; Waldmann, Thomas A; Quiñones, Octavio; Alvord, W Gregory

2009-08-31

A colorimetric cell proliferation assay using soluble tetrazolium salt [(CellTiter 96(R) Aqueous One Solution) cell proliferation reagent, containing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) and an electron coupling reagent phenazine ethosulfate], was optimized and qualified for quantitative determination of IL-15 dependent CTLL-2 cell proliferation activity. An in-house recombinant Human (rHu)IL-15 reference lot was standardized (IU/mg) against an international reference standard. Specificity of the assay for IL-15 was documented by illustrating the ability of neutralizing anti-IL-15 antibodies to block the product specific CTLL-2 cell proliferation and the lack of blocking effect with anti-IL-2 antibodies. Under the defined assay conditions, the linear dose-response concentration range was between 0.04 and 0.17ng/ml of the rHuIL-15 produced in-house and 0.5-3.0IU/ml for the international standard. Statistical analysis of the data was performed with the use of scripts written in the R Statistical Language and Environment utilizing a four-parameter logistic regression fit analysis procedure. The overall variation in the ED(50) values for the in-house reference standard from 55 independent estimates performed over the period of 1year was 12.3% of the average. Excellent intra-plate and within-day/inter-plate consistency was observed for all four parameter estimates in the model. Different preparations of rHuIL-15 showed excellent intra-plate consistency in the parameter estimates corresponding to the lower and upper asymptotes as well as to the 'slope' factor at the mid-point. The ED(50) values showed statistically significant differences for different lots and for control versus stressed samples. Three R-scripts improve data analysis capabilities allowing one to describe assay variations, to draw inferences between data sets from formal statistical tests, and to set up improved assay acceptance

Development and use of quantitative competitive PCR assays for relative quantifying rumen anaerobic fungal populations in both in vitro and in vivo systems.

PubMed

Sekhavati, Mohammad H; Mesgaran, Mohsen Danesh; Nassiri, Mohammad R; Mohammadabadi, Tahereh; Rezaii, Farkhondeh; Fani Maleki, Adham

2009-10-01

This paper describes the use of a quantitative competitive polymerase chain reaction (QC-PCR) assay; using PCR primers to the rRNA locus of rumen fungi and a standard-control DNA including design and validation. In order to test the efficiency of this method for quantifying anaerobic rumen fungi, it has been attempted to evaluate this method in in vitro conditions by comparing with an assay based on measuring cell wall chitin. The changes in fungal growth have been studied when they are grown in in vitro on either untreated (US) or sodium hydroxide treated wheat straw (TS). Results showed that rumen fungi growth was significantly higher in treated samples compared with untreated during the 12d incubation (P<0.05) and plotting the chitin assay's results against the competitive PCR's showed high positive correlation (R(2)> or =0.87). The low mean values of the coefficients of variance in repeatability in the QC-PCR method against the chitin assay demonstrated more reliability of this new approach. And finally, the efficiency of this method was investigated in in vivo conditions. Samples of rumen fluid were collected from four fistulated Holstein steers which were fed four different diets (basal diet, high starch, high sucrose and starch plus sucrose) in rotation. The results of QC-PCR showed that addition of these non-structural carbohydrates to the basal diets caused a significant decrease in rumen anaerobic fungi biomass. The QC-PCR method appears to be a reliable and can be used for rumen samples.

Cell-based Assays for Assessing Toxicity: A Basic Guide.

PubMed

Parboosing, Raveen; Mzobe, Gugulethu; Chonco, Louis; Moodley, Indres

2016-01-01

Assessment of toxicity is an important component of the drug discovery process. Cellbased assays are a popular choice for assessing cytotoxicity. However, these assays are complex because of the wide variety of formats and methods that are available, lack of standardization, confusing terminology and the inherent variability of biological systems and measurement. This review is intended as a guide on how to take these factors into account when planning, conducting and/or interpreting cell based toxicity assays. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

PubMed Central

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

2009-01-01

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

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

EPA Science Inventory

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

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

PubMed Central

Yoshida, Yoko; Miyata, Toshiyuki; Matsumoto, Masanori; Shirotani-Ikejima, Hiroko; Uchida, Yumiko; Ohyama, Yoshifumi; Kokubo, Tetsuro; Fujimura, Yoshihiro

2015-01-01

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. PMID:25951460

[Multicenter evaluation of h-FABP semi-quantitative assay (Cardio Detect) in central laboratory: the point in acute myocardial infarction diagnosis].

PubMed

Lefèvre, G; Fayet, J-M; Graïne, H; Berny, C; Maupas-Schwalm, F; Capolaghi, B; Morin, C

2007-01-01

The diagnostic performance of heart-Fatty Acid Binding Protein (h-FABP) (semi-quantitative CardioDetect test) and cardiac troponin I (TnIc) blood assays were compared in one hundred patients presenting with suspicion of acute coronary syndrome. Final patient diagnosis was "acute myocardial infarction" in 36 cases, "non ST myocardial infarction" in 25 cases and "non ischemic pathologies" in 39 cases. h-FABP results were positive in 26 patients, negative in 57 patients and ambiguous in 17 patients, the latter corresponding to the final diagnosis of "acute myocardial infarction" in 5 cases, "non ST myocardial infarction" in 2 cases and "non ischemic pathologies " in 10 cases. At admission, h-FABP and TnIc exhibiteda sensitivity of 54% an 66%, respectively and a specificity of 86% and 95%, respectively. Positive and negative predictive values were 81% and 64% for h-FABP, respectively and 92% and 75% for cTnI, respectively. h-FABP and cTnI demonstrated a similar diagnostic efficiency if admission delay is less than 4 hours after onset of chest pain (area under ROC curve TnIc = 0.767 +/- 0.091 ; area under ROC curve h-FABP = 0.622 +/- 0.109 ; p = 0.144). On the contrary, cTnI assay demonstrated a better efficiency than h-FABP (p< 0.005) for patients admitted in a delay of 4 to 12 hours after the onset of chest pain. If chosen cTnI cut-off corresponded to the recent consensus definition used for monitoring acute coronary syndrome patients, h-FABP semi-quantitative assay realized within central laboratory did not demonstrated a better diagnostic efficiency than cTnI.

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

PubMed

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.

Receptor-based screening assays for the detection of antibiotics residues - A review.

PubMed

Ahmed, Saeed; Ning, Jianan; Cheng, Guyue; Ahmad, Ijaz; Li, Jun; Mingyue, Liu; Qu, Wei; Iqbal, Mujahid; Shabbir, M A B; Yuan, Zonghui

2017-05-01

Consumer and regulatory agencies have a high concern to antibiotic residues in food producing animals, so appropriate screening assays of fast, sensitive, low cost, and easy sample preparation for the identification of these residues are essential for the food-safety insurance. Great efforts in the development of a high-throughput antibiotic screening assay have been made in recent years. Concerning the screening of antibiotic residue, this review elaborate an overview on the availability, advancement and applicability of antibiotic receptor based screening assays for the safety assessment of antibiotics usage (i.e. radio receptor assay, enzyme labeling assays, colloidal gold receptor assay, enzyme colorimetry assay and biosensor assay). This manuscript also tries to shed a light on the selection, preparation and future perspective of receptor protein for antibiotic residue detection. These assays have been introduced for the screening of numerous food samples. Receptor based screening technology for antibiotic detection has high accuracy. It has been concluded that at the same time, it can detect a class of drugs for certain receptor, and realize the multi-residue detection. These assays offer fast, easy and precise detection of antibiotics. Copyright © 2017 Elsevier B.V. All rights reserved.

Analytical Validation of a Highly Quantitative, Sensitive, Accurate, and Reproducible Assay (HERmark®) for the Measurement of HER2 Total Protein and HER2 Homodimers in FFPE Breast Cancer Tumor Specimens

PubMed Central

Larson, Jeffrey S.; Goodman, Laurie J.; Tan, Yuping; Defazio-Eli, Lisa; Paquet, Agnes C.; Cook, Jennifer W.; Rivera, Amber; Frankson, Kristi; Bose, Jolly; Chen, Lili; Cheung, Judy; Shi, Yining; Irwin, Sarah; Kiss, Linda D. B.; Huang, Weidong; Utter, Shannon; Sherwood, Thomas; Bates, Michael; Weidler, Jodi; Parry, Gordon; Winslow, John; Petropoulos, Christos J.; Whitcomb, Jeannette M.

2010-01-01

We report here the results of the analytical validation of assays that measure HER2 total protein (H2T) and HER2 homodimer (H2D) expression in Formalin Fixed Paraffin Embedded (FFPE) breast cancer tumors as well as cell line controls. The assays are based on the VeraTag technology platform and are commercially available through a central CAP-accredited clinical reference laboratory. The accuracy of H2T measurements spans a broad dynamic range (2-3 logs) as evaluated by comparison with cross-validating technologies. The measurement of H2T expression demonstrates a sensitivity that is approximately 7–10 times greater than conventional immunohistochemistry (IHC) (HercepTest). The HERmark assay is a quantitative assay that sensitively and reproducibly measures continuous H2T and H2D protein expression levels and therefore may have the potential to stratify patients more accurately with respect to response to HER2-targeted therapies than current methods which rely on semiquantitative protein measurements (IHC) or on indirect assessments of gene amplification (FISH). PMID:21151530

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

PubMed

Bigalke, Hans; Rummel, Andreas

2015-11-25

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

Flow cytometric HyPer-based assay for hydrogen peroxide.

PubMed

Lyublinskaya, O G; Antonov, S A; Gorokhovtsev, S G; Pugovkina, N A; Kornienko, Ju S; Ivanova, Ju S; Shatrova, A N; Aksenov, N D; Zenin, V V; Nikolsky, N N

2018-05-30

HyPer is a genetically encoded fluorogenic sensor for hydrogen peroxide which is generally used for the ratiometric imaging of H 2 O 2 fluxes in living cells. Here, we demonstrate the advantages of HyPer-based ratiometric flow cytometry assay for H 2 O 2 , by using K562 and human mesenchymal stem cell lines expressing HyPer. We show that flow cytometry analysis is suitable to detect HyPer response to submicromolar concentrations of extracellularly added H 2 O 2 that is much lower than concentrations addressed previously in the other HyPer-based assays (such as cell imaging or fluorimetry). Suggested technique is also much more sensitive to hydrogen peroxide than the widespread flow cytometry assay exploiting H 2 O 2 -reactive dye H 2 DCFDA and, contrary to the H 2 DCFDA-based assay, can be employed for the kinetic studies of H 2 O 2 utilization by cells, including measurements of the rate constants of H 2 O 2 removal. In addition, flow cytometry multi-parameter ratiometric measurements enable rapid and high-throughput detection of endogenously generated H 2 O 2 in different subpopulations of HyPer-expressing cells. To sum up, HyPer can be used in multi-parameter flow cytometry studies as a highly sensitive indicator of intracellular H 2 O 2 . Copyright © 2018. Published by Elsevier Inc.

Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories

USDA-ARS?s Scientific Manuscript database

Bead based multiplex assays (BBMA) also referred to as Luminex, MultiAnalyte Profiling or cytometric bead array (CBA) assays, are applicable for high throughput, simultaneous detection of multiple analytes in solution (from several, up to 50-500 analytes within a single, small sample volume). Curren...

Determination of antibacterial activity and minimum inhibitory concentration of larval extract of fly via resazurin-based turbidometric assay.

PubMed

Teh, Chien Huey; Nazni, Wasi Ahmad; Nurulhusna, Ab Hamid; Norazah, Ahmad; Lee, Han Lim

2017-02-16

Antimicrobial resistance is currently a major global issue. As the rate of emergence of antimicrobial resistance has superseded the rate of discovery and introduction of new effective drugs, the medical arsenal now is experiencing shortage of effective drugs to combat diseases, particularly against diseases caused by the dreadful multidrug-resistant strains, such as the methicillin-resistant Staphylococcus aureus (MRSA). The ability of fly larvae to thrive in septic habitats has prompted us to determine the antibacterial activity and minimum inhibitory concentrations (MICs) of larval extract of flies, namely Lucilia cuprina, Sarcophaga peregrina and Musca domestica against 4 pathogenic bacteria [Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa and Escherichia coli] via a simple and sensitive antibacterial assay, resazurin-based turbidometric (TB) assay as well as to demonstrate the preliminary chemical profile of larval extracts using gas chromatography-mass spectrophotometry (GC-MS). The resazurin-based TB assay demonstrated that the L. cuprina larval extract was inhibitory against all tested bacteria, whilst the larval extract of S. peregrina and M. domestica were only inhibitory against the MRSA, with a MIC of 100 mg ml -1 . Subsequent sub-culture of aliquots revealed that the larval extract of L. cuprina was bactericidal against MRSA whilst the larval extracts of S. peregrina and M. domestica were bacteriostatic against MRSA. The GC-MS analysis had quantitatively identified 20 organic compounds (fatty acids or their derivatives, aromatic acid esters, glycosides and phenol) from the larval extract of L. cuprina; and 5 fatty acid derivatives with known antimicrobial activities from S. peregrina and M. domestica. The resazurin-based turbidometric assay is a simple, reliable and feasible screening assay which evidently demonstrated the antibacterial activity of all fly larval extracts, primarily against the MRSA. The larval

An automated smartphone-based diagnostic assay for point-of-care semen analysis.

PubMed

Kanakasabapathy, Manoj Kumar; Sadasivam, Magesh; Singh, Anupriya; Preston, Collin; Thirumalaraju, Prudhvi; Venkataraman, Maanasa; Bormann, Charles L; Draz, Mohamed Shehata; Petrozza, John C; Shafiee, Hadi

2017-03-22

Male infertility affects up to 12% of the world's male population and is linked to various environmental and medical conditions. Manual microscope-based testing and computer-assisted semen analysis (CASA) are the current standard methods to diagnose male infertility; however, these methods are labor-intensive, expensive, and laboratory-based. Cultural and socially dominated stigma against male infertility testing hinders a large number of men from getting tested for infertility, especially in resource-limited African countries. We describe the development and clinical testing of an automated smartphone-based semen analyzer designed for quantitative measurement of sperm concentration and motility for point-of-care male infertility screening. Using a total of 350 clinical semen specimens at a fertility clinic, we have shown that our assay can analyze an unwashed, unprocessed liquefied semen sample with <5-s mean processing time and provide the user a semen quality evaluation based on the World Health Organization (WHO) guidelines with ~98% accuracy. The work suggests that the integration of microfluidics, optical sensing accessories, and advances in consumer electronics, particularly smartphone capabilities, can make remote semen quality testing accessible to people in both developed and developing countries who have access to smartphones. Copyright © 2017, American Association for the Advancement of Science.

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

PubMed

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

2009-08-15

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

A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets.

PubMed

Teste, Bruno; Ali-Cherif, Anaïs; Viovy, Jean Louis; Malaquin, Laurent

2013-06-21

Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.

Quantitative image analysis of cellular heterogeneity in breast tumors complements genomic profiling.

PubMed

Yuan, Yinyin; Failmezger, Henrik; Rueda, Oscar M; Ali, H Raza; Gräf, Stefan; Chin, Suet-Feung; Schwarz, Roland F; Curtis, Christina; Dunning, Mark J; Bardwell, Helen; Johnson, Nicola; Doyle, Sarah; Turashvili, Gulisa; Provenzano, Elena; Aparicio, Sam; Caldas, Carlos; Markowetz, Florian

2012-10-24

Solid tumors are heterogeneous tissues composed of a mixture of cancer and normal cells, which complicates the interpretation of their molecular profiles. Furthermore, tissue architecture is generally not reflected in molecular assays, rendering this rich information underused. To address these challenges, we developed a computational approach based on standard hematoxylin and eosin-stained tissue sections and demonstrated its power in a discovery and validation cohort of 323 and 241 breast tumors, respectively. To deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy number profiles between samples. We next devised a predictor for survival in estrogen receptor-negative breast cancer that integrated both image-based and gene expression analyses and significantly outperformed classifiers that use single data types, such as microarray expression signatures. Image processing also allowed us to describe and validate an independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative, image-based method could benefit any large-scale cancer study by refining and complementing molecular assays of tumor samples.

Improved soft-agar colony assay in a fluid processing apparatus.

PubMed

Forsman, A D; Herpich, A R; Chapes, S K

1999-01-01

The standard method for quantitating bone marrow precursor cells has been to count the number of colony-forming units that form in semisolid (0.3%) agar. Recently we adapted this assay for use in hardware, the Fluid Processing Apparatus, that is flown in standard payload lockers of the space shuttle. When mouse or rat macrophage colony-forming units were measured with this hardware in ground-based assays, we found significantly more colony growth than that seen in standard plate assays. The improved growth correlates with increased agar thickness but also appears to be due to properties inherent to the Fluid Processing Apparatus. This paper describes an improved method for determining bone marrow macrophage precursor numbers in semisolid agar.

Toward a microfluidic-based rapid amylase assay system.

PubMed

Holmes, Richard J; Summersgil, Philip; Ryan, Timothy; Brown, Bernard J Treves; Mockbil, Amal; Grieve, Bruce D; Fielden, Peter R

2009-08-01

This article describes work into a prototype system for the assay of amylase, using microfludic technologies. The new system has a significantly shorter cycle time than the current laboratory methods, which generally use microtitre plates, yet is capable of generating significantly superior results. As such, we have shown that sensitivity is enhanced by a factor of 10 in the standard assay trials, and by a factor of 2 in the real-sample lab trials. In both assays, the use of a microreactor system reduced the reaction time by a factor of 6.2, from 20 min incubation to 3.2 min. Basing the conclusion on the Megazyme Cerealpha Standard Method, and using the Cerealpha units as a measure of assay efficiency, the typical response for the microfluidic assay was shown to be 1.0 x 10(-3) CU/mL (standard deviation [SD] 2.5 x 10(-4) CU/mL), compared to 2.56 x 10(-4) CU/mL (SD 5.94 x 10(-5) CU/mL) for the standard macroassay. It is believed that this improvement in the reaction schematics is due to the inherent advantages of microfluidic devices such as superior mixing, higher thermal efficiency, and enhanced reaction kinetics.

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria.

PubMed

Gold, Ben; Roberts, Julia; Ling, Yan; Lopez Quezada, Landys; Glasheen, Jou; Ballinger, Elaine; Somersan-Karakaya, Selin; Warrier, Thulasi; Nathan, Carl

2016-12-14

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 °C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to ≥99% bacterial kill (MBC≥99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

Multiplexed detection of DNA sequences using a competitive displacement assay in a microfluidic SERRS-based device.

PubMed

Yazdi, Soroush H; Giles, Kristen L; White, Ian M

2013-11-05

We demonstrate sensitive and multiplexed detection of DNA sequences through a surface enhanced resonance Raman spectroscopy (SERRS)-based competitive displacement assay in an integrated microsystem. The use of the competitive displacement scheme, in which the target DNA sequence displaces a Raman-labeled reporter sequence that has lower affinity for the immobilized probe, enables detection of unlabeled target DNA sequences with a simple single-step procedure. In our implementation, the displacement reaction occurs in a microporous packed column of silica beads prefunctionalized with probe-reporter pairs. The use of a functionalized packed-bead column in a microfluidic channel provides two major advantages: (i) immobilization surface chemistry can be performed as a batch process instead of on a chip-by-chip basis, and (ii) the microporous network eliminates the diffusion limitations of a typical biological assay, which increases the sensitivity. Packed silica beads are also leveraged to improve the SERRS detection of the Raman-labeled reporter. Following displacement, the reporter adsorbs onto aggregated silver nanoparticles in a microfluidic mixer; the nanoparticle-reporter conjugates are then trapped and concentrated in the silica bead matrix, which leads to a significant increase in plasmonic nanoparticles and adsorbed Raman reporters within the detection volume as compared to an open microfluidic channel. The experimental results reported here demonstrate detection down to 100 pM of the target DNA sequence, and the experiments are shown to be specific, repeatable, and quantitative. Furthermore, we illustrate the advantage of using SERRS by demonstrating multiplexed detection. The sensitivity of the assay, combined with the advantages of multiplexed detection and single-step operation with unlabeled target sequences makes this method attractive for practical applications. Importantly, while we illustrate DNA sequence detection, the SERRS-based competitive

Impact of New Quick Gold Nanoparticle-Based Cortisol Assay During Adrenal Vein Sampling for Primary Aldosteronism.

PubMed

Yoneda, Takashi; Karashima, Shigehiro; Kometani, Mitsuhiro; Usukura, Mikiya; Demura, Masashi; Sanada, Junichirou; Minami, Tetsuya; Koda, Wataru; Gabata, Toshifumi; Matsui, Osamu; Idegami, Koutarou; Takamura, Yuzuru; Tamiya, Eiichi; Oe, Masashi; Nakai, Masuo; Mori, Shunsuke; Terayama, Noboru; Matsuda, Yuichi; Kamemura, Kouhei; Fujii, Sumie; Seta, Takashi; Sawamura, Toshitaka; Okuda, Rika; Takeda, Yoshimichi; Hayashi, Kenshi; Yamagishi, Masakazu; Takeda, Yoshiyu

2016-06-01

Adrenal vein sampling (AVS) is essential for identifying a surgically curable form of primary aldosteronism (PA), but accurate placement of the sampling catheter is technically challenging. Intraprocedural cortisol measurement can confirm the catheter's position, thereby increasing the AVS success rate. We developed a quick cortisol assay (QCA) that uses immunochromatography and gold nanoparticles and can be performed either semiquantitatively or quantitatively. The assay was evaluated in two studies. In a single-center study, PA patients were assigned to undergo AVS incorporating the semiquantitative QCA (n = 30), the quantitative QCA (n = 30), or without the QCA (n = 30), and the rates of successful AVS were determined. In a prospective multicenter randomized, controlled study, the success rates of AVS performed with (n = 148) or without (n = 145) the semiquantitative QCA were determined. Cortisol concentrations were measured during AVS in 6 minutes or less in the radiology suite, without additional technical assistance, and significantly correlated with a conventional reference assay (R(2) = 0.994; P < .001). In the single-center study, the differences in the AVS success rates associated with semiquantitative and quantitative QCAs were not significant (both 93%); however, the success rates were significantly higher than the rate of successful AVS performed without using the QCA (63%; P < .001). The success rate of AVS performed in the multicenter study was 94% for the semiquantitative QCA, which was significantly higher than the rate for the patients without QCA (60%; P < .001). Our novel QCA was rapidly and easily performed at the point of care and improved the rate of successful AVS.

[Study on ethnic medicine quantitative reference herb,Tibetan medicine fruits of Capsicum frutescens as a case].

PubMed

Zan, Ke; Cui, Gan; Guo, Li-Nong; Ma, Shuang-Cheng; Zheng, Jian

2018-05-01

High price and difficult to get of reference substance have become obstacles to HPLC assay of ethnic medicine. A new method based on quantitative reference herb (QRH) was proposed. Specific chromatograms in fruits of Capsicum frutescens were employed to determine peak positions, and HPLC quantitative reference herb was prepared from fruits of C. frutescens. The content of capsaicin and dihydrocapsaicin in the quantitative control herb was determined by HPLC. Eleven batches of fruits of C. frutescens were analyzed with quantitative reference herb and reference substance respectively. The results showed no difference. The present method is feasible for quality control of ethnic medicines and quantitative reference herb is suitable to replace reference substances in assay. Copyright© by the Chinese Pharmaceutical Association.

A rapid and ultrasensitive SERRS assay for histidine and tyrosine based on azo coupling.

PubMed

Sui, Huimin; Wang, Yue; Yu, Zhi; Cong, Qian; Han, Xiao Xia; Zhao, Bing

2016-10-01

A simple and highly sensitive surface-enhanced resonance Raman scattering (SERRS)-based approach coupled with azo coupling reaction has been put forward for quantitative analysis of histidine and tyrosine. The SERRS-based assay is simple and rapid by mixing the azo reaction products with silver nanoparticles (AgNPs) for measurements within 2min. The limits of detection (LODs) of the method are as low as 4.33×10(-11) and 8.80×10(-11)M for histidine and tyrosine, respectively. Moreover, the SERRS fingerprint information specific to corresponding amino acids guarantees the selective detection for the target histidine and tyrosine. The results from serum indicated the potential application of the proposed approach into biological samples. Compared with the methods ever reported, the main advantages of this methodology are simpleness, rapidity without time-consuming separation or pretreatment steps, high sensitivity, selectivity and the potential for determination of other molecules containing imidazole or phenol groups. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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

2014-01-01

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

High-throughput receptor-based assay for the detection of spirolides by chemiluminescence.

PubMed

Rodríguez, Laura P; Vilariño, Natalia; Molgó, Jordi; Aráoz, Rómulo; Botana, Luis M

2013-12-01

The spirolides are marine toxins that belong to a new class of macrocyclic imines produced by dinoflagellates. In this study a previously described solid-phase receptor-based assay for the detection of spirolides was optimized for high-throughput screening and prevalidated. This method is based on the competition between 13-desmethyl spirolide C and biotin-α-bungarotoxin immobilized on a streptavidin-coated surface, for binding to nicotinic acetylcholine receptors. In this inhibition assay the amount of nAChR bound to the well surface is quantified using a specific antibody, followed by a second anti-mouse IgG antibody labeled with horseradish peroxidase (HRP). The assay protocol was optimized for 384-well microplates, which allowed a reduction of the amount of reagents per sample and an increase of the number of samples per plate versus previously published receptor-based assays. The sensitivity of the assay for 13-desmethyl spirolide C ranged from 5 to 150 ng mL(-1). The performance of the assay in scallop extracts was adequate, with an estimated detection limit for 13-desmethyl spirolide C of 50 μg kg(-1) of shellfish meat. The recovery rate of 13-desmethyl spirolide C for spiked samples with this assay was 80% and the inter-assay coefficient of variation was 8%. This 384-well microplate, chemiluminescence method can be used as a high-throughput screening assay to detect 13-desmethyl spirolide C in shellfish meat in order to reduce the number of samples to be processed through bioassays or analytical methods. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development of a serology-based assay for efficacy evaluation of a lactococcicosis vaccine in Seriola fish.

PubMed

Nakajima, Nao; Kawanishi, Michiko; Imamura, Saiki; Hirano, Fumiya; Uchiyama, Mariko; Yamamoto, Kinya; Nagai, Hidetaka; Futami, Kunihiko; Katagiri, Takayuki; Maita, Masashi; Kijima, Mayumi

2014-05-01

Lactococcicosis is an infection caused by the bacterium Lactococcus garvieae and creates serious economic damage to cultured marine and fresh water fish industries. The use of the assay currently applied to evaluate the potency of the lactococcicosis vaccine is contingent upon meeting specific parameters after statistical analysis of the percent survival of the vaccinated yellowtail or greater amberjack fish after challenge with a virulent strain of L. garvieae. We found that measuring the serological response with a quantitative agglutinating antibody against the L. garvieae antigen (phenotype KG+) was an effective method of monitoring the potency of lactococcicosis vaccines. Vaccinated fish had significantly higher antibody titers than control fish when the L. garvieae Lg2-S strain was used as an antigen. Furthermore, the titer of the KG + agglutinating antibody was correlated with vaccine potency, and the cut-off titer was determined by comparing the data with those from the challenge test. An advantage of the proposed serology-based potency assay is that it will contribute to reduced numbers of animal deaths during vaccine potency evaluations. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

PubMed

2016-07-01

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 diagnostics and personalized treatment decisions. Here we describe the results of a community-wide benchmarking study comparing the performance of all widely used methods for DNA methylation analysis that are compatible with routine clinical use. We shipped 32 reference samples to 18 laboratories in seven different countries. Researchers in those laboratories collectively contributed 21 locus-specific assays for an average of 27 predefined genomic regions, as well as six global assays. We evaluated assay sensitivity on low-input samples and assessed the assays' ability to discriminate between cell types. Good agreement was observed across all tested methods, with amplicon bisulfite sequencing and bisulfite pyrosequencing showing the best all-round performance. Our technology comparison can inform the selection, optimization and use of DNA methylation assays in large-scale validation studies, biomarker development and clinical diagnostics.

A Novel Stopped-Flow Assay for Quantitating Carbonic-Anhydrase Activity and Assessing Red-Blood-Cell Hemolysis.

PubMed

Zhao, Pan; Geyer, R Ryan; Boron, Walter F

2017-01-01

We report a novel carbonic-anhydrase (CA) assay and its use for quantitating red-blood-cell (RBC) lysis during stopped-flow (SF) experiments. We combine two saline solutions, one containing HEPES/pH 7.03 and the other, ~1% CO 2 /44 mM [Formula: see text]/pH 8.41, to generate an out-of-equilibrium CO 2 /[Formula: see text] solution containing ~0.5% CO 2 /22 [Formula: see text]/pH ~7.25 (10°C) in the SF reaction cell. CA catalyzes relaxation of extracellular pH to ~7.50: [Formula: see text] + H + → CO 2 + H 2 O. Proof-of-concept studies (no intact RBCs) show that the pH-relaxation rate constant ( k ΔpH )-measured via pyranine fluorescence-rises linearly with increases in [bovine CAII] or [murine-RBC lysate]. The y-intercept (no CA) was k ΔpH = 0.0183 s -1 . Combining increasing amounts of murine-RBC lysate with ostensibly intact RBCs (pre-SF hemolysis ≅0.4%)-fixing total [hemoglobin] at 2.5 μM in the reaction cell to simulate hemolysis from ostensibly 0 to 100%-causes k ΔpH to increase linearly. This y-intercept (0% lysate/100% ostensibly intact RBCs) was k ΔpH = 0.0820 s -1 , and the maximal k ΔpH (100% lysate/0% intact RBCs) was 1.304 s -1 . Thus, mean percent hemolysis in the reaction cell was ~4.9%. Phenol-red absorbance assays yield indistinguishable results. The increase from 0.4 to 4.9% presumably reflects mechanical RBC disruption during rapid mixing. In all fluorescence studies, the CA blocker acetazolamide reduces k ΔpH to near-uncatalyzed values, implying that all CA activity is extracellular. Our lysis assay is simple, sensitive, and precise, and will be valuable for correcting for effects of lysis in physiological SF experiments. The underlying CA assay, applied to blood plasma, tissue-culture media, and organ perfusates could assess lysis in a variety of applications.

A Novel Stopped-Flow Assay for Quantitating Carbonic-Anhydrase Activity and Assessing Red-Blood-Cell Hemolysis

PubMed Central

Zhao, Pan; Geyer, R. Ryan; Boron, Walter F.

2017-01-01

We report a novel carbonic-anhydrase (CA) assay and its use for quantitating red-blood-cell (RBC) lysis during stopped-flow (SF) experiments. We combine two saline solutions, one containing HEPES/pH 7.03 and the other, ~1% CO2/44 mM HCO3-/pH 8.41, to generate an out-of-equilibrium CO2/HCO3- solution containing ~0.5% CO2/22 HCO3-/pH ~7.25 (10°C) in the SF reaction cell. CA catalyzes relaxation of extracellular pH to ~7.50: HCO3- + H+ → CO2 + H2O. Proof-of-concept studies (no intact RBCs) show that the pH-relaxation rate constant (kΔpH)—measured via pyranine fluorescence—rises linearly with increases in [bovine CAII] or [murine-RBC lysate]. The y-intercept (no CA) was kΔpH = 0.0183 s−1. Combining increasing amounts of murine-RBC lysate with ostensibly intact RBCs (pre-SF hemolysis ≅0.4%)—fixing total [hemoglobin] at 2.5 μM in the reaction cell to simulate hemolysis from ostensibly 0 to 100%—causes kΔpH to increase linearly. This y-intercept (0% lysate/100% ostensibly intact RBCs) was kΔpH = 0.0820 s−1, and the maximal kΔpH (100% lysate/0% intact RBCs) was 1.304 s−1. Thus, mean percent hemolysis in the reaction cell was ~4.9%. Phenol-red absorbance assays yield indistinguishable results. The increase from 0.4 to 4.9% presumably reflects mechanical RBC disruption during rapid mixing. In all fluorescence studies, the CA blocker acetazolamide reduces kΔpH to near-uncatalyzed values, implying that all CA activity is extracellular. Our lysis assay is simple, sensitive, and precise, and will be valuable for correcting for effects of lysis in physiological SF experiments. The underlying CA assay, applied to blood plasma, tissue-culture media, and organ perfusates could assess lysis in a variety of applications. PMID:28400735

Renilla luciferase-based quantitation of Potato virus A infection initiated with Agrobacterium infiltration of N. benthamiana leaves.

PubMed

Eskelin, K; Suntio, T; Hyvärinen, S; Hafren, A; Mäkinen, K

2010-03-01

A quantitation method based on the sensitive detection of Renilla luciferase (Rluc) activity was developed and optimized for Potato virus A (PVA; genus Potyviridae) gene expression. This system is based on infections initiated by Agrobacterium infiltration and subsequent detection of the translation of PVA::Rluc RNA, which is enhanced by viral replication, first within the cells infected initially and later by translation and replication within new cells after spread of the virus. Firefly luciferase (Fluc) was used as an internal control to normalize the Rluc activity. An approximately 10-fold difference in the Rluc/Fluc activity ratio between a movement-deficient and a replication-deficient mutant was observed starting from 48h post Agrobacterium infiltration (h.p.i.). The Rluc activity derived from wild type (wt) PVA increased significantly between 48 and 72h.p.i. and the Rluc/Fluc activity deviated clearly from that of the mutant viruses. Quantitation of the Rluc and Fluc mRNAs by semi-quantitative RT-PCR indicated that increases and decreases in the Renillareniformis luciferase (rluc) mRNA levels coincided with changes in Rluc activity. However, a subtle increase in the mRNA level led to pronounced changes in Rluc activity. PVA CP accumulation was quantitated by enzyme-linked immunosorbent assay. The increase in Rluc activity correlated closely with virus accumulation. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Evaluation of qPCR-Based Assays for Leprosy Diagnosis Directly in Clinical Specimens

PubMed Central

Sarno, Euzenir Nunes; Moraes, Milton Ozório

2011-01-01

The increased reliability and efficiency of the quantitative polymerase chain reaction (qPCR) makes it a promising tool for performing large-scale screening for infectious disease among high-risk individuals. To date, no study has evaluated the specificity and sensitivity of different qPCR assays for leprosy diagnosis using a range of clinical samples that could bias molecular results such as difficult-to-diagnose cases. In this study, qPCR assays amplifying different M. leprae gene targets, sodA, 16S rRNA, RLEP and Ag 85B were compared for leprosy differential diagnosis. qPCR assays were performed on frozen skin biopsy samples from a total of 62 patients: 21 untreated multibacillary (MB), 26 untreated paucibacillary (PB) leprosy patients, as well as 10 patients suffering from other dermatological diseases and 5 healthy donors. To develop standardized protocols and to overcome the bias resulted from using chromosome count cutoffs arbitrarily defined for different assays, decision tree classifiers were used to estimate optimum cutoffs and to evaluate the assays. As a result, we found a decreasing sensitivity for Ag 85B (66.1%), 16S rRNA (62.9%), and sodA (59.7%) optimized assay classifiers, but with similar maximum specificity for leprosy diagnosis. Conversely, the RLEP assay showed to be the most sensitive (87.1%). Moreover, RLEP assay was positive for 3 samples of patients originally not diagnosed as having leprosy, but these patients developed leprosy 5–10 years after the collection of the biopsy. In addition, 4 other samples of patients clinically classified as non-leprosy presented detectable chromosome counts in their samples by the RLEP assay suggesting that those patients either had leprosy that was misdiagnosed or a subclinical state of leprosy. Overall, these results are encouraging and suggest that RLEP assay could be useful as a sensitive diagnostic test to detect M. leprae infection before major clinical manifestations. PMID:22022631