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Sample records for highly specific detection

  1. Highly sensitive and specific colorimetric detection of cancer cells via dual-aptamer target binding strategy.

    PubMed

    Wang, Kun; Fan, Daoqing; Liu, Yaqing; Wang, Erkang

    2015-11-15

    Simple, rapid, sensitive and specific detection of cancer cells is of great importance for early and accurate cancer diagnostics and therapy. By coupling nanotechnology and dual-aptamer target binding strategies, we developed a colorimetric assay for visually detecting cancer cells with high sensitivity and specificity. The nanotechnology including high catalytic activity of PtAuNP and magnetic separation & concentration plays a vital role on the signal amplification and improvement of detection sensitivity. The color change caused by small amount of target cancer cells (10 cells/mL) can be clearly distinguished by naked eyes. The dual-aptamer target binding strategy guarantees the detection specificity that large amount of non-cancer cells and different cancer cells (10(4) cells/mL) cannot cause obvious color change. A detection limit as low as 10 cells/mL with detection linear range from 10 to 10(5) cells/mL was reached according to the experimental detections in phosphate buffer solution as well as serum sample. The developed enzyme-free and cost effective colorimetric assay is simple and no need of instrument while still provides excellent sensitivity, specificity and repeatability, having potential application on point-of-care cancer diagnosis. PMID:26042871

  2. Highly specific SNP detection using 2D graphene electronics and DNA strand displacement

    PubMed Central

    Hwang, Michael T.; Landon, Preston B.; Lee, Joon; Choi, Duyoung; Mo, Alexander H.; Glinsky, Gennadi; Lal, Ratnesh

    2016-01-01

    Single-nucleotide polymorphisms (SNPs) in a gene sequence are markers for a variety of human diseases. Detection of SNPs with high specificity and sensitivity is essential for effective practical implementation of personalized medicine. Current DNA sequencing, including SNP detection, primarily uses enzyme-based methods or fluorophore-labeled assays that are time-consuming, need laboratory-scale settings, and are expensive. Previously reported electrical charge-based SNP detectors have insufficient specificity and accuracy, limiting their effectiveness. Here, we demonstrate the use of a DNA strand displacement-based probe on a graphene field effect transistor (FET) for high-specificity, single-nucleotide mismatch detection. The single mismatch was detected by measuring strand displacement-induced resistance (and hence current) change and Dirac point shift in a graphene FET. SNP detection in large double-helix DNA strands (e.g., 47 nt) minimize false-positive results. Our electrical sensor-based SNP detection technology, without labeling and without apparent cross-hybridization artifacts, would allow fast, sensitive, and portable SNP detection with single-nucleotide resolution. The technology will have a wide range of applications in digital and implantable biosensors and high-throughput DNA genotyping, with transformative implications for personalized medicine. PMID:27298347

  3. Highly specific SNP detection using 2D graphene electronics and DNA strand displacement.

    PubMed

    Hwang, Michael T; Landon, Preston B; Lee, Joon; Choi, Duyoung; Mo, Alexander H; Glinsky, Gennadi; Lal, Ratnesh

    2016-06-28

    Single-nucleotide polymorphisms (SNPs) in a gene sequence are markers for a variety of human diseases. Detection of SNPs with high specificity and sensitivity is essential for effective practical implementation of personalized medicine. Current DNA sequencing, including SNP detection, primarily uses enzyme-based methods or fluorophore-labeled assays that are time-consuming, need laboratory-scale settings, and are expensive. Previously reported electrical charge-based SNP detectors have insufficient specificity and accuracy, limiting their effectiveness. Here, we demonstrate the use of a DNA strand displacement-based probe on a graphene field effect transistor (FET) for high-specificity, single-nucleotide mismatch detection. The single mismatch was detected by measuring strand displacement-induced resistance (and hence current) change and Dirac point shift in a graphene FET. SNP detection in large double-helix DNA strands (e.g., 47 nt) minimize false-positive results. Our electrical sensor-based SNP detection technology, without labeling and without apparent cross-hybridization artifacts, would allow fast, sensitive, and portable SNP detection with single-nucleotide resolution. The technology will have a wide range of applications in digital and implantable biosensors and high-throughput DNA genotyping, with transformative implications for personalized medicine. PMID:27298347

  4. A novel and highly specific phage endolysin cell wall binding domain for detection of Bacillus cereus.

    PubMed

    Kong, Minsuk; Sim, Jieun; Kang, Taejoon; Nguyen, Hoang Hiep; Park, Hyun Kyu; Chung, Bong Hyun; Ryu, Sangryeol

    2015-09-01

    Rapid, specific and sensitive detection of pathogenic bacteria is crucial for public health and safety. Bacillus cereus is harmful as it causes foodborne illness and a number of systemic and local infections. We report a novel phage endolysin cell wall-binding domain (CBD) for B. cereus and the development of a highly specific and sensitive surface plasmon resonance (SPR)-based B. cereus detection method using the CBD. The newly discovered CBD from endolysin of PBC1, a B. cereus-specific bacteriophage, provides high specificity and binding capacity to B. cereus. By using the CBD-modified SPR chips, B. cereus can be detected at the range of 10(5)-10(8) CFU/ml. More importantly, the detection limit can be improved to 10(2) CFU/ml by using a subtractive inhibition assay based on the pre-incubation of B. cereus and CBDs, removal of CBD-bound B. cereus, and SPR detection of the unbound CBDs. The present study suggests that the small and genetically engineered CBDs can be promising biological probes for B. cereus. We anticipate that the CBD-based SPR-sensing methods will be useful for the sensitive, selective, and rapid detection of B. cereus. PMID:26043681

  5. Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

    PubMed

    Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

    2015-01-01

    The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results. PMID:26345863

  6. Prostate specific antigen detection using AlGaN /GaN high electron mobility transistors

    NASA Astrophysics Data System (ADS)

    Kang, B. S.; Wang, H. T.; Lele, T. P.; Tseng, Y.; Ren, F.; Pearton, S. J.; Johnson, J. W.; Rajagopal, P.; Roberts, J. C.; Piner, E. L.; Linthicum, K. J.

    2007-09-01

    Antibody-functionalized Au-gated AlGaN /GaN high electron mobility transistors (HEMTs) were used to detect prostate specific antigen (PSA). The PSA antibody was anchored to the gate area through the formation of carboxylate succinimdyl ester bonds with immobilized thioglycolic acid. The AlGaN /GaN HEMT drain-source current showed a rapid response of less than 5s when target PSA in a buffer at clinical concentrations was added to the antibody-immobilized surface. The authors could detect a wide range of concentrations from 10pg/mlto1μg/ml. The lowest detectable concentration was two orders of magnitude lower than the cutoff value of PSA measurements for clinical detection of prostate cancer. These results clearly demonstrate the promise of portable electronic biological sensors based on AlGaN /GaN HEMTs for PSA screening.

  7. Highly sensitive nanoparticle-based immunoassays with elemental detection: Application to Prostate-Specific Antigen quantification.

    PubMed

    Garcia-Cortes, Marta; Encinar, Jorge Ruiz; Costa-Fernandez, Jose M; Sanz-Medel, Alfredo

    2016-11-15

    One of the major challenges in developing novel assay methods for the detection of biomolecules is achieving high sensitivity, because of the ultralow concentrations typically in clinical samples. Here, a Mn-doped ZnS quantum dots-based immunoassay platform is presented for highly sensitive detection of cancer biomarkers. Ultrahigh sensitivity is achieved through gold deposition on the surface of the nanoparticle tags acting as catalytic seeds, thus effectively amplifying the size of the metallic nanoparticles after the immunoassay and before the tag detection. Elemental mass spectrometry measurement of the gold content allowed detection of Prostate-Specific Antigen (PSA) at the low attog mL(-1) level. Moreover, the developed method showed not only an extremely high sensitivity for PSA detection but also a broad dynamic range, higher than 8 orders of magnitude, particularly useful for clinical studies involving quantitative detection of diverse biomarkers at their very different relevant concentration levels. Its applicability to discriminate small differences in PSA concentrations at low levels (few pgmL(-1)) in real serum samples was successfully evaluated. PMID:27162143

  8. Highly Specific Detection of Five Exotic Quarantine Plant Viruses using RT-PCR.

    PubMed

    Choi, Hoseong; Cho, Won Kyong; Yu, Jisuk; Lee, Jong-Seung; Kim, Kook-Hyung

    2013-03-01

    To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was 51.9°C. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virus-infected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea. PMID:25288934

  9. Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and specificity

    NASA Astrophysics Data System (ADS)

    Ning, Xinghai; Lee, Seungjun; Wang, Zhirui; Kim, Dongin; Stubblefield, Bryan; Gilbert, Eric; Murthy, Niren

    2011-08-01

    The diagnosis of bacterial infections remains a major challenge in medicine. Although numerous contrast agents have been developed to image bacteria, their clinical impact has been minimal because they are unable to detect small numbers of bacteria in vivo, and cannot distinguish infections from other pathologies such as cancer and inflammation. Here, we present a family of contrast agents, termed maltodextrin-based imaging probes (MDPs), which can detect bacteria in vivo with a sensitivity two orders of magnitude higher than previously reported, and can detect bacteria using a bacteria-specific mechanism that is independent of host response and secondary pathologies. MDPs are composed of a fluorescent dye conjugated to maltohexaose, and are rapidly internalized through the bacteria-specific maltodextrin transport pathway, endowing the MDPs with a unique combination of high sensitivity and specificity for bacteria. Here, we show that MDPs selectively accumulate within bacteria at millimolar concentrations, and are a thousand-fold more specific for bacteria than mammalian cells. Furthermore, we demonstrate that MDPs can image as few as 105 colony-forming units in vivo and can discriminate between active bacteria and inflammation induced by either lipopolysaccharides or metabolically inactive bacteria.

  10. Precise detection of L. monocytogenes hitting its highly conserved region possessing several specific antibody binding sites.

    PubMed

    Jahangiri, Abolfazl; Rasooli, Iraj; Reza Rahbar, Mohammad; Khalili, Saeed; Amani, Jafar; Ahmadi Zanoos, Kobra

    2012-07-21

    Listeria monocytogenes, a facultative intracellular fast-growing Gram-positive food-borne pathogen, can infect immunocompromised individuals leading to meningitis, meningoencephalitis and septicaemias. From the pool of virulence factors of the organism, ActA, a membrane protein, has a critical role in the life cycle of L. monocytogenes. High mortality rate of listeriosis necessitates a sensitive and rapid diagnostic test for precise identification of L. monocytogenes. We used bioinformatic tools to locate a specific conserved region of ActA for designing and developing an antibody-antigen based diagnostic test for the detection of L. monocytogenes. A number of databases were looked for ActA related sequences. Sequences were analyzed with several online software to find an appropriate region for our purpose. ActA protein was found specific to Listeria species with no homologs in other organisms. We finally introduced a highly conserved region within ActA sequence that possess several antibody binding sites specific to L. monocytogenes. This protein sequence can serve as an antigen for designing a relatively cheap, sensitive, and specific diagnostic test for detection of L. monocytogenes. PMID:22575546

  11. A Rapid In-Clinic Test Detects Acute Leptospirosis in Dogs with High Sensitivity and Specificity.

    PubMed

    Kodjo, Angeli; Calleja, Christophe; Loenser, Michael; Lin, Dan; Lizer, Joshua

    2016-01-01

    A rapid IgM-detection immunochromatographic test (WITNESS® Lepto, Zoetis) has recently become available to identify acute canine leptospirosis at the point of care. Diagnostic sensitivity and specificity of the test were evaluated by comparison with the microscopic agglutination assay (MAT), using a positive cut-off titer of ≥800. Banked serum samples from dogs exhibiting clinical signs and suspected leptospirosis were selected to form three groups based on MAT titer: (1) positive (n = 50); (2) borderline (n = 35); and (3) negative (n = 50). Using an analysis to weight group sizes to reflect French prevalence, the sensitivity and specificity were 98% and 93.5% (88.2% unweighted), respectively. This test rapidly identifies cases of acute canine leptospirosis with high levels of sensitivity and specificity with no interference from previous vaccination. PMID:27110562

  12. A Rapid In-Clinic Test Detects Acute Leptospirosis in Dogs with High Sensitivity and Specificity

    PubMed Central

    Kodjo, Angeli; Calleja, Christophe; Loenser, Michael; Lin, Dan; Lizer, Joshua

    2016-01-01

    A rapid IgM-detection immunochromatographic test (WITNESS® Lepto, Zoetis) has recently become available to identify acute canine leptospirosis at the point of care. Diagnostic sensitivity and specificity of the test were evaluated by comparison with the microscopic agglutination assay (MAT), using a positive cut-off titer of ≥800. Banked serum samples from dogs exhibiting clinical signs and suspected leptospirosis were selected to form three groups based on MAT titer: (1) positive (n = 50); (2) borderline (n = 35); and (3) negative (n = 50). Using an analysis to weight group sizes to reflect French prevalence, the sensitivity and specificity were 98% and 93.5% (88.2% unweighted), respectively. This test rapidly identifies cases of acute canine leptospirosis with high levels of sensitivity and specificity with no interference from previous vaccination. PMID:27110562

  13. Specific detection of Aspergillus parasiticus in wheat flour using a highly sensitive PCR assay.

    PubMed

    Sardiñas, Noelia; Vázquez, Covadonga; Gil-Serna, Jessica; González-Jaen, M Teresa; Patiño, Belén

    2010-06-01

    Aspergillus parasiticus is one of the most important aflatoxin-producing species that contaminates foodstuffs and beverages for human consumption. In this work, a specific and highly sensitive PCR protocol was developed to detect A. parasiticus using primers designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). The assay proved to be highly specific for A. parasiticus when tested on a wide range of related and other fungal species commonly found in commodities, and allowing discrimination from the closely related A. flavus. Accuracy of detection and quantification by conventional PCR were tested with genomic DNA obtained from wheat flour artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10(6) spore/g could be detected by the assay directly without prior incubation of the samples. The assay described is suitable for incorporation in routine analyses at critical points of the food chain within HACCP strategies. PMID:20486001

  14. Inexpensive Designer Antigen for Anti-HIV Antibody Detection with High Sensitivity and Specificity

    PubMed Central

    Talha, Sheikh M.; Salminen, Teppo; Chugh, Deepti A.; Swaminathan, Sathyamangalam; Soukka, Tero; Pettersson, Kim; Khanna, Navin

    2010-01-01

    A novel recombinant multiepitope protein (MEP) has been designed that consists of four linear, immunodominant, and phylogenetically conserved epitopes, taken from human immunodeficiency virus (HIV)-encoded antigens that are used in many third-generation immunoassay kits. This HIV-MEP has been evaluated for its diagnostic potential in the detection of anti-HIV antibodies in human sera. A synthetic MEP gene encoding these epitopes, joined by flexible peptide linkers in a single open reading frame, was designed and overexpressed in Escherichia coli. The recombinant HIV-MEP was purified using a single affinity step, yielding >20 mg pure protein/liter culture, and used as the coating antigen in an in-house immunoassay. Bound anti-HIV antibodies were detected by highly sensitive time-resolved fluorometry, using europium(III) chelate-labeled anti-human antibody. The sensitivity and specificity of the HIV-MEP were evaluated using Boston Biomedica worldwide HIV performance, HIV seroconversion, and viral coinfection panels and were found to be comparable with those of commercially available anti-HIV enzyme immunoassay (EIA) kits. The careful choice of epitopes, high epitope density, and an E. coli-based expression system, coupled with a simple purification protocol and the use of europium(III) chelate-labeled tracer, provide the capability for the development of an inexpensive diagnostic test with high degrees of sensitivity and specificity. PMID:20089793

  15. One-step species-specific high resolution melting analysis for nosocomial bacteria detection.

    PubMed

    Wong, Yeng Pooi; Chua, Kek Heng; Thong, Kwai Lin

    2014-12-01

    Nosocomial infections are a major public health concern worldwide. Early and accurate identification of nosocomial pathogens which are often multidrug resistant is crucial for prompt treatment. Hence, an alternative real-time polymerase chain reaction coupled with high resolution melting-curve analysis (HRMA) was developed for identification of five nosocomial bacteria. This assay targets species-specific regions of each nosocomial bacteria and produced five distinct melt curves with each representing a particular bacterial species. The melting curves were characterized by peaks of 78.8 ± 0.2 °C for Acinetobacter baumannii, 82.7 ± 0.2 °C for Escherichia coli, 86.3 ± 0.3 °C for Klebsiella pneumoniae, 88.8 ± 0.2 °C for Pseudomonas aeruginosa and 74.6 ± 02 °C for methicillin-resistant Staphylococcus aureus. The assay was able to specifically detect the five bacterial species with an overall detection limit of 2 × 10(-2) ng/μL. In conclusion, the HRM assay developed is a simple and rapid method for identification of the selected nosocomial pathogens. PMID:25307691

  16. In situ detection of basic fibroblast growth factor by highly specific antibodies.

    PubMed Central

    Schulze-Osthoff, K.; Risau, W.; Vollmer, E.; Sorg, C.

    1990-01-01

    Basic fibroblast growth factor (bFGF) is thought to be of major importance for fibrosis and angiogenesis. Despite intensive studies dealing with the biochemistry and multiple biologic effects of bFGF, the cellular distribution is virtually unknown. Therefore, using the indirect immunoperoxidase technique, we examined the effect of bFGF on a large pattern of normal, inflammatory, and tumorous human tissues. Staining was performed on cryostat sections with a highly specific affinity-purified antiserum. In normal tissues, especially those of the thymus and placenta, mainly dendritic cells contained the growth factor. High levels of bFGF were also detected in basal cells and gland epithelial cells of skin biopsies. A conspicuous expression was observed in chronic inflammatory tissues corresponding to a generally pronounced proliferation of fibroblasts and endothelial cells in these situations. Tumors revealed a very heterogenous staining pattern. In some lesions, bFGF was predominantly present in infiltrating and endothelial cells. In several, neoplasms tumor cells exhibited an intensive staining. In some, especially vascular tumors, bFGF could not be detected. From the staining results it is concluded that angiogenesis is not simply controlled by the presence of bFGF but is mediated by a balance of several angiogenic inducers and inhibitors. Images Figure 1 Figure 2 PMID:1695484

  17. A highly specific monoclonal antibody against monkeypox virus detects the heparin binding domain of A27.

    PubMed

    Hughes, Laura J; Goldstein, Jason; Pohl, Jan; Hooper, Jay W; Lee Pitts, R; Townsend, Michael B; Bagarozzi, Dennis; Damon, Inger K; Karem, Kevin L

    2014-09-01

    The eradication of smallpox and the cessation of global vaccination led to the increased prevalence of human infections in Central Africa. Serologic and protein-based diagnostic assay for MPXV detection is difficult due to cross-reactive antibodies that do not differentiate between diverse orthopoxvirus (OPXV) species. A previously characterized monoclonal antibody (mAb 69-126-3-7) against MPXV [1] was retested for cross-reactivity with various OPXVs. The 14.5 kDa band protein that reacted with mAb 69-126-3 was identified to be MPXV A29 protein (homolog of vaccinia virus Copenhagen A27). Amino acid sequence analysis of the MPXV A29 with other OPXV homologs identified four amino acid changes. Peptides corresponding to these regions were designed and evaluated for binding to mAb 69-126-3 by ELISA and BioLayer Interferometry (BLI). Further refinement and truncations mapped the specificity of this antibody to a single amino acid difference in a 30-mer peptide compared to other OPXV homologs. This particular residue is proposed to be essential for heparin binding by VACV A27 protein. Despite this substitution, MPXV A29 bound to heparin with similar affinity to that of VACV A27 protein, suggesting flexibility of this motif for heparin binding. Although binding of mAb 69-126-3-7 to MPXV A29 prevented interaction with heparin, it did not have any effect on the infectivity of MPXV. Characterization of 69-126-3-7 mAb antibody allows for the possibility of the generation of a serological based species-specific detection of OPXVs despite high proteomic homology. PMID:25108113

  18. Aptamer-MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen.

    PubMed

    Jolly, Pawan; Tamboli, Vibha; Harniman, Robert L; Estrela, Pedro; Allender, Chris J; Bowen, Jenna L

    2016-01-15

    This study reports the design and evaluation of a new synthetic receptor sensor based on the amalgamation of biomolecular recognition elements and molecular imprinting to overcome some of the challenges faced by conventional protein imprinting. A thiolated DNA aptamer with established affinity for prostate specific antigen (PSA) was complexed with PSA prior to being immobilised on the surface of a gold electrode. Controlled electropolymerisation of dopamine around the complex served to both entrap the complex, holding the aptamer in, or near to, it's binding conformation, and to localise the PSA binding sites at the sensor surface. Following removal of PSA, it was proposed that the molecularly imprinted polymer (MIP) cavity would act synergistically with the embedded aptamer to form a hybrid receptor (apta-MIP), displaying recognition properties superior to that of aptamer alone. Electrochemical impedance spectroscopy (EIS) was used to evaluate subsequent rebinding of PSA to the apta-MIP surface. The apta-MIP sensor showed high sensitivity with a linear response from 100pg/ml to 100ng/ml of PSA and a limit of detection of 1pg/ml, which was three-fold higher than aptamer alone sensor for PSA. Furthermore, the sensor demonstrated low cross-reactivity with a homologous protein (human Kallikrein 2) and low response to human serum albumin (HSA), suggesting possible resilience to the non-specific binding of serum proteins. PMID:26318788

  19. Highly Specific Plasmonic Biosensors for Ultrasensitive MicroRNA Detection in Plasma from Pancreatic Cancer Patients

    PubMed Central

    2015-01-01

    MicroRNAs (miRs) are small noncoding RNAs that regulate mRNA stability and/or translation. Because of their release into the circulation and their remarkable stability, miR levels in plasma and other biological fluids can serve as diagnostic and prognostic disease biomarkers. However, quantifying miRs in the circulation is challenging due to issues with sensitivity and specificity. This Letter describes for the first time the design and characterization of a regenerative, solid-state localized surface plasmon resonance (LSPR) sensor based on highly sensitive nanostructures (gold nanoprisms) that obviates the need for labels or amplification of the miRs. Our direct hybridization approach has enabled the detection of subfemtomolar concentration of miR-X (X = 21 and 10b) in human plasma in pancreatic cancer patients. Our LSPR-based measurements showed that the miR levels measured directly in patient plasma were at least 2-fold higher than following RNA extraction and quantification by reverse transcriptase-polymerase chain reaction. Through LSPR-based measurements we have shown nearly 4-fold higher concentrations of miR-10b than miR-21 in plasma of pancreatic cancer patients. We propose that our highly sensitive and selective detection approach for assaying miRs in plasma can be applied to many cancer types and disease states and should allow a rational approach for testing the utility of miRs as markers for early disease diagnosis and prognosis, which could allow for the design of effective individualized therapeutic approaches. PMID:25379951

  20. Highly specific DNA detection employing ligation on suspension bead array readout.

    PubMed

    Mezger, Anja; Kühnemund, Malte; Nilsson, Mats; Herthnek, David

    2015-09-25

    We show for the first time that monomerized rolling circle amplification (RCA) products can be directly detected with the Luminex suspension bead array readout without the need of PCR amplification. Furthermore, using monomerized RCA products to guide ligation of the detection oligonucleotide (DO) to barcode sequences on the magnetic Luminex beads, combined with efficient washing and increased measurement temperature, yields a higher signal to noise ratio. As a proof-of-principle, we demonstrate detection of pathogenic DNA sequences with high reproducibility, sensitivity and a dynamic range over four orders of magnitude. Using padlock probes in combination with bead suspension arrays opens up the possibility for highly multiplexed DNA targeting and readout. PMID:25681158

  1. High-Resolution and Specific Detection of Bacteria on Complex Surfaces Using Nanoparticle Probes and Electron Microscopy

    PubMed Central

    Ye, Jun; Nielsen, Shaun; Joseph, Stephen; Thomas, Torsten

    2015-01-01

    The study of the interaction of bacteria with surfaces requires the detection of specific bacterial groups with high spatial resolution. Here, we describe a method to rapidly and efficiently add nanogold particles to oligonucleotide probes, which target bacterial ribosomal RNA. These nanogold-labeled probes are then used in an in situ hybridization procedure that ensures both cellular integrity and high specificity. Electron microscopy subsequently enables the visualization of specific cells with high local precision on complex surface structures. This method will contribute to an increased understanding of how bacteria interact with surface structures on a sub-micron scale. PMID:26018431

  2. Hydrogen peroxide detection with high specificity in living cells and inflamed tissues.

    PubMed

    Rong, Lei; Zhang, Chi; Lei, Qi; Hu, Ming-Ming; Feng, Jun; Shu, Hong-Bing; Liu, Yi; Zhang, Xian-Zheng

    2016-12-01

    Hydrogen peroxide (H2O2) detection in biological systems is of significant importance, which act as critical second messenger in fundamental biological processes. Here, we report on a chemoselective fluorescent naphthylimide peroxide probe (NPP) for the H2O2 detection in vitro and in vivo. NPP is a phenylboronic acid-caged chromophore that selectively responds to H2O2 through a self-immolate mechanism. NPP exhibited high sensitivity and selectivity to H2O2 with distinctive fluorescence change due to the excellent two-photon excitation property, which permits the facile detection of inflammation produced H2O2 and offers chance to monitor the inflammatory stages in diseased cells. PMID:27482463

  3. Hydrogen peroxide detection with high specificity in living cells and inflamed tissues

    PubMed Central

    Rong, Lei; Zhang, Chi; Lei, Qi; Hu, Ming-Ming; Feng, Jun; Shu, Hong-Bing; Liu, Yi; Zhang, Xian-Zheng

    2016-01-01

    Hydrogen peroxide (H2O2) detection in biological systems is of significant importance, which act as critical second messenger in fundamental biological processes. Here, we report on a chemoselective fluorescent naphthylimide peroxide probe (NPP) for the H2O2 detection in vitro and in vivo. NPP is a phenylboronic acid-caged chromophore that selectively responds to H2O2 through a self-immolate mechanism. NPP exhibited high sensitivity and selectivity to H2O2 with distinctive fluorescence change due to the excellent two-photon excitation property, which permits the facile detection of inflammation produced H2O2 and offers chance to monitor the inflammatory stages in diseased cells. PMID:27482463

  4. Development of a Highly Specific Fluorescence Immunoassay for Detection of Diisobutyl Phthalate in Edible Oil Samples.

    PubMed

    Cui, Xiping; Wu, Panpan; Lai, Dan; Zheng, Shengwu; Chen, Yingshan; Eremin, Sergei A; Peng, Wei; Zhao, Suqing

    2015-10-28

    The diisobutyl phthalate (DiBP) hapten containing an amino group was synthesized successfully, and the polyclonal antibody against 4-amino phthalate-bovine serum albumin (BSA) was developed. On the basis of the polyclonal antibody, a rapid and sensitive indirect competitive fluorescence immunoassay (icFIA) has been established to detect DiBP in edible oil samples for the first time. Under the optimized conditions, the quantitative working range of the icFIA was from 10.47 to 357.06 ng/mL (R(2) = 0.991), exhibiting a detection limit of 5.82 ng/mL. In this assay, the specific results showed that other similar phthalates did not significantly interfere with the analysis, with the cross-reactivity less than 1.5%, except for that of DiBAP. Thereafter, DiBP contamination in edible oil samples was detected by icFIA, with the recovery being from 79 to 103%. Furthermore, the reliability of icFIA was validated by gas chromatography-mass spectrometry (GC-MS). Therefore, the developed icFIA is suitable for monitoring DiBP in some edible oil samples. PMID:26449794

  5. Highly Specific and Cost-Efficient Detection of Salmonella Paratyphi A Combining Aptamers with Single-Walled Carbon Nanotubes

    PubMed Central

    Yang, Ming; Peng, Zhihui; Ning, Yi; Chen, Yongzhe; Zhou, Qin; Deng, Le

    2013-01-01

    In this paper, a panel of single-stranded DNA aptamers with high affinity and specificity against Salmonella Paratyphi A was selected from an enriched oligonucleotide pool by a whole-cell-Systematic Evolution of Ligands by Exponential Enrichment (SELEX) procedure, during which four other Salmonella serovars were used as counter-selection targets. It was determined through a fluorescence assay that the selected aptamers had high binding ability and specificity to this pathogen. The dissociation constant of these aptamers were up to nanomolar range, and aptamer Apt22 with the lowest Kd (47 ± 3 nM) was used in cell imaging experiments. To detect this bacteria with high specificity and cost-efficiently, a novel useful detection method was also constructed based on the noncovalent self-assembly of single-walled carbon nanotubes (SWNTs) and DNAzyme-labeled aptamer detection probes. The amounts of target bacteria could be quantified by exploiting chemoluminescence intensity changes at 420 nm and the detection limit of the method was 103 cfu/mL. This study demonstrated the applicability of Salmonella specific aptamers and their potential for use in the detection of Salmonella in food, clinical and environmental samples. PMID:23698275

  6. Improved SERS-Active Nanoparticles with Various Shapes for CTC Detection without Enrichment Process with Supersensitivity and High Specificity.

    PubMed

    Wu, Xiaoxia; Xia, Yuanzhi; Huang, Youju; Li, Juan; Ruan, Huimin; Chen, Tianxiang; Luo, Liqiang; Shen, Zheyu; Wu, Aiguo

    2016-08-10

    Circulating tumor cells (CTCs) have received more and more attention in medical biology and clinical practice, especially diagnosis, prognosis, and cancer treatment monitoring. The detection of CTCs within the large number of healthy blood cells is a big challenge due to their rarity, which requires a detection method with supersensitivity and high specificity. In this study, we developed three kinds of new nanoparticles with the function of surface-enhanced Raman scattering (SERS) based on spherical gold nanoparticles (AuNPs), gold nanorods (AuNRs), and gold nanostars (AuNSs) with similar particle size, similar modifications, and different shapes for CTC detection without an enrichment process from the blood. The nanoparticles possess strong SERS signal due to modification of 4-mercaptobenzoic acid (4-MBA) (i.e., Raman reporter molecule), possess excellent specificity due to stabilization of reductive bovine serum albumin (rBSA) to reduce the nonspecific catching or uptake by healthy cells in blood, and possess high sensitivity due to conjugation of folic acid (FA) (i.e., a targeted ligand) to identify CTCs. Under the optimized experimental conditions, the results of detection demonstrate that these nanoparticles could all be utilized for CTC detection without enrichment process from the blood with high specificity, and the AuNS-MBA-rBSA-FA is the best one due to its supersensitivity, whose limit of detection (i.e., 1 cell/mL) is much lower than the currently reported lowest value (5 cells/mL). PMID:27434820

  7. 4H-SiC UV Photo Detector with Large Area and Very High Specific Detectivity

    NASA Technical Reports Server (NTRS)

    Yan, Feng; Shahid, Aslam; Franz, David; Xin, Xiaobin; Zhao, Jian H.; Zhao, Yuegang; Winer, Maurice

    2004-01-01

    Pt/4H-SiC Schottky photodiodes have been fabricated with the device areas up to 1 sq cm. The I-V characteristics and photo-response spectra have been measured and analyzed. For a 5 mm x 5 mm area device leakage current of 1 x 10(exp 15)A at zero bias and 1.2 x 10(exp 14)A at -IV have been established. The quantum efficiency is over 30% from 240nm to 320nm. The specific detectivity, D(sup *), has been calculated from the directly measured leakage current and quantum efficiency data and are shown to be higher than 10(exp 15) cmHz(sup 1/2)/W from 210nm to 350nm with a peak D(sup *) of 3.6 x 10(exp 15)cmH(sup 1/2)/W at 300nm.

  8. High Resolution X Chromosome-Specific Array-CGH Detects New CNVs in Infertile Males

    PubMed Central

    Krausz, Csilla; Giachini, Claudia; Lo Giacco, Deborah; Daguin, Fabrice; Chianese, Chiara; Ars, Elisabet; Ruiz-Castane, Eduard; Forti, Gianni; Rossi, Elena

    2012-01-01

    Context The role of CNVs in male infertility is poorly defined, and only those linked to the Y chromosome have been the object of extensive research. Although it has been predicted that the X chromosome is also enriched in spermatogenesis genes, no clinically relevant gene mutations have been identified so far. Objectives In order to advance our understanding of the role of X-linked genetic factors in male infertility, we applied high resolution X chromosome specific array-CGH in 199 men with different sperm count followed by the analysis of selected, patient-specific deletions in large groups of cases and normozoospermic controls. Results We identified 73 CNVs, among which 55 are novel, providing the largest collection of X-linked CNVs in relation to spermatogenesis. We found 12 patient-specific deletions with potential clinical implication. Cancer Testis Antigen gene family members were the most frequently affected genes, and represent new genetic targets in relationship with altered spermatogenesis. One of the most relevant findings of our study is the significantly higher global burden of deletions in patients compared to controls due to an excessive rate of deletions/person (0.57 versus 0.21, respectively; p = 8.785×10−6) and to a higher mean sequence loss/person (11.79 Kb and 8.13 Kb, respectively; p = 3.435×10−4). Conclusions By the analysis of the X chromosome at the highest resolution available to date, in a large group of subjects with known sperm count we observed a deletion burden in relation to spermatogenic impairment and the lack of highly recurrent deletions on the X chromosome. We identified a number of potentially important patient-specific CNVs and candidate spermatogenesis genes, which represent novel targets for future investigations. PMID:23056185

  9. Label free and high specific detection of mercury ions based on silver nano-liposome

    NASA Astrophysics Data System (ADS)

    Priyadarshini, Eepsita; Pradhan, Nilotpala; Pradhan, Arun K.; Pradhan, Pallavi

    2016-06-01

    Herein, we report an eco-friendly, mild and one-pot approach for synthesis of silver nanoparticles via a lipopeptide biosurfactant - CHBS. The biosurfactant forms liposome vesicles when dispersed in an aqueous medium. The amino acid groups of the biosurfactant assists in the reduction of Ag+ ions leading to the production of homogeneous silver nanoparticles, encapsulated within the liposome vesicle, as confirmed from TEM analysis. Rate of synthesis and size of particle were greatly dependent on pH and reaction temperature. Kinetic analysis suggests the involvement of an autocatalytic reaction and the observed rate constant (kobs) was found to decrease with temperature, suggesting faster reaction with increasing temperature. Furthermore, the silver nanoparticles served as excellent probes for highly selective and sensitive recognition of Hg2 + ions. Interaction with Hg2 + ions results in an immediate change in colour of nanoparticle solution form brownish red to milky white. With increasing Hg2 + ions concentration, a gradual disappearance of SPR peak was observed. A linear relationship (A420/660) with an R2 value of 0.97 was observed in the range of 20 to 100 ppm Hg2 + concentration. Hg2 + ions are reduced to their elemental forms which thereby interact with the vesicles, leading to aggregation and precipitation of particles. The detection method avoids the need of functionalizing ligands and favours Hg2 + detection in aqueous samples by visible range spectrophotometry and hence can be used for simple and rapid analysis.

  10. Label free and high specific detection of mercury ions based on silver nano-liposome.

    PubMed

    Priyadarshini, Eepsita; Pradhan, Nilotpala; Pradhan, Arun K; Pradhan, Pallavi

    2016-06-15

    Herein, we report an eco-friendly, mild and one-pot approach for synthesis of silver nanoparticles via a lipopeptide biosurfactant - CHBS. The biosurfactant forms liposome vesicles when dispersed in an aqueous medium. The amino acid groups of the biosurfactant assists in the reduction of Ag(+) ions leading to the production of homogeneous silver nanoparticles, encapsulated within the liposome vesicle, as confirmed from TEM analysis. Rate of synthesis and size of particle were greatly dependent on pH and reaction temperature. Kinetic analysis suggests the involvement of an autocatalytic reaction and the observed rate constant (kobs) was found to decrease with temperature, suggesting faster reaction with increasing temperature. Furthermore, the silver nanoparticles served as excellent probes for highly selective and sensitive recognition of Hg(2+) ions. Interaction with Hg(2+) ions results in an immediate change in colour of nanoparticle solution form brownish red to milky white. With increasing Hg(2+) ions concentration, a gradual disappearance of SPR peak was observed. A linear relationship (A420/660) with an R(2) value of 0.97 was observed in the range of 20 to 100ppm Hg(2+) concentration. Hg(2+) ions are reduced to their elemental forms which thereby interact with the vesicles, leading to aggregation and precipitation of particles. The detection method avoids the need of functionalizing ligands and favours Hg(2+) detection in aqueous samples by visible range spectrophotometry and hence can be used for simple and rapid analysis. PMID:27045785

  11. A novel label-free and reusable electrochemical cytosensor for highly sensitive detection and specific collection of CTCs.

    PubMed

    Shen, Huawei; Yang, Juan; Chen, Zhongping; Chen, Xueping; Wang, Li; Hu, Jing; Ji, Feihu; Xie, Guoming; Feng, Wenli

    2016-07-15

    Circulating tumor cells (CTCs) contain a great deal of information of tumor phenotype. Therefore, highly sensitive detection and specific enrichment of CTCs are of intense interest. Herein, a label-free electrochemical impedance spectroscopy cytosensor with effective surface recognition between specific epithelial cell adhesion molecules (EpCAM) over-expressed on the cell membrane and EpCAM aptamer was developed for the detection of CTCs. After immobilization of 6-mercapto-1-hexanol (MCH) onto the gold electrode, the capture probe can be directionally inserted in MCH interspaces, which can improve the sensitivity of the cytosensor. A wide detection range from 30 to 1×10(6)cellsmL(-1) with a detection limit as low as 10cellsmL(-1) is reached on the condition of acceptable stability and reproducibility. The cytosensor can easily distinguish CTCs from the real blood sample due to the specific combination of EpCAM and EpCAM aptamer. Furthermore, the cytosensor can be reused 8 times and enrich CTCs by Uracil DNA Excision Mix specific cleaving the deoxyuridines (dUs) of the aptamer. The collected CTCs can contribute to further study. Thus, we reported that this cytosensor is a promising technique for the early monitoring and therapy of cancer. PMID:27016910

  12. Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry.

    PubMed

    Hansbauer, Eva-Maria; Skiba, Martin; Endermann, Tanja; Weisemann, Jasmin; Stern, Daniel; Dorner, Martin B; Finkenwirth, Friedrich; Wolf, Jessica; Luginbühl, Werner; Messelhäußer, Ute; Bellanger, Laurent; Woudstra, Cédric; Rummel, Andreas; Fach, Patrick; Dorner, Brigitte G

    2016-09-21

    Botulinum neurotoxin (BoNT) serotypes C and D and their mosaic variants CD and DC cause severe cases of botulism in animal husbandry and wildlife. Epidemiological data on the exact serotype or toxin variant causing outbreaks are rarely available, mainly because of their high sequence identity and the lack of fast and specific screening tools to detect and differentiate the four similar toxins. To fill this gap, we developed four highly specific sandwich enzyme-linked immunosorbent assays (ELISAs) able to detect and differentiate botulinum neurotoxins type BoNT/C, D, CD, and DC based on four distinct combinations of specific monoclonal antibodies targeting both conserved and divergent subdomains of the four toxins. Here, highly sensitive detection with detection limits between 2 and 24 pg mL(-1) was achieved. The ELISAs were extensively validated and results were compared with data obtained by quantitative real-time PCR using a panel of Clostridium botulinum strains, real sample materials from veterinary botulism outbreaks, and non-BoNT-producing Clostridia. Additionally, in order to verify the results obtained by ELISA screening, the new monoclonal antibodies were used for BoNT enrichment and subsequent detection (i) on a functional level by endopeptidase mass spectrometry (Endopep-MS) assays and (ii) on a protein sequence level by LC-MS/MS spectrometry. Based on all technical information gathered in the validation study, the four differentiating ELISAs turned out to be highly reliable screening tools for the rapid analysis of veterinary botulism cases and should aid future field investigations of botulism outbreaks and the acquisition of epidemiological data. PMID:27353114

  13. A universal probe design for colorimetric detection of single-nucleotide variation with visible readout and high specificity

    PubMed Central

    Chen, Xueping; Zhou, Dandan; Shen, Huawei; Chen, Hui; Feng, Wenli; Xie, Guoming

    2016-01-01

    Single-nucleotide variation (SNV) is a crucial biomarker for drug resistance-related detection in cancer and bacterial infection. However, the unintended binding of DNA probes limits the specificity of SNV detection, and the need for redesigned sequences compromise the universality of SNV assay. Herein, we demonstrated a universal and low-cost assay for the colorimetric discrimination of drug-resistance related point mutation. By the use of a universal DNA probe and a split G-quadruplex, the signal could be recognized by naked eye at room temperature. The DNA probe was used as a signal reporter which not only improved the universality, but also enabled high specificity of probe hybridization. This assay was successfully applied in the detection of cancer-related SNV in the epidermal growth factor receptor (EGFR) gene, kirsten rat sarcoma viral oncogene homologue (KRAS), and tuberculosis drug-resistance related point mutation in RNA polymerase beta subunit gene (rpoB) with high specificity and visible readout. This method was simple, rapid, high-throughput and effective, which was suitable for point-of-care applications. PMID:26830326

  14. Highly specific fiber optic immunosensor coupled with immunomagnetic separation for detection of low levels of Listeria monocytogenes and L. ivanovii

    PubMed Central

    2012-01-01

    Background Immunomagnetic separation (IMS) and immunoassays are widely used for pathogen detection. However, novel technology platforms with highly selective antibodies are essential to improve detection sensitivity, specificity and performance. In this study, monoclonal antibodies (MAbs) against Internalin A (InlA) and p30 were generated and used on paramagnetic beads of varying diameters for concentration, as well as on fiber-optic sensor for detection. Results Anti-InlA MAb-2D12 (IgG2a subclass) was specific for Listeria monocytogenes and L. ivanovii, and p30-specific MAb-3F8 (IgM) was specific for the genus Listeria. At all bacterial concentrations (103–108 CFU/mL) tested in the IMS assay; the 1-μm diameter MyOne beads had significantly higher capture efficiency (P < 0.05) than the 2.8-μm diameter M-280 beads with both antibodies. The highest capture efficiency for MyOne-2D12 (49.2% for 105 CFU/mL) was significantly higher (P < 0.05) than that of MyOne-3F8 (16.6 %) and Dynabeads anti-Listeria antibody (9 %). Furthermore, capture efficiency for MyOne-2D12 was highly specific for L. monocytogenes and L. ivanovii. Subsequently, we captured L. monocytogenes by MyOne-2D12 and MyOne-3F8 from hotdogs inoculated with mono- or co-cultures of L. monocytogenes and L. innocua (10–40 CFU/g), enriched for 18 h and detected by fiber-optic sensor and confirmed by plating, light-scattering, and qPCR assays. The detection limit for L. monocytogenes and L. ivanovii by the fiber-optic immunosensor was 3 × 102 CFU/mL using MAb-2D12 as capture and reporter antibody. Selective media plating, light-scattering, and qPCR assays confirmed the IMS and fiber-optic results. Conclusions IMS coupled with a fiber-optic sensor using anti-InlA MAb is highly specific for L. monocytogenes and L. ivanovii and enabled detection of these pathogens at low levels from buffer or food. PMID:23176167

  15. A rapid and highly specific immunofluorescence method to detect Escherichia coli O157:H7 in infected meat samples.

    PubMed

    Balakrishnan, Baskar; Barizuddin, Syed; Wuliji, Tumen; El-Dweik, Majed

    2016-08-16

    Developing rapid and sensitive methods for the detection of pathogenic Escherichia coli O157:H7 remains a major challenge in food safety. The present study attempts to develop an immunofluorescence technique that uses Protein-A-coated, magnetic beads as the platform. The immunofluorescence technique described here is a direct detection method in which E. coli O157:H7 cells are labeled with tetramethylrhodamine (TRITC) fluorescent dye. TRITC-labeled bacteria are captured by the desired antibody (Ab), which is immobilized on the Protein-A magnetic beads. Fluorescence of the captured cells is recorded in a fluorescence spectrophotometer, where the fluorescence values are shown to be directly proportional to the number of bacteria captured on the immunobead. The formation of an immunocomplex is evidenced by the fluorescence of the beads under microscopy. The Ab immobilization procedure is also evidenced by microscopy using fluorescein isothiocyanate (FITC)-labeled Ab. The total experimental time, including preparation of the sample, is just 1h. The minimum bacterial concentration detected by this method is 1.2±0.06×10(3)CFUml(-1). The high specificity of this method was proved by using the specific monoclonal Ab (MAb) in the test. The proposed protocol was successfully validated with E. coli O157:H7-infected meat samples. This approach also opens the door for the detection of other bacterial pathogens using Protein-A magnetic beads as a detection platform. PMID:27209618

  16. Specific detection of mercury(II) irons using AlGaAs/InGaAs high electron mobility transistors

    NASA Astrophysics Data System (ADS)

    Wang, Chengyan; Zhang, Yang; Guan, Min; Cui, Lijie; Ding, Kai; Zhang, Bintian; Lin, Zhang; Huang, Feng; Zeng, Yiping

    2015-09-01

    As one of the most environmentally important cations, mercury(II) iron has the biological toxicity which impacts wild life ecology and human health heavily. A Hg2+ biosensor based on AlGaAs/InGaAs high electron mobility transistors with high sensitivity and short response time is demonstrated experimentally. To achieve highly specific detection of Hg2+, an one-end thiol-modified ssDNA with lots of T thymine is immobilized to the Au-coated gate area of the high electron mobility transistors by a covalent modification method. The introduction of Hg2+ to the gate of the high electron mobility transistors affects surface charges, which leads to a change in the concentration of the two-dimensional electron gas in the AlGaAs/InGaAs high electron mobility transistors. Thus, the saturation current curves can be shifted with the modification of the gate areas and varied concentrations of Hg2+. Under the bias of 100 mV, a detection limit for the Hg2+ as low as10 nM is achieved. Successful detection with minute quantity of the sample indicates that the sensor has great potential in practical screening for a wide population. In addition, the dimension of the active area of the sensor is 20×50 μm2 and that of the entire sensor chip is 1×2 mm2, which make the Hg2+ biosensor portable.

  17. Detection of lower limb deep venous thrombosis in asymptomatic high risk patients using a new radiolabelled thrombus specific agent

    SciTech Connect

    Butler, S.P.; Rahman, T.; Boyd S.J.

    1995-05-01

    Deep venous thrombosis is a serious consequence of major orthopaedic surgery and non invasive screening with either venous ultrasound or impedance plethysmography is unreliable for detecting or excluding DVT in this group. A new method of thrombus detection has been devised using Tc-99m labelled inhibited recombinant tissue plasminogen activator. The accuracy of scanning with this new radiopharmaceutical in asymptomatic high risk patients was evaluated using venography as the gold standard. 36 consecutive asymptomatic high risk patients (17 total hip, 19 total knee replacements) underwent both a contrast venogram on the operated leg and scintigraphic scan 7 days following operation. Scintigraphic imaging was performed at 4 hours post injection. For the purpose of this analysis, each venogram was divided into a proximal and a distal segment. Venograms were interpreted as being positive, negative or uninterpretable in each segment. Similar analysis of the scintigraphic scans was performed except that all segments were considered to be of diagnostic quality. 57 segments were able to be analysed. Of the 13 thrombosed segments (1 proximal, 12 calf), 12 had positive scans; in the 44 non thrombosed segments, 40 had negative scans. Thus in detecting lower limb thrombosis, scanning had a sensitivity of 92% and a specificity of 91%. Scintigraphic scanning with this new radiopharmaceutical permits accurate detection of thrombus in high risk patients.

  18. Highly specific fluorescence detection of T4 polynucleotide kinase activity via photo-induced electron transfer.

    PubMed

    Tao, Mangjuan; Shi, Zhilu; Cheng, Rui; Zhang, Jing; Li, Baoxin; Jin, Yan

    2015-09-15

    Sensitive and reliable study of the activity of polynucleotide kinase (PNK) and its potential inhibitors is of great importance for biochemical interaction related to DNA phosphorylation as well as development of kinase-targeted drug discovery. To achieve facile and reliable detection of PNK activity, we report here a novel fluorescence method for PNK assay based on a combination of exonuclease cleavage reaction and photo-induced electron transfer (PIET) by using T4 PNK as a model target. The fluorescence of 3'-carboxyfluorescein-labeled DNA probe (FDNA) is effectively quenched by deoxyguanosines at the 5' end of its complementary DNA (cDNA) due to an effective PIET between deoxyguanosines and fluorophore. Whereas FDNA/cDNA hybrid is phosphorylated by PNK and then immediately cleaved by lambda exonuclease (λ exo), fluorescence is greatly restored due to the break of PIET. This homogeneous PNK activity assay does not require a complex design by taking advantage of the quenching ability of deoxyguanosines, making the proposed strategy facile and cost-effective. The activity of PNK can be sensitively detected in the range of 0.005 to 10 U mL(-1) with a detection limit of 2.1×10(-3) U mL(-1). Research on inhibition efficiency of different inhibitors demonstrated that it can be explored to evaluate inhibition capacity of inhibitors. The application for detection of PNK activity in complex matrix achieved satisfactory results. Therefore, this PIET strategy opens a promising avenue for studying T4 PNK activity as well as evaluating PNK inhibitors, which is of great importance for discovering kinase-targeted drugs. PMID:26050629

  19. Highly specific detection of Cryptosporidium spp. oocysts in human stool samples by undemanding and inexpensive phase contrast microscopy.

    PubMed

    Ignatius, Ralf; Klemm, Thomas; Zander, Steffen; Gahutu, Jean Bosco; Kimmig, Peter; Mockenhaupt, Frank P; Regnath, Thomas

    2016-03-01

    To compare phase contrast microscopy (PCM) of unstained slides for the detection of Cryptosporidium spp. oocysts with a commercially available enzyme immunoassay (EIA) for the detection of cryptosporidial antigen in human stool samples, we prospectively analysed by both methods 463 fresh human stool samples obtained from diarrhoeic patients between July and October 2014. Compared with the EIA, the sensitivity, specificity, positive and negative predictive value of PCM were 88.9 % (95 % confidence interval (CI), 66.0-98.1 %), 100 % (95 % CI, 99.0-100 %), 100 % (95 % CI, 77.3-100 %) and 99.6 % (95 % CI, 98.3-100 %), respectively. Additionally, we retrospectively examined with PCM 65 fixed stool samples that had been collected in 2010 from mostly asymptomatic Rwandan children <5 years of age; 14 of these samples had previously yielded positive results with a highly sensitive real-time (RT)-PCR. PCM detected cryptosporidia in 5/14 RT-PCR-positive samples, and notably, also in one of 51 RT-PCR-negative samples, which was subsequently confirmed by acid-fast staining. Positive and negative percent agreement of PCM with RT-PCR were 35.7 % (95 % CI, 16.2-61.4 %) and 98.0 % (95 % CI, 88.7-100 %), respectively. Positive PCM results were associated with higher RT-PCR cycle threshold values (p = 0.044). In conclusion, PCM offers a highly specific, undemanding and inexpensive method for the laboratory diagnosis of acute human cryptosporidiosis independent of the causative Cryptosporidium species. PMID:26646397

  20. Sensitive solid-phase detection of donor-specific antibodies as an aid highly relevant to improving allograft outcomes.

    PubMed

    Schlaf, Gerald; Pollok-Kopp, Beatrix; Altermann, Wolfgang W

    2014-04-01

    Transplant recipients who have had sensitizing events such as pregnancies, blood transfusions and previous transplants often develop antibodies directed against human leukocyte antigen (HLA)-molecules of the donor tissue. These pre-formed donor-specific antibodies (DSA) represent a high risk of organ failure as a consequence of antibody-mediated hyper-acute or acute allograft rejection. As a first assay to detect DSA, the complement-dependent lymphocytotoxicity assay (CDC) was established more than 40 years ago. However, this assay is characterized by several drawbacks such as a low sensitivity and a high susceptibility to various artificial factors generally not leading to valid and reliable outcomes under several circumstances that are reviewed in this article. Furthermore, only those antibodies that exert complement-fixing activity are detected. As a consequence, novel procedures that act independently of the complement system and that do not represent functional assays were generated in the format of solid phase assays (SPAs) (bead- or ELISA-based). In this article, we review the pros and cons of these sensitive SPA in comparison with the detection of DSA through the use of the traditional methods such as CDC and flow cytometric analyses. Potential drawbacks of the alternative methodological approaches comprising high background reactivity, susceptibility to environmental factors and the possible influence of subjective operators' errors concerning the interpretation of the results are summarized and critically discussed for each method. We provide a forecast on the future role of SPAs reliably excluding highly deleterious DSA, thus leading to an improved graft survival. PMID:24170304

  1. Imaging of oxidation-specific epitopes with targeted nanoparticles to detect high-risk atherosclerotic lesions: Progress and future directions

    PubMed Central

    Briley-Saebo, Karen; Yeang, Calvin; Witztum, Joseph L.; Tsimikas, Sotirios

    2014-01-01

    Oxidation-specific epitopes (OSE) within developing atherosclerotic lesions are key antigens that drive innate and adaptive immune responses in atherosclerosis, leading to chronic inflammation. Oxidized phospholipids and malondialdehyde-lysine epitopes are well-characterized OSE present in human atherosclerotic lesions, particularly in pathologically defined vulnerable plaques. Using murine and human OSE-specific antibodies as targeting agents, we have developed radionuclide and magnetic resonance based nanoparticles, containing gadolinium, manganese or lipid-coated ultrasmall superparamagnetic iron oxide, to noninvasively image OSE within experimental atherosclerotic lesions. These methods quantitate plaque burden, allow detection of lesion progression and regression, plaque stabilization, and accumulation of OSE within macrophage-rich areas of the artery wall, suggesting they detect the most active lesions. Future studies will focus on using “natural” antibodies, lipopeptides and mimotopes for imaging applications. These approaches should enhance the clinical translation of this technique to image, monitor, evaluate efficacy of novel therapeutic agents and guide optimal therapy of high-risk atherosclerotic lesions. PMID:25297940

  2. Imaging of oxidation-specific epitopes with targeted nanoparticles to detect high-risk atherosclerotic lesions: progress and future directions.

    PubMed

    Briley-Saebo, Karen; Yeang, Calvin; Witztum, Joseph L; Tsimikas, Sotirios

    2014-11-01

    Oxidation-specific epitopes (OSE) within developing atherosclerotic lesions are key antigens that drive innate and adaptive immune responses in atherosclerosis, leading to chronic inflammation. Oxidized phospholipids and malondialdehyde-lysine epitopes are well-characterized OSE present in human atherosclerotic lesions, particularly in pathologically defined vulnerable plaques. Using murine and human OSE-specific antibodies as targeting agents, we have developed radionuclide and magnetic resonance based nanoparticles, containing gadolinium, manganese or lipid-coated ultrasmall superparamagnetic iron oxide, to non-invasively image OSE within experimental atherosclerotic lesions. These methods quantitate plaque burden, allow detection of lesion progression and regression, plaque stabilization, and accumulation of OSE within macrophage-rich areas of the artery wall, suggesting they detect the most active lesions. Future studies will focus on using "natural" antibodies, lipopeptides, and mimotopes for imaging applications. These approaches should enhance the clinical translation of this technique to image, monitor, evaluate efficacy of novel therapeutic agents, and guide optimal therapy of high-risk atherosclerotic lesions. PMID:25297940

  3. High specific detection and near-infrared photothermal therapy of lung cancer cells with high SERS active aptamer-silver-gold shell-core nanostructures.

    PubMed

    Wu, Ping; Gao, Yang; Lu, Yimei; Zhang, Hui; Cai, Chenxin

    2013-11-01

    Lung cancer is the leading cause of cancer death worldwide. Its early detection is of paramount importance for diagnosis, classification, treatment, and improvement of survivorship. However, current methods are not sensitive enough to detect lung cancer in its nascent stage. We reported an aptamer-Ag-Au shell-core nanostructure-based surface-enhanced Raman scattering (SERS) assay for sensitive and specific detection, and near-infrared (NIR) photothermal therapy of lung adenocarcinoma cells (A549 cells). The nanostructures target the cells with high affinity and specificity via the specific interaction between the aptamer (a 45-base oligonucleotide) and the cell, and distinguish A549 cells from other types of cancer cells (HeLa and MCF-7 cells) and subtypes of lung cancer cells (NCI-H157, NCI-H520, NCI-H1299, and NCI-H446 cells). The nanostructures have a high capability to absorb NIR irradiation and are able to perform photothermal therapy of the cells at a very low irradiation power density (0.20 W cm(-2)) without destroying the healthy cells and the surrounding normal tissues. In addition, the nanostructures exhibit a high SERS activity. Based on the SERS signal of the labeled Raman reporter (Rh6G molecules), we can specifically detect A549 cells at a very low abundance (~10 cells per mL) and monitor the therapy process of the cancer cells. Therefore, this nanostructure-based SERS assay has great potential in specific recognition, sensitive detection, and effective photothermal therapy of lung cancer. PMID:24040647

  4. Detection of prostate-specific antigen with biomolecule-gated AlGaN/GaN high electron mobility transistors

    NASA Astrophysics Data System (ADS)

    Li, Jia-dong; Cheng, Jun-jie; Miao, Bin; Wei, Xiao-wei; Xie, Jie; Zhang, Jin-cheng; Zhang, Zhi-qiang; Wu, Dong-min

    2014-07-01

    In order to improve the sensitivity of AlGaN/GaN high electron mobility transistor (HEMT) biosensors, a simple biomolecule-gated AlGaN/GaN HEMT structure was designed and successfully fabricated for prostate specific antigen (PSA) detection. UV/ozone was used to oxidize the GaN surface and then a 3-aminopropyl trimethoxysilane (APTES) self-assembled monolayer was bound to the sensing region. This monolayer serves as a binding layer for attachment of the prostate specific antibody (anti-PSA). The biomolecule-gated AlGaN/GaN HEMT sensor shows a rapid and sensitive response when the target prostate-specific antigen in buffer solution was added to the antibody-immobilized sensing area. The current change showed a logarithm relationship against the PSA concentration from 0.1 pg/ml to 0.993 ng/ml. The sensitivity of 0.215% is determined for 0.1 pg/ml PSA solution. The above experimental result of the biomolecule-gated AlGaN/GaN HEMT biosensor suggested that this biosensor might be a useful tool for prostate cancer screening.

  5. Highly Specific Detection of Myostatin Prodomain by an Immunoradiometric Sandwich Assay in Serum of Healthy Individuals and Patients

    PubMed Central

    Widera, Christian; Gottlieb, Jens; Vogel, Arndt; Schmidt, Sebastian; Brandes, Gudrun; Heuft, Hans-Gert; Lichtinghagen, Ralf; Kempf, Tibor; Wollert, Kai C.; Bauersachs, Johann; Heineke, Joerg

    2013-01-01

    Background Myostatin is a muscle derived factor that functions as a negative regulator of skeletal muscle growth. Induction of myostatin expression was observed in rodent models of muscle wasting and in cachectic patients with cancer or pulmonary disease. Therefore, there is an increasing interest to use serum myostatin as a biomarker. Methods We established an immunoradiometric sandwich assay (IRMA), which uses a commercially available chicken polyclonal, affinity purified antibody directed against human myostatin prodomain. We determined the serum concentrations of myostatin prodomain in 249 healthy individuals as well as 169 patients with heart failure, 53 patients with cancer and 44 patients with chronic pulmonary disease. Results The IRMA had a detection limit of 0.7ng/ml, an intraassay imprecision of ≤14.1% and an interassay imprecision of ≤ 18.9%. The specificity of our assay was demonstrated by size exclusion chromatography, detection of myostatin by Western-blotting and a SMAD-dependent transcriptional-reporter assay in the signal-rich serum fractions, as well as lack of interference by unspecific substances like albumin, hemoglobin or lipids. Myostatin prodomain was stable at room temperature and resistant to freeze-thaw cycles. Apparently healthy individuals over the age of 55 had a median myostatin prodomain serum concentration of 3.9ng/ml (25th-75th percentiles, 2-7ng/ml) and we could not detect increased levels in patients with stable chronic heart failure or cancer related weight loss. In contrast, we found strongly elevated concentrations of myostatin prodomain (median 26.9ng/ml, 25th-75th percentiles, 7-100ng/ml) in the serum of underweight patients with chronic pulmonary disease. Conclusions We established a highly specific IRMA for the quantification of myostatin prodomain concentration in human serum. Our assay could be useful to study myostatin as a biomarker for example in patients with chronic pulmonary disease, as we detected highly

  6. Hair Ethyl Glucuronide is Highly Sensitive and Specific for Detecting Moderate-to-Heavy Drinking in Patients with Liver Disease

    PubMed Central

    Stewart, Scott H.; Koch, David G.; Willner, Ira R.; Randall, Patrick K.; Reuben, Adrian

    2013-01-01

    Aims: Hair ethyl glucuronide (EtG) is a promising biomarker of moderate-to-heavy alcohol consumption and may have utility in detecting and monitoring alcohol use in clinical populations where alcohol use is of particular importance. This study evaluated the relationship between hair EtG and drinking in patients with liver disease. Methods: The subjects (n = 200) were patients with liver disease who presented for care at a university medical center. Alcohol use during the 3 months preceding participation in the study was assessed, and a sample of hair was obtained for EtG testing. Classification of drinking status (any drinking or averaging at least 28 g per day) by hair EtG was evaluated, as well as the effects of liver disease severity and demographic and hair care factors. Results: The area under the receiver operating characteristic curve for detecting an average of 28 g or more per day during the prior 90 days was 0.93. The corresponding sensitivity and specificity of hair EtG ≥8 pg/mg for averaging at least 28 g of ethanol per day were 92 and 87%, respectively. Cirrhosis and gender may have a modest influence on the relationship between drinking and hair EtG. Conclusion: Hair EtG was highly accurate in differentiating subjects with liver disease averaging at least 28 g of ethanol per day from abstainers and lighter drinkers. PMID:23015609

  7. Tetraphenylethylene conjugated with a specific peptide as a fluorescence turn-on bioprobe for the highly specific detection and tracing of tumor markers in live cancer cells.

    PubMed

    Huang, Yanyan; Hu, Fang; Zhao, Rui; Zhang, Guanxin; Yang, Hua; Zhang, Deqing

    2014-01-01

    Smart molecular probes and flexible methods are attracting remarkable interest for the visualization of cancer-related biological and chemical events. In this work, a new fluorescence turn-on probe with dual-recognition characteristics for the specific imaging of cancer cells is reported. This new bioprobe is rationally designed by linking tetraphenylethylene (TPE), an aggregation-induced emission (AIE) fluorophore, with the small peptide IHGHHIISVG (referred to as AP2H), a targeting ligand to the broad-spectrum cancer-related protein LAPTM4B. The binding of the probe TPE-AP2H with the target, both in solution and at the cellular level, switches on the fluorescence of TPE because of the inhibition of internal rotations within the TPE framework. Accordingly, this bioprobe allows the real-time monitoring and subcellular localization of LAPTM4B in cancer cells, with a very high target-to-background ratio for the imaging. Furthermore, brighter fluorescence images are detected after incubation of TPE-AP2H with tumor cells at lower pH values. Thus, this new bioprobe is more advantageous because it can simultaneously target the LAPTM4B protein and sense the characteristic low-pH environment of tumor cells. In addition, TPE-AP2H displays high photostability and low cytotoxicity. Therefore, this new bioprobe is promising for the more accurate and reliable imaging of tumor markers in live cancer cells. PMID:24516888

  8. A highly sensitive and specific method for the screening detection of genetically modified organisms based on digital PCR without pretreatment.

    PubMed

    Fu, Wei; Zhu, Pengyu; Wang, Chenguang; Huang, Kunlun; Du, Zhixin; Tian, Wenying; Wang, Qin; Wang, Huiyu; Xu, Wentao; Zhu, Shuifang

    2015-01-01

    Digital PCR has developed rapidly since it was first reported in the 1990 s. It was recently reported that an improved method facilitated the detection of genetically modified organisms (GMOs). However, to use this improved method, the samples must be pretreated, which could introduce inaccuracy into the results. In our study, we explored a pretreatment-free digital PCR detection method for the screening for GMOs. We chose the CaMV35s promoter and the NOS terminator as the templates in our assay. To determine the specificity of our method, 9 events of GMOs were collected, including MON810, MON863, TC1507, MIR604, MIR162, GA21, T25, NK603 and Bt176. Moreover, the sensitivity, intra-laboratory and inter-laboratory reproducibility of our detection method were assessed. The results showed that the limit of detection of our method was 0.1%, which was lower than the labeling threshold level of the EU. The specificity and stability among the 9 events were consistent, respectively. The intra-laboratory and inter-laboratory reproducibility were both good. Finally, the perfect fitness for the detection of eight double-blind samples indicated the good practicability of our method. In conclusion, the method in our study would allow more sensitive, specific and stable screening detection of the GMO content of international trading products. PMID:26239916

  9. A highly sensitive and specific method for the screening detection of genetically modified organisms based on digital PCR without pretreatment

    PubMed Central

    Fu, Wei; Zhu, Pengyu; Wang, Chenguang; Huang, Kunlun; Du, Zhixin; Tian, Wenying; Wang, Qin; Wang, Huiyu; Xu, Wentao; Zhu, Shuifang

    2015-01-01

    Digital PCR has developed rapidly since it was first reported in the 1990s. It was recently reported that an improved method facilitated the detection of genetically modified organisms (GMOs). However, to use this improved method, the samples must be pretreated, which could introduce inaccuracy into the results. In our study, we explored a pretreatment-free digital PCR detection method for the screening for GMOs. We chose the CaMV35s promoter and the NOS terminator as the templates in our assay. To determine the specificity of our method, 9 events of GMOs were collected, including MON810, MON863, TC1507, MIR604, MIR162, GA21, T25, NK603 and Bt176. Moreover, the sensitivity, intra-laboratory and inter-laboratory reproducibility of our detection method were assessed. The results showed that the limit of detection of our method was 0.1%, which was lower than the labeling threshold level of the EU. The specificity and stability among the 9 events were consistent, respectively. The intra-laboratory and inter-laboratory reproducibility were both good. Finally, the perfect fitness for the detection of eight double-blind samples indicated the good practicability of our method. In conclusion, the method in our study would allow more sensitive, specific and stable screening detection of the GMO content of international trading products. PMID:26239916

  10. Detection of four diabetes specific autoantibodies in a single radioimmunoassay: an innovative high-throughput approach for autoimmune diabetes screening

    PubMed Central

    Tiberti, C; Yu, L; Lucantoni, F; Panimolle, F; Spagnuolo, I; Lenzi, A; Eisenbarth, G S; Dotta, F

    2011-01-01

    Highly sensitive and specific radioimmunoassays have been validated for autoantibodies reacting with the four major autoantigens identified so far in autoimmune diabetes. However, the analysis of this large number of autoantigens has increased the costs and time necessary for complete autoantibody screenings. Our aim was to demonstrate that it is possible to detect the immunoreactivity against a combination of four different autoantigens by a single assay, this representing a rapid, low-cost first approach to evaluate humoral autoimmunity in diabetes. By using this novel multi-autoantigen radioimmunoassay (MAA), in subsequent steps we analysed 830 sera, 476 of known and 354 of unknown diabetes-specific immunoreactivity, collected from various groups of individuals including type 1 and type 2 diabetes patients, autoantibody-positive patients with a clinical diagnosis of type 2 diabetes (LADA), prediabetic subjects, individuals at risk to develop autoimmune diabetes, siblings of type 1 diabetic patients, coeliac patients and healthy control subjects. All sera reacting with one or more of the four autoantigens by single assays also resulted positive with MAA, as well as eight of 24 type 1 diabetic patients classified initially as autoantibody-negative at disease onset based on single autoantibody assays. In addition, MAA showed 92% sensitivity and 99% specificity by analysing 140 blinded sera from type 1 diabetic patients and control subjects provided in the 2010 Diabetes Autoantibody Standardization Program. MAA is the first combined method also able to evaluate, in addition to glutamic acid decarboxylase (GAD) and tyrosine phosphatase (IA)-2, insulin and islet beta-cell zinc cation efflux transporter (ZnT8) autoantibodies. It appears to be particularly appropriate as a first-line approach for large-scale population-based screenings of anti-islet autoimmunity. PMID:22059988

  11. High-throughput, low-cost, and event-specific polymerase chain reaction detection of herbicide tolerance in genetically modified soybean A2704-12.

    PubMed

    Ma, H; Li, H; Li, J; Wang, X F; Wei, P C; Li, L; Yang, J B

    2014-01-01

    The aim of this study was to develop an event-specific qualitative and real-time quantitative polymerase chain reaction (PCR) method for detection of herbicide-tolerance genetically modified (GM) soybean A2704-12. The event-specific PCR primers were designed, based on the 5'-flanking integration sequence in the soybean genome, to amplify the 239-bp target fragment. Employing the same event-specific primers, qualitative PCR and real-time quantitative PCR detection methods were successfully developed. The results showed that the A2704-12 event could be specifically distinguished from other GM soybean events. In the qualitative PCR assay, the limit of detection was 0.05%, and in the real-time quantitative PCR assay, the limit of detection was less than 0.01%. Moreover, our genomic DNA (gDNA) extraction protocol is high-throughput, safe, and low-cost. The event-specific PCR assay system is cost-efficient by using SYBR Green I in real-time PCR, and by using the same primers in both the qualitative and quantitative PCR assays. We therefore developed a high-throughput, low-cost, and event-specific qualitative and quantitative PCR detection method for GM soybean A2704-12. The method would be useful for market supervision and management of GM soybean A2704-12 due to its high specificity and sensitivity. PMID:24615034

  12. A highly sensitive and specific electrochemical sensing method for robust detection of Escherichia coli lac Z gene sequence.

    PubMed

    Li, Fengqin; Yu, Zhigang; Qu, Haichao; Zhang, Guiling; Yan, Hong; Liu, Xin; He, Xunjun

    2015-06-15

    Based on the interaction mode of capture probe-target-signal probe (CP-T-SP), a simple but efficient electrochemical sensing method is developed for robust detection of Escherichia coli (E. coli) lac Z gene sequence. The sensing system is simply composed of two probes with CP attached to the surface of gold electrode and methylene blue (MB)-labeled SP free in the detecting solution. Two duplexes are formed with the target DNA, an E. coli lac Z gene sequence, hybridizing with two ends of CP and SP separately. This structure can position the MB labels near to the electrode surface, thereby resulting in the change of detecting current. The proposed sensor exhibits a high sensitivity with a detection limit of ∼30 fM for target DNA and good mismatch discrimination ability (MDA). This method can also be applied to the construction of other sensors for the analysis of similar designated pathogenic bacteria gene sequence (PBGS). PMID:25562734

  13. Generation and purification of highly-specific antibodies for detecting post-translationally modified proteins in vivo

    PubMed Central

    Arur, Swathi; Schedl, Tim

    2014-01-01

    Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immuno-cytochemistry and immuno-precipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often non-specific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot and western blots assays are employed to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein specific antibody preparation. One full round of antibody purification and specificity testing takes 6 days of discontinuous time. PMID:24457330

  14. Highly sensitive silicon nanowire biosensor with novel liquid gate control for detection of specific single-stranded DNA molecules.

    PubMed

    Adam, Tijjani; Hashim, U

    2015-05-15

    The study demonstrates the development of a liquid-based gate-control silicon nanowire biosensor for detection of specific single-stranded DNA (ssDNA) molecules. The sensor was fabricated using conventional photolithography coupled with an inductively coupled plasma dry etching process. Prior to the application of DNA to the device, its linear response to pH was confirmed by serial dilution from pH 2 to pH 14. Then, the sensor surface was silanized and directly aminated with (3-aminopropyl) triethoxysilane to create a molecular binding chemistry for biofunctionalization. The resulting Si‒O‒Si‒ components were functionalized with receptor ssDNA, which interacted with the targeted ssDNA to create a field across the silicon nanowire and increase the current. The sensor shows selectivity for the target ssDNA in a linear range from target ssDNA concentrations of 100 pM to 25 nM. With its excellent detection capabilities, this sensor platform is promising for detection of specific biomarkers and other targeted proteins. PMID:25453738

  15. Silicon photonic crystal microarrays for high throughput label-free detection of lung cancer cell line lysates with sensitivity and specificity

    NASA Astrophysics Data System (ADS)

    Chakravarty, Swapnajit; Lai, Wei-Cheng; Zou, Yi; Gemmill, Robert M.; Chen, Ray T.

    2013-03-01

    Detection of biomolecules on microarrays based on label-free on-chip optical biosensors is very attractive since this format avoids complex chemistries caused by steric hindrance of labels. Application areas include the detection of cancers and allergens, and food-borne pathogens to name a few. We have demonstrated photonic crystal microcavity biosensors with high sensitivity down to 1pM concentrations (67pg/ml). High sensitivities were achieved by slow light engineering which reduced the radiation loss and increased the stored energy in the photonic crystal microcavity resonance mode. Resonances with high quality factor Q~26,760 in liquid ambient, coupled with larger optical mode volumes allowed enhanced interaction with the analyte biomolecules which resulted in sensitivities down to 10 cells per micro-liter to lung cancer cell lysates. The specificity of detection was ensured by multiplexed detections from multiple photonic crystal microcavities arrayed on the arms of a multimode interference power splitter. Specific binding interactions and control experiments were performed simultaneously at the same instant of time with the same 60 microliter sample volume. Specificity is further ensured by sandwich assay methods in the multiplexed experiment. Sandwich assay based amplification increased the sensitivity further resulting in the detection of lung cancer cell lysates down to concentrations of 2 cells per micro-liter. The miniaturization enabled by photonic crystal biosensors coupled with waveguide interconnected layout thus offers the potential of high throughput proteomics with high sensitivity and specificity.

  16. A highly sensitive and specific biosensor for ligation- and PCR-free detection of microRNAs.

    PubMed

    Gao, Zhiqiang; Peng, Yanfen

    2011-05-15

    A highly sensitive microRNA (miRNA) biosensor for both ligation- and PCR-free detection of miRNAs is described in this work. The biosensor was made of a monolayer of long oligonucleotide capture probes (CPs) comprising of miRNA capturing segments at the top (3' termini) and detection probe capturing segments at the bottom (5' termini). Following hybridization with a target miRNA, a cocktail of Surveyor(®) and exonuclease I in pH 7.4 phosphate buffered saline was applied to the biosensor. Unhybridized CP strands and mismatched miRNA/CP duplexes were digested while complementarily hybridized ones remained intact. Thereafter, electrochemically activated glucose oxidase-tagged peptide nucleic acid detection probes (GOx-DP) were hybridized to the bottom segments of the CPs on the biosensor. The number of GOx molecules found at the biosensor surface coincides with the number of miRNA strands hybridized and therefore correlates directly to the concentration of the target miRNA. A detection limit of 10 fM and a linear current-concentration relationship up to 10 pM were attained under optimized conditions. The biosensor effectively eliminated the needs for chemical/biological ligation and PCR. It was showed that there is little cross-hybridization among closely related miRNA family members even at single-base-mismatched levels. Successful attempts were made in applying the biosensor to the detection of miRNAs in total RNA extracted from cell lines. PMID:21420848

  17. Silver nanoprism etching-based plasmonic ELISA for the high sensitive detection of prostate-specific antigen.

    PubMed

    Liang, Jiajie; Yao, Cuize; Li, Xiuqing; Wu, Ze; Huang, Caihong; Fu, Qiangqiang; Lan, Caifeng; Cao, Donglin; Tang, Yong

    2015-07-15

    Ultrasensitive and quantitative detection using simple and low-cost assays is critical in clinical diagnostics. In this report, we developed a triangular silver nanoprism (AgNPRs) etching-based plasmonic biosensor for the detection of cancer biomarkers. The triangular AgNPRs-based plasmonic biosensor is an enzyme-linked immunosorbent assay combined with the enzyme-mediated surface plasmon resonance (SPR) of triangular AgNPRs. Triangular AgNPRs uses the immune response of prostate-specific antigen (PSA) to trigger the glucose oxidase (GOx)-catalysed oxidation of glucose (Glu), producing hydrogen peroxide. Hydrogen peroxide acts as an oxidant to etch the triangular AgNPRs into smaller spherical silver nanoparticles, which is accompanied by a substantial blueshift of the SPR peak and a colourimetric blue-to-purple change that can be observed by the naked eye. The SPR peak shift enables the quantitative assessment of PSA due to the remarkable colour change. The triangular AgNPRs-based plasmonic ELISA approach exhibited a quasilinear response to logarithmic PSA concentrations in the range of 10fg/mL to 100pg/mL with a limit of detection (LOD) of 4.1fg/mL. In addition, the LOD of PSA in this approach exceeds that of the conventional HRP-based ELISA (1.25ng/mL) approach by more than 5 orders of magnitude. Patient serum samples from 16 donors were assayed with triangular AgNPRs-based plasmonic ELISA. The results from the triangular AgNPRs-based immunoassay and the time-resolved fluorescence immunoassay showed excellent correlation, and there were no significant differences in the quantified amounts of PSA. The triangular AgNPRs-based plasmonic ELISA approach has advantages (ultrasensitive, cost-effective, ease of operation) that are expected to be of great interest in diagnostics and to be suitable for a point-of-care test. PMID:25721976

  18. High voltage-derived enhancement of electric conduction in nanogap devices for detection of prostate-specific antigen

    NASA Astrophysics Data System (ADS)

    Park, Hyung Ju; Chi, Young Shik; Choi, Insung S.; Yun, Wan Soo

    2010-07-01

    We report a simple method of enhancing electric conductance in nanogap devices without any additional treatments, such as silver-enhancing process. The low electric conductance after selective immobilization of biofunctionalized gold nanoparticles in the gap region was greatly enhanced by repeated I-V scans at relatively high voltage ranges of -5 to 5 V, which was attributed to the formation of a new conduction pathway across the gap. The higher conduction state of the nanogap device showed a very stable I-V curve, which was used as an excellent measure of the existence of prostate-specific antigen.

  19. Multi-primer qPCR assay capable of highly efficient and specific detection of the vast majority of all known Mycoplasma.

    PubMed

    Salling, H K; Bang-Christensen, S R

    2016-05-01

    Mycoplasma bacteria are able to pass through sterilizing grade filters due to their small size and lack of a cell wall, making them a common contaminant of biopharmaceutical productions. The classical method for detecting Mycoplasma is described in the European Pharmacopeia (Ph.Eur) 2.6.7. The method takes 28 days to perform, due to the slow growing nature of some Mycoplasma species. The Ph.Eur has described Nucleic Acid Testing (NAT) as a rapid alternative to the classical method. Here we present the development of a quantitative polymerase chain reaction (qPCR) assay capable of unambiguous detection of Mycoplasma with high sensitivity and specificity. The broadness of detection and the specificity towards Mycoplasma has been investigated by in silico analysis of the primer sequences followed by testing on purified Mycoplasma DNA as well as DNA from closely related genera. The assay will in all probability detect at least 356 species and strains of Mycoplasma, Spiroplasma and Acholeplasma with high sensitivity. To our knowledge this assay has the most uniform amplification efficiency over the broadest range of species and it is extremely specific towards Mycoplasma. With appropriate validation, the assay can be applied as a powerful tool for rapid Mycoplasma detection in the biopharmaceutical industry. PMID:27067447

  20. Development of a highly sensitive and specific enzyme-linked immunosorbent assay for detection of Sudan I in food samples.

    PubMed

    Han, Dan; Yu, Meng; Knopp, Dietmar; Niessner, Reinhard; Wu, Mei; Deng, Anping

    2007-08-01

    A highly selective and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) for Sudan I was developed. Two hapten derivatives with different lengths of carboxylic spacer at the azo-bound para-position were synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugates were used as immunogens, while the hapten-ovalbumin (OA) conjugates were applied as coating antigens. The antisera which were obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. At optimal experimental conditions it was found that IC50 and LOD values of seven pairs based on four antisera and two coating antigens were in the range of 0.3-2 ng/mL and 0.02-0.1 ng/mL, respectively. The most sensitive ELISA could be established with Sudan I-propionic acid-OA coating antigen and the antiserum which was obtained with the corresponding immunogen. The cross-reactivity values of the four antisera with Sudan II, III, and IV was estimated with 0.1-14.3%. No cross-reactivity was found with six edible colorants Sunset yellow, Amarant, Kermes, Indigotin, Bright blue and Lemon yellow, indicating high specificity for Sudan I. Six food samples were fortified with Sudan I and extracted by simple sample preparation. The methanolic extracts after dilution with methanol:water (5:95, v/v) were analyzed by the developed ELISA. Assay precision and accuracy was estimated by determination of three replicates. Acceptable recovery rates of 92.5-114% and intra-assay coefficients of variation of 5.9-24.8% were obtained. The data were validated by conventional HPLC method. As revealed, both methods were highly correlated (r = 0.9851, n = 7), demonstrating the applicability of the developed ELISA for Sudan I analysis in food samples. PMID:17622156

  1. Highly specific and efficient primers for in-house multiplex PCR detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum

    PubMed Central

    2014-01-01

    Background Although sophisticated methodologies are available, the use of endpoint polymerase chain reaction (PCR) to detect 16S rDNA genes remains a good approach for estimating the incidence and prevalence of specific infections and for monitoring infections. Considering the importance of the early diagnosis of sexually transmitted infections (STIs), the development of a sensitive and affordable method for identifying pathogens in clinical samples is needed. Highly specific and efficient primers for a multiplex polymerase chain reaction (m-PCR) system were designed in silico to detect the 16S rDNA genes of four bacteria that cause genital infections, and the PCR method was developed. Methods The Genosensor Probe Designer (GPD) (version 1.0a) software was initially used to design highly specific and efficient primers for in-house m-PCR. Single-locus PCR reactions were performed and standardised, and then primers for each locus in turn were added individually in subsequent amplifications until m-PCR was achieved. Amplicons of the expected size were obtained from each of the four bacterial gene fragments. Finally, the analytical specificity and limits of detection were tested. Results Because they did not amplify any product from non-STI tested species, the primers were specific. The detection limits for the Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum primer sets were 5.12 × 105, 3.9 × 103, 61.19 × 106 and 6.37 × 105 copies of a DNA template, respectively. Conclusions The methodology designed and standardised here could be applied satisfactorily for the simultaneous or individual detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma hominis and Ureaplasma urealyticum. This method is at least as efficient as other previously described methods; however, this method is more affordable for low-income countries. PMID:24997675

  2. A Highly Sensitive and Selective Competition Assay for the Detection of Cysteine Using Mercury-Specific DNA, Hg2+ and Sybr Green I

    PubMed Central

    Xu, Hui; Gao, Shuli; Liu, Quanwen; Pan, Dun; Wang, Lihua; Ren, Shuzhen; Ding, Min; Chen, Jingwen; Liu, Gang

    2011-01-01

    We here report a rapid, sensitive, selective and label-free fluorescence detection method for cysteine (Cys). The conformation of mercury-specific DNA (MSD) changes from a random coil form to a hairpin structure in the presence of Hg2+ due to the formation of a thymine-Hg2+-thymine (T-Hg2+-T) complex. Cys can selectively coordinate with Hg2+ and extract it from the thymine-Hg2+-thymine complex. The hairpin structure dehybridizes and the fluorescence intensity of Sybr Green I (SG) decreases upon addition of Cys because SG efficiently discriminates mercury-specific DNA and mercury-specific DNA/Hg2+ complex. The detection can be finished within 5 min with high sensitivity and selectivity. In addition, we can obtain variable dynamic ranges for Cys by changing the concentration of MSD/Hg2+. PMID:22346638

  3. Highly sensitive method for specific, brief, and economical detection of glycoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis by the synthesis of a new hydrazide derivative.

    PubMed

    Cong, Weitao; Zhou, Ayi; Liu, Zhiguo; Shen, Jiayi; Zhou, Xuan; Ye, Weijian; Zhu, Zhongxin; Zhu, Xinliang; Lin, Jianjun; Jin, Litai

    2015-02-01

    A new hydrazide derivative was synthesized and used for the first time as a specific, brief, and economical probe to selectively visualize glycoproteins in 1-D and 2-D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with high sensitivity. The detection limit of the newly developed staining method is 2- and 4-fold higher than that of the widely used Pro-Q Emerald 300 and 488 stains, respectively. PMID:25565298

  4. Low-cost and highly efficient DNA biosensor for heavy metal ion using specific DNAzyme-modified microplate and portable glucometer-based detection mode.

    PubMed

    Zhang, Jin; Tang, Ying; Teng, Liumei; Lu, Minghua; Tang, Dianping

    2015-06-15

    A simple and low-cost DNA sensing platform based on Pb(2+)-specific DNAzyme-modified microplate was successfully developed for highly sensitive monitoring of lead ion (Pb(2+), one kind of toxic heavy metal ion) in the environmental samples coupling with a portable personal glucometer (PGM)-based detection mode. The detection cell was first prepared simply by means of immobilizing the DNAzyme on the streptavidin-modified microplate. Gold nanoparticle labeled with single-stranded DNA and invertase (Enz-AuNP-DNA) was utilized as the signal-transduction tag to produce PGM substrate (glucose). Upon addition of lead ion into the microplate, the substrate strand of the immobilized DNAzyme was catalytically cleaved by target Pb(2+), and the newly generated single-strand DNA in the microplate could hybridize again with the single-stranded DNA on the Enz-AuNP-DNA. Accompanying with the Enz-AuNP-DNA, the carried invertase could convert sucrose into glucose. The as-produced glucose could be monitored by using a widely accessible PGM for in situ amplified digital readout. Based on Enz-AuNP-DNA amplification strategy, as low as 1.0 pM Pb(2+) could be detected under the optimal conditions. Moreover, the methodology also showed good reproducibility and high selectivity toward target Pb(2+) against other metal ions because of highly specific Pb(2+)-dependent DNAzyme, and was applicable for monitoring Pb(2+) in the naturally contaminated sewage and spiked drinking water samples. PMID:25576929

  5. Pseudoperonospora cubensis and P. humuli detection using species-specific probes and high definition melt curve analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three assays were developed for molecular differentiation of Pseudoperonospora cubensis and P. humuli, causal agents of cucurbit and hop downy mildew, respectively, for detection of airborne sporangia and diagnosis of symptomatic leaf tissue. The assays were based on previously identified single nuc...

  6. Detection of high mobility group A2 specific mRNA in the plasma of patients affected by epithelial ovarian cancer

    PubMed Central

    Pasquinelli, Rosa; Pignata, Sandro; Greggi, Stefano; Vuttariello, Emilia; Bello, Anna Maria; Calise, Celeste; Scaffa, Cono; Pisano, Carmela; Losito, Nunzia Simona; Fusco, Alfredo; Califano, Daniela; Chiappetta, Gennaro

    2015-01-01

    Ovarian cancer is the most lethal gynecological malignancy and the high mortality rate is associated with advanced-stage disease at the time of the diagnosis. In order to find new tools to make diagnosis of Epithelial Ovarian Cancer (EOC) at early stages we have analyzed the presence of specific HMGA2 mRNA in the plasma of patients affected by this neoplasm. HMGA2 overexpression represents a feature of several malignances including ovarian carcinomas. Notably, we detected HMGA2 specific mRNA in the plasma of 40 out 47 patients with EOC, but not in the plasma of healthy donors. All cases found positive for HMGA2 mRNA in the plasma showed HMGA2 protein expression in EOC tissues. Therefore, on the basis of these results, the analysis of circulating HMGA2 specific mRNA might be considered a very promising tool for the early diagnosis of EOC. PMID:25749380

  7. Generic and sequence-variant specific molecular assays for the detection of the highly variable Grapevine leafroll-associated virus 3.

    PubMed

    Chooi, Kar Mun; Cohen, Daniel; Pearson, Michael N

    2013-04-01

    Grapevine leafroll-associated virus 3 (GLRaV-3) is an economically important virus, which is found in all grapevine growing regions worldwide. Its accurate detection in nursery and field samples is of high importance for certification schemes and disease management programmes. To reduce false negatives that can be caused by sequence variability, a new universal primer pair was designed against a divergent sequence data set, targeting the open reading frame 4 (heat shock protein 70 homologue gene), and optimised for conventional one-step RT-PCR and one-step SYBR Green real-time RT-PCR assays. In addition, primer pairs for the simultaneous detection of specific GLRaV-3 variants from groups 1, 2, 6 (specifically NZ-1) and the outlier NZ2 variant, and the generic detection of variants from groups 1 to 5 were designed and optimised as a conventional one-step multiplex RT-PCR assay using the plant nad5 gene as an internal control (i.e. one-step hexaplex RT-PCR). Results showed that the generic and variant specific assays detected in vitro RNA transcripts from a range of 1×10(1)-1×10(8) copies of amplicon per μl diluted in healthy total RNA from Vitis vinifera cv. Cabernet Sauvignon. Furthermore, the assays were employed effectively to screen 157 germplasm and 159 commercial field samples. Thus results demonstrate that the GLRaV-3 generic and variant-specific assays are prospective tools that will be beneficial for certification schemes and disease management programmes, as well as biological and epidemiological studies of the divergent GLRaV-3 populations. PMID:23313884

  8. A New Restriction Endonuclease-Based Method for Highly-Specific Detection of DNA Targets from Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Smith, Maria W.; Ghindilis, Andrei L.; Seoudi, Ihab A.; Smith, Kenneth; Billharz, Rosalind; Simon, Holly M.

    2014-01-01

    PCR multiplexing has proven to be challenging, and thus has provided limited means for pathogen genotyping. We developed a new approach for analysis of PCR amplicons based on restriction endonuclease digestion. The first stage of the restriction enzyme assay is hybridization of a target DNA to immobilized complementary oligonucleotide probes that carry a molecular marker, horseradish peroxidase (HRP). At the second stage, a target-specific restriction enzyme is added, cleaving the target-probe duplex at the corresponding restriction site and releasing the HRP marker into solution, where it is quantified colorimetrically. The assay was tested for detection of the methicillin-resistant Staphylococcus aureus (MRSA) pathogen, using the mecA gene as a target. Calibration curves indicated that the limit of detection for both target oligonucleotide and PCR amplicon was approximately 1 nM. Sequences of target oligonucleotides were altered to demonstrate that (i) any mutation of the restriction site reduced the signal to zero; (ii) double and triple point mutations of sequences flanking the restriction site reduced restriction to 50–80% of the positive control; and (iii) a minimum of a 16-bp target-probe dsDNA hybrid was required for significant cleavage. Further experiments showed that the assay could detect the mecA amplicon from an unpurified PCR mixture with detection limits similar to those with standard fluorescence-based qPCR. Furthermore, addition of a large excess of heterologous genomic DNA did not affect amplicon detection. Specificity of the assay is very high because it involves two biorecognition steps. The proposed assay is low-cost and can be completed in less than 1 hour. Thus, we have demonstrated an efficient new approach for pathogen detection and amplicon genotyping in conjunction with various end-point and qPCR applications. The restriction enzyme assay may also be used for parallel analysis of multiple different amplicons from the same unpurified

  9. Detection of the sour-rot pathogen Geotrichum candidum in tomato fruit and juice by using a highly specific monoclonal antibody-based ELISA.

    PubMed

    Thornton, Christopher R; Slaughter, David C; Davis, R Michael

    2010-10-15

    Geotrichum candidum is a common soil-borne fungus that causes sour-rot of tomatoes, citrus fruits and vegetables, and is a major contaminant on tomato processing equipment. The aim of this work was to produce a monoclonal antibody and diagnostic assay for its detection in tomato fruit and juice. Using hybridoma technology, a cell line (FE10) was generated that produced a monoclonal antibody belonging to the immunoglobulin class M (IgM) that was specific to G. candidum and the closely related teleomorphic species Galactomyces geotrichum and anamorphic species Geotrichum europaeum and Geotrichum pseudocandidum in the G. geotrichum/G. candidum complex. The MAb did not cross-react with a wide range of unrelated fungi, including some likely to be encountered during crop production and processing. The MAb binds to an immunodominant high molecular mass (> 200 kDa) extracellular polysaccharide antigen that is present on the surface of arthroconidia and hyphae of G. candidum. The MAb was used in a highly specific enzyme-linked immunosorbent assay (ELISA) to accurately detect the fungus in infected tomato fruit and juice. Specificity of the ELISA was confirmed by sequencing of the internally transcribed spacer (ITS) 1-5.8S-ITS2 rRNA-encoding regions of fungi isolated from naturally-infected tomatoes. PMID:20850192

  10. Synthesis of grafted phosphorylcholine polymer layers as specific recognition ligands for C-reactive protein focused on grafting density and thickness to achieve highly sensitive detection.

    PubMed

    Kamon, Yuri; Kitayama, Yukiya; Itakura, Akiko N; Fukazawa, Kyoko; Ishihara, Kazuhiko; Takeuchi, Toshifumi

    2015-04-21

    We studied the effects of layer thickness and grafting density of poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) thin layers as specific ligands for the highly sensitive binding of C-reactive protein (CRP). PMPC layer thickness was controlled by surface-initiated activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP). PMPC grafting density was controlled by utilizing mixed self-assembled monolayers with different incorporation ratios of the bis[2-(2-bromoisobutyryloxy)undecyl] disulfide ATRP initiator, as modulated by altering the feed molar ratio with (11-mercaptoundecyl)tetra(ethylene glycol). X-ray photoelectron spectroscopy and ellipsometry measurements were used to characterize the modified surfaces. PMPC grafting densities were estimated from polymer thickness and the molecular weight obtained from sacrificial initiator during surface-initiated AGET ATRP. The effects of thickness and grafting density of the obtained PMPC layers on CRP binding performance were investigated using surface plasmon resonance employing a 10 mM Tris-HCl running buffer containing 140 mM NaCl and 2 mM CaCl2 (pH 7.4). Furthermore, the non-specific binding properties of the obtained layers were investigated using human serum albumin (HSA) as a reference protein. The PMPC layer which has 4.6 nm of thickness and 1.27 chains per nm(2) of grafting density showed highly sensitive CRP detection (limit of detection: 4.4 ng mL(-1)) with low non-specific HSA adsorption, which was improved 10 times than our previous report of 50 ng mL(-1). PMID:25783194

  11. Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples.

    PubMed

    Wang, Yuzhen; Wei, Dapeng; Yang, Hong; Yang, Yuan; Xing, Weiwei; Li, Yuan; Deng, Anping

    2009-03-15

    The use of Sudan I as an additive in food products has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I in food samples was developed. The hapten derivative with a three-carbon-atom length of carboxylic spacer at the azobound para-position was synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugate was used as an immunogen, while the hapten-ovalbumin (OVA) conjugate was applied as a coating antigen. The mAb against Sudan I was produced by hybridoma technique and the corresponding ELISA was characterized in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed in concentrations of 0.1-100 ngmL(-1). The values of IC(50) for nine standard curves were in the range of 1.1-2.0 ngmL(-1) and the LOD at a signal-to-noise ratio of 3 (S/N=3) was 0.07-0.14 ngmL(-1). The cross-reactivity values of the mAb with Sudan II, III and IV were 9.5%, 33.9% and 0.95%; no cross-reactivity was found with other six edible colorants: Lemon yellow, Bright blue, Indigotin, Kermes, Amarant and Sunset yellow, indicating the assay displays not only high sensitivity but also high specificity as well. The organic solvent effect on the assay was tested. It was observed that the ELISA was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC(50) value. Six food samples were spiked with Sudan I and the methanolic extracts after appropriate dilution were analyzed by ELISA. Acceptable recovery rates of 88.2-110.5% and coefficients of variation of 2.5-17.4% were obtained. The ELISA for nine spiked samples was confirmed by high-performance liquid chromatography (HPLC) with a high correlation coefficient of 0.9840 (n=9). The mAb-based ELISA proven to be a feasible quantitative

  12. Exoglycosidase purity and linkage specificity: assessment using oligosaccharide substrates and high-pH anion-exchange chromatography with pulsed amperometric detection.

    PubMed

    Tyagarajan, K; Forte, J G; Townsend, R R

    1996-01-01

    Simplified HPLC protocols to determine the activity and linkage specificity and to detect the most commonly-encountered contaminants in available exoglycosidase preparations (Jacob and Scudder, Methods Enzymol., 230, 280-300, 1994) were developed. Monosaccharides and oligosaccharides were analyzed in a single chromatographic step using high-pH anion-exchange chromatography with pulsed amperometric detection. All analyses were performed with underivatized oligosaccharide substrates and by direct injection of unprocessed, diluted enzyme digests into the chromatograph. The sialidase from Newcastle disease virus was found to release both alpha (2-->3)- and alpha (2-->6)-linked Neu5Ac from a triantennary, lactosamine-type oligosaccharide. The activity of alpha-galactosidase from green coffee beans was assayed using Gal alpha(1-->3)[Fuc-alpha(1ar2)]Gal by detection of Gal and Fuc alpha(1-->3)Gal. The linkage specificities of beta-galactosidases from Streptococcus pneumoniae and bovine testis were assessed using Gal beta(1-->3 or 4)GlcNAc beta(1-->3)beta(1-->4)Glc as substrates. Contaminating beta-N-acetylhexosaminidase activity in the beta-galactosidase preparation was assayed using an agalactobiantennary oligosaccharide. The alpha(1-->3 or 4) linkage specificity of fucosidase III from almond meal was confirmed (Scudder et al., J. Biol. Chem. 265, 16472-16477, 1990) by its inactivity against a biantennary oligosaccharide with all Fuc residues linked alpha(1-->6). An alpha-fucosidase from chicken liver was found to cleave alpha(1-->2,3 or 6)-linked Fuc residues from oligosaccharides. The activity of jack bean (Canavalia ensiformis) alpha-mannosidase was assayed with a relatively resistant substrate, Man alpha(1-->3)- Man beta(1-->4)GlcNAc. A GlcNAc beta(1-->4)-terminated triantennary oligosaccharide was used to assay for contaminating beta-N-acetylhexosaminidase activity in alpha-mannosidase preparations and to determine the linkage and branch specificity of beta

  13. A unique "turn-on" fluorescence signalling strategy for highly specific detection of ascorbic acid using carbon dots as sensing probe.

    PubMed

    Fong, Jessica Fung Yee; Chin, Suk Fun; Ng, Sing Muk

    2016-11-15

    Carbon dots (CDs) that showed strong blue fluorescence were successfully synthesised from sodium alginate via furnace pyrolysis. The single step pyrolytic synthesis was simple to perform while yielded CDs with high photostability, good water solubility and minimum by-products. In order to design the probe with "turn-on" sensing capability, the CDs were screened against a series of metal cations to first "turn-off" the fluorescence. It was found that ferric ions (Fe(3+)) were most responsive and effective in quenching the fluorescence of CDs. Based on this observation, the conditioning of the probe was performed to ensure the fluorescence was completely quenched, while not overloading the system with Fe(3+). At the optimised condition, the CDs-Fe(3+) mixture served as a highly specific detection probe for ascorbic acid (AA). The analytical potential of the probe was evaluated and showed a good linear range of response for AA concentration of 24-40μg/mL. The selectivity study against other possible co-existing species was carried out and proved that our unique "turn-on" fluorescence signalling strategy was highly effective and selective towards AA as the target analyte. The probe was demonstrated for quantification of AA in real samples, which was the commercially available vitamin C supplement. The result showed good accuracy with minimum deviation from standard method adopted for validation purpose. PMID:27290666

  14. A highly specific and sensitive loop-mediated isothermal amplification method for the detection of Escherichia coli O157:H7.

    PubMed

    Ravan, Hadi; Amandadi, Mojdeh; Sanadgol, Nima

    2016-02-01

    E. coli O157:H7 is one of the most important foodborne pathogen that causes some human illnesses such as bloody diarrhea, hemolytic-uremic syndrome, and kidney failure. We developed a loop-mediated isothermal amplification (LAMP) assay with six special primers that target a highly specific 299-bp region of the Z3276 gene for the detection of E. coli O157:H7. Among 117 bacterial strains tested in this study, positive results were only obtained from E. coli O157:H7 strains. The sensitivity level of the Z3276-LAMP assay was determined to be 5 CFU/reaction tube in pure bacterial culture. Moreover, the LAMP assay was successfully applied to artificially contaminated ground beef with a sensitivity level of 10(3) CFU/mL without pre-enrichment and 10 CFU/mL after a 4-h pre-enrichment. In conclusion, the present LAMP assay would be a useful and powerful tool for the rapid, sensitive, and specific diagnosis of E. coli O157:H7 strains in resource limited laboratories. PMID:26724736

  15. Accurate, rapid and high-throughput detection of strain-specific polymorphisms in Bacillus anthracis and Yersinia pestis by next-generation sequencing

    PubMed Central

    2010-01-01

    Background In the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD™) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization. Results To demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination

  16. High specific heat superconducting composite

    DOEpatents

    Steyert, Jr., William A.

    1979-01-01

    A composite superconductor formed from a high specific heat ceramic such as gadolinium oxide or gadolinium-aluminum oxide and a conventional metal conductor such as copper or aluminum which are insolubly mixed together to provide adiabatic stability in a superconducting mode of operation. The addition of a few percent of insoluble gadolinium-aluminum oxide powder or gadolinium oxide powder to copper, increases the measured specific heat of the composite by one to two orders of magnitude below the 5.degree. K. level while maintaining the high thermal and electrical conductivity of the conventional metal conductor.

  17. Method for site-specific detection of m6A nucleoside presence in RNA based on high-resolution melting (HRM) analysis.

    PubMed

    Golovina, Anna Y; Dzama, Margarita M; Petriukov, Kirill S; Zatsepin, Timofei S; Sergiev, Petr V; Bogdanov, Alexey A; Dontsova, Olga A

    2014-02-01

    Chemical landscape of natural RNA species is decorated with the large number of modified nucleosides. Some of those could easily be detected by reverse transcription, while others permit only high-performance liquid chromatography or mass-spectrometry detection. Presence of m(6)A nucleoside at a particular position of long RNA molecule is challenging to observe. Here we report an easy and high-throughput method for detection of m(6)A nucleosides in RNA based on high-resolution melting analysis. The method relies on the previous knowledge of the modified nucleoside position at a particular place of RNA and allows rapid screening for conditions or genes necessary for formation of that modification. PMID:24265225

  18. Molecules for Fluorescence Detection of Specific Chemicals

    NASA Technical Reports Server (NTRS)

    Fedor, Steve

    2008-01-01

    A family of fluorescent dye molecules has been developed for use in on-off fluorescence detection of specific chemicals. By themselves, these molecules do not fluoresce. However, when exposed to certain chemical analytes in liquid or vapor forms, they do fluoresce (see figure). These compounds are amenable to fixation on or in a variety of substrates for use in fluorescence-based detection devices: they can be chemically modified to anchor them to porous or non-porous solid supports or can be incorporated into polymer films. Potential applications for these compounds include detection of chemical warfare agents, sensing of acidity or alkalinity, and fluorescent tagging of proteins in pharmaceutical research and development. These molecules could also be exploited for use as two-photon materials for photodynamic therapy in the treatment of certain cancers and other diseases. A molecule in this family consists of a fluorescent core (such as an anthracene or pyrene) attached to two end groups that, when the dye is excited by absorption of light, transfer an electron to the core, thereby quenching the fluorescence. The end groups can be engineered so that they react chemically with certain analytes. Upon reaction, electrons on the end groups are no longer available for transfer to the core and, consequently, the fluorescence from the core is no longer quenched. The chemoselectivity of these molecules can be changed by changing the end groups. For example, aniline end groups afford a capability for sensing acids or acid halides (including those contained in chemical warfare agents). Pyridine or bipyridyl end groups would enable sensing of metal ions. Other chemicals that can be selectively detected through suitable choice of end groups include glucose and proteins. Moreover, the fluorescent cores can be changed to alter light-absorption and -emission characteristics: anthracene cores fluoresce at wavelengths around 500 nm, whereas perylene cores absorb and emit at

  19. Mumps virus infection in vaccinated patients can be detected by an increase in specific IgG antibodies to high titres: a retrospective study.

    PubMed

    Borgmann, S; Schwab, F; Santibanez, S; Mankertz, A

    2014-11-01

    Mumps outbreaks in highly vaccinated populations with genotype G have been reported repeatedly. Detection of these outbreaks can be difficult in a setting with relatively high vaccination coverage when acute cases of mumps are routinely diagnosed by IgM serology since this marker is not reliable for diagnosis of mumps re-infection. To learn whether diagnostic tests performed in a large private laboratory may be useful to detect mumps outbreaks retrospectively, we reviewed the results of almost 7000 mumps tests. Two groups were compared: group 1 comprised of 3438 samples from patients submitted by physicians and clinicians (it was assumed that these patients visited their doctor due to acute disease). Group 2 comprised of 3398 samples submitted from company medical officers and occupational physicians. Since these patients usually attend for routine check-ups and certification of immunity to vaccine-preventable diseases, these samples comprised a control group. From July 2010 to May 2011, a mumps virus outbreak with more than 300 cases occurred in Bavaria, Southeast Germany. Our study includes samples received for serological mumps tests from January 2009 until December 2011 (36 months). The two groups were analysed with regard to the number of IgM-positive cases per month and the level of IgG titre. We found a marked increase for both parameters in group 1 during the time of the outbreak, while the samples submitted by the occupational medical physicians did not display significant alterations. These parameters reflect the outbreak with high accuracy, indicating that a retrospective analysis of IgG titres may be a useful tool for detection of mumps outbreaks when, as was the case in Germany, (i) a nationwide notification system has not been implemented and (ii) a highly vaccinated population is affected. PMID:24423404

  20. High specific activity silicon-32

    DOEpatents

    Phillips, D.R.; Brzezinski, M.A.

    1996-06-11

    A process for preparation of silicon-32 is provided and includes contacting an irradiated potassium chloride target, including spallation products from a prior irradiation, with sufficient water, hydrochloric acid or potassium hydroxide to form a solution, filtering the solution, adjusting pH of the solution from about 5.5 to about 7.5, admixing sufficient molybdate-reagent to the solution to adjust the pH of the solution to about 1.5 and to form a silicon-molybdate complex, contacting the solution including the silicon-molybdate complex with a dextran-based material, washing the dextran-based material to remove residual contaminants such as sodium-22, separating the silicon-molybdate complex from the dextran-based material as another solution, adding sufficient hydrochloric acid and hydrogen peroxide to the solution to prevent reformation of the silicon-molybdate complex and to yield an oxidation state of the molybdate adapted for subsequent separation by an anion exchange material, contacting the solution with an anion exchange material whereby the molybdate is retained by the anion exchange material and the silicon remains in solution, and optionally adding sufficient alkali metal hydroxide to adjust the pH of the solution to about 12 to 13. Additionally, a high specific activity silicon-32 product having a high purity is provided.

  1. High specific activity silicon-32

    DOEpatents

    Phillips, Dennis R.; Brzezinski, Mark A.

    1996-01-01

    A process for preparation of silicon-32 is provided and includes contacting an irradiated potassium chloride target, including spallation products from a prior irradiation, with sufficient water, hydrochloric acid or potassium hydroxide to form a solution, filtering the solution, adjusting pH of the solution to from about 5.5 to about 7.5, admixing sufficient molybdate-reagent to the solution to adjust the pH of the solution to about 1.5 and to form a silicon-molybdate complex, contacting the solution including the silicon-molybdate complex with a dextran-based material, washing the dextran-based material to remove residual contaminants such as sodium-22, separating the silicon-molybdate complex from the dextran-based material as another solution, adding sufficient hydrochloric acid and hydrogen peroxide to the solution to prevent reformation of the silicon-molybdate complex and to yield an oxidization state of the molybdate adapted for subsequent separation by an anion exchange material, contacting the solution with an anion exchange material whereby the molybdate is retained by the anion exchange material and the silicon remains in solution, and optionally adding sufficient alkali metal hydroxide to adjust the pH of the solution to about 12 to 13. Additionally, a high specific activity silicon-32 product having a high purity is provided.

  2. Detection of RNA from a Novel West Nile-like Virus and High Prevalence of an Insect-specific Flavivirus in Mosquitoes in the Yucatan Peninsula of Mexico

    PubMed Central

    Farfan-Ale, Jose A.; Loroño-Pino, Maria A.; Garcia-Rejon, Julian E.; Hovav, Einat; Powers, Ann M.; Lin, Ming; Dorman, Karin S.; Platt, Kenneth B.; Bartholomay, Lyric C.; Soto, Victor; Beaty, Barry J.; Lanciotti, Robert S.; Blitvich, Bradley J.

    2009-01-01

    As part of our ongoing surveillance efforts for West Nile virus (WNV) in the Yucatan Peninsula of Mexico, 96,687 mosquitoes collected from January through December 2007 were assayed by virus isolation in mammalian cells. Three mosquito pools caused cytopathic effect. Two isolates were orthobunyaviruses (Cache Valley virus and Kairi virus) and the identity of the third infectious agent was not determined. A subset of mosquitoes was also tested by reverse transcription–polymerase chain reaction (RT-PCR) using WNV-, flavivirus-, alphavirus-, and orthobunyavirus-specific primers. A total of 7,009 Culex quinquefasciatus in 210 pools were analyzed. Flavivirus RNA was detected in 146 (70%) pools, and all PCR products were sequenced. The nucleotide sequence of one PCR product was most closely related (71–73% identity) with homologous regions of several other flaviviruses, including WNV, St. Louis encephalitis virus, and Ilheus virus. These data suggest that a novel flavivirus (tentatively named T’Ho virus) is present in Mexico. The other 145 PCR products correspond to Culex flavivirus, an insect-specific flavivirus first isolated in Japan in 2003. Culex flavivirus was isolated in mosquito cells from approximately one in four homogenates tested. The genomic sequence of one isolate was determined. Surprisingly, heterogeneous sequences were identified at the distal end of the 5′ untranslated region. PMID:19141845

  3. Specific detection of methionine 27 mutation in histone 3 variants (H3K27M) in fixed tissue from high-grade astrocytomas.

    PubMed

    Bechet, Denise; Gielen, Gerrit G H; Korshunov, Andrey; Pfister, Stefan M; Rousso, Caterina; Faury, Damien; Fiset, Pierre-Olivier; Benlimane, Naciba; Lewis, Peter W; Lu, Chao; David Allis, C; Kieran, Mark W; Ligon, Keith L; Pietsch, Torsten; Ellezam, Benjamin; Albrecht, Steffen; Jabado, Nada

    2014-11-01

    Studies in pediatric high-grade astrocytomas (HGA) by our group and others have uncovered recurrent somatic mutations affecting highly conserved residues in histone 3 (H3) variants. One of these mutations leads to analogous p.Lys27Met (K27M) mutations in both H3.3 and H3.1 variants, is associated with rapid fatal outcome, and occurs specifically in HGA of the midline in children and young adults. This includes diffuse intrinsic pontine gliomas (80 %) and thalamic or spinal HGA (>90 %), which are surgically challenging locations with often limited tumor material available and critical need for specific histopathological markers. Here, we analyzed formalin-fixed paraffin-embedded tissues from 143 pediatric HGA and 297 other primary brain tumors or normal brain. Immunohistochemical staining for H3K27M was compared to tumor genotype, and also compared to H3 tri-methylated lysine 27 (H3K27me3) staining, previously shown to be drastically decreased in samples carrying this mutation. There was a 100 % concordance between genotype and immunohistochemical analysis of H3K27M in tumor samples. Mutant H3K27M was expressed in the majority of tumor cells, indicating limited intra-tumor heterogeneity for this specific mutation within the limits of our dataset. Both H3.1 and H3.3K27M mutants were recognized by this antibody while non-neoplastic elements, such as endothelial and vascular smooth muscle cells or lymphocytes, did not stain. H3K27me3 immunoreactivity was largely mutually exclusive with H3K27M positivity. These results demonstrate that mutant H3K27M can be specifically identified with high specificity and sensitivity using an H3K27M antibody and immunohistochemistry. Use of this antibody in the clinical setting will prove very useful for diagnosis, especially in the context of small biopsies in challenging midline tumors and will help orient care in the context of the extremely poor prognosis associated with this mutation. PMID:25200321

  4. High Speed Edge Detection

    NASA Technical Reports Server (NTRS)

    Prokop, Norman F (Inventor)

    2016-01-01

    Analog circuits for detecting edges in pixel arrays are disclosed. A comparator may be configured to receive an all pass signal and a low pass signal for a pixel intensity in an array of pixels. A latch may be configured to receive a counter signal and a latching signal from the comparator. The comparator may be configured to send the latching signal to the latch when the all pass signal is below the low pass signal minus an offset. The latch may be configured to hold a last negative edge location when the latching signal is received from the comparator.

  5. High Speed Edge Detection

    NASA Technical Reports Server (NTRS)

    Prokop, Norman F (Inventor)

    2015-01-01

    Analog circuits for detecting edges in pixel arrays are disclosed. A comparator may be configured to receive an all pass signal and a low pass signal for a pixel intensity in an array of pixels. A latch may be configured to receive a counter signal and a latching signal from the comparator. The comparator may be configured to send the latching signal to the latch when the all pass signal is below the low pass signal minus an offset. The latch may be configured to hold a last negative edge location when the latching signal is received from the comparator.

  6. Multiplexed BioCD for prostate specific antigen detection

    NASA Astrophysics Data System (ADS)

    Wang, Xuefeng; Zhao, Ming; Nolte, David D.

    2008-02-01

    Specific protein concentrations in human body fluid can serve as diagnostic markers for some diseases, and a quantitative and high-throughput technique for multiplexed protein detection would speed up diagnosis and facilitate medical research. For this purpose, our group developed the BioCD, a spinning-disc interferometric biosensor on which antibody is immobilized. The detection system adopts a common-path scheme making it ultra stable. The scaling mass sensitivity is below 10 pg/mm for protein surface density. A 25000-spot antibody BioCD was fabricated to measure the concentration of prostate specific antigen (PSA), a protein indicating prostate cancer if its level is high. Statistical analysis of our immunoassay results projects that the detection limit of PSA would reach 20 pg/ml in a 2 mg/ml background solution. For future prospects, a multiplexed BioCD can be produced for simultaneous diagnosis of diverse diseases. For instance, 100 markers above 200 pg/ml could be measured on a single disc given that the detection limit is inversely proportional to square root of the number of spots.

  7. Sensitive, Fast, and Specific Immunoassays for Methyltestosterone Detection

    PubMed Central

    Kong, Na; Song, Shanshan; Peng, Juan; Liu, Liqiang; Kuang, Hua; Xu, Chuanlai

    2015-01-01

    An indirect competitive enzyme-linked immunosorbent assay (icELISA) and an immunochromatographic strip assay using a highly specific monoclonal antibody, were developed to detect methyltestosterone (MT) residues in animal feed. The optimized icELISA had a half-inhibition concentration value of 0.26 ng/mL and a limit of detection value of 0.045 ng/mL. There was no cross-reactivity with eight analogues, revealing high specificity for MT. Based on icELISA results, the recovery rate of MT in animal feed was 82.4%–100.6%. The results were in accordance with those obtained by gas chromatography-mass spectrometry. The developed immunochromatographic strip assay, as the first report for MT detection, had a visual cut-off value of 1 ng/mL in PBS, 2.5 ng/g in fish feed, and 2.5 ng/g in pig feed. Therefore, these immunoassays are useful and fast tools for MT residue detection in animal feed. PMID:25938198

  8. Sensitivity and Specificity in Child Abuse Detection.

    ERIC Educational Resources Information Center

    Gellert, George A.

    1995-01-01

    Discusses legal and psychological issues involved in erroneous reporting of child abuse. Addresses whether the child protection field will have the vision and initiative to address the problem proactively from within or whether regulation and restriction of activity will be imposed from without. Explores ideas to make child abuse detection more…

  9. Analytical bioconjugates, aptamers, enable specific quantitative detection of Listeria monocytogenes.

    PubMed

    Lee, Sang-Hee; Ahn, Ji-Young; Lee, Kyeong-Ah; Um, Hyun-Ju; Sekhon, Simranjeet Singh; Sun Park, Tae; Min, Jiho; Kim, Yang-Hoon

    2015-06-15

    As a major human pathogen in the Listeria genus, Listeria monocytogenes causes the bacterial disease listeriosis, which is a serious infection caused by eating food contaminated with the bacteria. We have developed an aptamer-based sandwich assay (ABSA) platform that demonstrates a promising potential for use in pathogen detection using aptamers as analytical bioconjugates. The whole-bacteria SELEX (WB-SELEX) strategy was adopted to generate aptamers with high affinity and specificity against live L. monocytogenes. Of the 35 aptamer candidates tested, LMCA2 and LMCA26 reacted to L. monocytogenes with high binding, and were consequently chosen as sensing probes. The ABSA platform can significantly enhance the sensitivity by employing a very specific aptamer pair for the sandwich complex. The ABSA platform exhibited a linear response over a wide concentration range of L. monocytogenes from 20 to 2×10(6) CFU per mL and was closely correlated with the following relationship: y=9533.3x+1542.3 (R(2)=0.99). Our proposed ABSA platform also provided excellent specificity for the tests to distinguish L. monocytogenes from other Listeria species and other bacterial genera (3 Listeria spp., 4 Salmonella spp., 2 Vibrio spp., 3 Escherichia coli and 3 Shigella spp.). Improvements in the sensitivity and specificity have not only facilitated the reliable detection of L. monocytogenes at extremely low concentrations, but also allowed for the development of a 96-well plate-based routine assay platform for multivalent diagnostics. PMID:25590973

  10. Specific Pathogen Detection Using Bioorthogonal Chemistry and Diagnostic Magnetic Resonance

    PubMed Central

    Liong, Monty; Fernandez-Suarez, Marta; Issadore, David; Min, Changwook; Tassa, Carlos; Reiner, Thomas; Fortune, Sarah M.; Toner, Mehmet; Lee, Hakho; Weissleder, Ralph

    2011-01-01

    The development of faster and more sensitive detection methods capable of identifying specific bacterial types and strains has remained a longstanding clinical challenge. Thus to date, the diagnosis of bacterial infections continues to rely on the performance of time-consuming cultures. Here, we demonstrate the use of bioorthogonal chemistry for magnetically labeling specific pathogens to enable their subsequent detection by nuclear magnetic resonance. Antibodies against a bacterial target of interest were first modified with trans-cyclooctene and then coupled to tetrazine-modified magnetic nanoprobes, directly on the bacteria. This labeling method was verified using surface plasmon resonance as well as by using a miniaturized diagnostic magnetic resonance device capable of highly specific detection of Staphylococcus aureus. Compared to other copper-free bioorthogonal chemistries, the cycloaddition reaction described displayed faster kinetics and yielded higher labeling efficiency. Considering the short assay times and the portability of the necessary instrumentation, it is feasible that this approach could be adapted for clinical use in resource-limited settings. PMID:22043803

  11. Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection.

    PubMed

    Arola, Henri O; Tullila, Antti; Kiljunen, Harri; Campbell, Katrina; Siitari, Harri; Nevanen, Tarja K

    2016-02-16

    Here we demonstrate a novel homogeneous one-step immunoassay, utilizing a pair of recombinant antibody antigen-binding fragments (Fab), that is specific for HT-2 toxin and has a positive readout. Advantages over the conventional competitive immunoassay formats such as enzyme-linked immunosorbent assay (ELISA) are the specificity, speed, and simplicity of the assay. Recombinant antibody HT2-10 Fab recognizing both HT-2 and T-2 toxins was developed from a phage display antibody library containing 6 × 10(7) different antibody clones. Specificity of the immunoassay was introduced by an anti-immune complex (IC) antibody binding the primary antibody-HT-2 toxin complex. When the noncompetitive immune complex assay was compared to the traditional competitive assay, an over 10-fold improvement in sensitivity was observed. Although the HT2-10 antibody has 100% cross-reactivity for HT-2 and T-2 toxins, the immune complex assay is highly specific for HT-2 alone. The assay performance with real samples was evaluated using naturally contaminated wheat reference material. The half-maximal effective concentration (EC50) value of the time-resolved fluorescence resonance energy transfer (TR-FRET) assay was 9.6 ng/mL, and the limit of detection (LOD) was 0.38 ng/mL (19 μg/kg). The labeled antibodies can be predried to the assay vials, e.g., microtiter plate wells, and readout is ready in 10 min after the sample application. PMID:26785138

  12. Highly specific and rapid immuno-fluorescent visualization and detection of E. coli O104:H4 with protein-A coated magnetic beads based LST-MUG assay.

    PubMed

    Barizuddin, Syed; Balakrishnan, Baskar; Stringer, R Cody; Dweik, Majed

    2015-08-01

    A method combining immunomagnetic separation and fluorescent sensing was developed to detect Escherichia coli (E. coli) O104:H4. The antibody specific to E. coli O104:H4 was immobilized on protein A-coated magnetic beads. This protein-A-anti E. coli O104:H4 complex was used to bind Fluorescein IsoThioCyanate (FITC) labeled E. coli O104:H4 antigen (whole cell) on it. The goal was to achieve a fluorescently detectable protein-A-anti E. coli O104:H4-E. coli O104:H4 complex on the magnetic beads. Fluorescent microscopy was used to image the magnetic beads. The resulting fluorescence on the beads was due to the FITC labeled antigen binding on the protein-A-anti E. coli O104:H4 immobilized magnetic beads. This visually proves the antigen-antibody binding. The fluorescent imaging results were obtained in 2 h if the minimum available bacteria in the sample were at least 10(5) CFU/ml. If no fluorescence was observed on the magnetic beads during fluorescent imaging, it indicates the bacterial concentration in the sample to be too low for it to have bound to the magnetic beads and hence no detection was possible. To detect bacterial concentration less than 10(5) CFU/ml in the sample, an additional step was required for detection. The magnetic bead complex was added to the LST-MUG (lauryl sulfate tryptose-4-methylumbelliferyl-β-D-glucuronide), a signaling reporter. The E. coli O104:H4 grows in LST-MUG and releases β-glucuronidase enzyme. This enzyme cleaves the MUG substrate that produces 4-methylumbelliferone, a highly fluorescent species. This fluorescence was detected using a spectrofluorometer. The emission peak in the fluorescent spectrum was found to be at 450 nm. The lower and upper detection range for this LST-MUG assay was found to be 2.05×10(5)-4.09×10(8) CFU/ml. The results for the LST-MUG assay for concentrations below 10(5) CFU/ml were ascertained in 8h. The advantages of this technique include the specific detection of bacteria without an enrichment step and

  13. Target-triggered three-way junction structure and polymerase/nicking enzyme synergetic isothermal quadratic DNA machine for highly specific, one-step, and rapid microRNA detection at attomolar level.

    PubMed

    Zhang, Qing; Chen, Feng; Xu, Feng; Zhao, Yongxi; Fan, Chunhai

    2014-08-19

    MicroRNAs (miRNAs) play important roles in many biological processes and are regarded as promising cancer biomarkers. Herein, a highly specific, one-step, and rapid miRNAs detection strategy with attomolar sensitivity has been developed on the basis of a target-triggered three-way junction (3-WJ) structure and polymerase/nicking enzyme synergetic isothermal quadratic DNA machine (ESQM). To this end, 3-WJ probes (primer and template) are designed to selectively recognize target miRNA and form the stable 3-WJ structure to trigger ESQM, resulting in a high quadratic amplified signal. A high specificity is demonstrated by the excellent discrimination of even single-base mismatched homologous sequences with mismatched bases in varied locations (close to the 3'-end, the 5'-end, and the middle). In addition, a low detection limit down to 2 amol was achieved within 30 min. This sensitivity is much higher than those of most linear amplification-based approaches and is even comparable to those of some exponential amplification-based methods. Furthermore, the applicability of this method in complex samples was demonstrated by the analysis of cancer cell small RNA extracts, results of which were in good agreement with those obtained by a commercial miRNA kit and previously published data. The miRNA with a 3' end modification (2'-O-methylation), such as plant miRNA, was also successfully detected, confirming the good universality of the proposed strategy. It is worthwhile to point out that several well-established methods using miRNA as primer for polymerization reaction are of relatively poor performance in the analysis of these modified miRNA. Therefore, these merits endow the developed strategy with powerful implications for biological research and an effective diagnostic assay. PMID:25072308

  14. Material-specific detection and classification of single nanoparticles.

    PubMed

    Person, Steven; Deutsch, Bradley; Mitra, Anirban; Novotny, Lukas

    2011-01-12

    Detection and classification of nanoparticles are important for environmental monitoring, contamination mitigation, biological label tracking, and biodefense. Detection techniques involve a trade-off between sensitivity, discrimination, and speed. This paper presents a material-specific dual-color common-path interferometric detection system. Two wavelengths are simultaneously used to discriminate between 60 nm silver and 80 nm diameter gold particles in solution with a detection time of τ ≈ 1 ms. The detection technique is applicable to situations where both particle size and material are of interest. PMID:21142033

  15. Material-specific detection and classification of single nanoparticles

    PubMed Central

    Person, Steven; Deutsch, Bradley; Mitra, Anirban; Novotny, Lukas

    2010-01-01

    Detection and classification of nanoparticles is important for environmental monitoring, contamination mitigation, biological label tracking, and bio-defense. Detection techniques involve a trade-off between sensitivity, discrimination, and speed. This paper presents a material-specific dual-color common-path interferometric detection system. Two wavelengths are simultaneously used to discriminate between 60 nm silver and 80 nm diameter gold particles in solution with a detection time of τ ≈ 1 ms. The detection technique is applicable to situations where both particle size and material are of interest. PMID:21142033

  16. SPECIES-SPECIFIC DETECTION OF HYDROCARBON UTILIZING BACTERIA. (R825810)

    EPA Science Inventory

    Rapid detection and quantitative assessment of specific microbial species in environmental samples is desirable for monitoring changes in ecosystems and for tracking natural or introduced microbial species during bioremediation of contaminated sites. In the interests of develo...

  17. High Detectivity Graphene-Silicon Heterojunction Photodetector.

    PubMed

    Li, Xinming; Zhu, Miao; Du, Mingde; Lv, Zheng; Zhang, Li; Li, Yuanchang; Yang, Yao; Yang, Tingting; Li, Xiao; Wang, Kunlin; Zhu, Hongwei; Fang, Ying

    2016-02-01

    A graphene/n-type silicon (n-Si) heterojunction has been demonstrated to exhibit strong rectifying behavior and high photoresponsivity, which can be utilized for the development of high-performance photodetectors. However, graphene/n-Si heterojunction photodetectors reported previously suffer from relatively low specific detectivity due to large dark current. Here, by introducing a thin interfacial oxide layer, the dark current of graphene/n-Si heterojunction has been reduced by two orders of magnitude at zero bias. At room temperature, the graphene/n-Si photodetector with interfacial oxide exhibits a specific detectivity up to 5.77 × 10(13) cm Hz(1/2) W(-1) at the peak wavelength of 890 nm in vacuum, which is highest reported detectivity at room temperature for planar graphene/Si heterojunction photodetectors. In addition, the improved graphene/n-Si heterojunction photodetectors possess high responsivity of 0.73 A W(-1) and high photo-to-dark current ratio of ≈10(7) . The current noise spectral density of the graphene/n-Si photodetector has been characterized under ambient and vacuum conditions, which shows that the dark current can be further suppressed in vacuum. These results demonstrate that graphene/Si heterojunction with interfacial oxide is promising for the development of high detectivity photodetectors. PMID:26643577

  18. PROBES FOR THE SPECIFIC DETECTION OF CRYPTOSPORIDIUM PARVUM

    EPA Science Inventory

    A probe set, consisting of two synthetic oligonucleotides each tagged with a fluorescent reporter molecule, has been developed for specific detection of Cryptosporidium parvum.Each probe strand detects ribosomal RNA from a range of isolates of this species, and the combination is...

  19. [Sensitivity and specificity of new commercial tests for the detection of specific Echinococcus antibodies].

    PubMed

    Auer, Herbert; Stöckl, Cornelia; Suhendra, Susanne; Schneider, Renate

    2009-10-01

    Cystic echinococcosis (CE), caused by Echinococcus granulosus, and alveolar echinococcosis (AE), caused by E. multilocularis belong to the most serious parasitic diseases. Both forms of echinococcosis occur in Austria; in addition, imported cases are diagnosed and treated regularly in Austria. Diagnosis of echinococcosis is based on clinical symptoms, imaging techniques and particularly on the detection of specific antibodies in serum specimens of patients. For decades several companies have been providing commercial Echinococcus antigens and echinococcosis tests based on different methods, i.e. complement fixation test (CFT) and electrophoretic methods (CIEP, IEP) in the past and enzyme-linked immunosorbent assays (ELISA), western blot assays (WB) and indirect haemagglutination assays (IHA) in recent years. During the last years two studies have been carried out in our laboratory in order to evaluate the sensitivity and specificity of two commercial E. granulosus antigens (the synthetic p176 antigen, arc 5 antigen) and three commercial testkits (IHA from Dade Behring, IHA from Fumouze, ELISA from Novagnost-Dade Behring). Sera of patients with histologically and/or molecular biologically confirmed cystic or alveolar echinococcosis, of patients with other parasitic infections and of healthy people were tested comparatively for specific Echinococcus antibodies. The synthetic p176 antigen proved to be a highly specific but a insensitive antigen, whereas both the indirect haemagglutination assay as well as the Novagnost-ELISA showed a much higher sensitivity but only moderate specificity. Our studies demonstrated that neither the commercial antigens nor the test kits tested should be used as a primary test in a routine laboratory for the diagnosis of cystic echinococcosis or of alveolar echinococcosis. PMID:19915815

  20. Development of Single-Stranded DNA Aptamers for Specific Bisphenol A Detection

    PubMed Central

    Jo, Minjoung; Ahn, Ji-Young; Lee, Joohyung; Lee, Seram; Hong, Sun Woo; Yoo, Jae-Wook; Kang, Jeehye; Dua, Pooja

    2011-01-01

    The development of reagents with high affinity and specificity to small molecules is crucial for the high-throughput detection of chemical compounds, such as toxicants or pollutants. Aptamers are short and single-stranded (ss) oligonucleotides able to recognize target molecules with high affinity. Here, we report the selection of ssDNA aptamers that bind to Bisphenol A (BPA), an environmental hormone. Using SELEX process, we isolated high affinity aptamers to BPA from a 1015 random library of 60 mer ssDNAs. The selected aptamers bound specifically to BPA, but not to structurally similar molecules, such as Bisphenol B with one methyl group difference, or 4,4′-Bisphenol with 2 methyl groups difference. Using these aptamers, we developed an aptamer-based sol–gel biochip and detected BPA dissolved in water. This novel BPA aptamer-based detection can be further applied to the universal and high-specificity detection of small molecules. PMID:21413891

  1. Patient-Specific Early Seizure Detection from Scalp EEG

    PubMed Central

    Minasyan, Georgiy R.; Chatten, John B.; Chatten, Martha Jane; Harner, Richard N.

    2010-01-01

    Objective Develop a method for automatic detection of seizures prior to or immediately after clinical onset using features derived from scalp EEG. Methods This detection method is patient-specific. It uses recurrent neural networks and a variety of input features. For each patient we trained and optimized the detection algorithm for two cases: 1) during the period immediately preceding seizure onset, and 2) during the period immediately following seizure onset. Continuous scalp EEG recordings (duration 15 – 62 h, median 25 h) from 25 patients, including a total of 86 seizures, were used in this study. Results Pre-onset detection was successful in 14 of the 25 patients. For these 14 patients, all of the testing seizures were detected prior to seizure onset with a median pre-onset time of 51 sec and false positive rate was 0.06/h. Post-onset detection had 100% sensitivity, 0.023/hr false positive rate and median delay of 4 sec after onset. Conclusions The unique results of this study relate to pre-onset detection. Significance Our results suggest that reliable pre-onset seizure detection may be achievable for a significant subset of epilepsy patients without use of invasive electrodes. PMID:20461014

  2. Chromosome-specific staining to detect genetic rearrangements

    DOEpatents

    Gray, Joe W.; Pinkel, Daniel; Tkachuk, Douglas; Westbrook, Carol

    2013-04-09

    Methods and compositions for staining based upon nucleic acid sequence that employ nucleic acid probes are provided. Said methods produce staining patterns that can be tailored for specific cytogenetic analyzes. Said probes are appropriate for in situ hybridization and stain both interphase and metaphase chromosomal material with reliable signals. The nucleic acid probes are typically of a complexity greater than 50 kb, the complexity depending upon the cytogenetic application. Methods and reagents are provided for the detection of genetic rearrangements. Probes and test kits are provided for use in detecting genetic rearrangements, particularly for use in tumor cytogenetics, in the detection of disease related loci, specifically cancer, such as chronic myelogenous leukemia (CML) and for biological dosimetry. Methods and reagents are described for cytogenetic research, for the differentiation of cytogenetically similar but genetically different diseases, and for many prognostic and diagnostic applications.

  3. Detection and Description of Soils with Specific Nematode Suppressiveness

    PubMed Central

    Westphal, Andreas

    2005-01-01

    Soils with specific suppressiveness to plant-parasitic nematodes are of interest to define the mechanisms that regulate population density. Suppressive soils prevent nematodes from establishing and from causing disease, and they diminish disease severity after initial nematode damage in continuous culturing of a host. A range of non-specific and specific soil treatments, followed by infestation with a target nematode, have been employed to identify nematode-suppressive soils. Biocidal treatments, soil transfer tests, and baiting approaches together with observations of the plant-parasitic nematode in the root zone of susceptible host plants have improved the understanding of nematode-suppressive soils. Techniques to demonstrate specific soil suppressiveness against plant-parasitic nematodes are compared in this review. The overlap of studies on soil suppressiveness with recent advances in soil health and quality is briefly discussed. The emphasis is on methods (or criteria) used to detect and identify soils that maintain specific soil suppressiveness to plant-parasitic nematodes. While biocidal treatments can detect general and specific soil suppressiveness, soil transfer studies, by definition, apply only to specific soil suppressiveness. Finally, potential strategies to exploit suppressive soils are presented. PMID:19262851

  4. Electrical detection of specific versus non-specific binding events in breast cancer cells

    NASA Astrophysics Data System (ADS)

    King, Benjamin C.; Clark, Michael; Burkhead, Thomas; Sethu, Palaniappan; Rai, Shesh; Kloecker, Goetz; Panchapakesan, Balaji

    2012-10-01

    Detection of circulating tumor cells (CTCs) from patient blood samples offers a desirable alternative to invasive tissue biopsies for screening of malignant carcinomas. A rigorous CTC detection method must identify CTCs from millions of other formed elements in blood and distinguish them from healthy tissue cells also present in the blood. CTCs are known to overexpress surface receptors, many of which aid them in invading other tissue, and these provide an avenue for their detection. We have developed carbon nanotube (CNT) thin film devices to specifically detect these receptors in intact cells. The CNT sidewalls are functionalized with antibodies specific to Epithelial Cell Adhesion Molecule (EpCAM), a marker overexpressed by breast and other carcinomas. Specific binding of EpCAM to anti-EpCAM antibodies causes a change in the local charge environment of the CNT surface which produces a characteristic electrical signal. Two cell lines were tested in the device: MCF7, a mammary adenocarcinoma line which overexpresses EpCAM, and MCF10A, a non-tumorigenic mammary epithelial line which does not. Introduction of MCF7s caused significant changes in the electrical conductance of the devices due to specific binding and associated charge environment change near the CNT sidewalls. Introduction of MCF10A displays a different profile due to purely nonspecific interactions. The profile of specific vs. nonspecific interaction signatures using carbon based devices will guide development of this diagnostic tool towards clinical sample volumes with wide variety of markers.

  5. Circulating tumor cell detection using carbon nanotube devices: specific versus non-specific interactions

    PubMed Central

    King, Benjamin C.; Burkhead, Thomas; Panchapakesan, Balaji

    2013-01-01

    Detection of circulating tumor cells (CTCs) from patient blood samples offers a desirable alternative to invasive tissue biopsies for screening of malignant carcinomas. A rigorous CTC detection method must identify CTCs from millions of other formed elements in blood and distinguish them from healthy tissue cells also present in the blood. CTCs are known to overexpress certain surface receptors, many of which aid them in invading other tissue, and these provide an avenue for their detection. We have developed carbon nanotube (CNT) thin film devices to specifically detect these receptors in intact cells. The CNT sidewalls are functionalized with antibodies specific to Epithelial Cell Adhesion Molecule (EpCAM), a marker overexpressed by breast and other carcinomas. Specific binding of EpCAM to anti-EpCAM causes a change in the local charge environment of the CNT surface which produces a characteristic electrical signal. Two cell lines are tested in the device: MCF7, a mammary adenocarcinoma line which overexpresses EpCAM, and MCF10A, a non-tumorigenic mammary epithelial line which does not. Introduction of MCF7s causes significant changes in the electrical conductance of the devices due to specific binding and associated charge environment change near the CNT sidewalls. Introduction of MCF10A displays a different profile due to purely nonspecific interactions. The profile of specific vs. nonspecific interaction signatures using carbon based devices will guide development of this diagnostic tool towards clinical sample volumes.

  6. Isotope-specific detection of low density materials with mono-energetic (gamma)-rays

    SciTech Connect

    Albert, F; Anderson, S G; Gibson, D J; Hagmann, C A; Johnson, M S; Messerly, M J; Semenov, V A; Shverdin, M Y; Tremaine, A M; Hartemann, F V; Siders, C W; McNabb, D P; Barty, C J

    2009-03-16

    The first demonstration of isotope-specific detection of a low-Z, low density object, shielded by a high-Z and high density material using mono-energetic gamma-rays is reported. Isotope-specific detection of LiH shielded by Pb and Al is accomplished using the nuclear resonance fluorescence line of {sup 7}Li at 0.478 MeV. Resonant photons are produced via laser-based Compton scattering. The detection techniques are general and the confidence level obtained is shown to be superior to that yielded by conventional x-ray/{gamma}-ray techniques in these situations.

  7. Can Hyperspectral Remote Sensing Detect Species Specific Biochemicals ?

    NASA Astrophysics Data System (ADS)

    Vanderbilt, V. C.; Daughtry, C. S.

    2011-12-01

    Discrimination of a few plants scattered among many plants is a goal common to detection of agricultural weeds, invasive plant species and illegal Cannabis clandestinely grown outdoors, the subject of this research. Remote sensing technology provides an automated, computer based, land cover classification capability that holds promise for improving upon the existing approaches to Cannabis detection. In this research, we investigated whether hyperspectral reflectance of recently harvested, fully turgid Cannabis leaves and buds depends upon the concentration of the psychoactive ingredient Tetrahydrocannabinol (THC) that, if present at sufficient concentration, presumably would allow species-specific identification of Cannabis.

  8. Production of high specific activity silicon-32

    SciTech Connect

    Phillips, D.R.; Brzezinski, M.A.

    1998-12-31

    This is the final report of a three-year, Laboratory Directed Research and Development Project (LDRD) at Los Alamos National Laboratory (LANL). There were two primary objectives for the work performed under this project. The first was to take advantage of capabilities and facilities at Los Alamos to produce the radionuclide {sup 32}Si in unusually high specific activity. The second was to combine the radioanalytical expertise at Los Alamos with the expertise at the University of California to develop methods for the application of {sup 32}Si in biological oceanographic research related to global climate modeling. The first objective was met by developing targetry for proton spallation production of {sup 32}Si in KCl targets and chemistry for its recovery in very high specific activity. The second objective was met by developing a validated field-useable, radioanalytical technique, based upon gas-flow proportional counting, to measure the dynamics of silicon uptake by naturally occurring diatoms.

  9. Development of High Specific Strength Envelope Materials

    NASA Astrophysics Data System (ADS)

    Komatsu, Keiji; Sano, Masa-Aki; Kakuta, Yoshiaki

    Progress in materials technology has produced a much more durable synthetic fabric envelope for the non-rigid airship. Flexible materials are required to form airship envelopes, ballonets, load curtains, gas bags and covering rigid structures. Polybenzoxazole fiber (Zylon) and polyalirate fiber (Vectran) show high specific tensile strength, so that we developed membrane using these high specific tensile strength fibers as a load carrier. The main material developed is a Zylon or Vectran load carrier sealed internally with a polyurethane bonded inner gas retention film (EVOH). The external surface provides weather protecting with, for instance, a titanium oxide integrated polyurethane or Tedlar film. The mechanical test results show that tensile strength 1,000 N/cm is attained with weight less than 230g/m2. In addition to the mechanical properties, temperature dependence of the joint strength and solar absorptivity and emissivity of the surface are measured. 

  10. Investigation of contactless detection using a giant magnetoresistance sensor for detecting prostate specific antigen.

    PubMed

    Sun, Xuecheng; Zhi, Shaotao; Lei, Chong; Zhou, Yong

    2016-08-01

    This paper presents a contactless detection method for detecting prostate specific antigen with a giant magnetoresistance sensor. In contactless detection case, the prostate specific antigen sample preparation was separated from the sensor that prevented the sensor from being immersed in chemical solvents, and made the sensor implementing in immediately reuse without wash. Experimental results showed that applied an external magnetic field in a range of 50 Oe to 90 Oe, Dynabeads with a concentration as low as 0.1 μg/mL can be detected by this system and could give an approximate quantitation to the logarithmic of Dynabeads concentration. Sandwich immunoassay was employed for preparing PSA samples. The PSA capture was implemented on a gold film modified with a self-assembled monolayer and using biotinylated secondary antibody against PSA and streptavidinylated Dynabeads. With DC magnetic field in the range of 50 to 90 Oe, PSA can be detected with a detection limit as low as 0.1 ng/mL. Samples spiked with different concentrations of PSA can be distinguished clearly. Due to the contactless detection method, the detection system exhibited advantages such as convenient manipulation, reusable, inexpensive, small weight. So, this detection method was a promising candidate in biomarker detection, especially in point of care detection. PMID:27379844

  11. Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma

    PubMed Central

    Harlé, Alexandre; Salleron, Julia; Franczak, Claire; Dubois, Cindy; Filhine-Tressarieu, Pierre; Leroux, Agnès; Merlin, Jean-Louis

    2016-01-01

    Background Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies. Methods Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM) PCR, Real-time allele-specific amplification (RT-ASA) PCR, Next generation sequencing (NGS), immunohistochemistry (IHC) and the fully-automated molecular diagnostics platform IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays. Results BRAF mutations were found in 28(47.5%), 29(49.2%), 31(52.5%), 29(49.2%) and 27(45.8%) samples with HRM, RT-ASA, NGS, IdyllaTM and IHC respectively. Twenty-six (81.2%) samples were found bearing a c.1799T>A (p.Val600Glu) mutation, three (9.4%) with a c.1798_1799delinsAA (p.Val600Lys) mutation and one with c.1789_1790delinsTC (p.Leu597Ser) mutation. Two samples were found bearing complex mutations. Conclusions HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays. PMID:27111917

  12. Species-specific real-time PCR assay for the detection of Streptococcus suis from clinical specimens.

    PubMed

    Srinivasan, Velusamy; McGee, Lesley; Njanpop-Lafourcade, Berthe-Marie; Moïsi, Jennifer; Beall, Bernard

    2016-06-01

    A real-time polymerase chain reaction was developed to detect all known strains of Streptococcus suis. The assay was highly specific, and sensitivity was <10 copies/assay for S. suis detection from clinical samples. PMID:27041105

  13. Electromagnetic properties of high specific surface minerals

    NASA Astrophysics Data System (ADS)

    Klein, Katherine Anne

    Interparticle electrical forces play a dominant role in the behaviour of high specific surface minerals, such as clays. This fact encourages the use of small electromagnetic perturbations to assess the microscale properties of these materials. Thus, this research focuses on using electromagnetic waves to understand fundamental particle-particle and particle-fluid interactions, and fabric formation in high specific surface mineral-fluid mixtures (particle size <~1 μm). Topics addressed in this study include: the role of specific surface and double layer phenomena in the engineering behaviour of clay-water-electrolyte mixtures; the interplay between surface conduction, double layer polarization, and interfacial polarization; the relationship between fabric, permittivity, shear wave velocity, and engineering properties in soft slurries; and the effect of ferromagnetic impurities on electromagnetic measurements. The critical role of specific surface on the engineering properties of fine-grained soils is demonstrated through fundamental principles and empirical correlations. Afterwards, the effect of specific surface on the electromagnetic properties of particulate materials is studied using simple microscale analyses of conduction and polarization phenomena in particle-fluid mixtures, and corroborated by experimentation. These results clarify the relative importance of specific surface, water content, electrolyte type, and ionic concentration on the electrical properties of particulate materials. The sensitivity of electromagnetic parameters to particle orientation is addressed in light of the potential assessment of anisotropy in engineering properties. It is shown that effective conductivity measurements provide a robust method to determine electrical anisotropy in particle-fluid mixtures. However, real relative dielectric measurements at frequencies below 1 MHz are unreliable due to electrode effects (especially in highly conductive mixtures). The relationship

  14. Nested methylation-specific polymerase chain reaction cancer detection method

    DOEpatents

    Belinsky, Steven A.; Palmisano, William A.

    2007-05-08

    A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection of lung and other cancers.

  15. Monoclonal Antibodies for Specific Detection of Encephalitozoon cuniculi

    PubMed Central

    Mo, Lan; Drancourt, Michel

    2004-01-01

    Seven species-specific monoclonal antibodies (MAbs) were produced against Encephalitozoon cuniculi and characterized. The MAbs were immunoglobulin G, and when used for indirect microimmunofluorescence microscopy and Western immunoblot assays, they detected E. cuniculi originating from clinical samples. They did not cross-react with other Encephalitozoon species (E. intestinalis and E. hellem) or with a collection of gram-negative bacteria, yeast, and other parasites. The MAbs reacted primarily with 121-, 56-, 45-, 43-, and 41-kDa protein epitopes of E. cuniculi. These epitopes were demonstrated to be E. cuniculi species specific by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We developed MAbs to strains of E. cuniculi that can be used successfully to distinguish E. cuniculi from other microsporidial species in cultures established from clinical specimens. These MAbs may provide a specific, simple, rapid, and low-cost tool for the identification and diagnosis of infections due to microsporidia. PMID:15539506

  16. Wp specific methylation of highly proliferated LCLs

    SciTech Connect

    Park, Jung-Hoon; Jeon, Jae-Pil; Shim, Sung-Mi; Nam, Hye-Young; Kim, Joon-Woo; Han, Bok-Ghee; Lee, Suman . E-mail: suman@cha.ac.kr

    2007-06-29

    The epigenetic regulation of viral genes may be important for the life cycle of EBV. We determined the methylation status of three viral promoters (Wp, Cp, Qp) from EBV B-lymphoblastoid cell lines (LCLs) by pyrosequencing. Our pyrosequencing data showed that the CpG region of Wp was methylated, but the others were not. Interestingly, Wp methylation was increased with proliferation of LCLs. Wp methylation was as high as 74.9% in late-passage LCLs, but 25.6% in early-passage LCLs. From two Burkitt's lymphoma cell lines, Wp specific hypermethylation was also found (>80%). Interestingly, the expression of EBNA2 gene which located directly next to Wp was associated with its methylation. Our data suggested that Wp specific methylation may be important for the indicator of the proliferation status of LCLs, and the epigenetic viral gene regulation of EBNA2 gene by Wp should be further defined possibly with other biological processes.

  17. Production Of High Specific Activity Copper-67

    DOEpatents

    Jamriska, Sr., David J.; Taylor, Wayne A.; Ott, Martin A.; Fowler, Malcolm; Heaton, Richard C.

    2003-10-28

    A process for the selective production and isolation of high specific activity Cu.sup.67 from proton-irradiated enriched Zn.sup.70 target comprises target fabrication, target irradiation with low energy (<25 MeV) protons, chemical separation of the Cu.sup.67 product from the target material and radioactive impurities of gallium, cobalt, iron, and stable aluminum via electrochemical methods or ion exchange using both anion and cation organic ion exchangers, chemical recovery of the enriched Zn.sup.70 target material, and fabrication of new targets for re-irradiation is disclosed.

  18. Production Of High Specific Activity Copper-67

    DOEpatents

    Jamriska, Sr., David J.; Taylor, Wayne A.; Ott, Martin A.; Fowler, Malcolm; Heaton, Richard C.

    2002-12-03

    A process for the selective production and isolation of high specific activity cu.sup.67 from proton-irradiated enriched Zn.sup.70 target comprises target fabrication, target irradiation with low energy (<25 MeV) protons, chemical separation of the Cu.sup.67 product from the target material and radioactive impurities of gallium, cobalt, iron, and stable aluminum via electrochemical methods or ion exchange using both anion and cation organic ion exchangers, chemical recovery of the enriched Zn.sup.70 target material, and fabrication of new targets for re-irradiation is disclosed.

  19. High specific energy, high capacity nickel-hydrogen cell design

    NASA Technical Reports Server (NTRS)

    Wheeler, James R.

    1993-01-01

    A 3.5 inch rabbit-ear-terminal nickel-hydrogen cell has been designed and tested to deliver high capacity at a C/1.5 discharge rate. Its specific energy yield of 60.6 wh/kg is believed to be the highest yet achieved in a slurry-process nickel-hydrogen cell, and its 10 C capacity of 113.9 AH the highest capacity yet made at a discharge rate this high in the 3.5 inch diameter size. The cell also demonstrated a pulse capability of 180 amps for 20 seconds. Specific cell parameters, performance, and future test plans are described.

  20. Noncovalent functionalization of carbon nanotubes for highly specific electronic biosensors

    NASA Astrophysics Data System (ADS)

    Chen, Robert J.; Bangsaruntip, Sarunya; Drouvalakis, Katerina A.; Wong Shi Kam, Nadine; Shim, Moonsub; Li, Yiming; Kim, Woong; Utz, Paul J.; Dai, Hongjie

    2003-04-01

    Novel nanomaterials for bioassay applications represent a rapidly progressing field of nanotechnology and nanobiotechnology. Here, we present an exploration of single-walled carbon nanotubes as a platform for investigating surface-protein and protein-protein binding and developing highly specific electronic biomolecule detectors. Nonspecific binding on nanotubes, a phenomenon found with a wide range of proteins, is overcome by immobilization of polyethylene oxide chains. A general approach is then advanced to enable the selective recognition and binding of target proteins by conjugation of their specific receptors to polyethylene oxide-functionalized nanotubes. This scheme, combined with the sensitivity of nanotube electronic devices, enables highly specific electronic sensors for detecting clinically important biomolecules such as antibodies associated with human autoimmune diseases.

  1. PCR-specific detection of recently described Lotmaria passim (Trypanosomatidae) in Chilean apiaries.

    PubMed

    Arismendi, Nolberto; Bruna, Alex; Zapata, Nelson; Vargas, Marisol

    2016-02-01

    The recently described trypanosome Lotmaria passim is currently considered the most predominant trypanosomatid in honey bees worldwide and could be a factor in honey bee declines. For a specific and quick detection of this pathogen, we developed primers based on the SSU rRNA and gGAPDH genes for the detection of L. passim in Chilean honey beehives. PCR products amplified and sequenced for these primers shared 99-100% identity with other sequences of L. passim. The designed primers were specific and we were able to detect a high prevalence (40-90%) of L. passim in bee hives distributed throughout Chile. Our described PCR-based method offers a feasible and specific detection of L. passim in any honey bee samples. PMID:26721451

  2. Nanostructured materials detect epidermal growth factor receptor, neuron specific enolase and carcinoembryonic antigen

    NASA Astrophysics Data System (ADS)

    Stefan-van Staden, Raluca-Ioana; Comnea-Stancu, Ionela Raluca; Surdu-Bob, Carmen Cristina; Badulescu, Marius

    2015-09-01

    New nanostructured materials based on thin films of Cu and Ni deposited on textile material (veil), as well as gold nanostructured microspheres were used for the design of new stochastic sensors. The stochastic sensors were able to detect simultaneously a panel of biomarkers comprising epidermal growth factor receptor, neuron specific enolase, and carcinoembryonic antigen from whole blood samples with high reliabilities - recovery tests higher than 97.00%, with a RSD (%) lower than 0.1%. The stochastic sensors had shown high sensitivities and low determination levels for the detection of the proposed panel of biomarkers making early detection of lung cancer possible by fast screening of whole blood.

  3. Detection device for high explosives

    DOEpatents

    Grey, Alan E.; Partin, Judy K.; Stone, Mark L.; Von Wandruszka, Ray M.; Reagen, William K.; Ingram, Jani C.; Lancaster, Gregory D.

    1992-01-01

    A portable fiber optic detector that senses the presence of specific target chemicals by electrostatically attracting the target chemical to an aromatic compound coating on an optical fiber. Attaching the target chemical to the coated fiber reduces the fluorescence so that a photon sensing detector records the reduced light level and activates an appropriate alarm or indicator.

  4. Detection device for high explosives

    DOEpatents

    Grey, A.E.; Partin, J.K.; Stone, M.L.; Von Wandruszka, R.M.; Reagen, W.K.; Ingram, J.C.; Lancaster, G.D.

    1992-10-20

    A portable fiber optic detector is described that senses the presence of specific target chemicals by electrostatically attracting the target chemical to an aromatic compound coating on an optical fiber. Attaching the target chemical to the coated fiber reduces the fluorescence so that a photon sensing detector records the reduced light level and activates an appropriate alarm or indicator. 5 figs.

  5. Production of high specific activity silicon-32

    DOEpatents

    Phillips, Dennis R.; Brzezinski, Mark A.

    1994-01-01

    A process for preparation of silicon-32 is provide and includes contacting an irradiated potassium chloride target, including spallation products from a prior irradiation, with sufficient water, hydrochloric acid or potassium hydroxide to form a solution, filtering the solution, adjusting pH of the solution to from about 5.5 to about 7.5, admixing sufficient molybdate-reagent to the solution to adjust the pH of the solution to about 1.5 and to form a silicon-molybdate complex, contacting the solution including the silicon-molybdate complex with a dextran-based material, washing the dextran-based material to remove residual contaminants such as sodium-22, separating the silicon-molybdate complex from the dextran-based material as another solution, adding sufficient hydrochloric acid and hydrogen peroxide to the solution to prevent reformation of the silicon-molybdate complex and to yield an oxidization state of the molybdate adapted for subsequent separation by an anion exchange material, contacting the solution with an anion exchange material whereby the molybdate is retained by the anion exchange material and the silicon remains in solution, and optionally adding sufficient alkali metal hydroxide to adjust the pH of the solution to about 12 to 13. Additionally, a high specific activity silicon-32 product having a high purity is provided.

  6. Production of high specific activity silicon-32

    SciTech Connect

    Phillips, D.R.; Brzezinski, M.A.

    1994-09-13

    A process for the preparation of silicon-32 is provided and includes contacting an irradiated potassium chloride target, including spallation products from a prior irradiation, with sufficient water, hydrochloric acid or potassium hydroxide to form a solution, filtering the solution, adjusting pH of the solution to from about 5.5 to about 7.5, admixing sufficient molybdate-reagent to the solution to adjust the pH of the solution to about 1.5 and to form a silicon-molybdate complex, contacting the solution including the silicon-molybdate complex with a dextran-based material, washing the dextran-based material to remove residual contaminants such as sodium-22, separating the silicon-molybdate complex from the dextran-based material as another solution, adding sufficient hydrochloric acid and hydrogen peroxide to the solution to prevent reformation of the silicon-molybdate complex and to yield an oxidization state of the molybdate adapted for subsequent separation by an anion exchange material, contacting the solution with an anion exchange material whereby the molybdate is retained by the anion exchange material and the silicon remains in solution, and optionally adding sufficient alkali metal hydroxide to adjust the pH of the solution to about 12 to 13. Additionally, a high specific activity silicon-32 product having a high purity is provided.

  7. Specific detection of banana residue in processed foods using polymerase chain reaction.

    PubMed

    Sakai, Yumiko; Ishihata, Kimie; Nakano, Shigeru; Yamada, Toshihiro; Yano, Takeo; Uchida, Kouji; Nakao, Yoshiki; Urisu, Atsuo; Adachi, Reiko; Teshima, Reiko; Akiyama, Hiroshi

    2010-07-28

    Specific polymerase chain reaction (PCR) methods were developed for the detection of banana residue in processed foods. For high banana specificity, the primer set BAN-F/BAN-R was designed on the basis of the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL) genes of chloroplasts and used to obtain amplified products specific to banana by both conventional and real-time PCR. To confirm the specificity of these methods, genomic DNA samples from 31 other species were examined; no amplification products were detected. Subsequently, eight kinds of processed foods containing banana were investigated using these methods to confirm the presence of banana DNA. Conventional PCR had a detection limit of 1 ppm (w/w) banana DNA spiked in 50 ng of salmon testis DNA, whereas SYBR Green I real-time semiquantitative PCR had a detection limit as low as 10 ppm banana DNA. Thus, both methods show high sensitivity and may be applicable as specific tools for the detection of trace amounts of banana in commercial food products. PMID:20604506

  8. New Concepts of Fluorescent Probes for Specific Detection of DNA Sequences: Bis-Modified Oligonucleotides in Excimer and Exciplex Detection

    PubMed Central

    Gbaj, A; Bichenkova, EV; Walsh, L; Savage, HE; Sardarian, AR; Etchells, LL; Gulati, A; Hawisa, S; Douglas, KT

    2009-01-01

    The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5′-bispyrene and 3′-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5′-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5′-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing. PMID:21483539

  9. Rapid and specific SPRi detection of L. pneumophila in complex environmental water samples.

    PubMed

    Foudeh, Amir M; Trigui, Hana; Mendis, Nilmini; Faucher, Sebastien P; Veres, Teodor; Tabrizian, Maryam

    2015-07-01

    Legionellosis is a very devastating disease worldwide mainly due to unpredictable outbreaks in man-made water systems. Developing a highly specific and sensitive rapid detection system that detects only metabolically active bacteria is a main priority for water quality assessment. We previously developed a versatile technique for sensitive and specific detection of synthetic RNA. In the present work, we further investigated the performance of the developed biosensor for detection of Legionella pneumophila in complex environmental samples, particularly those containing protozoa. The specificity and sensitivity of the detection system were verified using total RNA extracted from L. pneumophila in spiked water co-cultured with amoebae. We demonstrated that the expression level of ribosomal RNA (rRNA) is extremely dependent on the environmental conditions. The presence of amoebae with L. pneumophila, especially in nutrition-deprived samples, increased the amount of L. pneumophila 15-fold after 1 week as measured through the expression of 16s rRNA. Using the developed surface plasmon resonance imaging (SPRi) detection method, we were also able to successfully detect L. pneumophila within 3 h, both in the presence and absence of amoebae in the complex environmental samples obtained from a cooling water tower. These findings suggest that the developed biosensing system is a viable method for rapid, real-time and effective detection not only for L. pneumophila in environmental samples but also to assess the risk associated with the use of water contaminated with other pathogens. PMID:25935681

  10. Can Sample-Specific Simulations Help Detect Low Base-Rate Taxonicity?

    ERIC Educational Resources Information Center

    Beach, Steven R. H.; Amir, Nader; Bau, Jinn Jonp

    2005-01-01

    The authors examined the role of the sample-specific simulations (SSS; A. M. Ruscio & J. Ruscio, 2002; J. Ruscio & A. M. Ruscio, 2004) procedure in detecting low base-rate taxa that might otherwise prove elusive. The procedure preserved key distributional characteristics for moderate to high base-rate taxa, but it performed inadequately for low…

  11. Immunological-based assays for specific detection of shrimp viruses

    PubMed Central

    Chaivisuthangkura, Parin; Longyant, Siwaporn; Sithigorngul, Paisarn

    2014-01-01

    Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection. PMID:24567913

  12. Immunological-based assays for specific detection of shrimp viruses.

    PubMed

    Chaivisuthangkura, Parin; Longyant, Siwaporn; Sithigorngul, Paisarn

    2014-02-12

    Among shrimp viral pathogens, white spot syndrome virus (WSSV) and yellow head virus (YHV) are the most lethal agents, causing serious problems for both the whiteleg shrimp, Penaeus (Litopenaeus) vannamei, and the black tiger shrimp, Penaeus (Penaeus) monodon. Another important virus that infects P. vannamei is infectious myonecrosis virus (IMNV), which induces the white discoloration of affected muscle. In the cases of taura syndrome virus and Penaeus stylirostris densovirus (PstDNV; formerly known as infectious hypodermal and hematopoietic necrosis virus), their impacts were greatly diminished after the introduction of tolerant stocks of P. vannamei. Less important viruses are Penaeus monodon densovirus (PmDNV; formerly called hepatopancreatic parvovirus), and Penaeus monodon nucleopolyhedrovirus (PemoNPV; previously called monodon baculovirus). For freshwater prawn, Macrobrachium rosenbergii nodavirus and extra small virus are considered important viral pathogens. Monoclonal antibodies (MAbs) specific to the shrimp viruses described above have been generated and used as an alternative tool in various immunoassays such as enzyme-linked immunosorbent assay, dot blotting, Western blotting and immunohistochemistry. Some of these MAbs were further developed into immunochromatographic strip tests for the detection of WSSV, YHV, IMNV and PemoNPV and into a dual strip test for the simultaneous detection of WSSV/YHV. The strip test has the advantages of speed, as the result can be obtained within 15 min, and simplicity, as laboratory equipment and specialized skills are not required. Therefore, strip tests can be used by shrimp farmers for the pond-side monitoring of viral infection. PMID:24567913

  13. A Novel SERRS Sandwich-Hybridization Assay to Detect Specific DNA Target

    PubMed Central

    Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

    2011-01-01

    In this study, we have applied Surface Enhanced Resonance Raman Scattering (SERRS) technology to the specific detection of DNA. We present an innovative SERRS sandwich-hybridization assay that allows specific DNA detection without any enzymatic amplification, such as is the case with Polymerase Chain Reaction (PCR). In some substrates, such as ancient or processed remains, enzymatic amplification fails due to DNA alteration (degradation, chemical modification) or to the presence of inhibitors. Consequently, the development of a non-enzymatic method, allowing specific DNA detection, could avoid long, expensive and inconclusive amplification trials. Here, we report the proof of concept of a SERRS sandwich-hybridization assay that leads to the detection of a specific chamois DNA. This SERRS assay reveals its potential as a non-enzymatic alternative technology to DNA amplification methods (particularly the PCR method) with several applications for species detection. As the amount and type of damage highly depend on the preservation conditions, the present SERRS assay would enlarge the range of samples suitable for DNA analysis and ultimately would provide exciting new opportunities for the investigation of ancient DNA in the fields of evolutionary biology and molecular ecology, and of altered DNA in food frauds detection and forensics. PMID:21655320

  14. High Frequency QRS ECG Accurately Detects Cardiomyopathy

    NASA Technical Reports Server (NTRS)

    Schlegel, Todd T.; Arenare, Brian; Poulin, Gregory; Moser, Daniel R.; Delgado, Reynolds

    2005-01-01

    High frequency (HF, 150-250 Hz) analysis over the entire QRS interval of the ECG is more sensitive than conventional ECG for detecting myocardial ischemia. However, the accuracy of HF QRS ECG for detecting cardiomyopathy is unknown. We obtained simultaneous resting conventional and HF QRS 12-lead ECGs in 66 patients with cardiomyopathy (EF = 23.2 plus or minus 6.l%, mean plus or minus SD) and in 66 age- and gender-matched healthy controls using PC-based ECG software recently developed at NASA. The single most accurate ECG parameter for detecting cardiomyopathy was an HF QRS morphological score that takes into consideration the total number and severity of reduced amplitude zones (RAZs) present plus the clustering of RAZs together in contiguous leads. This RAZ score had an area under the receiver operator curve (ROC) of 0.91, and was 88% sensitive, 82% specific and 85% accurate for identifying cardiomyopathy at optimum score cut-off of 140 points. Although conventional ECG parameters such as the QRS and QTc intervals were also significantly longer in patients than controls (P less than 0.001, BBBs excluded), these conventional parameters were less accurate (area under the ROC = 0.77 and 0.77, respectively) than HF QRS morphological parameters for identifying underlying cardiomyopathy. The total amplitude of the HF QRS complexes, as measured by summed root mean square voltages (RMSVs), also differed between patients and controls (33.8 plus or minus 11.5 vs. 41.5 plus or minus 13.6 mV, respectively, P less than 0.003), but this parameter was even less accurate in distinguishing the two groups (area under ROC = 0.67) than the HF QRS morphologic and conventional ECG parameters. Diagnostic accuracy was optimal (86%) when the RAZ score from the HF QRS ECG and the QTc interval from the conventional ECG were used simultaneously with cut-offs of greater than or equal to 40 points and greater than or equal to 445 ms, respectively. In conclusion 12-lead HF QRS ECG employing

  15. High specific energy, high capacity nickel-hydrogen cell design

    NASA Technical Reports Server (NTRS)

    Wheeler, James R.

    1993-01-01

    A 3.5 inch rabbit-ear-terminal nickel-hydrogen cell was designed and tested to deliver high capacity at steady discharge rates up to and including a C rate. Its specific energy yield of 60.6 wh/kg is believed to be the highest yet achieved in a slurry-process nickel-hydrogen cell, and its 10 C capacity of 113.9 AH the highest capacity yet of any type in a 3.5 inch diameter size. The cell also demonstrated a pulse capability of 180 amps for 20 seconds. Specific cell parameters and performance are described. Also covered is an episode of capacity fading due to electrode swelling and its successful recovery by means of additional activation procedures.

  16. Characterization of Antibodies for Grain-Specific Gluten Detection.

    PubMed

    Sharma, Girdhari M; Rallabhandi, Prasad; Williams, Kristina M; Pahlavan, Autusa

    2016-03-01

    Gluten ingestion causes immunoglobulin E (IgE)-mediated allergy or celiac disease in sensitive individuals, and a strict gluten-free diet greatly limits food choices. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten-free foods. Anti-gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain-specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten-containing grain-specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti-gliadin, 11 to 15 and 72 to 95 kDa with anti-secalin, and 30 to 43 kDa with anti-hordein peptide antibodies, respectively. Minimal or no cross-reactivity with gluten from other grains was observed among these antibodies. The anti-consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross-reactivity of grain-specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation. PMID:26878584

  17. Trajectories for High Specific Impulse High Specific Power Deep Space Exploration

    NASA Technical Reports Server (NTRS)

    Polsgrove, T.; Adams, R. B.; Brady, Hugh J. (Technical Monitor)

    2002-01-01

    Preliminary results are presented for two methods to approximate the mission performance of high specific impulse high specific power vehicles. The first method is based on an analytical approximation derived by Williams and Shepherd and can be used to approximate mission performance to outer planets and interstellar space. The second method is based on a parametric analysis of trajectories created using the well known trajectory optimization code, VARITOP. This parametric analysis allows the reader to approximate payload ratios and optimal power requirements for both one-way and round-trip missions. While this second method only addresses missions to and from Jupiter, future work will encompass all of the outer planet destinations and some interstellar precursor missions.

  18. Specific detection of avidin-biotin binding using liquid crystal droplets.

    PubMed

    Khan, Mashooq; Park, Soo-Young

    2015-03-01

    Poly(acrylicacid-b-4-cynobiphenyl-4'-undecylacrylate) (PAA-b-LCP)-functionalized 4-cyano-4'-pentylbiphenyl (5CB) droplets were made by using microfluidic technique. The PAA chains on the 5CB droplets, were biotinylated, and used to specifically detect avidin-biotin binding at the 5CB/aqueous interface. The avidin-biotin binding was characterized by the configurational change (from radial to bipolar) of the 5CB droplets, as observed through a polarized optical microscope. The maximum biotinylation was obtained by injecting a >100 μg/mL biotin aqueous solution, which enabled a limit of detection of 0.5 μg/mL avidin. This droplet biosensor could specifically detect avidin against other proteins such as bovine serum albumin, lysozyme, hemoglobin, and chymotrypsinogen solutions. Avidin detection with 5CBPAA-biotin droplets having high sensitivity, specificity, and stability demonstrates new applications of the functionalized liquid crystal droplets that can detect specific proteins or other analytes through a ligand/receptor model. PMID:25689094

  19. Giant magnetoresistive sensor array for sensitive and specific multiplexed food allergen detection.

    PubMed

    Ng, Elaine; Nadeau, Kari C; Wang, Shan X

    2016-06-15

    Current common allergen detection methods, including enzyme-linked immunosorbent assays (ELISAs) and dip-stick methods, do not provide adequate levels of sensitivity and specificity for at-risk allergic patients. A method for performing highly sensitive and specific detection of multiple food allergens is thus imperative as food allergies are becoming increasingly recognized as a major healthcare concern, affecting an estimated 4% of the total population. We demonstrate first instance of sensitive and specific multiplexed detection of major peanut allergens Ara h 1 and Ara h 2, and wheat allergen Gliadin using giant magnetoresistive (GMR) sensor arrays. Commercialized ELISA kits for Ara h 1 and Ara h 2 report limits of detection (LODs) at 31.5 ng/mL and 0.2 ng/mL, respectively. In addition, the 96-well-based ELISA developed in-house for Gliadin was found to have a LOD of 40 ng/mL. Our multiplexed GMR-based assay demonstrates the ability to perform all three assays on the same chip specifically and with sensitivities at LODs about an order of magnitude lower than those of 96-well-based ELISAs. LODs of GMR-based assays developed for Ara h 1, Ara h 2, and Gliadin were 7.0 ng/mL, 0.2 ng/mL, and 1.5 ng/mL, respectively, with little to no cross-reactivity. These LODs are clinically important as some patients could react strongly against such low allergen levels. Given the limitations of current industrial detection technology, multiplexed GMR-based assays provide a method for highly sensitive and specific simultaneous detection of any combination of food-product allergens, thus protecting allergic patients from life-threatening events, including anaphylaxis, by unintentional consumption. PMID:26859787

  20. An Energy efficient application specific integrated circuit for electrocardiogram feature detection and its potential for ambulatory cardiovascular disease detection.

    PubMed

    Jain, Sanjeev Kumar; Bhaumik, Basabi

    2016-03-01

    A novel algorithm based on forward search is developed for real-time electrocardiogram (ECG) signal processing and implemented in application specific integrated circuit (ASIC) for QRS complex related cardiovascular disease diagnosis. The authors have evaluated their algorithm using MIT-BIH database and achieve sensitivity of 99.86% and specificity of 99.93% for QRS complex peak detection. In this Letter, Physionet PTB diagnostic ECG database is used for QRS complex related disease detection. An ASIC for cardiovascular disease detection is fabricated using 130-nm CMOS high-speed process technology. The area of the ASIC is 0.5 mm(2). The power dissipation is 1.73 μW at the operating frequency of 1 kHz with a supply voltage of 0.6 V. The output from the ASIC is fed to their Android application that generates diagnostic report and can be sent to a cardiologist through email. Their ASIC result shows average failed detection rate of 0.16% for six leads data of 290 patients in PTB diagnostic ECG database. They also have implemented a low-leakage version of their ASIC. The ASIC dissipates only 45 pJ with a supply voltage of 0.9 V. Their proposed ASIC is most suitable for energy efficient telemetry cardiovascular disease detection system. PMID:27284458

  1. An Energy efficient application specific integrated circuit for electrocardiogram feature detection and its potential for ambulatory cardiovascular disease detection

    PubMed Central

    Bhaumik, Basabi

    2016-01-01

    A novel algorithm based on forward search is developed for real-time electrocardiogram (ECG) signal processing and implemented in application specific integrated circuit (ASIC) for QRS complex related cardiovascular disease diagnosis. The authors have evaluated their algorithm using MIT-BIH database and achieve sensitivity of 99.86% and specificity of 99.93% for QRS complex peak detection. In this Letter, Physionet PTB diagnostic ECG database is used for QRS complex related disease detection. An ASIC for cardiovascular disease detection is fabricated using 130-nm CMOS high-speed process technology. The area of the ASIC is 0.5 mm2. The power dissipation is 1.73 μW at the operating frequency of 1 kHz with a supply voltage of 0.6 V. The output from the ASIC is fed to their Android application that generates diagnostic report and can be sent to a cardiologist through email. Their ASIC result shows average failed detection rate of 0.16% for six leads data of 290 patients in PTB diagnostic ECG database. They also have implemented a low-leakage version of their ASIC. The ASIC dissipates only 45 pJ with a supply voltage of 0.9 V. Their proposed ASIC is most suitable for energy efficient telemetry cardiovascular disease detection system. PMID:27284458

  2. Development of glycan specific lectin based immunoassay for detection of prostate specific antigen.

    PubMed

    Bhanushali, Paresh B; Badgujar, Shamkant B; Tripathi, Mukesh M; Gupta, Sanjeev; Murthy, Vedang; Krishnasastry, Musti V; Puri, Chander P

    2016-05-01

    We describe an analytical approach for the detection and verification of glycosylation patterns of prostate specific antigen (PSA), a key biomarker currently used for understanding the onset and prognosis of prostate cancer. PSA has been purified from the human seminal plasma and total PSA from prostate cancer sera. PSA is a monomeric glycoprotein with an apparent molecular mass 28040.467Da, which exhibits a characteristic protease activity against casein and gelatin. Its optimal protease activity is centered on neutral pH. Peptide mass fingerprint analysis of the purified PSA has yielded peptides that partially match with known database sequences (Uniprot ID P07288). Tryptic digestion profile of isolated PSA, infer the exclusive nature of PSA and may be additive molecule in the dictionary of seminal proteins. Surface plasmon resonance and lectin immunoassay revealed direct interaction between a newly developed anti-PSA monoclonal antibody (C4E6) and PSA. A lectin based immunoassay is reported here which was achieved with the C4E6 anti-PSA antibody and biotinylated plant lectins. This investigation provides an alternative method to isolate and quantify PSA with altered glycosylation which might be seen in the prostate cancer and developing a lectin based immunoassay to detect PSA in serum of prostate cancer patients. PMID:26840176

  3. Detection of High-Risk Atherosclerotic Plaque

    PubMed Central

    Fleg, Jerome L.; Stone, Gregg W.; Fayad, Zahi A.; Granada, Juan F.; Hatsukami, Thomas S.; Kolodgie, Frank D.; Ohayon, Jacques; Pettigrew, Roderic; Sabatine, Marc S.; Tearney, Guillermo; Waxman, Sergio; Domanski, Michael J.; Srinivas, Pothur R.; Narula, Jagat

    2013-01-01

    The leading cause of major morbidity and mortality in most countries around the world is atherosclerotic cardiovascular disease, most commonly caused by thrombotic occlusion of a high-risk coronary plaque resulting in myocardial infarction or cardiac death, or embolization from a high-risk carotid plaque resulting in stroke. The lesions prone to result in such clinical events are termed vulnerable or high-risk plaques, and their identification may lead to the development of pharmacological and mechanical intervention strategies to prevent such events. Autopsy studies from patients dying of acute myocardial infarction or sudden death have shown that such events typically arise from specific types of atherosclerotic plaques, most commonly the thin-cap fibroatheroma. However, the search in human beings for vulnerable plaques before their becoming symptomatic has been elusive. Recently, the PROSPECT (Providing Regional Observations to Study Predictors of Events in the Coronary Tree) study demonstrated that coronary plaques that are likely to cause future cardiac events, regardless of angiographic severity, are characterized by large plaque burden and small lumen area and/or are thin-cap fibroatheromas verified by radiofrequency intravascular ultrasound imaging. This study opened the door to identifying additional invasive and noninvasive imaging modalities that may improve detection of high-risk atherosclerotic lesions and patients. Beyond classic risk factors, novel biomarkers and genetic profiling may identify those patients in whom noninvasive imaging for vulnerable plaque screening, followed by invasive imaging for risk confirmation is warranted, and in whom future pharmacological and/or device-based focal or regional therapies may be applied to improve long-term prognosis. PMID:22974808

  4. Can hyperspectral remote sensing detect species specific biochemicals?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Discrimination of a few plants scattered among many plants is a goal common to detection of agricultural weeds and invasive species. Detection of clandestinely grown Cannabis sativa L. is in many ways a special case of weed detection. Remote sensing technology provides an automated, computer based,...

  5. A simple molecular beacon with duplex-specific nuclease amplification for detection of microRNA.

    PubMed

    Li, Yingcun; Zhang, Jiangyan; Zhao, Jingjing; Zhao, Likun; Cheng, Yongqiang; Li, Zhengping

    2016-02-01

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene activity, promoting or inhibiting cell proliferation, migration and apoptosis. Abnormal expression of miRNAs is associated with many diseases. Therefore, it is essential to establish a simple, rapid and sensitive miRNA detection method. In this paper, based on a simple molecular beacon (MB) and duplex-specific nuclease (DSN), we developed a target recycling amplification method for miRNA detection. By controlling the number of stem bases to 5, the MB probe used in this method can be prevented from hydrolysis by DSN without special modification. This assay is direct and simple to quantitatively detect miRNA with high sensitivity and specificity. The MB probe design provides a new strategy for nuclease-based amplification reaction. PMID:26688865

  6. Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection.

    PubMed

    Kc, R; Srivastava, A; Wilkowski, J M; Richter, C E; Shavit, J A; Burke, D T; Bielas, S L

    2016-01-01

    CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies. PMID:27557703

  7. Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection

    PubMed Central

    KC, R.; Srivastava, A.; Wilkowski, J. M.; Richter, C. E.; Shavit, J. A.; Burke, D. T.; Bielas, S. L.

    2016-01-01

    CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies. PMID:27557703

  8. High specific activity platinum-195m

    DOEpatents

    Mirzadeh, Saed; Du, Miting; Beets, Arnold L.; Knapp, Jr., Furn F.

    2004-10-12

    A new composition of matter includes .sup.195m Pt characterized by a specific activity of at least 30 mCi/mg Pt, generally made by method that includes the steps of: exposing .sup.193 Ir to a flux of neutrons sufficient to convert a portion of the .sup.193 Ir to .sup.195m Pt to form an irradiated material; dissolving the irradiated material to form an intermediate solution comprising Ir and Pt; and separating the Pt from the Ir by cation exchange chromatography to produce .sup.195m Pt.

  9. A highly specific test for periodicity

    SciTech Connect

    Ansmann, Gerrit

    2015-11-15

    We present a method that allows to distinguish between nearly periodic and strictly periodic time series. To this purpose, we employ a conservative criterion for periodicity, namely, that the time series can be interpolated by a periodic function whose local extrema are also present in the time series. Our method is intended for the analysis of time series generated by deterministic time-continuous dynamical systems, where it can help telling periodic dynamics from chaotic or transient ones. We empirically investigate our method's performance and compare it to an approach based on marker events (or Poincaré sections). We demonstrate that our method is capable of detecting small deviations from periodicity and outperforms the marker-event-based approach in typical situations. Our method requires no adjustment of parameters to the individual time series, yields the period length with a precision that exceeds the sampling rate, and its runtime grows asymptotically linear with the length of the time series.

  10. Sensitivity in error detection of patient specific QA tools for IMRT plans

    NASA Astrophysics Data System (ADS)

    Lat, S. Z.; Suriyapee, S.; Sanghangthum, T.

    2016-03-01

    The high complexity of dose calculation in treatment planning and accurate delivery of IMRT plan need high precision of verification method. The purpose of this study is to investigate error detection capability of patient specific QA tools for IMRT plans. The two H&N and two prostate IMRT plans with MapCHECK2 and portal dosimetry QA tools were studied. Measurements were undertaken for original and modified plans with errors introduced. The intentional errors composed of prescribed dose (±2 to ±6%) and position shifting in X-axis and Y-axis (±1 to ±5mm). After measurement, gamma pass between original and modified plans were compared. The average gamma pass for original H&N and prostate plans were 98.3% and 100% for MapCHECK2 and 95.9% and 99.8% for portal dosimetry, respectively. In H&N plan, MapCHECK2 can detect position shift errors starting from 3mm while portal dosimetry can detect errors started from 2mm. Both devices showed similar sensitivity in detection of position shift error in prostate plan. For H&N plan, MapCHECK2 can detect dose errors starting at ±4%, whereas portal dosimetry can detect from ±2%. For prostate plan, both devices can identify dose errors starting from ±4%. Sensitivity of error detection depends on type of errors and plan complexity.

  11. High sensitivity deflection detection of nanowires.

    PubMed

    Sanii, Babak; Ashby, Paul D

    2010-04-01

    A critical limitation of nanoelectromechanical systems (NEMS) is the lack of a high-sensitivity position detection mechanism. We introduce a noninterferometric optical approach to determine the position of nanowires with a high sensitivity and bandwidth. Its physical origins and limitations are determined by Mie scattering analysis. This enables a dramatic miniaturization of detectable cantilevers, with attendant reductions to the fundamental minimum force noise in highly damping environments. We measure the force noise of an 81+/-9 nm radius Ag(2)Ga nanowire cantilever in water at 6+/-3 fN/square root(Hz). PMID:20481957

  12. High Sensitivity deflection detection of nanowires

    SciTech Connect

    Sanii, Babak; Ashby, Paul

    2009-10-28

    A critical limitation of nanoelectromechanical systems (NEMS) is the lack of a high-sensitivity position detection mechanism. We introduce a noninterferometric optical approach to determine the position of nanowires with a high sensitivity and bandwidth. Its physical origins and limitations are determined by Mie scattering analysis. This enables a dramatic miniaturization of detectable cantilevers, with attendant reductions to the fundamental minimum force noise in highly damping environments. We measure the force noise of an 81{+-}9??nm radius Ag{sub 2}Ga nanowire cantilever in water at 6{+-}3??fN/{radical}Hz.

  13. SNP Detection in mRNA in Living Cells Using Allele Specific FRET Probes

    PubMed Central

    Dahan, Liya; Huang, Lingyan; Kedmi, Ranit; Behlke, Mark A.; Peer, Dan

    2013-01-01

    Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET) probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP) in murine Hras mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS) bonds, 2′OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes in vivo molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies. PMID:24039756

  14. Evaluation of mtp40 genomic fragment amplification for specific detection of Mycobacterium tuberculosis in clinical specimens.

    PubMed Central

    Herrera, E A; Segovia, M

    1996-01-01

    A PCR assay based on the species-specific mtp40 genomic fragment was developed for the specific detection and identification of Mycobacterium tuberculosis in different uncultured clinical specimens. The aim of the study was to evaluate the clinical applicability of this target DNA in comparison with those of conventional microbiological methods and to compare the results obtained with those obtained after amplification with the IS6110 repetitive element. Discrepant results were interpreted in conjunction with the patients' clinical data, medical histories, and response to therapy. A total of 172 specimens from 162 patients with respiratory symptoms were tested, 101 specimens were obtained from 92 patients clinically suspected of having tuberculosis, and 71 specimens were obtained from 70 patients without known mycobacterial infection. The results of our study suggest that PCR amplification with the mtp40 genomic fragment provides a highly sensitive and specific technique for the detection of M. tuberculosis strains in clinical samples. It allows for the differentiation between M. tuberculosis and other related mycobacteria, including M. bovis, and is more specific than the IS6110 target. For these and other reasons, we propose that the mtp40 assay is a possible alternative for the specific direct detection of M. tuberculosis in clinical laboratories. PMID:8727885

  15. Detecting exosomes specifically: a multiplexed device based on alternating current electrohydrodynamic induced nanoshearing.

    PubMed

    Vaidyanathan, Ramanathan; Naghibosadat, Maedeh; Rauf, Sakandar; Korbie, Darren; Carrascosa, Laura G; Shiddiky, Muhammad J A; Trau, Matt

    2014-11-18

    Exosomes show promise as noninvasive biomarkers for cancer, but their effective capture and specific detection is a significant challenge. Herein, we report a multiplexed microfluidic device for highly specific capture and detection of multiple exosome targets using a tunable alternating current electrohydrodynamic (ac-EHD) methodology, referred to as nanoshearing. In our system, electrical body forces generated by ac-EHD act within nanometers of an electrode surface (i.e., within the electrical layer) to generate nanoscaled fluid flow that enhances the specificity of capture and also reduce nonspecific adsorption of weakly bound molecules from the electrode surface. This approach demonstrates the analysis of exosomes derived from cells expressing human epidermal growth factor receptor 2 (HER2) and prostate specific antigen (PSA), and is also capable of specifically isolating exosomes from breast cancer patient samples. The device also exhibited a 3-fold enhancement in detection sensitivity in comparison to hydrodynamic flow based assays (LOD 2760 exosomes/μL for ac-EHD vs LOD 8300 exosomes/μL for hydrodynamic flow; (n = 3)). We propose this approach can potentially have relevance as a simple and rapid quantification tool to analyze exosome targets in biological applications. PMID:25324037

  16. Evaluation of Luminex xTAG Gastrointestinal Pathogen Analyte-Specific Reagents for High-Throughput, Simultaneous Detection of Bacteria, Viruses, and Parasites of Clinical and Public Health Importance

    PubMed Central

    Navidad, Jose F.; Griswold, David J.; Gradus, M. Stephen

    2013-01-01

    Acute diarrheal disease (ADD) can be caused by a range of pathogens, including bacteria, viruses, and parasites. Conventional diagnostic methods, such as culture, microscopy, biochemical assays, and enzyme-linked immunosorbent assays (ELISA), are laborious and time-consuming and lack sensitivity. Combined, the array of tests performed on a single specimen can increase the turnaround time (TAT) significantly. We validated a 19plex laboratory-developed gastrointestinal pathogen panel (GPP) using Luminex xTAG analyte-specific reagents (ASRs) to simultaneously screen directly in fecal specimens for diarrhea-causing pathogens, including bacteria (Campylobacter jejuni, Salmonella spp., Shigella spp., enterotoxigenic Escherichia coli [ETEC], Shiga toxin-producing E. coli [STEC], E. coli O157:H7, Vibrio cholerae, Yersinia enterocolitica, and toxigenic Clostridium difficile), parasites (Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica), and viruses (norovirus GI and GII, adenovirus 40/41, and rotavirus A). Performance characteristics of GPP ASRs were determined using 48 reference isolates and 254 clinical specimens. Stool specimens from individuals with diarrhea were tested for pathogens using conventional and molecular methods. Using the predictive methods as standards, the sensitivities of the GPP ASRs were 100% for adenovirus 40/41, norovirus, rotavirus A, Vibrio cholerae, Yersinia enterocolitica, Entamoeba histolytica, Cryptosporidium spp., and E. coli O157:H7; 95% for Giardia lamblia; 94% for ETEC and STEC; 93% for Shigella spp.; 92% for Salmonella spp.; 91% for C. difficile A/B toxins; and 90% for Campylobacter jejuni. The overall comparative performance of the GPP ASRs with conventional methods in clinical samples was 94.5% (range, 90% to 97%), with 99% (99.0% to 99.9%) specificity. Implementation of the GPP ASRs enables our public health laboratory to offer highly sensitive and specific screening and identification of the major ADD-causing pathogens

  17. Evaluation of Luminex xTAG gastrointestinal pathogen analyte-specific reagents for high-throughput, simultaneous detection of bacteria, viruses, and parasites of clinical and public health importance.

    PubMed

    Navidad, Jose F; Griswold, David J; Gradus, M Stephen; Bhattacharyya, Sanjib

    2013-09-01

    Acute diarrheal disease (ADD) can be caused by a range of pathogens, including bacteria, viruses, and parasites. Conventional diagnostic methods, such as culture, microscopy, biochemical assays, and enzyme-linked immunosorbent assays (ELISA), are laborious and time-consuming and lack sensitivity. Combined, the array of tests performed on a single specimen can increase the turnaround time (TAT) significantly. We validated a 19plex laboratory-developed gastrointestinal pathogen panel (GPP) using Luminex xTAG analyte-specific reagents (ASRs) to simultaneously screen directly in fecal specimens for diarrhea-causing pathogens, including bacteria (Campylobacter jejuni, Salmonella spp., Shigella spp., enterotoxigenic Escherichia coli [ETEC], Shiga toxin-producing E. coli [STEC], E. coli O157:H7, Vibrio cholerae, Yersinia enterocolitica, and toxigenic Clostridium difficile), parasites (Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica), and viruses (norovirus GI and GII, adenovirus 40/41, and rotavirus A). Performance characteristics of GPP ASRs were determined using 48 reference isolates and 254 clinical specimens. Stool specimens from individuals with diarrhea were tested for pathogens using conventional and molecular methods. Using the predictive methods as standards, the sensitivities of the GPP ASRs were 100% for adenovirus 40/41, norovirus, rotavirus A, Vibrio cholerae, Yersinia enterocolitica, Entamoeba histolytica, Cryptosporidium spp., and E. coli O157:H7; 95% for Giardia lamblia; 94% for ETEC and STEC; 93% for Shigella spp.; 92% for Salmonella spp.; 91% for C. difficile A/B toxins; and 90% for Campylobacter jejuni. The overall comparative performance of the GPP ASRs with conventional methods in clinical samples was 94.5% (range, 90% to 97%), with 99% (99.0% to 99.9%) specificity. Implementation of the GPP ASRs enables our public health laboratory to offer highly sensitive and specific screening and identification of the major ADD-causing pathogens

  18. Aeromonas detection and characterization using genus-specific PCR and single-strand conformation polymorphism (SSCP).

    PubMed

    Delamare, Ana Paula Longaray; Lucena, Roberto Francisco; Thomazi, Guilherme; Ferrarini, Shana; Zacaria, Jucimar; Echeverrigaray, Sergio

    2012-10-01

    Based on sequence alignment, oligonucleotide primers targeting the Aeromonas extracellular lipase gene were developed for PCR detection of member of the genus. A pair of primers designed for conserved regions of the gene amplified a 276 bp sequence in all Aeromonas species and tested strains, but did not have a positive result with other Gram-positive and Gram-negative bacteria, showing high specificity and sensitivity. Selective enrichment in alkaline peptone water, followed by centrifugation, and direct usage of cells suspension as template, detected initial populations of 10 c.f.u. ml⁻¹. Single-strand conformation polymorphism analysis of the PCR products allowed the characterization of Aeromonas strains with a high discriminatory power (Simpson's index = 0.988). The method presented here provides a useful tool for the rapid detection of Aeromonas and the characterization of Aeromonas isolates. PMID:22806741

  19. Direct molecule-specific glucose detection by Raman spectroscopy based on photonic crystal fiber.

    PubMed

    Yang, Xuan; Zhang, Alissa Y; Wheeler, Damon A; Bond, Tiziana C; Gu, Claire; Li, Yat

    2012-01-01

    This paper reports the first step toward the development of a glucose biosensor based on Raman spectroscopy and a photonic crystal fiber (PCF) probe. Historically, it has been very challenging to detect glucose directly by Raman spectroscopy due to its inherently small Raman scattering cross-section. In this work, we report the first quantitative glucose Raman detection in the physiological concentration range (0-25 mM) with a low laser power (2 mW), a short integration time (30 s), and an extremely small sampling volume (~50 nL) using the highly sensitive liquid-filled PCF probe. As a proof of concept, we also demonstrate the molecular specificity of this technique in the presence of a competing sugar, such as fructose. High sensitivity, flexibility, reproducibility, low cost, small sampling volume, and in situ remote sensing capability make PCF a very powerful platform for potential glucose detection based on Raman spectroscopy. PMID:22120042

  20. Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule.

    PubMed

    Yang, Litao; Guo, Jinchao; Pan, Aihu; Zhang, Haibo; Zhang, Kewei; Wang, Zhengming; Zhang, Dabing

    2007-01-10

    With the development of genetically modified organism (GMO) detection techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection, and real-time PCR is the most effective and important method for GMO quantification. An event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity. This study establishes the event-specific detection methods for TC1507 and CBH351 maizes. In addition, the event-specific TaqMan real-time PCR detection methods for another seven GM maize events (Bt11, Bt176, GA21, MON810, MON863, NK603, and T25) were systematically optimized and developed. In these PCR assays, the fluorescent quencher, TAMRA, was dyed on the T-base of the probe at the internal position to improve the intensity of the fluorescent signal. To overcome the difficulties in obtaining the certified reference materials of these GM maizes, one novel standard reference molecule containing all nine specific integration junction sequences of these GM maizes and the maize endogenous reference gene, zSSIIb, was constructed and used for quantitative analysis. The limits of detection of these methods were 20 copies for these different GM maizes, the limits of quantitation were about 20 copies, and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template. Furthermore, nine groups of the mixed maize samples of these nine GM maize events were quantitatively analyzed to evaluate the accuracy and precision. The accuracy expressed as bias varied from 0.67 to 28.00% for the nine tested groups of GM maize samples, and the precision expressed as relative standard deviations was from 0.83 to 26.20%. All of these indicated that the established event-specific real-time PCR detection systems and the reference molecule in this study are suitable for the identification and quantification of these GM

  1. Cellulose antibody films for highly specific evanescent wave immunosensors

    NASA Astrophysics Data System (ADS)

    Hartmann, Andreas; Bock, Daniel; Jaworek, Thomas; Kaul, Sepp; Schulze, Matthais; Tebbe, H.; Wegner, Gerhard; Seeger, Stefan

    1996-01-01

    For the production of recognition elements for evanescent wave immunosensors optical waveguides have to be coated with ultrathin stable antibody films. In the present work non amphiphilic alkylated cellulose and copolyglutamate films are tested as monolayer matrices for the antibody immobilization using the Langmuir-Blodgett technique. These films are transferred onto optical waveguides and serve as excellent matrices for the immobilization of antibodies in high density and specificity. In addition to the multi-step immobilization of immunoglobulin G(IgG) on photochemically crosslinked and oxidized polymer films, the direct one-step transfer of mixed antibody-polymer films is performed. Both planar waveguides and optical fibers are suitable substrates for the immobilization. The activity and specificity of immobilized antibodies is controlled by the enzyme-linked immunosorbent assay (ELISA) technique. As a result reduced non-specific interactions between antigens and the substrate surface are observed if cinnamoylbutyether-cellulose is used as the film matrix for the antibody immobilization. Using the evanescent wave senor (EWS) technology immunosensor assays are performed in order to determine both the non-specific adsorption of different coated polymethylmethacrylat (PMMA) fibers and the long-term stability of the antibody films. Specificities of one-step transferred IgG-cellulose films are drastically enhanced compared to IgG-copolyglutamate films. Cellulose IgG films are used in enzymatic sandwich assays using mucine as a clinical relevant antigen that is recognized by the antibodies BM2 and BM7. A mucine calibration measurement is recorded. So far the observed detection limit for mucine is about 8 ng/ml.

  2. HIGH ENERGY POLARIZATION OF BLAZARS: DETECTION PROSPECTS

    SciTech Connect

    Chakraborty, N.; Pavlidou, V.; Fields, B. D.

    2015-01-01

    Emission from blazar jets in the ultraviolet, optical, and infrared is polarized. If these low-energy photons were inverse-Compton scattered, the upscattered high-energy photons retain a fraction of the polarization. Current and future X-ray and gamma-ray polarimeters such as INTEGRAL-SPI, PoGOLITE, X-Calibur, Gamma-Ray Burst Polarimeter, GEMS-like missions, ASTRO-H, and POLARIX have the potential to discover polarized X-rays and gamma-rays from blazar jets for the first time. Detection of such polarization will open a qualitatively new window into high-energy blazar emission; actual measurements of polarization degree and angle will quantitatively test theories of jet emission mechanisms. We examine the detection prospects of blazars by these polarimetry missions using examples of 3C 279, PKS 1510-089, and 3C 454.3, bright sources with relatively high degrees of low-energy polarization. We conclude that while balloon polarimeters will be challenged to detect blazars within reasonable observational times (with X-Calibur offering the most promising prospects), space-based missions should detect the brightest blazars for polarization fractions down to a few percent. Typical flaring activity of blazars could boost the overall number of polarimetric detections by nearly a factor of five to six purely accounting for flux increase of the brightest of the comprehensive, all-sky, Fermi-LAT blazar distribution. The instantaneous increase in the number of detections is approximately a factor of two, assuming a duty cycle of 20% for every source. The detectability of particular blazars may be reduced if variations in the flux and polarization fraction are anticorrelated. Simultaneous use of variability and polarization trends could guide the selection of blazars for high-energy polarimetric observations.

  3. High Energy Polarization of Blazars: Detection Prospects

    NASA Astrophysics Data System (ADS)

    Chakraborty, N.; Pavlidou, V.; Fields, B. D.

    2015-01-01

    Emission from blazar jets in the ultraviolet, optical, and infrared is polarized. If these low-energy photons were inverse-Compton scattered, the upscattered high-energy photons retain a fraction of the polarization. Current and future X-ray and gamma-ray polarimeters such as INTEGRAL-SPI, PoGOLITE, X-Calibur, Gamma-Ray Burst Polarimeter, GEMS-like missions, ASTRO-H, and POLARIX have the potential to discover polarized X-rays and gamma-rays from blazar jets for the first time. Detection of such polarization will open a qualitatively new window into high-energy blazar emission; actual measurements of polarization degree and angle will quantitatively test theories of jet emission mechanisms. We examine the detection prospects of blazars by these polarimetry missions using examples of 3C 279, PKS 1510-089, and 3C 454.3, bright sources with relatively high degrees of low-energy polarization. We conclude that while balloon polarimeters will be challenged to detect blazars within reasonable observational times (with X-Calibur offering the most promising prospects), space-based missions should detect the brightest blazars for polarization fractions down to a few percent. Typical flaring activity of blazars could boost the overall number of polarimetric detections by nearly a factor of five to six purely accounting for flux increase of the brightest of the comprehensive, all-sky, Fermi-LAT blazar distribution. The instantaneous increase in the number of detections is approximately a factor of two, assuming a duty cycle of 20% for every source. The detectability of particular blazars may be reduced if variations in the flux and polarization fraction are anticorrelated. Simultaneous use of variability and polarization trends could guide the selection of blazars for high-energy polarimetric observations.

  4. Methods for detection, identification and specification of listerias

    DOEpatents

    Bochner, Barry

    1992-01-01

    The present invention relates generally to differential carbon source metabolism in the genus Listeria, metabolic, biochemical, immunological and genetic procedures to measure said differential carbon source metabolism and the use of these produces to detect, isolate and/or distinguish species of the genus Listeria as well as detect, isolate and/or distinguish strains of species of Listeria. The present invention also contemplates test kits and enrichment media to facilitate these procedures.

  5. Probe-free allele-specific copy number detection and analysis of tumors.

    PubMed

    Zhu, Ailin; Guan, Xiaowei; Gu, Xinbin; Xie, Guiqin

    2016-03-15

    Cancer development and progression frequently involve nucleotide mutations as well as amplifications and deletions of genomic segments. Quantification of allele-specific copy number is an important step in characterizing tumor genomes for precision medicine. Despite advances in approaches to high-throughput genomic DNA analysis, inexpensive and simple methods for analyzing complex nucleotide and copy number variants are still needed. Real-time polymerase chain reaction (PCR) methods for discovering and genotyping single nucleotide polymorphisms are becoming increasingly important in genetic analysis. In this study, we describe a simple, single-tube, probe-free method that combines SYBR Green I-based quantitative real-time PCR and quantitative melting curve analysis both to detect specific nucleotide variants and to quantify allele-specific copy number variants of tumors. The approach is based on the quantification of the targets of interest and the relative abundance of two alleles in a single tube. The specificity, sensitivity, and utility of the assay were demonstrated in detecting allele-specific copy number changes critical for carcinogenesis and therapeutic intervention. Our approach would be useful for allele-specific copy number analysis or precise genotyping. PMID:26743720

  6. Specific primers for the detection of freshwater alphaproteobacterial magnetotactic cocci.

    PubMed

    Lin, Wei; Pan, Yongxin

    2009-12-01

    Freshwater magnetotactic cocci within Alphaproteobacteria are of ecological interest due to their ubiquitous distribution in aquatic environments as well as their potential roles in iron cycling and the bulk magnetism of sediment. To effectively investigate the diversity and distribution of these cocci, specific primers (FMTCf and FMTCr) were developed. Their specificity, applicability, and effectiveness were then evaluated theoretically and empirically. PMID:20112228

  7. Rapid, sensitive, and specific detection of Clostridium tetani by loop-mediated isothermal amplification assay.

    PubMed

    Jiang, Dongneng; Pu, Xiaoyun; Wu, Jiehong; Li, Meng; Liu, Ping

    2013-01-01

    Tetanus is a specific infectious disease, which is often associated with catastrophic events such as earthquakes, traumas, and war wounds. The obligate anaerobe Clostridium tetani is the pathogen that causes tetanus. Once the infection of tetanus progresses to an advanced stage within the wounds of limbs, the rates of amputation and mortality increase manifold. Therefore, it is necessary to devise a rapid and sensitive point-of-care detection method for C. tetani so as to ensure an early diagnosis and clinical treatment of tetanus. In this study, we developed a detection method for C. tetani using loop-mediated isothermal amplification (LAMP) assay, wherein the C. tetani tetanus toxin gene was used as the target gene. The method was highly specific and sensitive, with a detection limit of 10 colony forming units (CFU)/ml, and allowed quantitative analysis. While detecting C. tetani in clinical samples, it was found that the LAMP results completely agreed with those of the traditional API 20A anaerobic bacteria identification test. As compared with the traditional API test and PCR assay, LAMP detection of C. tetani is simple and rapid, and the results can be identified through naked-eye observation. Therefore, it is an ideal and rapid point-of-care testing method for tetanus. PMID:23314360

  8. Procedure for detecting underground utilities with specific shape

    NASA Astrophysics Data System (ADS)

    Ristic, Aleksandar; Vrtunski, Milan; Govedarica, Miro; Bugarinovic, Zeljko

    2016-04-01

    Nowadays GPR technology is acknowledged as a reliable, fast, non-destructive remote sensing technology whose area of applications is wider every day. One of its most common applications is underground utility detection. Not only it is possible to detect the utility in the field, but using certain algorithms utilities which haven't been detected in the field can be detected in radargrams. There is a number of procedures for automated detection of utility in the radargrams. Further, there are procedures that can estimate certain parameters such as propagation velocity, diameter or even characteristics of the material. However, the majority of these procedures is designed to detect cylindrical shape utilities, which, in a radargram, are represented with hyperbolic reflection. According to geometry of hyperbola, utility parameters can be estimated. In this paper we present a procedure that is designed to estimate characteristics of non-cylindrical utilities. It is worth mentioning that these utilities are not so rare. Some underground tanks and sewage collectors are among them. Heat line is consisted of two insulated pipes of the same diameter, often placed in a concrete channel and covered with plates made from reinforced concrete. Therefore, it can be considered as non-cylindrical utility and such structure has characteristic signature in a radargram. The main idea of the proposed procedure is to detect this signature, and then, based on standardized parameters for the heat lines, to estimate the diameter of the pipes. The proposed procedure is based on artificial neural network. As a training set we made a number of radargrams collected on different locations which contain heat lines of various dimensions. Pipe diameters were in a range from 65 to 250 mm. 400MHz antenna was used since the depth hasn't exceeded 2m. After the network is trained it is validated using radargrams that haven't been used in the training set. Further tests were done with radargrams that

  9. Identification and characterization of species-specific nanobodies for the detection of Listeria monocytogenes in milk.

    PubMed

    Tu, Zhui; Chen, Qi; Li, Yanping; Xiong, Yonghua; Xu, Yang; Hu, Na; Tao, Yong

    2016-01-15

    Listeria monocytogenes (LM), one of the eight species belonging to the genus Listeria, is pathogenic for both humans and animals. In this study, two novel LM-specific clones, designated L5-78 and L5-79, were isolated from a phage display antibody library that was derived from the variable domain of heavy-chain antibodies (VHHs) of non-immunized alpaca. These two clones were expressed, purified, and characterized. Results showed that both isolated VHHs recognize three serotypes (1/2a, 1/2b, and 4b), which are responsible for more than 95% of documented human listeriosis cases. The recombinant VHHs possess high thermal stability, pH tolerance, and urea resistance. A sandwich enzyme-linked immunosorbent assay (ELISA) based on the VHH clone L5-79 and a monoclonal antibody was developed to detect LM in pasteurized milk, with a detection limit of 1 × 10(4) colony-forming units (CFU)/ml. These findings indicated that the species-specific VHHs could be directly isolated from the non-immunized library with a properly designed panning strategy and VHH could be a new source for possible diagnosis/detection of foodborne pathogens in food because it was shown to be highly specific and stable. PMID:26456330

  10. Specific NIST projects in support of the NIJ Concealed Weapon Detection and Imaging Program

    NASA Astrophysics Data System (ADS)

    Paulter, Nicholas G.

    1998-12-01

    The Electricity Division of the National Institute of Standards and Technology is developing revised performance standards for hand-held (HH) and walk-through (WT) metal weapon detectors, test procedures and systems for these detectors, and a detection/imaging system for finding concealed weapons. The revised standards will replace the existing National Institute of Justice (NIJ) standards for HH and WT devices and will include detection performance specifications as well as system specifications (environmental conditions, mechanical strength and safety, response reproducibility and repeatability, quality assurance, test reporting, etc.). These system requirements were obtained from the Law Enforcement and corrections Technology Advisory Council, an advisory council for the NIJ. Reproducible and repeatable test procedures and appropriate measurement systems will be developed for evaluating HH and WT detection performance. A guide to the technology and application of non- eddy-current-based detection/imaging methods (such as acoustic, passive millimeter-wave and microwave, active millimeter-wave and terahertz-wave, x-ray, etc.) Will be developed. The Electricity Division is also researching the development of a high- frequency/high-speed (300 GH to 1 THz) pulse-illuminated, stand- off, video-rate, concealed weapons/contraband imaging system.

  11. Sandwich fluorimetric method for specific detection of Staphylococcus aureus based on antibiotic-affinity strategy.

    PubMed

    Kong, Weijun; Xiong, Jie; Yue, Huan; Fu, Zhifeng

    2015-10-01

    A novel antibiotic-affinity strategy was designed for fluorimetric detection of pathogenic bacteria based on the strong affinity of antibiotic agent to the cell wall of bacteria. In this proof-of-concept work, vancocin, a glycopeptide antibiotic for Gram-positive bacteria, was used as a molecular recognition agent to anchor Staphylococcus aureus (S. aureus) cell. To improve the specificity of this method for S. aureus detection, IgG was adopted as the second recognition agent utilizing the binding between Fc region of IgG and S. aureus protein A in the cell wall, to form a sandwich complex. By using fluorescein isothiocyanate as the signal probe, S. aureus whole cells could be directly assayed within a linear range of 1.0 × 10(3)-1.0 × 10(9) CFU mL(-1) with a detection limit of 2.9 × 10(2) CFU mL(-1). The whole assay process could be completed within 130 min when a ready-for-use microplate was adopted. This proposed strategy for pathogenic bacteria detection possessed some attractive characteristics such as high sensitivity, wide linear range, simple manipulation, short assay time, and low cost. Furthermore, this sandwich mode also showed ideal specificity because vancocin and IgG bound with S. aureus at two distinct sites. It opened up a new pathway for high-throughput screening of pathogenic bacteria in medical diagnosis, food safety, bioterrorism defense, and drug discovery. PMID:26352835

  12. Detection of artifacts from high energy bursts in neonatal EEG.

    PubMed

    Bhattacharyya, Sourya; Biswas, Arunava; Mukherjee, Jayanta; Majumdar, Arun Kumar; Majumdar, Bandana; Mukherjee, Suchandra; Singh, Arun Kumar

    2013-11-01

    Detection of non-cerebral activities or artifacts, intermixed within the background EEG, is essential to discard them from subsequent pattern analysis. The problem is much harder in neonatal EEG, where the background EEG contains spikes, waves, and rapid fluctuations in amplitude and frequency. Existing artifact detection methods are mostly limited to detect only a subset of artifacts such as ocular, muscle or power line artifacts. Few methods integrate different modules, each for detection of one specific category of artifact. Furthermore, most of the reference approaches are implemented and tested on adult EEG recordings. Direct application of those methods on neonatal EEG causes performance deterioration, due to greater pattern variation and inherent complexity. A method for detection of a wide range of artifact categories in neonatal EEG is thus required. At the same time, the method should be specific enough to preserve the background EEG information. The current study describes a feature based classification approach to detect both repetitive (generated from ECG, EMG, pulse, respiration, etc.) and transient (generated from eye blinking, eye movement, patient movement, etc.) artifacts. It focuses on artifact detection within high energy burst patterns, instead of detecting artifacts within the complete background EEG with wide pattern variation. The objective is to find true burst patterns, which can later be used to identify the Burst-Suppression (BS) pattern, which is commonly observed during newborn seizure. Such selective artifact detection is proven to be more sensitive to artifacts and specific to bursts, compared to the existing artifact detection approaches applied on the complete background EEG. Several time domain, frequency domain, statistical features, and features generated by wavelet decomposition are analyzed to model the proposed bi-classification between burst and artifact segments. A feature selection method is also applied to select the

  13. Method for detecting pathogens attached to specific antibodies

    DOEpatents

    Miles, Robin R.; Venkateswaran, Kodumudi S.; Fuller, Christopher K.

    2005-01-25

    The use of impedance measurements to detect the presence of pathogens attached to antibody-coated beads. In a fluidic device antibodies are immobilized on a surface of a patterned interdigitated electrode. Pathogens in a sample fluid streaming past the electrode attach to the immobilized antibodies, which produces a change in impedance between two adjacent electrodes, which impedance change is measured and used to detect the presence of a pathogen. To amplify the signal, beads coated with antibodies are introduced and the beads would stick to the pathogen causing a greater change in impedance between the two adjacent electrodes.

  14. Detection of the specific binding on protein microarrays by oblique-incidence reflectivity difference method

    NASA Astrophysics Data System (ADS)

    Lu, Heng; Wen, Juan; Wang, Xu; Yuan, Kun; Li, Wei; Lu, Huibin; Zhou, Yueliang; Jin, Kuijuan; Ruan, Kangcheng; Yang, Guozhen

    2010-09-01

    The specific binding between Cy5-labeled goat anti-mouse Immunoglobulin G (IgG) and mouse IgG with a concentration range from 625 to 104 µg ml - 1 has been detected successfully by the oblique-incidence reflectivity difference (OI-RD) method in each procedure of microarray fabrication. The experimental data prove that the OI-RD method can be employed not only to distinguish the different concentrations in label-free fashion but also to detect the antibody-antigen capture. In addition, the differential treatment of the OI-RD signals can decrease the negative influences of glass slide as the microarray upholder. Therefore the OI-RD technique has promising applications for the label-free and high-throughput detection of protein microarrays.

  15. Modulation of glycan detection on specific glycoproteins by lectin multimerization

    PubMed Central

    Cao, Zheng; Partyka, Katie; McDonald, Mitchell; Brouhard, Elizabeth; Hincapie, Marina; Brand, Randall E.; Hancock, William S.; Haab, Brian B.

    2013-01-01

    Improved methods for studying glycans could spur significant advances in the understanding and application of glycobiology. The use of affinity reagents such as lectins and glycan-binding antibodies is a valuable complement to methods involving mass spectrometry and chromatography. Many lectins, however, are not useful as analytic tools due to low affinity in vitro. As an approach to increasing lectin avidity to targeted glycans, we tested the use of lectin multimerization. Several biotinylated lectins were linked together through streptavidin interactions. The binding of certain lectins for purified glycoproteins and glycoproteins captured directly out of biological solutions was increased using multimerization, resulting in the detection of lower concentrations of glycoprotein than possible using monomeric detection. The analysis of glycoproteins in plasma samples showed that the level of binding enhancement through multimerization was not equivalent across patient samples. Wheat germ agglutinin (WGA) reactive glycans on fibronectin and thrombospondin-5 were preferentially bound by multimers in pancreatic cancer patient samples relative to control samples, suggesting a cancer-associated change in glycan density that could be detected only through lectin multimerization. This strategy could lead to the more sensitive and informative detection of glycans in biological samples and a broader spectrum of lectins that are useful as analytical reagents. PMID:23286506

  16. Modulation of glycan detection on specific glycoproteins by lectin multimerization.

    PubMed

    Cao, Zheng; Partyka, Katie; McDonald, Mitchell; Brouhard, Elizabeth; Hincapie, Marina; Brand, Randall E; Hancock, William S; Haab, Brian B

    2013-02-01

    Improved methods for studying glycans could spur significant advances in the understanding and application of glycobiology. The use of affinity reagents such as lectins and glycan-binding antibodies is a valuable complement to methods involving mass spectrometry and chromatography. Many lectins, however, are not useful as analytic tools due to low affinity in vitro. As an approach to increasing lectin avidity to targeted glycans, we tested the use of lectin multimerization. Several biotinylated lectins were linked together through streptavidin interactions. The binding of certain lectins for purified glycoproteins and glycoproteins captured directly out of biological solutions was increased using multimerization, resulting in the detection of lower concentrations of glycoprotein than possible using monomeric detection. The analysis of glycoproteins in plasma samples showed that the level of binding enhancement through multimerization was not equivalent across patient samples. Wheat germ agglutinin (WGA) reactive glycans on fibronectin and thrombospondin-5 were preferentially bound by multimers in pancreatic cancer patient samples relative to control samples, suggesting a cancer-associated change in glycan density that could be detected only through lectin multimerization. This strategy could lead to the more sensitive and informative detection of glycans in biological samples and a broader spectrum of lectins that are useful as analytical reagents. PMID:23286506

  17. Simple, specific, sensitive and rapid loop-mediated method for detecting Yersinia enterocolitica.

    PubMed

    Xu, Yun-Ming; Liu, Xi-Lin; Ma, Jing; Li, Yan-Song; Hu, Pan; Zou, De-Ying; Guo, Xing; Chen, Xiao-Feng; Tang, Feng; Liu, Nan-Nan; Wei, Li-Bin; Zhou, Yu; Liu, Zeng-Shan; Ren, Hong-Lin; Lu, Shi-Ying

    2014-05-01

    Yersinia enterocolitica (YE) is a main pathogenic bacterium causing diarrhea and yersiniosis occurs in both developed and developing countries with high incidence. YE in contaminated food is able to survive for a long duration even under cold storage, thereby enhancing the risk of food infection. In this study, a new loop-mediated isothermal amplification (LAMP) method showing the characteristics of simplicity, rapidity, high specificity and sensitivity was established by targeting outL of pathogenic YE. Two inner-primers and outer-primers were designed and LAMP reaction was optimized for Mg2+, betaine, dNTPs and inner primers concentrations, reaction temperature and time. Sensitivity and specificity of the LAMP assay was evaluated using YE genomic DNA and those of 44 different bacteria strains, respectively. Validation of LAMP detection method was by employing meat samples spiked with varying CFU of YE. The optimized LAMP assay was specific, capable of detecting 97 fg of genomic DNA (equivalent to 37 genome copies) of YE (100-fold more sensitive than PCR) and 80 CFU/ml of YE-spiked meat samples based on ethidium bromide stained amplicon bands on agarose gel-electrophoresis and on GelRed fluorescence of the LAMP reaction solution, respectively. This rapid, sensitive and specific LAMP technique should enable application in field inspection of Y. enterocolitica in food. PMID:24974652

  18. Detection of specific antibodies to an antigenic mannoprotein for diagnosis of Penicillium marneffei penicilliosis.

    PubMed

    Cao, L; Chen, D L; Lee, C; Chan, C M; Chan, K M; Vanittanakom, N; Tsang, D N; Yuen, K Y

    1998-10-01

    The disseminated and progressive fungal disease Penicillium marneffei penicilliosis is one of the most common infectious diseases in AIDS patients in Southeast Asia. To diagnose systemic penicilliosis, we developed an enzyme-linked immunosorbent assay (ELISA)-based antibody test with Mp1p, a purified recombinant antigenic mannoprotein of P. marneffei. Evaluation of the test with guinea pig sera against P. marneffei and other pathogenic fungi indicated that this assay was specific for P. marneffei. Clinical evaluation revealed that high levels of specific antibody were detected in two immunocompetent penicilliosis patients. Furthermore, approximately 80% (14 of 17) of the documented penicilliosis patients with human immunodeficiency virus tested positive for the specific antibody. No false-positive results were found for serum samples from 90 healthy blood donors, 20 patients with typhoid fever, and 55 patients with tuberculosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Mp1p antibody can be of significant value as a diagnostic for penicilliosis. PMID:9738061

  19. Aptamer-nanobody based ELASA for specific detection of Acinetobacter baumannii isolates.

    PubMed

    Rasoulinejad, Samaneh; Gargari, Seyed Latif Mousavi

    2016-08-10

    Acinetobacter baumannii has turned into an important threat in nosocomial outbreak infections and multidrug resistance leading to high mortality rates in the 21st century. In recent years its mortality has increased by 15% which in part could be due to lack of a rapid and sensitive diagnostic test. In this work we introduced a new detection test for A. baumannii with two highly specific aptamer and nanobody molecules. High binding affinity DNA oligonucleotide aptamers toward A. baumannii were selected through 12 rounds of whole cell System Evolution of Ligands by EXponential enrichment process (SELEX). The SELEX procedures was monitored by flow cytometry. The dissociation constant and binding efficiency of the selected aptamer Aci49 was 7.547±1:353pM and 47.50%, respectively. A sandwich enzyme linked aptamer sorbent assay (ELASA) was designed with the biotinylated Aci49 aptamer and our previously developed nanobody against biofilm associated protein (Bap). The assay system was optimized with A. baumannii (ATCC 19606) and 47 clinical isolates of A. baumannii were tested. The threshold of detection in sandwich ELASA process was10(3) CFU/ml. The sensitivity of test toward the clinical isolates was 95.47%. Our results reveal that the sandwich ELASA is sensitive and specific enough for the rapid detection of A. baumannii from clinical isolates. PMID:27234880

  20. Robust Detection of Rare Species Using Environmental DNA: The Importance of Primer Specificity

    PubMed Central

    Wilcox, Taylor M.; McKelvey, Kevin S.; Young, Michael K.; Jane, Stephen F.; Lowe, Winsor H.; Whiteley, Andrew R.; Schwartz, Michael K.

    2013-01-01

    Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method’s sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design. PMID:23555689

  1. Robust detection of rare species using environmental DNA: the importance of primer specificity.

    PubMed

    Wilcox, Taylor M; McKelvey, Kevin S; Young, Michael K; Jane, Stephen F; Lowe, Winsor H; Whiteley, Andrew R; Schwartz, Michael K

    2013-01-01

    Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probe-based chemistries may represent a particularly powerful tool because of the method's sensitivity, specificity, and potential to quantify target DNA. However, there has been little work understanding the performance of these assays in the presence of closely related, sympatric taxa. If related species cause any cross-amplification or interference, false positives and negatives may be generated. These errors can be disastrous if false positives lead to overestimate the abundance of an endangered species or if false negatives prevent detection of an invasive species. In this study we test factors that influence the specificity and sensitivity of TaqMan MGB assays using co-occurring, closely related brook trout (Salvelinus fontinalis) and bull trout (S. confluentus) as a case study. We found qPCR to be substantially more sensitive than traditional PCR, with a high probability of detection at concentrations as low as 0.5 target copies/µl. We also found that number and placement of base pair mismatches between the Taqman MGB assay and non-target templates was important to target specificity, and that specificity was most influenced by base pair mismatches in the primers, rather than in the probe. We found that insufficient specificity can result in both false positive and false negative results, particularly in the presence of abundant related species. Our results highlight the utility of qPCR as a highly sensitive eDNA tool, and underscore the importance of careful assay design. PMID:23555689

  2. Determination of total sulfur content via sulfur-specific chemiluminescence detection

    SciTech Connect

    Kubala, S.W.; Campbell, D.N.; DiSanzo, F.P.

    1995-12-31

    A specially designed system, based upon sulfur-specific chemiluminescence detection (SSCD), was developed to permit the determination of total sulfur content in a variety of samples. This type of detection system possesses several advantages such as excellent linearity and selectivity, low minimum detectable levels, and an equimolar response to various sulfur compounds. This paper will focus on the design and application of a sulfur-specific chemiluminescence detection system for use in determining total sulfur content in gasoline.

  3. Detection of single-nucleotide polymorphisms using gold nanoparticles and single-strand-specific nucleases.

    PubMed

    Chen, Yen-Ting; Hsu, Chiao-Ling; Hou, Shao-Yi

    2008-04-15

    The current study reports an assay approach that can detect single-nucleotide polymorphisms (SNPs) and identify the position of the point mutation through a single-strand-specific nuclease reaction and a gold nanoparticle assembly. The assay can be implemented via three steps: a single-strand-specific nuclease reaction that allows the enzyme to truncate the mutant DNA; a purification step that uses capture probe-gold nanoparticles and centrifugation; and a hybridization reaction that induces detector probe-gold nanoparticles, capture probe-gold nanoparticles, and the target DNA to form large DNA-linked three-dimensional aggregates of gold nanoparticles. At high temperature (63 degrees C in the current case), the purple color of the perfect match solution would not change to red, whereas a mismatched solution becomes red as the assembled gold nanoparticles separate. Using melting analysis, the position of the point mutation could be identified. This assay provides a convenient colorimetric detection that enables point mutation identification without the need for expensive mass spectrometry. To our knowledge, this is the first report concerning SNP detection based on a single-strand-specific nuclease reaction and a gold nanoparticle assembly. PMID:18211817

  4. Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations

    PubMed Central

    Zonta, Eleonora; Garlan, Fanny; Pécuchet, Nicolas; Perez-Toralla, Karla; Caen, Ouriel; Milbury, Coren; Didelot, Audrey; Fabre, Elizabeth; Blons, Hélène; Laurent-Puig, Pierre; Taly, Valérie

    2016-01-01

    In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients. PMID:27416070

  5. Specific and Rapid Detection of Mycobacterium tuberculosis Complex in Clinical Samples by Polymerase Chain Reaction

    PubMed Central

    Singh, Anamika; Kashyap, Vijendra Kumar

    2012-01-01

    Background. Tuberculosis, a global health problem and highly prevalent in India, has always been a serious problem with respect to definitive diagnosis. Polymerase chain reaction (PCR) techniques are now widely used for early detection and species differentiation of mycobacteria, but mostly with their own limitations. We aim to detect and differentiate Mycobacterium tuberculosis (Mtb) infections by choosing appropriate target sequences, ideally present in all mycobacterial species (MTB complex) and absent in others. Methods. Amplification of three target sequences from unrelated genes, namely, hsp 65 (165 bp), dnaJ (365 bp), and insertion element IS 6110 (541 bp) by PCR was carried out in clinical samples from suspected cases of tuberculosis/ mycobacterioses and healthy controls. Results. The sensitivity of this method ranged from 73.33% to 84.61%, and the specificity was 80%. The PCR method was significantly better (P = 0.03 and P = 0.009) than both smear and culture methods. Conclusion. Our trimarker-based PCR method could specifically detect M. tuberculosis and MTB complex infection from that of major pathogenic NTM and nonpathogenic mycobacteria. This method, by well distinguishing between MTB complex and NTM, presented a fast and accurate method to detect and diagnose mycobacterial infections more efficiently and could thereby help in better patient management particularly considering the increase in mycobacterial infections due to emergence of NTM over the past decades. PMID:23093958

  6. High-sensitivity detection of TNT

    PubMed Central

    Pushkarsky, Michael B.; Dunayevskiy, Ilya G.; Prasanna, Manu; Tsekoun, Alexei G.; Go, Rowel; Patel, C. Kumar N.

    2006-01-01

    We report high-sensitivity detection of 2,4,6-trinitrotoluene (TNT) by using laser photoacoustic spectroscopy where the laser radiation is obtained from a continuous-wave room temperature high-power quantum cascade laser in an external grating cavity geometry. The external grating cavity quantum cascade laser is continuously tunable over ≈400 nm around 7.3 μm and produces a maximum continuous-wave power of ≈200 mW. The IR spectroscopic signature of TNT is sufficiently different from that of nitroglycerine so that unambiguous detection of TNT without false positives from traces of nitroglycerine is possible. We also report the results of spectroscopy of acetylene in the 7.3-μm region to demonstrate continuous tunability of the IR source. PMID:17164325

  7. Colored thin films for specific metal ion detection.

    PubMed

    Schauer, Caroline L; Chen, Mu-San; Price, Ronald R; Schoen, Paul E; Ligler, Frances S

    2004-08-15

    This paper describes the investigation of chitosan and poly(allylamine) (PAH) for the creation of a multi-film, color-based dipstick for the detection of metal ions in solution. Thin, colored films of chitosan and PAH cross-linked with hexamethylene 1,6-di(aminocarboxysulfonate) (HDACS) are created where color is due to film thickness and optical interference effects. The films are investigated for their ability to selectively detect aqueous metal ions via changes in thickness and/or color. Chitosan-HDACS films were selective for Cr(VI) over all other metal ions tested including Cr(acac)3 and Cr(NO3)3 x 9H2O, and PAH-HDACS films were selective for Cu(II) and Cu(I) salts over all other metal ions tested. The irreversible, selective changes due to metal ion solutions were not caused by varying the pH. Potomac River water was also tested using the two films, with results indicating the presence of Cu(II) in the aqueous sample. PMID:15382871

  8. Detection of a Specific Biomarker for Epstein-Barr Virus Using a Polymer-Based Genosensor

    PubMed Central

    Balvedi, Renata P. A.; Castro, Ana C. H.; Madurro, João M.; Brito-Madurro, Ana G.

    2014-01-01

    This paper describes methodology for direct and indirect detections of a specific oligonucleotide for Epstein-Barr virus (EBV) using electrochemical techniques. The sequence of oligonucleotide probe (EBV1) revealed a high sequence identity (100%) with the EBV genome. For the development of the genosensor, EBV1 was grafted to the platform sensitized with poly(4-aminothiophenol). After that, the hybridization reaction was carried out with the complementary target (EBV2) on the modified electrode surface using ethidium bromide as DNA intercalator. The oxidation peak currents of ethidium bromide increased linearly with the values of the concentration of the complementary sequences in the range from 3.78 to 756 μmol·L−1. In nonstringent experimental conditions, this genosensor can detect 17.32 nmol·L−1 (three independent experiments) of oligonucleotide target, discriminating between complementary and non-complementary oligonucleotides, as well as differentiating one-base mismatch, as required for detection of genetic diseases caused by point mutations. The biosensor also displayed high specificity to the EBV target with elimination of interference from mix (alanine, glucose, uric acid, ascorbic acid, bovine serum albumin (BSA), glutamate and glycine) and good stability (120 days). In addition, it was possible to observe differences between hybridized and non-hybridized surfaces through atomic force microscopy. PMID:24853286

  9. Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

    PubMed

    Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

    2015-07-01

    Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world. PMID:26008649

  10. A Rapid and Specific Method for the Detection of Indole in Complex Biological Samples

    PubMed Central

    Chappell, Cynthia; Gonzales, Christopher; Okhuysen, Pablo

    2015-01-01

    Indole, a bacterial product of tryptophan degradation, has a variety of important applications in the pharmaceutical industry and is a biomarker in biological and clinical specimens. Yet, specific assays to quantitate indole are complex and require expensive equipment and a high level of training. Thus, indole in biological samples is often estimated using the simple and rapid Kovács assay, which nonspecifically detects a variety of commonly occurring indole analogs. We demonstrate here a sensitive, specific, and rapid method for measuring indole in complex biological samples using a specific reaction between unsubstituted indole and hydroxylamine. We compared the hydroxylamine-based indole assay (HIA) to the Kovács assay and confirmed that the two assays are capable of detecting microgram amounts of indole. However, the HIA is specific to indole and does not detect other naturally occurring indole analogs. We further demonstrated the utility of the HIA in measuring indole levels in clinically relevant biological materials, such as fecal samples and bacterial cultures. Mean and median fecal indole concentrations from 53 healthy adults were 2.59 mM and 2.73 mM, respectively, but varied widely (0.30 mM to 6.64 mM) among individuals. We also determined that enterotoxigenic Escherichia coli strain H10407 produces 3.3 ± 0.22 mM indole during a 24-h period in the presence of 5 mM tryptophan. The sensitive and specific HIA should be of value in a variety of settings, such as the evaluation of various clinical samples and the study of indole-producing bacterial species in the gut microbiota. PMID:26386049

  11. Specific and sensitive detection of the conifer pathogen Gremmeniella abietina by nested PCR

    PubMed Central

    Zeng, Qing-Yin; Hansson, Per; Wang, Xiao-Ru

    2005-01-01

    Background Gremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The fungus is widespread and causes severe damage to forest plantations in Europe, North America and Asia. To facilitate early diagnosis and improve measures to control the spread of the disease, rapid, specific and sensitive detection methods for G. abietina in conifer hosts are needed. Results We designed two pairs of specific primers for G. abietina based on the 18S rDNA sequence variation pattern. These primers were validated against a wide range of fungi and 14 potential conifer hosts. Based on these specific primers, two nested PCR systems were developed. The first system employed universal fungal primers to enrich the fungal DNA targets in the first round, followed by a second round selective amplification of the pathogen. The other system employed G. abietina-specific primers in both PCR steps. Both approaches can detect the presence of G. abietina in composite samples with high sensitivity, as little as 7.5 fg G. abietina DNA in the host genomic background. Conclusion The methods described here are rapid and can be applied directly to a wide range of conifer species, without the need for fungal isolation and cultivation. Therefore, it represents a promising alternative to disease inspection in forest nurseries, plantations and quarantine control facilities. PMID:16280082

  12. A Molecular Assay for Sensitive Detection of Pathogen-Specific T-Cells

    PubMed Central

    Kasprowicz, Victoria O.; Mitchell, Jessica E.; Chetty, Shivan; Govender, Pamla; Huang, Kuan-Hsiang Gary; Fletcher, Helen A.; Webster, Daniel P.; Brown, Sebastian; Kasmar, Anne; Millington, Kerry; Day, Cheryl L.; Mkhwanazi, Nompumelelo; McClurg, Cheryl; Chonco, Fundisiwe; Lalvani, Ajit; Walker, Bruce D.; Ndung'u, Thumbi; Klenerman, Paul

    2011-01-01

    Here we describe the development and validation of a highly sensitive assay of antigen-specific IFN-γ production using real time quantitative PCR (qPCR) for two reporters - monokine-induced by IFN-γ (MIG) and the IFN-γ inducible protein-10 (IP10). We developed and validated the assay and applied it to the detection of CMV, HIV and Mycobacterium tuberculosis (MTB) specific responses, in a cohort of HIV co-infected patients. We compared the sensitivity of this assay to that of the ex vivo RD1 (ESAT-6 and CFP-10)-specific IFN-γ Elispot assay. We observed a clear quantitative correlation between the two assays (P<0.001). Our assay proved to be a sensitive assay for the detection of MTB-specific T cells, could be performed on whole blood samples of fingerprick (50 uL) volumes, and was not affected by HIV-mediated immunosuppression. This assay platform is potentially of utility in diagnosis of infection in this and other clinical settings. PMID:21853018

  13. High School Educational Specifications: Facilities Planning Standards. Edition I.

    ERIC Educational Resources Information Center

    Jefferson County School District R-1, Denver, CO.

    The Jefferson County School District (Colorado) has developed a manual of high school specifications for Design Advisory Groups and consultants to use for planning and designing the district's high school facilities. The specifications are provided to help build facilities that best meet the educational needs of the students to be served.…

  14. Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution

    SciTech Connect

    Kreader, C.A.

    1995-04-01

    Because Bacteroides spp. are obligate anaerobes that dominate the human fecal flora, and because some species may live only in the human intestine, these bacteria might be useful to distinguish human from nonhuman sources of fecal pollution. To test this hypothesis, PCR primers specific for 16S rRNA gene sequences of Bacteroides distasonis, B. thetaiotaomicron, and B. vulgatus were designed. Hybridization with species-specific internal probes was used to detect the intended PCR products. Extracts from 66 known Bacteroides strains, representing 10 related species, were used to confirm the specificity of these PCR-hybridization assays. To test for specificity in feces, procedures were developed to prepare DNA of sufficient purity for PCR. Extracts of feces from 9 humans and 70 nonhumans (cats, dogs, cattle, hogs, horses, sheep, goats, and chickens) were each analyzed with and without an internal positive control to verify that PCR amplification was not inhibited by substances in the extract. In addition, serial dilutions from each extract that tested positive were assayed to estimate the relative abundance of target Bacteroides spp. in the sample. Depending on the primer-probe set used, either 78 or 67% of the human fecal extracts tested had high levels of target DNA. On the other hand, only 7 to 11% of the nonhuman extracts tested had similarly high levels of target DNA. An additional 12 to 20% of the nonhuman extracts had levels of target DNA that were 100- to 1,000-fold lower than those found in humans. Although the B. vulgatus probes detected high levels of their target DNA in most of the house pets, similarly high levels of target DNA were found only in a few individuals from other groups of nonhumans. Therefore, the results indicate that these probes can distinguish human from non human feces in many cases. 50 refs., 5 figs., 2 tabs.

  15. [Comparative research into sensitivity and specificity of immune-enzyme analysis with chemiluminescence and colorimetric detection for detecting antigens and antibodies to avian influenza viruses and newcastle disease].

    PubMed

    Vitkova, O N; Kapustina, T P; Mikhailova, V V; Safonov, G A; Vlasova, N N; Belousova, R V

    2015-01-01

    The goal of this work was to demonstrate the results of the development of the enzyme-linked immunosorbent tests with chemiluminescence detection and colorimetric detection of specific viral antigens and antibodies for identifying the avian influenza and the Newcastle disease viruses: high sensitivity and specificity of the immuno- chemiluminescence assay, which are 10-50 times higher than those of the ELISA colorimetric method. The high effectiveness of the results and the automation of the process of laboratory testing (using a luminometer) allow these methods to be recommended for including in primary screening tests for these infectious diseases. PMID:27024917

  16. Application of photostable quantum dots for indirect immunofluorescent detection of specific bacterial serotypes on small marine animals

    NASA Astrophysics Data System (ADS)

    Decho, Alan W.; Beckman, Erin M.; Chandler, G. Thomas; Kawaguchi, Tomohiro

    2008-06-01

    An indirect immunofluorescence approach was developed using semiconductor quantum dot nanocrystals to label and detect a specific bacterial serotype of the bacterial human pathogen Vibrio parahaemolyticus, attached to small marine animals (i.e. benthic harpacticoid copepods), which are suspected pathogen carriers. This photostable labeling method using nanotechnology will potentially allow specific serotypes of other bacterial pathogens to be detected with high sensitivity in a range of systems, and can be easily applied for sensitive detection to other Vibrio species such as Vibrio cholerae.

  17. Group-Specific Multiplex PCR Detection Systems for the Identification of Flying Insect Prey

    PubMed Central

    Sint, Daniela; Niederklapfer, Bettina; Kaufmann, Ruediger; Traugott, Michael

    2014-01-01

    The applicability of species-specific primers to study feeding interactions is restricted to those ecosystems where the targeted prey species occur. Therefore, group-specific primer pairs, targeting higher taxonomic levels, are often desired to investigate interactions in a range of habitats that do not share the same species but the same groups of prey. Such primers are also valuable to study the diet of generalist predators when next generation sequencing approaches cannot be applied beneficially. Moreover, due to the large range of prey consumed by generalists, it is impossible to investigate the breadth of their diet with species-specific primers, even if multiplexing them. However, only few group-specific primers are available to date and important groups of prey such as flying insects have rarely been targeted. Our aim was to fill this gap and develop group-specific primers suitable to detect and identify the DNA of common taxa of flying insects. The primers were combined in two multiplex PCR systems, which allow a time- and cost-effective screening of samples for DNA of the dipteran subsection Calyptratae (including Anthomyiidae, Calliphoridae, Muscidae), other common dipteran families (Phoridae, Syrphidae, Bibionidae, Chironomidae, Sciaridae, Tipulidae), three orders of flying insects (Hymenoptera, Lepidoptera, Plecoptera) and coniferous aphids within the genus Cinara. The two PCR assays were highly specific and sensitive and their suitability to detect prey was confirmed by testing field-collected dietary samples from arthropods and vertebrates. The PCR assays presented here allow targeting prey at higher taxonomic levels such as family or order and therefore improve our ability to assess (trophic) interactions with flying insects in terrestrial and aquatic habitats. PMID:25525799

  18. ULTRASENSITIVE HIGH-TEMPERATURE SELECTIVE GAS DETECTION USING PIEZOELECTRIC MICROCANTILEVERS

    SciTech Connect

    Wan Y. Shih; Tejas Patil; Qiang Zhao; Yi-Shi Chiu; Wei-Heng Shih

    2004-03-05

    We have obtained very promising results in the Phase I study. Specifically, for temperature effects, we have established that piezoelectric cantilever sensors could retain their resonance peak strength at high temperatures, i.e., the Q values of the resonance peaks remained above 10 even when the temperature was very close to the Curie temperature. This confirms that a piezoelectric cantilever sensor can be used as a sensor up to its Curie temperature. Furthermore, we have shown that the mass detection sensitivity remained unchanged at different temperatures. For selective gas detection, we have demonstrated selective NH{sub 3} detection using piezoelectric cantilever sensors coated with mesoporous SiO{sub 2}. For high-temperature sensor materials development, we have achieved highly oriented Sr-doped lead titanate thin films that possessed superior dielectric and ferroelectric properties. Such highly oriented films can be microfabricated into high-performance piezoelectric microcantilever sensors that can be used up to 490 C. We have accomplished the goal of Phase I study in exploring the various aspects of a high-temperature gas sensor. We propose to continue the study in Phase II to develop a sensor that is suitable for high-temperature applications using piezoelectrics with a high Curie temperature and by controlling the effects of temperature. The lead titanate based thin film developed in Phase I is good for applications up to 490 C. In phase II, we will develop lithium niobate thin film based cantilevers for applications up to 1000 C.

  19. High voltage spark carbon fiber detection system

    NASA Technical Reports Server (NTRS)

    Yang, L. C.

    1980-01-01

    The pulse discharge technique was used to determine the length and density of carbon fibers released from fiber composite materials during a fire or aircraft accident. Specifications are given for the system which uses the ability of a carbon fiber to initiate spark discharge across a high voltage biased grid to achieve accurate counting and sizing of fibers. The design of the system was optimized, and prototype hardware proved satisfactory in laboratory and field tests.

  20. Rapid detection of specific polyclonal and monoclonal antibodies against bovine leukemia virus.

    PubMed

    Llames, L; Goyache, J; Domenech, A; de Avila, A; Suarez, G; Gomez-Lucia, E

    1999-10-01

    ELISA and Western blot have been used for detecting specific antibodies or antigens for routine diagnostic laboratory tests and experimental protocols, as well as for screening hybridomas secreting antibodies. Although these techniques are sensitive, some slow growing hybridomas are identified as positive only when they are grown slowly long time. We standardized the dot-ELISA, a more sensitive technique, for the detection of antibodies against BLV. The main advantages of the dot-ELISA described in this study are (a) its sensitivity, detecting hybridomas which would otherwise be considered negative and discarded from the results of indirect ELISA and/or Western blot; and (b) the possibility of economizing reagents using as little as 1 microl of the antigen and 0.5 microl of antibody and conjugate. Different BLV-antigen preparations were bound to nitrocellulose membranes (NC), including cells lysed chemically (LYS) or by sonication (SOC), semi-purified virus (PV), and supernatant from infected cultures, either without treatment (SUP) or sonicated (SOS). The antigen preparations most adequate for detecting monoclonal antibodies against BLV and polyclonal antibodies in cattle sera were undiluted cell lysates (LYS) and semi-purified BLV (PV). When testing bovine sera, the supernatant (SUP) and sonicated supernatant (SOS) antigens gave a high background due to the presence of FCS which reacted with the anti-bovine labeled antibodies. In this study, 59 BLV specific antibody secreting hybridomas were identified using the dot-ELISA, compared to only 20 detected using iELISA, and doubtful reactions due to nonspecific binding to fetal calf serum (FCS) and cellular components were measured. The results of the improved dot-ELISA described may be stored at room temperature for future reference. Results were consistently reproducible in coated nitrocellulose membranes kept at different storage temperatures (-20 degrees C, 4 degrees C, and 25-30 degrees C) 48 h, 1 week and 5

  1. A microarray platform for detecting disease-specific circulating miRNA in human serum.

    PubMed

    Roy, Somenath; Soh, Jun Hui; Ying, Jackie Y

    2016-01-15

    Circulating microRNAs (miRNAs) are emerging as potential blood-based biomarkers for cancer and other critical diseases. To profile the expression levels of these tiny molecules, especially in a point-of-care setting, it is imperative to quantify them directly in complex biological fluids. Herein, we report the development of a microarray platform with carboxyl-polyethylene glycol (PEG) as a functional layer and aminated hairpin nucleic acid molecules as target-specific capture probes (CPs). Due to the anti-fouling effect conferred by the carboxyl-PEG layer, we could directly detect as little as 10fM of miRNA targets in 20µl of unprocessed human serum. In contrast to the conventional miRNA microarrays, our platform does not require RNA extraction, labeling and target amplification, thus significantly reducing both the sample preparation steps as well as the total assay duration. The use of specially designed hairpin CPs entails reliable discrimination of miRNA sequences with high sequence homology. A nanoparticle-based detection technique, with the help of differential interference contrast (DIC) microscopy, offers excellent resolution down to a single molecule. With the capability of detecting disease-specific miRNA targets directly in human serum, our microarray platform has potential applications in rapid, minimally invasive clinical diagnostic assays. PMID:26319167

  2. Low Noise, High Detectivity Photodetectors based on Organic Materials

    NASA Astrophysics Data System (ADS)

    Guo, Fawen

    Organic photodetectors (OPDs) are potentially useful in many applications because of their light weight, flexibility and good form factors. Despite the high detectivities that have been frequently reported for OPDs recently, the application of these OPDs for weak light detection has been rarely demonstrated. In this thesis, low noise, high gain photodetectors based on organic and ZnO nanoparticles were proposed and demonstrated for highly sensitive UV light detection. The nanocomposite photodetector works in a hybrid mode of photodiode and photoconductor with the transition controlled by the UV light illumination. The nanocomposite detector shows two orders of magnitude higher sensitivity than silicon detectors in the UV range, which is the first time an organic, solution-processed detector has been shown to significantly outperform the inorganic photonic devices. In the fullerene-based photodetector, the dark-current has been successfully reduced by a cross-linked TPD (C-TPD) buffer layer. The high detectivity of 3.6 x 1011 cm Hz½ W-1 (Jones) at 370 nm and the wide Linear dynamic range (LDR) of 90 dB, along with a response speed faster than 20 kHz, suggests that the fullerene-based organic photodetectors proposed here can open the way for many potential applications. The ZnO nanoparticles have been introduced into the C-TPD buffer layer of the fullerene-based photodetector to increase the photoconductive gain and reduce the noise current. The peak external quantum efficiency (EQE) value of approximately 400% and the peak specific detectivity of 6.5 x 10 12 Jones at the wavelength of 390 nm, along with the record high LDR of 120 dB, enable the photodetector to be used in wide range of applications such as imaging, communication, and defense. The extremely high sensitivity of the photodetector also makes it particularly attractive for very weak light detection.

  3. Development of a Dipstick Assay for Detection of Leishmania-Specific Canine Antibodies

    PubMed Central

    Schallig, Henk D. F. H.; Cardoso, Luís; Hommers, Marieke; Kroon, Nel; Belling, Guus; Rodrigues, Manuela; Semião-Santos, Saul J.; Vetter, Hans

    2004-01-01

    A dipstick assay, based on Leishmania infantum antigen, for the rapid detection of Leishmania-specific antibodies in canine serum samples was developed and evaluated. After determination of optimal dipstick test conditions, test performance was compared with two existing serological tests, i.e., the direct agglutination test (DAT) and the fast agglutination screening test (FAST). In the present study the dipstick test had a sensitivity of 99.2% and a specificity of 87.9%. The DAT had a sensitivity of 97.7% and a specificity of 95.2%, whereas the FAST had also a sensitivity of 97.7% and a specificity of 93.0%. High degrees of agreement were observed between the dipstick test and DAT (93.7%; κ value, 0.86), between the dipstick test and FAST (91.8%; κ value, 0.82), and between the DAT and FAST (95.2%; κ value, 0.90). The high sensitivity and ease of performance make the dipstick test very suitable for surveillance surveys. PMID:14715752

  4. An Analytic Approximation to Very High Specific Impulse and Specific Power Interplanetary Space Mission Analysis

    NASA Technical Reports Server (NTRS)

    Williams, Craig Hamilton

    1995-01-01

    A simple, analytic approximation is derived to calculate trip time and performance for propulsion systems of very high specific impulse (50,000 to 200,000 seconds) and very high specific power (10 to 1000 kW/kg) for human interplanetary space missions. The approach assumed field-free space, constant thrust/constant specific power, and near straight line (radial) trajectories between the planets. Closed form, one dimensional equations of motion for two-burn rendezvous and four-burn round trip missions are derived as a function of specific impulse, specific power, and propellant mass ratio. The equations are coupled to an optimizing parameter that maximizes performance and minimizes trip time. Data generated for hypothetical one-way and round trip human missions to Jupiter were found to be within 1% and 6% accuracy of integrated solutions respectively, verifying that for these systems, credible analysis does not require computationally intensive numerical techniques.

  5. Detection of Allergen Specific Antibodies From Nasal Secretion of Allergic Rhinitis Patients

    PubMed Central

    Yoon, Moon-Gyeong; Seo, Dae-Hong; Kim, Bong-Sun; Ban, Ga-Young; Ye, Young-Min; Shin, Yoo Seob

    2016-01-01

    Purpose Allergic rhinitis (AR) is a common and increasing disease in which Dermatophagoides (D.) farinae is one of the most common causative allergens. The aims of this study were to confirm the presence of locally produced antibodies to D. farinae in nasal secretions between nasal provocation test (NPT)-positive and -negative groups of AR patients, to evaluate their relationships with the levels of inflammatory mediators, and to determine adaptive and innate immune responses in nasal mucosa. Methods Sixty AR patients sensitive to house dust mites confirmed by skin prick test or serum specific IgE to D. farinae underwent NPT for D. farinae. Nasal packs were placed in both nasal cavities of the patients for 5 minutes to obtain nasal secretions after NPT. The levels of total IgE, specific IgE to D. farinae, eosinophil cationic protein (ECP), and tryptase in nasal secretions were detected by using ImmunoCAP. The levels of specific IgE, IgA, and secretory IgA antibodies to D. farinae in nasal secretions were measured by using ELISA. The levels of IL-8, VEGF, IL-25, and IL-33 were also measured by using ELISA. Results High levels of total IgE, specific IgE, specific IgA, and secretory IgA to D. farinae, as well as inflammatory mediators, such as ECP, IL-8, VEGF and tryptase, were detected in nasal secretions, although the differences were not statistically significant between the NPT-positive and NPT-negative groups. Levels of all immunoglobulins measured in this study significantly correlated with ECP, IL-8, and VEGF (P<0.05), but not with tryptase (P>0.05). IL-33 and IL-25 were also detected, and IL-25 level significantly correlated with IL-8 (r=0.625, P<0.001). Conclusions These findings confirmed the presence of locally produced specific antibodies, including D. farinae-specific IgE and IgA, in nasal secretions collected from D. farinae-sensitive AR patients in both the NPT-positive and NPT-negative groups, and close correlations were noted between antibodies and

  6. A protein multiplex microarray substrate with high sensitivity and specificity

    PubMed Central

    Fici, Dolores A.; McCormick, William; Brown, David W.; Herrmann, John E.; Kumar, Vikram; Awdeh, Zuheir L.

    2010-01-01

    The problems that have been associated with protein multiplex microarray immunoassay substrates and existing technology platforms include: binding, sensitivity, a low signal to noise ratio, target immobilization and the optimal simultaneous detection of diverse protein targets. Current commercial substrates for planar multiplex microarrays rely on protein attachment chemistries that range from covalent attachment to affinity ligand capture, to simple adsorption. In this pilot study, experimental performance parameters for direct monoclonal mouse IgG detection were compared for available two and three dimensional slide surface coatings with a new colloidal nitrocellulose substrate. New technology multiplex microarrays were also developed and evaluated for the detection of pathogen specific antibodies in human serum and the direct detection of enteric viral antigens. Data supports the nitrocellulose colloid as an effective reagent with the capacity to immobilize sufficient diverse protein target quantities for increased specificory signal without compromising authentic protein structure. The nitrocellulose colloid reagent is compatible with the array spotters and scanners routinely used for microarray preparation and processing. More importantly, as an alternate to fluorescence, colorimetric chemistries may be used for specific and sensitive protein target detection. The advantages of the nitrocellulose colloid platform indicate that this technology may be a valuable tool for the further development and expansion of multiplex microarray immunoassays in both the clinical and research laborat environment. PMID:20974147

  7. B cells Using Calcium Signaling for Specific and Rapid Detection of Escherichia coli O157:H7.

    PubMed

    Wang, Ling; Wang, Ronghui; Kong, Byung-Whi; Jin, Sha; Ye, Kaiming; Fang, Weihuan; Li, Yanbin

    2015-01-01

    A rapid and sensitive detection technology is highly desirable for specific detection of E. coli O157:H7, one of the leading bacterial pathogens causing foodborne illness. In this study, we reported the rapid detection of E. coli O157:H7 by using calcium signaling of the B cell upon cellular membrane anchors anti-E. coli O157:H7 IgM. The binding of E. coli O157:H7 to the IgM on B cell surface activates the B cell receptor (BCR)-induced Ca(2+) signaling pathway and results in the release of Ca(2+) within seconds. The elevated intracellular Ca(2+) triggers Fura-2, a fluorescent Ca(2+) indicator, for reporting the presence of pathogens. The Fura-2 is transferred to B cells before detection. The study demonstrated that the developed B cell based biosensor was able to specifically detect E. coli O157:H7 at the low concentration within 10 min in pure culture samples. Finally, the B cell based biosensor was used for the detection of E. coli O157:H7 in ground beef samples. With its short detection time and high sensitivity at the low concentration of the target bacteria, this B cell biosensor shows promise in future application of the high throughput and rapid food detection, biosafety and environmental monitoring. PMID:26034978

  8. B cells Using Calcium Signaling for Specific and Rapid Detection of Escherichia coli O157:H7

    PubMed Central

    Wang, Ling; Wang, Ronghui; Kong, Byung-Whi; Jin, Sha; Ye, Kaiming; Fang, Weihuan; Li, Yanbin

    2015-01-01

    A rapid and sensitive detection technology is highly desirable for specific detection of E. coli O157:H7, one of the leading bacterial pathogens causing foodborne illness. In this study, we reported the rapid detection of E. coli O157:H7 by using calcium signaling of the B cell upon cellular membrane anchors anti-E. coli O157:H7 IgM. The binding of E. coli O157:H7 to the IgM on B cell surface activates the B cell receptor (BCR)-induced Ca2+ signaling pathway and results in the release of Ca2+ within seconds. The elevated intracellular Ca2+ triggers Fura-2, a fluorescent Ca2+ indicator, for reporting the presence of pathogens. The Fura-2 is transferred to B cells before detection. The study demonstrated that the developed B cell based biosensor was able to specifically detect E. coli O157:H7 at the low concentration within 10 min in pure culture samples. Finally, the B cell based biosensor was used for the detection of E. coli O157:H7 in ground beef samples. With its short detection time and high sensitivity at the low concentration of the target bacteria, this B cell biosensor shows promise in future application of the high throughput and rapid food detection, biosafety and environmental monitoring. PMID:26034978

  9. A new specific method to detect cyanide in body fluids, especially whole blood, by fluorimetry.

    PubMed

    Felscher, D; Wulfmeyer, M

    1998-09-01

    This study shows a simple, rapid, and specific method for the quantitative determination of cyanide ion in body fluids, especially blood, by fluorimetry. It is based upon the transformation of cyanide ion into hydrocyanic acid, which then reacts with 2,3-naphthalenedialdehyde and taurine in a self-contained system. The 1-cyano-2-benzoisoindole derivate thus formed is suitable for fluorimetric measurement (lambdaEX = 418 nm; lambdaEM = 460 nm). The fluorescence intensity can be determined by spectrophotometry or by high-performance liquid chromatography (HPLC) with fluorescence detection. The detection limit is 0.002 microg/mL. Linearity was excellent from 0.002 to 1 microg/mL for spectrophotometry and from 0.002 to 5 microg/mL for HPLC with fluorescence detection. The coefficient of variation for repeatability was 8% or less. Thiocyanate and sulfide did not interfere, even at high concentrations (200 microg/mL). The method was applicable to whole blood, so it should be suitable for both clinical and forensic purposes. PMID:9737330

  10. Aptamer-Functionalized Fluorescent Silica Nanoparticles for Highly Sensitive Detection of Leukemia Cells.

    PubMed

    Tan, Juntao; Yang, Nuo; Hu, Zixi; Su, Jing; Zhong, Jianhong; Yang, Yang; Yu, Yating; Zhu, Jianmeng; Xue, Dabin; Huang, Yingying; Lai, Zongqiang; Huang, Yong; Lu, Xiaoling; Zhao, Yongxiang

    2016-12-01

    A simple, highly sensitive method to detect leukemia cells has been developed based on aptamer-modified fluorescent silica nanoparticles (FSNPs). In this strategy, the amine-labeled Sgc8 aptamer was conjugated to carboxyl-modified FSNPs via amide coupling between amino and carboxyl groups. Sensitivity and specificity of Sgc8-FSNPs were assessed using flow cytometry and fluorescence microscopy. These results showed that Sgc8-FSNPs detected leukemia cells with high sensitivity and specificity. Aptamer-modified FSNPs hold promise for sensitive and specific detection of leukemia cells. Changing the aptamer may allow the FSNPs to detect other types of cancer cells. PMID:27299653

  11. Aptamer-Functionalized Fluorescent Silica Nanoparticles for Highly Sensitive Detection of Leukemia Cells

    NASA Astrophysics Data System (ADS)

    Tan, Juntao; Yang, Nuo; Hu, Zixi; Su, Jing; Zhong, Jianhong; Yang, Yang; Yu, Yating; Zhu, Jianmeng; Xue, Dabin; Huang, Yingying; Lai, Zongqiang; Huang, Yong; Lu, Xiaoling; Zhao, Yongxiang

    2016-06-01

    A simple, highly sensitive method to detect leukemia cells has been developed based on aptamer-modified fluorescent silica nanoparticles (FSNPs). In this strategy, the amine-labeled Sgc8 aptamer was conjugated to carboxyl-modified FSNPs via amide coupling between amino and carboxyl groups. Sensitivity and specificity of Sgc8-FSNPs were assessed using flow cytometry and fluorescence microscopy. These results showed that Sgc8-FSNPs detected leukemia cells with high sensitivity and specificity. Aptamer-modified FSNPs hold promise for sensitive and specific detection of leukemia cells. Changing the aptamer may allow the FSNPs to detect other types of cancer cells.

  12. Trajectories for High Specific Impulse High Specific Power Deep Space Exploration

    NASA Technical Reports Server (NTRS)

    Polsgrove, Tara; Adams, Robert B.; Brady, Hugh J. (Technical Monitor)

    2002-01-01

    Flight times and deliverable masses for electric and fusion propulsion systems are difficult to approximate. Numerical integration is required for these continuous thrust systems. Many scientists are not equipped with the tools and expertise to conduct interplanetary and interstellar trajectory analysis for their concepts. Several charts plotting the results of well-known trajectory simulation codes were developed and are contained in this paper. These charts illustrate the dependence of time of flight and payload ratio on jet power, initial mass, specific impulse and specific power. These charts are intended to be a tool by which people in the propulsion community can explore the possibilities of their propulsion system concepts. Trajectories were simulated using the tools VARITOP and IPOST. VARITOP is a well known trajectory optimization code that involves numerical integration based on calculus of variations. IPOST has several methods of trajectory simulation; the one used in this paper is Cowell's method for full integration of the equations of motion. An analytical method derived in the companion paper was also evaluated. The accuracy of this method is discussed in the paper.

  13. Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction

    SciTech Connect

    Day, D.J.; Barany, F.; Speiser, P.W.

    1995-09-01

    Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol that occurs in 1 in 10,000-15,000 births. Affected females are born with ambiguous genitalia, a condition that can be ameliorated by administering dexamethasone to the mother for most of gestation. Prenatal diagnosis is required for accurate treatment of affected females as well as for genetic counseling purposes. Approximately 95% of mutations causing this disorder result from recombinations between the gene encoding the 21-hydroxylase enzyme (CYP21) and a linked, highly homologous pseudogene (CYP21P). Approximately 20% of these mutations are gene deletions, and the remainder are gene conversions that transfer any of nine deleterious mutations from the CYP21P pseudogene to CYP21. We describe a methodology for genetic diagnosis of 21-hydroxylase deficiency that utilizes gene-specific PCR amplification in conjunction with thermostable DNA ligase to discriminate single nucleotide variations in a multiplexed ligation detection assay. The assay has been designed to be used with either fluorescent or radioactive detection of ligation products by electrophoresis on denaturing acrylamide gels and is readily adaptable for use in other disease systems. 30 refs., 5 figs.

  14. Efficient and Specific Detection of Salmonella in Food Samples Using a stn-Based Loop-Mediated Isothermal Amplification Method

    PubMed Central

    Srisawat, Mevaree; Panbangred, Watanalai

    2015-01-01

    The Salmonella enterotoxin (stn) gene exhibits high homology among S. enterica serovars and S. bongori. A set of 6 specific primers targeting the stn gene were designed for detection of Salmonella spp. using the loop-mediated isothermal amplification (LAMP) method. The primers amplified target sequences in all 102 strains of 87 serovars of Salmonella tested and no products were detected in 57 non-Salmonella strains. The detection limit in pure cultures was 5 fg DNA/reaction when amplified at 65°C for 25 min. The LAMP assay could detect Salmonella in artificially contaminated food samples as low as 220 cells/g of food without a preenrichment step. However, the sensitivity was increased 100-fold (~2 cells/g) following 5 hr preenrichment at 35°C. The LAMP technique, with a preenrichment step for 5 and 16 hr, was shown to give 100% specificity with food samples compared to the reference culture method in which 67 out of 90 food samples gave positive results. Different food matrixes did not interfere with LAMP detection which employed a simple boiling method for DNA template preparation. The results indicate that the LAMP method, targeting the stn gene, has great potential for detection of Salmonella in food samples with both high specificity and high sensitivity. PMID:26543859

  15. Development of a high-specific-speed centrifugal compressor

    SciTech Connect

    Rodgers, C.

    1997-07-01

    This paper describes the development of a subscale single-stage centrifugal compressor with a dimensionless specific speed (Ns) of 1.8, originally designed for full-size application as a high volume flow, low pressure ratio, gas booster compressor. The specific stage is noteworthy in that it provides a benchmark representing the performance potential of very high-specific-speed compressors, of which limited information is found in the open literature. Stage and component test performance characteristics are presented together with traverse results at the impeller exit. Traverse test results were compared with recent CFD computational predictions for an exploratory analytical calibration of a very high-specific-speed impeller geometry. The tested subscale (0.583) compressor essentially satisfied design performance expectations with an overall stage efficiency of 74% including, excessive exit casing losses. It was estimated that stage efficiency could be increased to 81% with exit casing losses halved.

  16. Preparation of genosensor for detection of specific DNA sequence of the hepatitis B virus

    NASA Astrophysics Data System (ADS)

    Honorato Castro, Ana C.; França, Erick G.; de Paula, Lucas F.; Soares, Marcia M. C. N.; Goulart, Luiz R.; Madurro, João M.; Brito-Madurro, Ana G.

    2014-09-01

    An electrochemical genosensor was constructed for detection of specific DNA sequence of the hepatitis B virus, based on graphite electrodes modified with poly(4-aminophenol) and incorporating a specific oligonucleotide probe. The modified electrode containing the probe was evaluated by differential pulse voltammetry, before and after incubation with the complementary oligonucleotide target. Detection was performed by monitoring oxidizable DNA bases (direct detection) or using ethidium bromide as indicator of the hybridization process (indirect detection). The device showed a detection limit for the oligonucleotide target of 2.61 nmol L-1. Indirect detection using ethidium bromide was promising in discriminating mismatches, which is a very desirable attribute for detection of disease-related point mutations. In addition, it was possible to observe differences between hybridized and non-hybridized surfaces by atomic force microscopy.

  17. Improvement of Prostate Cancer Diagnosis by Detecting PSA Glycosylation-Specific Changes

    PubMed Central

    Llop, Esther; Ferrer-Batallé, Montserrat; Barrabés, Sílvia; Guerrero, Pedro Enrique; Ramírez, Manel; Saldova, Radka; Rudd, Pauline M.; Aleixandre, Rosa N.; Comet, Josep; de Llorens, Rafael; Peracaula, Rosa

    2016-01-01

    New markers based on PSA isoforms have recently been developed to improve prostate cancer (PCa) diagnosis. However, novel approaches are still required to differentiate aggressive from non-aggressive PCa to improve decision making for patients. PSA glycoforms have been shown to be differentially expressed in PCa. In particular, changes in the extent of core fucosylation and sialylation of PSA N-glycans in PCa patients compared to healthy controls or BPH patients have been reported. The objective of this study was to determine these specific glycan structures in serum PSA to analyze their potential value as markers for discriminating between BPH and PCa of different aggressiveness. In the present work, we have established two methodologies to analyze the core fucosylation and the sialic acid linkage of PSA N-glycans in serum samples from BPH (29) and PCa (44) patients with different degrees of aggressiveness. We detected a significant decrease in the core fucose and an increase in the α2,3-sialic acid percentage of PSA in high-risk PCa that differentiated BPH and low-risk PCa from high-risk PCa patients. In particular, a cut-off value of 0.86 of the PSA core fucose ratio, could distinguish high-risk PCa patients from BPH with 90% sensitivity and 95% specificity, with an AUC of 0.94. In the case of the α2,3-sialic acid percentage of PSA, the cut-off value of 30% discriminated between high-risk PCa and the group of BPH, low-, and intermediate-risk PCa with a sensitivity and specificity of 85.7% and 95.5%, respectively, with an AUC of 0.97. The latter marker exhibited high performance in differentiating between aggressive and non-aggressive PCa and has the potential for translational application in the clinic. PMID:27279911

  18. Improvement of Prostate Cancer Diagnosis by Detecting PSA Glycosylation-Specific Changes.

    PubMed

    Llop, Esther; Ferrer-Batallé, Montserrat; Barrabés, Sílvia; Guerrero, Pedro Enrique; Ramírez, Manel; Saldova, Radka; Rudd, Pauline M; Aleixandre, Rosa N; Comet, Josep; de Llorens, Rafael; Peracaula, Rosa

    2016-01-01

    New markers based on PSA isoforms have recently been developed to improve prostate cancer (PCa) diagnosis. However, novel approaches are still required to differentiate aggressive from non-aggressive PCa to improve decision making for patients. PSA glycoforms have been shown to be differentially expressed in PCa. In particular, changes in the extent of core fucosylation and sialylation of PSA N-glycans in PCa patients compared to healthy controls or BPH patients have been reported. The objective of this study was to determine these specific glycan structures in serum PSA to analyze their potential value as markers for discriminating between BPH and PCa of different aggressiveness. In the present work, we have established two methodologies to analyze the core fucosylation and the sialic acid linkage of PSA N-glycans in serum samples from BPH (29) and PCa (44) patients with different degrees of aggressiveness. We detected a significant decrease in the core fucose and an increase in the α2,3-sialic acid percentage of PSA in high-risk PCa that differentiated BPH and low-risk PCa from high-risk PCa patients. In particular, a cut-off value of 0.86 of the PSA core fucose ratio, could distinguish high-risk PCa patients from BPH with 90% sensitivity and 95% specificity, with an AUC of 0.94. In the case of the α2,3-sialic acid percentage of PSA, the cut-off value of 30% discriminated between high-risk PCa and the group of BPH, low-, and intermediate-risk PCa with a sensitivity and specificity of 85.7% and 95.5%, respectively, with an AUC of 0.97. The latter marker exhibited high performance in differentiating between aggressive and non-aggressive PCa and has the potential for translational application in the clinic. PMID:27279911

  19. Phage-Based Fluorescent Biosensor Prototypes to Specifically Detect Enteric Bacteria Such as E. coli and Salmonella enterica Typhimurium

    PubMed Central

    Vinay, Manon; Franche, Nathalie; Grégori, Gérald; Fantino, Jean-Raphaël; Pouillot, Flavie; Ansaldi, Mireille

    2015-01-01

    Water safety is a major concern for public health and for natural environment preservation. We propose to use bacteriophages to develop biosensor tools able to detect human and animal pathogens present in water. For this purpose, we take advantage of the highly discriminating properties of the bacteriophages, which specifically infect their bacterial hosts. The challenge is to use a fluorescent reporter protein that will be synthesized, and thus detected, only once the specific recognition step between a genetically modified temperate bacteriophage and its bacterial host has occurred. To ensure the accuracy and the execution speed of our system, we developed a test that does not require bacterial growth, since a simple 1-hour infection step is required. To ensure a high sensitivity of our tool and in order to detect up to a single bacterium, fluorescence is measured using a portable flow cytometer, also allowing on-site detection. In this study, we have constructed and characterized several "phagosensor" prototypes using the HK620 bacteriophage and its host Escherichia coli TD2158 and we successfully adapted this method to Salmonella detection. We show that the method is fast, robust and sensitive, allowing the detection of as few as 10 bacteria per ml with no concentration nor enrichment step. Moreover, the test is functional in sea water and allows the detection of alive bacteria. Further development will aim to develop phagosensors adapted on demand to the detection of any human or animal pathogen that may be present in water. PMID:26186207

  20. Phage-Based Fluorescent Biosensor Prototypes to Specifically Detect Enteric Bacteria Such as E. coli and Salmonella enterica Typhimurium.

    PubMed

    Vinay, Manon; Franche, Nathalie; Grégori, Gérald; Fantino, Jean-Raphaël; Pouillot, Flavie; Ansaldi, Mireille

    2015-01-01

    Water safety is a major concern for public health and for natural environment preservation. We propose to use bacteriophages to develop biosensor tools able to detect human and animal pathogens present in water. For this purpose, we take advantage of the highly discriminating properties of the bacteriophages, which specifically infect their bacterial hosts. The challenge is to use a fluorescent reporter protein that will be synthesized, and thus detected, only once the specific recognition step between a genetically modified temperate bacteriophage and its bacterial host has occurred. To ensure the accuracy and the execution speed of our system, we developed a test that does not require bacterial growth, since a simple 1-hour infection step is required. To ensure a high sensitivity of our tool and in order to detect up to a single bacterium, fluorescence is measured using a portable flow cytometer, also allowing on-site detection. In this study, we have constructed and characterized several "phagosensor" prototypes using the HK620 bacteriophage and its host Escherichia coli TD2158 and we successfully adapted this method to Salmonella detection. We show that the method is fast, robust and sensitive, allowing the detection of as few as 10 bacteria per ml with no concentration nor enrichment step. Moreover, the test is functional in sea water and allows the detection of alive bacteria. Further development will aim to develop phagosensors adapted on demand to the detection of any human or animal pathogen that may be present in water. PMID:26186207

  1. Rapid and specific influenza virus detection by functionalized magnetic nanoparticles and mass spectrometry

    PubMed Central

    2011-01-01

    Background The timely and accurate diagnosis of specific influenza virus strains is crucial to effective prophylaxis, vaccine preparation and early antiviral therapy. The detection of influenza A viruses is mainly accomplished using polymerase chain reaction (PCR) techniques or antibody-based assays. In conjugation with the immunoassay utilizing monoclonal antibody, mass spectrometry is an alternative to identify proteins derived from a target influenza virus. Taking advantage of the large surface area-to-volume ratio, antibody-conjugated magnetic nanoparticles can act as an effective probe to extract influenza virus for sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and on-bead mass spectrometric analysis. Results Iron oxide magnetic nanoparticles (MNP) were functionalized with H5N2 viral antibodies targeting the hemagglutinin protein and capped with methoxy-terminated ethylene glycol to suppress nonspecific binding. The antibody-conjugated MNPs possessed a high specificity to H5N2 virus without cross-reactivity with recombinant H5N1 viruses. The unambiguous identification of the captured hemagglutinin on magnetic nanoparticles was realized by SDS-PAGE visualization and peptide sequence identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Conclusions The assay combining efficient magnetic separation and MALDI-MS readout offers a rapid and sensitive method for virus screening. Direct on-MNP detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provided high sensitivity (~103 EID50 per mL) and a timely diagnosis within one hour. The magnetic nanoparticles encapsulated with monoclonal antibodies could be used as a specific probe to distinguish different subtypes of influenza. PMID:22088100

  2. Direct, Specific and Rapid Detection of Staphylococcal Proteins and Exotoxins Using a Multiplex Antibody Microarray

    PubMed Central

    Stieber, Bettina; Monecke, Stefan; Müller, Elke; Büchler, Joseph; Ehricht, Ralf

    2015-01-01

    Background S. aureus is a pathogen in humans and animals that harbors a wide variety of virulence factors and resistance genes. This bacterium can cause a wide range of mild to life-threatening diseases. In the latter case, fast diagnostic procedures are important. In routine diagnostic laboratories, several genotypic and phenotypic methods are available to identify S. aureus strains and determine their resistances. However, there is a demand for multiplex routine diagnostic tests to directly detect staphylococcal toxins and proteins. Methods In this study, an antibody microarray based assay was established and validated for the rapid detection of staphylococcal markers and exotoxins. The following targets were included: staphylococcal protein A, penicillin binding protein 2a, alpha- and beta-hemolysins, Panton Valentine leukocidin, toxic shock syndrome toxin, enterotoxins A and B as well as staphylokinase. All were detected simultaneously within a single experiment, starting from a clonal culture on standard media. The detection of bound proteins was performed using a new fluorescence reading device for microarrays. Results 110 reference strains and clinical isolates were analyzed using this assay, with a DNA microarray for genotypic characterization performed in parallel. The results showed a general high concordance of genotypic and phenotypic data. However, genotypic analysis found the hla gene present in all S. aureus isolates but its expression under given conditions depended on the clonal complex affiliation of the actual isolate. Conclusions The multiplex antibody assay described herein allowed a rapid and reliable detection of clinically relevant staphylococcal toxins as well as resistance- and species-specific markers. PMID:26624622

  3. An ultrasensitive electrogenerated chemiluminescence-based immunoassay for specific detection of Zika virus

    PubMed Central

    Acharya, Dhiraj; Bastola, Pradip; Le, Linda; Paul, Amber M.; Fernandez, Estefania; Diamond, Michael S.; Miao, Wujian; Bai, Fengwei

    2016-01-01

    Zika virus (ZIKV) is a globally emerging mosquito-transmitted flavivirus that can cause severe fetal abnormalities, including microcephaly. As such, highly sensitive, specific, and cost-effective diagnostic methods are urgently needed. Here, we report a novel electrogenerated chemiluminescence (ECL)-based immunoassay for ultrasensitive and specific detection of ZIKV in human biological fluids. We loaded polystyrene beads (PSB) with a large number of ECL labels and conjugated them with anti-ZIKV monoclonal antibodies to generate anti-ZIKV-PSBs. These anti-ZIKV-PSBs efficiently captured ZIKV in solution forming ZIKV-anti-ZIKV-PSB complexes, which were subjected to measurement of ECL intensity after further magnetic beads separation. Our results show that the anti-ZIKV-PSBs can capture as little as 1 PFU of ZIKV in 100 μl of saline, human plasma, or human urine. This platform has the potential for development as a cost-effective, rapid and ultrasensitive assay for the detection of ZIKV and possibly other viruses in clinical diagnosis, epidemiologic and vector surveillance, and laboratory research. PMID:27554037

  4. An ultrasensitive electrogenerated chemiluminescence-based immunoassay for specific detection of Zika virus.

    PubMed

    Acharya, Dhiraj; Bastola, Pradip; Le, Linda; Paul, Amber M; Fernandez, Estefania; Diamond, Michael S; Miao, Wujian; Bai, Fengwei

    2016-01-01

    Zika virus (ZIKV) is a globally emerging mosquito-transmitted flavivirus that can cause severe fetal abnormalities, including microcephaly. As such, highly sensitive, specific, and cost-effective diagnostic methods are urgently needed. Here, we report a novel electrogenerated chemiluminescence (ECL)-based immunoassay for ultrasensitive and specific detection of ZIKV in human biological fluids. We loaded polystyrene beads (PSB) with a large number of ECL labels and conjugated them with anti-ZIKV monoclonal antibodies to generate anti-ZIKV-PSBs. These anti-ZIKV-PSBs efficiently captured ZIKV in solution forming ZIKV-anti-ZIKV-PSB complexes, which were subjected to measurement of ECL intensity after further magnetic beads separation. Our results show that the anti-ZIKV-PSBs can capture as little as 1 PFU of ZIKV in 100 μl of saline, human plasma, or human urine. This platform has the potential for development as a cost-effective, rapid and ultrasensitive assay for the detection of ZIKV and possibly other viruses in clinical diagnosis, epidemiologic and vector surveillance, and laboratory research. PMID:27554037

  5. Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

    PubMed Central

    Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta’ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

    2016-01-01

    The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology. PMID:27435636

  6. Development of a Specific Latex Agglutination Test to Detect Antibodies of Enterovirus 71.

    PubMed

    Qin, Bo; Zhang, Jianhua; Xie, Wenhao; Liu, Xuehong; He, Tingting; Chen, Jinkun; Dong, Xuejun

    2015-10-01

    A latex agglutination test (LAT) was developed for the rapid detection of antibodies against the VP1 or VP1 proteins of Enterovirus 71 (EV71). The proteins of interest including prokaryotically expressed VP1 and two strains of anti-VP1 monoclonal antibody (McAb) against EV71 were covalently linked to carboxylated latex using ethyl-dimethyl-amino-propyl carbodiimide (EDC) to prepare sensitized latex beads. LAT was evaluated by an enzyme-linked immunosorbent assay (ELISA) as a reference test. The VP1-LAT showed a sensitivity of 87.0%, specificity of 88.9%, and an agreement ratio of 90.0% in detecting VP1 in 100 serum samples from experimentally infected mice, whereas these values were 86.8, 96.7, and 93.3%, respectively, for 608 clinical human serum samples. The VP1-LAT has advantages over other assays in terms of low cost, rapidity, chemical stability, high sensitivity, repeatability, and specificity. The LAT established in the present study is a rapid and simple test suitable for field monitoring of antibodies against VP1-EV71. PMID:26363276

  7. Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA.

    PubMed

    Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta'ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

    2016-01-01

    The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology. PMID:27435636

  8. Measuring the Electronic Properties of DNA-Specific Schottky Diodes Towards Detecting and Identifying Basidiomycetes DNA

    NASA Astrophysics Data System (ADS)

    Periasamy, Vengadesh; Rizan, Nastaran; Al-Ta’Ii, Hassan Maktuff Jaber; Tan, Yee Shin; Tajuddin, Hairul Annuar; Iwamoto, Mitsumasa

    2016-07-01

    The discovery of semiconducting behavior of deoxyribonucleic acid (DNA) has resulted in a large number of literatures in the study of DNA electronics. Sequence-specific electronic response provides a platform towards understanding charge transfer mechanism and therefore the electronic properties of DNA. It is possible to utilize these characteristic properties to identify/detect DNA. In this current work, we demonstrate a novel method of DNA-based identification of basidiomycetes using current-voltage (I-V) profiles obtained from DNA-specific Schottky barrier diodes. Electronic properties such as ideality factor, barrier height, shunt resistance, series resistance, turn-on voltage, knee-voltage, breakdown voltage and breakdown current were calculated and used to quantify the identification process as compared to morphological and molecular characterization techniques. The use of these techniques is necessary in order to study biodiversity, but sometimes it can be misleading and unreliable and is not sufficiently useful for the identification of fungi genera. Many of these methods have failed when it comes to identification of closely related species of certain genus like Pleurotus. Our electronics profiles, both in the negative and positive bias regions were however found to be highly characteristic according to the base-pair sequences. We believe that this simple, low-cost and practical method could be useful towards identifying and detecting DNA in biotechnology and pathology.

  9. A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

    NASA Astrophysics Data System (ADS)

    Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

    2016-05-01

    The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar

  10. Modular microfluidic system fabricated in thermoplastics for the strain-specific detection of bacterial pathogens.

    PubMed

    Chen, Yi-Wen; Wang, Hong; Hupert, Mateusz; Witek, Makgorzata; Dharmasiri, Udara; Pingle, Maneesh R; Barany, Francis; Soper, Steven A

    2012-09-21

    The recent outbreaks of a lethal E. coli strain in Germany have aroused renewed interest in developing rapid, specific and accurate systems for detecting and characterizing bacterial pathogens in suspected contaminated food and/or water supplies. To address this need, we have designed, fabricated and tested an integrated modular-based microfluidic system and the accompanying assay for the strain-specific identification of bacterial pathogens. The system can carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and detect single nucleotide variations in selected genes to allow for the identification of the bacterial species, even its strain with high specificity. The unique aspect of this fluidic cartridge is its modular format with task-specific modules interconnected to a fluidic motherboard to permit the selection of the target material. In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and packaged into a small footprint instrument (1 ft(3)). The fluidic cartridge was capable of performing cell enrichment, cell lysis, solid-phase extraction (SPE) of genomic DNA, continuous flow (CF) PCR, CF ligase detection reaction (LDR) and universal DNA array readout. The cartridge was comprised of modules situated on a fluidic motherboard; the motherboard was made from polycarbonate, PC, and used for cell lysis, SPE, CF PCR and CF LDR. The modules were task-specific units and performed universal zip-code array readout or affinity enrichment of the target cells with both made from poly(methylmethacrylate), PMMA. Two genes, uidA and sipB/C, were used to discriminate between E. coli and Salmonella, and evaluated as a model system. Results showed that the fluidic

  11. Modular microfluidic system fabricated in thermoplastics for the strain-specific detection of bacterial pathogens†

    PubMed Central

    Chen, Yi-Wen; Wang, Hong; Hupert, Mateusz; Witek, Makgorzata; Dharmasiri, Udara; Pingle, Maneesh R.; Barany, Francis

    2015-01-01

    The recent outbreaks of a lethal E. coli strain in Germany have aroused renewed interest in developing rapid, specific and accurate systems for detecting and characterizing bacterial pathogens in suspected contaminated food and/or water supplies. To address this need, we have designed, fabricated and tested an integrated modular-based microfluidic system and the accompanying assay for the strain-specific identification of bacterial pathogens. The system can carry out the entire molecular processing pipeline in a single disposable fluidic cartridge and detect single nucleotide variations in selected genes to allow for the identification of the bacterial species, even its strain with high specificity. The unique aspect of this fluidic cartridge is its modular format with task-specific modules interconnected to a fluidic motherboard to permit the selection of the target material. In addition, to minimize the amount of finishing steps for assembling the fluidic cartridge, many of the functional components were produced during the polymer molding step used to create the fluidic network. The operation of the cartridge was provided by electronic, mechanical, optical and hydraulic controls located off-chip and packaged into a small footprint instrument (1 ft3). The fluidic cartridge was capable of performing cell enrichment, cell lysis, solid-phase extraction (SPE) of genomic DNA, continuous flow (CF) PCR, CF ligase detection reaction (LDR) and universal DNA array readout. The cartridge was comprised of modules situated on a fluidic motherboard; the motherboard was made from polycarbonate, PC, and used for cell lysis, SPE, CF PCR and CF LDR. The modules were task-specific units and performed universal zip-code array readout or affinity enrichment of the target cells with both made from poly(methylmethacrylate), PMMA. Two genes, uidA and sipB/C, were used to discriminate between E. coli and Salmonella, and evaluated as a model system. Results showed that the fluidic system

  12. Technical specifications manual for the MARK-1 pulsed ionizing radiation detection system. Volume 1

    SciTech Connect

    Lawrence, R.S.; Harker, Y.D.; Jones, J.L.; Hoggan, J.M.

    1993-03-01

    The MARK-1 detection system was developed by the Idaho National Engineering Laboratory for the US Department of Energy Office of Arms Control and Nonproliferation. The completely portable system was designed for the detection and analysis of intense photon emissions from pulsed ionizing radiation sources. This manual presents the technical design specifications for the MARK-1 detection system and was written primarily to assist the support or service technician in the service, calibration, and repair of the system. The manual presents the general detection system theory, the MARK-1 component design specifications, the acquisition and control software, the data processing sequence, and the system calibration procedure. A second manual entitled: Volume 2: Operations Manual for the MARK-1 Pulsed Ionizing Radiation Detection System (USDOE Report WINCO-1108, September 1992) provides a general operational description of the MARK-1 detection system. The Operations Manual was written primarily to assist the field operator in system operations and analysis of the data.

  13. A Simple Method for the Immunocytochemical Detection of Proteins Inside Nuclear Structures That Are Inaccessible to Specific Antibodies

    PubMed Central

    Svistunova, Darya M.; Musinova, Yana R.; Polyakov, Vladimir Yu.

    2012-01-01

    It has been demonstrated elsewhere that a high concentration of an antigen within the nucleolus may prevent its proper recognition by specific antibodies. In this study, the authors found that a short proteinase treatment allowed for the detection of antigens in the nucleoli. The described approach is compatible with the simultaneous observation of proteins fused to fluorescent tags and with preembedding electron microscopy. It appears that the described method can be useful in situations when the proper recognition of antigens by specific antibodies is disturbed by a high density of cellular structures or a high concentration of antigens inside these structures. PMID:22114257

  14. Prenatal detection of congenital high airway obstruction syndrome with encephalocele

    PubMed Central

    Padmanabhan, Laxmi Devi; Nampoothiri, Sheela

    2016-01-01

    Congenital high airway obstruction syndrome (CHAOS) causes secondary morphological changes which can be detected on ultrasound. Here we report a case of congenital high airway obstruction with an occipital encephalocele detected at 23 weeks of gestation. PMID:27081227

  15. Nucleotide sequences specific to Brucella and methods for the detection of Brucella

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.

    2009-02-24

    Nucleotide sequences specific to Brucella that serves as a marker or signature for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  16. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Vitalis, Elizabeth A

    2007-02-06

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  17. Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Vitalis, Elizabeth A

    2009-02-24

    Described herein is the identification of nucleotide sequences specific to Francisella tularensis that serves as a marker or signature for identification of this bacterium. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  18. Nucleotide sequences specific to Yersinia pestis and methods for the detection of Yersinia pestis

    DOEpatents

    McCready, Paula M.; Radnedge, Lyndsay; Andersen, Gary L.; Ott, Linda L.; Slezak, Thomas R.; Kuczmarski, Thomas A.; Motin, Vladinir L.

    2009-02-24

    Nucleotide sequences specific to Yersinia pestis that serve as markers or signatures for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

  19. A Specific Qualitative Detection Method for Peanut (Arachis Hypogagea) in Foods Using Polymerase Chain Reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A qualitative method for detection of peanuts in foods using polymerase chain reaction was developed. A universal primer pair CP 03-5 /CP 03-3 was designed to confirm the validity of the DNAs for PCR. The plant-specific amplified fragments were detected from 13 kinds of plants using the universal pr...

  20. A Specific Qualitative Detection Method for Peanut (Arachis Hypogaea) in Foods Using Polymerase Chain Reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed a qualitative detection method for peanuts in foods using polymerase chain reaction (PCR). We designed a universal primer pair CP 03-5’/ CP 03-3’ to confirm the validity of the DNAs for PCR. The plant specific amplified fragments were detected from 13 kinds of plants using the universal...

  1. Open-Source Radiation Exposure Extraction Engine (RE3) with Patient-Specific Outlier Detection.

    PubMed

    Weisenthal, Samuel J; Folio, Les; Kovacs, William; Seff, Ari; Derderian, Vana; Summers, Ronald M; Yao, Jianhua

    2016-08-01

    We present an open-source, picture archiving and communication system (PACS)-integrated radiation exposure extraction engine (RE3) that provides study-, series-, and slice-specific data for automated monitoring of computed tomography (CT) radiation exposure. RE3 was built using open-source components and seamlessly integrates with the PACS. RE3 calculations of dose length product (DLP) from the Digital imaging and communications in medicine (DICOM) headers showed high agreement (R (2) = 0.99) with the vendor dose pages. For study-specific outlier detection, RE3 constructs robust, automatically updating multivariable regression models to predict DLP in the context of patient gender and age, scan length, water-equivalent diameter (D w), and scanned body volume (SBV). As proof of concept, the model was trained on 811 CT chest, abdomen + pelvis (CAP) exams and 29 outliers were detected. The continuous variables used in the outlier detection model were scan length (R (2)  = 0.45), D w (R (2) = 0.70), SBV (R (2) = 0.80), and age (R (2) = 0.01). The categorical variables were gender (male average 1182.7 ± 26.3 and female 1047.1 ± 26.9 mGy cm) and pediatric status (pediatric average 710.7 ± 73.6 mGy cm and adult 1134.5 ± 19.3 mGy cm). PMID:26644157

  2. Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

    PubMed Central

    Roma, Cristin; Esposito, Claudia; Rachiglio, Anna Maria; Pasquale, Raffaella; Chicchinelli, Nicoletta; Mancini, Rita; Pisconti, Salvatore; Botti, Gerardo; Morabito, Alessandro

    2013-01-01

    Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct = 37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples. PMID:24364033

  3. Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution.

    PubMed Central

    Kreader, C A

    1995-01-01

    Because Bacteroides spp. are obligate anaerobes that dominate the human fecal flora, and because some species may live only in the human intestine, these bacteria might be useful to distinguish human from nonhuman sources of fecal pollution. To test this hypothesis, PCR primers specific for 16S rRNA gene sequences of Bacteroides distasonis, B. thetaiotaomicron, and B. vulgatus were designed. Hybridization with species-specific internal probes was used to detect the intended PCR products. Extracts from 66 known Bacteroides strains, representing 10 related species, were used to confirm the specificity of these PCR-hybridization assays. To test for specificity in feces, procedures were developed to prepare DNA of sufficient purity for PCR. Extracts of feces from 9 humans and 70 nonhumans (cats, dogs, cattle, hogs, horses, sheep, goats, and chickens) were each analyzed with and without an internal positive control to verify that PCR amplification was not inhibited by substances in the extract. In addition, serial dilutions from each extract that tested positive were assayed to estimate the relative abundance of target Bacteroides spp. in the sample. Depending on the primer-probe set used, either 78 or 67% of the human fecal extracts tested had high levels of target DNA. On the other hand, only 7 to 11% of the nonhuman extracts tested had similarly high levels of target DNA. An additional 12 to 20% of the nonhuman extracts had levels of target DNA that were 100- to 1,000-fold lower than those found in humans.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7538270

  4. Development of single dilution immunoassay to detect E2 protein specific classical swine fever virus antibody.

    PubMed

    Kumar, Rakesh; Barman, Nagendra N; Khatoon, Elina; Kumar, Sachin

    2016-04-01

    Classical swine fever virus (CSFV) is the causative agent of a highly contagious disease in swine. The disease is endemic in different parts of the world and vaccination is the only way to protect pigs from CSFV infection. The virus surface protein E2 is the major immunogenic protein eliciting protective immunity against CSFV infection in swine. The whole virus antigen cannot differentiate CSFV from other pestiviruses as it cross reacts with border disease and bovine viral diarrhoea viruses. Commercial available ELISA is based on the whole CSFV particle and can lead to false positive results. Moreover, the available commercial ELISA is not cost effective. In the present study, a recombinant E2 protein based single serum dilution ELISA was developed which showed enhanced sensitivity, specificity and accuracy as compared to commercial CSFV detection ELISA. The recombinant E2 protein based ELISA could be an alternate to existing diagnostics against CSFV infection in pigs. PMID:27032503

  5. Space-time airborne disease mapping applied to detect specific behaviour of varicella in Valencia, Spain.

    PubMed

    Iftimi, Adina; Montes, Francisco; Santiyán, Ana Míguez; Martínez-Ruiz, Francisco

    2015-01-01

    Airborne diseases are one of humanity's most feared sicknesses and have regularly caused concern among specialists. Varicella is an airborne disease which usually affects children before the age of 10. Because of its nature, varicella gives rise to interesting spatial, temporal and spatio-temporal patterns. This paper studies spatio-temporal exploratory analysis tools to detect specific behaviour of varicella in the city of Valencia, Spain, from 2008 to 2013. These methods have shown a significant association between the spatial and the temporal component, confirmed by the space-time models applied to the data. High relative risk of varicella is observed in economically disadvantaged regions, areas less involved in vaccination programmes. PMID:26530821

  6. NAIP proteins are required for cytosolic detection of specific bacterial ligands in vivo.

    PubMed

    Rauch, Isabella; Tenthorey, Jeannette L; Nichols, Randilea D; Al Moussawi, Khatoun; Kang, James J; Kang, Chulho; Kazmierczak, Barbara I; Vance, Russell E

    2016-05-01

    NLRs (nucleotide-binding domain [NBD] leucine-rich repeat [LRR]-containing proteins) exhibit diverse functions in innate and adaptive immunity. NAIPs (NLR family, apoptosis inhibitory proteins) are NLRs that appear to function as cytosolic immunoreceptors for specific bacterial proteins, including flagellin and the inner rod and needle proteins of bacterial type III secretion systems (T3SSs). Despite strong biochemical evidence implicating NAIPs in specific detection of bacterial ligands, genetic evidence has been lacking. Here we report the use of CRISPR/Cas9 to generate Naip1(-/-) and Naip2(-/-) mice, as well as Naip1-6(Δ/Δ) mice lacking all functional Naip genes. By challenging Naip1(-/-) or Naip2(-/-) mice with specific bacterial ligands in vivo, we demonstrate that Naip1 is uniquely required to detect T3SS needle protein and Naip2 is uniquely required to detect T3SS inner rod protein, but neither Naip1 nor Naip2 is required for detection of flagellin. Previously generated Naip5(-/-) mice retain some residual responsiveness to flagellin in vivo, whereas Naip1-6(Δ/Δ) mice fail to respond to cytosolic flagellin, consistent with previous biochemical data implicating NAIP6 in flagellin detection. Our results provide genetic evidence that specific NAIP proteins function to detect specific bacterial proteins in vivo. PMID:27045008

  7. The evolutionary development of high specific impulse electric thruster technology

    NASA Technical Reports Server (NTRS)

    Sovey, James S.; Hamley, John A.; Patterson, Michael J.; Rawlin, Vincent K.; Myers, Roger M.

    1992-01-01

    Electric propulsion flight and technology demonstrations conducted primarily by Europe, Japan, China, the U.S., and the USSR are reviewed. Evolutionary mission applications for high specific impulse electric thruster systems are discussed, and the status of arcjet, ion, and magnetoplasmadynamic thrusters and associated power processor technologies are summarized.

  8. The evolutionary development of high specific impulse electric thruster technology

    SciTech Connect

    Sovey, J.S.; Hamley, J.A.; Patterson, M.J.; Rawlin, V.K.; Meyers, R.M.

    1992-03-01

    Electric propulsion flight and technology demonstrations conducted primarily by Europe, Japan, Peoples Republic of China, USA, and USSR are reviewed. Evolutionary mission applications for high specific impulse electric thruster systems are discussed, and the status of arcjet, ion, and magnetoplasmadynamic thruster and associated power processor technologies are summarized.

  9. A Ratio Test of Interrater Agreement with High Specificity

    ERIC Educational Resources Information Center

    Cousineau, Denis; Laurencelle, Louis

    2015-01-01

    Existing tests of interrater agreements have high statistical power; however, they lack specificity. If the ratings of the two raters do not show agreement but are not random, the current tests, some of which are based on Cohen's kappa, will often reject the null hypothesis, leading to the wrong conclusion that agreement is present. A new test of…

  10. Event-specific detection of seven genetically modified soybean and maizes using multiplex-PCR coupled with oligonucleotide microarray.

    PubMed

    Xu, Jia; Zhu, Shuifang; Miao, Haizhen; Huang, Wensheng; Qiu, Minyan; Huang, Yan; Fu, Xuping; Li, Yao

    2007-07-11

    With the increasing development of genetically modified organism (GMO) detection techniques, the polymerase chain reaction (PCR) technique has been the mainstay for GMO detection. An oligonucleotide microarray is a glass chip to the surface of which an array of oligonucleotides was fixed as spots, each containing numerous copies of a sequence-specific probe that is complementary to a gene of interest. So it is used to detect ten or more targets synchronously. In this research, an event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity using multiplex-PCR together with oligonucleotide microarray. A commercial GM soybean (GTS 40-3-2) and six GM maize events (MON810, MON863, Bt176, Bt11, GA21, and T25) were detected by this method. The results indicate that it is a suitable method for the identification of these GM soybean and maizes. PMID:17559227