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Sample records for highly specific detection

  1. Heterodyne method for high specificity gas detection.

    NASA Technical Reports Server (NTRS)

    Dimeff, J.; Donaldson, R. W.; Gunter, W. D., Jr.; Jaynes, D. N.; Margozzi, A. P.; Deboo, G. J.; Mcclatchie, E. A.; Williams, K. G.

    1971-01-01

    This paper describes a new technique for measuring trace quantities of gases. The technique involves the use of a reference cell (containing a known amount of the gas being sought) and a sample cell (containing an unknown amount of the same gas) wherein the gas densities are modulated. Light passing through the two cells in sequence is modulated in intensity at the vibrational-rotational lines characteristic of the absorption spectrum for the gas of interest. Since the absorption process is nonlinear, modulating the two absorption cells at two different frequencies gives rise to a heterodyning effect, which in turn introduces sum and difference frequencies in the detected signal. Measuring the ratio of the difference frequency signal for example, to the signal introduced by the reference cell provides a normalized measure of the amount of the gas in the sample cell. The readings produced are thereby independent of source intensity, window transparency, and detector sensitivity. Experimental evaluation of the technique suggests that it should be applicable to a wide range of gases, that it should be able to reject spurious signals due to unwanted gases, and that it should be sensitive to concentrations of the order of 10 to the minus 8th power when used with a sample cell of only 20 cm length.

  2. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  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.

  4. 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

  5. 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.

  6. Detection of pork adulteration by highly-specific PCR assay of mitochondrial D-loop.

    PubMed

    Karabasanavar, Nagappa S; Singh, S P; Kumar, Deepak; Shebannavar, Sunil N

    2014-02-15

    We describe a highly specific PCR assay for the authentic identification of pork. Accurate detection of tissues derived from pig (Sus scrofa) was accomplished by using newly designed primers targeting porcine mitochondrial displacement (D-loop) region that yielded an unique amplicon of 712 base pairs (bp). Possibility of cross-amplification was precluded by testing as many as 24 animal species (mammals, birds, rodent and fish). Suitability of PCR assay was confirmed in raw (n = 20), cooked (60, 80 and 100 °C), autoclaved (121 °C) and micro-oven processed pork. Sensitivity of detection of pork in other species meat using unique pig-specific PCR was established to be at 0.1%; limit of detection (LOD) of pig DNA was 10 pg (pico grams). The technique can be used for the authentication of raw, processed and adulterated pork and products under the circumstances of food adulteration related disputes or forensic detection of origin of pig species.

  7. Highly Sensitive, Highly Specific Whole-Cell Bioreporters for the Detection of Chromate in Environmental Samples

    PubMed Central

    Branco, Rita; Cristóvão, Armando; Morais, Paula V.

    2013-01-01

    Microbial bioreporters offer excellent potentialities for the detection of the bioavailable portion of pollutants in contaminated environments, which currently cannot be easily measured. This paper describes the construction and evaluation of two microbial bioreporters designed to detect the bioavailable chromate in contaminated water samples. The developed bioreporters are based on the expression of gfp under the control of the chr promoter and the chrB regulator gene of TnOtChr determinant from Ochrobactrum tritici 5bvl1. pCHRGFP1 Escherichia coli reporter proved to be specific and sensitive, with minimum detectable concentration of 100 nM chromate and did not react with other heavy metals or chemical compounds analysed. In order to have a bioreporter able to be used under different environmental toxics, O. tritici type strain was also engineered to fluoresce in the presence of micromolar levels of chromate and showed to be as specific as the first reporter. Their applicability on environmental samples (spiked Portuguese river water) was also demonstrated using either freshly grown or cryo-preserved cells, a treatment which constitutes an operational advantage. These reporter strains can provide on-demand usability in the field and in a near future may become a powerful tool in identification of chromate-contaminated sites. PMID:23326558

  8. Highly specific and sensitive electrochemical genotyping via gap ligation reaction and surface hybridization detection.

    PubMed

    Huang, Yong; Zhang, Yan-Li; Xu, Xiangmin; Jiang, Jian-Hui; Shen, Guo-Li; Yu, Ru-Qin

    2009-02-25

    This paper developed a novel electrochemical genotyping strategy based on gap ligation reaction with surface hybridization detection. This strategy utilized homogeneous enzymatic reactions to generate molecular beacon-structured allele-specific products that could be cooperatively annealed to capture probes stably immobilized on the surface via disulfide anchors, thus allowing ultrasensitive surface hybridization detection of the allele-specific products through redox tags in close proximity to the electrode. Such a unique biphasic architecture provided a universal methodology for incorporating enzymatic discrimination reactions in electrochemical genotyping with desirable reproducibility, high efficiency and no interferences from interficial steric hindrance. The developed technique was demonstrated to show intrinsic high sensitivity for direct genomic analysis, and excellent specificity with discriminativity of single nucleotide variations.

  9. Molecular inversion probe: a new tool for highly specific detection of plant pathogens.

    PubMed

    Lau, Han Yih; Palanisamy, Ramkumar; Trau, Matt; Botella, Jose R

    2014-01-01

    Highly specific detection methods, capable of reliably identifying plant pathogens are crucial in plant disease management strategies to reduce losses in agriculture by preventing the spread of diseases. We describe a novel molecular inversion probe (MIP) assay that can be potentially developed into a robust multiplex platform to detect and identify plant pathogens. A MIP has been designed for the plant pathogenic fungus Fusarium oxysporum f.sp. conglutinans and the proof of concept for the efficiency of this technology is provided. We demonstrate that this methodology can detect as little as 2.5 ng of pathogen DNA and is highly specific, being able to accurately differentiate Fusarium oxysporum f.sp. conglutinans from other fungal pathogens such as Botrytis cinerea and even pathogens of the same species such as Fusarium oxysporum f.sp. lycopersici. The MIP assay was able to detect the presence of the pathogen in infected Arabidopsis thaliana plants as soon as the tissues contained minimal amounts of pathogen. MIP methods are intrinsically highly multiplexable and future development of specific MIPs could lead to the establishment of a diagnostic method that could potentially screen infected plants for hundreds of pathogens in a single assay.

  10. Novel fluorescently labeled peptide compounds for detection of oxidized low-density lipoprotein at high specificity.

    PubMed

    Sato, Akira; Yamanaka, Hikaru; Oe, Keitaro; Yamazaki, Yoji; Ebina, Keiichi

    2014-10-01

    The probes for specific detection of oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to be useful for the identification, diagnosis, prevention, and treatment for atherosclerosis. In this study, to develop a fluorescent peptide probe for specific detection of ox-LDL, we investigated the interaction of fluorescein isothiocyanate (FITC)-labeled peptides with ox-LDL using polyacrylamide gel electrophoresis. Two heptapeptides (KWYKDGD and KP6) coupled through the ε-amino group of K at the N-terminus to FITC in the presence/absence of 6-amino-n-caproic acid (AC) linker to FITC--(FITC-AC)KP6 and (FITC)KP6--both bound with high specificity to ox-LDL in a dose-dependent manner. In contrast, a tetrapeptide (YKDG) labeled with FITC at the N-terminus and a pentapeptide (YKDGK) coupled through the ε-amino group of K at the C-terminus to FITC did not bind selectively to ox-LDL. Furthermore, (FITC)KP6 and (FITC-AC)KP6 bound with high specificity to the protein in mouse plasma (probably ox-LDL fraction). These findings strongly suggest that (FITC)KP6 and (FITC-AC)KP6 may be effective novel fluorescent probes for specific detection of ox-LDL.

  11. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Fast and highly specific DNA-based multiplex detection on a solid support.

    PubMed

    Barišić, Ivan; Kamleithner, Verena; Schönthaler, Silvia; Wiesinger-Mayr, Herbert

    2015-01-01

    Highly specific and fast multiplex detection methods are essential to conduct reasonable DNA-based diagnostics and are especially important to characterise infectious diseases. More than 1000 genetic targets such as antibiotic resistance genes, virulence factors and phylogenetic markers have to be identified as fast as possible to facilitate the correct treatment of a patient. In the present work, we developed a novel ligation-based DNA probe concept that was combined with the microarray technology and used it for the detection of bacterial pathogens. The novel linear chain (LNC) probes identified all tested species correctly within 1 h based on their 16S rRNA gene in a 25-multiplex reaction. Genomic DNA was used directly as template in the ligation reaction identifying as little as 10(7) cells without any pre-amplification. The high specificity was further demonstrated characterising a single nucleotide polymorphism leading to no false positive fluorescence signals of the untargeted single nucleotide polymorphism (SNP) variants. In comparison to conventional microarray probes, the sensitivity of the novel LNC3 probes was higher by a factor of 10 or more. In summary, we present a fast, simple, highly specific and sensitive multiplex detection method adaptable for a wide range of applications.

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

    PubMed Central

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

    2013-01-01

    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

  14. 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.

  15. 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

  16. 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.

  17. MOF-Bacteriophage Biosensor for Highly Sensitive and Specific Detection of Staphylococcus aureus.

    PubMed

    Bhardwaj, Neha; Bhardwaj, Sanjeev K; Mehta, Jyotsana; Kim, Ki-Hyun; Deep, Akash

    2017-09-21

    To produce a sensitive and specific biosensor for Staphylococcus aureus, bacteriophages have been interfaced with a water-dispersible and environmentally stable metal-organic framework (MOF), NH2-MIL-53(Fe). The conjugation of the MOF with bacteriophages has been achieved through the use of glutaraldehyde as cross-linker. Highly sensitive detection of S. aureus in both synthetic and real samples was realized by the proposed MOF-bacteriophage biosensor based on the photoluminescence quenching phenomena: limit of detection (31 CFU/mL) and range of detection (40 to 4 × 10(8) CFU/mL). This is the first report exploiting the use of an MOF-bacteriophage complex for the biosensing of S. aureus. The results of our study highlight that the proposed biosensor is more sensitive than most of the previous methods while exhibiting some advanced features like specificity, regenerability, extended range of linear detection, and stability for long-term storage (even at room temperature).

  18. STEAK: A specific tool for transposable elements and retrovirus detection in high-throughput sequencing data

    PubMed Central

    Gambron, Philippe; Marchi, Emanuele; Karamitros, Timokratis; Katzourakis, Aris

    2017-01-01

    Abstract The advancements of high-throughput genomics have unveiled much about the human genome highlighting the importance of variations between individuals and their contribution to disease. Even though numerous software have been developed to make sense of large genomics datasets, a major short falling of these has been the inability to cope with repetitive regions, specifically to validate structural variants and accordingly assess their role in disease. Here we describe our program STEAK, a massively parallel software designed to detect chimeric reads in high-throughput sequencing data for a broad number of applications such as identifying presence/absence, as well as discovery of transposable elements (TEs), and retroviral integrations. We highlight the capabilities of STEAK by comparing its efficacy in locating HERV-K HML-2 in clinical whole genome projects, target enrichment sequences, and in the 1000 Genomes CEU Trio to the performance of other TE and virus detecting tools. We show that STEAK outperforms other software in terms of computational efficiency, sensitivity, and specificity. We demonstrate that STEAK is a robust tool, which allows analysts to flexibly detect and evaluate TE and retroviral integrations in a diverse range of sequencing projects for both research and clinical purposes. PMID:28948042

  19. High Sensitivity and High Detection Specificity of Gold-Nanoparticle-Grafted Nanostructured Silicon Mass Spectrometry for Glucose Analysis.

    PubMed

    Tsao, Chia-Wen; Yang, Zhi-Jie

    2015-10-14

    Desorption/ionization on silicon (DIOS) is a high-performance matrix-free mass spectrometry (MS) analysis method that involves using silicon nanostructures as a matrix for MS desorption/ionization. In this study, gold nanoparticles grafted onto a nanostructured silicon (AuNPs-nSi) surface were demonstrated as a DIOS-MS analysis approach with high sensitivity and high detection specificity for glucose detection. A glucose sample deposited on the AuNPs-nSi surface was directly catalyzed to negatively charged gluconic acid molecules on a single AuNPs-nSi chip for MS analysis. The AuNPs-nSi surface was fabricated using two electroless deposition steps and one electroless etching step. The effects of the electroless fabrication parameters on the glucose detection efficiency were evaluated. Practical application of AuNPs-nSi MS glucose analysis in urine samples was also demonstrated in this study.

  20. Highly specific detection of IL-8 protein using combination tapered fiber-optic biosensor dip-probe

    NASA Astrophysics Data System (ADS)

    Wang, Chun-Wei; Kapoor, Rakesh

    2010-02-01

    We are reporting detection of IL-8 in a mixed protein solution, using combination tapered fiber-optic biosensor (CTFOB) dip-probe. Sandwich immunoassay was used as the detection technique. The specificity of the sensor was established by using two types of negative control probes. It is demonstrated that with the help of these CTFOB dipprobe we could successfully detect IL-8 with high specificity in protein mixture. The lowest detected concentration of IL-8 was 150 pM.

  1. An RNA-based signature enables high specificity detection of circulating tumor cells in hepatocellular carcinoma

    PubMed Central

    Kalinich, Mark; Bhan, Irun; Kwan, Tanya T.; Miyamoto, David T.; Javaid, Sarah; LiCausi, Joseph A.; Milner, John D.; Hong, Xin; Goyal, Lipika; Sil, Srinjoy; Choz, Melissa; Ho, Uyen; Kapur, Ravi; Muzikansky, Alona; Zhang, Huidan; Weitz, David A.; Sequist, Lecia V.; Ryan, David P.; Chung, Raymond T.; Zhu, Andrew X.; Isselbacher, Kurt J.; Ting, David T.; Toner, Mehmet; Maheswaran, Shyamala; Haber, Daniel A.

    2017-01-01

    Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. We recently described a high-throughput microfluidic CTC-iChip, which efficiently depletes hematopoietic cells from blood specimens and enriches for CTCs with well-preserved RNA. Application of RNA-based digital PCR to detect CTC-derived signatures may thus enable highly accurate tissue lineage-based cancer detection in blood specimens. As proof of principle, we examined hepatocellular carcinoma (HCC), a cancer that is derived from liver cells bearing a unique gene expression profile. After identifying a digital signature of 10 liver-specific transcripts, we used a cross-validated logistic regression model to identify the presence of HCC-derived CTCs in nine of 16 (56%) untreated patients with HCC versus one of 31 (3%) patients with nonmalignant liver disease at risk for developing HCC (P < 0.0001). Positive CTC scores declined in treated patients: Nine of 32 (28%) patients receiving therapy and only one of 15 (7%) patients who had undergone curative-intent ablation, surgery, or liver transplantation were positive. RNA-based digital CTC scoring was not correlated with the standard HCC serum protein marker alpha fetoprotein (P = 0.57). Modeling the sequential use of these two orthogonal markers for liver cancer screening in patients with high-risk cirrhosis generates positive and negative predictive values of 80% and 86%, respectively. Thus, digital RNA quantitation constitutes a sensitive and specific CTC readout, enabling high-throughput clinical applications, such as noninvasive screening for HCC in populations where viral hepatitis and cirrhosis are prevalent. PMID:28096363

  2. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. 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

  4. Rapid and Highly Sensitive Non-Competitive Immunoassay for Specific Detection of Nodularin.

    PubMed

    Akter, Sultana; Vehniäinen, Markus; Kankaanpää, Harri T; Lamminmäki, Urpo

    2017-09-12

    Nodularin (NOD) is a cyclic penta-peptide hepatotoxin mainly produced by Nodularia spumigena, reported from the brackish water bodies of various parts of the world. It can accumulate in the food chain and, for safety reasons, levels of NOD not only in water bodies but also in food matrices are of interest. Here, we report on a non-competitive immunoassay for the specific detection of NOD. A phage display technique was utilized to interrogate a synthetic antibody phage library for binders recognizing NOD bound to an anti-ADDA (3-Amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4(E),6(E)-dienoic acid) monoclonal antibody (Mab). One of the obtained immunocomplex binders, designated SA32C11, showed very high specificity towards nodularin-R (NOD-R) over to the tested 10 different microcystins (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF, -LW, -LA, -WR). It was expressed in Escherichia coli as a single chain antibody fragment (scFv) fusion protein and used to establish a time-resolved fluorometry-based assay in combination with the anti-ADDA Mab. The detection limit (blank + 3SD) of the immunoassay, with a total assay time of 1 h 10 min, is 0.03 µg/L of NOD-R. This represents the most sensitive immunoassay method for the specific detection of NOD reported so far. The assay was tested for its performance to detect NOD using spiked (0.1 to 3 µg/L of NOD-R) water samples including brackish sea and coastal water and the recovery ranged from 79 to 127%. Furthermore, a panel of environmental samples, including water from different sources, fish and other marine tissue specimens, were analyzed for NOD using the assay. The assay has potential as a rapid screening tool for the analysis of a large number of water samples for the presence of NOD. It can also find applications in the analysis of the bioaccumulation of NOD in marine organisms and in the food chain.

  5. Digital obstructive arterial disease can be detected by laser Doppler measurements with high sensitivity and specificity.

    PubMed

    Mahe, Guillaume; Liedl, David A; McCarter, Charlene; Shepherd, Roger; Gloviczki, Peter; McPhail, Ian R; Rooke, Thom W; Wennberg, Paul W

    2014-04-01

    This study was conducted to determine the sensitivity and specificity of laser Doppler flowmetry (LDF) measurements for digital obstructive arterial disease (DOAD) using angiography as the reference standard and to compare the accuracy of different classical tests used to assess DOAD. Diagnosis of vascular abnormalities at the digital level is challenging. Angiography is the gold standard for assessment of DOAD but is invasive and expensive to perform. We performed a retrospective analysis of consecutive patients referred at Mayo Clinic (Rochester, Minn) for upper extremity arterial assessment during a 27-month period. Finger-brachial index, skin blood flow (in arbitrary units [a.u.]), and skin temperature (in degrees Celsius) were recorded in each digit on the pulp at baseline and after a thermal challenge test (hand placed in a thermal box at 47.0°C for 15 minutes). Angiogram analysis was blinded and performed by a radiologist using a vascularization scale ranging from 0 (no vessel) to 4 (normal). The receiver operating characteristic curve was used to define a specific cutoff point to detect DOAD. Twenty-two patients had LDF measurements and complete angiograms. A total of 185 digits were analyzed because some patients had only analysis of one hand. The best area under the curve (AUC) was 0.98 (range, 0.94-0.99) for postwarming skin blood flow, with a cutoff point of ≤206 a.u. This AUC was statistically different from AUCs of all the other tests (P < .01). Sensitivity and specificity were 93% (95% confidence interval, 85%-97%) and 96% (95% confidence interval, 90%-99%), respectively. LDF combined with a thermal challenge is highly accurate, safe, and noninvasive means to detect DOAD. Copyright © 2014 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.

  6. Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples

    PubMed Central

    Calvani, Nichola Eliza Davies; Windsor, Peter Andrew; Bush, Russell David

    2017-01-01

    Background Fasciolosis, due to Fasciola hepatica and Fasciola gigantica, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and prone to sensitivity errors when a large number of samples must be processed or if the operator lacks sufficient experience. Additionally, diagnosis can only be made once the 12-week pre-patent period has passed. Recently, a commercially available coprological antigen ELISA has enabled detection of F. hepatica prior to the completion of the pre-patent period, providing earlier diagnosis and increased throughput, although species differentiation is not possible in areas of parasite sympatry. Real-time PCR offers the combined benefits of highly sensitive species differentiation for medium to large sample sizes. However, no molecular diagnostic workflow currently exists for the identification of Fasciola spp. in faecal samples. Methodology/Principal findings A new molecular diagnostic workflow for the highly-sensitive detection and quantification of Fasciola spp. in faecal samples was developed. The technique involves sedimenting and pelleting the samples prior to DNA isolation in order to concentrate the eggs, followed by disruption by bead-beating in a benchtop homogeniser to ensure access to DNA. Although both the new molecular workflow and the traditional sedimentation technique were sensitive and specific, the new molecular workflow enabled faster sample throughput in medium to large epidemiological studies, and provided the additional benefit of speciation. Further, good correlation (R2 = 0.74–0.76) was observed between the real-time PCR values and the faecal egg count (FEC) using the new molecular workflow for all herds and sampling periods. Finally, no effect of storage in 70% ethanol was detected on sedimentation and DNA isolation outcomes; enabling

  7. A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists.

    PubMed

    Bovee, Toine F H; Helsdingen, Richard J R; Hamers, Astrid R M; van Duursen, Majorie B M; Nielen, Michel W F; Hoogenboom, Ron L A P

    2007-11-01

    Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17beta-testosterone, the concentration where half-maximal activation is reached (EC(50)) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC(50) of 17beta-testosterone and the EC(50) of the compound, of 5alpha-dihydrotestosterone, methyltrienolone, and 17beta-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action.

  8. A new highly specific and robust yeast androgen bioassay for the detection of agonists and antagonists

    PubMed Central

    Helsdingen, Richard J. R.; Hamers, Astrid R. M.; van Duursen, Majorie B. M.; Nielen, Michel W. F.; Hoogenboom, Ron L. A. P.

    2007-01-01

    Public concern about the presence of natural and anthropogenic compounds which affect human health by modulating normal endocrine functions is continuously growing. Fast and simple high-throughput screening methods for the detection of hormone activities are thus indispensable. During the last two decades, a panel of different in vitro assays has been developed, mainly for compounds with an estrogenic mode of action. Here we describe the development of an androgen transcription activation assay that is easy to use in routine screening. Recombinant yeast cells were constructed that express the human androgen receptor and yeast enhanced green fluorescent protein (yEGFP), the latter in response to androgens. Compared with other reporters, the yEGFP reporter protein is very convenient because it is directly measurable in intact living cells, i.e., cell wall disruption and the addition of a substrate are not needed. When yeast was exposed to 17β-testosterone, the concentration where half-maximal activation is reached (EC50) was 50 nM. The relative androgenic potencies, defined as the ratio between the EC50 of 17β-testosterone and the EC50 of the compound, of 5α-dihydrotestosterone, methyltrienolone, and 17β-boldenone are 2.3, 1.4, and 0.15 respectively. The results presented in this paper demonstrate that this new yeast androgen bioassay is fast, sensitive, and very specific and also suited to detect compounds that have an antiandrogenic mode of action. PMID:17849102

  9. 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

  10. Label-Free Isothermal Amplification Assay for Specific and Highly Sensitive Colorimetric miRNA Detection

    PubMed Central

    2016-01-01

    We describe a new method for the detection of miRNA in biological samples. This technology is based on the isothermal nicking enzyme amplification reaction and subsequent hybridization of the amplification product with gold nanoparticles and magnetic microparticles (barcode system) to achieve naked-eye colorimetric detection. This platform was used to detect a specific miRNA (miRNA-10b) associated with breast cancer, and attomolar sensitivity was demonstrated. The assay was validated in cell culture lysates from breast cancer cells and in serum from a mouse model of breast cancer. PMID:27713932

  11. 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

  12. 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

  13. High-resolution and specific detection of bacteria on complex surfaces using nanoparticle probes and electron microscopy.

    PubMed

    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.

  14. Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions

    PubMed Central

    Haab, Brian B; Dunham, Maitreya J; Brown, Patrick O

    2001-01-01

    Background: We have developed and tested a method for printing protein microarrays and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, the other representing an experimental sample in which the protein quantities were to be measured, were labeled by covalent attachment of spectrally resolvable fluorescent dyes. Results: Specific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signal representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To test the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs. 50% of the arrayed antigens and 20% of the arrayed antibodies provided specific and accurate measurements of their cognate ligands at or below concentrations of 0.34 μg/ml and 1.6 μg/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of 1 part in 106, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples. Conclusions: These results suggest that protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications. PMID:11182887

  15. 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.

  16. 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

  17. 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.

  18. 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. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Real time detection of anthrax spores using highly specific anti-EA1 recombinant antibodies produced by competitive panning.

    PubMed

    Love, Tracey E; Redmond, Caroline; Mayers, Carl N

    2008-05-20

    We describe a targeted approach for the production of biological recognition elements capable of fast, specific detection of anthrax spores on biosensor surfaces. The aim was to produce single chain antibodies (scFvs) to EA1, a Bacillus anthracis S-layer protein that is also present, although not identical, in related to Bacillus species. The aim of the work was to produce antibodies that would detect B. anthracis EA1 protein and intact spores with a high degree of specificity, but would not detect other Bacillus species. Existing monoclonal antibodies were evaluated and found to recognise B. anthracis EA1 and S-layer proteins from other closely related Bacillus species. Recombinant anti-EA1 scFvs were isolated from B. anthracis immune library that contained antibody genes raised against B. anthracis spores and purified exosporium. Two approaches for scFv selection were used; standard (non-competitive) panning, and competitive panning. The non-competitive biopanning strategy isolated scFvs that recognised EA1 from B. anthracis, but also cross-reacted with other Bacillus species. In contrast, the competitive panning approach used S-layer proteins from other Bacillus species to generate scFvs that were highly specific to B. anthracis EA1 and demonstrated apparent nanomolar binding affinities. Specific, real time detection of B. anthracis spores was demonstrated with these scFvs using an evanescent wave biosensor, the Resonant Mirror. The approach described can be used to generate specific antibodies to any desired target where homologous proteins also exist in closely related species, and demonstrates clear advantages to using recombinant technology to produce biological recognition elements for detection of biological threat agents.

  20. 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.

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

    PubMed

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

    2016-02-02

    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.

  2. 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

  3. Evaluation of any or type-specific persistence of high-risk human papillomavirus for detecting cervical precancer.

    PubMed

    Marks, Morgan A; Castle, Philip E; Schiffman, Mark; Gravitt, Patti E

    2012-02-01

    High-risk human papillomavirus (HR-HPV) testing is increasingly important. We therefore examined the impact on accuracy of repeated versus one-time testing, type-specific versus pooled detection, and assay analytic sensitivity. By using a nested case-control design from the ASCUS-LSIL Triage Study, we selected women with incident cervical intraepithelial neoplasia grade 2 or grade 3 (CIN2/3; n = 325) and a random sample of women with specificity for different permutations of multiple measurements were compared to a single measurement using marginal regression models. We found that repeat detection of any HR-HPV (by HC2, LA, or LBA) and of type-specific persistence (by LA or LBA) were significantly more specific but less sensitive than use of a single time point measurement of any HR-HPV. Sensitivity decreased and specificity increased further when testing intervals were increased from 12 to 24 months. Including detection of borderline carcinogenic/noncarcinogenic HPV types with HR-HPV types decreased specificity for repeat measures of HPV with no impact on sensitivity. Similar patterns were observed when we used a CIN3 end point. We conclude that assay performance for detecting incident CIN2/3 was affected by which types were included, the analytic sensitivity of the assay, and the testing interval. These trade-offs need to be considered when assessing the potential overall clinical utility of repeated testing for HR-HPV DNA to identify women at risk for CIN2/3.

  4. Immunocytochemical detection of ERG expression in exfoliated urinary cells identifies with high specificity patients with prostate cancer.

    PubMed

    Pal, Raj P; Kockelbergh, Roger C; Pringle, John Howard; Cresswell, Lara; Hew, Roger; Dormer, John P; Cooper, Colin; Mellon, John Kilian; Barwell, Julian G; Hollox, Edward J

    2016-04-01

    To evaluate the immunocytochemical detection of ERG protein in exfoliated cells as a means of identifying patients with prostate cancer (PCa) before prostate biopsy. Urine samples (30 mL) were collected after digital rectal examination (DRE) from 159 patients with an elevated age-specific prostate-specific antigen (PSA) and/or an abnormal DRE who underwent prostate biopsy. In all cases, exfoliated urinary cells from half of the urine sample underwent immunocytochemical assessment for ERG protein expression. Exfoliated cells in the remaining half underwent assessment of TMPRSS2:ERG status using either nested reverse-transcriptase (RT)-PCR (151 cases) or fluorescence in situ hybridization (FISH; eight cases). Corresponding tissue samples were evaluated using FISH to determine chromosomal gene fusion tissue status and immunohistochemistry (IHC) to determine ERG protein expression. Results were correlated with clinicopathological variables. The sensitivity and specificity of urinary ERG immunocytochemistry (ICC) for PCa were 22.7 and 100%, respectively. ERG ICC results correlated with advanced tumour grade, stage and higher serum PSA. In comparison, urine TMPRSS2:ERG transcript analysis had 27% sensitivity and 98% specificity for PCa detection. On tissue IHC, ERG staining was highly specific for PCa. In all, 52% of cancers harboured foci of ERG staining; however, only 46% of cancers that were found to have ERG overexpression were positive on urine ICC. The ERG ICC results showed strong concordance with urinary RT-PCR and FISH, and tissue IHC and FISH. This is the first study to show that cytological gene fusion detection using ICC is feasible and identifies patients with adverse disease markers. ERG ICC was highly specific, but this technique was less sensitive than RT-PCR. © 2015 The Authors BJU International © 2015 BJU International Published by John Wiley & Sons Ltd.

  5. Highly specific detection of thrombin using an aptamer-based suspension array and the interaction analysis via microscale thermophoresis.

    PubMed

    Liu, Yanan; Liu, Nan; Ma, Xinhua; Li, Xiaoli; Ma, Jia; Li, Ya; Zhou, Zhijiang; Gao, Zhixian

    2015-04-21

    A novel aptamer-based suspension array detection platform was designed for the sensitive, specific and rapid detection of human α-thrombin as a model. Thrombin was first recognized by a 29-mer biotinylated thrombin-binding aptamer (TBA) in solution. Then 15-mer TBA modified magnetic beads (MBs) captured the former TBA-thrombin to form an aptamer-thrombin-aptamer sandwich complex. The median fluorescence intensity obtained via suspension array technology was positively correlated with the thrombin concentration. The interactions between TBAs and thrombin were analyzed using microscale thermophoresis (MST). The dissociation constants could be respectively achieved to be 44.2 ± 1.36 nM (TBA1-thrombin) and 15.5 ± 0.637 nM (TBA2-thrombin), which demonstrated the high affinities of TBA-thrombin and greatly coincided with previous reports. Interaction conditions such as temperature, reaction time, and coupling protocol were optimized. The dynamic quantitative working range of the aptamer-based suspension array was 18.37-554.31 nM, and the coefficients of determination R(2) were greater than 0.9975. The lowest detection limit of thrombin was 5.4 nM. This method was highly specific for thrombin without being affected by other analogs and interfering proteins. The recoveries of thrombin spiked in diluted human serum were in the range 82.6-114.2%. This innovative aptamer-based suspension array detection platform not only exhibits good sensitivity based on MBs facilitating highly efficient separation and amplification, but also suggests high specificity by the selective aptamer binding, thereby suggesting the expansive application prospects in research and clinical fields.

  6. Highly specific confirmatory western blot test for African swine fever virus antibody detection using the recombinant virus protein p54.

    PubMed

    Alcaraz, C; Rodriguez, F; Oviedo, J M; Eiras, A; De Diego, M; Alonso, C; Escribano, J M

    1995-03-01

    A Western blot technique using a recombinant protein has been developed to confirm positive results obtained in African swine fever (ASF)-specific antibody detection by ELISA. The new confirmatory Western blot is based on the use of protein p54, one of the most antigenic ASF virus structural proteins, expressed in Escherichia coli fused to the N-terminus of MS2 polymerase. The recombinant Western blot assay was highly specific and equally sensitive for ASF virus-infected pigs detection as the conventional Western blot, which uses virus-induced proteins ranging in molecular weight between 23 and 35 kDa. The novel Western blot assay provides a simpler interpretation of the test, eliminates the possibility of false-positive reactions produced by cellular compounds that contaminate the antigen employed in the conventional technique, and avoids the use of live virus in antigen production.

  7. Specific, sensitive, high-resolution detection of protein molecules in eukaryotic cells using metal-tagging transmission electron microscopy

    PubMed Central

    Risco, Cristina; Sanmartín-Conesa, Eva; Tzeng, Wen-Pin; Frey, Teryl K.; Seybold, Volker; de Groot, Raoul J.

    2012-01-01

    Summary More than any other methodology, transmission electron microscopy (TEM) has contributed to our understanding of the architecture and organization of cells. With current detection limits approaching atomic resolution, it will ultimately become possible to ultrastructurally image intracellular macromolecular assemblies in situ. Presently, however, methods to unambiguously identify proteins within the crowded environment of the cell’s interior are lagging behind. We describe a novel approach, metal-tagging TEM (METTEM) that allows detection of intracellular proteins in mammalian cells with high specificity, exceptional sensitivity and at molecular scale resolution. In live cells treated with gold salts, proteins bearing a small metal-binding tag will form 1-nm gold nanoclusters, readily detectable in electron micrographs. The applicability and strength of METTEM is demonstrated by a study of Rubella virus replicase and capsid proteins, which revealed virus-induced cell structures not seen before. PMID:22579245

  8. 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.

  9. Label-Free Electrical Immunosensor for Highly Sensitive and Specific Detection of Microcystin-LR in Water Samples.

    PubMed

    Tan, Feng; Saucedo, Nuvia Maria; Ramnani, Pankaj; Mulchandani, Ashok

    2015-08-04

    Microcystin-LR (MCLR) is one of the most commonly detected and toxic cyclic heptapeptide cyanotoxins released by cyanobacterial blooms in surface waters, for which sensitive and specific detection methods are necessary to carry out its recognition and quantification. Here, we present a single-walled carbon nanotube (SWCNTs)-based label-free chemiresistive immunosensor for highly sensitive and specific detection of MCLR in different source waters. MCLR was initially immobilized on SWCNTs modified interdigitated electrode, followed by incubation with monoclonal anti-MCLR antibody. The competitive binding of MCLR in sample solutions induced departure of the antibody from the antibody-antigen complexes formed on SWCNTs, resulting in change in the conductivity between source and drain of the sensor. The displacement assay greatly improved the sensitivity of the sensor compared with direct immunoassay on the same device. The immunosensor exhibited a wide linear response to log value of MCLR concentration ranging from 1 to 1000 ng/L, with a detection limit of 0.6 ng/L. This method showed good reproducibility, stability and recovery. The proposed method provides a powerful tool for rapid and sensitive monitoring of MCLR in environmental samples.

  10. Rapid and specific detection of porcine parvovirus using real-time PCR and high resolution melting (HRM) analysis.

    PubMed

    Yu, Hai-Qiong; Cai, Xian-Quan; Lin, Zhi-Xiong; Li, Xiang-Li; Yue, Qiao-Yun; Li, Rong; Zhu, Xing-Quan

    2015-02-28

    Porcine parvovirus (PPV) is the important causative agent for infectious infertility, which is a fairly tough virus that multiplies normally in the intestine of pigs without causing clinical signs in the world. We developed an assay integrating real-time PCR and high resolution melting (HRM) analysis for the detection of PPV. Primers targeting the VP gene were highly specific, as evidenced by the negative amplification of closely related viruses, such as porcine circovirus 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), classical swine fever virus (CSFV), or Japanese encephalitis virus (JEV). The performance of unlabeled real time PCR was compared to TaqMan real time PCR, and the detection limits of the two methods were nearly equal. Moreover, there was good correlation between Cp and diluted genomic DNA when tested with the two methods. The assay has the accuracy of 100% in reference to labeled real time PCR, when it was tested on 45 clinical samples. The present study demonstrated that the established assay integrating real-time PCR and HRM is relatively cost-effective and more stable, which provides an alternative tool for rapid, simple, specific and sensitive detection of PPV.

  11. Sequence-Specific Covalent Capture Coupled with High-Contrast Nanopore Detection of a Disease-Derived Nucleic Acid Sequence.

    PubMed

    Nejad, Maryam Imani; Shi, Ruicheng; Zhang, Xinyue; Gu, Li-Qun; Gates, Kent S

    2017-07-18

    Hybridization-based methods for the detection of nucleic acid sequences are important in research and medicine. Short probes provide sequence specificity, but do not always provide a durable signal. Sequence-specific covalent crosslink formation can anchor probes to target DNA and might also provide an additional layer of target selectivity. Here, we developed a new crosslinking reaction for the covalent capture of specific nucleic acid sequences. This process involved reaction of an abasic (Ap) site in a probe strand with an adenine residue in the target strand and was used for the detection of a disease-relevant T→A mutation at position 1799 of the human BRAF kinase gene sequence. Ap-containing probes were easily prepared and displayed excellent specificity for the mutant sequence under isothermal assay conditions. It was further shown that nanopore technology provides a high contrast-in essence, digital-signal that enables sensitive, single-molecule sensing of the cross-linked duplexes. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. A silicon-based electrochemical sensor for highly sensitive, specific, label-free and real-time DNA detection.

    PubMed

    Guo, Yuanyuan; Su, Shao; Wei, Xinpan; Zhong, Yiling; Su, Yuanyuan; Huang, Qing; Fan, Chunhai; He, Yao

    2013-11-08

    We herein present a new kind of silicon-based electrochemical sensor using a gold nanoparticles-decorated silicon wafer (AuNPs@Si) as a high-performance electrode, which is facilely prepared via in situ AuNPs growth on a silicon wafer. Particularly significantly, the resultant electrochemical sensor is efficacious for label-free DNA detection with high sensitivity due to the unique merits of the prepared silicon-based electrode. Typically, DNA at remarkably low concentrations (1-10 fM) could be readily detected without requiring additional signal-amplification procedures, which is better than or comparable to the lowest DNA concentration ever detected via well-studied signal-amplification-assisted electrochemical sensors. Moreover, the silicon-based sensor features high specificity, allowing unambiguous discrimination of single-based mismatches. We further show that real-time DNA assembly is readily monitored via recording the intensity changes of current signals due to the robust thermal stability of the silicon-based electrode. The unprecedented advantages of the silicon-based electrochemical sensor would offer new opportunities for myriad sensing applications.

  13. Highly sensitive and specific detection of E. coli by a SERS nanobiosensor chip utilizing metallic nanosculptured thin films.

    PubMed

    Srivastava, Sachin K; Hamo, Hilla Ben; Kushmaro, Ariel; Marks, Robert S; Grüner, Christoph; Rauschenbach, Bernd; Abdulhalim, Ibrahim

    2015-05-07

    A nanobiosensor chip, utilizing surface enhanced Raman spectroscopy (SERS) on nanosculptured thin films (nSTFs) of silver, was shown to detect Escherichia coli (E. coli) bacteria down to the concentration level of a single bacterium. The sensor utilizes highly enhanced plasmonic nSTFs of silver on a silicon platform for the enhancement of Raman bands as checked with adsorbed 4-aminothiophenol molecules. T-4 bacteriophages were immobilized on the aforementioned surface of the chip for the specific capture of target E. coli bacteria. To demonstrate that no significant non-specific immobilization of other bacteria occurs, three different, additional bacterial strains, Chromobacterium violaceum, Paracoccus denitrificans and Pseudomonas aeruginosa were used. Furthermore, experiments performed on an additional strain of E. coli to address the specificity and reusability of the sensor showed that the sensor operates for different strains of E. coli and is reusable. Time resolved phase contrast microscopy of the E. coli-T4 bacteriophage chip was performed to study its interaction with bacteria over time. Results showed that the present sensor performs a fast, accurate and stable detection of E. coli with ultra-small concentrations of bacteria down to the level of a single bacterium in 10 μl volume of the sample.

  14. Nanozyme-based bio-barcode assay for high sensitive and logic-controlled specific detection of multiple DNAs.

    PubMed

    Lin, Xiaodong; Liu, Yaqing; Tao, Zhanhui; Gao, Jinting; Deng, Jiankang; Yin, Jinjin; Wang, Shuo

    2017-08-15

    Since HCV and HIV share a common transmission path, high sensitive detection of HIV and HCV gene is of significant importance to improve diagnosis accuracy and cure rate at early stage for HIV virus-infected patients. In our investigation, a novel nanozyme-based bio-barcode fluorescence amplified assay is successfully developed for simultaneous detection of HIV and HCV DNAs with excellent sensitivity in an enzyme-free and label-free condition. Here, bimetallic nanoparticles, PtAuNPs, present outstanding peroxidase-like activity and act as barcode to catalyze oxidation of nonfluorescent substrate of amplex red (AR) into fluorescent resorufin generating stable and sensitive "Turn On" fluorescent output signal, which is for the first time to be integrated with bio-barcode strategy for fluorescence detection DNA. Furthermore, the provided strategy presents excellent specificity and can distinguish single-base mismatched mutant from target DNA. What interesting is that cascaded INHIBIT-OR logic gate is integrated with biosensors for the first time to distinguish individual target DNA from each other under logic function control, which presents great application in development of rapid and intelligent detection. Copyright © 2017. Published by Elsevier B.V.

  15. Degenerate and Nested PCR: a Highly Sensitive and Specific Method for Detection of Human Papillomavirus Infection in Cutaneous Warts

    PubMed Central

    Harwood, Catherine A.; Spink, Patricia J.; Surentheran, T.; Leigh, Irene M.; de Villiers, Ethel-Michele; McGregor, Jane M.; Proby, Charlotte M.; Breuer, Judith

    1999-01-01

    The role of human papillomavirus (HPV) in anogenital carcinogenesis is firmly established, but evidence that supports a similar role in skin remains speculative. Immunosuppressed renal transplant recipients have an increased incidence of viral warts and nonmelanoma skin cancer, and the presence of HPV DNA in these lesions, especially types associated with the condition epidermodysplasia verruciformis (EV), has led to suggestions that HPV may play a pathogenic role. However, differences in the specificities and sensitivities of techniques used to detect HPV in skin have led to wide discrepancies in the spectrum of HPV types reported. We describe a degenerate nested PCR technique with the capacity to detect a broad spectrum of cutaneous, mucosal, and EV HPV types. In a series of 51 warts from 23 renal transplant recipients, this method detected HPV DNA in all lesions, representing a significant improvement over many previously published studies. Cutaneous types were found in 84.3% of warts and EV types were found in 80.4% of warts, whereas mucosal types were detected in 27.4% of warts. In addition, the method allowed codetection of two or more distinct HPV types in 94.1% of lesions. In contrast, single HPV types were detected in all but 1 of 20 warts from 15 immunocompetent individuals. In summary, we have established a highly sensitive and comprehensive degenerate PCR methodology for detection and genotyping of HPV from the skin and have demonstrated a diverse spectrum of multiple HPV types in cutaneous warts from transplant recipients. Studies designed to assess the significance of these findings to cutaneous carcinogenesis are under way. PMID:10523550

  16. Specific detection of Flt3 point mutations by highly sensitive real-time polymerase chain reaction in acute myeloid leukemia.

    PubMed

    Scholl, Sebastian; Krause, Claudia; Loncarevic, Ivan F; Müller, Rouven; Kunert, Christa; Wedding, Ulrich; Sayer, Herbert G; Clement, Joachim H; Höffken, Klaus

    2005-06-01

    Among activating class III receptor tyrosine kinase (Flt3) mutations, internal tandem duplications of Flt3 (Flt3-ITD) are detected in about 25% of patients with acute myeloid leukemia (AML). In contrast, mutations within the tyrosine kinase domain of Flt3 (Flt3-TKD mutations) are less frequent (approximately 7%), and there are only limited data on the frequency of recently demonstrated activating Flt3 point mutation at codon 592 (Flt3-V592A mutation). We evaluated a new approach for rapid screening of Flt3-TKD and Flt3-V592A mutations using the fluorescence resonance energy transfer (FRET) principle in a group of 122 patients. Based on individual Flt3-TKD mutations, we designed patient-specific primers to perform a highly sensitive polymerase chain reaction (PCR) assay for rapid detection of minimal residual disease (MRD). We also used a model system with MonoMac-6 cells carrying the Flt3-V592A mutation to establish a mutation-specific real-time PCR approach also for this molecular aberration. We identified 9 cases (8%) of Flt3-TKD mutations (5 cases of mutation D835Y, 3 cases of mutation D835H, and 1 case of mutation Del836), and no cases of Flt3-V592A mutation. Screening for Flt3-TKD mutations with fluorescent probes is equivalent to conventional screening using standard PCR followed by EcoRV restriction. We present a real-time PCR protocol that can be used for MRD analyses based on individual Flt3-TKD mutations. Examples of MRD analyses are presented for all 3 subtypes of Flt3-TKD mutation identified in this study. In summary, we demonstrate new methodological approaches for rapid screening of Flt3 point mutations and for detection of MRD based on patient-specific Flt3-TKD mutations.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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

  2. 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

    2015-08-04

    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.

  3. Morin Stain Detects Aluminum-Containing Macrophages in Macrophagic Myofasciitis and Vaccination Granuloma With High Sensitivity and Specificity.

    PubMed

    Chkheidze, Rati; Burns, Dennis K; White, Charles L; Castro, Diana; Fuller, Julie; Cai, Chunyu

    2017-04-01

    Macrophagic myofasciitis (MMF) is an inflammatory condition associated with the intramuscular (i.m.) injection of aluminum adjuvant-containing vaccines. It is clinically characterized by myalgia, weakness, and chronic fatigue and histologically by aggregates of cohesive macrophages with abundant basophilic, periodic acid-Schiff (PAS)-positive, diastase-resistant granules that percolate through the peri- and endomysium without eliciting substantial myofiber damage. The definitive diagnosis of MMF requires demonstration of aluminum within these macrophages. We evaluated the Morin stain, a simple, 2-step histochemical stain for aluminum, as a confirmatory diagnostic tool for MMF. Among 2270 muscle biopsies processed at UTSW between 2010 and 2015, a total of 12 MMF cases and 1 subcutaneous vaccination granuloma case were identified (11 pediatric, 2 adults). With the Morin stain, all 13 cases showed strong granular reactivity within the cytoplasm of macrophages but not in myofibers or connective tissue. Three cases of inflammatory myopathy with abundant macrophages (IMAM), 8 cases of granulomatous inflammation and 23 other deltoid muscle biopsies used as controls were all negative. Morin stain could be used in both formalin-fixed paraffin-embedded and cryostat sections. Thus, Morin stain detects aluminum with high sensitivity and specificity in human muscle and soft tissue and may improve the diagnostic yield of MMF and vaccination granuloma. © 2017 American Association of Neuropathologists, Inc.

  4. Activated phosphonated trifunctional chelates for highly sensitive lanthanide-based FRET immunoassays applied to total prostate specific antigen detection.

    PubMed

    Nchimi-Nono, Katia; Wegner, K David; Lindén, Stina; Lecointre, Alexandre; Ehret-Sabatier, Laurence; Shakir, Shakir; Hildebrandt, Niko; Charbonnière, Loïc J

    2013-10-14

    The first example of an activated phosphonated trifunctional chelate (TFC) is presented, which combines a non-macrocyclic coordination site for lanthanide coordination based on two aminobis-methylphosphonate coordinating arms, a central bispyrazolylpyridyl antenna and an N-hydroxysuccinimide ester in para position of the central pyridine as an activated function for the labeling of biomaterial. The synthesis of the TFC is presented together with photo-physical studies of the related Tb and Eu complexes. Excited state lifetime measurements in H2O and D2O confirmed an excellent shielding of the cation from water molecules with a hydration number of zero. The Tb complex provides a high photoluminescence (PL) quantum yield of 24% in aqueous solutions (0.01 M Tris-HCl, pH 7.4) and a very long luminescence lifetime of 2.6 ms. The activated ligand was conjugated to different biological compounds such as streptavidin, and a monoclonal antibody against total prostate specific antigen (TPSA). In combination with AlexaFluor647 (AF647) and crosslinked allophycocyanin (XL665) antibody (ABs) conjugates, homogeneous time-resolved Fluorescence Resonance Energy Transfer (FRET) immunoassays of TPSA were performed in serum samples. The Tb donor-dye acceptor FRET pairs provided large Förster distances of 5.3 nm (AF647) and 7.1 nm (XL665). A detailed time-resolved FRET analysis of Tb donor and dye acceptor PL decays revealed average donor-acceptor distances of 4.2 nm (AF647) and 6.3 nm (XL665) within the sandwich immunocomplex and FRET efficiencies of 0.79 and 0.68, respectively. Very low detection limits of 1.4 ng mL(-1) (43 pM) and 2.4 ng mL(-1) (74 pM) TPSA were determined using a KRYPTOR fluorescence immunoanalyzer. These results demonstrate the applicability of our novel Tb-bioconjugates for highly sensitive clinical diagnostics.

  5. 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

  6. 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.

  7. Highly sensitive fluorescence quantitative detection of specific DNA sequences with molecular beacons and nucleic acid dye SYBR Green I.

    PubMed

    Xiang, Dongshan; Zhai, Kun; Xiang, Wenjun; Wang, Lianzhi

    2014-11-01

    A highly sensitive fluorescence method of quantitative detection for specific DNA sequence is developed based on molecular beacon (MB) and nucleic acid dye SYBR Green I by synchronous fluorescence analysis. It is demonstrated by an oligonucleotide sequence of wild-type HBV (target DNA) as a model system. In this strategy, the fluorophore of MB is designed to be 6-carboxyfluorescein group (FAM), and the maximum excitation wavelength and maximum emission wavelength are both very close to that of SYBR Green I. In the presence of targets DNA, the MBs hybridize with the targets DNA and form double-strand DNA (dsDNA), the fluorophore FAM is separated from the quencher BHQ-1, thus the fluorophore emit fluorescence. At the same time, SYBR Green I binds to dsDNA, the fluorescence intensity of SYBR Green I is significantly enhanced. When targets DNA are detected by synchronous fluorescence analysis, the fluorescence peaks of FAM and SYBR Green I overlap completely, so the fluorescence signal of system will be significantly enhanced. Thus, highly sensitive fluorescence quantitative detection for DNA can be realized. Under the optimum conditions, the total fluorescence intensity of FAM and SYBR Green I exhibits good linear dependence on concentration of targets DNA in the range from 2×10(-11) to 2.5×10(-9)M. The detection limit of target DNA is estimated to be 9×10(-12)M (3σ). Compared with previously reported methods of detection DNA with MB, the proposed method can significantly enhance the detection sensitivity.

  8. High specificity targeting and detection of human neuroblastoma using multifunctional anti-GD2 iron-oxide nanoparticles

    PubMed Central

    Baiu, Dana C.; Artz, Nathan S.; McElreath, Meghan R.; Menapace, Bryan D.; Hernando, Diego; Reeder, Scott B.; Grüttner, Cordula; Otto, Mario

    2015-01-01

    Aim To develop biocompatible, tumor-specific multifunctional iron oxide nanoconstructs targeting neuroblastoma, an aggressive pediatric malignancy. Materials & methods Clinical-grade humanized monoclonal antibody (hu14.18K322A), designed to target GD2 antigen on neuroblastoma with reduced non-specific immune interactions, was conjugated to hydroxyethyl starch-coated iron oxide nanoparticles. Targeting capability in vitro and in vivo was assessed by immunofluorescence, electron microscopy, analytical spectrophotometry, histochemistry and magnetic resonance R2* relaxometry. Results The biocompatible nanoconstructs demonstrated high tumor-specificity in vitro and in vivo, and low background uptake in a mouse flank xenograft model. Specific accumulation in tumors enabled particle visualization and quantification by magnetic resonance R2* mapping. Conclusions Our findings support the further development towards clinical application of this anti-GD2 iron-oxide nanoconstruct as diagnostic and therapeutic scaffold for neuroblastoma and potentially other GD2 positive malignancies. PMID:26420448

  9. [Establishment of a high sensitive indirect ELISA for detecting specific antibodies against H9 subtype avian influenza virus].

    PubMed

    Zhang, Wei; Hou, Lidan; Song, Jie; Zhang, Shuang; Li, Yun; Li, Jing; Sun, Lei; Fan, Wenhui; Liu, Wenjun

    2017-08-25

    H9 subtype avian influenza virus causes worldwide epidemic, resulting in enormous economic losses of poultry production. In the present study, an indirect ELISA method was established for more accurate and specific detection. The recombinant protein of the globular head domain of HA of H9 subtype avian influenza virus was used as antigen. Specific blocking buffers and dilution buffers were determined to increase the sensitivity and specificity. The sensitivity of ELISA was higher than that of hemagglutination inhibition (HI) test. The coating antigen is very specific and no cross-reactivity with positive serum against H3N2, H5N2 and H7N9 subtype influenza viruses, Newcastle disease virus, avian infectious bronchitis virus, avian infectious disease virus, and egg drop syndrome virus. Two hundred of clinical sera samples were examined. The results indicate the coincidence rate between ELISA and HI test reached 97%. In addition, there was a positive correlation between OD450 values and the logarithm of HI titer to the base 2 of an individual serum sample (R2=0.981 1).

  10. Towards functional group-specific detection in high-performance liquid chromatography using mid-infrared quantum cascade lasers.

    PubMed

    Edelmann, A; Ruzicka, C; Frank, J; Lend, B; Schrenk, W; Gornik, E; Strasser, G

    2001-11-16

    A distributed feedback quantum cascade laser was applied for the first time as a powerful light source for mid-infrared (MIR) detection in liquid chromatography. Fructose and glucose in red wine were separated with an isocratic HPLC system, which was connected to a custom-made flow cell. This flow cell was constructed of two diamond windows with adjustable spacing and two hollow wave-guides for guiding the incoming and outgoing light. The HPLC column based on an ion-exchange resin with calcium(II) counter ion was run at 80 degrees C with 0.04% formic acid as the mobile phase. Under these conditions the carbohydrates could not be completely separated from the organic acids also present in wine. However, the emission of the laser at 1067 cm(-1) matches the absorption maximum of fructose and glucose, whereas the organic acids do not absorb appreciably at this wavenumber. Thus group-specific detection could be achieved. Additionally, the optical path length could be increased from 25 to 125 microm, which is very promising in gaining enhanced sensitivity compared to Fourier transform IR detection.

  11. Immunohistochemistry is highly sensitive and specific for detecting the BRAF V600E mutation in papillary thyroid carcinoma.

    PubMed

    Sun, Jian; Zhang, Jing; Lu, Junliang; Gao, Jie; Lu, Tao; Ren, Xinyu; Duan, Huanli; Liang, Zhiyong

    2015-01-01

    The V600E mutation in the B-type Raf kinase (BRAF) gene is a common genetic change in cases of papillary thyroid carcinoma (PTC) that appears to play a key role in the development and progression of this disease. We sought to assess the sensitivity and specificity of immunohistochemical detection of this mutation with a V600E mutated BRAF antibody in a Chinese PTC cohort. In this study, we used fully automated immunohistochemistry (IHC) assay with a BRAF V600E (VE1) mouse monoclonal primary antibody to screen for the BRAF V600E mutation in 556 cases of PTC. Moreover, to verify the IHC staining results, real-time PCR was applied to detect this mutation in the same patient cohort. Among the 556 cases in the examined primary PTC cohort, 414 (74.5%) cases and 419 (75.4%) cases were positive for the BRAF V600E mutation by IHC staining and by real-time PCR, respectively. The real-time PCR results indicated that the sensitivity and specificity of IHC staining for the BRAF V600E mutation were 98.8% and 100%, respectively. The BRAF V600E mutation was common among Chinese patients with primary PTC, and was strongly correlated with older patient age and the conventional subtype of PTC but was not associated with parameters of clinicopathological aggressiveness. The fully automated IHC is a reliable technique that can serve as an alternative to molecular biological approaches for the routine detection of the BRAF V600E mutation in PTC patients.

  12. Silver nanoclusters with enhanced fluorescence and specific ion recognition capability triggered by alcohol solvents: a highly selective fluorimetric strategy for detecting iodide ions in urine.

    PubMed

    Feng, Luping; Sun, Zongzhao; Liu, Huan; Liu, Min; Jiang, Yao; Fan, Chuan; Cai, Yuanyuan; Zhang, Sheng; Xu, Jihong; Wang, Hua

    2017-08-22

    Alcohol solvents especially isopropanol were demonstrated for the first time to endow silver nanoclusters (AgNCs) in water with dramatically enhanced red fluorescence. More importantly, the specific iodide recognition capability of the AgNCs could thus be obtained towards a highly selective fluorimetric assay for detecting iodide ions in urine.

  13. A novel signal-amplified strategy based on assembly reactivation for highly specific and sensitive detection of chair-like antiparallel G-quadruplex.

    PubMed

    Gai, Wei; Yang, Qianfan; Xiang, Junfeng; Jiang, Wei; Li, Qian; Sun, Hongxia; Yu, Lijia; Shang, Qian; Guan, Aijiao; Zhang, Hong; Tang, Yalin

    2013-02-21

    Specific and sensitive identification of special G-quadruplex structures is an important issue and attracts increasing interest. In this paper, a novel, signal-amplified strategy is proposed for the specific detection of G-quadruplexes, which is very different from those currently-dominating methods based on single-molecular fluorescent probes. In this strategy, a 'special designed' cyanine dye loses the ability of self-assembly in solution but still keeps its assembly potential. When the G-quadruplex with a specific structure is present, the assembly potential of the dye is reactivated so that it can assemble to J-aggregates. Other DNA motifs without this specific structure cannot activate its assembly potential no matter if they are double-stranded or quadruplex DNA. Since the assembly is quite specific to structure, the induced procedure of the aggregates provides high specificity for this strategy. In addition, the attached aggregates exponentially amplify the signal due to the signal stacking of those monomers within the aggregates, which then significantly enhances the sensitivity of this strategy. As a result, this strategy exhibits a highly specific and sensitive detection ability for specific G-quadruplex structures both between quadruplexes and non-quadruplexes and among diverse quadruplex motifs.

  14. 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.

  15. Carbapenem inactivation: a very affordable and highly specific method for phenotypic detection of carbapenemase-producing Pseudomonas aeruginosa isolates compared with other methods.

    PubMed

    Akhi, Mohammad Taghi; Khalili, Younes; Ghotaslou, Reza; Kafil, Hossein Samadi; Yousefi, Saber; Nagili, Behroz; Goli, Hamid Reza

    2016-07-22

    This investigation was undertaken to compare phenotypic and molecular methods for detection of carbapenemase-producing Pseudomonas aeruginosa. A total of 245 non-duplicated isolates of P. aeruginosa were collected from hospitalized patients. Disc diffusion method was used to identify carbapenem-resistant bacteria. Three phenotypic methods, including Modified Hodge Test (MHT), Modified Carba NP (MCNP) test and Carbapenem Inactivation Method (CIM) were used for investigation of carbapenemase production. In addition, polymerase chain reaction (PCR) was used to detect carbapenemase encoding genes. Of 245 P. aeruginosa isolates investigated, 121 isolates were carbapenem-resistant. Among carbapenem-resistant isolates, 40, 39 and 35 isolates exhibited positive results using MHT, MCNP test and CIM, respectively. PCR indicated the presence of carbapenemase genes in 35 of carbapenem-resistant isolates. MHT showed low sensitivity and specificity for carbapenemase detection among P. aeruginosa isolates in comparison to PCR. CIM was most affordable and highly specific than MCNP test compared with the molecular method.

  16. 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

  17. High throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry

    PubMed Central

    Porichis, Filippos; Hart, Meghan G.; Griesbeck, Morgane; Everett, Holly L.; Hassan, Muska; Baxter, Amy E.; Lindqvist, Madelene; Miller, Sara M.; Soghoian, Damien Z.; Kavanagh, Daniel G.; Reynolds, Susan; Norris, Brett; Mordecai, Scott K.; Nguyen, Quan; Lai, Chunfai; Kaufmann, Daniel E.

    2014-01-01

    Fluorescent in situ hybridization (FISH) is a method that uses fluorescent probes to detect specific nucleic acid sequences at the single cell level. Here we describe optimized protocols that exploit a highly sensitive FISH method based on branched DNA technology to detect mRNA and miRNA in human leukocytes. This technique can be multiplexed and combined with fluorescent antibody protein staining to addressa variety of questions in heterogeneous cell populations. We demonstrate antigen-specific upregulation of IFNγ and IL-2 mRNAs in HIV- and CMV-specific T cells. We show simultaneous detection of cytokine mRNA and corresponding protein in single cells. We apply this method to detect mRNAs for which flow antibodies against the corresponding proteins are poor or are not available. We use this technique to show modulation of a microRNA critical for T cell function, miR-155. We adapt this assay for simultaneous detection of mRNA and proteins by Image Stream technology. PMID:25472703

  18. Highly sensitive optical detection of specific protein in breast cancer cells using microstructured fiber in extremely low sample volume

    NASA Astrophysics Data System (ADS)

    Padmanabhan, Saraswathi; Shinoj, Vengalathunadakal K.; Murukeshan, Vadakke M.; Padmanabhan, Parasuraman

    2010-01-01

    A simple optical method using hollow-core photonic crystal fiber for protein detection has been described. In this study, estrogen receptor (ER) from a MCF-7 breast carcinoma cell lysates immobilized inside a hollow-core photonic crystal fiber was detected using anti-ER primary antibody with either Alexa™ Fluor 488 (green fluorescent dye) or 555 (red Fluorescent dye) labeled Goat anti-rabbit IgG as the secondary antibody. The fluorescence fingerprints of the ERα protein were observed under fluorescence microscope, and its optical characteristics were analyzed. The ERα protein detection by this proposed method is based on immuno binding from sample volume as low as 50 nL. This method is expected to offer great potential as a biosensor for medical diagnostics and therapeutics applications.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  20. Highly stable, fluorescence-labeled heptapeptides substituted with a D-amino acid for the specific detection of oxidized low-density lipoprotein in plasma.

    PubMed

    Sato, Akira; Yamanaka, Hikaru; Oe, Keitaro; Yokoyama, Izumi; Yamazaki, Yoji; Ebina, Keiichi

    2015-03-01

    Probes that can detect oxidized low-density lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques can be useful for the diagnosis, prevention, and treatment of atherosclerosis. Recently, we have reported that two heptapeptides (Lys-Trp-Tyr-Lys-Asp-Gly-Asp, KP6) coupled to fluorescein isothiocyanate (FITC) through the ε-amino group of N-terminus Lys in the absence/presence of 6-amino-n-caproic acid (AC) linker to FITC-(FITC)KP6 and (FITC-AC)KP6-can be useful as fluorescent probes for the specific detection of ox-LDL. In this study, to develop the fluorescent peptides with high plasma stability for the specific detection of ox-LDL, we investigated the interaction of (FITC)KP6 and (FITC-AC)KP6 substituted with D-Lys at the N-terminus-(FITC)dKP6 and (FITC-AC)dKP6-with ox-LDL, and the in vitro stability of these peptides in mouse plasma. (FITC)dKP6 and (FITC-AC)dKP6 bound with high specificity to ox-LDL in a dose-dependent manner, and also to ox-LDL in the mouse plasma. Furthermore, (FITC)dKP6 was more stable than (FITC)KP6 in mouse plasma (102.1% versus 69.0% remained after 1 h). These findings strongly suggest that (FITC)dKP6 and (FITC-AC)dKP6 may be effective fluorescent probes with higher plasma stability than (FITC)KP6 and (FITC-AC)KP6 for the specific detection of ox-LDL.

  1. A non-variable L1-peptide displays high sensitivity and specificity for detecting women having human papillomavirus-associated cervical lesions.

    PubMed

    Urquiza, Mauricio; Guevara, Tatiana; Sanchez, Ricardo; Vanegas, Magnolia; Patarroyo, Manuel E

    2008-06-01

    Anti-human papillomavirus (HPV) antibody detection is promising technique for detecting women at risk of suffering cervical cancer, since potentially oncogenic, persistent, long-term HPV-infections elicit an antibody response which is rarely detected in transitory HPV-infection patients. We have identified a non-variable C-terminus L1-peptide, belonging to an alpha-helix surface exposed on L1-protein, specifically recognized by antibodies from HPV-associated cervical lesion patients. This peptide tested against 313 sera presented higher reactivity with antibodies from cervical cancer (OD mean 0.43+/-0.13) or cervical lesion patients (OD mean 0.41+/-0.17) than antibodies from normal cytology patients (OD mean 0.17+/-0.03). High-risk HPV-infected patients presented higher antibody reactivity (OD mean 0.36+/-0.17) than high-risk HPV-non-infected patients (OD mean 0.22+/-0.11). This peptide showed 88.36% sensitivity, 99.39% specificity and 94.21% correct classification of high risk-HPV cervical lesion or cervical cancer patients. This peptide should be taken into account for designing serological screening or diagnostic tests for use in a clinical scenario.

  2. 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.

  3. A computational imaging target specific detectivity metric

    NASA Astrophysics Data System (ADS)

    Preece, Bradley L.; Nehmetallah, George

    2017-05-01

    Due to the large quantity of low-cost, high-speed computational processing available today, computational imaging (CI) systems are expected to have a major role for next generation multifunctional cameras. The purpose of this work is to quantify the performance of theses CI systems in a standardized manner. Due to the diversity of CI system designs that are available today or proposed in the near future, significant challenges in modeling and calculating a standardized detection signal-to-noise ratio (SNR) to measure the performance of these systems. In this paper, we developed a path forward for a standardized detectivity metric for CI systems. The detectivity metric is designed to evaluate the performance of a CI system searching for a specific known target or signal of interest, and is defined as the optimal linear matched filter SNR, similar to the Hotelling SNR, calculated in computational space with special considerations for standardization. Therefore, the detectivity metric is designed to be flexible, in order to handle various types of CI systems and specific targets, while keeping the complexity and assumptions of the systems to a minimum.

  4. 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

  5. Prostatic cancer surveillance following whole-gland high-intensity focused ultrasound: comparison of MRI and prostate-specific antigen for detection of residual or recurrent disease

    PubMed Central

    Punwani, S; Emberton, M; Walkden, M; Sohaib, A; Freeman, A; Ahmed, H; Allen, C; Kirkham, A

    2012-01-01

    Objective This retrospective study compares dynamic contrast-enhanced (DCE) MRI with the serial prostate-specific antigen (PSA) measurement for detection of residual disease following whole-gland high-intensity focused ultrasound (HIFU) therapy of prostate cancer. Methods Patients in whom post-HIFU DCE-MRI was followed within 3 months by ultrasound-guided transrectal biopsy were selected from a local database. 26 patients met the study inclusion criteria. Serial PSA levels following HIFU and post-HIFU follow-up MRI were retrieved for each patient. Three radiologists unaware of other investigative results independently assessed post-HIFU MRI studies for the presence of cancer, scoring on a four-point scale (1, no disease; 2, probably no disease; 3, probably residual disease; and 4, residual disease). Sensitivity, specificity and receiver operating characteristic (ROC) analysis were performed for each reader, post-HIFU PSA nadir and pre-biopsy PSA level thresholds of >0.2 and >0.5 ng ml−1. Results The sensitivity of DCE-MRI for detection of residual disease for the three readers ranged between 73% and 87%, and the specificity between 73% and 82%. There was good agreement between readers (κ=0.69–0.77). The sensitivity and specificity of PSA thresholds was 60–87% and 73–100%, respectively. The area under the ROC curve was greatest for pre-biopsy PSA (0.95). Conclusion DCE-MRI performed following whole-gland HIFU has similar sensitivity and specificity and ROC performance to serial PSA measurements for detection of residual or recurrent disease. PMID:22253342

  6. Automation of the ELISpot assay for high-throughput detection of antigen-specific T-cell responses.

    PubMed

    Almeida, Coral-Ann M; Roberts, Steven G; Laird, Rebecca; McKinnon, Elizabeth; Ahmed, Imran; Pfafferott, Katja; Turley, Joanne; Keane, Niamh M; Lucas, Andrew; Rushton, Ben; Chopra, Abha; Mallal, Simon; John, Mina

    2009-05-15

    The enzyme linked immunospot (ELISpot) assay is a fundamental tool in cellular immunology, providing both quantitative and qualitative information on cellular cytokine responses to defined antigens. It enables the comprehensive screening of patient derived peripheral blood mononuclear cells to reveal the antigenic restriction of T-cell responses and is an emerging technique in clinical laboratory investigation of certain infectious diseases. As with all cellular-based assays, the final results of the assay are dependent on a number of technical variables that may impact precision if not highly standardised between operators. When studies that are large scale or using multiple antigens are set up manually, these assays may be labour intensive, have many manual handling steps, are subject to data and sample integrity failure and may show large inter-operator variability. Here we describe the successful automated performance of the interferon (IFN)-gamma ELISpot assay from cell counting through to electronic capture of cytokine quantitation and present the results of a comparison between automated and manual performance of the ELISpot assay. The mean number of spot forming units enumerated by both methods for limiting dilutions of CMV, EBV and influenza (CEF)-derived peptides in six healthy individuals were highly correlated (r>0.83, p<0.05). The precision results from the automated system compared favourably with the manual ELISpot and further ensured electronic tracking, increased through-put and reduced turnaround time.

  7. Use of high-resolution melting and melting temperature-shift assays for specific detection and identification of Bacillus anthracis based on single nucleotide discrimination.

    PubMed

    Derzelle, Sylviane; Mendy, Christiane; Laroche, Séverine; Madani, Nora

    2011-11-01

    Single nucleotide polymorphisms (SNPs) are important diagnostic markers for the detection and differentiation of Bacillus anthracis. High-Resolution Melting (HRM) and Melting Temperature (Tm)-shift methods are two approaches that enable SNP detection without the need for expensive labeled probes. We evaluated the potential diagnostic capability of those methods to discriminate B. anthracis from the other members of the B. cereus group. Two assays targeting B. anthracis-specific SNPs in the plcR and gyrA genes were designed for each method and used to genotype a panel of 155 Bacilli strains. All B. anthracis isolates (n=65) were correctly and unambiguously identified. Assays also proved to be appropriate for the direct genotyping of biological samples. They could reliably detect B. anthracis in contaminated organs containing as little as 10(3)CFU/ml, corresponding to a few genome equivalents per reaction. The HRM and Tm-shift applications described here represent valuable tools for specific identification of B. anthracis at reduced cost.

  8. Highly sensitive and specific detection of histamine via the formation of a self-assembled magic number cluster with thymine by mass spectrometry.

    PubMed

    Sun, Jiamu; Qin, Zhen; Liu, Jia; Zhang, Chengsen; Luo, Hai

    2014-06-21

    A novel method for the detection of histamine (HIM) via the formation of a self-assembled magic number cluster with thymine (T) by electrospray ionization tandem mass spectrometry (ESI-MS/MS) is described. The formation of the magic number cluster [T17 + HIM + 2H](2+) shifts the MS signal of histamine to the interference-free higher mass range and the signal intensity is increased by four orders of magnitude. In addition, the formation of [T17 + HIM + 2H](2+) is highly specific to histamine compared with its metabolite and other similar biogenic amines, which may be attributed to both of its amino and imidazole groups. The linear dynamic range of the method is in the range of 1 nM-20 μM, and the limit of detection can be as low as 0.1 nM. The feasibility of this method is further demonstrated by the quantitative analysis of histamine in a red wine sample. Since little sample preparation or separation is required before the analysis, this method provides a rapid new way for the sensitive and specific detection of histamine by MS.

  9. The first specific detection of a highly pathogenic avian influenza virus (H5N1) in Ivory Coast.

    PubMed

    Couacy-Hymann, E; Danho, T; Keita, D; Bodjo, S C; Kouakou, C; Koffi, Y M; Beudje, F; Tripodi, A; de Benedictis, P; Cattoli, G

    2009-02-01

    The Virology Laboratory of the Central Laboratory of Animal Diseases in Ivory Coast at Bingerville received samples of wild and domestic avian species between February and December 2006. An RT-PCR technique was used to test for avian influenza (AI) and highly pathogenic AI subtype viruses. Among 2125 samples, 16 were type A positive; of which, 12 were later confirmed to be H5N1. Fifteen of these 16 type A positive samples were inoculated into the chorioallantoic cavity of 11-day-old embryonated hens' eggs for virus isolation. Eight produced virus with hemagglutination titres from 1/64 to 1/512. The 4/16 M-RT-PCR positive samples, which were H5N1 negative, were shown to be H7 subtype negative. The diagnostic efficiency of the laboratory for the surveillance of H5N1 in Ivory Coast was demonstrated. The positive cases of H5N1 were from a sparrowhawk (Accipter nisus); live market poultry and in free-range poultry, where the mortality rate was approximately 20% (2/10) and 96.7% (29/30) respectively. Currently, investigations into intensive poultry farms have proved negative for H5N1. No human cases have been reported this time.

  10. Development of a highly sensitive and robust Cor a 9 specific enzyme-linked immunosorbent assay for the detection of hazelnut traces.

    PubMed

    Trashin, Stanislav A; Cucu, Tatiana; Devreese, Bart; Adriaens, Annemie; De Meulenaer, Bruno

    2011-12-05

    Allergy to tree nuts represents an acute health problem. Sensitized people can be inadvertently exposed to hidden allergens resulting from cross-contamination of foods. For this reason, reliable and highly sensitive analytical methods are needed to be developed for control and labeling of food ingredients and products. In the present paper we have proposed a new allergen specific sandwich-ELISA for hazelnut operated in optical and electrochemical modes. The ELISA was based on chicken egg yolk antibodies raised against a major hazelnut allergen, Cor a 9. The developed ELISA has a limit of detection in phosphate buffer of 4 ng mL(-1). No significant cross-reactivity with peanut, wheat or other food ingredients has been detected. Extracts of blank control cookies did not show any false positive response and the limit of detection in cookies was estimated to be 0.1 μg of hazelnut protein per g of food (0.1 ppm). The ELISA protocol was successfully adapted to operate in electrochemical mode and it was applied for the detection of hazelnut traces in cookies.

  11. High Concentrations of Angiopoietin-Like Protein 4 Detected in Serum from Patients with Rheumatoid Arthritis Can Be Explained by Non-Specific Antibody Reactivity

    PubMed Central

    Makoveichuk, Elena; Ruge, Toralph; Nilsson, Solveig; Södergren, Anna

    2017-01-01

    Angiopoietin-like protein 4 (ANGPTL4) is suggested to be a master regulator of plasma triglyceride metabolism. Our aim was to study whether the previously reported high levels of ANGPTL4 detected in serum from patients with rheumatoid arthritis (RA) by ELISA was due to any specific molecular form of this protein (oligomers, monomers or fragments). ANGPTL4 levels were first determined in serum from 68 RA patients and 43 age and sex matched control subjects and the mean values differed by a factor of 5.0. Then, ANGPTL4 was analyzed after size exclusion chromatography (SEC) of serum samples. With serum from one of the RA patients with high levels of ANGPTL4, the dominant reactivity was found in fractions corresponding to high-molecular weight proteins. In addition, a minor peak of reactivity eluting late from the column was found both in the patient and in controls. By the use of HeteroBlock®, and by careful selection of antibodies, we documented non-specific reactions for ANGPTL4 in 39% of samples from the RA patients, most likely due to cross-reactivity of the antibodies with rheumatoid factor (RF). The corresponding figure for control subjects was 6.3%. After corrections for non-specific reactions, the mean level of ANGPTL4 in serum from RA patients was still significantly higher than in control individuals (mean levels were 101±62 and 67±39 ng/ml respectively, P = 0.02). We re-analyzed samples from our previously published studies on ANGPL4 levels in patients on hemodialysis and patients with diabetes type 2. These samples did not show false positive reactions. The levels of ANGPTL4 were comparable to those detected previously. PMID:28107351

  12. 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).

  13. Fast specific field detection of RHDVb.

    PubMed

    Dalton, K P; Nicieza, I; Podadera, A; de Llano, D; Martin Alonso, J M; de Los Toyos, J R; García Ocaña, M; Vázquez-Villa, F; Velasco, B; Landeta, O; Parra, F

    2017-02-28

    This work describes a simple and rapid test for field detection of the emerging rabbit pathogen RHDVb. The assay is specific for RHDVb, showing no cross-reactivity with other RHDV types giving a specific result in under 10 min using rabbit liquid exudates or liver homogenate samples taken at necropsy.

  14. Results of a Zika Virus (ZIKV) Immunoglobulin M-Specific Diagnostic Assay Are Highly Correlated With Detection of Neutralizing Anti-ZIKV Antibodies in Neonates With Congenital Disease.

    PubMed

    Cordeiro, Marli T; Brito, Carlos A A; Pena, Lindomar J; Castanha, Priscila M S; Gil, Laura H V G; Lopes, Kennya G S; Dhalia, Rafael; Meneses, Jucille A; Ishigami, Ana C; Mello, Luisa M; Alencar, Liciana X E; Guarines, Klarissa M; Rodrigues, Laura C; Marques, Ernesto T A

    2016-12-15

     Usually, immunoglobulin M (IgM) serologic analysis is not sufficiently specific to confirm Zika virus (ZIKV) infection. However, since IgM does not cross the placenta, it may be a good marker of infection in neonates.  We tested blood from 42 mothers and neonates with microcephaly and collected cerebrospinal fluid (CSF) specimens from 30 neonates. Molecular assays were performed for detection of ZIKV, dengue virus, and chikungunya virus; IgM enzyme-linked immunosorbent assays and plaque-reduction neutralization tests (PRNTs) were performed to detect ZIKV and dengue virus. No control neonates without microcephaly were evaluated.  Among neonates, all 42 tested positive for ZIKV IgM: 38 of 42 serum specimens (90.5%) were positive, whereas 30 of 30 CSF specimens (100%) were positive. ZIKV IgM-specific ELISA ratios, calculated as the mean optical density (OD) of the test sample when reacted on viral antigen divided by the mean OD of the negative control when reacted with viral antigen, were higher in CSF specimens (median, 14.9 [range, 9.3-16.4]) than in serum (median, 8.9 [range, 2.1-20.6]; P = .0003). All ZIKV IgM-positive results among the neonates were confirmed by the detection of neutralizing antibodies. Mother/neonate pairs with primary ZIKV infection had neutralizing antibodies to ZIKV only, and mother/neonate pairs with ZIKV virus infection secondary to infection with another flavivirus had high titers of neutralizing antibodies to ZIKV. Among secondary infections, median titers in serum were 2072 (range, 232-12 980) for mothers and 2730 (range, 398-12 980) for neonates (P < .0001), and the median titer in CSF was 93 (range, 40-578) among neonates (P < .0001).  Among neonates, detection of ZIKV IgM in serum is confirmatory of congenital ZIKV infection, and detection of ZIKV IgM in CSF is confirmatory of neurologic infection. Therefore, we recommend testing for ZIKV IgM in neonates suspected of having congenital ZIKV infection and performance of PRNTs

  15. Enzyme-mimetic catalyst-modified nanoporous SiO2-cellulose hybrid composites with high specific surface area for rapid H2O2 detection.

    PubMed

    Jiang, Yijun; Wang, Wei; Li, Xiutao; Wang, Xicheng; Zhou, Jianwei; Mu, Xindong

    2013-03-01

    Mesoporous silica-cellulose hybrid composites were prepared by surface sol-gel coating process on nature cellulose substance. The template CTAB (hexadecyl trimethyl ammonium Bromide) in the silica film can be removed by extraction to obtain high specific surface area (80.7 m(2) g(-1)), which is 2 orders of magnitude higher than that of raw cellulose. In the following, the enzyme-mimetic catalyst and chromogenic agent were introduced onto the hybrid system. Just as the peroxidase, the resultant hybrid material exhibits extraordinary sensitivity for the H2O2 and shows an immediate and obvious color change. The detection limit is about 1 μmol L(-1) by the naked eye.

  16. Effect of diabetes mellitus on high-grade prostate cancer detection among Japanese obese patients with prostate-specific antigen less than 10 ng/mL.

    PubMed

    Fukushima, Hiroshi; Masuda, Hitoshi; Kawakami, Satoru; Ito, Masaya; Sakura, Mizuaki; Numao, Noboru; Koga, Fumitaka; Saito, Kazutaka; Fujii, Yasuhisa; Yamamoto, Shinya; Yonese, Junji; Fukui, Iwao; Kihara, Kazunori

    2012-06-01

    To investigate the association of diabetes mellitus (DM) with prostate cancer (PCa) risk and grade among Japanese patients undergoing extended biopsy and to investigate how obesity modifies these relationships. We retrospectively evaluated the data from 2038 patients with a prostate-specific antigen (PSA) level <10 ng/mL undergoing initial extended biopsy at our institutions. The DM history was determined by self-report and medication use. Multivariate analyses of DM for PCa risk and grade were done using logistic regression. Moreover, we examined whether these associations were modified by the body mass index using subgroup analyses (nonobese <25 kg/m(2) or obese ≥25 kg/m(2)) and interaction tests. Cancer grade was classified according to the Gleason score (GS): low-grade (GS ≤6), intermediate-grade (GS 7), and high-grade (GS 8-10). Of 2038 patients, obesity and DM was observed in 606 (30%) and 213 (11%), respectively. Also, 836 patients (41%) had positive biopsy findings. On multivariate analysis, we found no significant association of DM with the risk of overall PCa (P = .106) or the risk of low-grade (P = .735), intermediate-grade (P = .119), or high-grade (P = .110) disease. When stratified by obesity, the relative risk (RR) of PCa detection for diabetic men apparently increased with higher cancer grade (low grade, RR = 1.19, P = .71; intermediate grade, RR = 2.01, P = .099; high-grade, RR = 4.03, P = .025). However, in the nonobese men, no association was noted between DM and PCa risk, irrespective of grade. Obesity modified the effect of DM on high-grade disease risk with a trend (P for interaction = .087). DM was associated with more aggressive PCa detection among Japanese obese patients with gray-zone PSA levels undergoing extended biopsy. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. A panel of TMPRSS2:ERG fusion transcript markers for urine-based prostate cancer detection with high specificity and sensitivity.

    PubMed

    Nguyen, Phuong-Nam; Violette, Philippe; Chan, Sam; Tanguay, Simon; Kassouf, Wassim; Aprikian, Armen; Chen, Junjian Z

    2011-03-01

    The TMPRSS2:ERG fusion is both prevalent and unique to prostate cancer (PCa) and has great potential for noninvasive diagnosis of PCa in bodily fluids. To evaluate the specificity and sensitivity of the TMPRSS2:ERG fusion in urine from diverse clinical contexts and to explore potential clinical applications. A total of 101 subjects were enrolled in 2008 from urologic oncology clinics to form three study groups: 44 PCa free, 46 confirmed PCa, and 11 negative prostate biopsies. The PCa-free group included females, healthy young men, and post-radical prostatectomy (RP) patients. The confirmed PCa group was composed of patients under active surveillance, scheduled for treatment, or with metastatic disease. Urine was collected after attentive digital rectal exam (DRE) and coded to blind group allocation for laboratory test. RNA from urine sediments was analyzed using a panel of four TMPRSS2:ERG fusion markers with quantitative polymerase chain reaction (qPCR). Our fusion markers demonstrated very high technical specificity and sensitivity for detecting a single fusion-positive cancer cell (VCaP) in the presence of at least 3000 cells in urine sediments. In clinical analysis, there were no fusion-positive samples in the PCa-free group (0 of 44 samples), while there were 16 of 46 (34.8%) fusion-positive samples in the confirmed PCa group. The fusion incidence varied significantly among the three PCa subgroups. The clinical sensitivity increased to 45.4% in cancer patients prior to treatments. The fusion markers were detected in 2 of 11 (18.2%) biopsy-negative patients, suggesting potentially false negative biopsies. This study is not prospective and is limited in sample sizes. Our novel panel of TMPRSS2:ERG fusion markers provided a very specific and sensitive tool for urine-based detection of PCa. Theses markers can potentially be used to diagnose patients with PCa who have negative biopsies. Copyright © 2010 European Association of Urology. Published by Elsevier B

  18. Sensitivity and specificity for detecting early glaucoma in eyes with high myopia from normative database of macular ganglion cell complex thickness obtained from normal non-myopic or highly myopic Asian eyes.

    PubMed

    Nakanishi, Hideo; Akagi, Tadamichi; Hangai, Masanori; Kimura, Yugo; Suda, Kenji; Kumagai, Kyoko Kawashima; Morooka, Satoshi; Ikeda, Hanako Ohashi; Yoshimura, Nagahisa

    2015-07-01

    We aimed to determine the sensitivity and specificity of the normative database of non-myopic and highly myopic eyes of the macular ganglion cell complex (mGCC) thickness embedded in the NIDEK RS-3000 spectral-domain optical coherence tomography (SD-OCT) for detecting early glaucoma in highly myopic eyes. Forty-seven highly myopic eyes (axial length ≥26.0 mm) of 47 subjects were studied. The SD-OCT images were used to determine the mGCC thickness within a 9-mm diameter circle centered on the fovea. The sensitivity and specificity of the non-myopic database were compared to that of the highly myopic database for distinguishing the early glaucomatous eyes from the non-glaucomatous eyes. The mGCC scans were classified as abnormal if at least one of the eight sectors of the significance map was < 1 % of the normative thickness. Twenty-one eyes were diagnosed to be non-glaucomatous and 26 eyes to have early glaucoma. . The average mGCC thickness was significantly thinner (80.9 ± 8.5 μm) in the early glaucoma group than in the non-glaucomatous group (91.2 ± 7.5 μm; p <1 × 10(-4)). The sensitivity was 96.2 % and specificity was 47.6 % when the non-myopic database was used, and the sensitivity was 92.3 % and the specificity was 90.5 % when the highly myopic database was used. The difference in the specificity was significant (p < 0.01). The significantly higher specificity of the myopic normative database for detecting early glaucoma in highly myopic eyes will lead to fewer false positive diagnoses. The database obtained from highly myopic eyes should be used when evaluating the mGCC thickness of highly myopic eyes.

  19. 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.

  20. Determination of pesticide residues in coconut water by liquid-liquid extraction and gas chromatography with electron-capture plus thermionic specific detection and solid-phase extraction and high-performance liquid chromatography with ultraviolet detection.

    PubMed

    Brito, N M; Navickiene, S; Polese, L; Jardim, E F G; Abakerli, R B; Ribeiro, M L

    2002-05-31

    Two simple methods were developed to determine 11 pesticides in coconut water, a natural isotonic drink rich in salts, sugars and vitamins consumed by the people and athletes. The first procedure involves solid-phase extraction using Sep-Pak Vac C18 disposable cartridges with methanol for elution. Isocratic analysis was carried out by means of high-performance liquid chromatography with ultraviolet detection at 254 nm to analyse captan, chlorothalonil, carbendazim, lufenuron and diafenthiuron. The other procedure is based on liquid-liquid extraction with hexane-dichloromethane (1:1, v/v), followed by gas chromatographic analysis with effluent splitting to electron-capture detection for determination of endosulfan, captan, tetradifon and trichlorfon and thermionic specific detection for determination of malathion, parathion-methyl and monocrotophos. The methods were validated with fortified samples at different concentration levels (0.01-12.0 mg/kg). Average recoveries ranged from 75 to 104% with relative standard deviations between 1.4 and 11.5%. Each recovery analysis was repeated at least five times. Limits of detection ranged from 0.002 to 2.0 mg/kg. The analytical procedures were applied to 15 samples and no detectable amounts of the pesticides were found in any samples under the conditions described.

  1. High bandwidth EDMR detection

    NASA Astrophysics Data System (ADS)

    Huebl, H.; Willems van Beveren, L. H.; Starrett, R. P.; McCamey, D. R.; Ferguson, A. J.

    2009-03-01

    Several proposals discuss the realization of quantum computation with the help of the spin degree of freedom in semiconductors. Electrically detected magnetic resonance (EDMR) provides a well established tool to investigate spin states in semiconductors which was recently extended to investigate the spin dynamics of phosphorus donors in silicon. Typically, the detection bandwidth of EDMR is limited by the characteristic RC time constant of the sample. In this contribution we show that by embedding the sample in a LRC resonant circuit, a so-called tank circuit, it is possible to overcome this limitations. Here, we investigate a silicon MOSFET where the microwave magnetic field to induce the spin transitions is generated on chip by a shorted coplanar stripline[1]. We monitor the MOSFET resistance with a current preamplifier and in-situ by the response of the LRC resonant circuit and observe a spin resonance signature in both cases. Investigating the detection bandwidth by using frequency modulation of the microwaves applied indicates that the spin signature observed with the tank circuit is limited at the high end currently by the experimental setup. This shows that this method has the expected high bandwidth opening the view to faster phenomena in EDMR in a more direct manner. [1] Willems van Beveren et al., APL 93, 072102 (2008)

  2. 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.

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

    PubMed Central

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

    2014-01-01

    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 m6A 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 m6A 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

  4. OR2M3: A Highly Specific and Narrowly Tuned Human Odorant Receptor for the Sensitive Detection of Onion Key Food Odorant 3-Mercapto-2-methylpentan-1-ol.

    PubMed

    Noe, Franziska; Polster, Johannes; Geithe, Christiane; Kotthoff, Matthias; Schieberle, Peter; Krautwurst, Dietmar

    2016-12-04

    The detection of key food odorants appears to be an important capability of odorant receptors. Here, thiols occupy an outstanding position among the 230 known key food odorants because of their very low odor thresholds. Members of the homologous series of 3-mercapto-2-methylalkan-1-ols have been described as onion key food odorants or food constituents and are detected at logarithmically different thresholds. 3-Mercapto-2-methylpentan-1-ol being the only key food odorant within this series also has the lowest odor threshold. Most odorants typically activate combinations of odorant receptors, which may be narrowly or broadly tuned. Consequently, a specific receptor activation pattern will define an odor quality. In contrast, here we show that just 1 of the 391 human odorant receptors, OR2M3, responded exclusively to 3-mercapto-2-methylpentan-1-ol of the 190 key food odorants tested, with a half maximal effective concentration at submicromolar concentration. Moreover, neither the Denisovan OR2M3 nor the closest OR2M3 homologs from five species did respond to this compound. This outstanding specificity of extremely narrowly tuned human OR2M3 can explain both odor qualities and odor threshold trend within a homologous series of 3-mercapto-2-methylalkan-1-ols and suggests a modern human-specific, food-related function of OR2M3 in detecting a single onion key food odorant.

  5. Specific detection by PCR of Streptococcus agalactiae in milk.

    PubMed

    Martinez, G; Harel, J; Gottschalk, M

    2001-01-01

    The aim of this study was to develop a simple and specific method for direct detection of Streptococcus agalactiae from cow's milk. The method was based on polymerase chain reaction (PCR) using species-specific and universal primers derived from the 16S rRNA gene. The amplification product was verified by restriction endonuclease digest and sequencing. Specific identification was proven on a collection of 147 S. agalactiae isolates of bovine and human origin. In addition, 17 strains belonging to different bacterial species that potentially can be found in milk samples also tested negative. The PCR developed was used for direct detection of S. agalactiae in milk, using for the first time with gram-positive bacteria the nucleic acid-binding properties of diatomaceous earth. The test, which has high specificity, high sensitivity (100 cfu/mL), and can be carried out in less than 24 h, represents an innovative diagnostic tool for the detection of S. agalactiae in milk.

  6. A new label-free electrochemical immunosensor based on dendritic core-shell AuPd@Au nanocrystals for highly sensitive detection of prostate specific antigen.

    PubMed

    Wang, Rui; Liu, Wei-Dong; Wang, Ai-Jun; Xue, Yadong; Wu, Liang; Feng, Jiu-Ju

    2018-01-15

    Herein, bimetallic dendritic core-shell AuPd@Au nanocrystals (AuPd@Au NCs) were prepared by a simple one-pot aqueous method using xanthine as a green growth-directing agent. By virtue of the enhanced peak currents in the H2O2 reduction catalyzed by AuPd@Au NCs, a label-free immunosensor was constructed for the detection of prostate specific antigen (PSA). The immunosensor exhibited significantly improved analytical performance for the assay of PSA with wide linear range of 0.1-50ngmL(-1) and low detection limit of 0.078ngmL(-1) (S/N = 3), coupled with the improved stability, reproducibility and selectivity. It provides a promising platform for clinical research and diagnosis. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Circulating cell-free DNA has a high degree of specificity to detect exon 19 deletions and the single-point substitution mutation L858R in non-small cell lung cancer.

    PubMed

    Qian, Xin; Liu, Jia; Sun, Yuhui; Wang, Meifang; Lei, Huaiding; Luo, Guoshi; Liu, Xianjun; Xiong, Chang; Liu, Dan; Liu, Jie; Tang, Yijun

    2016-05-17

    Detection of an epidermal growth factor receptor (EGFR) mutation in circulating cell-free DNA (cfDNA) is a noninvasive method to collect genetic information to guide treatment of lung cancer with tyrosine-kinase inhibitors (TKIs). However, the association between cfDNA and detection of EGFR mutations in tumor tissue remains unclear. Here, a meta-analysis was performed to determine whether cfDNA could serve as a substitute for tissue specimens for the detection of EGFR mutations. The pooled sensitivity, specificity, and areas under the curve of cfDNA were 0.60, 0.94, and 0.9208 for the detection of EGFR mutations, 0.64, 0.99, and 0.9583 for detection of the exon 19 deletion, and 0.57, 0.99, and 0.9605 for the detection of the L858R mutation, respectively. Our results showed that cfDNA has a high degree of specificity to detect exon 19 deletions and L858R mutation. Due to its high specificity and noninvasive characteristics, cfDNA analysis presents a promising method to screen for mutations in NSCLC and predict patient response to EGFR-TKI treatment, dynamically assess treatment outcome, and facilitate early detection of resistance mutations.

  8. Sensitive electrochemical aptamer cytosensor for highly specific detection of cancer cells based on the hybrid nanoelectrocatalysts and enzyme for signal amplification.

    PubMed

    Sun, Duanping; Lu, Jing; Zhong, Yuwen; Yu, Yanyan; Wang, Yu; Zhang, Beibei; Chen, Zuanguang

    2016-01-15

    Human cancer is becoming a leading cause of death in the world and the development of a straightforward strategy for early detection of cancer is urgently required. Herein, a sandwich-type electrochemical aptamer cytosensor was developed for detection of human liver hepatocellular carcinoma cells (HepG2) based on the hybrid nanoelectrocatalysts and enzyme for signal amplification. The thiolated TLS11a aptamers were used as a selective bio-recognition element, attached to the gold nanoparticles (AuNPs) modified the glassy carbon electrode (GCE) surface. Meanwhile, the electrochemical nanoprobes were fabricated through the G-quadruplex/hemin/aptamer complexes and horseradish peroxidase (HRP) immobilized on the surfaces of Au@Pd core-shell nanoparticle-modified magnetic Fe3O4/MnO2 beads (Fe3O4/MnO2/Au@Pd). After the target cells were captured, the hybrid nanoprobes were further assembled to form an aptamer-cell-nanoprobes sandwich-like system on the electrode surface. Then, hybrid Fe3O4/MnO2/Au@Pd nanoelectrocatalysts, G-quadruplex/hemin HRP-mimicking DNAzymes and the natural HRP enzyme efficiently catalyzed the oxidation of hydroquinone (HQ) with H2O2, amplifying the electrochemical signals and improving the detection sensitivity. This electrochemical cytosensor delivered a wide detection range of 1×10(2)-1×10(7)cellsmL(-1), high sensitivity with a low detection limit of 15cellsmL(-1), good selectivity and repeatability. Finally, an electrochemical reductive desorption method was performed to break gold-thiol bond and desorb the components on the AuNPs/GCE for regenerating the cytosensor. These results have demonstrated that the electrochemical cytosensor has the potential to be a feasible tool for cost-effective cancer cell detection in early cancer diagnosis.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. Soft Salt-Mannitol Agar–Cloxacillin Test: a Highly Specific Bedside Screening Test for Detection of Colonization with Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Mir, Nuria; Sánchez, Miguel; Baquero, Fernando; López, Blanca; Calderón, Celia; Cantón, Rafael

    1998-01-01

    The early detection of colonization with methicillin-resistant Staphylococcus aureus (MRSA) of patients in intensive-care units is an essential step in the strategy for preventing MRSA epidemics. In this study, tubes containing soft salt-mannitol agar with cloxacillin (6 μg/ml) (SSMAC) were prepared for inoculation of clinical samples at patients’ bedsides by personnel of an intensive-care unit. A total of 1,914 swabs from different sample sites of 81 patients were dipped into SSMAC tubes, and after 24 h of incubation (in an incubator located near the intensive-care unit), an evident color change was considered by the intensive-care-unit personnel to be an MRSA alarm. Sixty-three (3.3%) SSMAC tubes were considered positive for MRSA, 1,827 (95.4%) were considered negative, and 24 (1.2%) were considered intermediate. Compared with values for parallel conventional surveillance cultures for MRSA, excluding tubes with intermediate results, the SSMAC test had a sensitivity of 72.7%, a specificity of 99.2%, a positive predictive value of 76.2%, and a negative predictive value of 99.0%. When intermediate tubes were considered positive, the corresponding values were 75.3, 98.2, 63.2, and 99.0%, respectively. The sensitivity and specificity values of the test to identify MRSA-colonized patients were 89.4 and 100%, respectively. Oropharyngeal and naris specimens were the most reliable samples for MRSA detection. False-negative results were frequent in bronchial aspirates with low (<103 to 106 CFU/ml) MRSA counts. False-positive results were mainly due to methicillin-resistant Staphylococcus haemolyticus. The SSMAC tube is a useful, rapid, and inexpensive tool for the early identification of MRSA-colonized patients and, consequently, for the implementation of measures to prevent the spread of MRSA. PMID:9542922

  14. 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

  15. 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.

  16. 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.

  17. Easy-to-Fabricate and High-Sensitivity LSPR Type Specific Protein Detection Sensor Using AAO Nano-Pore Size Control.

    PubMed

    Kim, Sae-Wan; Lee, Jae-Sung; Lee, Sang-Won; Kang, Byoung-Ho; Kwon, Jin-Beom; Kim, Ok-Sik; Kim, Ju-Seong; Kim, Eung-Soo; Kwon, Dae-Hyuk; Kang, Shin-Won

    2017-04-13

    In this study, we developed a pore size/pore area-controlled optical biosensor-based anodic aluminum oxide (AAO) nanostructure. As the pore size of AAO increases, the unit cell of AAO increases, which also increases the non-pore area to which the antibody binds. The increase in the number of antibodies immobilized on the surface of the AAO enables effective detection of trace amounts of antigen, because increased antigen-antibody bonding results in a larger surface refractive index change. High sensitivity was thus achieved through amplification of the interference wave of two vertically-incident reflected waves through the localized surface plasmon resonance phenomenon. The sensitivity of the fabricated sensor was evaluated by measuring the change in wavelength with the change in the refractive index of the device surface, and sensitivity was increased with increasing pore-size and non-pore area. The sensitivity of the fabricated sensor was improved and up to 11.8 ag/mL serum amyloid A1 antigen was detected. In addition, the selectivity of the fabricated sensor was confirmed through a reaction with a heterogeneous substance, C-reactive protein antigen. By using hard anodization during fabrication of the AAO, the fabrication time of the device was reduced and the AAO chip was fabricated quickly and easily.

  18. Easy-to-Fabricate and High-Sensitivity LSPR Type Specific Protein Detection Sensor Using AAO Nano-Pore Size Control

    PubMed Central

    Kim, Sae-Wan; Lee, Jae-Sung; Lee, Sang-Won; Kang, Byoung-Ho; Kwon, Jin-Beom; Kim, Ok-Sik; Kim, Ju-Seong; Kim, Eung-Soo; Kwon, Dae-Hyuk; Kang, Shin-Won

    2017-01-01

    In this study, we developed a pore size/pore area-controlled optical biosensor-based anodic aluminum oxide (AAO) nanostructure. As the pore size of AAO increases, the unit cell of AAO increases, which also increases the non-pore area to which the antibody binds. The increase in the number of antibodies immobilized on the surface of the AAO enables effective detection of trace amounts of antigen, because increased antigen-antibody bonding results in a larger surface refractive index change. High sensitivity was thus achieved through amplification of the interference wave of two vertically-incident reflected waves through the localized surface plasmon resonance phenomenon. The sensitivity of the fabricated sensor was evaluated by measuring the change in wavelength with the change in the refractive index of the device surface, and sensitivity was increased with increasing pore-size and non-pore area. The sensitivity of the fabricated sensor was improved and up to 11.8 ag/mL serum amyloid A1 antigen was detected. In addition, the selectivity of the fabricated sensor was confirmed through a reaction with a heterogeneous substance, C-reactive protein antigen. By using hard anodization during fabrication of the AAO, the fabrication time of the device was reduced and the AAO chip was fabricated quickly and easily. PMID:28406469

  19. [Detecting high risk pregnancy].

    PubMed

    Doret, Muriel; Gaucherand, Pascal

    2009-12-20

    Antenatal care is aiming to reduce maternal land foetal mortality and morbidity. Maternal and foetal mortality can be due to different causes. Their knowledge allows identifying pregnancy (high risk pregnancy) with factors associated with an increased risk for maternal and/or foetal mortality and serious morbidity. Identification of high risk pregnancies and initiation of appropriate treatment and/or surveillance should improve maternal and/or foetal outcome. New risk factors are continuously described thanks to improvement in antenatal care and development in biology and cytopathology, increasing complexity in identifying high risk pregnancies. Level of risk can change all over the pregnancy. Ideally, it should be evaluated prior to the pregnancy and at each antenatal visit. Clinical examination is able to screen for intra-uterin growth restriction, pre-eclampsia, threatened for preterm labour; ultrasounds help in the diagnosis of foetal morphological anomalies, foetal chromosomal anomalies, placenta praevia and abnormal foetal growth; biological exams are used to screen for pre-eclampsia, gestational diabetes, trisomy 21 (for which screening method just changed), rhesus immunisation, seroconversion for toxoplasmosis or rubeola, unknown infectious disease (syphilis, hepatitis B, VIH). During pregnancy, most of the preventive strategies have to be initiated during the first trimester or even before conception. Prevention for neural-tube defects, neonatal hypocalcemia and listeriosis should be performed for all women. On the opposite, some measures are concerning only women with risk factors such as prevention for toxoplasmosis, rhesus immunization (which recently changed), tobacco complications and pre-eclampsia and intra-uterine growth factor restriction.

  20. IsoDOT Detects Differential RNA-isoform Expression/Usage with respect to a Categorical or Continuous Covariate with High Sensitivity and Specificity

    PubMed Central

    Sun, Wei; Liu, Yufeng; Crowley, James J.; Chen, Ting-Hued; Zhou, Hua; Chu, Haitao; Huang, Shunping; Kuan, Pei-Fen; Li, Yuan; Miller, Darla R.; Shaw, Ginger D.; Wu, Yichao; Zhabotynsky, Vasyl; McMillan, Leonard; Zou, Fei; Sullivan, Patrick F.; de Villena, Fernando Pardo-Manuel

    2015-01-01

    We have developed a statistical method named IsoDOT to assess differential isoform expression (DIE) and differential isoform usage (DIU) using RNA-seq data. Here isoform usage refers to relative isoform expression given the total expression of the corresponding gene. IsoDOT performs two tasks that cannot be accomplished by existing methods: to test DIE/DIU with respect to a continuous covariate, and to test DIE/DIU for one case versus one control. The latter task is not an uncommon situation in practice, e.g., comparing the paternal and maternal alleles of one individual or comparing tumor and normal samples of one cancer patient. Simulation studies demonstrate the high sensitivity and specificity of IsoDOT. We apply IsoDOT to study the effects of haloperidol treatment on the mouse transcriptome and identify a group of genes whose isoform usages respond to haloperidol treatment. PMID:26617424

  1. IsoDOT Detects Differential RNA-isoform Expression/Usage with respect to a Categorical or Continuous Covariate with High Sensitivity and Specificity.

    PubMed

    Sun, Wei; Liu, Yufeng; Crowley, James J; Chen, Ting-Hued; Zhou, Hua; Chu, Haitao; Huang, Shunping; Kuan, Pei-Fen; Li, Yuan; Miller, Darla R; Shaw, Ginger D; Wu, Yichao; Zhabotynsky, Vasyl; McMillan, Leonard; Zou, Fei; Sullivan, Patrick F; de Villena, Fernando Pardo-Manuel

    We have developed a statistical method named IsoDOT to assess differential isoform expression (DIE) and differential isoform usage (DIU) using RNA-seq data. Here isoform usage refers to relative isoform expression given the total expression of the corresponding gene. IsoDOT performs two tasks that cannot be accomplished by existing methods: to test DIE/DIU with respect to a continuous covariate, and to test DIE/DIU for one case versus one control. The latter task is not an uncommon situation in practice, e.g., comparing the paternal and maternal alleles of one individual or comparing tumor and normal samples of one cancer patient. Simulation studies demonstrate the high sensitivity and specificity of IsoDOT. We apply IsoDOT to study the effects of haloperidol treatment on the mouse transcriptome and identify a group of genes whose isoform usages respond to haloperidol treatment.

  2. MassARRAY Spectrometry Is More Sensitive than PreTect HPV-Proofer and Consensus PCR for Type-Specific Detection of High-Risk Oncogenic Human Papillomavirus Genotypes in Cervical Cancer▿

    PubMed Central

    Basu, Partha; Chandna, Puneet; Bamezai, R. N. K.; Siddiqi, Maqsood; Saranath, Dhananjaya; Lear, Adrian; Ratnam, Sam

    2011-01-01

    Type-specific detection of human papillomavirus (HPV) is indicated for better risk stratification and clinical management of women testing positive for HPV and for epidemiologic surveillance. MassARRAY spectrometry (MassARRAY; Sequenom) is a novel method for type-specific detection of 15 high-risk oncogenic HPV types: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73. PreTect HPV-Proofer (Proofer; Norchip) is a type-specific assay that detects E6/E7 mRNA from five high-risk oncogenic HPV types: HPV-16, -18, -31, -33, and -45. The performance of these tests for type-specific identification of HPV was assessed with cervical specimens from 192 cases of cervical cancer in comparison with consensus MY09/MY11 PCR followed by nucleotide sequencing (consensus PCR). The overall HPV detection rates were 94.8% (95% confidence interval [CI], 91.7, 97.9), 83.3% (95% CI, 78.1, 88.5), and 86.5% (95% CI, 81.7, 91.3) for MassARRAY, Proofer, and consensus PCR, respectively. All tests were negative in six (3.1%) of the 192 cases. Considering only the specimens that contained at least one of the five types targeted by Proofer, the detection rates were 96.6%, 91.4%, and 86.9% for MassARRAY, Proofer, and consensus PCR, respectively. MassARRAY detected multiple infections in 14.1%, Proofer detected multiple infections in 3.6%, and consensus PCR failed to detect any multiple infections. The agreement was highest at 86.0% (kappa = 0.76) between MassARRAY and Proofer and lowest at 81.8% (kappa = 0.69) between Proofer and consensus PCR. In conclusion, MassARRAY is a highly sensitive and accurate method for type-specific detection of oncogenic HPV in cervical cancer, with Proofer showing impressive performance. PMID:21813716

  3. MassARRAY spectrometry is more sensitive than PreTect HPV-Proofer and consensus PCR for type-specific detection of high-risk oncogenic human papillomavirus genotypes in cervical cancer.

    PubMed

    Basu, Partha; Chandna, Puneet; Bamezai, R N K; Siddiqi, Maqsood; Saranath, Dhananjaya; Lear, Adrian; Ratnam, Sam

    2011-10-01

    Type-specific detection of human papillomavirus (HPV) is indicated for better risk stratification and clinical management of women testing positive for HPV and for epidemiologic surveillance. MassARRAY spectrometry (MassARRAY; Sequenom) is a novel method for type-specific detection of 15 high-risk oncogenic HPV types: HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -68, and -73. PreTect HPV-Proofer (Proofer; Norchip) is a type-specific assay that detects E6/E7 mRNA from five high-risk oncogenic HPV types: HPV-16, -18, -31, -33, and -45. The performance of these tests for type-specific identification of HPV was assessed with cervical specimens from 192 cases of cervical cancer in comparison with consensus MY09/MY11 PCR followed by nucleotide sequencing (consensus PCR). The overall HPV detection rates were 94.8% (95% confidence interval [CI], 91.7, 97.9), 83.3% (95% CI, 78.1, 88.5), and 86.5% (95% CI, 81.7, 91.3) for MassARRAY, Proofer, and consensus PCR, respectively. All tests were negative in six (3.1%) of the 192 cases. Considering only the specimens that contained at least one of the five types targeted by Proofer, the detection rates were 96.6%, 91.4%, and 86.9% for MassARRAY, Proofer, and consensus PCR, respectively. MassARRAY detected multiple infections in 14.1%, Proofer detected multiple infections in 3.6%, and consensus PCR failed to detect any multiple infections. The agreement was highest at 86.0% (kappa = 0.76) between MassARRAY and Proofer and lowest at 81.8% (kappa = 0.69) between Proofer and consensus PCR. In conclusion, MassARRAY is a highly sensitive and accurate method for type-specific detection of oncogenic HPV in cervical cancer, with Proofer showing impressive performance.

  4. Development of an Analytic Method for Sulfur Compounds in Aged Garlic Extract with the Use of a Postcolumn High Performance Liquid Chromatography Method with Sulfur-Specific Detection.

    PubMed

    Matsutomo, Toshiaki; Kodera, Yukihiro

    2016-02-01

    Garlic and its processed preparations contain numerous sulfur compounds that are difficult to analyze in a single run using HPLC. The aim of this study was to develop a rapid and convenient sulfur-specific HPLC method to analyze sulfur compounds in aged garlic extract (AGE). We modified a conventional postcolumn HPLC method by employing a hexaiodoplatinate reagent. Identification and structural analysis of sulfur compounds were conducted by LC-mass spectrometry (LC-MS) and nuclear magnetic resonance. The production mechanisms of cis-S-1-propenylcysteine (cis-S1PC) and S-allylmercaptocysteine (SAMC) were examined by model reactions. Our method has the following advantages: less interference from nonsulfur compounds, high sensitivity, good correlation coefficients (r > 0.98), and high resolution that can separate >20 sulfur compounds, including several isomers, in garlic preparations in a single run. This method was adapted for LC-MS analysis. We identified cis-S1PC and γ-glutamyl-S-allyl-mercaptocysteine in AGE. The results of model reactions suggest that cis-S1PC is produced from trans-S1PC through an isomerization reaction and that SAMC is produced by a reaction involving S-allylcysteine/S1PC and diallyldisulfide during the aging period. We developed a rapid postcolumn HPLC method for both qualitative and quantitative analyses of sulfur compounds, and this method helped elucidate a potential mechanism of cis-S1PC and SAMC action in AGE. © 2016 American Society for Nutrition.

  5. Universal and specific quantitative detection of botulinum neurotoxin genes

    PubMed Central

    2010-01-01

    Background Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA)-accepted assay to detect and type botulinum neurotoxins (BoNTs) is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR) technology to determine the specific serotype of the neurotoxin. Results We assayed purified C. botulinum DNA and crude toxin preparations, as well as food and stool from healthy individuals spiked with purified BoNT DNA, and one stool sample from a case of infant botulism for the presence of the NTNH gene, which is part of the BoNT gene cluster, and for the presence of serotype-specific BoNT genes. The PCR surpassed the mouse bioassay both in specificity and sensitivity, detecting positive signals in BoNT preparations containing well below the 1 LD50 required for detection via the mouse bioassay. These results were type-specific and we were reliably able to quantify as few as 10 genomic copies. Conclusions While other studies have reported

  6. Sensitive, fast, and specific immunoassays for methyltestosterone detection.

    PubMed

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

    2015-04-29

    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.

  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. A comparison of high resolution melting, allele-specific priming and Sanger sequencing for the detection of BRAFV600E mutation in hairy cell leukaemia from different haematological specimens.

    PubMed

    So, Chi-Chiu; Chan, Amy; Chung, Lap-Ping; Siu, Lisa; Wong, Wai-Shan; Wong, Kit-Fai

    2014-10-01

    The BRAFV600E mutation is a highly sensitive and specific marker for the diagnosis of hairy cell leukaemia (HCL) and a potential therapeutic target. We assessed the performance of high resolution melting (HRM), allele-specific priming (ASP) and Sanger sequencing (SS) for BRAFV600E detection in 17 unenriched samples from 15 HCL patients: blood (n = 7), marrow aspirate (n = 3), ethylenediaminetetraacetic acid (EDTA)-decalcified trephine biopsy (n = 2), formic acid (FA)-decalcified trephine biopsy (n = 5). Our results showed that for blood and marrow aspirate samples, both HRM and ASP had a very high analytical sensitivity (1%) in clinical specimens and excellent diagnostic sensitivity (100%) and specificity (100%) in analysable samples. Sanger sequencing had a lower analytical sensitivity (4%), resulting in false-negative analysis in samples with a low tumour cell percentage. High resolution melting was technically the simplest and had the shortest turn-around time (2 hours). Analysis of decalcified trephine biopsies was more difficult because of suboptimal DNA preservation. Although Sanger sequencing was least demanding on sample DNA quality for a successful analysis, none of the three techniques showed satisfactory diagnostic performance on trephine biopsies. Therefore, careful selection of a suitable sample type and testing platform is important to optimise the detection of this important mutation in HCL.

  9. [Prostate histopathology of NIH category IV prostatitis detected by sextant prostate needle biopsy from the patients with high prostatic specific antigen].

    PubMed

    Shimomura, Tatsuya; Kiyota, Hiroshi; Takahashi, Hiroyuki; Madarame, Jun; Kimura, Takahiro; Onodera, Shouichi

    2003-08-01

    Asymptomatic prostatitis is classified as category IV in NIH classification of prostatitis syndrome (1999). No report concerning this category has been present. We investigated this category histopathologically and clinically, in order to clarify the histopathological distribution and its correlation to the clinical features, in this study. Among 785 patients who were suspected prostate cancer because of their high prostatic specific antigen (PSA) values and to have a sextant prostate needle biopsy was performed between January, 1996 and December, 2000, 88 patients (11.2%) were diagnosed as NIH category IV prostatitis (asymptomatic prostatitis). We observed all pathological specimens stained with Hematoxylin-Eosine, and classified them into subtypes according to the classification criteria for prostatitis defined by True et al. (1999). We also investigated the relationship between histopathological distribution and clinical features such as PSA values, PSA density, the incidence of pyuria or bacteriuria. In the histopathological study, grade distributions were 12.5% (11/88) in mild, 71.6% (63/88) in moderate, and 15.9% (14/88) in severe. Location distributions were 2.3% (2/88) in glandular, 68.2% (60/88) in periglandular, and 29.5% (26/88) in stromal. No relationship between these subtypes and clinical features was recognized statistically. However, 7 patients (7.95%) were diagnosed as prostate cancers, later. Pyuria was found in 29.1% (23/79). Bacteriuria was present in 14.3% (11/77). Isolated bacteria were 4 strains of Enterococcus faccalis, 2 strains of each of Pseudomonas aeruginosa and Staphylococcus aureus, and one strain of each of Escherichia coli, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Staphylococcus haemolyticus, and Staphylococcus epidermidis. Gram positive rod, and Candida sp. No relationship between these subtypes and bacterial species was recognized. These results indicated that the incidence of NIII category IV prostatits

  10. Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection.

    PubMed

    Oakley, Brian B; Line, J Eric; Berrang, Mark E; Johnson, Jessica M; Buhr, R Jeff; Cox, Nelson A; Hiett, Kelli L; Seal, Bruce S

    2012-02-01

    Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in <1 h. Pyrosequencing results show the assay to be extremely (>99%) sensitive, and spike-back experiments demonstrated a detection threshold of <10(2) CFU mL(-1). Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications.

  11. Linear-After-The-Exponential (LATE)-PCR: Primer design criteria for high yields of specific single-stranded DNA and improved real-time detection

    PubMed Central

    Pierce, Kenneth E.; Sanchez, J. Aquiles; Rice, John E.; Wangh, Lawrence J.

    2005-01-01

    Traditional asymmetric PCR uses conventional PCR primers at unequal concentrations to generate single-stranded DNA. This method, however, is difficult to optimize, often inefficient, and tends to promote nonspecific amplification. An alternative approach, Linear-After-The-Exponential (LATE)-PCR, solves these problems by using primer pairs deliberately designed for use at unequal concentrations. The present report systematically examines the primer design parameters that affect the exponential and linear phases of LATE-PCR amplification. In particular, we investigated how altering the concentration-adjusted melting temperature (Tm) of the limiting primer (TmL) relative to that of the excess primer (TmX) affects both amplification efficiency and specificity during the exponential phase of LATE-PCR. The highest reaction efficiency and specificity were observed when TmL - TmX ≥ 5°C. We also investigated how altering TmX relative to the higher Tm of the double-stranded amplicon (TmA) affects the rate and extent of linear amplification. Excess primers with TmX closer to TmA yielded higher rates of linear amplification and stronger signals from a hybridization probe. These design criteria maximize the yield of specific single-stranded DNA products and make LATE-PCR more robust and easier to implement. The conclusions were validated by using primer pairs that amplify sequences within the cystic fibrosis transmembrane regulator (CFTR) gene, mutations of which are responsible for cystic fibrosis. PMID:15937116

  12. Monodispersed nanoparticles of conjugated polyelectrolyte brush with high charge density for rapid, specific and label-free detection of tumor marker.

    PubMed

    Liu, Xingfen; Shi, Lin; Zhang, Zhiyong; Fan, Quli; Huang, Yanqin; Su, Shao; Fan, Chunhai; Wang, Lianhui; Huang, Wei

    2015-03-21

    Highly charged nanoparticles of a conjugated polyelectrolyte brush were used to sense the human α-fetoprotein (AFP) by observing selective superquenching in several minutes. The unique property of nanoparticles that the self-aggregation causes an unchanged or enhanced fluorescence can reduce the interference from non-target substance significantly.

  13. Development of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of phenylethanolamine A in tissue and feed samples and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS).

    PubMed

    Cao, Biyun; He, Guangzhao; Yang, Hong; Chang, Huafang; Li, Shuqun; Deng, Anping

    2013-10-15

    Phenylethanolamine A (PA) is a new emerged β-adrenergic agonist illegally used as feed additives for growth promotion. In this study, a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of PA in tissue and feed samples was developed and confirmed by liquid chromatography tandem mass spectrometry (LC-MS/MS). By reduction of nitryl group to amino group, the PA derivative was synthesized and coupled to carrier proteins with diazobenzidine method. The antisera obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. All antisera displayed high sensitivity with IC50 values lower than 0.48 ng mL(-1). The most sensitive ELISA was established with IC50 and limit of detection (LOD) values of 0.049 ng mL(-1) and 0.003 ng mL(-1), respectively. The cross-reactivity (CR) values of the antisera with three frequently used β-adrenergic agonists (clenbuterol, salbutamol and ractopamine) were lesser than 0.39%; there was no CR of the antisera with other six compounds including two structurally related substances (isoproterenol, phenylephrine). To investigate the accuracy and precision of the assay, swine kidney, liver, meat and feed samples were fortified with PA at different content and analyzed by ELISA. Acceptable recovery rates of 92.2-113.7% and intra-assay coefficients of variation of 3.8-10.9% (n=3) were achieved. Seven spiked samples were simultaneously analyzed by ELISA and LC-MS/MS. There was a high correlation coefficient of 0.9956 (n=7) between the two methods. The proposed ELISA proven to be a feasible quantitative/screening method for PA analysis in tissue and feed samples with the properties of high sensitivity and specificity, high sample throughput and low expensive.

  14. Aptamer-guided silver-gold bimetallic nanostructures with highly active surface-enhanced Raman scattering for specific detection and near-infrared photothermal therapy of human breast cancer cells.

    PubMed

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

    2012-09-18

    The aptamer (S2.2)-guided Ag-Au nanostructures (aptamer-Ag-Au) have been synthesized by photoreduction and validated by ultraviolet-visible light (UV-vis) spectra and transmission electron microscopy (TEM) images. Differential interference contrast (DIC), fluorescence, and TEM images, and surface-enhanced Raman scattering (SERS) spectra indicated that the aptamer-Ag-Au nanostructures can target the surface of human breast cancer cells (MCF-7) with high affinity and specificity. This targeting is completed via the specific interaction between S2.2 aptamer (a 25-base oligonucleotide) and MUC1 mucin (a large transmembrane glycoprotein, whose expression increased at least 10-fold at MCF-7 cells in primary and metastatic breast cancers). However, the nanostructures cannot target HepG2 (human liver cancer cells) or MCF-10A cells (human normal breast epithelial cells), because these cells are MUC1-negative expressed. Moreover, the synthesized nanostructures exhibited a high SERS activity. Based on these results, a new assay for specifically detecting MCF-7 cells has been proposed. This assay can also discriminate MCF-7 cells from MCF-10A cells and different cancer cell lines, such as HepG2 cells. In addition, the aptamer-Ag-Au nanostructures have a high capability of adsorpting near-infrared (NIR) irradiation and are able to perform photothermal therapy of MCF-7 cells at a very low irradiation power density (0.25 W/cm(2)) without destroying the healthy cells and the surrounding normal tissue. Therefore, the proposed assay is significant for the diagnosis of tumors in their nascent stage. The synthesized nanostructures could offer a protocol to specifically recognize and sensitively detect the cancer cells, and would have great potential for application in the photothermal therapy of the cancers.

  15. Specific and ultrasensitive ciprofloxacin detection by responsive photonic crystal sensor.

    PubMed

    Zhang, Rong; Wang, Yong; Yu, Li-Ping

    2014-09-15

    A new approach for specific and ultrasensitive measurement of ciprofloxacin has been developed by integrating ternary complexes into responsive photonic crystal (RPC). Tryptophan was first immobilized within the polyacrylamide hydrogel substrates of RPC. The determination of ciprofloxacin was via the existence of zinc(II) ions that function as a 'bridge' to form specific tryptophan-zinc(II)-ciprofloxacin complexes step by step, which resulted in a stepwise red-shift of the diffraction wavelength. A maximum wavelength shift from 798 to 870 nm for ciprofloxacin was observed when the RPC film was immersed in 10(-4)M ciprofloxacin. A linear relationship has been obtained between the Δλ of diffraction peak and logarithm of ciprofloxacin concentration at pH 5.0 in the range of 10(-10) to 10(-4)M. And the least detectable concentration in present work is about 5 × 10(-11)M. The results demonstrated that the as-designed ternary complexes-based RPC sensor exhibited high sensitivity, satisfactory specificity and excellent recoverability for sensing of ciprofloxacin in aqueous media and were validated by detecting ciprofloxacin in the eye-drop sample.

  16. Separation methods for high specific activity radioarsenic

    NASA Astrophysics Data System (ADS)

    Jurisson, S. S.; Wycoff, D. E.; DeGraffenreid, A.; Embree, M. F.; Ketring, A. R.; Cutler, C. S.; Fassbender, M. E.; Ballard, B.

    2012-12-01

    Radiopharmaceuticals require the use of high specific activity radionuclides, especially when targeting limited numbers of receptors on tumor surfaces. Two radioisotopes of arsenic (72As and 77As) are potentially useful in diagnostic and therapeutic radiopharmaceuticals. Methods for the production, separation, and isolation of high specific activity 72As and 77As are presented.

  17. A novel immunosensing platform for highly sensitive prostate specific antigen detection based on dual-quenching of photocurrent from CdSe sensitized TiO2 electrode by gold nanoparticles decorated polydopamine nanospheres.

    PubMed

    Dong, Yu-Xiang; Cao, Jun-Tao; Liu, Yan-Ming; Ma, Shu-Hui

    2017-05-15

    Herein, a novel photoelectrochemical (PEC) immunosensing platform for highly sensitive detection of prostate specific antigen (PSA) was constructed based on dual-quenching of photocurrent from CdSe sensitized TiO2 electrode by gold nanoparticles decorated dopamine-melanin nanospheres (AuNPs-Dpa-melanin CNSs). In this proposal, CdSe sensitized TiO2 was used as photoelectrochemical matrix and the functional AuNPs-Dpa-melanin CNSs were used as signal quenching element. The dual quenching of the gold nanoparticles decorated Dpa-melanin CNSs to the CdSe sensitized TiO2 was achieved as follows: (i) the strong energy transfer between the CdSe quantum dots (QDs) and Au NPs diminishes the photocurrent signal of CdSe QDs; (ii) the steric hindrance of AuNPs-Dpa-melanin CNSs partly obstructs the diffusion of the electron donor, i.e. ascorbic acid, to the surface of photoelectrode, which make the depleting efficiency of the photogenerated holes decrease, leading to a declined photocurrent intensity. On the basis of the dual quenching effect of AuNPs-Dpa-melanin CNSs, a competitive immunosensing platform for PSA was designed upon the specific binding of anti-PSA to PSA and PSA functionalized AuNPs-Dpa-melanin CNSs conjugates. This proposed immunosensor possesses wide linear range from 1.0×10(-11)gmL(-1) to 1.0×10(-7)gmL(-1) with the detection limit of 2.7pgmL(-1). Moreover, the applicability of the present method was demonstrated in the determination of PSA in human serum. The strategy creates new paradigms for PSA and other tumor markers detection and demonstrates high sensitivity, good specificity, and satisfied applicability in complex biological samples.

  18. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. 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

  20. The establishment of a highly sensitive ELISA for detecting bovine serum albumin (BSA) based on a specific pair of monoclonal antibodies (mAb) and its application in vaccine quality control.

    PubMed

    Zhang, Kui; Song, Chaojun; Li, Qi; Li, Yongming; Sun, Yuanjie; Yang, Kun; Jin, Boquan

    2010-08-01

    A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for quantifying BSA was established, based on two mAbs that recognize different epitopes on a BSA molecule. Our ELISA system was used to detect BSA concentrations in several vaccines, such as the MMR (measles, mumps and rubella) vaccine, hepatitis A vaccine, and hepatitis B vaccine. Moreover, we compared the mAb ELISA and the present pAb ELISA by detecting BSA standards and bovine serum samples. The results showed that our ELISA system was in good accordance with the pAb ELISA system. A pair of mAbs (FMU-BSA NO.6 and FMU-BSA NO.11) from 11 murine hybridomas secreting BSA-specific mAbs was selected for the development of the sandwich ELISA. The detection limit of this quantitative assay reaches 0.38 μg/L, which is 10-fold more sensitive than those previously reported. The quantitative range of BSA concentration is from 0.5 to 40 μg/L, which is comparable to the currently used polyclonal antibody (pAb) ELISA. Intra-assay and inter-assay coefficient variations are both lower than 10% at the three concentrations used (10, 20, and 40 μg/L). Thus, the mAb sandwich ELISA developed herein may provide a stable, precise, and highly sensitive method for quantifying BSA, which is very useful in the quality control of some vaccines.

  1. Detection and Tracking of NY-ESO-1-Specific CD8+ T Cells by High-Throughput T Cell Receptor β (TCRB) Gene Rearrangements Sequencing in a Peptide-Vaccinated Patient.

    PubMed

    Miyai, Manami; Eikawa, Shingo; Hosoi, Akihiro; Iino, Tamaki; Matsushita, Hirokazu; Isobe, Midori; Uenaka, Akiko; Udono, Heiichiro; Nakajima, Jun; Nakayama, Eiichi; Kakimi, Kazuhiro

    2015-01-01

    Comprehensive immunological evaluation is crucial for monitoring patients undergoing antigen-specific cancer immunotherapy. The identification and quantification of T cell responses is most important for the further development of such therapies. Using well-characterized clinical samples from a high responder patient (TK-f01) in an NY-ESO-1f peptide vaccine study, we performed high-throughput T cell receptor β-chain (TCRB) gene next generation sequencing (NGS) to monitor the frequency of NY-ESO-1-specific CD8+ T cells. We compared these results with those of conventional immunological assays, such as IFN-γ capture, tetramer binding and limiting dilution clonality assays. We sequenced human TCRB complementarity-determining region 3 (CDR3) rearrangements of two NY-ESO-1f-specific CD8+ T cell clones, 6-8L and 2F6, as well as PBMCs over the course of peptide vaccination. Clone 6-8L possessed the TCRB CDR3 gene TCRBV11-03*01 and BJ02-01*01 with amino acid sequence CASSLRGNEQFF, whereas 2F6 possessed TCRBV05-08*01 and BJ02-04*01 (CASSLVGTNIQYF). Using these two sequences as models, we evaluated the frequency of NY-ESO-1-specific CD8+ T cells in PBMCs ex vivo. The 6-8L CDR3 sequence was the second most frequent in PBMC and was present at high frequency (0.7133%) even prior to vaccination, and sustained over the course of vaccination. Despite a marked expansion of NY-ESO-1-specific CD8+ T cells detected from the first through 6th vaccination by tetramer staining and IFN-γ capture assays, as evaluated by CDR3 sequencing the frequency did not increase with increasing rounds of peptide vaccination. By clonal analysis using 12 day in vitro stimulation, the frequency of B*52:01-restricted NY-ESO-1f peptide-specific CD8+ T cells in PBMCs was estimated as only 0.0023%, far below the 0.7133% by NGS sequencing. Thus, assays requiring in vitro stimulation might be underestimating the frequency of clones with lower proliferation potential. High-throughput TCRB sequencing using NGS

  2. Educational Specifications: University City High School.

    ERIC Educational Resources Information Center

    Philadelphia School District, PA.

    Educational specifications are presented delineating instructional space requirements and relationships for a new high school in Philadelphia, Pennsylvania. These specifications comprise a set of written instructions from which the architect can derive a design concept compatible with current educational needs and adaptable to future changes in…

  3. 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...

  4. 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...

  5. Development of a highly sensitive and specific monoclonal antibody based enzyme-linked immunosorbent assay for the detection of a new β-agonist, phenylethanolamine A, in food samples.

    PubMed

    Jiang, Danni; Cao, Biyun; Wang, Meiyu; Yang, Hong; Zhao, Kang; Li, Jianguo; Li, Mingxin; Sun, Lulu; Deng, Anping

    2017-02-01

    All β-agonists are banned as feed additives for growth promotion in animals due to toxic effects on humans after consuming the β-agonist contaminated meats. Phenylethanolamine A (PA) is a newly emerged β-agonist. Thus there is a need to develop highly sensitive and specific analytical methods for the detection of PA in food samples. In this study, the monoclonal antibody (mAb) against PA was produced by hybridoma technology and used for the development of enzyme-linked immunosorbent assay (ELISA). The IC50 values and limits of detection (LODs) of the ELISA using homogeneous combination of coating antigen/antibody for PA were 0.16 ng mL(-1) and 0.011 ng mL(-1) , respectively. The cross-reactive (CR) values of the assay with 14 structurally related β-agonists were lower than 0.59%. Swine liver and meat samples were spiked with PA at different content and analysed by ELISA. Acceptable recovery rates of 91.40-105.51% and intra-assay coefficients of variation of 1.56-9.92% (n = 3) were obtained. The ELISA for seven spiked samples was confirmed by LC-MS/MS with a high correlation coefficient of 0.9881. The proposed mAb-based ELISA was highly sensitive and specific for PA and could be used as a quantitative/screening method for PA analysis in food samples. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  6. Ferrocene-graphene sheets for high-efficiency quenching of electrochemiluminescence from Au nanoparticles functionalized cadmium sulfide flower-like three dimensional assemblies and sensitive detection of prostate specific antigen.

    PubMed

    Yang, Jiu-Jun; Cao, Jun-Tao; Wang, Hui; Liu, Yan-Ming; Ren, Shu-Wei

    2017-05-15

    A signal-switchable electrochemiluminescence (ECL) aptasensor was presented for sensitive prostate specific antigen (PSA) assay using ferrocene-graphene sheets (Fc-GNs) for high-efficiency quenching of ECL from Au nanoparticles functionalized cadmium sulfide flower-like three dimensional (3D) assemblies (Au-CdS flower-like 3D assemblies). Au-CdS flower-like 3D assemblies were synthesized and employed as luminophore, exhibiting strong and stable ECL intensity, and followed by assembling captured DNA (cDNA) and hybridizing it with half of base sequence of PSA aptamer on the Au-CdS flower-like 3D assemblies modified electrode. The remaining part of the non-complementary base of the aptamer could preferentially adsorb GN with the signal switched "off" state. While in the presence of the PSA, the binding of PSA with aptamer caused desorption of aptamer from the surface of Fc-GNs and was then released from electrode surface, thus allowing the ECL signal enhancement. With the transformation of luminescence signal from "off" to "on", the aptasensor displays high sensitivity for PSA detection with a linear range from 1pgmL(-1) to 25ngmL(-1) and a detection limit of 0.38pgmL(-1)S/N=3). Moreover, this developed method could be successfully applied to the determination of PSA in human serum samples with recoveries of 85.8-104.0%, suggesting great potential applications in biochemical analysis.

  7. Detection of specific immunoglobulin E in patients with toxoplasmosis.

    PubMed Central

    Pinon, J M; Toubas, D; Marx, C; Mougeot, G; Bonnin, A; Bonhomme, A; Villaume, M; Foudrinier, F; Lepan, H

    1990-01-01

    An immunocapture assay was developed to detect Toxoplasma gondii-specific immunoglobulin E (IgE) in sera from adults with acute acquired infection or reactivation and from babies with congenital toxoplasmosis. The components of this assay were monoclonal antibody to human IgE, samples from patients, and T. gondii tachyzoites treated with Formalin. When T. gondii-specific IgE antibodies were present, visually detectable agglutination occurred. Sera, umbilical cord blood, fetal blood, cerebrospinal fluid, and amniotic fluid were tested by this method. Specific IgE antibodies were detected in sera from 25 (86%) of 29 adults who developed specific IgG antibody during pregnancy or had specific IgA and IgM antibodies. Specific IgE was present early during infection, at the time that IgM antibodies were present, and slightly preceding the presence of specific IgA antibodies. In 23 patients tested serially, IgE antibodies never persisted for longer than 4 months. No nonspecific anti-T. gondii IgE was detected in sera from uninfected individuals. Maternal IgE antibodies did not cross the placenta. In sera of patients with congenital toxoplasmosis, specific IgE antibodies were found at birth, during the first year of life, and during immunologic recrudescence following discontinuation of pyrimethamine-sulfonamide therapy. The IgE immunocapture assay is simple to perform. It is especially useful for determining when T. gondii was acquired by recently infected pregnant women. PMID:2203811

  8. Development of peptide nucleic acid mediated polymerase chain reaction clamping (PMPC)--direct sequencing method for detecting lamivudine-resistant hepatitis B virus (HBV) variants with high sensitivity and specificity.

    PubMed

    Ogata, Norio; Ichida, Takafumi; Aoyagi, Yutaka; Kitajima, Isao

    2003-04-01

    Reduced sensitivity of HBV to lamivudine, which causes a viral breakthrough during treatment, is attributed to mutations within the tyrosine-methionine-aspartate(YMDD) locus in the reverse transcriptase(rt) domain of HBV polymerase, mainly a methionine(rtM204) substitution. The sensitive detection of such mutations before or early in treatment could assist in optimizing antiviral treatment. For this purpose, we developed peptide nucleic acid(PNA) mediated polymerase chain reaction(PCR) clamping(PMPC) with a PNA probe targeting the YMDD locus. We first tested this method for its sensitivity and specificity in detecting a mutant on HBV DNA standards consisting of serial copy number ratios of a known lamivudine-resistant, mutant clone with rtM204I(ATT) to a wild-type clone with rtM204(ATG). The sensitivity was 0.1 to 0.01% in the coexistence of wild-type clones and the specificity was guaranteed by direct sequencing of the products. We next applied this method to HBV DNA specimens extracted from serum from 4 chronic hepatitis B patients treated with lamivudine. Two of these exhibited a break-through of the HBV mutant with rtM204I(ATT), while the other 2 did not. Before treatment, all 4 patients showed HBV with rtM204I encoded by ATA. During treatment, HBV with the rtM204I(ATT) emerged in the 2 breakthrough patients more than 3 months before the breakthrough, whereas this and other known lamivudine-resistant viruses did not appear in the 2 non-breakthrough patients. Thus, our PMPC-direct sequencing method is highly sensitive and reliable for the early identification of lamivudine-resistant HBV that causes a viral breakthrough.

  9. Design of a New Fluorescent Oligonucleotide-Based Assay for a Highly Specific Real-Time Detection of Apurinic/Apyrimidinic Site Cleavage by Tyrosyl-DNA Phosphodiesterase 1.

    PubMed

    Lebedeva, Natalia A; Anarbaev, Rashid O; Kupryushkin, Maxim S; Rechkunova, Nadejda I; Pyshnyi, Dmitrii V; Stetsenko, Dmitry A; Lavrik, Olga I

    2015-10-21

    Tyrosyl-DNA phosphodiesterase 1 (Tdp1) promotes catalytic scission of a phosphodiester bond between the 3'-end of DNA and the hydroxyl group of a tyrosine residue, as well as cleaving off a variety of other 3'-terminal phosphate-linked DNA substituents. We have shown recently that Tdp1 can initiate an apurinic/apyrimidinic (AP) site repair pathway that is independent from the one mediated by AP endonuclease 1 (APE1). Until recently, there was no method available of tracking the AP-site cleaving activity of Tdp1 by real-time fluorescence assay. In the present study we demonstrate a highly specific real-time detection of the AP-site cleaving activity of Tdp1 which allows one to distinguish it from the activity of APE1 by using a short hairpin oligonucleotide with a 1,12-dodecanediol loop, a 5'-fluorophore, and a 3'-quencher. Specific phosphodiesterase activity of Tdp1, which is usually able to remove quencher from the 3'-end of DNA, was suppressed in our approach by introducing a noncleavable phosphate group mimic between the 3'-end and the quencher. As a nondigestible 3'-phosphate analogue, we have used a new uncharged tetramethyl phosphoryl guanidine (Tmg) group, which is resistant to 3'-phosphodiesterase cleavage.

  10. Sensitive and specific DNA probe for detection of Plasmodium falciparum.

    PubMed Central

    Enea, V

    1986-01-01

    The isolation and some characteristics of a very sensitive DNA probe for the detection of Plasmodium falciparum are described. The probe is species specific and represents a large, albeit variable, fraction of the genome in all the strains tested. In addition to its immediate practical uses for the detection and quantitation of parasites, the probe defines an interesting family of repeated sequences. Images PMID:3023833

  11. Development and characterization of a highly specific and sensitive SYBR green reverse transcriptase PCR assay for detection of the 2009 pandemic H1N1 influenza virus on the basis of sequence signatures.

    PubMed

    Medina, Rafael A; Rojas, Mark; Tuin, Astrid; Huff, Stephen; Ferres, Marcela; Martinez-Valdebenito, Constanza; Godoy, Paula; García-Sastre, Adolfo; Fofanov, Yuriy; SantaLucia, John

    2011-01-01

    The emergence and rapid spread of the 2009 H1N1 pandemic influenza virus showed that many diagnostic tests were unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the U.S. Centers for Disease Control and Prevention was used for diagnostic purposes. The substantial sequence data that became available during the course of the pandemic created the opportunity to utilize bioinformatics tools to evaluate the unique sequence properties of this virus for the development of diagnostic tests. We used a comprehensive computational approach to examine conserved 2009 H1N1 sequence signatures that are at least 20 nucleotides long and contain at least two mismatches compared to any other known H1N1 genome. We found that the hemagglutinin (HA) and neuraminidase (NA) genes contained sequence signatures that are highly conserved among 2009 H1N1 isolates. Based on the NA gene signatures, we used Visual-OMP to design primers with optimal hybridization affinity and we used ThermoBLAST to minimize amplification artifacts. This procedure resulted in a highly sensitive and discriminatory 2009 H1N1 detection assay. Importantly, we found that the primer set can be used reliably in both a conventional TaqMan and a SYBR green reverse transcriptase (RT)-PCR assay with no loss of specificity or sensitivity. We validated the diagnostic accuracy of the NA SYBR green assay with 125 clinical specimens obtained between May and August 2009 in Chile, and we showed diagnostic efficacy comparable to the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic.

  12. Development and Characterization of a Highly Specific and Sensitive SYBR Green Reverse Transcriptase PCR Assay for Detection of the 2009 Pandemic H1N1 Influenza Virus on the Basis of Sequence Signatures▿ †

    PubMed Central

    Medina, Rafael A.; Rojas, Mark; Tuin, Astrid; Huff, Stephen; Ferres, Marcela; Martinez-Valdebenito, Constanza; Godoy, Paula; García-Sastre, Adolfo; Fofanov, Yuriy; SantaLucia, John

    2011-01-01

    The emergence and rapid spread of the 2009 H1N1 pandemic influenza virus showed that many diagnostic tests were unsuitable for detecting the novel virus isolates. In most countries the probe-based TaqMan assay developed by the U.S. Centers for Disease Control and Prevention was used for diagnostic purposes. The substantial sequence data that became available during the course of the pandemic created the opportunity to utilize bioinformatics tools to evaluate the unique sequence properties of this virus for the development of diagnostic tests. We used a comprehensive computational approach to examine conserved 2009 H1N1 sequence signatures that are at least 20 nucleotides long and contain at least two mismatches compared to any other known H1N1 genome. We found that the hemagglutinin (HA) and neuraminidase (NA) genes contained sequence signatures that are highly conserved among 2009 H1N1 isolates. Based on the NA gene signatures, we used Visual-OMP to design primers with optimal hybridization affinity and we used ThermoBLAST to minimize amplification artifacts. This procedure resulted in a highly sensitive and discriminatory 2009 H1N1 detection assay. Importantly, we found that the primer set can be used reliably in both a conventional TaqMan and a SYBR green reverse transcriptase (RT)-PCR assay with no loss of specificity or sensitivity. We validated the diagnostic accuracy of the NA SYBR green assay with 125 clinical specimens obtained between May and August 2009 in Chile, and we showed diagnostic efficacy comparable to the CDC assay. Our approach highlights the use of systematic computational approaches to develop robust diagnostic tests during a viral pandemic. PMID:21084522

  13. 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

  14. Chromosome-specific staining to detect genetic rearrangements

    SciTech Connect

    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.

  15. Detection and description of soils with specific nematode suppressiveness.

    PubMed

    Westphal, Andreas

    2005-03-01

    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.

  16. 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

  17. 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.

  18. 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. 

  19. Research on High-Specific-Heat Dielectrics

    DTIC Science & Technology

    1990-01-31

    wellp as related thermodynamic properties , we infer the following conclusions: 1. The exceptionally high C peaks for ZnCr204 andp 2 CdCr204 in the 2...which determine the electric, magnetic, and thermodynamic properties of the system. In addition, we have found from this microscopic analysis that... properties of this lattice will therefore be dominated by the properties of the cluster. The 3 thermodynamic properties such as the energy, the specific

  20. 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.

  1. 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.

  2. 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.

  3. Specific detection of GII-1 lineage of infectious bronchitis virus.

    PubMed

    Domanska-Blicharz, K; Lisowska, A; Pikuła, A; Sajewicz-Krukowska, J

    2017-08-01

    Infectious bronchitis virus (IBV) is a worldwide prevalent RNA virus that causes highly contagious and economically devastating disease in chicken. The virus exists in many different genetic forms which made the disease control very difficult. The present study describes the development and validation of TaqMan probe-based real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) targeting the S1 coding region of S gene characteristic for the GII-1 lineage (formerly the D1466-like variant) of IBV. These strains are quite different from other European IBV belonging to different lineages of the GI genotype. The developed method was 30-fold more sensitive than used so far for standard nested RT-PCR with detection limit of 56 RNA copies per reaction. The specificity of the assay was also evaluated with a panel of different poultry pathogens. Repeatability and reproducibility of the method was very high with coefficients of variation lower than 4%. One hundred and twenty-seven IBV-positive samples were tested by this method and GII-1 strains were detected in four of them (3·15%) which indicate a decrease in the GII-1 IBV prevalence in Poland. The assay was proven to be a valuable tool for rapid diagnosis of GII-1 lineage of IBV strains and moreover it enabled the monitoring of viral loads which can be used to assess disease progression. This study reports a TaqMan probe-based real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) for rapid and accurate identification of GII-1 lineage (formerly D1466-like variant) of infectious bronchitis virus (IBV). The assay revealed to be more sensitive than standard nested RT-PCR assay, previously used for this purpose. The developed assay has been tested on numerous field samples and revealed 3·15% prevalence of this lineage of IBV in Polish chicken population. Moreover, this new assay enables the assessment of viral load measurement which might be useful for epidemiology and pathogenesis

  4. 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.

  5. 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.

  6. 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.

  7. Mechanisms of visual threat detection in specific phobia.

    PubMed

    Weierich, Mariann R; Treat, Teresa A

    2015-01-01

    People with anxiety or stress-related disorders attend differently to threat-relevant compared with non-threat stimuli, yet the temporal mechanisms of differential allocation of attention are not well understood. We investigated two independent mechanisms of temporal processing of visual threat by comparing spider-phobic and non-fearful participants using a rapid serial visual presentation task. Consistent with prior literature, spider phobics, but not non-fearful controls, displayed threat-specific facilitated detection of spider stimuli relative to negative stimuli and neutral stimuli. Further, signal detection analyses revealed that facilitated threat detection in spider-phobic participants was driven by greater sensitivity to threat stimulus features and a trend towards a lower threshold for detecting spider stimuli. However, phobic participants did not display reliably slowed temporal disengagement from threat-relevant stimuli. These findings advance our understanding of threat feature processing that might contribute to the onset and maintenance of symptoms in specific phobia and disorders that involve visual threat information more generally.

  8. Mechanisms of Visual Threat Detection in Specific Phobia

    PubMed Central

    Weierich, Mariann R.; Treat, Teresa A.

    2014-01-01

    People with anxiety or stress-related disorders attend differently to threat-relevant compared with non-threat stimuli, yet the temporal mechanisms of differential allocation of attention are not well-understood. We investigated two independent mechanisms of temporal processing of visual threat by comparing spider-phobic and non-fearful participants using a rapid serial visual presentation task. Consistent with prior literature, spider phobics, but not non-fearful controls, displayed threat-specific facilitated detection of spider stimuli relative to negative stimuli and neutral stimuli. Further, signal detection analyses revealed that facilitated threat detection in spider-phobic participants was driven by greater sensitivity to threat stimulus features and a trend toward a lower threshold for detecting spider stimuli. However, phobic participants did not display reliably slowed temporal disengagement from threat-relevant stimuli. These findings advance our understanding of threat feature processing that might contribute to the onset and maintenance of symptoms in specific phobia and disorders that involve visual threat information more generally. PMID:25251896

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Electrochemical genosensor for specific detection of the food-borne pathogen, Vibrio cholerae.

    PubMed

    Low, Kim-Fatt; Chuenrangsikul, Kritsanaporn; Rijiravanich, Patsamon; Surareungchai, Werasak; Chan, Yean-Yean

    2012-04-01

    A disposable horseradish peroxidase (HRP)-based electrochemical genosensor was developed for chronoamperometric detection of single-stranded asymmetric lolB gene PCR amplicon (118 bp in length) of the food-borne pathogen, Vibrio cholerae. A two-step sandwich-type hybridization strategy using two specific probes was employed for specific detection of the target single-stranded DNA (ssDNA). The analytical performances of the detection platform have been evaluated using a synthetic ssDNA (ST3) which was identical to the target single-stranded amplicon and a total of 19 bacterial strains. Under optimal condition, ST3 was calibrated with a dynamic range of 0.4883-15.6250 nM. By coupling asymmetric PCR amplification, the probe-based electrochemical genosensor was highly specific to the target organism (100% specificity) and able to detect as little as 0.85 ng/μl of V. cholerae genomic DNA.

  15. 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.

  16. A high-throughput multiplex method adapted for GMO detection.

    PubMed

    Chaouachi, Maher; Chupeau, Gaëlle; Berard, Aurélie; McKhann, Heather; Romaniuk, Marcel; Giancola, Sandra; Laval, Valérie; Bertheau, Yves; Brunel, Dominique

    2008-12-24

    A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

  17. 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.

  18. Immunological screening method to detect specific translation products.

    PubMed Central

    Broome, S; Gilbert, W

    1978-01-01

    We describe a very sensitive method to detect as antigens the presence of specific proteins within phage plaques or bacterial colonies. We coat plastic sheets with antibody molecules, expose the sheet to lysed bacteria so that a released antigen can bind, and then label the immobilized antigen with radioiodinated antibodies. Thus, the antigen is sandwiched between the antibodies attached to the plastic sheet and those carrying the radioactive label. Autoradiography then shows the positions of antigen-containing colonies or phage plaques. A few molecules of antigen released from each bacterial cell generatean adequate signal. Images PMID:96441

  19. Patient specific Parkinson's disease detection for adaptive deep brain stimulation.

    PubMed

    Mohammed, Ameer; Zamani, Majid; Bayford, Richard; Demosthenous, Andreas

    2015-08-01

    Continuous deep brain stimulation for Parkinson's disease (PD) patients results in side effects and shortening of the pacemaker battery life. This can be remedied using adaptive stimulation. To achieve adaptive DBS, patient customized PD detection is required due to the inconsistency associated with biomarkers across patients and time. This paper proposes the use of patient specific feature extraction together with adaptive support vector machine (SVM) classifiers to create a patient customized detector for PD. The patient specific feature extraction is obtained using the extrema of the ratio between the PD and non-PD spectra bands of each patient as features, while the adaptive SVM classifier adjusts its decision boundary until a suitable model is obtained. This yields individualised features and classifier pairs for each patient. Datasets containing local field potentials of PD patients were used to validate the method. Six of the nine patient datasets tested achieved a classification accuracy greater than 98%. The adaptive detector is suitable for realization on chip.

  20. Sensitive and selective detection of prostate-specific antigen using a photonic crystal nanolaser.

    PubMed

    Hachuda, Shoji; Watanabe, Takumi; Takahashi, Daichi; Baba, Toshihiko

    2016-06-13

    The detection of low-concentration biomarkers is expected to facilitate the early diagnosis of severe diseases, including malignant tumors. Using photonic crystal nanolaser sensors, we detected prostate-specific antigen (PSA) from a concentration of 1 fM, which is difficult to detect by conventional enzyme-linked immunosorbent assay. The signal intensity and stability were improved by using a surfactant (i.e., ethanolamine). Even when a contaminant such as bovine serum albumin was mixed into the PSA sample, thereby increasing the concentration of the contaminant ten billion times, it was still possible to maintain a high level of detection.

  1. Scalable graphene field-effect sensors for specific protein detection.

    PubMed

    Saltzgaber, Grant; Wojcik, Peter; Sharf, Tal; Leyden, Matthew R; Wardini, Jenna L; Heist, Christopher A; Adenuga, Adeniyi A; Remcho, Vincent T; Minot, Ethan D

    2013-09-06

    We demonstrate that micron-scale graphene field-effect transistor biosensors can be fabricated in a scalable fashion from large-area chemical vapor deposition derived graphene. We electrically detect the real-time binding and unbinding of a protein biomarker, thrombin, to and from aptamer-coated graphene surfaces. Our sensors have low background noise and high transconductance, comparable to exfoliated graphene devices. The devices are reusable and have a shelf-life greater than one week.

  2. 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.

  3. 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

  4. 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

  5. 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.

  6. High flux isotope reactor technical specifications

    SciTech Connect

    Not Available

    1982-04-01

    Technical specifications are presented concerning safety limits and limiting safety system settings; limiting conditions for operation; surveillance requirements; design features; administrative controls; and accidents and anticipated transients.

  7. 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

  8. Scene-specific pedestrian detection for static video surveillance.

    PubMed

    Wang, Xiaogang; Wang, Meng; Li, Wei

    2014-02-01

    The performance of a generic pedestrian detector may drop significantly when it is applied to a specific scene due to the mismatch between the source training set and samples from the target scene. We propose a new approach of automatically transferring a generic pedestrian detector to a scene-specific detector in static video surveillance without manually labeling samples from the target scene. The proposed transfer learning framework consists of four steps. 1) Through exploring the indegrees from target samples to source samples on a visual affinity graph, the source samples are weighted to match the distribution of target samples. 2) It explores a set of context cues to automatically select samples from the target scene, predicts their labels, and computes confidence scores to guide transfer learning. 3) The confidence scores propagate among target samples according to their underlying visual structures. 4) Target samples with higher confidence scores have larger influence on training scene-specific detectors. All these considerations are formulated under a single objective function called confidence-encoded SVM, which avoids hard thresholding on confidence scores. During test, only the appearance-based detector is used without context cues. The effectiveness is demonstrated through experiments on two video surveillance data sets. Compared with a generic detector, it improves the detection rates by 48 and 36 percent at one false positive per image (FPPI) on the two data sets, respectively. The training process converges after one or two iterations on the data sets in experiments.

  9. Multi-Harmony: detecting functional specificity from sequence alignment.

    PubMed

    Brandt, Bernd W; Feenstra, K Anton; Heringa, Jaap

    2010-07-01

    Many protein families contain sub-families with functional specialization, such as binding different ligands or being involved in different protein-protein interactions. A small number of amino acids generally determine functional specificity. The identification of these residues can aid the understanding of protein function and help finding targets for experimental analysis. Here, we present multi-Harmony, an interactive web sever for detecting sub-type-specific sites in proteins starting from a multiple sequence alignment. Combining our Sequence Harmony (SH) and multi-Relief (mR) methods in one web server allows simultaneous analysis and comparison of specificity residues; furthermore, both methods have been significantly improved and extended. SH has been extended to cope with more than two sub-groups. mR has been changed from a sampling implementation to a deterministic one, making it more consistent and user friendly. For both methods Z-scores are reported. The multi-Harmony web server produces a dynamic output page, which includes interactive connections to the Jalview and Jmol applets, thereby allowing interactive analysis of the results. Multi-Harmony is available at http://www.ibi.vu.nl/ programs/shmrwww.

  10. Multi-Harmony: detecting functional specificity from sequence alignment

    PubMed Central

    Brandt, Bernd W.; Feenstra, K. Anton; Heringa, Jaap

    2010-01-01

    Many protein families contain sub-families with functional specialization, such as binding different ligands or being involved in different protein–protein interactions. A small number of amino acids generally determine functional specificity. The identification of these residues can aid the understanding of protein function and help finding targets for experimental analysis. Here, we present multi-Harmony, an interactive web sever for detecting sub-type-specific sites in proteins starting from a multiple sequence alignment. Combining our Sequence Harmony (SH) and multi-Relief (mR) methods in one web server allows simultaneous analysis and comparison of specificity residues; furthermore, both methods have been significantly improved and extended. SH has been extended to cope with more than two sub-groups. mR has been changed from a sampling implementation to a deterministic one, making it more consistent and user friendly. For both methods Z-scores are reported. The multi-Harmony web server produces a dynamic output page, which includes interactive connections to the Jalview and Jmol applets, thereby allowing interactive analysis of the results. Multi-Harmony is available at http://www.ibi.vu.nl/ programs/shmrwww. PMID:20525785

  11. 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.

  12. Non-specific sensor arrays for chemical detection

    NASA Astrophysics Data System (ADS)

    Johnson, Kevin; Minor, Christian

    2015-05-01

    Non-specific chemical sensor arrays have been the subject of considerable research efforts over the past thirty years with the idea that, by analogy to vertebrate olfaction, they are potentially capable of rendering complex chemical assessments with relatively modest logistical footprints. However, the actual implementation of such devices in challenging "real world" scenarios has arguably continued to fall short of these expectations. This work examines the inherent limitations of such devices for complex chemical sensing scenarios, placing them on a continuum between simple univariate sensors and complex multivariate analytical instrumentation and analyzing their utility in general-purpose chemical detection and accurate chemical sensing in the presence of unknown "unknowns." Results with simulated and acquired data sets are presented with discussion of the implications in development of chemical sensor arrays suitable for complex scenarios.

  13. EPCA-2: a highly specific serum marker for prostate cancer.

    PubMed

    Leman, Eddy S; Cannon, Grant W; Trock, Bruce J; Sokoll, Lori J; Chan, Daniel W; Mangold, Leslie; Partin, Alan W; Getzenberg, Robert H

    2007-04-01

    To describe the initial assessment of early prostate cancer antigen (EPCA)-2 as a serum marker for the detection of prostate cancer and to examine its sensitivity and specificity. Serum samples were obtained from 385 men: those with prostate-specific antigen (PSA) levels less than 2.5 ng/mL, PSA levels of 2.5 ng/mL or greater with negative biopsy findings, benign prostatic hyperplasia, organ-confined prostate cancer, non-organ-confined disease, and prostate cancer with PSA levels less than 2.5 ng/mL. In addition, a diverse group of controls was assessed with an enzyme-linked immunosorbent assay to detect an epitope of the EPCA-2 protein, EPCA-2.22. Using a cutoff of 30 ng/mL, the EPCA-2.22 assay had a 92% specificity (95% confidence interval 85% to 96%) for healthy men and men with benign prostatic hyperplasia and 94% sensitivity (95% confidence interval [CI] 93% to 99%) for overall prostate cancer. The specificity for PSA in these selected groups of patients was 65% (95% CI 55% to 75%). Additionally, EPCA-2.22 was highly accurate in differentiating between localized and extracapsular disease (area under the curve 0.89, 95% CI 0.82 to 0.97, P <0.0001) in contrast to PSA (area under the curve 0.62, 95% CI 0.50 to 0.75, P = 0.05). The results of our study have shown that EPCA-2 is a novel biomarker associated with prostate cancer that has high sensitivity and specificity and accurately differentiates between men with organ-confined and non-organ-confined disease.

  14. 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

  15. Measuring Specific Heats at High Temperatures

    NASA Technical Reports Server (NTRS)

    Vandersande, Jan W.; Zoltan, Andrew; Wood, Charles

    1987-01-01

    Flash apparatus for measuring thermal diffusivities at temperatures from 300 to 1,000 degrees C modified; measures specific heats of samples to accuracy of 4 to 5 percent. Specific heat and thermal diffusivity of sample measured. Xenon flash emits pulse of radiation, absorbed by sputtered graphite coating on sample. Sample temperature measured with thermocouple, and temperature rise due to pulse measured by InSb detector.

  16. 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…

  17. 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…

  18. Sensitivity and Specificity of Histoplasma Antigen Detection by Enzyme Immunoassay.

    PubMed

    Cunningham, Lauren; Cook, Audrey; Hanzlicek, Andrew; Harkin, Kenneth; Wheat, Joseph; Goad, Carla; Kirsch, Emily

    2015-01-01

    The objective of this study was to evaluate the sensitivity and specificity of an antigen enzyme immunoassay (EIA) on urine samples for the diagnosis of histoplasmosis in dogs. This retrospective medical records review included canine cases with urine samples submitted for Histoplasma EIA antigen assay between 2007 and 2011 from three veterinary institutions. Cases for which urine samples were submitted for Histoplasma antigen testing were reviewed and compared to the gold standard of finding Histoplasma organisms or an alternative diagnosis on cytology or histopathology. Sensitivity, specificity, negative predictive value, positive predictive value, and the kappa coefficient and associated confidence interval were calculated for the EIA-based Histoplasma antigen assay. Sixty cases met the inclusion criteria. Seventeen cases were considered true positives based on identification of the organism, and 41 cases were considered true negatives with an alternative definitive diagnosis. Two cases were considered false negatives, and there were no false positives. Sensitivity was 89.47% and the negative predictive value was 95.35%. Specificity and the positive predictive value were both 100%. The kappa coefficient was 0.9207 (95% confidence interval, 0.8131-1). The Histoplasma antigen EIA test demonstrated high specificity and sensitivity for the diagnosis of histoplasmosis in dogs.

  19. Reassessing the Detection of B-Virus–Specific Serum Antibodies

    PubMed Central

    Katz, David; Shi, Wei; Wildes, Martin J; Krug, Peter W; Hilliard, Julia K

    2012-01-01

    B virus, a natural pathogen of macaques, can cause a fatal zoonotic disease in humans. Serologic screening of macaques by titration ELISA (tELISA, screening test) and by Western blot analysis (WBA, confirmatory test) is one of the principle measures to prevent human infection. Here we slightly modified these 2 tests and reevaluated their correlation. We developed a high-throughput tELISA and used it to screen 278 sera simultaneously against the homologous BV antigen and the heterologous antigens of Papiine herpesvirus 2 and Human herpesvirus 1. More sera (35.6%) were positive by the BV-ELISA than by the HVP2-ELISA (21.6%) or HSV1-ELISA (19.8%). The superiority of the homologous tELISA over the heterologous tELISA was prominent in low-titer sera. WBA confirmed only 21% of the tELISA-positive sera with low or intermediate antibody titers. These sera might have contained antibodies to conformational epitopes that could not be detected by WBA, in which denatured antigens are used, but that could be detected by tELISA, which detects both linear and conformational epitopes. WBA confirmed 82% of the tELISA high-titer sera. However, WBA defined the remaining 18% of sera, which were negative by tELISA, as nonnegative. This finding can be attributed to the difficulties encountered with the subjective interpretation of results by WBA. Together, the current results indicate the inadequacy of WBA as a confirmatory assay for sera with low antibody titers. PMID:23561886

  20. 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.

  1. Specific determination of benzene in urine using dynamic headspace and mass-selective detection.

    PubMed

    Ljungkvist, G; Lärstad, M; Mathiasson, L

    1999-01-08

    A method for the determination of benzene in urine was developed, based on dynamic headspace and preconcentration of the analyte on a solid sorbent. The subsequent analysis by thermal desorption of the sorbent, capillary gas chromatography and mass-selective detection ascertained a low limit of detection (6.5 ng/l) and a highly specific determination. The limit of detection is an order of magnitude lower than that reported earlier and allows reliable quantitation of occupational exposure and of most environmental exposures. Samples could be stored frozen for at least a month without significant loss.

  2. The detection of a precartilage, blastema-specific marker.

    PubMed

    Aulthouse, A L; Solursh, M

    1987-04-01

    Mesenchymal cell aggregates, termed blastema in vivo, precede cartilage differentiation in vivo and in high-density cell cultures. The galactose specific lectin, peanut agglutinin (PNA), has been shown to be blastema specific (B. Zimmermann and M. Thies, 1984, Histochemistry 81, 353-361). PNA appears to be a marker for precartilage cellular aggregates both in vivo and in vitro. Frozen sections of stage 24 chick wing buds were double stained with PNA-rhodamine and by indirect immunofluorescence with antibody directed against type II collagen. The PNA stained the humeral blastema intensely and extended distal to the level of type II collagen. High-density cultures of stage 24 chick wing buds were also evaluated for the distribution of PNA binding. Sixteen-hour cultures showed the earliest consistent appearance of PNA binding. The PNA-stained areas coincided with hematoxylin-stained cell aggregates. PNA staining was inhibited by 50 mM D(+)-galactose and was not sensitive to 1% testicular hyaluronidase pretreatment. No Alcian blue-staining nodules were present yet at 16 hr. The presence of a precartilage, blastema-specific marker in situ, as well as in precartilage aggregates in cultures, suggests the similarities in chondrogenesis between these two conditions. Stage 19 limb bud cultures did not form nodules but did form aggregates that were PNA positive. Furthermore, single cells that differentiated into chondrocytes on collagen gels or after cytochalasin D treatment lacked PNA-binding material. These results suggest that this material is specific to precartilage aggregates. The PNA-positive material was extracellular in distribution and was removed after brief extraction with 0.5 M guanidine hydrochloride.

  3. Automated type specific ELISA probe detection of amplified NS3 gene products of dengue viruses.

    PubMed Central

    Chow, V T; Yong, R Y; Ngoh, B L; Chan, Y C

    1997-01-01

    AIM: To apply an automated system of nucleic acid hybridisation coupled with the enzyme linked immunosorbent assay (ELISA) for the type specific detection of amplification products of dengue viruses. METHODS: Non-structural 3 (NS3) gene targets of reference strains of all four dengue and other flaviviruses, as well as dengue patient viraemic sera, were subjected to reverse transcription and polymerase chain reaction using consensus and dengue type specific primers and digoxigenin-11-dUTP label incorporation. The amplification products were detected by biotinylated type specific primers which served as ELISA capture probes bound to streptavidin coated tubes. RESULTS: Significantly high spectrophotometric absorbance readings were obtained by hybridisation of the consensus and seminested amplification products of all four dengue viruses with their respective capture probes. In contrast, extremely low absorbances were observed for consensus products of Japanese encephalitis, yellow fever, and Kunjin viruses, which served as negative controls. These ELISA data correlated well with agarose gel electrophoresis of dengue type specific amplified products of diagnostic sizes. CONCLUSIONS: The combination of in vitro amplification and antibody based detection offers rapid, type specific, high throughput, and gel-free detection of amplified products of dengue viruses. Images PMID:9215155

  4. 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.

  5. 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.

  6. High specific activity platinum-195m

    SciTech Connect

    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.

  7. 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.

  8. 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.

  9. 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.

  10. Colorimetric Immuno-Protein Phosphatase Inhibition Assay for Specific Detection of Microcystins and Nodularins of Cyanobacteria

    PubMed Central

    Metcalf, James S.; Bell, Steven G.; Codd, Geoffrey A.

    2001-01-01

    A novel immunoassay was developed for specific detection of cyanobacterial cyclic peptide hepatotoxins which inhibit protein phosphatases. Immunoassay methods currently used for microcystin and nodularin detection and analysis do not provide information on the toxicity of microcystin and/or nodularin variants. Furthermore, protein phosphatase inhibition-based assays for these toxins are not specific and respond to other environmental protein phosphatase inhibitors, such as okadaic acid, calyculin A, and tautomycin. We addressed the problem of specificity in the analysis of protein phosphatase inhibitors by combining immunoassay-based detection of the toxins with a colorimetric protein phosphatase inhibition system in a single assay, designated the colorimetric immuno-protein phosphatase inhibition assay (CIPPIA). Polyclonal antibodies against microcystin-LR were used in conjunction with protein phosphatase inhibition, which enabled seven purified microcystin variants (microcystin-LR, -D-Asp3-RR, -LA, -LF, -LY, -LW, and -YR) and nodularin to be distinguished from okadaic acid, calyculin A, and tautomycin. A range of microcystin- and nodularin-containing laboratory strains and environmental samples of cyanobacteria were assayed by CIPPIA, and the results showed good correlation (R2 = 0.94, P < 0.00001) with the results of high-performance liquid chromatography with diode array detection for toxin analysis. The CIPPIA procedure combines ease of use and detection of low concentrations with toxicity assessment and specificity for analysis of microcystins and nodularins. PMID:11157261

  11. 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.

  12. Can hyperspectral remote sensing detect species specific biochemicals?

    USDA-ARS?s Scientific Manuscript database

    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,...

  13. 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.

  14. 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.

  15. Sequence-Specific DNA Detection at 10 fM by Electromechanical Signal Transduction

    PubMed Central

    2015-01-01

    Target DNA fragments at 10 fM concentration (approximately 6 × 105 molecules) were detected against a DNA background simulating the noncomplementary genomic DNA present in real samples using a simple, PCR-free, optics-free approach based on electromechanical signal transduction. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is highly desired for a range of diverse applications. We previously described a potentially low-cost device for sequence-specific nucleic acid detection based on conductance change measurement of a pore blocked by electrophoretically mobilized bead-(peptide nucleic acid probe) conjugates upon hybridization with target nucleic acid. Here, we demonstrate the operation of our device with longer DNA targets, and we describe the resulting improvement in the limit of detection (LOD). We investigated the detection of DNA oligomers of 110, 235, 419, and 1613 nucleotides at 1 pM to 1 fM and found that the LOD decreased as DNA length increased, with 419 and 1613 nucleotide oligomers detectable down to 10 fM. In addition, no false positive responses were obtained with noncomplementary, control DNA fragments of similar length. The 1613-base DNA oligomer is similar in size to 16S rRNA, which suggests that our device may be useful for detection of pathogenic bacteria at clinically relevant concentrations based on recognition of species-specific 16S rRNA sequences. PMID:25203740

  16. High Density Jet Fuel Supply and Specifications

    DTIC Science & Technology

    1986-01-01

    same shortcomings. Perhaps different LAK blends using heavy reformate or heavy cat cracker naphtha (both high in aromatics and isoparaffins) could... catalytic cracking (FCC) process. Subsequent investigations funded by the U. S. Air Force concentrated on producing a similar fuel from the...cut (19% overhead) and adding heavy naphtha (320-440F) from a nearby paraffinic crude (40"API Wyoming Sweet) an excellent JP-8X can be created. Table 5

  17. 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

  18. 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.

  19. 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

  20. Specific PCR detection of tiger, leopard, and lion ingredients from test samples.

    PubMed

    Cao, Jijuan; Xu, Junyi; Liu, Ran; Yu, Ke; Wang, Changwen

    2011-01-01

    A PCR method was developed for specific detection of tiger, leopard, and lion DNA from test specimens for inspection and quarantine or for law-enforced animal protection. Three pairs of specific primers were designed based on the mitochondrial cytochrome b gene of tiger, leopard, and lion and used in the PCR testing. To mimic the effect of food processing on the sensitivity of the test, the tiger muscle and bovine bonemeal powder samples were treated at 133 degrees C for 30 min. At this processing condition, the method was sensitive enough to detect as low as 0.05% of tiger-derived ingredients from the mixed bonemeal powders. The data demonstrate that our PCR method is convenient and economic, with high sensitivity and repeatability, and can be used to detect and identify tiger, leopard, and lion ingredients from various test samples.

  1. Sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of HIV-1.

    PubMed

    Curtis, Kelly A; Rudolph, Donna L; Owen, S Michele

    2009-06-01

    HIV diagnosis at the point-of-care or in resource-limited settings poses considerable challenges due to time and cost limitations. Currently, nucleic acid-based tests are the only reliable method for diagnosing recent infections during the window period post-infection and pre-seroconversion, but these tests are only suitable for well-equipped laboratory settings. The reverse transcription loop-mediated isothermal amplification (RT-LAMP) technology exhibits characteristics that are ideal for the development of a rapid, cost-effective nucleic acid-based test for detection of HIV DNA and RNA. In this study, a sequence-specific detection method was developed for immediate, naked-eye visualization of RT-LAMP products with high sensitivity and specificity. The rapid detection method was incorporated into the HIV-1-specific RT-LAMP assay and validated using minute volumes of whole blood from HIV-1-infected individuals. Together with the minimal sample preparation time and one-step, isothermal amplification reaction, the sequence-specific detection method adds to the overall versatility of the RT-LAMP assay and enhances the applicability for use at point-of-care or resource-limited sites.

  2. Broad-spectrum protein biosensors for class-specific detection of antibiotics.

    PubMed

    Weber, Cornelia C; Link, Nils; Fux, Cornelia; Zisch, Andreas H; Weber, Wilfried; Fussenegger, Martin

    2005-01-05

    The dramatically increasing prevalence of multi-drug-resistant human pathogenic bacteria and related mortality requires two key actions: (i) decisive initiatives for the detection of novel antibiotics and (ii) a global ban for use of antibiotics as growth promotants in stock farming. Both key actions entail technology for precise, high-sensitive detection of antibiotic substances either to detect and validate novel anti-infective structures or to enforce the non-use of clinically relevant antibiotics. We have engineered prokaryotic antibiotic response regulators into a molecular biosensor configuration able to detect tetracycline, streptogramin, and macrolide antibiotics in spiked liquids including milk and serum at ng/mL concentrations and up to 2 orders of magnitude below current Swiss and EC threshold values. This broad-spectrum, class-specific, biosensor-based assay has been optimized for use in a storable ready-to-use and high-throughput-compatible ELISA-type format. At the center of the assay is an antibiotic sensor protein whose interaction with specific DNA fragments is responsive to a particular class of antibiotics. Binding of biosensor protein to the cognate DNA chemically linked to a solid surface is converted into an immuno-based colorimetric readout correlating with specific antibiotics concentrations.

  3. Highly specific urine-based marker of bladder cancer.

    PubMed

    Van Le, Thu-Suong; Miller, Raymond; Barder, Timothy; Babjuk, Marko; Potter, Douglas M; Getzenberg, Robert H

    2005-12-01

    Bladder cancer represents a major health problem throughout the world, but advances in tumor biomarker development are revolutionizing how physicians diagnose the disease. We previously used an indirect immunoassay to demonstrate that the bladder cancer specific biomarker, BLCA-4, is present in urine samples from patients with bladder cancer, but not in samples from healthy individuals. In this study, a sandwich immunoassay was used to measure BLCA-4 in urine samples from patient populations with various urologic conditions and healthy individuals. Urine was collected from healthy individuals and from patients with bladder cancer, benign urologic conditions, or prostate cancer. BLCA-4 levels were evaluated by a sandwich immunoassay using two antibodies directed against distinct epitopes on BLCA-4. Using a prospectively determined cutoff of an absorbance unit (OD) of 0.04, 67 of the 75 samples from patients with bladder cancer were positive for BLCA-4, resulting in an assay sensitivity of 89%. Also, 62 of the 65 samples from individuals without bladder cancer were negative for BLCA-4, resulting in an assay specificity of 95%. The high sensitivity and specificity of the sandwich BLCA-4 immunoassay may allow for earlier detection and treatment of disease, thus greatly improving patient care.

  4. 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.

  5. Highly sensitive terahertz sensor for glucose detection

    NASA Astrophysics Data System (ADS)

    Kim, Hyo-Suk; Lee, Dong-Kyu; Lee, Seok; Chung, Youngchul; Seo, Minah

    2015-07-01

    In this report, we present a new type of non-contact detection method for glucose molecule using nano antenna array based bio sensing chip that operates at terahertz frequency range (0.5 - 2.5 THz). Localized and hugely enhanced transmitted terahertz field by nano antenna array in the sensing chip induced enhancement of absorption coefficient of glucose molecule that enables us to detect even very small volume of molecules. Nano antenna based terahertz sensing chip can be expected to offer accurate identification of glucose level as a non-invasive and painless sensing tool with high sensitivity.

  6. 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

  7. Improving qPCR methodology for detection of foaming bacteria by analysis of broad-spectrum primers and a highly specific probe for quantification of Nocardia spp. in activated sludge.

    PubMed

    Asvapathanagul, P; Olson, B H

    2017-01-01

    To develop qPCR broad-spectrum primers combined with a Nocardia genus-specific probe for the identification of a broad spectrum of Nocardia spp. and to analyse the effects of using this developed primer and probe set on the ability to quantify Nocardia spp. in mixed DNA. The consequences of using a degenerative primer set and species-specific probe for the genus Nocardia on qPCR assays were examined using DNA extracts of pure cultures and activated sludge. The mixed DNA extracts where the target organism Nocardia flavorosea concentration ranged from 5 × 10(2) to 5 × 10(6) copies per reaction, while the background organism's DNA (Mycobacterium bovis) concentration was held at 5 × 10(6) copies per reaction, only produced comparable cycle threshold florescence levels when N. flavorosea concentration was greater than or equal to the background organism concentration. When concentrations of N. flavorosea were lowered in increments of 1 log, while holding M. bovis concentrations constant at 5 × 10(6) copies per reaction, all assays demonstrated delayed cycle threshold values with a maximum 34·6-fold decrease in cycle threshold at a ratio of 10(6) M. bovis: 10(2) N. flavorosea copies per reaction. The data presented in this study indicated that increasing the ability of a primer set to capture a broad group of organisms can affect the accuracy of quantification even when a highly specific probe is used. This study examined several applications of molecular tools in complex communities such as evaluating the effect of mispriming vs interference. It also elucidates the importance of understanding the community genetic make-up on primer design. Degenerative primers are very useful in amplifying bacterial DNA across genera, but reduce the efficiency of qPCR reactions. Therefore, standards that address closely related background species must be used to obtain accurate qPCR results. © 2016 The Society for Applied Microbiology.

  8. 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.

  9. Mass Spectrometry Detection of Isolevuglandin Adduction to Specific Protein Residues

    PubMed Central

    Charvet, Casey D.; Pikuleva, Irina A.

    2014-01-01

    The aging process seems to be associated with oxidative stress and hence increased production of lipid peroxidation products, including isolevuglandins (isoLGs). The latter are highly reactive γ-ketoaldehydes which can form covalent adducts with primary amino groups of enzymes and proteins and alter the properties of these biomolecules. Yet, little is currently known about amino acid-containing compounds affected by isoLG modification in different age-related pathological processes. To facilitate the detection of these biomolecules, we developed a strategy in which the purified enzyme (or protein) of interest is first treated with authentic isoLG in vitro to evaluate whether it contains reactive lysine residues prone to modification with isoLGs. The data obtained serve as a basis for making the “GO/NO GO” decision as to whether to pursue a further search of this isoLG modification in a biological sample. In this chapter, we describe the conditions for the in vitro isoLG modification assay and how to use mass spectrometry to identify the isoLG-modified peptides and amino acid residues. Our studies were carried out on cytochrome P450 27A1, an important metabolic enzyme, and utilized iso[4]levuglandin E2 as a prototypical isoLG. The isoLG-treated cytochrome P450 was subjected to proteolysis followed by liquid chromatography-tandem mass spectrometry for peptide separation and analysis by Mascot, a proteomics search engine, for the presence of modified peptides. The developed protocol could be applied to characterization of other enzymes/proteins and other types of unconventional post-translational protein modification. PMID:25323515

  10. 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.

  11. 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

  12. High-sensitivity detection of TNT.

    PubMed

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

    2006-12-26

    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 approximately 400 nm around 7.3 microm and produces a maximum continuous-wave power of approximately 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-microm region to demonstrate continuous tunability of the IR source.

  13. Specific detection of biomolecules in physiological solutions using graphene transistor biosensors.

    PubMed

    Gao, Ning; Gao, Teng; Yang, Xiao; Dai, Xiaochuan; Zhou, Wei; Zhang, Anqi; Lieber, Charles M

    2016-12-20

    Nanomaterial-based field-effect transistor (FET) sensors are capable of label-free real-time chemical and biological detection with high sensitivity and spatial resolution, although direct measurements in high-ionic-strength physiological solutions remain challenging due to the Debye screening effect. Recently, we demonstrated a general strategy to overcome this challenge by incorporating a biomolecule-permeable polymer layer on the surface of silicon nanowire FET sensors. The permeable polymer layer can increase the effective screening length immediately adjacent to the device surface and thereby enable real-time detection of biomolecules in high-ionic-strength solutions. Here, we describe studies demonstrating both the generality of this concept and application to specific protein detection using graphene FET sensors. Concentration-dependent measurements made with polyethylene glycol (PEG)-modified graphene devices exhibited real-time reversible detection of prostate specific antigen (PSA) from 1 to 1,000 nM in 100 mM phosphate buffer. In addition, comodification of graphene devices with PEG and DNA aptamers yielded specific irreversible binding and detection of PSA in pH 7.4 1x PBS solutions, whereas control experiments with proteins that do not bind to the aptamer showed smaller reversible signals. In addition, the active aptamer receptor of the modified graphene devices could be regenerated to yield multiuse selective PSA sensing under physiological conditions. The current work presents an important concept toward the application of nanomaterial-based FET sensors for biochemical sensing in physiological environments and thus could lead to powerful tools for basic research and healthcare.

  14. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. 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

  16. High frequency of SLC22A12 variants causing renal hypouricemia 1 in the Czech and Slovak Roma population; simple and rapid detection method by allele-specific polymerase chain reaction.

    PubMed

    Gabrikova, Dana; Bernasovska, Jarmila; Sokolova, Jitka; Stiburkova, Blanka

    2015-10-01

    Renal hypouricemia is a rare heterogeneous inherited disorder characterized by impaired tubular uric acid transport with severe complications, such as acute kidney injury. Type 1 and 2 are caused by loss-of-function mutations in the SLC22A12 and SLC2A9 gene, respectively. A cohort of 881 randomly chosen ethnic Roma from two regions in Eastern Slovakia and two regions in the Czech Republic participated. Genomic DNA was isolated from buccal swabs and/or from blood samples. The c.1245_1253del and c.1400C>T genotypes were determined using polymerase chain reaction with allele-specific primers in a multiplex arrangement and/or direct sequencing of exon 7 and 9. Allele frequencies and genotypes were tested for Hardy-Weinberg equilibrium using the Chi-square test. 25 subjects were heterozygous and three were homozygous for the c.1245_1253del, while 92 subjects were heterozygous and two were homozygous for the c.1400C>T. Moreover, two participants were compound heterozygotes. Frequencies of the c.1245_1253del and c.1400C>T variants were 1.87 and 5.56 %, respectively. Our finding confirms an uneven geographical and ethnic distribution of SLC22A12 mutant variants. We found that the c.1245_1253del and c.1400C>T variants were present in the Czech and Slovak Roma population at unexpectedly high frequencies. Renal hypouricemia should be kept in mind during differential diagnostic on Roma patients with low serum uric acid concentrations.

  17. 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.

  18. A reusable sensor for the label-free detection of specific oligonucleotides by surface plasmon fluorescence spectroscopy.

    PubMed

    Nöll, Gilbert; Su, Qiang; Heidel, Björn; Yu, Yaming

    2014-01-01

    The development of a reusable molecular beacon (MB)-based sensor for the label-free detection of specific oligonucleotides using surface plasmon fluorescence spectroscopy (SPFS) as the readout method is described. The MBs are chemisorbed at planar gold surfaces serving as fluorescence quenching units. Target oligonucleotides of 24 bases can be detected within a few minutes at high single-mismatch discrimination rates.

  19. 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.

  20. 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.

  1. 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.

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

    PubMed Central

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

    2011-01-01

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

  3. Investigations of Novel Sensor Technology for Explosive Specific Detection

    DTIC Science & Technology

    2009-12-01

    considered impractical due to oxidation , however, a zinc analogue was synthesized to improve the photostability. [67] They concluded that AcrH2 is...of 2,4-dinitrotoluene in a γ- CD/metal oxide matrix and its sensitive detection via a cyclic surface polarization impedance (cSPI) method”, Chemistry...sensor ........................................................................................... 40 6.3.13 Nanofibrous membranes

  4. 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

    2013-02-05

    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.

  5. Comparison of techniques for the detection of monocyte specific antigens.

    PubMed

    Jager, M J; Thompson, J S; Claas, F H; Van Rood, J J

    1985-10-10

    Monocyte specific antigens are relevant in renal and bone marrow transplantation, but a reproducible monocyte-antigen system has not yet been recognized. In order to establish a sensitive test system with reproducible results in monocyte serology, 3 different monocyte cytotoxicity techniques were compared. In our hands the two-colour fluorescence test on post-Ficoll total leukocyte suspensions fulfilled the criteria. This technique was used to screen sera from multiparous women and renal transplant recipients for the presence of monocyte specific antibodies. By testing sera on cells from individuals who were HLA compatible with the serum donors, anti-HLA reactions were excluded. Several promising sera containing monocyte specific antibodies were identified, thus indicating the success of our approach.

  6. 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.

  7. An RNAi-Enhanced Logic Circuit for Cancer Specific Detection and Destruction

    DTIC Science & Technology

    2013-02-01

    cancer specific detection and destruction. PRINCIPAL INVESTIGATOR: Ron Weiss...2013 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER An RNAi-enhanced logic circuit for cancer specific detection and destruction. 5b. GRANT NUMBER...ABSTRACT Modern breast cancer therapies utilize non-specific approaches to kill or remove cancerous cells, inflicting significant collateral damage to

  8. High resolution detection system of capillary electrophoresis

    NASA Astrophysics Data System (ADS)

    Wang, Jie; Wang, Li Qiang; Shi, Yan; Zheng, Hua; Lu, Zu Kang

    2007-12-01

    The capillary electrophoresis (CE) with laser induced fluorescence detection (LIFD) system was founded according to confocal theory. The 3-D adjustment of the exciting and collecting optical paths was realized. The photomultiplier tube (PMT) is used and the signals are processed by a software designed by ourselves. Under computer control, high voltage is applied to appropriate reservoirs and to inject and separate DNA samples respectively. Two fluorescent dyes Thiazole Orange (TO) and SYBR Green I were contrasted. With both of the dyes, high signals-to-noise images were obtained with the CE-LIFD system. The single-bases can be distinguished from the electrophoretogram and high resolution of DNA sample separation was obtained.

  9. Sensitive, Specific Complementary - Strand Optical Detection of Viral RNA

    DTIC Science & Technology

    1997-06-01

    sensing. 14. SUBJECT TERMS 15. NUMBER OF PAGES RNA, enterovirii, biosensor, evanscent waves, refractometry , 31 phase detection, complementary strand...the investigator(s) adhered to the CDC-NIH Guide for Biosafety in Microbiological and Biomedical Laboratories. (,- u r,’ Dt Table of Contents Overview...Nonetheless, the physical basis of the Georgia Tech signal is T ( u 20 30 40 50 identical to ours. They have also developed a single-stranded,Time (minutes

  10. Improving Indel Detection Specificity of the Ion Torrent PGM Benchtop Sequencer

    PubMed Central

    Yeo, Zhen Xuan; Chan, Maurice; Yap, Yoon Sim; Ang, Peter; Rozen, Steve; Lee, Ann Siew Gek

    2012-01-01

    The emergence of benchtop sequencers has made clinical genetic testing using next-generation sequencing more feasible. Ion Torrent's PGMTM is one such benchtop sequencer that shows clinical promise in detecting single nucleotide variations (SNVs) and microindel variations (indels). However, the large number of false positive indels caused by the high frequency of homopolymer sequencing errors has impeded PGMTM's usage for clinical genetic testing. An extensive analysis of PGMTM data from the sequencing reads of the well-characterized genome of the Escherichia coli DH10B strain and sequences of the BRCA1 and BRCA2 genes from six germline samples was done. Three commonly used variant detection tools, SAMtools, Dindel, and GATK's Unified Genotyper, all had substantial false positive rates for indels. By incorporating filters on two major measures we could dramatically improve false positive rates without sacrificing sensitivity. The two measures were: B-Allele Frequency (BAF) and VARiation of the Width of gaps and inserts (VARW) per indel position. A BAF threshold applied to indels detected by UnifiedGenotyper removed ∼99% of the indel errors detected in both the DH10B and BRCA sequences. The optimum BAF threshold for BRCA sequences was determined by requiring 100% detection sensitivity and minimum false discovery rate, using variants detected from Sanger sequencing as reference. This resulted in 15 indel errors remaining, of which 7 indel errors were removed by selecting a VARW threshold of zero. VARW specific errors increased in frequency with higher read depth in the BRCA datasets, suggesting that homopolymer-associated indel errors cannot be reduced by increasing the depth of coverage. Thus, using a VARW threshold is likely to be important in reducing indel errors from data with higher coverage. In conclusion, BAF and VARW thresholds provide simple and effective filtering criteria that can improve the specificity of indel detection in PGMTM data without

  11. 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.

  12. A specific oligonucleotide primer for the rapid detection of Lactobacillus lindneri by polymerase chain reaction.

    PubMed

    Yasui, T; Okamoto, T; Taguchi, H

    1997-02-01

    A polymerase chain reaction (PCR) method was developed for the rapid detection of the beer-spoilage heterofermentative lactic acid bacterium Lactobacillus lindneri. Three strains, the Chinese brewery isolate DA1, the Japanese commercial beer isolate BG2, and the Japanese brewery isolate SE3, which were serologically classified as belonging to L. lindneri, were used in this study. After sequencing the 16S rDNA of the isolates DA1 and BG2 and the typical beer-spoilage heterofermentative Lactobacillus brevis L63, these sequences were compared with published data. A L. lindneri specific PCR primer, DA-40, was then constructed based on the V1 variable region of 16S rDNA. The specificity of PCR using the L. lindneri specific primer DA-40 and the universal primer 907r was examined using five L. lidneri strains: the three isolates described above and two strains from culture collection, DSM 20690 and DSM 20692. A variety of beer-spoilage lactic acid bacteria, including 71 Lactobacillus strains and 13 Pediococcus strains, were also included in this examination. No PCR product was obtained from any DNA with the exception of the five L. lindneri strains, indicating that the L. lindneri specific primer DA-40 was highly specific. The detection limit for L. lindneri in beer was 63 CFU/100 mL of beer.

  13. 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

  14. 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.

  15. Specificity tests of an oligonucleotide probe against food-outbreak salmonella for biosensor detection

    NASA Astrophysics Data System (ADS)

    Chen, I.-H.; Horikawa, S.; Xi, J.; Wikle, H. C.; Barbaree, J. M.; Chin, B. A.

    2017-05-01

    Phage based magneto-elastic (ME) biosensors have been shown to be able to rapidly detect Salmonella in various food systems to serve food pathogen monitoring purposes. In this ME biosensor platform, the free-standing strip-shaped magneto-elastic sensor is the transducer and the phage probe that recognizes Salmonella in food serves as the bio-recognition element. According to Sorokulova et al. at 2005, a developed oligonucleotide probe E2 was reported to have high specificity to Salmonella enterica Typhimurium. In the report, the specificity tests were focused in most of Enterobacterace groups outside of Salmonella family. Here, to understand the specificity of phage E2 to different Salmonella enterica serotypes within Salmonella Family, we further tested the specificity of the phage probe to thirty-two Salmonella serotypes that were present in the major foodborne outbreaks during the past ten years (according to Centers for Disease Control and Prevention). The tests were conducted through an Enzyme linked Immunosorbent Assay (ELISA) format. This assay can mimic probe immobilized conditions on the magnetoelastic biosensor platform and also enable to study the binding specificity of oligonucleotide probes toward different Salmonella while avoiding phage/ sensor lot variations. Test results confirmed that this oligonucleotide probe E2 was high specific to Salmonella Typhimurium cells but showed cross reactivity to Salmonella Tennessee and four other serotypes among the thirty-two tested Salmonella serotypes.

  16. Detecting General and Specific Errors in Expository Texts.

    ERIC Educational Resources Information Center

    Yussen, Steven R.; Smith, M. Cecil

    1990-01-01

    In three experiments, a total of 148 college students read or listened to expository passages containing general or specific errors. In all 3 experiments, students were more likely to spot general errors. Results do not indicate that monitoring skills were substantially different for listening or reading. (SLD)

  17. 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.…

  18. Crimean-Congo Hemorrhagic Fever Virus-Specific Antibody Detection in Cattle in Mauritania.

    PubMed

    Sas, Miriam A; Mertens, Marc; Isselmou, Ekaterina; Reimer, Nicole; El Mamy, Bezeid O; Doumbia, Baba; Groschup, Martin H

    2017-08-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) was detected for the first time in Mauritania in 1983 and several CCHFV outbreaks were reported in the following years. The last human case was diagnosed in 2015. However, no recent data exist about the prevalence of CCHFV in animals, although it is already described that prevalence studies in animals serve as good risk indicators. CCHFV can cause a severe hemorrhagic fever with a high case fatality rate in humans. Therefore, a precise risk assessment on the basis of updated data is very important. This article gives an overview about the current CCHFV prevalence in cattle in Mauritania. A seroprevalence study was carried out using 495 cattle sera from Mauritania, which were collected in the year 2013. The sera were analyzed by an inhouse CCHFV-IgG-ELISA. As second screening test, an adapted commercial CCHFV-IgG-ELISA was performed. Inconclusive sera were additionally tested by a modified commercial CCHFV-IgG-IFA. All assays showed high diagnostic sensitivity (>95%) and specificity (>98%). The overall prevalence of CCHFV-specific antibodies found in Mauritanian cattle was 67%, ranging from 56% to 90% in different provinces. This study shows a very high CCHFV-specific antibody prevalence in cattle in Mauritania. It is the highest seroprevalence detected in Mauritania so far. This strengthens the hypothesis that CCHFV is a serious and ongoing threat for public health in Mauritania.

  19. 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.

  20. Surface plasmon-enhanced fluorescence on Au nanohole array for prostate-specific antigen detection

    PubMed Central

    Zhang, Qingwen; Wu, Lin; Wong, Ten It; Zhang, Jinling; Liu, Xiaohu; Zhou, Xiaodong; Bai, Ping; Liedberg, Bo; Wang, Yi

    2017-01-01

    Localized surface plasmon (LSP) has been widely applied for the enhancement of fluorescence emission for biosensing owing to its potential for strong field enhancement. However, due to its small penetration depth, LSP offers limited fluorescence enhancement over a whole sensor chip and, therefore, insufficient sensitivity for the detection of biomolecules, especially large molecules. We demonstrate the simultaneous excitation of LSP and propagating surface plasmon (PSP) on an Au nanohole array under Kretschmann configuration for the detection of prostate-specific antigen with a sandwich immunoassay. The proposed method combines the advantages of high field enhancement by LSP and large surface area probed by PSP field. The simulated results indicated that a maximum enhancement of electric field intensity up to 1,600 times can be achieved under the simultaneous excitation of LSP and PSP modes. The sandwich assay of PSA carried out on gold nanohole array substrate showed a limit of detection of 140 fM supporting coexcitation of LSP and PSP modes. The limit of detection was approximately sevenfold lower than that when only LSP was resonantly excited on the same substrate. The results of this study demonstrate high fluorescence enhancement through the coexcitation of LSP and PSP modes and pave a way for its implementation as a highly sensitive bioassay. PMID:28392689

  1. Surface plasmon-enhanced fluorescence on Au nanohole array for prostate-specific antigen detection.

    PubMed

    Zhang, Qingwen; Wu, Lin; Wong, Ten It; Zhang, Jinling; Liu, Xiaohu; Zhou, Xiaodong; Bai, Ping; Liedberg, Bo; Wang, Yi

    2017-01-01

    Localized surface plasmon (LSP) has been widely applied for the enhancement of fluorescence emission for biosensing owing to its potential for strong field enhancement. However, due to its small penetration depth, LSP offers limited fluorescence enhancement over a whole sensor chip and, therefore, insufficient sensitivity for the detection of biomolecules, especially large molecules. We demonstrate the simultaneous excitation of LSP and propagating surface plasmon (PSP) on an Au nanohole array under Kretschmann configuration for the detection of prostate-specific antigen with a sandwich immunoassay. The proposed method combines the advantages of high field enhancement by LSP and large surface area probed by PSP field. The simulated results indicated that a maximum enhancement of electric field intensity up to 1,600 times can be achieved under the simultaneous excitation of LSP and PSP modes. The sandwich assay of PSA carried out on gold nanohole array substrate showed a limit of detection of 140 fM supporting coexcitation of LSP and PSP modes. The limit of detection was approximately sevenfold lower than that when only LSP was resonantly excited on the same substrate. The results of this study demonstrate high fluorescence enhancement through the coexcitation of LSP and PSP modes and pave a way for its implementation as a highly sensitive bioassay.

  2. The Drosophila immune system detects bacteria through specific peptidoglycan recognition.

    PubMed

    Leulier, François; Parquet, Claudine; Pili-Floury, Sebastien; Ryu, Ji-Hwan; Caroff, Martine; Lee, Won-Jae; Mengin-Lecreulx, Dominique; Lemaitre, Bruno

    2003-05-01

    The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions through selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist infection by Gram-negative bacteria. The bacterial components recognized by these pathways remain to be defined. Here we report that Gram-negative diaminopimelic acid-type peptidoglycan is the most potent inducer of the Imd pathway and that the Toll pathway is predominantly activated by Gram-positive lysine-type peptidoglycan. Thus, the ability of Drosophila to discriminate between Gram-positive and Gram-negative bacteria relies on the recognition of specific forms of peptidoglycan.

  3. Site-Specific Detection and Management of Nematodes

    USDA-ARS?s Scientific Manuscript database

    Nematode distribution varies significantly throughout a field and is highly correlated to soil texture and other edaphic factors. Field-wide application results in nematicides being applied to areas without nematodes and the application of sub-effective levels in areas with high nematode densities. ...

  4. Improved Sensitivity and Specificity for Detection of Prostate Cancer

    DTIC Science & Technology

    2007-11-01

    SUBJECT TERMS Prostate, spectroscopy, MRI 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF...assessment of the correlation between MRI /MRSI and histopathology. Specific Aim 2: The development of a tumor index based on individual MRI /MRSI...Overall Status of the Project: The goal of the study is to diagnose prostate cancer more effectively using various MRI techniques, with the ultimate

  5. Analysis of methylation microarray for tissue specific detection.

    PubMed

    Muangsub, Tachapol; Samsuwan, Jarunya; Tongyoo, Pumipat; Kitkumthorn, Nakarin; Mutirangura, Apiwat

    2014-12-10

    The role of human DNA methylation has been extensively studied in genomic imprinting, X-inactivation, and disease. However, studies of tissue-specific methylation remain limited. In this study, we use bioinformatics methods to analyze methylation data and reveal loci that are exclusively methylated or unmethylated in individual tissues. We collect 39 previously published DNA methylation profiles using an Illumina® HumanMethylation 27 BeadChip Kit containing 22 common tissues and involving 27,578 CpG loci across the human genome. We found 86 positions of tissue specific methylation CpG (TSM) that encompass 34 hypermethylated TSMs (31 genes) and 52 hypomethylated TSMs (47 genes). Tissues were found to contain 1 to 25 TSM loci, with the majority in the liver (25), testis (18), and brain (16). Fewer TSM loci were found in the muscle (8), ovary (7), adrenal gland (3), pancreas (2-4), kidney, spleen, and stomach (1 each). TSMs are predominantly located 0-300 base pairs in the 3' direction after the transcription start site. Similar to known promoters of methylation, hypermethylated TSM genes suppress transcription, while hypomethylated TSMs allow gene transcription. The majority of hypermethylated TSM genes encode membrane proteins and receptors, while hypomethylated TSM genes primarily encode signal peptides and tissue-specific proteins. In summary, the database of TSM loci produced herein is useful for the selection of tissue-specific DNA markers as diagnostic tools, as well as for the further study of the mechanisms and roles of TSM. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Scene-Specific Pedestrian Detection for Static Video Surveillance.

    PubMed

    Wang, Xiaogang; Wang, Meng; Li, Wei

    2013-06-21

    The performance of a generic pedestrian detector may drop significantly when it is applied to a specific scene due to the mismatch between the source training set and samples from the target scene. We propose a new approach of automatically transferring a generic pedestrian detector to a scene-specific detector in static video surveillance without manually labeling samples from the target scene. The proposed transfer learning framework consists of four steps. (1) Through exploring the indegrees from target samples to source samples on a visual affinity graph, the source samples are weighted to match the distribution of target samples. (2) It explores a set of context cues to automatically select samples from the target scene, predicts their labels, and computes confidence scores to guide transfer learning. (3) The confidence scores propagate among target samples according to their underlying visual structures. (4) Target samples with higher confidence scores have larger influence on training scene-specific detectors. All these considerations are formulated under a single objective function called Confidence-Encoded SVM, which avoids hard thresholding on confidence scores. During test, only the appearance-based detector is used without context cues. The effectiveness is demonstrated through experiments on two video surveillance datasets.

  7. A stacking flow immunoassay for the detection of dengue-specific immunoglobulins in salivary fluid.

    PubMed

    Zhang, Yi; Bai, Jianhao; Ying, Jackie Y

    2015-03-21

    Paper-based immunoassays, usually in the form of lateral flow tests, are currently the standard platform for home diagnostics. However, conventional lateral tests are often complicated by severe non-specific adsorption of detector particles when applied to test samples containing salivary fluid. It is believed that a high concentration of proteinaceous substances in salivary fluid causes particle aggregation and adhesion. In this study, we developed a stacking flow platform for single-step detection of a target antibody in salivary fluid. Stacking flow circumvents the need for separate sample pre-treatments, such as filtration or centrifugation, which are often required prior to testing saliva samples using paper-based immunoassays. This is achieved by guiding the samples and reagents to the test strip through different paths. By doing so, salivary substances that interfere with the particle-based sensing system are removed before they come into contact with the detection reagents, which greatly reduces the background. In addition, the stacking flow configuration enables uniform flow with a unique flow regulator, which leads to even test lines with good quantification capability, enabling the detection of ~20 ng mL(-1) α-fetoprotein in the serum. We have successfully applied the stacking flow device to detect dengue-specific immunoglobulins that are present in salivary fluid.

  8. Application of Food-specific IgG Antibody Detection in Allergy Dermatosis

    PubMed Central

    Yine, Hu; Shufang, Dai; Bin, Wang; Wei, Qu; Ashraf, Muhammad Aqeel; Junling, Gao

    2015-01-01

    The application of food-specific IgG antibody detection in allergy dermatoses was explored. 181 patients with allergy dermatoses were diagnosed from January to September 2014 and 20 healthy subjects were selected. Fourteen kinds of food-specific IgG antibodies were detected by ELISA method among all the subjects. The positive rates of IgG antibody of the patient group and the healthy group were respectively 65.2% and 5.0%. The positive rates of IgG antibody of egg, milk, shrimp and crab took a large proportion in three groups of patients with three kinds of allergy dermatoses of urticaria, eczema and allergic dermatitis, the proportion of which was respectively 70.2%, 77.8% and 71.7%. Among urticaria and allergic dermatitis patients with positive antibody, the positive rate of children was significantly higher than that of adults (p<0.05) while there was no significant difference between children and adults among eczema patients with positive antibody (p>0.05). Allergy dermatoses are closely related to food-specific IgG antibodies, and the allergy dermatoses patients have a high incidence rate of food intolerance; detecting IgG antibody in the serum of patients is of great significance for the diagnosis and treatment of allergy dermatoses.

  9. 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.

  10. 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.

  11. Nanopore based sequence specific detection of duplex DNA for genomic profiling.

    PubMed

    Singer, Alon; Wanunu, Meni; Morrison, Will; Kuhn, Heiko; Frank-Kamenetskii, Maxim; Meller, Amit

    2010-02-10

    We demonstrate a purely electrical method for the single-molecule detection of specific DNA sequences, achieved by hybridizing double-stranded DNA (dsDNA) with peptide nucleic acid (PNA) probes and electrophoretically threading the DNA through sub-5 nm silicon nitride pores. Bis-PNAs were used as the tagging probes in order to achieve high affinity and sequence specificity. Sequence detection is performed by reading the ion current traces of individual translocating DNA molecules, which display a characteristic secondary blockade level, absent in untagged molecules. The potential for barcoding DNA is demonstrated through nanopore analysis of once-tagged and twice-tagged DNA at different locations on the same genomic fragment. Our high-throughput, long-read length method can be used to identify key sequences embedded in individual DNA molecules, without the need for amplification or fluorescent/radio labeling. This opens up a wide range of possibilities in human genomics as well as in pathogen detection for fighting infectious diseases.

  12. [Inducible specific lux-biosensors for the detection of antibiotics: construction and main parameters].

    PubMed

    Kotova, V Iu; Ryzhenkova, K V; Manukhov, I V; Zavil'gel'skiĭ, G B

    2014-01-01

    Based on Escherichia coli, highly sensitive specific lux-biosensors for the detection of tetracycline and beta-lactam antibiotics, quinolones, and aminoglycosides have been obtained. To make biosensors, bacteria were used that contained fungal plasmids pTetA'::lux, pAmpC'::lux, pColD'::lux, and plbpA'::lux, in which transcription of the reporter Photorhabdus luminescens luxCDABE genes occurred from the inducible promoters of the tetA, ampC, cda, and ibpA genes, respectively. The main parameters (threshold sensitivity and response time) of lux-biosensors were measured. The high specificity of biosensors responding only to antibiotics of a certain type was demonstrated.

  13. 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.

  14. 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

  15. 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

  16. A rapid and specific method for the detection of indole in complex biological samples.

    PubMed

    Darkoh, Charles; Chappell, Cynthia; Gonzales, Christopher; Okhuysen, Pablo

    2015-12-01

    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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Anisakis simplex recombinant allergens increase diagnosis specificity preserving high sensitivity.

    PubMed

    Caballero, María Luisa; Umpierrez, Ana; Perez-Piñar, Teresa; Moneo, Ignacio; de Burgos, Carmen; Asturias, Juan A; Rodríguez-Pérez, Rosa

    2012-01-01

    So far, the frequency of Anisakis simplex-specific IgE antibodies has been determined by skin prick tests (SPTs) and the ImmunoCAP system. These commercial methods have good sensitivity, but their specificity is poor because they use complete parasite extracts. Our aim was to determine the frequency of sensitization to A. simplex using recombinant Ani s 1, Ani s 3, Ani s 5, Ani s 9 and Ani s 10 and to evaluate these allergens for diagnosis, comparing their performance with the commercial methods. We conducted a descriptive, cross-sectional validation study performed in an allergy outpatient hospital clinic. Patients without fish-related allergy (tolerant patients, n = 99), and A. simplex-allergic patients (n = 35) were studied by SPTs, ImmunoCAP assays and detection of specific IgE to A. simplex recombinant allergens by dot blotting. SPTs and ImmunoCAP assays were positive in 18 and 17% of tolerant patients, respectively. All A. simplex-allergic patients had positive SPTs and ImmunoCAP assays. Specific IgE against at least one of the A. simplex recombinant allergens tested was detected in 15% of sera from tolerant patients and in 100% of sera from A. simplex-allergic patients. Detection of at least one A. simplex recombinant allergen by dot blotting and ImmunoCAP assay using complete extract showed a diagnostic sensitivity of 100% with both methods. However, the specificity of dot blotting with A. simplex recombinant allergens was higher compared with ImmunoCAP (84.85 vs. 82.83%). There are 15% of tolerant patients with specific IgE against important A. simplex allergens. The recombinant allergens studied here increase the specificity of A. simplex diagnosis while keeping the highest sensitivity. A. simplex recombinant allergens should be included with A. simplex allergy diagnostic tests to improve their specificity. Copyright © 2012 S. Karger AG, Basel.

  18. [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.

  19. Diagnosis of typhoid fever: detection of Salmonella typhi porins-specific antibodies by inhibition ELISA.

    PubMed Central

    Nandakumar, K S; Palanivel, V; Muthukkaruppan, V

    1993-01-01

    Porins are highly immunogenic outer membrane proteins of Salmonella. Sera from typhoid patients contained a high level of IgG antibodies directed to porins of Salm. typhi. Since porins are highly conserved proteins, anti-porins antibodies both from typhoid patients and healthy normals reacted with porins from several Gram-negative bacteria. Therefore, in order to improve the specificity of detecting Salm. typhi porins-specific antibodies, an inhibition ELISA was developed using enzyme-conjugated MoAbs (MP1 and MPN4) specific to Salm. typhi porins. Sera from typhoid patients with positive haemoculture (16 out of 17) inhibited the binding of MP1 to porins, thus showing a positive test for typhoid, whereas sera from patients with other Gram-negative bacterial infections (n = 7) and from healthy volunteers (66 out of 67) were found to be negative. The sensitivity, specificity, accuracy, positive predictive value and negative predictive value of this assay were 94.1, 98.7, 97.8, 94.1 and 98.7% respectively. The validity of our inhibition ELISA for typhoid was higher than that of the Widal test. The diagnosis of typhoid fever as early as 3 days after the onset of fever, using a single specimen is possible. PMID:8222322

  20. 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.

  1. Detection of classical Hodgkin lymphoma specific sequence in peripheral blood using a next-generation sequencing approach

    PubMed Central

    Oki, Yasuhiro; Neelapu, Sattva S.; Fanale, Michelle; Kwak, Larry W.; Fayad, Luis; Rodriguez, Maria A.; Wallace, Michael; Klinger, Mark; Carlton, Victoria; Kong, Katherine; Faham, Malek; Younes, Anas

    2017-01-01

    Summary We applied a highly sensitive next-generation sequencing method to identify lymphoma-specific immunoglobulin gene segments in classical Hodgkin lymphoma (CHL) at initial diagnosis or recurrence, and assessed the ability of detecting such lymphoma-specific sequences in peripheral blood (PB). Seventeen CHL cases were tested and lymphoma-specific sequences were identified in 12 of the primary tumour biopsies. In 11 of these patients whose paired PB samples were available, tumour-specific clonotypes were detected in PB in eight patients. This data demonstrates the feasibility of detecting circulating tumour-specific sequences, creating an unprecedented opportunity to optimize the future treatment and monitoring strategies for patients with CHL. PMID:25818067

  2. 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.

  3. Optical detection and ionization of donors in specific electronic and nuclear spin States.

    PubMed

    Yang, A; Steger, M; Karaiskaj, D; Thewalt, M L W; Cardona, M; Itoh, K M; Riemann, H; Abrosimov, N V; Churbanov, M F; Gusev, A V; Bulanov, A D; Kaliteevskii, A K; Godisov, O N; Becker, P; Pohl, H-J; Ager, J W; Haller, E E

    2006-12-01

    We resolve the remarkably sharp bound exciton transitions of highly enriched 28Si using a single-frequency laser and photoluminescence excitation spectroscopy, as well as photocurrent spectroscopy. Well-resolved doublets in the spectrum of the 31P donor reflect the hyperfine coupling of the electronic and nuclear donor spins. The optical detection of the nuclear spin state, and selective pumping and ionization of donors in specific electronic and nuclear spin states, suggests a number of new possibilities which could be useful for the realization of silicon-based quantum computers.

  4. Highly Sensitive Detection of Protein Biomarkers with Organic Electrochemical Transistors.

    PubMed

    Fu, Ying; Wang, Naixiang; Yang, Anneng; Law, Helen Ka-Wai; Li, Li; Yan, Feng

    2017-09-18

    The analysis of protein biomarkers is of great importance in the diagnosis of diseases. Although many convenient and low-cost electrochemical approaches have been extensively investigated, they are not sensitive enough in the detection of protein biomarkers with low concentrations in physiological environments. Here, this study reports a novel organic-electrochemical-transistor-based biosensor that can successfully detect cancer protein biomarkers with ultrahigh sensitivity. The devices are operated by detecting electrochemical activity on gate electrodes, which is dependent on the concentrations of proteins labeled with catalytic nanoprobes. The protein sensors can specifically detect a cancer biomarker, human epidermal growth factor receptor 2, down to the concentration of 10(-14) g mL(-1) , which is several orders of magnitude lower than the detection limits of previously reported electrochemical approaches. Moreover, the devices can successfully differentiate breast cancer cells from normal cells at various concentrations. The ultrahigh sensitivity of the protein sensors is attributed to the inherent amplification function of the organic electrochemical transistors. This work paves a way for developing highly sensitive and low-cost biosensors for the detection of various protein biomarkers in clinical analysis in the future. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Strategy for Sensitive and Specific Detection of Yersinia pestis in Skeletons of the Black Death Pandemic

    PubMed Central

    Seifert, Lisa; Harbeck, Michaela; Thomas, Astrid; Hoke, Nadja; Zöller, Lothar; Wiechmann, Ingrid; Grupe, Gisela; Scholz, Holger C.; Riehm, Julia M.

    2013-01-01

    Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14th century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study. PMID:24069445

  6. Strategy for sensitive and specific detection of Yersinia pestis in skeletons of the black death pandemic.

    PubMed

    Seifert, Lisa; Harbeck, Michaela; Thomas, Astrid; Hoke, Nadja; Zöller, Lothar; Wiechmann, Ingrid; Grupe, Gisela; Scholz, Holger C; Riehm, Julia M

    2013-01-01

    Yersinia pestis has been identified as the causative agent of the Black Death pandemic in the 14(th) century. However, retrospective diagnostics in human skeletons after more than 600 years are critical. We describe a strategy following a modern diagnostic algorithm and working under strict ancient DNA regime for the identification of medieval human plague victims. An initial screening and DNA quantification assay detected the Y. pestis specific pla gene of the high copy number plasmid pPCP1. Results were confirmed by conventional PCR and sequence analysis targeting both Y. pestis specific virulence plasmids pPCP1 and pMT1. All assays were meticulously validated according to human clinical diagnostics requirements (ISO 15189) regarding efficiency, sensitivity, specificity, and limit of detection (LOD). Assay specificity was 100% tested on 41 clinically relevant bacteria and 29 Y. pseudotuberculosis strains as well as for DNA of 22 Y. pestis strains and 30 previously confirmed clinical human plague samples. The optimized LOD was down to 4 gene copies. 29 individuals from three different multiple inhumations were initially assessed as possible victims of the Black Death pandemic. 7 samples (24%) were positive in the pPCP1 specific screening assay. Confirmation through second target pMT1 specific PCR was successful for 4 of the positive individuals (14%). A maximum of 700 and 560 copies per µl aDNA were quantified in two of the samples. Those were positive in all assays including all repetitions, and are candidates for future continuative investigations such as whole genome sequencing. We discuss that all precautions taken here for the work with aDNA are sufficient to prevent external sample contamination and fulfill the criteria of authenticity. With regard to retrospective diagnostics of a human pathogen and the uniqueness of ancient material we strongly recommend using a careful strategy and validated assays as presented in our study.

  7. 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

  8. Double-immunodiffusion assay for detecting specific antibodies.

    PubMed

    Hornbeck, P

    2001-05-01

    Double immunodiffusion owes its success to the unique nature of antibody-antigen interactions. When polyvalent antibodies with moderate-to-high intrinsic affinities are mixed with antigen at the right ratio (called the zone of equivalence) lattices of antibody-antigen complexes form and precipitate out of solution. When, as described in this unit, gradients of antigen and antibody are established by diffusion from adjacent wells in a bed of agar, a line of practically insoluble precipitation forms at the equivalence zone (precipitin lines).

  9. 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.

  10. 21 CFR 866.3332 - Reagents for detection of specific novel influenza A viruses.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... A viruses. 866.3332 Section 866.3332 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Reagents § 866.3332 Reagents for detection of specific novel influenza A viruses. (a) Identification. Reagents for detection of specific novel influenza A viruses are devices that are intended for use in a...

  11. 21 CFR 866.3332 - Reagents for detection of specific novel influenza A viruses.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... A viruses. 866.3332 Section 866.3332 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Reagents § 866.3332 Reagents for detection of specific novel influenza A viruses. (a) Identification. Reagents for detection of specific novel influenza A viruses are devices that are intended for use in a...

  12. 21 CFR 866.3332 - Reagents for detection of specific novel influenza A viruses.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... A viruses. 866.3332 Section 866.3332 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Reagents § 866.3332 Reagents for detection of specific novel influenza A viruses. (a) Identification. Reagents for detection of specific novel influenza A viruses are devices that are intended for use in a...

  13. 21 CFR 866.3332 - Reagents for detection of specific novel influenza A viruses.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... A viruses. 866.3332 Section 866.3332 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Reagents § 866.3332 Reagents for detection of specific novel influenza A viruses. (a) Identification. Reagents for detection of specific novel influenza A viruses are devices that are intended for use in a...

  14. 21 CFR 866.3332 - Reagents for detection of specific novel influenza A viruses.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... A viruses. 866.3332 Section 866.3332 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF... Reagents § 866.3332 Reagents for detection of specific novel influenza A viruses. (a) Identification. Reagents for detection of specific novel influenza A viruses are devices that are intended for use in a...

  15. 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.

  16. 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.

  17. Comparison of the specificity of implantable dual chamber defibrillator detection algorithms.

    PubMed

    Hintringer, Florian; Deibl, Martina; Berger, Thomas; Pachinger, Otmar; Roithinger, Franz Xaver

    2004-07-01

    The aim of the study was to compare the specificity of dual chamber ICDs detection algorithms for correct classification of supraventricular tachyarrhythmias derived from clinical studies according to their size to detect an impact of sample size on the specificity. Furthermore, the study sought to compare the specificities of detection algorithms calculated from clinical data with the specificity calculated from simulations of tachyarrhythmias. A survey was conducted of all available sources providing data regarding the specificity of five dual chamber ICDs. The specificity was correlated with the number of patients included, number of episodes, and number of supraventricular tachyarrhythmias recorded. The simulation was performed using tachyarrhythmias recorded in the electrophysiology laboratory. The range of the number of patients included into the studies was 78-1,029, the range of the total number of episodes recorded was 362-5,788, and the range of the number of supraventricular tachyarrhythmias used for calculation of the specificity for correct detection of these arrhythmias was 100 (Biotronik) to 1662 (Medtronic). The specificity for correct detection of supraventricular tachyarrhythmias was 90% (Biotronik), 89% (ELA Medical), 89% (Guidant), 68% (Medtronic), and 76% (St. Jude Medical). There was an inverse correlation (r = -0.9, P = 0.037) between the specificity for correct classification of supraventricular tachyarrhythmias and the number of patients. The specificity for correct detection of supraventricular tachyarrhythmias calculated from the simulation after correction for the clinical prevalence of the simulated tachyarrhythmias was 95% (Biotronik), 99% (ELA Medical), 94% (Guidant), 93% (Medtronic), and 92% (St. Jude Medical). In conclusion, the specificity of ICD detection algorithms calculated from clinical studies or registries may depend on the number of patients studied. Therefore, a direct comparison between different detection algorithms

  18. Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer

    PubMed Central

    Huang, Jun; Zeng, Xun; Sigal, Natalia; Lund, Peder J.; Su, Laura F.; Huang, Huang; Chien, Yueh-hsiu; Davis, Mark M.

    2016-01-01

    Here we report a peptide-MHC (pMHC) dodecamer as a “next generation” technology that is a significantly more sensitive and versatile alternative to pMHC tetramers for the detection, isolation, and phenotypic analysis of antigen-specific T cells. In particular, dodecamers are able to detect two- to fivefold more antigen-specific T cells in both human and murine CD4+ and CD8+ αβ T-cell compartments compared with the equivalent tetramers. The low-affinity, tetramer-negative, dodecamer-positive T cells showed comparable effector cytokine responses as those of high-affinity, tetramer-positive T cells. Dodecamers are able to detect early stage CD4+CD8+ double-positive thymocytes on which T-cell receptors are 10- to 30-fold less dense than mature T cells. Dodecamers also show utility in the analysis of γδ T cells and in cytometry by time-of-flight applications. This construct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or other molecules. The dodecamer is straightforward and inexpensive to produce and is compatible with current tetramer technology and commercially available streptavidin conjugates. PMID:26979955

  19. Marking of specific sequences in double-stranded DNA molecules—SNP detection and direct observation

    PubMed Central

    Shigemori, Yasushi; Haruta, Hirotaka; Okada, Takao; Oishi, Michio

    2004-01-01

    In this study, we describe a simple method to mark specific sequences in double-stranded DNA molecules. For the marking, we used two specifically designed oligonucleotides, one of which is complementary to the sequence to be marked and the other, serving as a splint, to make the marking stable and detectable by subsequent various analytical means. In the presence of the two deoxyoligonucleotides, whereas RecA protein-mediated reaction converts the sequence to be marked to a regional triple-stranded structure with the complementary (probing) oligonucleotide, DNA ligase transforms it to a stable multi- (possibly quintuple) stranded structure with the splint oligonucleotide. The whole marking process is simple and completed in a single reaction mixture. Because RecA protein makes the marking to proceed with high fidelity, we were able to mark (detect) SNPs in complex genomes like human's. Furthermore, the structure of the marked sequence is stable and quite distinct enough to be readily detectable by biochemical means or direct observation by scanning probe microscopy. PMID:15574826

  20. Improved detection specificity for plasma proteins by targeting cysteine-containing peptides with photo-SRM.

    PubMed

    Enjalbert, Quentin; Girod, Marion; Simon, Romain; Jeudy, Jérémy; Chirot, Fabien; Salvador, Arnaud; Antoine, Rodolphe; Dugourd, Philippe; Lemoine, Jérôme

    2013-03-01

    Targeted mass spectrometry using selected reaction monitoring (SRM) has emerged as an alternative to immunoassays for protein quantification owing to faster development time and higher multiplexing capability. However, the SRM strategy is faced with the high complexity of peptide mixtures after trypsin digestion of whole plasma or the cellular proteome that most of the time causes contamination, irremediably, by interfering compounds in the transition channels monitored. This problem becomes increasingly acute when the targeted protein is present at a low concentration. In this work, the merit of laser-induced photo-dissociation in the visible region at 473 nm implemented in an hybrid quadrupole linear ion-trap mass spectrometer (photo-SRM) was evaluated for detection specificity of cysteine-containing peptides in a group of plasma proteins after tagging with a dabcyl chromophore. Compared with conventional SRM, photo-SRM chromatograms have improved detection specificity for most of peptides monitored. Comparison of the signals obtained for the best proteotypic peptides in SRM mode and those recorded by photo-SRM of cysteine-containing peptides for the same proteins reveals either increased (up to 10-fold) or similar signal to photo-SRM detection. Finally, photo-SRM has extended response linearity across a calibration plot obtained by diluting human plasma in rat plasma, down to the lowest concentrations. Hence, photo-SRM may advantageously complement conventional SRM in assay of proteins in complex biological matrices.

  1. FRET-based detection of isozyme-specific activities of transglutaminases.

    PubMed

    Tatsukawa, Hideki; Liu, Hong Hong; Oba, Shota; Kamiya, Noriho; Nakanishi, Yoichi; Hitomi, Kiyotaka

    2017-03-01

    Transglutaminases (TGs) comprise a protein family in which the members catalyze the formation of isopeptide bonds between glutamine and lysine residues in various proteins. Expression studies on its three major members, FXIII, TG1, and TG2, have been performed in a relatively large number of mammalian tissues in comparison with those on the other isozymes. We previously identified a highly reactive substrate peptide, including glutamine, for each isozyme from a phage display library and developed a method for detecting isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. Here, we describe genetically encoded Förster resonance energy transfer (FRET)-based probes composed of each fluorescence protein (Cerulean and EVenus) fused with substrate peptides. The probe pairs, designated as Trac-MTG (His-CerΔ11-LQ/EV-K-His) containing linker and substrate peptide sequence for microbial TG (MTG), increased the EVenus:Cerulean fluorescence intensity ratio by more than 1.5-fold. Furthermore, we demonstrated that Trac-TG1 (His-CerΔ11-K5) and Trac-TG2 (His-CerΔ11-T26) containing substrate peptide sequence for mammalian TGs successfully detected the isozyme-specific activity of TG1 and TG2, respectively. In this study, we developed a rapid and convenient experimental system for measuring the isozyme-specific activity of TGs. The application of these probes for analyses in cells and tissues will be helpful for elucidating the physiological and pathological functions of TGs.

  2. Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis

    PubMed Central

    Roth, Aleeza J.; Ooi, Joshua D.; Hess, Jacob J.; van Timmeren, Mirjan M.; Berg, Elisabeth A.; Poulton, Caroline E.; McGregor, JulieAnne; Burkart, Madelyn; Hogan, Susan L.; Hu, Yichun; Winnik, Witold; Nachman, Patrick H.; Stegeman, Coen A.; Niles, John; Heeringa, Peter; Kitching, A. Richard; Holdsworth, Stephen; Jennette, J. Charles; Preston, Gloria A.; Falk, Ronald J.

    2013-01-01

    Anti-neutrophil cytoplasmic antibody–associated (ANCA-associated) small vessel necrotizing vasculitis is caused by immune-mediated inflammation of the vessel wall and is diagnosed in some cases by the presence of myeloperoxidase-specific antibodies (MPO-ANCA). This multicenter study sought to determine whether differences in ANCA epitope specificity explain why, in some cases, conventional serologic assays do not correlate with disease activity, why naturally occurring anti-MPO autoantibodies can exist in disease-free individuals, and why ANCA are undetected in patients with ANCA-negative disease. Autoantibodies from human and murine samples were epitope mapped using a highly sensitive epitope excision/mass spectrometry approach. Data indicated that MPO autoantibodies from healthy individuals had epitope specificities different from those present in ANCA disease. Importantly, this methodology led to the discovery of MPO-ANCA in ANCA-negative disease that reacted against a sole linear sequence. Autoantibodies against this epitope had pathogenic properties, as demonstrated by their capacity to activate neutrophils in vitro and to induce nephritis in mice. The confounder for serological detection of these autoantibodies was the presence of a fragment of ceruloplasmin in serum, which was eliminated in purified IgG, allowing detection. These findings implicate immunodominant epitopes in the pathology of ANCA-associated vasculitis and suggest that autoantibody diversity may be common to other autoimmune diseases. PMID:23549081

  3. Detection of human papillomavirus types 45 and 51 by type-specific polymerase chain reaction.

    PubMed

    Weyn, Christine; Boulenouar, Selma; Mathys, Vanessa; Vanhoolandt, Julie; Bernis, Aurore; Fontaine, Véronique

    2007-12-01

    Human papillomavirus (HPV) types 45 and 51 are both considered as high risk types for the development of human cervical cancer. To optimize the detection of these two types in clinical samples, HPV-45 and HPV-51 specific primers were designed to amplify respectively a 141bp and a 266bp fragment from the L1 gene by polymerase chain reaction (PCR). The sensitivity and the specificity of these two PCR reactions were determined using varying amounts of HPV DNA containing plasmids and negative and positive controls. Overall, the sensitivity for the HPV-45 plasmid DNA is 10fg, while for HPV-51 the sensitivity is 1fg. This is equivalent to approximately 100 and 10 HPV genome copies per PCR reaction, respectively.

  4. Mining of novel species-specific primers for PCR detection of Listeria monocytogenes based on genomic approach.

    PubMed

    Tao, Tingting; Chen, Qiming; Bie, Xiaomei; Lu, Fengxia; Lu, Zhaoxin

    2015-12-01

    Listeria monocytogenes in contaminated food is considered as a serious health threat for consumers due to its high mortality rate. The objective of this study was to obtain novel species-specific target-genes and primers for the molecular detection of L. monocytogenes using a comparative genomic approach. By comparative analysis of L. monocytogenes and non-L. monocytogenes genome sequences in the GenBank database with BLAST program, 26 specific target sequences were used as candidates and the primers were designed for L. monocytogenes species-specificity verification by using PCR assay. Finally, the three genes LMOf2365_0970, LMOf2365_2721 and mpl were identified to have L. monocytogenes species-specificity and be unique as detection targets for diagnostic application. The species-specific primer Lm8 of gene LMOf2365_0970, Lm13 of gene LMOf2365_2721 and Lm20 of gene mpl showed better specificity and sensitivity than the primers described previously. The PCR detection limits of the three specific primer sets were 430, 43, 4.3 fg/μL for genomic DNA, and 5 × 10(3), 50, 5 cfu/mL for pure culture of L. monocytogenes. There was no interference in specificity of detecting L. monocytogenes by co-culture with other foodborne pathogens in high concentration. Moreover, after 6-8 h of enrichment, L. monocytogenes in the artificially contaminated milk samples at an inoculum dose of 38 cfu/10 mL milk could be detected successfully with the studied three primers. Therefore, the three specific genes and primers can be applied to establish a novel rapid and accurate method for detecting L. monocytogenes in food materials.

  5. Antibodies specific for nucleic acids and applications in genomic detection and clinical diagnostics.

    PubMed

    Hu, Zonglin; Leppla, Stephen H; Li, Baoguang; Elkins, Christopher A

    2014-09-01

    Detection of nucleic acids using antibodies is uncommon. This is in part because nucleic acids are poor immunogens and it is difficult to elicit antibodies having high affinity to each type of nucleic acid while lacking cross-reactivity to others. We describe the origins and applications of a variety of anti-nucleic acid antibodies, including ones reacting with modified nucleosides and nucleotides, single-stranded DNA, double-stranded DNA, RNA, DNA:RNA hybrids, locked-nucleic acids or peptide nucleic acid:nucleic acid hybrids. Carefully selected antibodies can be excellent reagents for detecting bacteria, viruses, small RNAs, microRNAs, R-loops, cancer cells, stem cells, apoptotic cells and so on. The detection may be sensitive, simple, rapid, specific, reproducible, quantitative and cost-effective. Current microarray and diagnostic methods that depend on cDNA or cRNA can be replaced by using antibody detection of nucleic acids. Therefore, development should be encouraged to explore new utilities and create a robust arsenal of new anti-nucleic acid antibodies.

  6. Detection of summer truffle (Tuber aestivum Vittad.) in ectomycorrhizae and in soil using specific primers.

    PubMed

    Gryndler, Milan; Hršelová, Hana; Soukupová, Lucie; Streiblová, Eva; Valda, Slavomír; Borovička, Jan; Gryndlerová, Hana; Gažo, Ján; Miko, Marián

    2011-05-01

    Tuber aestivum is becoming an important commodity of great economical value in some European countries. At the same time, it is a highly protected organism in other countries, where it needs careful treatment. A reliable method of detection in roots and soil is thus needed for assessment of geographic distribution, ecological studies and inoculation efficiency testing in man-made experiments. A PCR-based method of detection of T. aestivum using specific primers was therefore developed. A pair of PCR primers Tu1sekvF/Tu2sekvR selective for T. aestivum and some genotypes of Tuber mesentericum was designed on the basis of the known internal transcribed spacer T. aestivum sequences. TaiI restriction cleavage was then used to distinguish the two species. The selectivity of the designed primer pair was evaluated using DNA extracted from specimens of a further 13 Tuber spp. Subsequently, the selectivity and robustness to false-positive results with nontarget DNA of the designed primers was compared with two other primer pairs (UncI/UncII and BTAE-F/BTAEMB-R). The occurrence of T. aestivum in soil and ectomycorrhizae collected in its native habitat has been successfully detected using the designed primers and nested PCR. The method is reliable and thus suitable for detection of T. aestivum in the field.

  7. Radio Detection of Ultra High Energy Neutrinos

    NASA Astrophysics Data System (ADS)

    Beatty, James J.

    2011-05-01

    Ultra high energy cosmic rays interact with the cosmic microwave background radiation, resulting in the production of energetic pions. These interactions result in energy loss by the incident cosmic ray leading to the Greisen-Zatsepin-Kuzmin (GZK) feature in the cosmic ray spectrum at about 4×10^19 eV, and the decay of the charged pions produced in these interactions results in neutrinos known as Berezinskii-Zatsepin (BZ) neutrinos. These neutrinos interact only via the weak interaction, with negligible absorption over cosmic distances but interaction lengths in the Earth of a few hundred kilometers. When these neutrinos interact in a dense medium, the electromagnetic component of the resulting shower develops a negative charge excess due to Compton scattering of the electrons from the medium and depletion of positrons by in-flight annihilation. This macroscopic charge excess moves at nearly the speed of light, and its passage through a dielectric medium results in coherent Cherenkov radiation at radio wavelengths longer than the size of the radiating region. This process is known as the Askaryan mechanism, and has been observed in accelerator experiments. The radio pulse is impulsive, and can be detected over large volumes in materials with long radio attenuation lengths, most notably the cold ice in the Antarctic ice sheet. Upper limits on the neutrino flux obtained by the balloon-borne instrument ANITA are now approaching the expected flux, and prototype in-ice antenna arrays are now being deployed. Prospects for large detectors capable of detecting hundreds of these neutrinos will be discussed. This work is supported by NASA under grants NNX08AC17G and NNX11AC45G, by the NSF under grant PHY-0758082, and by the Ohio State Center for Cosmology and Particle Astrophysics (CCAPP).

  8. Simultaneous detection of many T-cell specificities using combinatorial tetramer staining

    PubMed Central

    Newell, Evan W.; Klein, Lawrence O.; Yu, Wong; Davis, Mark M.

    2009-01-01

    The direct detection of antigen-specific T cells using tetramers of soluble peptide-major histocompatibilty complex (pMHC) molecules is widely used in both basic and clinical immunology. However, the number of specificities that can be assessed simultaneously has been a major limitation. Here we describe and validate a method using combinations of fluorescent pMHC tetramers to simultaneously detect large numbers (≥ 15) of T cell specificities in a single human blood sample. PMID:19543286

  9. The performance of an online osteoporosis detection system a sensitivity and specificity analysis.

    PubMed

    Chang, Shu Fang; Hong, Chin Ming; Yang, Rong Sen

    2014-07-01

    To develop an online system for the detection of osteoporosis risk and to test its accuracy. Osteoporosis is a silent killer; usually, there are no symptoms, such as pain, until bone erosion and fracture occur. The risks of osteoporosis have been underestimated and neglected; as a result, osteoporosis can be as dangerous as heart diseases and cancers that lead to a healthcare crisis. Cross-sectional study. The study participants were individuals presenting for routine health examinations at a medical centre in Taiwan from 2006-2007. Women over 30 years of age who underwent dual-energy X-ray absorptiometry scanning for measurement of bone mineral density were eligible for this study. The system for osteoporosis detection and health risk, which was developed in this study, was analysed. The findings indicated a high sensitivity of 75%, specificity of 75%, positive predictive value of 75% and negative predictive value of 75%. In addition, the online osteoporosis detective system had a higher predictive power (24·2% vs. 11%) and a similar cut-off point (33% vs. 27%) compared with the tool designed by the International Osteoporosis Foundation. The online system for detection of osteoporosis risk had excellent reliability and validity. It performed well in predicting osteoporosis and the cut-off point used for identifying the risk among women at risk of developing osteoporosis. Therefore, it is suitable for the Asian women and can help women achieve the goals of early detection and health promotion. Early detection is the only way to prevent osteoporosis. Professional nurses should apply effective technology to promote health care in community-dwelling people. © 2013 John Wiley & Sons Ltd.

  10. A generic real-time TaqMan assay for specific detection of lapinized Chinese vaccines against classical swine fever.

    PubMed

    Liu, Lihong; Xia, Hongyan; Everett, Helen; Sosan, Olubukola; Crooke, Helen; Meindl-Böhmer, Alexandra; Qiu, Hua-Ji; Moennig, Volker; Belák, Sándor; Widén, Frederik

    2011-08-01

    Classical swine fever (CSF) is a highly contagious disease, causing severe economic losses in the pig industry worldwide. Vaccination of pigs with lapinized Chinese vaccines is still practised in some regions of the world, where the virus is enzootic, in order to prevent and control the disease. However, a single real-time assay that can detect all lapinized Chinese vaccines used widely, namely, Lapinized Philippines Coronel (LPC), Hog Cholera Lapinized virus (HCLV) and the Riems C-strain is still lacking. This study describes a real-time RT-PCR assay, targeting the N(pro) gene region, for specific detection of these lapinized vaccine strains. The assay is highly sensitive, with a detection limit of 10 genome copies per reaction for HCLV and Riems C-strain and highly specific, as more than 100 strains of wild type CSFV representing all major genotypes were not detected. The assay is also highly repeatable: the coefficient of variation of Ct values in three runs was 2.77% for the detection of 10 copies of the vaccine viral RNA. This study provides a potentially useful tool for specific detection of the lapinized Chinese vaccines, HCLV and C-strain, and the differentiation of these vaccines from wild type CSFV.

  11. Neighbor Detection Induces Organ-Specific Transcriptomes, Revealing Patterns Underlying Hypocotyl-Specific Growth.

    PubMed

    Kohnen, Markus V; Schmid-Siegert, Emanuel; Trevisan, Martine; Petrolati, Laure Allenbach; Sénéchal, Fabien; Müller-Moulé, Patricia; Maloof, Julin; Xenarios, Ioannis; Fankhauser, Christian

    2016-12-01

    In response to neighbor proximity, plants increase the growth of specific organs (e.g., hypocotyls) to enhance access to sunlight. Shade enhances the activity of Phytochrome Interacting Factors (PIFs) by releasing these bHLH transcription factors from phytochrome B-mediated inhibition. PIFs promote elongation by inducing auxin production in cotyledons. In order to elucidate spatiotemporal aspects of the neighbor proximity response, we separately analyzed gene expression patterns in the major light-sensing organ (cotyledons) and in rapidly elongating hypocotyls of Arabidopsis thaliana PIFs initiate transcriptional reprogramming in both organs within 15 min, comprising regulated expression of several early auxin response genes. This suggests that hypocotyl growth is elicited by both local and distal auxin signals. We show that cotyledon-derived auxin is both necessary and sufficient to initiate hypocotyl growth, but we also provide evidence for the functional importance of the local PIF-induced response. With time, the transcriptional response diverges increasingly between organs. We identify genes whose differential expression may underlie organ-specific elongation. Finally, we uncover a growth promotion gene expression signature shared between different developmentally regulated growth processes and responses to the environment in different organs. © 2016 American Society of Plant Biologists. All rights reserved.

  12. Neighbor Detection Induces Organ-Specific Transcriptomes, Revealing Patterns Underlying Hypocotyl-Specific Growth[OPEN

    PubMed Central

    Trevisan, Martine; Petrolati, Laure Allenbach

    2016-01-01

    In response to neighbor proximity, plants increase the growth of specific organs (e.g., hypocotyls) to enhance access to sunlight. Shade enhances the activity of Phytochrome Interacting Factors (PIFs) by releasing these bHLH transcription factors from phytochrome B-mediated inhibition. PIFs promote elongation by inducing auxin production in cotyledons. In order to elucidate spatiotemporal aspects of the neighbor proximity response, we separately analyzed gene expression patterns in the major light-sensing organ (cotyledons) and in rapidly elongating hypocotyls of Arabidopsis thaliana. PIFs initiate transcriptional reprogramming in both organs within 15 min, comprising regulated expression of several early auxin response genes. This suggests that hypocotyl growth is elicited by both local and distal auxin signals. We show that cotyledon-derived auxin is both necessary and sufficient to initiate hypocotyl growth, but we also provide evidence for the functional importance of the local PIF-induced response. With time, the transcriptional response diverges increasingly between organs. We identify genes whose differential expression may underlie organ-specific elongation. Finally, we uncover a growth promotion gene expression signature shared between different developmentally regulated growth processes and responses to the environment in different organs. PMID:27923878

  13. New Mexico High School Competency Examination. Domain Specifications.

    ERIC Educational Resources Information Center

    New Mexico State Dept. of Education, Santa Fe. Assessment and Evaluation Unit.

    The State Department of Education is releasing the domain specifications of the New Mexico High School Competency Examination (NMHSCE) for educator use because the Spring 1996 administration of the test will be in a different form. The NMHSCE is a high school graduation requirement in New Mexico. It assesses students' competence in writing,…

  14. 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

  15. Preparation of Labeled Aflatoxins with High Specific Activities

    PubMed Central

    Hsieh, D. P. H.; Mateles, R. I.

    1971-01-01

    Resting cells of Aspergillus parasiticus ATCC 15517 were used to prepare highly labeled aflatoxins from labeled acetate. High synthetic activity in growing cells was evidenced only during 40 to 70 hr of incubation. Glucose was required for high incorporation efficiency, whereas the concentration of the labeled acetate determined the specific activity of the product. When labeled acetate was continuously added to maintain a concentration near but not exceeding 10 mm, in a culture containing 30 g of glucose per liter, 2% of its labels could be recovered in the purified aflatoxins which have a specific activity more than three times that of the labeled acetate. PMID:4329435

  16. Detection of Bovine viral diarrhea virus-specific neutralizing antibodies in fresh colostrum: a modification of the virus neutralization test.

    PubMed

    Bedekovic, Tomislav; Mihaljevic, Zeljko; Jungic, Andreja; Lemo, Nina; Lojkic, Ivana; Cvetnic, Zeljko; Cac, Zeljko

    2013-03-01

    To eliminate cytotoxic effects of colostrum on cells, a modified virus neutralization test (VNT) for the detection of Bovine viral diarrhea virus-specific neutralizing antibodies in colostrum was developed. The new test was compared to the World Organization for Animal Health-recommended VNT and the results evaluated. The agreement of the new test compared to the standard VNT was determined to be 98%, whereas sensitivity and specificity of the modified VNT compared to the standard VNT were 100%. Bovine viral diarrhea virus-specific antibodies were detected in 42 sera samples and 38 colostrum samples. The antibody titers in serum and colostrum showed a high correlation (n = 56, r = 0.9719, P < 0.001). The modified virus neutralization technique described herein succeeds in eliminating cytotoxic effects and can be readily applied for the detection of specific antibodies against other infectious agents in colostrum.

  17. 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.

  18. Detection of drug specific circulating immune complexes from in vivo cynomolgus monkey serum samples.

    PubMed

    Pierog, Piotr; Krishna, Murli; Yamniuk, Aaron; Chauhan, Anil; DeSilva, Binodh

    2015-01-01

    Administration of a biotherapeutic can result in the formation of anti-drug antibodies (ADAs). The resulting ADA can potentially form immune complexes (ICs) with the drug leading to altered pharmacokinetic (PK) profiles and/or adverse events. Furthermore the presence of such complexes may interfere with accurate PK assessment, and/or detection of ADA in immunogenicity assays. Here, we present two assays to detect the presence of drug-ADA immune complexes in cynomolgus monkeys. Serum samples were analyzed for IC formation in vivo. 8/8 tested animals were positive for drug specific IC. Depending on the time point tested 4/8 or 7/8 animals tested positive for ADA during drug dosing. All 8 animals were confirmed positive for ADA during the washout phase, indicating drug interference in the bridging assay. Relative amount of IC over time was determined and its correlation with PK and ADA was then assessed. Multivariate data analysis demonstrates good correlation between signals obtained from the anti-drug and FcγRIIIa based capture assays, although due to its biological characteristic FcγRIIIa based assay captured only a subset of drug specific IC. In one animal IC remained in circulation even when the drug levels decreased below detection limit. Results from this study indicate the presence of IC during administration of an immunogenic biotherapeutic. Potential application of these assays includes detection of ADA in an IC during high drug levels. The results on the kinetics of IC formation during ADA response can complement the understanding of PK and ADA profiles. Moreover, the presence of IC indicates possible ADA interference in standard PK assays and potential underestimation of total drug exposure in toxicology studies. In addition this study also highlights the need to understand downstream in vivo consequences of drug-ADA IC as no animals under investigation developed adverse events. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Advanced Tribological Coatings for High Specific Strength Alloys

    DTIC Science & Technology

    1989-09-29

    Hard Anodised 4 HSSA12 (SHT) Plasma Nitrided 1 HSSA13 (H&G) Plasma Nitrided 2 HSSA14 (SHT) High Temperature Nitrocarburized 1 HSSA15 (H&G) Nitrox 1...HSSA26 ( High Temperature Plasma Nitriding) has recently arrived, and is currently undergoing metallographic examination. The remaining samples are still...Report No 3789/607 Advanced Tribological Coatings For High Specific Strength Alloys, R&D 5876-MS-01 Contract DAJ A45-87-C-0044 5th Interim Report

  20. Detecting circulating antibodies by controlled surface modification with specific target proteins: Application to malaria.

    PubMed

    Cardoso, Ana R; Cabral-Miranda, Gustavo; Reyes-Sandoval, Arturo; Bachmann, Martin F; Sales, M Goreti F

    2017-05-15

    Sensitive detection of specific antibodies by biosensors has become of major importance for monitoring and controlling epidemics. Here we report a development of a biosensor able to specifically measure antibodies in a drop of unmodified blood serum. Within minutes, the detection system measures presence of antibodies against Plasmodium vivax, a causing agent for malaria. The biosensor consists of a layer of carbon nanotubes (CNTs) which were casted on a carbon working electrode area of a three-electrode system and oxidized. An amine layer was produced next by modifying the surface with EDAC/NHS followed by reaction with a diamine compound. Finally, the protein fragments derived from P. vivax containing well-known antigen sequences were casted on this layer and bound through electrostatic interactions, involving hydrogen and ionic bonding. All these chemical changes occurring at the carbon surface along the biosensor assembly were followed and confirmed by Fourier Transformed Infrared s pectrometry (FTIR) and Raman spectroscopy. The presence of antibodies in serum was detected by monitoring the electrical properties of the layer, making use of cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV), against a standard iron probe. Overall, the charge-transfer resistance decreased after antibody binding, because there was an additional amount of protein bound to the surface. This hindered the access of the iron redox probe to the conductive support at the electrode surface. Electrical changes could be measured at antibody concentration as low as ~6-50pg/L (concentrations in the range of 10-15M) and as high as ~70μg/L. Specific measurement with low background was even possible in undiluted serum. Hence, this novel biosensor allows assessing serum antibody levels in real time and in un-manipulated serum samples on-site where needed. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Ultrasensitive Electrochemical Detection of miRNA-21 Using a Zinc Finger Protein Specific to DNA-RNA Hybrids.

    PubMed

    Fang, Chiew San; Kim, Kwang-Sun; Yu, Byeongjun; Jon, Sangyong; Kim, Moon-Soo; Yang, Haesik

    2017-02-07

    Both high sensitivity and high specificity are crucial for detection of miRNAs that have emerged as important clinical biomarkers. Just Another Zinc finger proteins (JAZ, ZNF346) bind preferably (but nonsequence-specifically) to DNA-RNA hybrids over single-stranded RNAs, single-stranded DNAs, and double-stranded DNAs. We present an ultrasensitive and highly specific electrochemical method for miRNA-21 detection based on the selective binding of JAZ to the DNA-RNA hybrid formed between a DNA capture probe and a target miRNA-21. This enables us to use chemically stable DNA as a capture probe instead of RNA as well as to apply a standard sandwich-type assay format to miRNA detection. High signal amplification is obtained by (i) enzymatic amplification by alkaline phosphatase (ALP) coupled with (ii) electrochemical-chemical-chemical (ECC) redox cycling involving an ALP product (hydroquinone). Low nonspecific adsorption of ALP-conjugated JAZ is obtained using a polymeric self-assembled-monolayer-modified and casein-treated indium-tin oxide electrode. The detection method can discriminate between target miRNA-21 and nontarget nucleic acids (DNA-DNA hybrid, single-stranded DNA, miRNA-125b, miRNA-155, single-base mismatched miRNA, and three-base mismatched miRNA). The detection limits for miRNA-21 in buffer and 10-fold diluted serum are approximately 2 and 30 fM, respectively, indicating that the detection method is ultrasensitive. This detection method can be readily extended to multiplex detection of miRNAs with only one ALP-conjugated JAZ probe due to its nonsequence-specific binding character. We also believe that the method could offer a promising solution for point-of-care testing of miRNAs in body fluids.

  2. Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

    PubMed Central

    Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

    2013-01-01

    Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19+ tumor targets. This clone can be used to detect CD19-specific CAR+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy. PMID:23469246

  3. Chimeric antigen receptor (CAR)-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

    PubMed

    Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A; Champlin, Richard E; Cooper, Laurence J N

    2013-01-01

    Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR(+) T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+) tumor targets. This clone can be used to detect CD19-specific CAR(+) T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR(+) T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

  4. Rapid Detection of Hepatitis B Virus in Blood Plasma by a Specific and Sensitive Loop-Mediated Isothermal Amplification Assay

    PubMed Central

    Nyan, Dougbeh-Chris; Ulitzky, Laura E.; Cehan, Nicoleta; Williamson, Phillip; Winkelman, Valerie; Rios, Maria; Taylor, Deborah R.

    2014-01-01

    Background. Hepatitis B virus (HBV) is an important blood-borne pathogen that causes hepatic inflammation and can lead to liver cirrhosis and hepatocellular carcinoma. Conventional methods of HBV detection are time consuming and require highly trained personnel and elaborate equipment. This report describes the development of a rapid, simple, specific, and sensitive loop-mediated isothermal amplification assay (LAMP) for detection of HBV genotypes A, B, C, D, E, and F in blood samples. Methods. HBV standard plasma panels and clinical donor plasma specimens were used for the development and validation of the LAMP assay. Amplification was performed at 60°C for 60 minutes using extracted DNA or heat-treated plasma specimens without DNA extraction. The assay was evaluated for its ability to detect various HBV genotypes and for its sensitivity, specificity, and time-point of detection. Results. The LAMP assay detected HBV genotypes A–F and demonstrated a sensitivity of 10–100 IU per reaction of HBV DNA. The assay also detected 69 of 75 (92%) HBV-positive donor plasma specimens tested and demonstrated a specificity of 100%. Conclusions. These results demonstrate that our HBV-LAMP assay is rapid, sensitive and specific, and capable of detecting the major HBV genotypes. This assay could be used in clinical point-of-care settings, mainly in endemic and resource-limited environments for HBV diagnostics, donor screening, epidemiological studies, and therapeutic monitoring of patients undergoing antiviral treatment. PMID:24704724

  5. [Early detection of specific reading disorder through response to intervention:a cohort study].

    PubMed

    Koeda, Tatsuya; Seki, Ayumi; Tanaka, Daisuke; Uchiyama, Hitoshi

    2014-07-01

    The present study examined the effectiveness of response to intervention (RTI) for the early detection and intervention for specific reading disorder. RTIs were applied to seventy-seven first graders (36 boys and 41 girls) for the early detection and intervention of specific reading disorders. The outcomes were examined when the children reached the third grade. Reading difficulties were detected in four children at the beginning of the first grade. Decoding training and vocabulary learning were conducted with these children as interventions for reading difficulties. Three children exhibited improvements in reading difficulties at the end of the first grade. The fourth child responded poorly and was diagnosed with specific reading disorder. The assessment of outcomes at the third grade showed that this child still had reading difficulties, whereas no other child displayed symptoms for being diagnosed with specific reading disorder. RTI is effective for the early detection and intervention of specific reading disorder.

  6. Rapid and specific detection of the thermostable direct hemolysin gene in Vibrio parahaemolyticus by loop-mediated isothermal amplification.

    PubMed

    Nemoto, Jiro; Sugawara, Chiyo; Akahane, Kenji; Hashimoto, Keiji; Kojima, Tadashi; Ikedo, Masanari; Konuma, Hirotaka; Hara-Kudo, Yukiko

    2009-04-01

    Several investigators have reported that thermostable direct hemolysin (TDH) and TDH-related hemolysin are important virulence factors of Vibrio parahaemolyticus, but it has been difficult to detect these factors rapidly in seafood and other environmental samples. A novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity and rapidity under isothermal conditions, was applied. In this study, we designed tdh gene-specific LAMP primers for detection of TDH-producing V. parahaemolyticus. The specificity of this assay was evaluated with 32 strains of TDH-producing V. parahaemolyticus, one strain of TDH-producing Grimontia hollisae, 10 strains of TDH-nonproducing V. parahaemolyticus, and 94 strains of TDH-nonproducing bacteria, and the sensitivity was high enough to detect one cell per test. Moreover, to investigate the detection of TDH-producing V. parahaemolyticus in oysters, the LAMP assay was performed with enrichment culture in alkaline peptone water of oyster samples inoculated with TDH-producing V. parahaemolyticus and TDH-nonproducing V. parahaemolyticus and V. alginolyticus after enrichment in alkaline peptone water. These results suggest that the LAMP assay targeting tdh gene has high sensitivity and specificity and is useful to detect TDH-producing V. parahaemolyticus in oyster after enrichment.

  7. 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.

  8. Large bowel carcinoma-specific antigens detected by the lectin, Griffonia simplicifolia agglutinin-II.

    PubMed

    Nakayama, J; Katsuyama, T; Ono, K; Honda, T; Akamatsu, T; Hattori, H

    1985-11-01

    A lectin reactivity specific to human bowel carcinoma is reported. Twenty-six cases of carcinoma of the large intestine were examined. Normal as well as transitional mucosa and carcinoma tissues were removed from surgical specimens, and paraffin sections were stained with a battery of histochemical methods to characterize glycoconjugates, including high iron diamine-Alcian blue pH 2.5, modified PAS reaction to detect various sialic acids, paradoxical concanavalin A (Con A) staining, and stainings with 10 species of lectins labeled with horseradish peroxidase (HRP). Among the techniques employed, only Griffonia simplicifolia agglutinin-II (GS-II, specific to glucosamine)-HRP staining revealed highly selective affinity to the carcinoma tissues; the apical surface of the carcinoma cells stained most intensely. GS-II reactivity of the cells persisted after prior periodate oxidation, but was significantly enhanced by neuraminidase digestion. Comparison with two other lectin stainings with the same sugar specificity, viz. paradoxical concanavalin A staining and wheat germ agglutinin (WGA)-HRP staining, showed that the GS-II reactive sites lacked class III Con A reactivity but were possibly included in WGA reactive sites. The GS-II-HRP staining should be helpful in the identification of carcinoma tissue and for analysis of carcinoma-associated antigens.

  9. A Protocol Specification-Based Intrusion Detection System for VoIP and Its Evaluation

    NASA Astrophysics Data System (ADS)

    Phit, Thyda; Abe, Kôki

    We propose an architecture of Intrusion Detection System (IDS) for VoIP using a protocol specification-based detection method to monitor the network traffics and alert administrator for further analysis of and response to suspicious activities. The protocol behaviors and their interactions are described by state machines. Traffic that behaves differently from the standard specifications are considered to be suspicious. The IDS has been implemented and simulated using OPNET Modeler, and verified to detect attacks. It was found that our system can detect typical attacks within a reasonable amount of delay time.

  10. 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

  11. 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

  12. Intraneuronal Aβ detection in 5xFAD mice by a new Aβ-specific antibody

    PubMed Central

    2012-01-01

    Background The form(s) of amyloid-β peptide (Aβ) associated with the pathology characteristic of Alzheimer's disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal Aβ accumulation is an issue of considerable controversy; even the existence of Aβ deposits within neurons has recently been challenged by Winton and co-workers. These authors purport that it is actually intraneuronal APP that is being detected by antibodies thought to be specific for Aβ. To further address this issue, an anti-Aβ antibody was developed (MOAB-2) that specifically detects Aβ, but not APP. This antibody allows for the further evaluation of the early accumulation of intraneuronal Aβ in transgenic mice with increased levels of human Aβ in 5xFAD and 3xTg mice. Results MOAB-2 (mouse IgG2b) is a pan-specific, high-titer antibody to Aβ residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), particularly compared to 6E10 (a commonly used commercial antibody to Aβ residues 3-8). MOAB-2 did not detect APP or APP-CTFs in cell culture media/lysates (HEK-APPSwe or HEK-APPSwe/BACE1) or in brain homogenates from transgenic mice expressing 5 familial AD (FAD) mutation (5xFAD mice). Using IHC on 5xFAD brain tissue, MOAB-2 immunoreactivity co-localized with C-terminal antibodies specific for Aβ40 and Aβ42. MOAB-2 did not co-localize with either N- or C-terminal antibodies to APP. In addition, no MOAB-2-immunreactivity was observed in the brains of 5xFAD/BACE-/- mice, although significant amounts of APP were detected by N- and C-terminal antibodies to APP, as well as by 6E10. In both 5xFAD and 3xTg mouse brain tissue, MOAB-2 co-localized with cathepsin-D, a marker for acidic organelles, further evidence for intraneuronal Aβ, distinct from Aβ associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues. Conclusions Both intraneuronal Aβ accumulation and

  13. 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

  14. Specific detection of tetanus toxoid using an aptamer-based matrix.

    PubMed

    Modh, Harshvardhan B; Bhadra, Ankan K; Patel, Kinjal A; Chaudhary, Rajeev K; Jain, Nishant K; Roy, Ipsita

    2016-11-20

    Batch-to-batch variation of therapeutic proteins produced by biological means requires rigorous monitoring at all stages of the production process. A large number of animals are employed for risk assessment of biologicals, which has low ethical and economic acceptability. Research is now focussed on the validation of in vitro and ex vivo tests to replace live challenges. Among in vitro methods, enzyme-linked immunosorbent assay (ELISA) is considered to be the gold standard for estimation of integrity of tetanus toxoid. ELISA utilizes antibodies for detection, which, because of their biological origin and limited modifiability, may have low stability and result in irreproducibility. We have developed a method using highly specific and selective RNA aptamers for detection of tetanus toxoid. Using displacement assay, we first identified aptamers which bind to different aptatopes on the surface of the toxoid. Pairs of these aptamers were employed as capture-detection ligands in a sandwich-ALISA (aptamer-linked immobilized sorbent assay) format. The binding efficiency was confirmed by the fluorescence intensity in each microtire plate well. Using aptamers alone, detection of tetanus toxoid was possible with the same level of sensitivity as antibody. Aptamers were also used in the capture ALISA format. Adjuvanted tetanus toxoid was subjected to accelerated stress testing, including thermal, mechanical and freeze-thawing stress conditions. The loss in antigenicity of the preparation determined by ALISA in each case was found to be similar to that determined by conventional ELISA. Thus, it is possible to replace antibodies with aptamers to develop a more robust detection tool for tetanus toxoid.

  15. 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

  16. 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.

  17. 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

    2016-07-20

    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.

  18. 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

  19. Sensitivity and specificity of PS/AA-modified nanoparticles used in malaria detection.

    PubMed

    Thiramanas, Raweewan; Jangpatarapongsa, Kulachart; Asawapirom, Udom; Tangboriboonrat, Pramuan; Polpanich, Duangporn

    2013-07-01

    Polystyrene (PS) nanoparticle (NP) copolymerized with acrylic acid (AA) and coloured monomer, i.e. 2,3,6,7-tetra(2,2'-bithiophene)-1,4,5,8-naphthalenetetracarboxylic-N,N'-di(2-methylallyl)-bisimide (ALN8T), was synthesized via the miniemulsion polymerization. Before applying for malaria antigen detection, the blue NP was conjugated with human polyclonal malaria IgG antibody (Ab) specific to Plasmodium falciparum. For the conjugation, three methods, i.e. physical adsorption, covalent coupling and affinity binding via streptavidin (SA) and biotin interaction, were employed. The optimum ratio of Ab to NPs used in each immobilization procedure and the latex agglutination test based on the reaction between Ab conjugated NPs and malaria patient plasma were investigated. All Ab-latex conjugates provided the high sensitivity for the detection of P. falciparum malaria plasma. The highest specificity to P. falciparum was obtained from using Ab-NPs conjugated via the SA-biotin interaction. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  20. Sensitivity and specificity of PS/AA-modified nanoparticles used in malaria detection

    PubMed Central

    Thiramanas, Raweewan; Jangpatarapongsa, Kulachart; Asawapirom, Udom; Tangboriboonrat, Pramuan; Polpanich, Duangporn

    2013-01-01

    Summary Polystyrene (PS) nanoparticle (NP) copolymerized with acrylic acid (AA) and coloured monomer, i.e. 2,3,6,7-tetra(2,2′-bithiophene)-1,4,5,8-naphthalenetetracarboxylic-N,N′-di(2-methylallyl)-bisimide (ALN8T), was synthesized via the miniemulsion polymerization. Before applying for malaria antigen detection, the blue NP was conjugated with human polyclonal malaria IgG antibody (Ab) specific to Plasmodium falciparum. For the conjugation, three methods, i.e. physical adsorption, covalent coupling and affinity binding via streptavidin (SA) and biotin interaction, were employed. The optimum ratio of Ab to NPs used in each immobilization procedure and the latex agglutination test based on the reaction between Ab conjugated NPs and malaria patient plasma were investigated. All Ab–latex conjugates provided the high sensitivity for the detection of P. falciparum malaria plasma. The highest specificity to P. falciparum was obtained from using Ab–NPs conjugated via the SA–biotin interaction. PMID:23298152

  1. Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases.

    PubMed

    Mangayil, Rahul; Karp, Matti; Lamminmäki, Urpo; Santala, Ville

    2016-10-27

    Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases.

  2. 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

  3. Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases

    PubMed Central

    Mangayil, Rahul; Karp, Matti; Lamminmäki, Urpo; Santala, Ville

    2016-01-01

    Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases. PMID:27786270

  4. E6-Specific Detection and Typing of Human Papillomaviruses in Oral Cavity Specimens from Iranian Patients

    PubMed

    Razavi Nikoo, Hadi; Ardebili, Abdollah; Ravanshad, Mehrdad; Rezaei, Fatemeh; Teimoori, Ali; Khanizadeh, Sayyad; Pouriayevali, Mohammad Hassan; Ajorloo, Mehdi

    2017-05-02

    Detection and quantification of human Papillomavirus (HPV) genome in oral carcinoma play an important role in diagnosis, as well as implications for progression of disease. We evaluated tissues from 50 esopharyngeal cancers collected from different regions of Iran for HPV E6 using the two type-specific primers sets. E6 gene of HPV genotypes was amplified by specific primers. The sensitivity of PCR assay was analyzed and determined using HPV-DNA-containing plasmids. Real-time PCR was utilized to determine the prevalence and HPV viral load in patients with oral cavity squamous cell carcinoma. Eighteen (36%) specimens were positive for HPV. Among the 18 positive specimens, 10 showed HPV-18 (55.55%), and 8 specimens were positive for HPV-11 (44.44%). Of the 18 infected specimens, 6 (33.32%) and 12 (66.65%) were identified as high-titer and low-titer viral load, respectively. The PCR-based assay, developed in the current study, could be used for HPV detection, quantification, and genotyping in epidemiological and clinical studies.

  5. 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

  6. 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

  7. Electrochemical immunosensor for competitive detection of neuron specific enolase using functional carbon nanotubes and gold nanoprobe.

    PubMed

    Yu, Tianxiao; Cheng, Wei; Li, Qing; Luo, Caihui; Yan, Li; Zhang, Decai; Yin, Yibing; Ding, Shijia; Ju, Huangxian

    2012-05-15

    An electrochemical immunosensor for detection of neuron specific enolase (NSE) was designed by immobilizing NSE covalently functionalized single-walled carbon nanotubes (NSE-SWNTs) on a glassy carbon electrode. The NSE-SWNTs not only enhanced electrochemical signal but also presented abundant antigen domains for competitive immunological recognition to anti-NSE primary antibody and then gold nanoprobes labeled with alkaline phosphatase conjugated secondary antibody (AP-anti-IgG/AuNPs). The AP-anti-IgG/AuNPs exhibited highly catalytic activity toward enzyme substrate and significantly amplified the amperometric signal for target molecule detection. Based on the dual signal amplification of SWNTs and gold nanoprobe, the immunosensor could response down to 0.033 ng mL(-1) NSE with a linear range from 0.1 ng mL(-1) to 2 μg mL(-1), and showed acceptable precision and reproducibility. The designed immunosensor was amenable to direct quantification of target protein with a wide range of concentration in complex clinical serum specimens. The assay results were in a good agreement with the reference values. The proposed electrochemical immunosensor provided a pragmatic platform for convenient detection of tumor markers in clinical diagnosis. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Multiplex Detection of Protease Activity with Quantum Dot Nanosensors Prepared by Intein-Mediated Specific Bioconjugation

    PubMed Central

    Xia, Zuyong; Xing, Yun; So, Min-Kyung; Koh, Ai Leen; Sinclair, Robert; Rao, Jianghong

    2009-01-01

    We report here a protease sensing nanoplatform based on semiconductor nanocrystals or quantum dots (QDs) and bioluminescence resonance energy transfer (QD-BRET) to detect the protease activity in complex biological samples. These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and protease substrates sandwiched between the two as a sensing group. An intein-mediated conjugation strategy was developed for site-specific conjugation of proteins to QDs in preparing these QD nanosensors. In this traceless ligation, the intein itself is spliced out and excluded from the final conjugation product. With this method, we have synthesized a series of QD nanosensors for highly sensitive detection of an important class of protease matrix metalloproteinase (MMP) activity. We demonstrated that these nanosensors can detect the MMP activity in buffers and in mouse serum with the sensitivity to a few ng/ml, and secreted proteases by tumor cells. The suitability of these nanosensors for a multiplex protease assay has also been shown. PMID:18922019

  9. Potential of treatment-specific protein biomarker profiles for detection of hormone abuse in cattle.

    PubMed

    Ludwig, Susann Katrina Julie; Smits, Nathalie Gabriëlle Esther; Cannizzo, Francesca Tiziana; Nielen, Michel Wilhelmus Franciscus

    2013-05-15

    Targeted protein biomarker profiling is suggested as a fast screening approach for detection of illegal hormone treatment in meat production. The advantage of using biomarkers is that they mark the biological response and, thus, are responsive to a panel of substances with similar effects. In a preliminary feasibility study, a 4-plex protein biomarker flow cytometric immunoassay (FCIA) previously developed for the detection of recombinant bovine somatotropin (rbST) was applied to cattle treated with steroids, such as estradiol, dexamethasone, and prednisolone. Each treatment resulted in a specific plasma biomarker profile for insulin-like growth factor-1 (IGF-1), IGF binding protein 2, osteocalcin, and anti-rbST antibodies, which could be distinguished from the profile of untreated animals. In summary, the 4-plex biomarker FCIA is, apart from rbST, also capable of detecting treatment with other growth-promoting agents and therefore clearly shows the potential of biomarker profiling as a screening method in veterinary control. It is proposed to perform additional validation studies covering high numbers of treated and untreated animals to support inclusion or adaptation of protein biomarker approaches in future monitoring regulations.

  10. Bactrian camel nanobody-based immunoassay for specific and sensitive detection of Cry1Fa toxin.

    PubMed

    Wang, Pingyan; Li, Guanghui; Yan, Junrong; Hu, Yonghong; Zhang, Cunzheng; Liu, Xianjin; Wan, Yakun

    2014-12-15

    The variable domain of the heavy-chain-only antibody (VHH) or nanobody (Nb), derived from camelids, begins to play an important role on the detection of protein markers. In this study, we constructed a phage-displayed library of VHHs against Cry1Fa by immunizing a healthy Bactrian camel with Cry1Fa toxin. After a series of bio-panning and screening by phage display technology, three anti-Cry1Fa nanobodies (Nbs) with great difference in complementarity determining region 3 (CDR3) were obtained and they were highly specific to Cry1Fa as well as showed full of activity when exposed to 70 °C for 3 h. Through modifying Nbs with Horseradish Peroxidase (HRP) and biotin, two Nbs which can recognize the different epitopes of Cry1Fa were determined and they were used to establish a novel sandwich immune ELISA based on biotin-SA interaction for Cry1Fa detection. The immunoassay exhibited a linear range from 1 to 100 ng/mL with a detection limit of 0.88 ng/mL. The recoveries from spiked corn and soybean samples were ranged from 83.33 to 117.17%, with a coefficient of variation (C.V) less than 6.0%. All together, the proposed immunoassay will be a promising way for sensitive and accurate determination of Cry1Fa toxin.

  11. Stimulus-specific adaptation and deviance detection in the inferior colliculus

    PubMed Central

    Ayala, Yaneri A.; Malmierca, Manuel S.

    2013-01-01

    Deviancy detection in the continuous flow of sensory information into the central nervous system is of vital importance for animals. The task requires neuronal mechanisms that allow for an efficient representation of the environment by removing statistically redundant signals. Recently, the neuronal principles of auditory deviance detection have been approached by studying the phenomenon of stimulus-specific adaptation (SSA). SSA is a reduction in the responsiveness of a neuron to a common or repetitive sound while the neuron remains highly sensitive to rare sounds (Ulanovsky et al., 2003). This phenomenon could enhance the saliency of unexpected, deviant stimuli against a background of repetitive signals. SSA shares many similarities with the evoked potential known as the “mismatch negativity,” (MMN) and it has been linked to cognitive process such as auditory memory and scene analysis (Winkler et al., 2009) as well as to behavioral habituation (Netser et al., 2011). Neurons exhibiting SSA can be found at several levels of the auditory pathway, from the inferior colliculus (IC) up to the auditory cortex (AC). In this review, we offer an account of the state-of-the art of SSA studies in the IC with the aim of contributing to the growing interest in the single-neuron electrophysiology of auditory deviance detection. The dependence of neuronal SSA on various stimulus features, e.g., probability of the deviant stimulus and repetition rate, and the roles of the AC and inhibition in shaping SSA at the level of the IC are addressed. PMID:23335883

  12. 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

  13. High sensitivity leak detection method and apparatus

    DOEpatents

    Myneni, G.R.

    1994-09-06

    An improved leak detection method is provided that utilizes the cyclic adsorption and desorption of accumulated helium on a non-porous metallic surface. The method provides reliable leak detection at superfluid helium temperatures. The zero drift that is associated with residual gas analyzers in common leak detectors is virtually eliminated by utilizing a time integration technique. The sensitivity of the apparatus of this disclosure is capable of detecting leaks as small as 1 [times] 10[sup [minus]18] atm cc sec[sup [minus]1]. 2 figs.

  14. High sensitivity leak detection method and apparatus

    DOEpatents

    Myneni, Ganapatic R.

    1994-01-01

    An improved leak detection method is provided that utilizes the cyclic adsorption and desorption of accumulated helium on a non-porous metallic surface. The method provides reliable leak detection at superfluid helium temperatures. The zero drift that is associated with residual gas analyzers in common leak detectors is virtually eliminated by utilizing a time integration technique. The sensitivity of the apparatus of this disclosure is capable of detecting leaks as small as 1.times.10.sup.-18 atm cc sec.sup.-1.

  15. Novel Bacteroides host strains for detection of human- and animal-specific bacteriophages in water.

    PubMed

    Wicki, Melanie; Auckenthaler, Adrian; Felleisen, Richard; Tanner, Marcel; Baumgartner, Andreas

    2011-03-01

    Bacteriophages active against specific Bacteroides host strains were shown to be suitable for detection of human faecal pollution. However, the practical application of this finding is limited because some specific host strains were restricted to certain geographic regions. In this study, novel Bacteroides host strains were isolated that discriminate human and animal faecal pollution in Switzerland. Two strains specific for bacteriophages present in human faecal contamination and three strains specific for bacteriophages indicating animal faecal contamination were evaluated. Bacteriophages infecting human strains were exclusively found in human wastewater, whereas animal strains detected bacteriophages only in animal waste. The newly isolated host strains could be used to determine the source of surface and spring water faecal contamination in field situations. Applying the newly isolated host Bacteroides thetaiotaomicron ARABA 84 for detection of bacteriophages allowed the detection of human faecal contamination in spring water.

  16. High specific energy and specific power aluminum/air battery for micro air vehicles

    NASA Astrophysics Data System (ADS)

    Kindler, A.; Matthies, L.

    2014-06-01

    Micro air vehicles developed under the Army's Micro Autonomous Systems and Technology program generally need a specific energy of 300 - 550 watt-hrs/kg and 300 -550 watts/kg to operate for about 1 hour. At present, no commercial cell can fulfill this need. The best available commercial technology is the Lithium-ion battery or its derivative, the Li- Polymer cell. This chemistry generally provides around 15 minutes flying time. One alternative to the State-of-the Art is the Al/air cell, a primary battery that is actually half fuel cell. It has a high energy battery like aluminum anode, and fuel cell like air electrode that can extract oxygen out of the ambient air rather than carrying it. Both of these features tend to contribute to a high specific energy (watt-hrs/kg). High specific power (watts/kg) is supported by high concentration KOH electrolyte, a high quality commercial air electrode, and forced air convection from the vehicles rotors. The performance of this cell with these attributes is projected to be 500 watt-hrs/kg and 500 watts/kg based on simple model. It is expected to support a flying time of approximately 1 hour in any vehicle in which the usual limit is 15 minutes.

  17. An RNAi-enhanced Logic Circuit for Cancer Specific Detection and Destruction

    DTIC Science & Technology

    2010-07-01

    Bcl-2 family: mBax ( Mus musculus ), hBax (Homo sapiens), and its mutant hBax-S184A [4]. A plasmid containing the tested gene was transfected into HEK...0240 TITLE: An RNAi-enhanced logic circuit for cancer specific detection and destruction PRINCIPAL INVESTIGATOR: Ron Weiss...From - To) 1 Jul 2009 – 30 Jun 2010 4. TITLE AND SUBTITLE An RNAi-enhanced logic circuit for cancer specific detection and destruction

  18. 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.

  19. 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…

  20. 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.

  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).

  2. Detection of EGFR mutations by TaqMan mutation detection assays powered by competitive allele-specific TaqMan PCR technology.

    PubMed

    Roma, Cristin; Esposito, Claudia; Rachiglio, Anna Maria; Pasquale, Raffaella; Iannaccone, Alessia; Chicchinelli, Nicoletta; Franco, Renato; Mancini, Rita; Pisconti, Salvatore; De Luca, Antonella; Botti, Gerardo; Morabito, Alessandro; Normanno, Nicola

    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.

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

    DOEpatents

    McCready, Paula M [Tracy, CA; Radnedge, Lyndsay [San Mateo, CA; Andersen, Gary L [Berkeley, CA; Ott, Linda L [Livermore, CA; Slezak, Thomas R [Livermore, CA; Kuczmarski, Thomas A [Livermore, CA; Motin, Vladinir L [League City, TX

    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.

  4. 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.

  5. 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.

  6. 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.

  7. Dual labeling of a binding protein allows for specific fluorescence detection of native protein.

    PubMed

    Karlström, A; Nygren, P A

    2001-08-01

    Fluorescence resonance energy transfer has been investigated in the context of specific detection of unlabeled proteins. A model system based on the staphylococcal protein A (SPA)-IgG interaction was designed, in which a single domain was engineered to facilitate site-specific incorporation of fluorophores. An Asn23Cys mutant of the B domain from SPA was expressed in Escherichia coli and subsequently labeled at the introduced unique thiol and at an amino group, using N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and succinimidyl 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoate (NBD-X, SE), respectively. Biosensor analysis of purified doubly labeled protein showed that high-affinity binding to the Fc region of IgG was retained. The fluorescence emission spectrum of the doubly labeled protein showed a shift in the relative emission of the two fluorophores in the presence of Fc3(1) fragments, which bind specifically to the B domain. In addition, the fluorescence emission ratio 480/525 nm was shown to increase with increasing concentration of Fc3(1), whereas the presence of a control protein did not affect the emission ratio over the same concentration range.

  8. Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

    PubMed

    Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

    2015-07-01

    The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides.

  9. [Evaluation of simultaneous detection of specific antinuclear antibodies using multiplexed technology].

    PubMed

    Uto, Kenichi; Hayashi, Nobuhide; Kinoshita, Shouhiro; Kawano, Seiji; Kumagai, Shunichi

    2009-10-01

    BioPlex2200 is a fully automated analyzer using multiplexed technology and BioPlex2200 ANA Screen can analyze 11 antinuclear antibodies (ANA). We evaluated simultaneous detection of 11 ANA and clinical utility as ANA screening test. We collected sera from 317 connective tissue disease (CTD) patients, 166 healthy subjects, and 105 sera in which anti dsDNA antibody(RIA) is requested. Detection of 11 ANA were measured by BioPlex2200 ANA Screen using BioPlex2200 along with conventional methods. We evaluated positive ratio for healthy subjects and CTD patients and concordance rate between BioPlex2200 and IF. The prevalence of disease specific ANA in CTD patients were comparable with general occurrence rate except anti dsDNA antibody (39.3%) in SLE. Concordance rate between BioPlex2200 and conventional methods were high(89.0-99.7%) except anti dsDNA antibody(ELISA: 68.8%, RIA: 58.1%). Discordant sera of Bioplex2200+/DID- for anti SS-A antibodies were observed in 30 sera, and 20 sera were positive only for anti 52kD SS-A/Ro antibody. As ANA screening test, positive ratio was low (7.2%) in healthy subjects, and comparable with that of IF(1:160). Concordance rate between BioPlex2200 and IF was low (75.1%). However, 44 sera of BioPlex2200+/IF- contained 6 samples positive for anti Jo-1 antibodies and 29 samples positive for anti-52kD SS-A/Ro antibodies. Diagnostic accuracy of the Medical Decision Support Software (MDSS) by BioPlex2200 as compared to clinical diagnosis is good for specificity. Taken together, BioPlex2200 can appropriately perform simultaneous detection of 11 ANA and detect independently anti-52kD SS-A/Ro antibody. However, detection for anti dsDNA antibody of low titers is needed to be improved.

  10. Highly specific protein-protein interactions, evolution and negative design.

    PubMed

    Sear, Richard P

    2004-12-01

    We consider highly specific protein-protein interactions in proteomes of simple model proteins. We are inspired by the work of Zarrinpar et al (2003 Nature 426 676). They took a binding domain in a signalling pathway in yeast and replaced it with domains of the same class but from different organisms. They found that the probability of a protein binding to a protein from the proteome of a different organism is rather high, around one half. We calculate the probability of a model protein from one proteome binding to the protein of a different proteome. These proteomes are obtained by sampling the space of functional proteomes uniformly. In agreement with Zarrinpar et al we find that the probability of a protein binding a protein from another proteome is rather high, of order one tenth. Our results, together with those of Zarrinpar et al, suggest that designing, say, a peptide to block or reconstitute a single signalling pathway, without affecting any other pathways, requires knowledge of all the partners of the class of binding domains the peptide is designed to mimic. This knowledge is required to use negative design to explicitly design out interactions of the peptide with proteins other than its target. We also found that patches that are required to bind with high specificity evolve more slowly than those that are required only to not bind to any other patch. This is consistent with some analysis of sequence data for proteins engaged in highly specific interactions.

  11. Specific detection of Pectobacterium carotovorum by loop-mediated isothermal amplification.

    PubMed

    Yasuhara-Bell, Jarred; Marrero, Glorimar; De Silva, Asoka; Alvarez, Anne M

    2016-12-01

    Potatoes are an important agroeconomic crop worldwide and maceration diseases caused by pectolytic bacterial pathogens result in significant pre- and post-harvest losses. Pectobacterium carotovorum shares a common host range with other Pectobacterium spp. and other members of the Enterobacteriaceae, such as Dickeya spp. As these pathogens cannot be clearly differentiated on the basis of the symptoms they cause, improved methods of identification are critical for the determination of sources of contamination. Current standardized methods for the differentiation of pectolytic species are time consuming and require trained personnel, as they rely on traditional bacteriological practices that do not always produce conclusive results. In this growing world market, there is a need for rapid diagnostic tests that can differentiate between pectolytic pathogens, as well as separate them from non-pectolytic enteric bacteria associated with soft rots of potato. An assay has been designed previously to detect the temperate pathogen Pectobacterium atrosepticum, but there is currently no recognized rapid assay for the detection of the tropical/subtropical counterpart, Pectobacterium carotovorum. This report describes the development of a loop-mediated isothermal amplification (LAMP) assay that detects P. carotovorum with high specificity. The assay was evaluated using all known species of Pectobacterium and only showed positive reactions for P. carotovorum. This assay was also tested against 15 non-target genera of plant-associated bacteria and did not produce any false positives. The LAMP assay described here can be used as a rapid test for the differentiation of P. carotovorum from other pectolytic pathogens, and its gene target can be the basis for the development of other molecular-based detection assays.

  12. Benchmarking Procedures for High-Throughput Context Specific Reconstruction Algorithms

    PubMed Central

    Pacheco, Maria P.; Pfau, Thomas; Sauter, Thomas

    2016-01-01

    Recent progress in high-throughput data acquisition has shifted the focus from data generation to processing and understanding of how to integrate collected information. Context specific reconstruction based on generic genome scale models like ReconX or HMR has the potential to become a diagnostic and treatment tool tailored to the analysis of specific individuals. The respective computational algorithms require a high level of predictive power, robustness and sensitivity. Although multiple context specific reconstruction algorithms were published in the last 10 years, only a fraction of them is suitable for model building based on human high-throughput data. Beside other reasons, this might be due to problems arising from the limitation to only one metabolic target function or arbitrary thresholding. This review describes and analyses common validation methods used for testing model building algorithms. Two major methods can be distinguished: consistency testing and comparison based testing. The first is concerned with robustness against noise, e.g., missing data due to the impossibility to distinguish between the signal and the background of non-specific binding of probes in a microarray experiment, and whether distinct sets of input expressed genes corresponding to i.e., different tissues yield distinct models. The latter covers methods comparing sets of functionalities, comparison with existing networks or additional databases. We test those methods on several available algorithms and deduce properties of these algorithms that can be compared with future developments. The set of tests performed, can therefore serve as a benchmarking procedure for future algorithms. PMID:26834640

  13. Power spectral density specifications for high-power laser systems

    SciTech Connect

    Lawson, J.K.; Aikens, D.A.; English, R.E. Jr.; Wolfe, C.R.

    1996-04-22

    This paper describes the use of Fourier techniques to characterize the transmitted and reflected wavefront of optical components. Specifically, a power spectral density, (PSD), approach is used. High power solid-state lasers exhibit non-linear amplification of specific spatial frequencies. Thus, specifications that limit the amplitude of these spatial frequencies are necessary in the design of these systems. Further, NIF optical components have square, rectangular or irregularly shaped apertures with major dimensions up-to 800 mm. Components with non-circular apertures can not be analyzed correctly with Zernicke polynomials since these functions are an orthogonal set for circular apertures only. A more complete and powerful representation of the optical wavefront can be obtained by Fourier analysis in 1 or 2 dimensions. The PSD is obtained from the amplitude of frequency components present in the Fourier spectrum. The shape of a resultant wavefront or the focal spot of a complex multicomponent laser system can be calculated and optimized using PSDs of the individual optical components which comprise the system. Surface roughness can be calculated over a range of spatial scale-lengths by integrating the PSD. Finally, since the optical transfer function (OTF) of the instruments used to measure the wavefront degrades at high spatial frequencies, the PSD of an optical component is underestimated. We can correct for this error by modifying the PSD function to restore high spatial frequency information. The strengths of PSD analysis are leading us to develop optical specifications incorporating this function for the planned National Ignition Facility (NIF).

  14. Specific and Nonspecific Staining in Detecting Staphylococcal Alpha Toxin on Erythrocytes by Immunofluorescence

    PubMed Central

    Kaplan, Milton T.

    1972-01-01

    Specifically stained staphylococcal alpha toxin particles were detected on unstained and nonspecifically stained ghost membranes when rabbit erythrocytes were exposed to alpha toxin and stained with fluorescein isothiocyanate-labeled specific antitoxin. Fading of the nonspecific staining was observed and was attributed to the degradation of the membranes by alpha toxin. Images PMID:4118044

  15. Rapid Generation of Highly Specific Aptamers via Micromagnetic Selection

    PubMed Central

    Qian, Jiangrong; Lou, Xinhui; Zhang, Yanting; Xiao, Yi; Soh, H. Tom

    2009-01-01

    Aptamers are nucleic acid-based reagents that bind to target molecules with high affinity and specificity. However, methods for generating aptamers from random combinatorial libraries (e.g., SELEX) are often labor-intensive and time-consuming. Recent studies suggest that microfluidic SELEX (M-SELEX) technology can accelerate aptamer isolation by enabling highly stringent selection conditions through the use of very small amounts of target molecules. We present here an alternative M-SELEX method, which employs a disposable microfluidic chip to rapidly generate aptamers with high affinity and specificity. The Micro-Magnetic Separation (MMS) chip integrates microfabricated ferromagnetic structures to reproducibly generate large magnetic field gradients within its microchannel that efficiently trap magnetic bead-bound aptamers. Operation of the MMS device is facile, robust and demonstrates high recovery of the beads (99.5%), such that picomolar amounts of target molecule can be used. Importantly, the device demonstrates exceptional separation efficiency in removing weakly-bound and unbound ssDNA to rapidly enrich target-specific aptamers. As a model, we demonstrate here the generation of DNA aptamers against streptavidin in three rounds of positive selection. We further enhanced the specificity of the selected aptamers via a round of negative selection in the same device against bovine serum albumin (BSA). The resulting aptamers displayed dissociation constants ranging from 25 to 65 nM for streptavidin but negligible affinity for BSA. Since a wide spectrum of molecular targets can be readily conjugated on magnetic beads, MMS-based SELEX should provide a general platform for rapid generation of specific aptamers. PMID:19480397

  16. Improved PCR assay for the specific detection and quantitation of Escherichia coli serotype O157 in water.

    PubMed

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

    2014-09-01

    Escherichia coli serotype O157 is still a major global healthcare problem. However, only limited information is now available on the molecular and serological detection of pathogenic bacteria. Therefore, the development of appropriate strategies for their rapid identification and monitoring is still needed. In general, the sequence analysis based on stx, slt, eae, hlyA, rfb, and fliCh7 genes is widely employed for the identification of E. coli serotype O157; but there have been critical defects in the diagnosis and identification of E. coli serotype O157, in that they are also present in other E. coli serogroups. In this study, NCBI-BLAST searches using the nucleotide sequences of the putative regulatory protein gene from E. coli O157:H7 str. Sakai found sequence difference at the serotype level. The specific primers from the putative regulatory protein gene were designed and investigated for their sensitivity and specificity for detecting the pathogen in environment water samples. The specificity of the primer set was evaluated using genomic DNA from 8 isolates of E. coli serotype O157 and 32 other reference strains. In addition, the sensitivity and specificity of this assay were confirmed by successful identification of E. coli serotype O157 in environmental water samples. In conclusion, this study showed that the newly developed quantitative serotype-specific PCR method is a highly specific and efficient tool for the surveillance and rapid detection of high-risk E. coli serotype O157.

  17. Highly Sensitive Colorimetric Detection of Ochratoxin A by a Label-Free Aptamer and Gold Nanoparticles

    PubMed Central

    Luan, Yunxia; Chen, Jiayi; Li, Cheng; Xie, Gang; Fu, Hailong; Ma, Zhihong; Lu, Anxiang

    2015-01-01

    A label-free aptamer-based assay for the highly sensitive and specific detection of Ochratoxin A (OTA) was developed using a cationic polymer and gold nanoparticles (AuNPs). The OTA aptamer was used as a recognition element for the colorimetric detection of OTA based on the aggregation of AuNPs by the cationic polymer. By spectroscopic quantitative analysis, the colorimetric assay could detect OTA down to 0.009 ng/mL with high selectivity in the presence of other interfering toxins. This study offers a new alternative in visual detection methods that is rapid and sensitive for OTA detection. PMID:26690477

  18. Highly Sensitive Colorimetric Detection of Ochratoxin A by a Label-Free Aptamer and Gold Nanoparticles.

    PubMed

    Luan, Yunxia; Chen, Jiayi; Li, Cheng; Xie, Gang; Fu, Hailong; Ma, Zhihong; Lu, Anxiang

    2015-12-10

    A label-free aptamer-based assay for the highly sensitive and specific detection of Ochratoxin A (OTA) was developed using a cationic polymer and gold nanoparticles (AuNPs). The OTA aptamer was used as a recognition element for the colorimetric detection of OTA based on the aggregation of AuNPs by the cationic polymer. By spectroscopic quantitative analysis, the colorimetric assay could detect OTA down to 0.009 ng/mL with high selectivity in the presence of other interfering toxins. This study offers a new alternative in visual detection methods that is rapid and sensitive for OTA detection.

  19. Detection and source identification of faecal pollution in non-sewered catchment by means of host-specific molecular markers.

    PubMed

    Ahmed, W; Powell, D; Goonetilleke, A; Gardner, T

    2008-01-01

    Multiple host-specific molecular markers were used to detect the sources of faecal pollution in a mixed land use non-sewered catchment in Southeast Queensland, Australia. These markers included human-specific Bacteroides (HF183 and HF134), cattle-specific Bacteroides (CF128), dog-specific Bacteroides (BacCan) and human-specific enterococci surface protein (esp) markers. The sensitivity and specificity of these markers were determined by testing 197 faecal samples from 13 host groups. The overall sensitivity and specificity of these markers was high (sensitivity>/=85% and specificity>/=93%) indicating their suitability for detecting the sources of faecal pollution. Of the 16 samples collected from the study area, 14 (87%) were positive for at least one of the molecular marker tested. Amongst all the markers, cattle-specific CF128 was more prevalent than others, followed by human-specific HF183 which was consistently detected in samples collected from sites within close proximity to urban development. Significant correlations were found between E. coli and enterococci concentrations with the positive/negative results of human-specific Bacteroides HF183 (p<0.001, p<0.0001) and HF134 (p<0.001, p<0.004) markers. No correlations were found between faecal indicators (E. coli or enterococci) with the CF128 or BacCan markers. A significant correlation was also found between enterococci concentrations and the presence/absence of the esp marker (p<0.02). Based on the results, it appears that the host-specific markers such as HF183 and esp are a sensitive measure of sources of human faecal pollution in surface waters in Southeast Queensland, Australia.

  20. High specificity in plant leaf metabolic responses to arbuscular mycorrhiza.

    PubMed

    Schweiger, Rabea; Baier, Markus C; Persicke, Marcus; Müller, Caroline

    2014-05-22

    The chemical composition of plants (phytometabolome) is dynamic and modified by environmental factors. Understanding its modulation allows to improve crop quality and decode mechanisms underlying plant-pest interactions. Many studies that investigate metabolic responses to the environment focus on single model species and/or few target metabolites. However, comparative studies using environmental metabolomics are needed to evaluate commonalities of chemical responses to certain challenges. We assessed the specificity of foliar metabolic responses of five plant species to the widespread, ancient symbiosis with a generalist arbuscular mycorrhizal fungus. Here we show that plant species share a large 'core metabolome' but nevertheless the phytometabolomes are modulated highly species/taxon-specifically. Such a low conservation of responses across species highlights the importance to consider plant metabolic prerequisites and the long time of specific plant-fungus coevolution. Thus, the transferability of findings regarding phytometabolome modulation by an identical AM symbiont is severely limited even between closely related species.

  1. Breast-Specific γ-Imaging for the Detection of Mammographically Occult Breast Cancer in Women at Increased Risk.

    PubMed

    Brem, Rachel F; Ruda, Rachel C; Yang, Jialu L; Coffey, Caitrín M; Rapelyea, Jocelyn A

    2016-05-01

    Breast-specific γ-imaging (BSGI) is a physiologic imaging modality that can detect subcentimeter and mammographically occult breast cancer, with a sensitivity and specificity comparable to MRI. The purpose of this study was to determine the incremental increase in breast cancer detection when BSGI is used as an adjunct to mammography in women at increased risk for breast cancer. All patients undergoing BSGI from April 2010 through January 2014 were retrospectively reviewed. Eligible patients were identified as women at increased risk for breast cancer and whose most recent mammogram was benign. Examinations exhibiting focally increased radiotracer uptake were considered positive. Incremental increase in cancer detection was calculated as the percentage of mammographically occult BSGI-detected breast cancer and the number of mammographically occult breast cancers detected per 1,000 women screened. Included in this study were 849 patients in whom 14 BSGI examinations detected mammographically occult breast cancer. Patients ranged in age from 26 to 83 y, with a mean age of 57 y. Eleven of 14 cancers were detected in women with dense breasts. The addition of BSGI to the annual breast screen of asymptomatic women at increased risk for breast cancer yields 16.5 cancers per 1,000 women screened. When high-risk lesions and cancers were combined, BSGI detected 33.0 high-risk lesions and cancers per 1,000 women screened. BSGI is a reliable adjunct modality to screening mammography that increases breast cancer detection by 1.7% (14/849) in women at increased risk for breast cancer, comparable to results reported for breast MRI. BSGI is beneficial in breast cancer detection in women at increased risk, particularly in those with dense breasts. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  2. 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.

  3. Multimode nondestructive detecting method for high-speed rail defects

    NASA Astrophysics Data System (ADS)

    Sun, Mingjian; Cheng, Xingzhen; Wan, Guangnan; Liu, Ting; Fu, Ying; Wang, Yan

    2015-11-01

    It is very important to detect the surface defects of the high-speed rail for security concerns. A multimode detecting method, which integrates high resolution of optical image, high precision of photoacoustic detection and strong penetration of ultrasound detecting, is proposed for the rail defect detection. Utilizing the surface defect characteristics obtained from optical signal, the photoacoustic and ultrasound scanning region could be determined, and rail shallow and internal defect characteristics can be acquired subsequently. Eventually, fusing three modal signals mentioned above, the information of the entire rail defect, including type, extension trend and depth can be detected. It has been proved that the multimode method can improve the detecting efficiency, and enlarge the detection range in the meantime.

  4. High-Collection-Efficiency Fluorescence Detection Cell

    NASA Technical Reports Server (NTRS)

    Hanisco, Thomas; Cazorla, Maria; Swanson, Andrew

    2013-01-01

    A new fluorescence cell has been developed for the laser induced fluorescence (LIF) detection of formaldehyde. The cell is used to sample a flow of air that contains trace concentrations of formaldehyde. The cell provides a hermetically sealed volume in which a flow of air containing formaldehyde can be illuminated by a laser. The cell includes the optics for transmitting the laser beam that is used to excite the formaldehyde and for collecting the resulting fluorescence. The novelty of the cell is its small size and simple design that provides a more robust and cheaper alternative to the state of the art. Despite its simplicity, the cell provides the same sensitivity to detection as larger, more complicated cells.

  5. Dual Immunomagnetic Nanobeads-Based Lateral Flow Test Strip for Simultaneous Quantitative Detection of Carcinoembryonic Antigen and Neuron Specific Enolase

    PubMed Central

    Lu, Wenting; Wang, Kan; Xiao, Kun; Qin, Weijian; Hou, Yafei; Xu, Hao; Yan, Xinyu; Chen, Yanrong; Cui, Daxiang; He, Jinghua

    2017-01-01

    A novel immunomagnetic nanobeads -based lateral flow test strip was developed for the simultaneous quantitative detection of neuron specific enolase (NSE) and carcinoembryonic antigen (CEA), which are sensitive and specific in the clinical diagnosis of small cell lung cancer. Using this nanoscale method, high saturation magnetization, carboxyl-modified magnetic nanobeads were successfully synthesized. To obtain the immunomagnetic probes, a covalent bioconjugation of the magnetic nanobeads with the antibody of NSE and CEA was carried out. The detection area contained test line 1 and test line 2 which captured the immune complexes sensitively and formed sandwich complexes. In this assay, cross-reactivity results were negative and both NSE and CEA were detected simultaneously with no obvious influence on each other. The magnetic signal intensity of the nitrocellulose membrane was measured by a magnetic assay reader. For quantitative analysis, the calculated limit of detection was 0.094 ng/mL for NSE and 0.045 ng/mL for CEA. One hundred thirty clinical samples were used to validate the test strip which exhibited high sensitivity and specificity. This dual lateral flow test strip not only provided an easy, rapid, simultaneous quantitative detection strategy for NSE and CEA, but may also be valuable in automated and portable diagnostic applications. PMID:28186176

  6. A novel antigen capture ELISA for the specific detection of IgG antibodies to elephant endotheliotropic herpes virus.

    PubMed

    van den Doel, Petra B; Prieto, Víctor Rodríguez; van Rossum-Fikkert, Sarah E; Schaftenaar, Willem; Latimer, Erin; Howard, Lauren; Chapman, Sarah; Masters, Nic; Osterhaus, Albert D M E; Ling, Paul D; Dastjerdi, Akbar; Martina, Byron

    2015-08-14

    Elephants are classified as critically endangered animals by the International Union for Conservation of Species (IUCN). Elephant endotheliotropic herpesvirus (EEHV) poses a large threat to breeding programs of captive Asian elephants by causing fatal haemorrhagic disease. EEHV infection is detected by PCR in samples from both clinically ill and asymptomatic elephants with an active infection, whereas latent carriers can be distinguished exclusively via serological assays. To date, identification of latent carriers has been challenging, since there are no serological assays capable of detecting seropositive elephants. Here we describe a novel ELISA that specifically detects EEHV antibodies circulating in Asian elephant plasma/serum. Approximately 80 % of PCR positive elephants display EEHV-specific antibodies. Monitoring three Asian elephant herds from European zoos revealed that the serostatus of elephants within a herd varied from non-detectable to high titers. The antibody titers showed typical herpes-like rise-and-fall patterns in time which occur in all seropositive animals in the herd more or less simultaneously. This study shows that the developed ELISA is suitable to detect antibodies specific to EEHV. It allows study of EEHV seroprevalence in Asian elephants. Results confirm that EEHV prevalence among Asian elephants (whether captive-born or wild-caught) is high.

  7. [Species-specific detection of Proteus vulgaris and Proteus mirabilis by the polymerase chain reaction].

    PubMed

    Limanskiĭ, A; Minukhin, V; Limanskaia, O; Pavlenko, N; Mishina, M; Tsygenenko, A

    2005-01-01

    Sets of primers for the species-specific detection of P. mirabilis and P. vulgaris by the polymerase chain reaction (PCR) were developed. As targets for these primers beta-lactamase and 16S rRNA gene fragments were chosen on the basis of the multiple leveling of the sequences of the DNA of all known P. mirabilis and P. vulgaris isolates. For differential detection oligonucleotides were selected in such a way that primers, specific for P. vulgaris, contained the non-paired nucleotide for P. mirabilis isolate at the 3'-end, and all other nucleotides were complementary to the beta-lactamase gene fragment. Primers, specific for gene 16S rRNA of P. mirabilis, contained the non-paired nucleotide for P. vulgaris isolates at the 3'-end. Standard PCR was carried out for 6 P. mirabilis and P. vulgaris strains. The use of PCR species-specific primers to P. vulgaris DNA made it possible to amplify the DNA fragment of the expected length only for P. vulgaris isolates, while the result of PCR for P. mirabilis was negative. PCR with primers specific to P. mirabilis permitted the detection of amplicon sized 101 nucleotides pairs only for P. mirabilis strains. These primers were optimized so as to use them in the specific differentiation of closely related P. mirabilis and P. vulgaris species by multiplex PCR. Genus-specific primers permitted the detection of bacterial gyrB gene of the genus Proteus were developed also.

  8. Stimulus change detection in phasic auditory units in the frog midbrain: frequency and ear specific adaptation

    PubMed Central

    Ponnath, Abhilash; Hoke, Kim L.

    2013-01-01

    Neural adaptation, a reduction in the response to a maintained stimulus, is an important mechanism for detecting stimulus change. Contributing to change detection is the fact that adaptation is often stimulus specific: adaptation to a particular stimulus reduces excitability to a specific subset of stimuli, while the ability to respond to other stimuli is unaffected. Phasic cells (e.g., cells responding to stimulus onset) are good candidates for detecting the most rapid changes in natural auditory scenes, as they exhibit fast and complete adaptation to an initial stimulus presentation. We made recordings of single phasic auditory units in the frog midbrain to determine if adaptation was specific to stimulus frequency and ear of input. In response to an instantaneous frequency step in a tone, 28 % of phasic cells exhibited frequency specific adaptation based on a relative frequency change (delta-f = ±16 %). Frequency specific adaptation was not limited to frequency steps, however, as adaptation was also overcome during continuous frequency modulated stimuli and in response to spectral transients interrupting tones. The results suggest that adaptation is separated for peripheral (e.g., frequency) channels. This was tested directly using dichotic stimuli. In 45 % of binaural phasic units, adaptation was ear specific: adaptation to stimulation of one ear did not affect responses to stimulation of the other ear. Thus, adaptation exhibited specificity for stimulus frequency and lateralization at the level of the midbrain. This mechanism could be employed to detect rapid stimulus change within and between sound sources in complex acoustic environments. PMID:23344947

  9. Molecular characterization and specific detection of Anaplasma species (AP-sd) in sika deer and its first detection in wild brown bears and rodents in Hokkaido, Japan.

    PubMed

    Moustafa, Mohamed Abdallah Mohamed; Lee, Kyunglee; Taylor, Kyle; Nakao, Ryo; Sashika, Mariko; Shimozuru, Michito; Tsubota, Toshio

    2015-12-01

    A previously undescribed Anaplasma species (herein referred to as AP-sd) has been detected in sika deer, cattle and ticks in Japan. Despite being highly similar to some strains of A. phagocytophilum, AP-sd has never been detected in humans. Its ambiguous epidemiology and the lack of tools for its specific detection make it difficult to understand and interpret the prevalence of this Anaplasma species. We developed a method for specific detection, and examined AP-sd prevalence in Hokkaido wildlife. Our study included 250 sika deer (Cervus nippon yesoensis), 13 brown bears (Ursus arctos yesoensis) and 252 rodents including 138 (Apodemus speciosus), 45 (Apodemus argenteus), 42 (Myodes rufocanus) and 27 (Myodes rutilus) were collected from Hokkaido island, northern Japan, collected during 2010 to 2015. A 770 bp and 382 bp segment of the 16S rRNA and gltA genes, respectively, were amplified by nested PCR. Results were confirmed by cloning and sequencing of the positive PCR products. A reverse line blot hybridization (RLB) based on the 16S rRNA gene was then developed for the specific detection of AP-sd. The prevalence of AP-sd by nested PCR in sika deer was 51% (128/250). We detected this Anaplasma sp. for the first time in wild brown bears and rodents with a prevalence of 15% (2/13) and 2.4% (6/252), respectively. The sequencing results of the 16S rRNA and gltA gene amplicons were divergent from the selected A. phagocytophilum sequences in GenBank. Using a newly designed AP-sd specific probe for RLB has enabled us to specifically detect this Anaplasma species. Besides sika deer and cattle, wild brown bears and rodents were identified as potential reservoir hosts for AP-sd. This study provided a high throughput molecular method that specifically detects AP-sd, and which can be used to investigate its ecology and its potential as a threat to humans in Japan.

  10. Diagnostic Efficacy of Vessel Specific Coronary Calcium Score in Detection of Coronary Artery Stenosis

    PubMed Central

    Motevalli, Marzieh; Ghanaati, Hossein; Firouznia, Kavous; Kargar, Jalal; Aliyari Ghasabeh, Mounes; Shahriari, Mona; Jalali, Amir Hosein; Shakiba, Madjid

    2014-01-01

    Background: Coronary artery calcification which is determined quantitatively by coronary calcium scoring has been known as a sign of coronary stenosis and thus future cardiac events; hence it has been noticed on spotlight of researchers in recent years. Developing different method for early and optimal detection of coronary artery disease (CAD) is really essential as CAD are the first cause of death in population. Objectives: To evaluate predictive value of vessel specific coronary artery calcium (CAC) score in predicting obstructive coronary artery disease. Patients and Methods: In this diagnostic test study we evaluated patients with coronary computed tomography angiography (CCTA) and CAC score which had been referred to two referral radiology center in Tehran, Iran and finally we selected 2525 patients in a single and sequential pattern to create a diagnostic study. The whole-heart CAC scores and vessel specific CAC scores were calculated individually for the 4 major epicardial coronary arteries in 2 distinct group; group A ( patients with previous history of CABG) and group B (patients without history of CABG). For evaluation of obstruction tree cut off points were described: 0 > ; at least 1 segment with any kind of stenosis, ≥ 50; at least 1 segment with stenosis ≥ 50, ≥ 70; at least 1 segment with stenosis ≥ 70. Results: Mean of coronary calcium scores in terms of each coronary artery vessel increase by increasing coronary stenosis grade in group B; LAD, RCA, LCX respectively have mean CAC score 6.06, 6.21 and 5.04 in normal patients and 221.6, 226.7 and 106.6 in patients with complete stenosis. As expected these findings don't work for group A. Also By increasing calcium score cutoff in all four vessels sensitivity decreased and specificity increased but steal LAD had higher sensitivity than other vessels and LM had higher specificity. Thus using calcium score method is useful for ruling out stenosis in LAD while calcium score of LM can predict

  11. Rapid and specific detection of H3 swine influenza virus using reverse transcription loop-mediated isothermal amplification method.

    PubMed

    Gu, H; Qi, X; Li, X; Jiang, H; Wang, Y; Liu, F; Lu, S; Yang, Y; Liu, F

    2010-04-01

    The main objective of our study is to develop a reverse transcriptase loop-mediated isothermal amplification (RT-LAMP)-based system for rapid and specific detection of H3 swine influenza virus (SIV). The system, H3 RT-LAMP, contained a set of six novel primers that targeted eight distinct regions of the viral haemagglutinin (HA) gene that are highly conserved among H3 influenza A viruses but not between H3 and other subtypes. H3 RT-LAMP accurately and specifically detected H3 SIV of different isolates from culture and from swine lung samples. The system is at least 10-fold more sensitive than the conventional RT-PCR assay and even comparable to the real-time RT-PCR method, with the detection limit of about one plaque-forming unit per reaction. Of 27 swine lung samples tested, 11 samples were positive in reactions with the RT-LAMP and real-time RT-PCR methods, while only 7 were positive with the conventional RT-PCR assay. Importantly, the assay can be completed within 45 min and is faster than the conventional RT-PCR and real-time RT-PCR approaches. Our results provide the first direct evidence that RT-LAMP is highly specific and sensitive for detecting H3 SIV. These results suggest that LAMP offers a promising alternative tool for rapid, inexpensive and specific diagnosis of influenza virus infection of swine and other animals in frontline settings.

  12. Accelerator Production and Separations for High Specific Activity Rhenium-186

    SciTech Connect

    Jurisson, Silvia S.; Wilbur, D. Scott

    2016-04-01

    Tungsten and osmium targets were evaluated for the production of high specific activity rhenium-186. Rhenium-186 has potential applications in radiotherapy for the treatment of a variety of diseases, including targeting with monoclonal antibodies and peptides. Methods were evaluated using tungsten metal, tungsten dioxide, tungsten disulfide and osmium disulfide. Separation of the rhenium-186 produced and recycling of the enriched tungsten-186 and osmium-189 enriched targets were developed.

  13. Method of preparing high specific activity platinum-195m

    SciTech Connect

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

    2004-06-15

    A method of preparing high-specific-activity .sup.195m Pt 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.

  14. Method for preparing high specific activity 177Lu

    DOEpatents

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

    2004-04-06

    A method of separating lutetium from a solution containing Lu and Yb, particularly reactor-produced .sup.177 Lu and .sup.177 Yb, includes the steps of: providing a chromatographic separation apparatus containing LN resin; loading the apparatus with a solution containing Lu and Yb; and eluting the apparatus to chromatographically separate the Lu and the Yb in order to produce high-specific-activity .sup.177 Yb.

  15. Outlier detection in high-density surface electromyographic signals.

    PubMed

    Marateb, Hamid R; Rojas-Martínez, Monica; Mansourian, Marjan; Merletti, Roberto; Villanueva, Miguel A Mañanas

    2012-01-01

    Recently developed techniques allow the analysis of surface EMG in multiple locations over the skin surface (high-density surface electromyography, HDsEMG). The detected signal includes information from a greater proportion of the muscle of interest than conventional clinical EMG. However, recording with many electrodes simultaneously often implies bad-contacts, which introduce large power-line interference in the corresponding channels, and short-circuits that cause near-zero single differential signals when using gel. Such signals are called 'outliers' in data mining. In this work, outlier detection (focusing on bad contacts) is discussed for monopolar HDsEMG signals and a new method is proposed to identify 'bad' channels. The overall performance of this method was tested using the agreement rate against three experts' opinions. Three other outlier detection methods were used for comparison. The training and test sets for such methods were selected from HDsEMG signals recorded in Triceps and Biceps Brachii in the upper arm and Brachioradialis, Anconeus, and Pronator Teres in the forearm. The sensitivity and specificity of this algorithm were, respectively, 96.9 ± 6.2 and 96.4 ± 2.5 in percent in the test set (signals registered with twenty 2D electrode arrays corresponding to a total of 2322 channels), showing that this method is promising.

  16. Development and Evaluation of Species-Specific PCR for Detection of Nine Acinetobacter Species.

    PubMed

    Li, Xue Min; Choi, Ji Ae; Choi, In Sun; Kook, Joong Ki; Chang, Young-Hyo; Park, Geon; Jang, Sook Jin; Kang, Seong Ho; Moon, Dae Soo

    2016-05-01

    Molecular methods have the potential to improve the speed and accuracy of Acinetobacter species identification in clinical settings. The goal of this study is to develop species-specific PCR assays based on differences in the RNA polymerase beta-subunit gene (rpoB) to detect nine commonly isolated Acinetobacter species including Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter pittii, Acinetobacter nosocomialis, Acinetobacter lwoffii, Acinetobacter ursingii, Acinetobacter bereziniae, Acinetobacter haemolyticus, and Acinetobacter schindleri. The sensitivity and specificity of these nine assays were measured using genomic DNA templates from 55 reference strains and from 474 Acinetobacter clinical isolates. The sensitivity of A. baumannii-specific PCR assay was 98.9%, and the sensitivity of species-specific PCR assays for all other species was 100%. The specificities of A. lwoffii- and A. schindleri-specific PCR were 97.8 and 98.9%, respectively. The specificity of species-specific PCR for all other tested Acinetobacter species was 100%. The lower limit of detection for the nine species-specific PCR assays developed in this study was 20 or 200 pg of genomic DNA from type strains of each species. The Acinetobacter species-specific PCR assay would be useful to determine the correct species among suggested candidate Acinetobacter species when conventional methods including MALDI-TOF MS identify Acinetobacter only to the genus level. The species-specific assay can be used to screen large numbers of clinical and environmental samples obtained for epidemiologic study of Acinetobacter for the presence of target species.

  17. Specific detection and confirmation of Campylobacter jejuni by DNA hybridization and PCR.

    PubMed Central

    Ng, L K; Kingombe, C I; Yan, W; Taylor, D E; Hiratsuka, K; Malik, N; Garcia, M M

    1997-01-01

    Conventional detection and confirmation methods for Campylobacter jejuni are lengthy and tedious. A rapid hybridization protocol in which a 1,475-bp chromogen-labelled DNA probe (pDT1720) and Campylobacter strains filtered and grown on 0.22-micron-pore-size hydrophobic grid membrane filters (HGMFs) are used was developed. Among the environmental and clinical isolates of C. jejuni, Campylobacter coli, Campylobacter jejuni subsp. doylei, Campylobacter lari, and Arcobacter nitrofigilis and a panel of 310 unrelated bacterial strains tested, only C. jejuni and C. jejuni subsp. doylei isolates hybridized with the probe under stringent conditions. The specificity of the probe was confirmed when the protocol was applied to spiked skim milk and chicken rinse samples. Based on the nucleotide sequence of pDT1720, a pair of oligonucleotide primers was designed for PCR amplification of DNA from Campylobacter spp. and other food pathogens grown overnight in selective Mueller-Hinton broth with cefoperazone and growth supplements. All C. jejuni strains tested, including DNase-producing strains and C. jejuni subsp. doylei, produced a specific 402-bp amplicon, as confirmed by restriction and Southern blot analysis. The detection range of the assay was as low as 3 CFU per PCR to as high as 10(5) CFU per PCR for pure cultures. Overnight enrichment of chicken rinse samples spiked initially with as little as approximately 10 CFU/ml produced amplicons after the PCR. No amplicon was detected with any of the other bacterial strains tested or from the chicken background microflora. Since C. jejuni is responsible for 99% of Campylobacter contamination in poultry, PCR and HGMF hybridization were performed on naturally contaminated chicken rinse samples, and the results were compared with the results of conventional cultural isolation on Preston agar. All samples confirmed to be culture positive for C. jejuni were also identified by DNA hybridization and PCR amplification, thus confirming that

  18. Specific detection and confirmation of Campylobacter jejuni by DNA hybridization and PCR.

    PubMed

    Ng, L K; Kingombe, C I; Yan, W; Taylor, D E; Hiratsuka, K; Malik, N; Garcia, M M

    1997-11-01

    Conventional detection and confirmation methods for Campylobacter jejuni are lengthy and tedious. A rapid hybridization protocol in which a 1,475-bp chromogen-labelled DNA probe (pDT1720) and Campylobacter strains filtered and grown on 0.22-micron-pore-size hydrophobic grid membrane filters (HGMFs) are used was developed. Among the environmental and clinical isolates of C. jejuni, Campylobacter coli, Campylobacter jejuni subsp. doylei, Campylobacter lari, and Arcobacter nitrofigilis and a panel of 310 unrelated bacterial strains tested, only C. jejuni and C. jejuni subsp. doylei isolates hybridized with the probe under stringent conditions. The specificity of the probe was confirmed when the protocol was applied to spiked skim milk and chicken rinse samples. Based on the nucleotide sequence of pDT1720, a pair of oligonucleotide primers was designed for PCR amplification of DNA from Campylobacter spp. and other food pathogens grown overnight in selective Mueller-Hinton broth with cefoperazone and growth supplements. All C. jejuni strains tested, including DNase-producing strains and C. jejuni subsp. doylei, produced a specific 402-bp amplicon, as confirmed by restriction and Southern blot analysis. The detection range of the assay was as low as 3 CFU per PCR to as high as 10(5) CFU per PCR for pure cultures. Overnight enrichment of chicken rinse samples spiked initially with as little as approximately 10 CFU/ml produced amplicons after the PCR. No amplicon was detected with any of the other bacterial strains tested or from the chicken background microflora. Since C. jejuni is responsible for 99% of Campylobacter contamination in poultry, PCR and HGMF hybridization were performed on naturally contaminated chicken rinse samples, and the results were compared with the results of conventional cultural isolation on Preston agar. All samples confirmed to be culture positive for C. jejuni were also identified by DNA hybridization and PCR amplification, thus confirming that

  19. Alignment-Free Methods for the Detection and Specificity Prediction of Adenylation Domains.

    PubMed

    Agüero-Chapin, Guillermin; Pérez-Machado, Gisselle; Sánchez-Rodríguez, Aminael; Santos, Miguel Machado; Antunes, Agostinho

    2016-01-01

    Identifying adenylation domains (A-domains) and their substrate specificity can aid the detection of nonribosomal peptide synthetases (NRPS) at genome/proteome level and allow inferring the structure of oligopeptides with relevant biological activities. However, that is challenging task due to the high sequence diversity of A-domains (~10-40 % of amino acid identity) and their selectivity for 50 different natural/unnatural amino acids. Altogether these characteristics make their detection and the prediction of their substrate specificity a real challenge when using traditional sequence alignment methods, e.g., BLAST searches. In this chapter we describe two workflows based on alignment-free methods intended for the identification and substrate specificity prediction of A-domains. To identify A-domains we introduce a graphical-numerical method, implemented in TI2BioP version 2.0 (topological indices to biopolymers), which in a first step uses protein four-color maps to represent A-domains. In a second step, simple topological indices (TIs), called spectral moments, are derived from the graphical representations of known A-domains (positive dataset) and of unrelated but well-characterized sequences (negative set). Spectral moments are then used as input predictors for statistical classification techniques to build alignment-free models. Finally, the resulting alignment-free models can be used to explore entire proteomes for unannotated A-domains. In addition, this graphical-numerical methodology works as a sequence-search method that can be ensemble with homology-based tools to deeply explore the A-domain signature and cope with the diversity of this class (Aguero-Chapin et al., PLoS One 8(7):e65926, 2013). The second workflow for the prediction of A-domain's substrate specificity is based on alignment-free models constructed by transductive support vector machines (TSVMs) that incorporate information of uncharacterized A-domains. The construction of the models was

  20. Solar-powered rocket engine optimization for high specific impulse

    NASA Astrophysics Data System (ADS)

    Pande, J. Bradley

    1993-11-01

    Hercules Aerospace is currently developing a solar-powered rocket engine (SPRE) design optimized for high specific impulse (Isp). The SPRE features a low loss geometry in its light-gathering cavity, which includes an integral secondary concentrator. The simple one-piece heat exchanger is made from refractory metal and/or ceramic open-celled foam. The foam's high surface-area-to-volume ratio will efficiently transfer the thermal energy to the hydrogen propellant. The single-pass flow of propellant through the heat exchanger further boosts thermal efficiency by regeneratively cooling surfaces near the entrance of the optical cavity. These surfaces would otherwise reradiate a significant portion of the captured solar energy back out of the solar entrance. Such design elements promote a high overall thermal efficiency and hence, a high operating Isp

  1. Validation of improved vessel-specific leads (VSLs) for detecting acute myocardial ischemia.

    PubMed

    Wang, John J; Title, Lawrence M; Martin, Thomas N; Wagner, Galen S; Warren, James W; Horáček, B Milan; Sapp, John L

    2015-01-01

    Existing criteria recommended by ACC/ESC for identifying patients with ST elevation myocardial infarction (STEMI) from the 12-lead ECG perform with high specificity (SP), but low sensitivity (SE). In our previous studies, we found that the SE of ischemia detection can be markedly improved without any loss of SP by calculating, from the 12-lead ECG, ST deviation in 3 "optimal" vessel-specific leads (VSLs). Our original VSLs, based on ΔST body-surface potential maps (BSPMs), have been modified by using the more appropriate J-point BSPMs at peak ischemia (without subtraction of pre-occlusion distributions). The aim of the present study was to compare the performance of these new VSLs with that achieved by the STEMI criteria used in current practice. Two independent datasets of 12-lead ECGs were used: the STAFF III dataset acquired during ischemic episodes caused by balloon inflation in LAD (n=35), RCA (n=47), and LCx (n=17) coronary arteries, and the Glasgow dataset comprising admission 12-lead ECGs of 116 patients who were hospitalized for chest pain and underwent contrast-enhanced cardiac MRI that confirmed AMI in 58 patients (50%). We found that, in the STAFF III dataset, the detection of ischemic state by the STEMI criteria attained SE/SP of 60/97%, whereas SE/SP values of VSLs were 72/98%. In the Glasgow dataset, STEMI criteria yielded SE/SP of 43/98%, whereas the VSLs improved SE/SP to 60/98%. The most significant increase in diagnostic performance appeared in patients with LCx coronary artery occlusion: in STAFF III data (n=17) SE achieved by STEMI criteria was improved by the VSLs from 35% to 71%; in Glasgow data (n=12) SE of 31% achieved by STEMI criteria was improved by the VSLs to 69%. In our study population, existing ACC/ESC STEMI criteria complemented by the new VSLs yielded much improved sensitivity of ischemia detection without any detrimental effect on specificity. This finding needs to be corroborated on a larger chest-pain patient population with

  2. Sensitivity and specificity for detecting basal cell carcinomas in Mohs excisions with confocal fluorescence mosaicing microscopy.

    PubMed

    Gareau, Daniel S; Karen, Julie K; Dusza, Stephen W; Tudisco, Marie; Nehal, Kishwer S; Rajadhyaksha, Milind

    2009-01-01

    Recent studies have demonstrated the ability of confocal fluorescence mosaicing microscopy to rapidly detect basal cell carcinomas (BCCs) directly in thick and fresh Mohs surgical excisions. Mosaics of confocal images display large areas of tissue with high resolution and magnification equivalent to 2x, which is the standard magnification when examining pathology. Comparison of mosaics to Mohs frozen histopathology was shown to be excellent for all types of BCCs. However, comparisons in the previous studies were visual and qualitative. In this work, we report the results of a semiquantitative preclinical study in which 45 confocal mosaics are blindly evaluated for the presence (or absence) of BCC tumor. The evaluations are made by two clinicians: a senior Mohs surgeon with prior expertise in interpreting confocal images, and a novice Mohs fellow with limited experience. The blinded evaluation is compared to the gold standard of frozen histopathology. BCCs are detected with an overall sensitivity of 96.6%, specificity of 89.2%, positive predictive value of 93.0%, and negative predictive value of 94.7%. The results demonstrate the potential clinical utility of confocal mosaicing microscopy toward rapid surgical pathology at the bedside to expedite and guide surgery.

  3. Species specific PCR based detection of Escherichia coli from Indian foods.

    PubMed

    Godambe, Lipika Pansare; Bandekar, Jayant; Shashidhar, Ravindranath

    2017-06-01

    Escherichia coli is a faecal indicator and certain virotypes are known as pathogens. Therefore, detection and prevention of E. coli in food is very important. The existing rapid methods concentrate on detecting the pathogenic E. coli instead of total E. coli population. Present study evaluates the use of two molecular markers (uidA and flanking region of uspA) specific for the E. coli in combination with microbiological method for confirmation. Majority of the isolates (77%) were positive for both the genes tested. However, 22% of the E. coli isolates were positive for any one of the two primer sets [uidA (9%) and flanking region of uspA (13%)]. High levels of E. coli incidences (92% samples) were observed in beef while low occurrence (19% samples) was found in sprouts. Low percentage (7.3%) of E. coli isolates was positive for virulence genes tested (lt, ipaH, aggR, eaeA, stx1 and stx2). Two isolates were positive for stx genes. However, none of the isolates including stx positive isolates were E. coli 0157:H7. Maximum number of the E. coli (44%) isolates was characterized under phylogenetic group B2. This phylogenetic group comprises of extra intestinal and virulent E. coli strains.

  4. Multiplexing detection of IgG against Plasmodium falciparum pregnancy-specific antigens

    PubMed Central

    Fonseca, Ana Maria; Quinto, Llorenç; Jiménez, Alfons; González, Raquel; Bardají, Azucena; Maculuve, Sonia; Dobaño, Carlota; Rupérez, Maria; Vala, Anifa; Aponte, John J.; Sevene, Esperanza; Macete, Eusebio; Menéndez, Clara

    2017-01-01

    Background Pregnant women exposed to Plasmodium falciparum generate antibodies against VAR2CSA, the parasite protein that mediates adhesion of infected erythrocytes to the placenta. There is a need of high-throughput tools to determine the fine specificity of these antibodies that can be used to identify immune correlates of protection and exposure. Here we aimed at developing a multiplex-immunoassay to detect antibodies against VAR2CSA antigens. Methods and findings We constructed two multiplex-bead arrays, one composed of 3 VAR2CSA recombinant-domains (DBL3X, DBL5Ɛ and DBL6Ɛ) and another composed of 46 new peptides covering VAR2CSA conserved and semi-conserved regions. IgG reactivity was similar in multiplexed and singleplexed determinations (Pearson correlation, protein array: R2 = 0.99 and peptide array: R2 = 0.87). IgG recognition of 25 out of 46 peptides and all recombinant-domains was higher in pregnant Mozambican women (n = 106) than in Mozambican men (n = 102) and Spanish individuals (n = 101; p<0.05). Agreement of IgG levels detected in cryopreserved plasma and in elutions from dried blood spots was good after exclusion of inappropriate filter papers. Under heterogeneous levels of exposure to malaria, similar seropositivity cutoffs were obtained using finite mixture models applied to antibodies measured on pregnant Mozambican women and average of antibodies measured on pregnant Spanish women never exposed to malaria. The application of the multiplex-bead array developed here, allowed the assessment of higher IgG levels and seroprevalences against VAR2CSA-derived antigens in women pregnant during 2003–2005 than during 2010–2012, in accordance with the levels of malaria transmission reported for these years in Mozambique. Conclusions The multiplex bead-based immunoassay to detect antibodies against selected 25 VAR2CSA new-peptides and recombinant-domains was successfully implemented. Analysis of field samples showed that responses were specific among

  5. Multiplexing detection of IgG against Plasmodium falciparum pregnancy-specific antigens.

    PubMed

    Fonseca, Ana Maria; Quinto, Llorenç; Jiménez, Alfons; González, Raquel; Bardají, Azucena; Maculuve, Sonia; Dobaño, Carlota; Rupérez, Maria; Vala, Anifa; Aponte, John J; Sevene, Esperanza; Macete, Eusebio; Menéndez, Clara; Mayor, Alfredo

    2017-01-01

    Pregnant women exposed to Plasmodium falciparum generate antibodies against VAR2CSA, the parasite protein that mediates adhesion of infected erythrocytes to the placenta. There is a need of high-throughput tools to determine the fine specificity of these antibodies that can be used to identify immune correlates of protection and exposure. Here we aimed at developing a multiplex-immunoassay to detect antibodies against VAR2CSA antigens. We constructed two multiplex-bead arrays, one composed of 3 VAR2CSA recombinant-domains (DBL3X, DBL5Ɛ and DBL6Ɛ) and another composed of 46 new peptides covering VAR2CSA conserved and semi-conserved regions. IgG reactivity was similar in multiplexed and singleplexed determinations (Pearson correlation, protein array: R2 = 0.99 and peptide array: R2 = 0.87). IgG recognition of 25 out of 46 peptides and all recombinant-domains was higher in pregnant Mozambican women (n = 106) than in Mozambican men (n = 102) and Spanish individuals (n = 101; p<0.05). Agreement of IgG levels detected in cryopreserved plasma and in elutions from dried blood spots was good after exclusion of inappropriate filter papers. Under heterogeneous levels of exposure to malaria, similar seropositivity cutoffs were obtained using finite mixture models applied to antibodies measured on pregnant Mozambican women and average of antibodies measured on pregnant Spanish women never exposed to malaria. The application of the multiplex-bead array developed here, allowed the assessment of higher IgG levels and seroprevalences against VAR2CSA-derived antigens in women pregnant during 2003-2005 than during 2010-2012, in accordance with the levels of malaria transmission reported for these years in Mozambique. The multiplex bead-based immunoassay to detect antibodies against selected 25 VAR2CSA new-peptides and recombinant-domains was successfully implemented. Analysis of field samples showed that responses were specific among pregnant women and dependent on the level of

  6. Sensitive and specific colorimetric DNA detection by invasive reaction coupled with nicking endonuclease-assisted nanoparticles amplification.

    PubMed

    Zou, Bingjie; Cao, Xiaomei; Wu, Haiping; Song, Qinxin; Wang, Jianping; Kajiyama, Tomoharu; Kambara, Hideki; Zhou, Guohua

    2015-04-15

    Colorimetric DNA detection is preferable to methods in clinical molecular diagnostics, because no expensive equipment is required. Although many gold nanoparticle-based colorimetric DNA detection strategies have been developed to analyze DNA sequences of interest, few of them can detect somatic mutations due to their insufficient specificity. In this study, we proposed a colorimetric DNA detection method by coupling invasive reaction with nicking endonuclease-assisted nanoparticles amplification (IR-NEANA). A target DNA firstly produces many flaps by invasive reaction. Then the flaps are converted to targets of nicking reaction-assisted nanoparticles amplification by ligation reaction to produce the color change of AuNPs, which can be observed by naked eyes. The detection limit of IR-NEANA was determined as 1pM. Most importantly, the specificity of the method is high enough to pick up as low as 1% mutant from a large amount of wild-type DNA backgrounds. The EGFR gene mutated at c.2573 T>G in 9 tissue samples from non-small cell lung cancer patients were successfully detected by using IR-NEANA, suggesting that our proposed method can be used to detect somatic mutations in biological samples.

  7. Design and Implementation of an On-Chip Patient-Specific Closed-Loop Seizure Onset and Termination Detection System.

    PubMed

    Zhang, Chen; Bin Altaf, Muhammad Awais; Yoo, Jerald

    2016-07-01

    This paper presents the design of an area- and energy-efficient closed-loop machine learning-based patient-specific seizure onset and termination detection algorithm, and its on-chip hardware implementation. Application- and scenario-based tradeoffs are compared and reviewed for seizure detection and suppression algorithm and system which comprises electroencephalography (EEG) data acquisition, feature extraction, classification, and stimulation. Support vector machine achieves a good tradeoff among power, area, patient specificity, latency, and classification accuracy for long-term monitoring of patients with limited training seizure patterns. Design challenges of EEG data acquisition on a multichannel wearable environment for a patch-type sensor are also discussed in detail. Dual-detector architecture incorporates two area-efficient linear support vector machine classifiers along with a weight-and-average algorithm to target high sensitivity and good specificity at once. On-chip implementation issues for a patient-specific transcranial electrical stimulation are also discussed. The system design is verified using CHB-MIT EEG database [1] with a comprehensive measurement criteria which achieves high sensitivity and specificity of 95.1% and 96.2%, respectively, with a small latency of 1 s. It also achieves seizure onset and termination detection delay of 2.98 and 3.82 s, respectively, with seizure length estimation error of 4.07 s.

  8. Detecting location-specific neuronal firing rate increases in the hippocampus of freely-moving monkeys.

    PubMed

    Ludvig, Nandor; Tang, Hai M; Gohil, Baiju C; Botero, Juan M

    2004-07-16

    The spatial properties of the firing of hippocampal neurons have mainly been studied in (a) freely moving rodents, (b) non-human primates seated in a moveable primate chair with head fixed, and (c) epileptic patients subjected to virtual navigation. Although these studies have all revealed the ability of hippocampal neurons to generate spatially selective discharges, the detected firing patterns have been found to be considerably different, even conflicting, in many respects. The present cellular electrophysiological study employed squirrel monkeys (Saimiri sciureus), which moved freely on the walls and floor of a large test chamber. This permitted the examination of the spatial firing of hippocampal neurons in nearly ideal conditions, similar to those used in rodents, yet in a species that belongs to the primate Suborder Anthropoidea. The major findings were that: (1) a group of slow-firing complex-spike cells increased their basal, awake firing rate more than 20-fold, often above 30 spikes/s, when the monkey was in a particular location in the chamber, (2) these location-specific discharges occurred consistently, forming 4-25 s action potential volleys, and (3) fast-firing cells displayed no such electrical activity. Thus, during free movement in three dimensions, primate hippocampal complex-spike cells do generate high-frequency, location-specific action potential volleys. Since these cells are components of the medial temporal lobe memory system, their uncovered firing pattern may well be involved in the formation of declarative memories on places.

  9. Ultra-high sensitivity radiation detection apparatus and method

    DOEpatents

    Gross, Kenneth C.; Valentine, John D.; Markum, Francis; Zawadzki, Mary; Dickerman, Charles

    1999-01-01

    A method and apparatus are provided to concentrate and detect very low levels of radioactive noble gases from the atmosphere. More specifically the invention provides a method and apparatus to concentrate xenon, krypton and radon in an organic fluid and to detect these gases by the radioactive emissions.

  10. A simple and reliable assay for detecting specific nucleotide sequences in plants using optical thin-film biosensor chips.

    PubMed

    Bai, Su-Lan; Zhong, Xiaobo; Ma, Ligeng; Zheng, Wenjie; Fan, Liu-Min; Wei, Ning; Deng, Xing Wang

    2007-01-01

    Here we report the adaptation and optimization of an efficient, accurate and inexpensive assay that employs custom-designed silicon-based optical thin-film biosensor chips to detect unique transgenes in genetically modified (GM) crops and SNP markers in model plant genomes. Briefly, aldehyde-attached sequence-specific single-stranded oligonucleotide probes are arrayed and covalently attached to a hydrazine-derivatized biosensor chip surface. Unique DNA sequences (or genes) are detected by hybridizing biotinylated PCR amplicons of the DNA sequences to probes on the chip surface. In the SNP assay, target sequences (PCR amplicons) are hybridized in the presence of a mixture of biotinylated detector probes and a thermostable DNA ligase. Only perfect matches between the probe and target sequences, but not those with even a single nucleotide mismatch, can be covalently fixed on the chip surface. In both cases, the presence of specific target sequences is signified by a color change on the chip surface (gold to blue/purple) after brief incubation with an anti-biotin IgG horseradish peroxidase (HRP) to generate a precipitable product from an HRP substrate. Highly sensitive and accurate identification of PCR targets can be completed within 30 min. This assay is extremely robust, exhibits high sensitivity and specificity, and is flexible from low to high throughput and very economical. This technology can be customized for any nucleotide sequence-based identification assay and widely applied in crop breeding, trait mapping, and other work requiring positive detection of specific nucleotide sequences.

  11. High detection rate of dog circovirus in diarrheal dogs.

    PubMed

    Hsu, Han-Siang; Lin, Ting-Han; Wu, Hung-Yi; Lin, Lee-Shuan; Chung, Cheng-Shu; Chiou, Ming-Tang; Lin, Chao-Nan

    2016-06-17

    Diarrhea is one of the most common clinical symptoms reported in companion animal clinics. Dog circovirus (DogCV) is a new mammalian circovirus that is considered to be a cause of alimentary syndromes such as diarrhea, vomiting and hemorrhagic enteritis. DogCV has previously only been identified in the United States, Italy, Germany (GeneBank accession number: KF887949) and China (GeneBank accession number: KT946839). Therefore, the aims of this study were to determine the prevalence of DogCV in Taiwan and to explore the correlation between diarrhea and DogCV infection. Clinical specimens were collected between 2012 and 2014 from 207 dogs suffering from diarrhea and 160 healthy dogs. In this study, we developed a sensitive and specific SYBR Green-based real-time PCR assays to detected DogCV in naturally infected animals. Of the analyzed fecal samples from diarrheal dogs and health dogs, 58 (28.0 %) and 19 (11.9 %), respectively, were DogCV positive. The difference in DogCV prevalence was highly significant (P = 0.0002755) in diarrheal dogs. This is the first study to reveal that DogCV is currently circulating in domestic dogs in Taiwan and to demonstrate its high detection rate in dogs with diarrhea.

  12. High-Throughput Variation Detection and Genotyping Using Microarrays

    PubMed Central

    Cutler, David J.; Zwick, Michael E.; Carrasquillo, Minerva M.; Yohn, Christopher T.; Tobin, Katherine P.; Kashuk, Carl; Mathews, Debra J.; Shah, Nila A.; Eichler, Evan E.; Warrington, Janet A.; Chakravarti, Aravinda

    2001-01-01

    The genetic dissection of complex traits may ultimately require a large number of SNPs to be genotyped in multiple individuals who exhibit phenotypic variation in a trait of interest. Microarray technology can enable rapid genotyping of variation specific to study samples. To facilitate their use, we have developed an automated statistical method (ABACUS) to analyze microarray hybridization data and applied this method to Affymetrix Variation Detection Arrays (VDAs). ABACUS provides a quality score to individual genotypes, allowing investigators to focus their attention on sites that give accurate information. We have applied ABACUS to an experiment encompassing 32 autosomal and eight X-linked genomic regions, each consisting of ∼50 kb of unique sequence spanning a 100-kb region, in 40 humans. At sufficiently high-quality scores, we are able to read ∼80% of all sites. To assess the accuracy of SNP detection, 108 of 108 SNPs have been experimentally confirmed; an additional 371 SNPs have been confirmed electronically. To access the accuracy of diploid genotypes at segregating autosomal sites, we confirmed 1515 of 1515 homozygous calls, and 420 of 423 (99.29%) heterozygotes. In replicate experiments, consisting of independent amplification of identical samples followed by hybridization to distinct microarrays of the same design, genotyping is highly repeatable. In an autosomal replicate experiment, 813,295 of 813,295 genotypes are called identically (including 351 heterozygotes); at an X-linked locus in males (haploid), 841,236 of 841,236 sites are called identically. PMID:11691856

  13. Validation of an Immunodiagnostic Assay for Detection of 13 Streptococcus pneumoniae Serotype-Specific Polysaccharides in Human Urine

    PubMed Central

    Huijts, Susanne M.; Wu, Kangjian; Souza, Victor; Passador, Sherry; Tinder, Chunyan; Song, Esther; Elfassy, Arik; McNeil, Lisa; Menton, Ronald; French, Roger; Callahan, Janice; Webber, Chris; Gruber, William C.; Bonten, Marc J. M.; Jansen, Kathrin U.

    2012-01-01

    To improve the clinical diagnosis of pneumococcal infection in bacteremic and nonbacteremic community-acquired pneumonia (CAP), a Luminex technology-based multiplex urinary antigen detection (UAD) diagnostic assay was developed and validated. The UAD assay can simultaneously detect 13 different serotypes of Streptococcus pneumoniae by capturing serotype-specific S. pneumoniae polysaccharides (PnPSs) secreted in human urine. Assay specificity is achieved by capturing the polysaccharides with serotype-specific monoclonal antibodies (MAbs) on spectrally unique microspheres. Positivity for each serotype was based on positivity cutoff values calculated from a standard curve run on each assay plate together with positive- and negative-control urine samples. The assay is highly specific, since significant signals are detected only when each PnPS was paired with its homologous MAb-coated microspheres. Validation experiments demonstrated excellent accuracy and precision. The UAD assay and corresponding positivity cutoff values were clinically validated by assessing 776 urine specimens obtained from patients with X-ray-confirmed CAP. The UAD assay demonstrated 97% sensitivity and 100% specificity using samples obtained from patients with bacteremic, blood culture-positive CAP. Importantly, the UAD assay identified Streptococcus pneumoniae (13 serotypes) in a proportion of individuals with nonbacteremic CAP, a patient population for which the pneumococcal etiology of CAP was previously difficult to assess. Therefore, the UAD assay provides a specific, noninvasive, sensitive, and reproducible tool to support vaccine efficacy as well as epidemiological evaluation of pneumococcal disease, including CAP, in adults. PMID:22675155

  14. Validation of an immunodiagnostic assay for detection of 13 Streptococcus pneumoniae serotype-specific polysaccharides in human urine.

    PubMed

    Pride, Michael W; Huijts, Susanne M; Wu, Kangjian; Souza, Victor; Passador, Sherry; Tinder, Chunyan; Song, Esther; Elfassy, Arik; McNeil, Lisa; Menton, Ronald; French, Roger; Callahan, Janice; Webber, Chris; Gruber, William C; Bonten, Marc J M; Jansen, Kathrin U

    2012-08-01

    To improve the clinical diagnosis of pneumococcal infection in bacteremic and nonbacteremic community-acquired pneumonia (CAP), a Luminex technology-based multiplex urinary antigen detection (UAD) diagnostic assay was developed and validated. The UAD assay can simultaneously detect 13 different serotypes of Streptococcus pneumoniae by capturing serotype-specific S. pneumoniae polysaccharides (PnPSs) secreted in human urine. Assay specificity is achieved by capturing the polysaccharides with serotype-specific monoclonal antibodies (MAbs) on spectrally unique microspheres. Positivity for each serotype was based on positivity cutoff values calculated from a standard curve run on each assay plate together with positive- and negative-control urine samples. The assay is highly specific, since significant signals are detected only when each PnPS was paired with its homologous MAb-coated microspheres. Validation experiments demonstrated excellent accuracy and precision. The UAD assay and corresponding positivity cutoff values were clinically validated by assessing 776 urine specimens obtained from patients with X-ray-confirmed CAP. The UAD assay demonstrated 97% sensitivity and 100% specificity using samples obtained from patients with bacteremic, blood culture-positive CAP. Importantly, the UAD assay identified Streptococcus pneumoniae (13 serotypes) in a proportion of individuals with nonbacteremic CAP, a patient population for which the pneumococcal etiology of CAP was previously difficult to assess. Therefore, the UAD assay provides a specific, noninvasive, sensitive, and reproducible tool to support vaccine efficacy as well as epidemiological evaluation of pneumococcal disease, including CAP, in adults.

  15. Detection of Leptospira-Specific Antibodies Using a Recombinant Antigen-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Chen, Hua-Wei; Zhang, Zhiwen; Halsey, Eric S.; Guevara, Carolina; Canal, Enrique; Hall, Eric; Maves, Ryan; Tilley, Drake H.; Kochel, Tadeusz J.; Ching, Wei-Mei

    2013-01-01

    We produced three highly purified recombinant antigens rLipL32, rLipL41, and rLigA-Rep (leptospiral immunoglobulin-like A repeat region) for the detection of Leptospira-specific antibodies in an enzyme-linked immunosorbent assay (ELISA). The performance of these recombinant antigens was evaluated using 121 human sera. Among them, 63 sera were microscopic agglutination test (MAT)-confirmed positive sera from febrile patients in Peru, 22 sera were indigenous MAT-negative febrile patient sera, and 36 sera were from patients with other febrile diseases from Southeast Asia, where leptospirosis is also endemic. Combining the results of immunoglobulin M (IgM) and IgG detection from these three antigens, the overall sensitivity is close to 90% based on the MAT. These results suggest that an ELISA using multiple recombinant antigens may be used as an alternative method for the detection of Leptospira-specific antibodies. PMID:24166046

  16. Nucleic acid spot hybridization based species-specific detection of Sclerotium rolfsii associated with collar rot disease of Amorphophallus paeoniifolius.

    PubMed

    Pravi, V; Jeeva, M L; Archana, P V

    2015-02-01

    Collar rot is one of the most destructive and prevalent disease of Amorphophallus paeoniifolius, resulting in heavy yield losses. The causative organism, Sclerotium rolfsii is a soil-borne polyphagous fungus characterized by prolific growth and ability to produce persistent sclerotia. The pathogen propagules surviving in soil and planting material are the major sources of inoculum. This study presents the suitability of DNA hybridization technique for species specific detection of S. rolfsii in soil and planting material. The detection limit of the probe was 10-15 pg of pure pathogen DNA. The developed probe was found to be highly specific and could be used for accurate identification of pathogen up to the species level. The protocol was standardized for detection of the pathogen in naturally infected field samples.

  17. High Energy Electron Detection with ATIC

    NASA Technical Reports Server (NTRS)

    Chang, J.; Schmidt, W. K. H.; Adams, James H., Jr.; Ahn, H.; Ampe, J.; Whitaker, Ann F. (Technical Monitor)

    2001-01-01

    The ATIC (Advanced Thin Ionization Calorimeter) balloon-borne ionization calorimeter is well suited to record and identify high energy cosmic ray electrons. The instrument was exposed to high-energy beams at CERN H2 bean-dine in September of 1999. We have simulated the performance of the instrument, and compare the simulations with actual high energy electron exposures at the CERN accelerator. Simulations and measurements do not compare exactly, in detail, but overall the simulations have predicted actual measured behavior quite well.

  18. [Quantitative specific detection of Staphylococcus aureus based on recombinant lysostaphin and ATP bioluminescence].

    PubMed

    Li, Yuyuan; Mi, Zhiqiang; An, Xiaoping; Zhou, Yusen; Tong, Yigang

    2014-08-01

    Quantitative specific detection of Staphylococcus aureus is based on recombinant lysostaphin and ATP bioluminescence. To produce recombinant lysostaphin, the lysostaphin gene was chemically synthesized and inserted it into prokaryotic expression vector pQE30, and the resulting expression plasmid pQE30-Lys was transformed into E. coli M15 for expressing lysostaphin with IPTG induction. The recombinant protein was purified by Ni(2+)-NTA affinity chromatography. Staphylococcus aureus was detected by the recombinant lysostaphin with ATP bioluminescence, and plate count method. The results of the two methods were compared. The recombinant lysostaphin was successfully expressed, and a method of quantitative specific detection of S. aureus has been established, which showed a significant linear correlation with the colony counting. The detection method developed has good perspective to quantify S. aureus.

  19. Detecting Faults In High-Voltage Transformers

    NASA Technical Reports Server (NTRS)

    Blow, Raymond K.

    1988-01-01

    Simple fixture quickly shows whether high-voltage transformer has excessive voids in dielectric materials and whether high-voltage lead wires too close to transformer case. Fixture is "go/no-go" indicator; corona appears if transformer contains such faults. Nests in wire mesh supported by cap of clear epoxy. If transformer has defects, blue glow of corona appears in mesh and is seen through cap.

  20. Detecting Faults In High-Voltage Transformers

    NASA Technical Reports Server (NTRS)

    Blow, Raymond K.

    1988-01-01

    Simple fixture quickly shows whether high-voltage transformer has excessive voids in dielectric materials and whether high-voltage lead wires too close to transformer case. Fixture is "go/no-go" indicator; corona appears if transformer contains such faults. Nests in wire mesh supported by cap of clear epoxy. If transformer has defects, blue glow of corona appears in mesh and is seen through cap.

  1. Whole genome sequence analysis of unidentified genetically modified papaya for development of a specific detection method.

    PubMed

    Nakamura, Kosuke; Kondo, Kazunari; Akiyama, Hiroshi; Ishigaki, Takumi; Noguchi, Akio; Katsumata, Hiroshi; Takasaki, Kazuto; Futo, Satoshi; Sakata, Kozue; Fukuda, Nozomi; Mano, Junichi; Kitta, Kazumi; Tanaka, Hidenori; Akashi, Ryo; Nishimaki-Mogami, Tomoko

    2016-08-15

    Identification of transgenic sequences in an unknown genetically modified (GM) papaya (Carica papaya L.) by whole genome sequence analysis was demonstrated. Whole genome sequence data were generated for a GM-positive fresh papaya fruit commodity detected in monitoring using real-time polymerase chain reaction (PCR). The sequences obtained were mapped against an open database for papaya genome sequence. Transgenic construct- and event-specific sequences were identified as a GM papaya developed to resist infection from a Papaya ringspot virus. Based on the transgenic sequences, a specific real-time PCR detection method for GM papaya applicable to various food commodities was developed. Whole genome sequence analysis enabled identifying unknown transgenic construct- and event-specific sequences in GM papaya and development of a reliable method for detecting them in papaya food commodities. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Coomassie blue is sufficient for specific protein detection of aptamer-conjugated chips.

    PubMed

    Lee, Gwan-Ho; Hah, Sang Soo

    2012-02-15

    An aptamer-based biochip for protein detection and quantitation which combines the recent biochip technology and the conventional staining methods, is described. Using a model system comprising His-tagged proteins as the analyte and single-stranded RNA aptamers specific for His-tagged proteins as immobilized ligands on chips, we could demonstrate that aptamers were equivalent or superior to antibodies in terms of specificity and sensitivity, respectively. The sensor has the characteristics of good stability, reproducibility and reusability, with detection limit as low as 85 ng/mL His-tagged protein. It has been demonstrated that the sensor can be stored for at least 4 weeks and reused with reasonable reduction rate of staining intensity. In conclusion, we could show the suitability of nucleic acid aptamers as low molecular weight receptors on biochips for sensitive and specific protein detection and quantitation. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Detecting Dual AGN at High Redshift

    NASA Astrophysics Data System (ADS)

    Barrows, Robert S.

    2012-01-01

    The existence of supermassive black holes (SMBHs) in most, if not all, galaxies, along with observations of galaxy mergers, suggests that pairs of SMBHs should exist for some time in the merger remnant. Observational evidence for these systems at kpc-scale separations (i.e. dual AGN) has dramatically increased recently through a combination of spectral and morphological selections. I discuss observations of CXOXBJ142607.6+353351 (CXOJ1426+35), a candidate dual AGN at z=1.175, and put its properties, including significant obscuration, within the context of other candidate/confirmed dual AGN at lower redshifts. Though dual AGN are expected to be more common at higher redshifts, they are more difficult to detect. Furthermore, adding to the difficulties of detection are a number of other physical mechanisms which can mimic the spectroscopic signature of two Type 2 AGN. In particular, I will discuss the possibility of strong outflows from an AGN. These outflow phenomena can be an important feedback mechanism in galaxies and are apparently common in AGN, making them a viable alternative to the dual AGN scenario. Based on our candidate's luminosity and emission line intensities, we find that an outflow is a possibility. If this is the case, such an outflow would be especially strong and has implications for AGN feedback in galaxies. However, the dual AGN scenario cannot be ruled out, and at z=1.175, the two putative AGN could potentially be resolved with Chandra. Other candidate dual AGN at similar redshifts and with significant obscuration could also be confirmed this way. This research was sponsored by the Strategic University Research Partnership Program, the National Aeronautics and Space Administration and the Arkansas NASA EPSCoR program.

  4. High efficiency cell-specific targeting of cytokine activity

    NASA Astrophysics Data System (ADS)

    Garcin, Geneviève; Paul, Franciane; Staufenbiel, Markus; Bordat, Yann; van der Heyden, José; Wilmes, Stephan; Cartron, Guillaume; Apparailly, Florence; de Koker, Stefaan; Piehler, Jacob; Tavernier, Jan; Uzé, Gilles

    2014-01-01

    Systemic toxicity currently prevents exploiting the huge potential of many cytokines for medical applications. Here we present a novel strategy to engineer immunocytokines with very high targeting efficacies. The method lies in the use of mutants of toxic cytokines that markedly reduce their receptor-binding affinities, and that are thus rendered essentially inactive. Upon fusion to nanobodies specifically binding to marker proteins, activity of these cytokines is selectively restored for cell populations expressing this marker. This ‘activity-by-targeting’ concept was validated for type I interferons and leptin. In the case of interferon, activity can be directed to target cells in vitro and to selected cell populations in mice, with up to 1,000-fold increased specific activity. This targeting strategy holds promise to revitalize the clinical potential of many cytokines.

  5. Study of Prominence Detection Based on Various Phone-Specific Features

    NASA Astrophysics Data System (ADS)

    Kim, Sung Soo; Han, Chang Woo; Kim, Nam Soo

    In this letter, we present useful features accounting for pronunciation prominence and propose a classification technique for prominence detection. A set of phone-specific features are extracted based on a forced alignment of the test pronunciation provided by a speech recognition system. These features are then applied to the traditional classifiers such as the support vector machine (SVM), artificial neural network (ANN) and adaptive boosting (Adaboost) for detecting the place of prominence.

  6. DNA detection on transistor arrays following mutation-specific enzymatic amplification

    NASA Astrophysics Data System (ADS)

    Pouthas, F.; Gentil, C.; Côte, D.; Bockelmann, U.

    2004-03-01

    An integrated array of silicon field-effect transistor structures is used for electronic detection of label-free DNA. Measurements of the dc current-voltage characteristics of the transistors gives us access to reproducible detection of single- and double-stranded DNA, locally adsorbed on the surface of the device. We combine this approach with allele-specific polymerase chain reaction, to test for the 35delG mutation, a frequent mutation related to prelingual nonsyndromic deafness.

  7. Specificity of PCR and serological assays in the detection of Escherichia coli Shiga toxin subtypes.

    PubMed

    Feng, Peter C H; Jinneman, Karen; Scheutz, Flemming; Monday, Steven R

    2011-09-01

    Specificity analysis for stx or Stx subtypes in Escherichia coli showed that the PCR assays we tested did not detect stx(1d) and stx(2f), and some also missed stx(2b) and stx(2g). Most of the serological assays examined did not detect Stx2c, Stx2e, Stx2f, and Stx2g, and some strain-to-strain variation in reactivity was observed for Stx2b.

  8. Specific detection of benzimidazole resistance in Colletotrichum gloeosporioides from fruit crops by PCR-RFLP.

    PubMed

    Chung, Wen-Hsin; Chung, Wen-Chuan; Peng, Mun-Tsu; Yang, Hong-Ren; Huang, Jenn-Wen

    2010-02-28

    Anthracnose diseases, caused by Colletotrichum gloeosporioides, are a worldwide problem and are especially important in Taiwan owing to the severe economic damage they cause to tropical fruits that are grown for local consumption and export. Benzimidazoles are systemic fungicides widely used for controlling these diseases in Taiwan. Thirty-one isolates of C. gloeosporioides from mango and strawberry grown in Taiwan were examined for their sensitivity to benzimidazole fungicides. The responses of the isolates grown on benzimidazole-amended culture media were characterized as sensitive, moderately resistant, resistant or highly resistant. Analysis of point mutations in the beta-tubulin gene by DNA sequencing of PCR-amplified fragments revealed a substitution of GCG for GAG at codon 198 in resistant and highly resistant isolates and a substitution of TAC for TTC at codon 200 in moderately resistant isolates. A set of specific primers, TubGF1 and TubGR, was designed to amplify a portion of the beta-tubulin gene for the detection of benzimidazole-resistant C. gloeosporioides. Bsh1236I restriction maps of the amplified beta-tubulin gene showed that the resistant isolate sequence, but not the sensitive isolate sequence, was cut. The PCR restriction fragment length polymorphism (PCR-RFLP) was validated to detect benzimidazole-resistant and benzimidazole-sensitive C. gloeosporioides isolates recovered from avocado, banana, carambola, dragon fruit, grape, guava, jujube, lychee, papaya, passion fruit and wax apple. This method has the potential to become a valuable tool for monitoring the occurrence of benzimidazole-resistant C. gloeosporioides and for assessment of the need for alternative management practices. Copyright 2009 Elsevier B.V. All rights reserved.

  9. Immuno-PCR: Very sensitive antigen detection by means of specific antibody-DNA conjugates

    SciTech Connect

    Sano, T.; Smith, C.L.; Cantor, C.R. )

    1992-10-02

    An antigen detection system, termed immuno-polymerase chain reaction (immuno-PCR), was developed in which a specific DNA molecule is used as the marker. A streptavidin-protein A chimera that possesses tight and specific binding affinity both for biotin and immunoglobulin G was used to attach a biotinylated DNA specifically to antigen-monoclonal antibody complexes that had been immobilized on microtiter plate wells. Then, a segment of the attached DNA was amplified by PCR. Analysis of the PCR products by agarose gel electrophoresis after staining with ethidium bromide allowed as few as 580 antigen molecules to be readily and reproducibly detected. Direct comparison with enzyme-linked immunosorbent assay with the use of a chimera-alkaline phosphatase conjugate demonstrates that enhancement in detection sensitivity was obtained with the use of immuno-PCR. Given the enormous amplification capability and specificity of PCR, this immuno-PCR technology has a sensitivity greater than any existing antigen detection system and, in principle, could be applied to the detection of single antigen molecules.

  10. International multidimensional authenticity specification (IMAS) algorithm for detection of commercial pomegranate juice adulteration.

    PubMed

    Zhang, Yanjun; Krueger, Dana; Durst, Robert; Lee, Rupo; Wang, David; Seeram, Navindra; Heber, David

    2009-03-25

    The pomegranate fruit ( Punica granatum ) has become an international high-value crop for the production of commercial pomegranate juice (PJ). The perceived consumer value of PJ is due in large part to its potential health benefits based on a significant body of medical research conducted with authentic PJ. To establish criteria for authenticating PJ, a new International Multidimensional Authenticity Specifications (IMAS) algorithm was developed through consideration of existing databases and comprehensive chemical characterization of 45 commercial juice samples from 23 different manufacturers in the United States. In addition to analysis of commercial juice samples obtained in the United States, data from other analyses of pomegranate juice and fruits including samples from Iran, Turkey, Azerbaijan, Syria, India, and China were considered in developing this protocol. There is universal agreement that the presence of a highly constant group of six anthocyanins together with punicalagins characterizes polyphenols in PJ. At a total sugar concentration of 16 degrees Brix, PJ contains characteristic sugars including mannitol at >0.3 g/100 mL. Ratios of glucose to mannitol of 4-15 and of glucose to fructose of 0.8-1.0 are also characteristic of PJ. In addition, no sucrose should be present because of isomerase activity during commercial processing. Stable isotope ratio mass spectrometry as > -25 per thousand assures that there is no added corn or cane sugar added to PJ. Sorbitol was present at <0.025 g/100 mL; maltose and tartaric acid were not detected. The presence of the amino acid proline at >25 mg/L is indicative of added grape products. Malic acid at >0.1 g/100 mL indicates adulteration with apple, pear, grape, cherry, plum, or aronia juice. Other adulteration methods include the addition of highly concentrated aronia, blueberry, or blackberry juices or natural grape pigments to poor-quality juices to imitate the color of pomegranate juice, which results in

  11. [Establishment of a high-throughput respiratory virus detection technology without RNA purification and reverse transcription].

    PubMed

    Yang, Dan-li; Tian, Xiao-yi; Shi, Wei-xian; Zheng, Zhi

    2013-02-01

    To establish a convenient and high-throughput respiratory virus detection method to facilitate epidemiological viral monitoring. We used high-throughput microsphere-based flexible multi-analyte profiling technology (xMAP) coupled with signal amplification molecules to simultaneously detect RNAs of 8 viruses including influenza viruses A and B, parainfluenza viruses type 1, 2 and 3, respiratory syncytial viruses A and B, and metapneumovirus in a 96-well plate format. The sensitivity and specificity of the method for the synthetic viral RNAs were evaluated. There was no cross-reactivity among the 8 respiratory viral target RNAs. The detection limits for the 8 viral in intro-transcribed RNAs ranged from 1204 to 4695 RNA copies. We establish a sensitive, specific, convenient, and high-throughput multiplex detection method suitable for detecting multiple respiratory viral RNAs for epidemiological viral monitoring.

  12. Detection of the Cracks using High-Tc SQUID

    NASA Astrophysics Data System (ADS)

    Fujii, Tatsuhiko; Hyun-Sung, Tae; Takamatsu, Tsuyoshi; Sakuta, Ken; Itozaki, Hideo

    Eddy current non-distractive evaluation (NDE) is very useful technique for detection of cracks. We use the high-Tc SQUID in this system. First, we respect the result of this measurement by the finite element method. We can detect of the signal from the hole by NDE system with high-Tc SQUID. This result is reasonable to compare with the result of simulation. Finaly, we can detect of the hidden defect under 5mm depth from the sample surface.

  13. Enzyme-linked immunosorbent assay for detection of filovirus species-specific antibodies.

    PubMed

    Nakayama, Eri; Yokoyama, Ayaka; Miyamoto, Hiroko; Igarashi, Manabu; Kishida, Noriko; Matsuno, Keita; Marzi, Andrea; Feldmann, Heinz; Ito, Kimihito; Saijo, Masayuki; Takada, Ayato

    2010-11-01

    Several enzyme-linked immunosorbent assays (ELISAs) for the detection of filovirus-specific antibodies have been developed. However, diagnostic methods to distinguish antibodies specific to the respective species of filoviruses, which provide the basis for serological classification, are not readily available. We established an ELISA using His-tagged secreted forms of the transmembrane glycoproteins (GPs) of five different Ebola virus (EBOV) species and one Marburg virus (MARV) strain as antigens for the detection of filovirus species-specific antibodies. The GP-based ELISA was evaluated by testing antisera collected from mice immunized with virus-like particles as well as from humans and nonhuman primates infected with EBOV or MARV. In our ELISA, little cross-reactivity of IgG antibodies was observed in most of the mouse antisera. Although sera and plasma from some patients and monkeys showed notable cross-reactivity with the GPs from multiple filovirus species, the highest reactions of IgG were uniformly detected against the GP antigen homologous to the virus species that infected individuals. We further confirmed that MARV-specific IgM antibodies were specifically detected in specimens collected from patients during the acute phase of infection. These results demonstrate the usefulness of our ELISA for diagnostics as well as ecological and serosurvey studies.

  14. Screening DNA chip and event-specific multiplex PCR detection methods for biotech crops.

    PubMed

    Lee, Seong-Hun

    2014-11-01

    There are about 80 biotech crop events that have been approved by safety assessment in Korea. They have been controlled by genetically modified organism (GMO) and living modified organism (LMO) labeling systems. The DNA-based detection method has been used as an efficient scientific management tool. Recently, the multiplex polymerase chain reaction (PCR) and DNA chip have been developed as simultaneous detection methods for several biotech crops' events. The event-specific multiplex PCR method was developed to detect five biotech maize events: MIR604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The specificity was confirmed and the sensitivity was 0.5%. The screening DNA chip was developed from four endogenous genes of soybean, maize, cotton and canola respectively along with two regulatory elements and seven genes: P35S, tNOS, pat, bar, epsps1, epsps2, pmi, cry1Ac and cry3B. The specificity was confirmed and the sensitivity was 0.5% for four crops' 12 events: one soybean, six maize, three cotton and two canola events. The multiplex PCR and DNA chip can be available for screening, gene-specific and event-specific analysis of biotech crops as efficient detection methods by saving on workload and time. © 2014 Society of Chemical Industry. © 2014 Society of Chemical Industry.

  15. Detection of fungal development in closed spaces through the determination of specific chemical targets.

    PubMed

    Moularat, Stéphane; Robine, Enric; Ramalho, Olivier; Oturan, Mehmet A

    2008-05-01

    In addition to the biodegradation problems encountered in buildings, exposure of their occupants to moulds is responsible for numerous diseases: infections (invasive nosocomial aspergillosis), immediate or delayed allergies, food-borne infections and different types of irritation. In this context, the aim of our work has been to determine specific chemical tracers for fungal development on construction materials. More generally, by detecting a specific chemical fingerprint of fungal development, our objective was to propose a microbiological alert system which could control systems and/or procedures for the microbiological treatment of indoor areas. We therefore characterized the chemical emissions from six types of construction material contaminated artificially by moulds. Chemical fingerprints were established for 19 compounds arising specifically from fungal metabolism: 2-ethylhexanoic acid methyl ester, 1-octen-3-ol, 3-heptanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 1,3-octadiene, 2-(5H)-furanone, 2-heptene, alpha-pinene, 2-methylisoborneol, 4-heptanone, 2-methylfuran, 3-methylfuran, dimethyldisulfide, methoxybenzene, a terpenoid and three sesquiterpenes. Determining the origin of these compounds and their specific links with a growth substrate or fungal species made it possible to judge the pertinence of choosing these compounds as tracers. Thus the detecting specific volatile organic compounds emitted as from the second day of fungal growth demonstrated that this approach had the advantage of detecting fungal development both reliably and rapidly before any visible signs of contamination could be detected.

  16. Denatured G-protein coupled receptors as immunogens to generate highly specific antibodies.

    PubMed

    Talmont, Franck; Moulédous, Lionel; Boué, Jérôme; Mollereau, Catherine; Dietrich, Gilles

    2012-01-01

    G-protein coupled receptors (GPCRs) play a major role in a number of physiological and pathological processes. Thus, GPCRs have become the most frequent targets for development of new therapeutic drugs. In this context, the availability of highly specific antibodies may be decisive to obtain reliable findings on localization, function and medical relevance of GPCRs. However, the rapid and easy generation of highly selective anti-GPCR antibodies is still a challenge. Herein, we report that highly specific antibodies suitable for detection of GPCRs in native and unfolded forms can be elicited by immunizing animals against purified full length denatured recombinant GPCRs. Contrasting with the currently admitted postulate, our study shows that an active and well-folded GPCR is not required for the production of specific anti-GPCR antibodies. This new immunizing strategy validated with three different human GPCR (μ-opioid, κ-opioid, neuropeptide FF2 receptors) might be generalized to other members of the GPCR family.

  17. Reverse enzyme immunoassay for detection of specific anti-Toxoplasma immunoglobulin M antibodies.

    PubMed Central

    Franco, E L; Walls, K W; Sulzer, A J

    1981-01-01

    A reverse enzyme immunoassay (R-EIA) is described, in which polystyrene muplates are sensitized with anti-immunoglobulin M (IgM) (mu chain) antibodies and then sequentially allowed to react with patient's serum, peroxidase-labeled Toxoplasma gondii soluble antigen, and substrate. Measurement of activity of the solid-phase bound enzyme conjugate was done by colorimetric reading of the final developed color and kinetically by the initial rate of color development. This R-EIA allowed full resolution between absorbance values of a group of 36 sera which presented positive results in the Toxoplasma IgM immunofluorescence test and the remaining groups, which consisted of 39 normal individuals, 22 rheumatoid factor-positive sera, 8 Waldenstrom's macroglobulinemic sera, 3 infectious mononucleosis samples, and 6 high-titered IgG anti-T. gondii sera. No interference of rheumatoid factor IgM or inhibition by high-titered specific IgG was detected, even in the false IgM immunofluorescence-positive rheumatoid factor samples. Likewise, false-negative IgM immunofluorescence samples gave positive R-EIA even without adsorption with Staphylococcus aureus protein A. The possibility of direct tagging of the antigen with the enzyme eliminates the need for using antigen and anti-antigen conjugates as separate layers, therefore eliminating one step in the assay. PMID:7016911

  18. Highly active and selective endopeptidases with programmed substrate specificities

    PubMed Central

    Varadarajan, Navin; Rodriguez, Sarah; Hwang, Bum-Yeol; Georgiou, George; Iverson, Brent L

    2009-01-01

    A family of engineered endopeptidases has been created that is capable of cleaving a diverse array of peptide sequences with high selectivity and catalytic efficiency (kcat/KM > 104 M−1 s−1). By screening libraries with a selection-counterselection substrate method, protease variants were programmed to recognize amino acids having altered charge, size and hydrophobicity properties adjacent to the scissile bond of the substrate, including Glu↓Arg, a specificity that to our knowledge has not been observed among natural proteases. Members of this artificial protease family resulted from a relatively small number of amino acid substitutions that (at least in one case) proved to be epistatic. PMID:18391948

  19. On-chip microbial culture for the specific detection of very low levels of bacteria.

    PubMed

    Bouguelia, Sihem; Roupioz, Yoann; Slimani, Sami; Mondani, Laure; Casabona, Maria G; Durmort, Claire; Vernet, Thierry; Calemczuk, Roberto; Livache, Thierry

    2013-10-21

    Microbial culture continues to be the most common protocol for bacterial detection and identification in medicine and agronomics. Using this process may take days to identify a specific pathogen for most bacterial strains. Surface Plasmon Resonance (SPR) detection is an emerging alternative technology that can be used for the detection of bacteria using protein microarrays although typical limits of detection are in the range of 10(3)-10(6) cfu mL(-1), which is not compatible with most Food Safety regulation requirements. In this work, we combine concomitant "on-chip" microbial culture with sensitive SPR detection of bacteria thus allowing rapid specific detection of bacteria pathogens - including Salmonella enterica serovar Enteritidis, Streptococcus pneumoniae and Escherichia coli O157:H7 - cultured on a protein microarray. This Culture-Capture-Measure (CCM) approach significantly decreases both the number of processing steps and the overall assay time for bacterial detection. Signal analysis of SPR responses allowed the fast and quantitative assessment of bacterial concentrations initially present in the sample as low as 2.8 ± 19.6 cfu per milliliter. Altogether, our results show how simple, easy-to-operate, fluidic-less and lo-tec microarrays can be used with unprocessed samples and yield - in a single assay - both qualitative and quantitative information regarding bacterial contamination.

  20. Engineering the stereochemistry of cephalosporin for specific detection of pathogenic carbapenemase-expressing bacteria.

    PubMed

    Shi, Haibin; Cheng, Yunfeng; Lee, Kyung Hyun; Luo, Robert F; Banaei, Niaz; Rao, Jianghong

    2014-07-28

    Reported herein is the design of fluorogenic probes specific for carbapenem-resistant Enterobacteriaceae (CRE) and they were designed based on stereochemically modified cephalosporin having a 6,7-trans configuration. Through experiments using recombinant β-lactamase enzymes and live bacterial species, these probes demonstrate the potential for use in the specific detection of carbapenemases, including metallo-β-lactamases in active bacterial pathogens.

  1. Comparison of MY09/11 consensus PCR and type-specific PCRs in the detection of oncogenic HPV types.

    PubMed

    Depuydt, C E; Boulet, G A V; Horvath, C A J; Benoy, I H; Vereecken, A J; Bogers, J J

    2007-01-01

    The causal relationship between persistent infection with high-risk HPV and cervical cancer has resulted in the development of HPV DNA detection systems. The widely used MY09/11 consensus PCR targets a 450bp conserved sequence in the HPV L1 gene, and can therefore amplify a broad spectrum of HPV types. However, limitations of these consensus primers are evident, particularly in regard to the variability in detection sensitivity among different HPV types. This study compared MY09/11 PCR with type-specific PCRs in the detection of oncogenic HPV types. The study population comprised 15, 774 patients. Consensus PCR failed to detect 522 (10.9%) HPV infections indicated by type-specific PCRs. A significant correlation between failure of consensus PCR and HPV type was found. HPV types 51, 68 and 45 were missed most frequently. The clinical relevance of the HPV infections missed by MY09/11 PCR was reflected in the fraction of cases with cytological abnormalities and in follow-up, showing 104 (25.4%) CIN2+ cases. The MY09/11 false negativity could be the result of poor sensitivity, mismatch of MY09/11 primers or disruption of L1 target by HPV integration or DNA degradation. Furthermore, MY09/11 PCR lacked specificity for oncogenic HPVs. Diagnostic accuracy of the PCR systems, in terms of sensitivity (MY09/11 PCR: 87.9%; type-specific PCRs: 98.3%) and specificity (MY09/11 PCR: 38.7%; type-specific PCRs: 76.14%), and predictive values for histologically confirmed CIN2+, suggest that type-specific PCRs could be used in a clinical setting as a reliable screening tool.

  2. RNase H-dependent PCR (rhPCR): improved specificity and single nucleotide polymorphism detection using blocked cleavable primers

    PubMed Central

    2011-01-01

    Background The polymerase chain reaction (PCR) is commonly used to detect the presence of nucleic acid sequences both in research and diagnostic settings. While high specificity is often achieved, biological requirements sometimes necessitate that primers are placed in suboptimal locations which lead to problems with the formation of primer dimers and/or misamplification of homologous sequences. Results Pyrococcus abyssi (P.a.) RNase H2 was used to enable PCR to be performed using blocked primers containing a single ribonucleotide residue which are activated via cleavage by the enzyme (rhPCR). Cleavage occurs 5'-to the RNA base following primer hybridization to the target DNA. The requirement of the primer to first hybridize with the target sequence to gain activity eliminates the formation of primer-dimers and greatly reduces misamplification of closely related sequences. Mismatches near the scissile linkage decrease the efficiency of cleavage by RNase H2, further increasing the specificity of the assay. When applied to the detection of single nucleotide polymorphisms (SNPs), rhPCR was found to be far more sensitive than standard allele-specific PCR. In general, the best discrimination occurs when the mismatch is placed at the RNA:DNA base pair. Conclusion rhPCR eliminates the formation of primer dimers and markedly improves the specificity of PCR with respect to off-target amplification. These advantages of the assay should find utility in challenging qPCR applications such as genotyping, high level multiplex assays and rare allele detection. PMID:21831278

  3. [High output stoma: detection and approach].

    PubMed

    Arenas Villafranca, Jose Javier; Abilés, Jimena; Moreno, Gloria; Tortajada Goitia, Begoña; Utrilla Navarro, Pilar; Gándara Adán, Norberto

    2014-12-01

    High output stoma is a frequent complication in patients with ileostomies that is not well identified and is not often properly addressed by clinicians. It has not been described properly, and can vary between debits of 2.000ml in 24 h to 1.500 ml in 3-5 days, according to different authors. Frequently presents both short-term and long-term negative implications for patients and is associated with readmissions. We present a review of published literature focusing in surgical resection-related factors that influence a later appearance of this complication, causes involved in its development, the need to establish a clear and objective concept of high ouput as well as the negative implications it presents. Also we develop how should we the management of these patients regarding treatment and nutritional approach.

  4. Design optimization of a high specific speed Francis turbine runner

    NASA Astrophysics Data System (ADS)

    Enomoto, Y.; Kurosawa, S.; Kawajiri, H.

    2012-11-01

    Francis turbine is used in many hydroelectric power stations. This paper presents the development of hydraulic performance in a high specific speed Francis turbine runner. In order to achieve the improvements of turbine efficiency throughout a wide operating range, a new runner design method which combines the latest Computational Fluid Dynamics (CFD) and a multi objective optimization method with an existing design system was applied in this study. The validity of the new design system was evaluated by model performance tests. As the results, it was confirmed that the optimized runner presented higher efficiency compared with an originally designed runner. Besides optimization of runner, instability vibration which occurred at high part load operating condition was investigated by model test and gas-liquid two-phase flow analysis. As the results, it was confirmed that the instability vibration was caused by oval cross section whirl which was caused by recirculation flow near runner cone wall.

  5. Detection and quantification of drug-specific T cells in penicillin allergy.

    PubMed

    Rozieres, A; Hennino, A; Rodet, K; Gutowski, M-C; Gunera-Saad, N; Berard, F; Cozon, G; Bienvenu, J; Nicolas, J-F

    2009-04-01

    Drug allergic reactions presenting as maculo-papular exanthema (MPE) are mediated by drug-specific T cells. In this study, the frequency of circulating specific T cells was analyzed by interferon-gamma (IFN-gamma) enzyme-linked immunospot assay in 22 patients with an allergic MPE to amoxicillin (amox). Amox-specific circulating T cells were detected in 20/22 patients with frequencies ranging from 1 : 8000 to 1 : 30 000 circulating leucocytes. No reactivity was observed in 46 control patients, including 15 patients with immunoglobulin E-mediated allergy to amoxicillin, 11 patients with a history of drug-induced MPE but tolerant to amoxicillin and 20 healthy individuals. Furthermore, amox-specific T cells were still detectable several years after the occurrence of the allergic reaction even after strict drug avoidance. Finally, analysis of drug-specific T cells in one patient allergic to ticarcillin (a penicillin antibiotic distinct from amox) revealed the presence of IFN-gamma-producing T cells reactive to ticarcillin and several other betalactam antibiotics, suggesting that the IFN-gamma ELISPOT assay is able to detect T cell cross-reactivity against chemically related drugs. These findings confirm that drug-induced MPE is associated with the presence of specific T cells in blood and further suggest that the IFN-gamma ELISPOT is a sensitive assay which could improve the diagnosis of betalactam allergy.

  6. Tissue-Specific DNA Methylation Patterns in Forensic Samples Detected by Pyrosequencing®.

    PubMed

    Antunes, Joana; Balamurugan, Kuppareddi; Duncan, George; McCord, Bruce

    2015-01-01

    In certain circumstances the outcome of a trial may hinge on the ability of a forensic laboratory to determine the identity of biological stains present at crime scenes. An example of such a situation would be the detection of blood, saliva, vaginal fluid, or other body fluid in a specific location whereby its presence would reinforce the victim's or suspect's version of the events that happened during the commission of a crime. However, current serological methods used for identifying body fluids may lack the sensitivity and specificity to identify these fluids, particularly for trace levels. New procedures using proteomic methods and RNA-based gene expression show promise in addressing this issue; however, concerns about stability and relative levels of gene expression remain. An alternative approach is to utilize patterns of epigenetic DNA methylation. DNA methylation is an epigenetic mechanism that regulates the specificity of genes being expressed or silenced in cells. Regions in the human genome referred to as tissue-specific differentially methylated regions account for unique patterns of DNA methylation that are specific for each cell type. This chapter addresses the application of bisulfite-modified PCR combined with Pyrosequencing(®) to detect tissue-specific DNA methylation patterns and perform trace serological analysis. The quantitative nature and precision available with Pyrosequencing presents major advantages in these studies as it permits detection of and contrast between cells with differential levels of methylation. The procedure can be applied to a variety of biological fluids which may be present at crime scenes.

  7. Plate-specific gain map correction for the improvement of detective quantum efficiency in computed radiography.

    PubMed

    Schnell, Erich A; Samei, Ehsan; Dobbins, James T

    2012-03-01

    The purpose of this work is to improve the noise power spectrum (NPS), and thus the detective quantum efficiency (DQE), of computed radiography (CR) images by correcting for spatial gain variations specific to individual imaging plates. CR devices have not traditionally employed gain-map corrections, unlike the case with flat-panel detectors, because of the multiplicity of plates used with each reader. The lack of gain-map correction has limited the DQE(f) at higher exposures with CR. This current work describes a feasible solution to generating plate-specific gain maps. Ten high-exposure open field images were taken with an RQA5 spectrum, using a sixth generation CR plate suspended in air without a cassette. Image values were converted to exposure, the plates registered using fiducial dots on the plate, the ten images averaged, and then high-pass filtered to remove low frequency contributions from field inhomogeneity. A gain-map was then produced by converting all pixel values in the average into fractions with mean of one. The resultant gain-map of the plate was used to normalize subsequent single images to correct for spatial gain fluctuation. To validate performance, the normalized NPS (NNPS) for all images was calculated both with and without the gain-map correction. Variations in the quality of correction due to exposure levels, beam voltage/spectrum, CR reader used, and registration were investigated. The NNPS with plate-specific gain-map correction showed improvement over the noncorrected case over the range of frequencies from 0.15 to 2.5 mm(-1). At high exposure (40 mR), NNPS was 50%-90% better with gain-map correction than without. A small further improvement in NNPS was seen from carefully registering the gain-map with subsequent images using small fiducial dots, because of slight misregistration during scanning. Further improvement was seen in the NNPS from scaling the gain map about the mean to account for different beam spectra. This study

  8. Plate-specific gain map correction for the improvement of detective quantum efficiency in computed radiography

    SciTech Connect

    Schnell, Erich A.; Samei, Ehsan; Dobbins, James T.

    2012-03-15

    Purpose: The purpose of this work is to improve the noise power spectrum (NPS), and thus the detective quantum efficiency (DQE), of computed radiography (CR) images by correcting for spatial gain variations specific to individual imaging plates. CR devices have not traditionally employed gain-map corrections, unlike the case with flat-panel detectors, because of the multiplicity of plates used with each reader. The lack of gain-map correction has limited the DQE(f) at higher exposures with CR. This current work describes a feasible solution to generating plate-specific gain maps. Methods: Ten high-exposure open field images were taken with an RQA5 spectrum, using a sixth generation CR plate suspended in air without a cassette. Image values were converted to exposure, the plates registered using fiducial dots on the plate, the ten images averaged, and then high-pass filtered to remove low frequency contributions from field inhomogeneity. A gain-map was then produced by converting all pixel values in the average into fractions with mean of one. The resultant gain-map of the plate was used to normalize subsequent single images to correct for spatial gain fluctuation. To validate performance, the normalized NPS (NNPS) for all images was calculated both with and without the gain-map correction. Variations in the quality of correction due to exposure levels, beam voltage/spectrum, CR reader used, and registration were investigated. Results: The NNPS with plate-specific gain-map correction showed improvement over the noncorrected case over the range of frequencies from 0.15 to 2.5 mm{sup -1}. At high exposure (40 mR), NNPS was 50%-90% better with gain-map correction than without. A small further improvement in NNPS was seen from carefully registering the gain-map with subsequent images using small fiducial dots, because of slight misregistration during scanning. Further improvement was seen in the NNPS from scaling the gain map about the mean to account for different beam

  9. Flexible and Lightweight Fuel Cell with High Specific Power Density.

    PubMed

    Ning, Fandi; He, Xudong; Shen, Yangbin; Jin, Hehua; Li, Qingwen; Li, Da; Li, Shuping; Zhan, Yulu; Du, Ying; Jiang, Jingjing; Yang, Hui; Zhou, Xiaochun

    2017-06-27

    Flexible devices have been attracting great attention recently due to their numerous advantages. But the energy densities of current energy sources are still not high enough to support flexible devices for a satisfactory length of time. Although proton exchange membrane fuel cells (PEMFCs) do have a high-energy density, traditional PEMFCs are usually too heavy, rigid, and bulky to be used in flexible devices. In this research, we successfully invented a light and flexible air-breathing PEMFC by using a new design of PEMFC and a flexible composite electrode. The flexible air-breathing PEMFC with 1 × 1 cm(2) working area can be as light as 0.065 g and as thin as 0.22 mm. This new PEMFC exhibits an amazing specific volume power density as high as 5190 W L(-1), which is much higher than traditional (air-breathing) PEMFCs. Also outstanding is that the flexible PEMFC retains 89.1% of its original performance after being bent 600 times, and it retains its original performance after being dropped five times from a height of 30 m. Moreover, the research has demonstrated that when stacked, the flexible PEMFCs are also useful in mobile applications such as mobile phones. Therefore, our research shows that PEMFCs can be made light, flexible, and suitable for applications in flexible devices. These innovative flexible PEMFCs may also notably advance the progress in the PEMFC field, because flexible PEMFCs can achieve high specific power density with small size, small volume, low weight, and much lower cost; they are also much easier to mass produce.

  10. Efficiency Analysis of a High-Specific Impulse Hall Thruster

    NASA Technical Reports Server (NTRS)

    Jacobson, David (Technical Monitor); Hofer, Richard R.; Gallimore, Alec D.

    2004-01-01

    Performance and plasma measurements of the high-specific impulse NASA-173Mv2 Hall thruster were analyzed using a phenomenological performance model that accounts for a partially-ionized plasma containing multiply-charged ions. Between discharge voltages of 300 to 900 V, the results showed that although the net decrease of efficiency due to multiply-charged ions was only 1.5 to 3.0 percent, the effects of multiply-charged ions on the ion and electron currents could not be neglected. Between 300 to 900 V, the increase of the discharge current was attributed to the increasing fraction of multiply-charged ions, while the maximum deviation of the electron current from its average value was only +5/-14 percent. These findings revealed how efficient operation at high-specific impulse was enabled through the regulation of the electron current with the applied magnetic field. Between 300 to 900 V, the voltage utilization ranged from 89 to 97 percent, the mass utilization from 86 to 90 percent, and the current utilization from 77 to 81 percent. Therefore, the anode efficiency was largely determined by the current utilization. The electron Hall parameter was nearly constant with voltage, decreasing from an average of 210 at 300 V to an average of 160 between 400 to 900 V. These results confirmed our claim that efficient operation can be achieved only over a limited range of Hall parameters.

  11. Vision-based in-line fabric defect detection using yarn-specific shape features

    NASA Astrophysics Data System (ADS)

    Schneider, Dorian; Aach, Til

    2012-01-01

    We develop a methodology for automatic in-line flaw detection in industrial woven fabrics. Where state of the art detection algorithms apply texture analysis methods to operate on low-resolved ({200 ppi) image data, we describe here a process flow to segment single yarns in high-resolved ({1000 ppi) textile images. Four yarn shape features are extracted, allowing a precise detection and measurement of defects. The degree of precision reached allows a classification of detected defects according to their nature, providing an innovation in the field of automatic fabric flaw detection. The design has been carried out to meet real time requirements and face adverse conditions caused by loom vibrations and dirt. The entire process flow is discussed followed by an evaluation using a database with real-life industrial fabric images. This work pertains to the construction of an on-loom defect detection system to be used in manufacturing practice.

  12. Heterojunction phototransistor for highly sensitive infrared detection

    NASA Astrophysics Data System (ADS)

    Rezaei, Mohsen; Park, Min-Su; Tan, Chee Leong; Rabinowitz, Cobi; Wheaton, Skyler; Mohseni, Hooman

    2017-02-01

    In this work, we have proposed a model for the ultimate physical limit on the sensitivity of the heterojunction bipolar phototransistors (HPTs). Based on our modeling we have extracted the design criteria for the HPT for high sensitivity application. HPT with the submicron emitter and base area has the potential to be used for the low number photon resolving in near-infrared (NIR) wavelength. However, in practice, the quality of materials, processing, and the passivation plays an important role in the realization of the highly sensitive HPT. For short wave infrared (SWIR) HPTs based on lattice matched InGaAs to InP is studied. For these devices, conditions to reach to the highest possible sensitivity is examined. We have made an HPT based on InGaAs collector and base on the InP substrate. After developing proper processing combination of wet and dry etching and the surface passivation for the device we made an imager with 320x256 pixels based with a 30m pixel pitch. The imager shows the sensitivity less the 30 photons for each pixel with the frame rate more than 1K frames per second.

  13. Development of taxon-specific sequences of common wheat for the detection of genetically modified wheat.

    PubMed

    Iida, Mayu; Yamashiro, Satomi; Yamakawa, Hirohito; Hayakawa, Katsuyuki; Kuribara, Hideo; Kodama, Takashi; Furui, Satoshi; Akiyama, Hiroshi; Maitani, Tamio; Hino, Akihiro

    2005-08-10

    Qualitative and quantitative Polymerase Chain Reaction (PCR) systems aimed at the specific detection and quantification of common wheat DNA are described. Many countries have issued regulations to label foods that include genetically modified organisms (GMOs). PCR technology is widely recognized as a reliable and useful technique for the qualitative and quantitative detection of GMOs. Detection methods are needed to amplify a target GM gene, and the amplified results should be compared with those of the corresponding taxon-specific reference gene to obtain reliable results. This paper describes the development of a specific DNA sequence in the waxy-D1 gene for common wheat (Triticum aestivum L.) and the design of a specific primer pair and TaqMan probe on the waxy-D1 gene for PCR analysis. The primers amplified a product (Wx012) of 102 bp. It is indicated that the Wx012 DNA sequence is specific to common wheat, showing homogeneity in qualitative PCR results and very similar quantification accuracy along 19 distantly related common wheat varieties. In Southern blot and real-time PCR analyses, this sequence showed either a single or a low number of copy genes. In addition, by qualitative and quantitative PCR using wx012 primers and a wx012-T probe, the limits of detection of the common wheat genome were found to be about 15 copies, and the reproducibility was reliable. In consequence, the PCR system using wx012 primers and wx012-T probe is considered to be suitable for use as a common wheat-specific taxon-specific reference gene in DNA analyses, including GMO tests.

  14. Highly sensitive detection of dipicolinic acid with a water-dispersible terbium-metal organic framework.

    PubMed

    Bhardwaj, Neha; Bhardwaj, Sanjeev; Mehta, Jyotsana; Kim, Ki-Hyun; Deep, Akash

    2016-12-15

    The sensitive detection of dipicolinic acid (DPA) is strongly associated with the sensing of bacterial organisms in food and many types of environmental samples. To date, the demand for a sensitive detection method for bacterial toxicity has increased remarkably. Herein, we investigated the DPA detection potential of a water-dispersible terbium-metal organic framework (Tb-MOF) based on the fluorescence quenching mechanism. The Tb-MOF showed a highly sensitive ability to detect DPA at a limit of detection of 0.04nM (linear range of detection: 1nM to 5µM) and also offered enhanced selectivity from other commonly associated organic molecules. The present study provides a basis for the application of Tb-MOF for direct, convenient, highly sensitive, and specific detection of DPA in the actual samples.

  15. Novel primers and PCR protocols for the specific detection and quantification of Sphingobium suberifaciens in situ

    USDA-ARS?s Scientific Manuscript database

    The pathogen causing corky root on lettuce, Sphingobium suberifaciens, is recalcitrant to standard epidemiological methods. Primers were selected from 16S rDNA sequences useful for the specific detection and quantification of S. suberifaciens. Conventional (PCR) and quantitative (qPCR) PCR protocols...

  16. Robust detection of rare species using environmental DNA: The importance of primer specificity

    Treesearch

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

    2013-01-01

    Environmental DNA (eDNA) is being rapidly adopted as a tool to detect rare animals. Quantitative PCR (qPCR) using probebased 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...

  17. The detection of specific biomolecular interactions with micro-Hall magnetic sensors.

    PubMed

    Manandhar, Pradeep; Chen, Kan-Sheng; Aledealat, Khaled; Mihajlović, Goran; Yun, C Steven; Field, Mark; Sullivan, Gerard J; Strouse, Geoffrey F; Chase, P Bryant; von Molnár, Stephan; Xiong, Peng

    2009-09-02

    The detection of reagent-free specific biomolecular interactions through sensing of nanoscopic magnetic labels provides one of the most promising routes to biosensing with solid-state devices. In particular, Hall sensors based on semiconductor heterostructures have shown exceptional magnetic moment sensitivity over a large dynamic field range suitable for magnetic biosensing using superparamagnetic labels. Here we demonstrate the capability of such micro-Hall sensors to detect specific molecular binding using biotin-streptavidin as a model system. We apply dip-pen nanolithography to selectively biotinylate the active areas of InAs micro-Hall devices with nanoscale precision. Specific binding of complementarily functionalized streptavidin-coated superparamagnetic beads to the Hall crosses occurs via molecular recognition, and magnetic detection of the assembled beads is achieved at room temperature using phase sensitive micro-Hall magnetometry. The experiment constitutes the first unambiguous demonstration of magnetic detection of specific biomolecular interactions with semiconductor micro-Hall sensors, and the selective molecular functionalization and resulting localized bead assembly demonstrate the possibility of multiplexed sensing of multiple target molecules using a single device with an array of micro-Hall sensors.

  18. TM7 detection in human microbiome: are PCR primers and FISH probes specific enough?

    PubMed Central

    Sizova, Maria V.; Doerfert, Sebastian N.; Gavrish, Ekaterina; Epstein, Slava S.

    2015-01-01

    TM7 appears important and omnipresent because it is repeatedly detected by molecular techniques in diverse environments. Here we report that most of primers and FISH probes thought to be TM7-specific do hybridize with multiple species from oral and vaginal cavity. This calls for re-examination of TM7 distribution and abundance. PMID:25957511

  19. Development of an Enzyme Linked Immunosorbent Assay to Detect Chicken Parvovirus Specific Antibodies

    USDA-ARS?s Scientific Manuscript database

    Here we report the development and application of an enzyme linked immunosorbent assay to detect parvovirus-specific antibodies in chicken sera. We used an approach previously described for other parvoviruses to clone and express viral structural proteins in insect cells from recombinant baculovirus...

  20. Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis

    EPA Science Inventory

    ABSTRACT BACKGROUND Anti-neutrophil cytoplasmic autoantibodies (ANCA) specific for myeloperoxidase (MPO) or proteinase 3 (PR3) are detectable in >90% of patients with ANCA-associated vasculitis (AAV). ANCA titers do not correlate well with disease activity. In vivo and in vi...

  1. Early detection of prostate cancer. Role of prostate-specific antigen.

    PubMed Central

    Prabhakaran, V. M.

    1996-01-01

    Pressure to request prostate-specific antigen (PSA) tests for early detection of prostate cancer in middle-aged and older men is increasing. However, current scientific data suggest that such testing does more harm than good. Most professional groups do not advise routine screening for prostate cancer. This paper reviews the current status of PSA testing. PMID:8653039

  2. Construction of specific DNA probe for the detection of Salmonella in food.

    PubMed

    Pilantanapak, A; Jayanetra, P; Panbangred, W; Klungthong, C; Bangtrakulnonth, A

    1997-03-01

    The Salmonella specific DNA fragment from genomic DNA of S. typhimurium ATCC 23566 was cloned in E. coli and successfully used as a digoxigenin labeled probe for detecting the presence of Salmonella serotypes in both artificially contaminated food and natural contaminated food samples.

  3. Sextant-Specific Analysis of Detection and Tumor Volume by HistoScanning™.

    PubMed

    Porres, Daniel; Kuru, Timur Hasan; Epplen, Robin; Eck, Andreas; Zugor, Vahudin; Kennes, Lieven Niels; Afram, Samir; Braunschweig, Till; Knüchel-Clarke, Ruth; Pfister, David; Heidenreich, Axel

    2016-01-01

    Published results of HistoScanning™ (HS) for prostate cancer (PCa) diagnostics are inconsistent and their value remains unclear. We prospectively analyzed the detection rate and tumor volume concordance in PCa patients. Two hundred and eighty-two patients with biopsy-proven PCa scheduled for radical prostatectomy (RP) were included. All patients underwent ultrasonographical examination by HS prior to surgery. HS was evaluated compared to RP specimen as to (1) the prediction of overall tumor volume and (2) accuracy of HS in detection of PCa lesions larger than 0.2/0.5 ml, separated for each sextant. For each sextant, receiver operating characteristic (ROC)-analysis and area under the curve were determined. Sensitivity and specificity were calculated and visualized in ROC-curves. HS tends to underestimate volume of cancerous lesions, particularly larger lesions >8 ml. Using a 0.2 ml detection threshold, specificity and sensitivity of HS were between 29-68% and 46-78%. For a 0.5 ml detection threshold, sextant-specific specificity increased to 59-92% and sensitivity decreased to 16-54%. Stratification according to pre-operational PSA values did not improve performance characteristics of HS. Our results do not support a significant contribution of HS to PCa diagnostics. © 2015 S. Karger AG, Basel.

  4. Epitope specificity determines pathogenicity and detectability in ANCA-associated vasculitis

    EPA Science Inventory

    ABSTRACT BACKGROUND Anti-neutrophil cytoplasmic autoantibodies (ANCA) specific for myeloperoxidase (MPO) or proteinase 3 (PR3) are detectable in >90% of patients with ANCA-associated vasculitis (AAV). ANCA titers do not correlate well with disease activity. In vivo and in vi...

  5. Simultaneous, specific and real-time detection of biothreat and frequently encountered food-borne pathogens

    PubMed Central

    Woubit, Abdela Salah; Yehualaeshet, Teshome; Habtemariam, Tsegaye; Samuel, Temesgen

    2012-01-01

    The bacterial genera Escherichia, Salmonella, Shigella, Vibrio, Yersinia and Francisella include important food safety and biothreat agents causing food-related and other human illnesses worldwide. We aimed to develop rapid methods with the capability to simultaneously and differentially detect all six pathogens in one run. Our initial experiments to use previously reported sets of primers revealed non-specificity of some of the sequences when tested against a broader array of pathogens, or proved not optimal for simultaneous detection parameters. By extensive mining of the whole genome and protein databases of diverse closely and distantly related bacterial species and strains, we have identified unique genome regions, which we utilized to develop a detection platform. Twelve of the specific genomic targets we have identified to design the primers in F. tularensis ssp. tularensis, F. tularensis ssp. novicida, S. dysentriae, S. typhimurium, V. cholera, Y. pestis, and Y. pseudotuberculosis contained either hypothetical or putative proteins, the functions of which have not been clearly defined. Corresponding primer sets were designed from the target regions for use in real-time PCR assays to detect specific biothreat pathogens at species or strain levels. The primer sets were first tested by in-silico PCR against whole genome sequences of different species, sub-species, or strains and then by in vitro PCR against genomic DNA preparations from 23 strains representing six biothreat agents (E.coli O157:H7 strain EDL 933, Shigella dysentriae, Salmonella typhi, Francisella tularensis ssp. tularensis, Vibrio cholera, and Yersinia pestis) and six foodborne pathogens (Salmonella typhimurium, Salmonella saintpaul, Shigella sonnei, Francisella novicida, Vibrio parahemolytica and Yersinia pseudotuberculosis). Each pathogen was specifically identifiable at the genus and species levels. Sensitivity assays performed using purified DNA showed the lowest detection limit of 640 fg

  6. Characterization of grain-specific peptide markers for the detection of gluten by mass spectrometry.

    PubMed

    Fiedler, Katherine L; McGrath, Sara C; Callahan, John H; Ross, Mark M

    2014-06-25

    Global and targeted mass spectrometry-based proteomic approaches were developed to discover, evaluate, and apply gluten peptide markers to detect low parts per million (ppm) wheat contamination of oats. Prolamins were extracted from wheat, barley, rye, and oat flours and then reduced, alkylated, and digested with chymotrypsin. The resulting peptides were subjected to LC-MS/MS analysis and database matching. No peptide markers common to wheat, barley, and rye were identified that could be used for global gluten detection. However, many grain-specific peptide markers were identified, and a set of these markers was selected for gluten detection and grain differentiation. Wheat flour was spiked into gluten-free oat flour at concentrations of 1-100,000 ppm and analyzed to determine the lowest concentration at which the wheat "contaminant" could be confidently detected in the mixture. The same 2D ion trap instrument that was used for the global proteomics approach was used for the targeted proteomics approach, providing a seamless transition from target discovery to application. A powerful, targeted MS/MS method enabled detection of two wheat peptide markers at the 10 ppm wheat flour-in-oat flour concentration. Because gluten comprises approximately 10% of wheat flour protein, the reported wheat gluten-specific peptides can enable detection of approximately 1 ppm of wheat gluten in oats.

  7. Detection of the MYD88 mutation by the combination of the allele-specific PCR and quenching probe methods.

    PubMed

    Nogami, S; Kawaguchi-Ihara, N; Shiratori, E; Ohtaka, M; Itoh, M; Tohda, S

    2017-04-01

    The MYD88 missense mutation c.794T>C, p.Leu265Pro, is found in patients with Waldenstörm's macroglobulinemia and lymphoma. Direct sequencing, allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and high-resolution melting analysis (HRM) are currently used to detect the mutation; however, they are either time-consuming or have low detection sensitivity. Here, we developed a novel highly sensitive and rapid detection method based on the quenching probe (QP) technique and AS-PCR. A lymphoma cell line heterozygous for the MYD88 mutation, two wild-type cell lines, and two samples from Waldenstörm's macroglobulinemia patients were analyzed by AS-PCR, PCR-RFLP, HRM, and QP, and their detection sensitivity was examined using the mixtures of the mutant and wild-type DNA. For mutation-carrying heterozygous samples, the QP method produced W-shaped melting profiles presenting curves derived from the wild-type and mutant alleles. The QP analysis was performed in 2 h and demonstrated the detection limit of 5%, which was similar to that of the other methods. However, the combination of AS-PCR and QP (AS-QP) improved the sensitivity to 0.62% of the mutant allele. The AS-QP analysis is rapid and minimally improves detection sensitivity compared to the AS-PCR. © 2016 John Wiley & Sons Ltd.

  8. Microdevices for detecting locus-specific DNA methylation at CpG resolution.

    PubMed

    Koo, Kevin M; Wee, Eugene J H; Rauf, Sakandar; Shiddiky, Muhammad J A; Trau, Matt

    2014-06-15

    Simple, rapid, and inexpensive strategies for detecting DNA methylation could facilitate routine patient diagnostics. Herein, we describe a microdevice based electrochemical assay for the detection of locus-specific DNA methylation at single CpG dinucleotide resolution after bisulfite conversion of a target DNA sequence. This is achieved by using the ligase chain reaction (LCR) to recognize and amplify a C to T base change at a CpG site and measuring the change electrochemically (eLCR). Unlike other electrochemical detection methods for DNA methylation, methylation specific (MS)-eLCR can potentially interrogate any CpG of interest in the genome. MS-eLCR also distinguishes itself from other electrochemical LCR detection schemes by integrating a peroxidase-mimicking DNAzyme sequence into the LCR amplification probes design which in turn, serves as a redox moiety when bound with a hemin cofactor. Following hybridization to surface-bound capture probes, the DNAzyme-linked LCR products induce electrocatalytic responses that are proportional to the methylation levels of the gene locus in the presence of hydrogen peroxide. The performance of the assay was evaluated by simultaneously detecting C to T changes at a locus associated with cancer metastasis in breast cancer cell lines and serum-derived samples. MS-eLCR required as little as 0.04 pM of starting material and was sensitive to 10-15% methylation change with good reproducibility (RSD=7.9%, n=3). Most importantly, the accuracy of the method in quantifying locus-specific methylation was comparable to both fluorescence-based and Next Generation Sequencing approaches. We thus believe that the proposed assay could potentially be a low cost alternative to current technologies for DNA methylation detection of specific CpG sites. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Specific Heat of High Temperature Superconductors: a Review

    NASA Astrophysics Data System (ADS)

    Junod, Alain

    The following sections are included: * INTRODUCTION * EXPERIMENTAL * LATTICE SPECIFIC HEAT * NORMAL-STATE ELECTRONIC SPECIFIC HEAT * SUPERCONDUCTING STATE * BEHAVIOR AT T→0 * CONCLUSION * ACKNOWLEDGEMENTS * APPENDIX * REFERENCES

  10. [Sensitivity and specificity of the ELISA Kit for the detection of antidobies to Junin virus].

    PubMed

    Pirozhkov, A P; Timofeev, M A; Borisevich, I V; Syromiatnikova, S I; Shatokhina, I V; Pantyukhov, V B; Kovalchuk, A V; Borisevich, S V

    2015-01-01

    The goal of this work was to describe methodological approaches to determination of sensitivity and specificity of the enzyme-linked immunosorbent assay kit (ELISA Kit) for detection of the specific anti-Junin virus (JV) antibody. Comparison of ELISA to plaque reduction neutralization test (PRNT) showed direct relationship between antibody titers in the samples of serum of immunized animals, determined by either PRNT or ELISA methods. The obtained results provided an opportunity to form the panels of positive and negative serum samples to determine the sensitivity and specificity of the ELISA Kit. Sensitivity of the ELISA Kit was at least 98% when studying the samples of serum of immunized guinea pigs and rabbits (determined as positive in PRNT). The sensitivity of the ELISA Kit was at least 68% when studying the samples determined by PNRT as uncertain positive. The specificity was 98%. The specificity of the ELISA Kit was 98%.

  11. Glaucoma progression detection: agreement, sensitivity, and specificity of expert visual field evaluation, event analysis, and trend analysis.

    PubMed

    Antón, Alfonso; Pazos, Marta; Martín, Belén; Navero, José Manuel; Ayala, Miriam Eleonora; Castany, Marta; Martínez, Patricia; Bardavío, Javier

    2013-01-01

    To assess sensitivity, specificity, and agreement among automated event analysis, automated trend analysis, and expert evaluation to detect glaucoma progression. This was a prospective study that included 37 eyes with a follow-up of 36 months. All had glaucomatous disks and fields and performed reliable visual fields every 6 months. Each series of fields was assessed with 3 different methods: subjective assessment by 2 independent teams of glaucoma experts, glaucoma/guided progression analysis (GPA) event analysis, and GPA (visual field index-based) trend analysis. Kappa agreement coefficient between methods and sensitivity and specificity for each method using expert opinion as gold standard were calculated. The incidence of glaucoma progression was 16% to 18% in 3 years but only 3 cases showed progression with all 3 methods. Kappa agreement coefficient was high (k=0.82) between subjective expert assessment and GPA event analysis, and only moderate between these two and GPA trend analysis (k=0.57). Sensitivity and specificity for GPA event and GPA trend analysis were 71% and 96%, and 57% and 93%, respectively. The 3 methods detected similar numbers of progressing cases. The GPA event analysis and expert subjective assessment showed high agreement between them and moderate agreement with GPA trend analysis. In a period of 3 years, both methods of GPA analysis offered high specificity, e