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Sample records for resolution melting application

  1. High Resolution Melting Applications for Clinical Laboratory Medicine

    PubMed Central

    Erali, Maria; Voelkerding, Karl V.; Wittwer, Carl T.

    2008-01-01

    Separation of the two strands of DNA with heat (melting) is a fundamental property of DNA that is conveniently monitored with fluorescence. Conventional melting is performed after PCR on any real-time instrument to monitor product purity (dsDNA dyes) and sequence (hybridization probes). Recent advances include high-resolution instruments and saturating DNA dyes that distinguish many different species. For example, mutation scanning (identifying heterozygotes) by melting is closed-tube and has similar or superior sensitivity and specificity compared to methods that require physical separation. With high resolution melting, SNPs can be genotyped without probes and more complex regions can be typed with unlabeled hybridization probes. Highly polymorphic HLA loci can be melted to establish sequence identity for transplantation matching. Simultaneous genotyping with one or more unlabeled probes and mutation scanning of the entire amplicon can be performed at the same time in the same tube, vastly decreasing or eliminating the need for re-sequencing in genetic analysis. High-resolution PCR product melting is homogeneous, closed-tube, rapid (1–5 min), non-destructive and does not require covalently-labeled fluorescent probes. In the clinical laboratory, it is an ideal format for in-house testing, with minimal cost and time requirements for new assay development. PMID:18502416

  2. Application of high-resolution melting analysis for differentiation of spoilage yeasts.

    PubMed

    Erdem, Mine; Kesmen, Zülal; Özbekar, Esra; Çetin, Bülent; Yetim, Hasan

    2016-09-01

    A new method based on high resolution melting (HRM) analysis was developed for the differentiation and classification of the yeast species that cause food spoilage. A total 134 strains belonging to 21 different yeast species were examined to evaluate the discriminative power of HRM analysis. Two different highly variable DNA regions on the 26 rRNA gene were targeted to produce the HRM profiles of each strain. HRM-based grouping was compared and confirmed by (GTG)5 rep-PCR fingerprinting analysis. All of the yeast species belonging to the genera Pichia, Candida, Kazachstania, Kluyveromyces, Debaryomyces, Dekkera, Saccharomyces, Torulaspora, Ustilago, and Yarrowia, which were produced as species-specific HRM profiles, allowed discrimination at species and/or strain level. The HRM analysis of both target regions provided successful discrimination that correlated with rep-PCR fingerprinting analysis. Consequently, the HRM analysis has the potential for use in the rapid and accurate classification and typing of yeast species isolated from different foods to determine their sources and routes as well as to prevent contamination. PMID:27572511

  3. High Resolution Melting (HRM) applied to wine authenticity.

    PubMed

    Pereira, Leonor; Gomes, Sónia; Castro, Cláudia; Eiras-Dias, José Eduardo; Brazão, João; Graça, António; Fernandes, José R; Martins-Lopes, Paula

    2017-02-01

    Wine authenticity methods are in increasing demand mainly in Denomination of Origin designations. The DNA-based methodologies are a reliable means of tracking food/wine varietal composition. The main aim of this work was the study of High Resolution Melting (HRM) application as a screening method for must and wine authenticity. Three sample types (leaf, must and wine) were used to validate the three developed HRM assays (Vv1-705bp; Vv2-375bp; and Vv3-119bp). The Vv1 HRM assay was only successful when applied to leaf and must samples. The Vv2 HRM assay successfully amplified all sample types, allowing genotype discrimination based on melting temperature values. The smallest amplicon, Vv3, produced a coincident melting curve shape in all sample types (leaf and wine) with corresponding genotypes. This study presents sensitive, rapid and efficient HRM assays applied for the first time to wine samples suitable for wine authenticity purposes. PMID:27596395

  4. High-Resolution DNA Melting Analysis in Plant Research.

    PubMed

    Simko, Ivan

    2016-06-01

    Genetic and genomic studies provide valuable insight into the inheritance, structure, organization, and function of genes. The knowledge gained from the analysis of plant genes is beneficial to all aspects of plant research, including crop improvement. New methods and tools are continually being developed to facilitate rapid and accurate mapping, sequencing, and analyzing of genes. Here, I review the recent progress in the application of high-resolution melting (HRM) analysis of DNA, a method that allows detecting polymorphism in double-stranded DNA by comparing profiles of melting curves. Use of HRM has expanded considerably in the past few years as the method was successfully applied for high-throughput genotyping, mapping genes, testing food products and seeds, and other areas of plant research. PMID:26827247

  5. Ribo HRM--detection of inter- and intra-species polymorphisms within ribosomal DNA by high resolution melting analysis supported by application of artificial allelic standards.

    PubMed

    Masny, Aleksander; Jagiełło, Agata; Płucienniczak, Grażyna; Golab, Elzbieta

    2012-09-01

    Ribo HRM, a single-tube PCR and high resolution melting (HRM) assay for detection of polymorphisms in the large subunit ribosomal DNA expansion segment V, was developed on a Trichinella model. Four Trichinella species: T. spiralis (isolates ISS3 and ISS160), T. nativa (isolates ISS10 and ISS70), T. britovi (isolates ISS2 and ISS392) and T. pseudospiralis (isolates ISS13 and ISS1348) were genotyped. Cloned allelic variants of the expansion segment V were used as standards to prepare reference HRM curves characteristic for single sequences and mixtures of several cloned sequences imitating allelic composition detected in Trichinella isolates. Using the primer pair Tsr1 and Trich1bi, it was possible to amplify a fragment of the ESV and detect PCR products obtained from the genomic DNA of pools of larvae belonging to the four investigated species: T. pseudospiralis, T. spiralis, T. britovi and T. nativa, in a single tube Real-Time PCR reaction. Differences in the shape of the HRM curves of Trichinella isolates suggested the presence of differences between examined isolates of T. nativa, T. britovi and T. pseudospiralis species. No differences were observed between T. spiralis isolates. The presence of polymorphisms within the amplified ESV sequence fragment of T. nativa T. britovi and T. pseudospiralis was confirmed by sequencing of the cloned PCR products. Novel sequences were discovered and deposited in GenBank (GenBank IDs: JN971020-JN971027, JN120902.1, JN120903.1, JN120904.1, JN120906.1, JN120905.1). Screening the ESV region of Trichinella for polymorphism is possible using the genotyping assay Ribo HRM at the current state of its development. The Ribo HRM assay could be useful in phylogenetic studies of the Trichinella genus. PMID:22766326

  6. High resolution melting analysis for the differentiation of Mycobacterium species.

    PubMed

    Issa, Rahizan; Abdul, Hatijah; Hashim, Siti Hasmah; Seradja, Valentinus H; Shaili, Nurul 'Aishah; Hassan, Nurul Akma Mohd

    2014-10-01

    A quantitative real-time PCR (qPCR) followed by high resolution melting (HRM) analysis was developed for the differentiation of Mycobacterium species. Rapid differentiation of Mycobacterium species is necessary for the effective diagnosis and management of tuberculosis. In this study, the 16S rRNA gene was tested as the target since this has been identified as a suitable target for the identification of mycobacteria species. During the temperature gradient and primer optimization process, the melting peak (Tm) analysis was determined at a concentration of 50 ng DNA template and 0.3, 0.4 and 0.5 µM primer. The qPCR assay for the detection of other mycobacterial species was done at the Tm and primer concentration of 62 °C and 0.4 µM, respectively. The HRM analysis generated cluster patterns that were specific and sensitive to distinguished small sequence differences of the Mycobacterium species. This study suggests that the 16S rRNA-based real-time PCR followed by HRM analysis produced unique cluster patterns for species of Mycobacterium and could differentiate the closely related mycobacteria species. PMID:25038139

  7. Mutation Scanning and Genotyping in Plants by High Resolution DNA Melting

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High-resolution melting analysis after PCR allows closed-tube mutation scanning and genotyping without processing, labeled probes, real-time monitoring or allele-specific amplification. PCR is performed in the presence of the saturating dye, LCGreen® Plus, with subsequent high-resolution melting ana...

  8. Consequences and Resolution of Lunar Lower Mantle Partial Melt

    NASA Astrophysics Data System (ADS)

    Fuqua, H.; Bremner, P. M.; Diamond, M. R.; Garapic, G.; Lock, S. J.; Mallik, A.; Nishikawa, Y.; Panovska, S.; Shahar, A.; Lognonne, P. H.; Panero, W. R.; Faul, U.; Panning, M. P.; Jimenez-Perez, H.; Schmerr, N. C.; Williams, Q. C.

    2014-12-01

    Existence of a partially molten layer at depth has been proposed to explain the lack of observed farside deep moonquakes, the observation of reflected phases from deep moonquakes, and the dissipation of tidal energy within the lunar interior. However, subsequent models explore the possibility that dissipation due to elevated temperatures alone can explain the observed dissipation factor (Q) and tidal love numbers. We have explored the hypothesis that high titanium melt compositions associated with lunar mantle overturn may sink to the base of the mantle, locally or regionally. We have performed forward calculations varying composition and thickness of layers to evaluate if a partially molten layer at the base of the mantle is well constrained by the observational data. Self-consistent physical parameters are calculated for each compositional model that are then compared against the observed data to determine a subset of permissible models. The data constraints considered by this study include bulk density, moment of inertia, real and imaginary parts of the Love numbers, seismic travel times, and electrical conductivity. Dynamic calculations using ASPECT have also been considered to determine the implications of early lunar mantle convection for the survivability of the partially molten layer. Finally, and as a perspective for a future NASA New Frontiers Geophysical Network, we present 1D synthetic seismograms calculated for each proposed structure of the Moon to investigate the future seismological resolution of these deep lunar structure features. This work was initiated at the CIDER 2014 program.

  9. Y-STR genetic screening by high-resolution melting analysis.

    PubMed

    Deng, J Q; Liu, B Q; Wang, Y; Liu, W; Cai, J F; Long, R; Li, W H

    2016-01-01

    Currently, the widely used automated capillary electrophoresis-based short tandem repeat (STR) genotyping method for genetic screening in forensic practice is laborious, time-consuming, expensive, and technically challenging in some cases. Thus, new molecular-based strategies for conclusively identifying forensically relevant biological evidence are required. Here, we used high-resolution melting analysis (HRM) for Y-chromosome STR genotyping for forensic genetic screening. The reproducibility of the melting profile over dilution, sensitivity, discrimination power, and other factors was preliminarily studied in 10 Y-STR loci. The results showed that HRM-based approaches revealed more genotypes (compared to capillary electrophoresis), showed higher uniformity in replicate tests and diluted samples, and enabled successful detection of DNA at concentrations as low as 0.25 ng. For mixed samples, the melting curve profiles discriminated between mixed samples based on reference samples with high efficiency. The triplex Y-chromosome STR HRM assay was performed and provided a foundation for further studies such as a multiplex HRM assay. The HRM approach is a one-step application and the entire procedure can be completed within 2 h at a low cost. In conclusion, our findings demonstrate that the HRM-based Y-STR assay is a useful screening tool that can be used in forensic practice. PMID:26909950

  10. Kinetics of Melting and Applications to Chondrules

    NASA Astrophysics Data System (ADS)

    Greenwood, James Paul

    1997-12-01

    The congruent melting kinetics of Amelia albite were experimentally determined at 1125o C,/ 1150o C,/ 1175o C, and 1200o C. It was determined that congruent melting is a heterogeneous process. Melting is initiated at external surfaces and cleavage planes. Melting kinetics of albite are best described using a normal growth model. Congruent melting of albite was found to be interface controlled, and rates of melting are directly proportional to the amount of superheat, and inversely proportional to viscosity. Comparison of the results obtained here with previous studies of melting kinetics on other materials (oxides and silicates) finds that the normal growth model can be used to predict melting rates within an order of magnitude. The normal growth model was used to predict congruent melting rates of forsterite and enstatite as well as other minerals which may have been present in the chondrule forming region of the solar nebula. Constraints on the peak temperatures of chondrule formation are thus obtained. Specifically, chondrules containing relict grains of forsteritic olivine and enstatitic pyroxene could not have been heated above 1901o C and 1577o C, respectively, for more than a few seconds. Reanalyses of Na-Al-rich chondrule glasses by EPMA have found that previous EPMA work resulted in loss of Na from the activated volume due to migration in an electrical potential gradient. The Na-Al-rich chondrules have Na/Al ratios of unity, suggesting that they did not lose alkalis during flash heating. Experiments reproduced the chondrule glasses and determined the formational constraints of these chondrules. Specifically, the chondrules needed to have been cooled at low rates (<6o C/hr) at the lower temperature end of chondrule formation.

  11. High resolution melting analysis as a new approach to discriminate gluten-containing cereals.

    PubMed

    Martín-Fernández, Begoña; Costa, Joana; de-Los-Santos-Álvarez, Noemí; López-Ruiz, Beatriz; Oliveira, M Beatriz P P; Mafra, Isabel

    2016-11-15

    With this work, it is intended to propose a novel approach based on high resolution melting (HRM) analysis to detect wheat and discriminate it from other gluten-containing cereals. The method consisted of a real-time PCR assay targeting the gene encoding for the germ agglutinin isolectin A protein (Tri a 18 allergen), using the fluorescent Evagreen dye combined with HRM analysis. The results enabled wheat differentiation from other phylogenetically related cereals, namely barley, rye and oat with high level of confidence. Additionally, a quantitative real-time PCR approach was proposed, allowing detecting and quantifying wheat down to 20mg/kg in rice flour and 20pg of wheat DNA (∼1.1 DNA copies). Its application was successfully achieved in the analysis of processed foods to verify labelling compliance, being considered as a cost-effective tool for the specific detection of cereals in gluten-free foods. PMID:27283646

  12. Silicon purification melting for photovoltaic applications

    SciTech Connect

    VAN DEN AVYLE,JAMES A.; HO,PAULINE; GEE,JAMES M.

    2000-04-01

    The availability of polysilicon feedstock has become a major issue for the photovoltaic (PV) industry in recent years. Most of the current polysilicon feedstock is derived from rejected material from the semiconductor industry. However, the reject material can become scarce and more expensive during periods of expansion in the integrated-circuit industry. Continued rapid expansion of the PV crystalline-silicon industry will eventually require a dedicated supply of polysilicon feedstock to produce solar cells at lower costs. The photovoltaic industry can accept a lower purity polysilicon feedstock (solar-grade) compared to the semiconductor industry. The purity requirements and potential production techniques for solar-grade polysilicon have been reviewed. One interesting process from previous research involves reactive gas blowing of the molten silicon charge. As an example, Dosaj et all reported a reduction of metal and boron impurities from silicon melts using reactive gas blowing with 0{sub 2} and Cl{sub 2}. The same authors later reassessed their data and the literature, and concluded that Cl{sub 2}and 0{sub 2}/Cl{sub 2} gas blowing are only effective for removing Al, Ca, and Mg from the silicon melt. Researchers from Kawasaki Steel Corp. reported removal of B and C from silicon melts using reactive gas blowing with an 0{sub 2}/Ar plasma torch. Processes that purify the silicon melt are believed to be potentially much lower cost compared to present production methods that purify gas species.

  13. Simple models for disequilibrium fractional melting and batch melting with application to REE fractionation in abyssal peridotites

    NASA Astrophysics Data System (ADS)

    Liang, Yan; Liu, Boda

    2016-01-01

    Disequilibrium melting arises when the kinetics of chemical exchange between a residual mineral and partial melt is sluggish compare to the rate of melting. To better understand the role of a finite crystal-melt exchange rate on trace element fractionation during mantle melting, we have developed a disequilibrium melting model for partial melting in an upwelling steady-state column. We use linear kinetics to approximate crystal-melt mass exchange rate and obtain simple analytical solutions for cases of perfect fractional melting and batch melting. A key parameter determining the extent of chemical disequilibrium during partial melting is an element specific dimensionless ratio (ε) defined as the melting rate relative to the solid-melt chemical exchange rate for the trace element of interest. In the case of diffusion in mineral limited chemical exchange, ε is inversely proportional to diffusivity of the element of interest. Disequilibrium melting is important for the trace element when ε is comparable to or greater than the bulk solid-melt partition coefficient for the trace element (k). The disequilibrium fractional melting model is reduced to the equilibrium perfect fractional melting model when ε is much smaller than k. Hence highly incompatible trace elements with smaller mobilities in minerals are more susceptible to disequilibrium melting than moderately incompatible and compatible trace elements. Effect of chemical disequilibrium is to hinder the extent of fractionation between residual solid and partial melt, making the residual solid less depleted and the accumulated melt more depleted in incompatible trace element abundances relative the case of equilibrium melting. Application of the disequilibrium fractional melting model to REE and Y abundances in clinopyroxene in abyssal peridotites from the Central Indian Ridge and the Vema Lithospheric Section, Mid-Atlantic Ridge revealed a positive correlation between the disequilibrium parameter ε and the

  14. Differentiating between monozygotic twins through DNA methylation-specific high-resolution melt curve analysis.

    PubMed

    Stewart, Leander; Evans, Neil; Bexon, Kimberley J; van der Meer, Dieudonne J; Williams, Graham A

    2015-05-01

    Although short tandem repeat profiling is extremely powerful in identifying individuals from crime scene stains, it is unable to differentiate between monozygotic (MZ) twins. Efforts to address this include mutation analysis through whole genome sequencing and through DNA methylation studies. Methylation of DNA is affected by environmental factors; thus, as MZ twins age, their DNA methylation patterns change. This can be characterized by bisulfite treatment followed by pyrosequencing. However, this can be time-consuming and expensive; thus, it is unlikely to be widely used by investigators. If the sequences are different, then in theory the melting temperature should be different. Thus, the aim of this study was to assess whether high-resolution melt curve analysis can be used to differentiate between MZ twins. Five sets of MZ twins provided buccal swabs that underwent extraction, quantification, bisulfite treatment, polymerase chain reaction amplification and high-resolution melting curve analysis targeting two markers, Alu-E2F3 and Alu-SP. Significant differences were observed between all MZ twins targeting Alu-E2F3 and in four of five MZ twins targeting Alu-SP (P<0.05). Thus, it has been demonstrated that bisulfite treatment followed by high-resolution melting curve analysis could be used to differentiate between MZ twins. PMID:25677265

  15. High resolution melting (HRM) analysis of DNA--its role and potential in food analysis.

    PubMed

    Druml, Barbara; Cichna-Markl, Margit

    2014-09-01

    DNA based methods play an increasing role in food safety control and food adulteration detection. Recent papers show that high resolution melting (HRM) analysis is an interesting approach. It involves amplification of the target of interest in the presence of a saturation dye by the polymerase chain reaction (PCR) and subsequent melting of the amplicons by gradually increasing the temperature. Since the melting profile depends on the GC content, length, sequence and strand complementarity of the product, HRM analysis is highly suitable for the detection of single-base variants and small insertions or deletions. The review gives an introduction into HRM analysis, covers important aspects in the development of an HRM analysis method and describes how HRM data are analysed and interpreted. Then we discuss the potential of HRM analysis based methods in food analysis, i.e. for the identification of closely related species and cultivars and the identification of pathogenic microorganisms. PMID:24731338

  16. Submarine Melting of Icebergs from Repeat High-Resolution Digital Elevation Models

    NASA Astrophysics Data System (ADS)

    Enderlin, E. M.; Hamilton, G. S.; Straneo, F.; Cenedese, C.

    2014-12-01

    Icebergs calved from tidewater glaciers act as distributed freshwater sources as they transit through fjords to the surrounding ocean basins. Glacier discharge estimates provide a crude approximation of the total iceberg discharge on inter-annual timescales, but the liquid freshwater flux from icebergs in glacial fjords is largely unknown. Here we use repeat high-resolution digital elevation models (DEMs) to derive meltwater fluxes for 18 icebergs in Sermilik Fjord, East Greenland, during the 2011-2013 boreal summers, and for 33 comparably-sized icebergs in Ilulissat Fjord, West Greenland, during March-April 2011 and July 2012. We find that iceberg melt rates for Sermilik Fjord are in good agreement with simulated melt rates along the vertical terminus of Helheim Glacier in winter, i.e. when melting at the glacier front is not enhanced by subglacial discharge, providing an independent validation of our technique. Variations in meltwater fluxes from icebergs are primarily related to differences in the submerged area of individual icebergs, which is consistent with theory. The stratification of water masses in fjords has a noticeable effect on summertime-derived melt estimates, with lower melt rates (and meltwater fluxes) observed in the relatively cold and fresh Polar Water layer and higher melt rates in the underlying warmer and more saline Atlantic Water layer. The meltwater flux dependence on submerged area, particularly within the deeper Atlantic Water layer, suggests that changes in the characteristics of icebergs (size/shape/keel-depth) calved from a tidewater glacier will alter the magnitude and distribution of meltwater fluxes within the fjord, which may in turn influence fjord circulation and the heat content delivered to the glacier terminus.

  17. Single-melt beta C for spring and fastener applications

    NASA Astrophysics Data System (ADS)

    Yu, K. O.; Crist, E. M.; Pesa, R.; Cecchini, N.; Bugle, C. M.

    2005-12-01

    Beta C is a high-strength metastable titanium alloy widely used for spring and fastener applications. Input stock of these springs and fasteners is currently made by rolling billets forged from conventional double-melt VAR (2×VAR) ingots. Recent advances in plasma arc melting (PAM) single-melt technology offer the potential to reduce the input stock cost by directly rolling as-cast near-net shape PAM ingots instead of the conventional forged billets. A 127 mm (5 in.) diameter as-cast Beta C PAM ingot was rolled into 15 mm (0.6 in.) bars for springs and fasteners. Microstructures and mechanical properties of the bar product are evaluated in this paper.

  18. Evaluation of High Resolution Melting for MTHFR C677T Genotyping in Congenital Heart Disease

    PubMed Central

    Yue, Shuying; Zhang, Kun; Wang, Hui; Dong, Rui; Yang, Xiaomeng; Liu, Yi; Ma, Yanhui

    2016-01-01

    Background High resolution melting (HRM) is a simple, flexible and low-cost mutation screening technique. The methylenetetrahydrofolate reductase (MTHFR) gene encoding a critical enzyme, potentially affects susceptibility to some congenital defects like congenital heart disease (CHD). We evaluate the performance of HRM for genotyping of the MTHFR gene C677T locus in CHD cases and healthy controls of Chinese Han population. Methods A total of 315 blood samples from 147 CHD patients (male72, female 75) and 168 healthy controls (male 92, female 76) were enrolled in the study. HRM was utilized to genotype MTHFR C677T locus of all the samples. The results were compared to that of PCR-RFLP and Sanger sequencing. The association of the MTHFR C677T genotypes and the risk of CHD was analyzed using odds ratio with their 95% confidence interval (CIs) from unconditional logistic regression. Results All the samples were successfully genotyped by HRM within 1 hour and 30 minutes while at least 6 hours were needed for PCR-RFLP and sequencing. The genotypes of MTHFR C677T CC, CT, and TT were 9.52%, 49.66%, and 40.82% in CHD group but 29.17%, 50% and 20.83% in control group, which were identical using both methods of HRM and PCR-RFLP, demonstrating the sensitivity and specificity of HRM were all 100%. Conclusion MTHFR C677T is a potential risk factor for CHD in our local residents of Shandong province in China. HRM is a fast, sensitive, specific and reliable method for clinical application of genotyping. PMID:26990189

  19. High-Resolution Melting Analysis of MED12 Mutations in Uterine Leiomyomas in Chinese Patients

    PubMed Central

    Wang, Hua; Qian, Hua; Zhou, Ruifang; Jiang, Jun; Ye, Lihua

    2015-01-01

    Objectives: Somatic mutations in mediator complex subunit 12 (MED12) have emerged as a critical genetic change in the development of uterine leiomyomas. Studies, however, have focused largely on cohorts consisting of Caucasian patients. In this study, uterine leiomyomas from Chinese patients were examined for MED12 mutations. In addition, polymerase chain reaction (PCR)-based high-resolution melting analysis (HRMA) was compared with direct sequencing as a potentially more sensitive method for the detection of MED12 mutations. Methods: Tissue samples with the pathologies of uterine leiomyoma (n=181) and other endometrial diseases (n=157) were collected from Chinese patients at the Taizhou People's Hospital and Taizhou Polytechnic College (Taizhou City, China). Genomic DNA was prepared from all samples. Both PCR-based HRMA and PCR-based direct sequencing were used to detect MED12 mutations. Results: PCR-based HRMA and direct sequencing revealed MED12 mutations in 95/181 (52.5%) and 93/181 (51.4%) uterine leiomyomas, respectively. Nearly half of these mutations (46/93) were found in a single codon, codon 131. The coincidence rate between the two methods was 98.9% (179/181) so that no statistically significant difference was evident in the application of the methodologies (χ2=0.011, p=0.916). In addition, MED12 mutations were identified in 1/157 (4.17%) case of other endometrial pathologies by both methods. Conclusions: MED12 mutations were closely associated with the development of uterine leiomyomas, as opposed to other uterine pathologies in Chinese patients, and PCR-based HRMA was found to be a reliable method for the detection of MED12 mutations. PMID:25615570

  20. Automated Classification and Cluster Visualization of Genotypes Derived from High Resolution Melt Curves

    PubMed Central

    Kanderian, Sami; Jiang, Lingxia; Knight, Ivor

    2015-01-01

    Introduction High Resolution Melting (HRM) following PCR has been used to identify DNA genotypes. Fluorescent dyes bounded to double strand DNA lose their fluorescence with increasing temperature, yielding different signatures for different genotypes. Recent software tools have been made available to aid in the distinction of different genotypes, but they are not fully automated, used only for research purposes, or require some level of interaction or confirmation from an analyst. Materials and Methods We describe a fully automated machine learning software algorithm that classifies unknown genotypes. Dynamic melt curves are transformed to multidimensional clusters of points whereby a training set is used to establish the distribution of genotype clusters. Subsequently, probabilistic and statistical methods were used to classify the genotypes of unknown DNA samples on 4 different assays (40 VKORC1, CYP2C9*2, CYP2C9*3 samples in triplicate, and 49 MTHFR c.665C>T samples in triplicate) run on the Roche LC480. Melt curves of each of the triplicates were genotyped separately. Results Automated genotyping called 100% of VKORC1, CYP2C9*3 and MTHFR c.665C>T samples correctly. 97.5% of CYP2C9*2 melt curves were genotyped correctly with the remaining 2.5% given a no call due to the inability to decipher 3 melt curves in close proximity as either homozygous mutant or wild-type with greater than 99.5% posterior probability. Conclusions We demonstrate the ability to fully automate DNA genotyping from HRM curves systematically and accurately without requiring any user interpretation or interaction with the data. Visualization of genotype clusters and quantification of the expected misclassification rate is also available to provide feedback to assay scientists and engineers as changes are made to the assay or instrument. PMID:26605797

  1. Probing the atomic structure of basaltic melts generated by partial melting of upper mantle peridotite (KLB-1): Insights from high-resolution solid-state NMR study

    NASA Astrophysics Data System (ADS)

    Park, S. Y.; Lee, S. K.

    2015-12-01

    Probing the structural disorder in multi-component silicate glasses and melts with varying composition is essential to reveal the change of macroscopic properties in natural silicate melts. While a number of NMR studies for the structure of multi-component silicate glasses and melts including basaltic and andesitic glasses have been reported (e.g., Park and Lee, Geochim. Cosmochim. Acta, 2012, 80, 125; Park and Lee, Geochim. Cosmochim. Acta, 2014, 26, 42), many challenges still remain. The composition of multi-component basaltic melts vary with temperature, pressure, and melt fraction (Kushiro, Annu. Rev. Earth Planet. Sci., 2001, 71, 107). Especially, the eutectic point (the composition of first melt) of nepheline-forsterite-quartz (the simplest model of basaltic melts) moves with pressure from silica-saturated to highly undersaturated and alkaline melts. The composition of basaltic melts generated by partial melting of upper mantle peridotite (KLB-1, the xenolith from Kilbourne Hole) also vary with pressure. In this study we report experimental results for the effects of composition on the atomic structure of Na2O-MgO-Al2O3-SiO2 (NMAS) glasses in nepheline (NaAlSiO4)-forsterite (Mg2SiO4)-quartz (SiO2) eutectic composition and basaltic glasses generated by partial melting of upper mantle peridotite (KLB-1) using high-resolution multi-nuclear solid-state NMR. The Al-27 3QMAS (triple quantum magic angle spinning) NMR spectra of NMAS glasses in nepheline-forsterite-quartz eutectic composition show only [4]Al. The Al-27 3QMAS NMR spectra of KLB-1 basaltic glasses show mostly [4]Al and a non-negligible fraction of [5]Al. The fraction of [5]Al, the degree of configurational disorder, increases from 0 at XMgO [MgO/(MgO+Al2O3)]=0.55 to ~3% at XMgO=0.79 in KLB-1 basaltic glasses while only [4]Al are observed in nepheline-forsterite-quartz eutectic composition. The current experimental results provide that the fraction of [5]Al abruptly increases by the effect of

  2. Rapid and inexpensive species differentiation using a multiplex real-time polymerase chain reaction high-resolution melt assay.

    PubMed

    Elkins, Kelly M; Perez, Anjelica C U; Sweetin, Katherine C

    2016-05-01

    We demonstrate a method for developing real-time polymerase chain reaction (PCR) high-resolution melt (HRM) assays to identify multiple species present in a mixture simultaneously using LCGreen Plus and melt temperatures. Highly specific PCR primers are designed to yield amplicons with different melt temperatures for simple routine species identification compared with differentiating melt curve kinetics traces or difference plots. This method is robust and automatable, and it leads to savings in time and reagent costs, is easily modified to probe any species of interest, eliminates the need for post-PCR gel or capillary electrophoresis in routine assays, and requires no expensive dye-labeled primers. PMID:26836486

  3. Identification case of evidence in timber tracing of Pinus radiate, using high-resolution melting (HRM) analysis.

    PubMed

    Solano, Jaime; Anabalón, Leonardo; Encina, Francisco

    2016-03-01

    Fast, accurate detection of plant species and their hybrids using molecular tools will facilitate assessment and monitoring of timber tracing evidence. In this study the origin of unknown pine samples is determined for a case of timber theft in the region of Araucania southern Chile. We evaluate the utility of the trnL marker region for species identification applied to pine wood based on High Resolution Melting. This efficient tracing methods can be incorporated into forestry applications such as certification of origin. The object of this work was genotype identification using high-resolution melting (HRM) and trnL approaches for Pinus radiata (Don) in timber tracing evidence. Our results indicate that trnL is a very sensitive marker for delimiting species and HRM analysis was used successfully for genotyping Pinus samples for timber tracing purposes. Genotyping samples by HRM analysis with the trnL1 approach allowed us to differentiate two wood samples from the Pinaceae family: Pinus radiata (Don) and Pseudotsuga menziesii (Mirb.) Franco. The same approach with Pinus trnL wood was not able to discriminate between samples of Pinus radiata, indicating that the samples were genetically indistinguishable, possibly because they have the same genotype at this locus. Timber tracing with HRM analysis is expected to contribute to future forest certification schemes, control of illegal trading, and molecular traceability of Pinus spp. PMID:26626827

  4. Improved Protocol for Rapid Identification of Certain Spa Types Using High Resolution Melting Curve Analysis

    PubMed Central

    Mayerhofer, Benjamin; Stöger, Anna; Pietzka, Ariane T.; Fernandez, Haizpea Lasa; Prewein, Bernhard; Sorschag, Sieglinde; Kunert, Renate; Allerberger, Franz; Ruppitsch, Werner

    2015-01-01

    Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital. PMID:25768007

  5. Improved protocol for rapid identification of certain spa types using high resolution melting curve analysis.

    PubMed

    Mayerhofer, Benjamin; Stöger, Anna; Pietzka, Ariane T; Fernandez, Haizpea Lasa; Prewein, Bernhard; Sorschag, Sieglinde; Kunert, Renate; Allerberger, Franz; Ruppitsch, Werner

    2015-01-01

    Methicillin-resistant Staphylococcus aureus is one of the most significant pathogens associated with health care. For efficient surveillance, control and outbreak investigation, S. aureus typing is essential. A high resolution melting curve analysis was developed and evaluated for rapid identification of the most frequent spa types found in an Austrian hospital consortium covering 2,435 beds. Among 557 methicillin-resistant Staphylococcus aureus isolates 38 different spa types were identified by sequence analysis of the hypervariable region X of the protein A gene (spa). Identification of spa types through their characteristic high resolution melting curve profiles was considerably improved by double spiking with genomic DNA from spa type t030 and spa type t003 and allowed unambiguous and fast identification of the ten most frequent spa types t001 (58%), t003 (12%), t190 (9%), t041 (5%), t022 (2%), t032 (2%), t008 (2%), t002 (1%), t5712 (1%) and t2203 (1%), representing 93% of all isolates within this hospital consortium. The performance of the assay was evaluated by testing samples with unknown spa types from the daily routine and by testing three different high resolution melting curve analysis real-time PCR instruments. The ten most frequent spa types were identified from all samples and on all instruments with 100% specificity and 100% sensitivity. Compared to classical spa typing by sequence analysis, this gene scanning assay is faster, cheaper and can be performed in a single closed tube assay format. Therefore it is an optimal screening tool to detect the most frequent endemic spa types and to exclude non-endemic spa types within a hospital. PMID:25768007

  6. Detection of Indel Mutations in Drosophila by High-Resolution Melt Analysis (HRMA).

    PubMed

    Housden, Benjamin E; Perrimon, Norbert

    2016-01-01

    Although CRISPR technology allows specific genome alterations to be created with relative ease, detection of these events can be problematic. For example, CRISPR-induced double-strand breaks are often repaired imprecisely to generate unpredictable short indel mutations. Detection of these events requires the use of molecular screening techniques such as endonuclease assays, restriction profiling, or high-resolution melt analysis (HRMA). Here, we provide detailed protocols for HRMA-based mutation screening in Drosophila and analysis of the resulting data using the online tool HRMAnalyzer. PMID:27587781

  7. Rapid Identification of Medically Important Candida Isolates Using High Resolution Melting Analysis

    PubMed Central

    Nemcova, Eva; Cernochova, Michaela; Ruzicka, Filip; Malisova, Barbora

    2015-01-01

    An increasing trend in non albicans infections and various susceptibility patterns to antifungal agents implies a requirement for the quick and reliable identification of a number of medically important Candida species. Real-time PCR followed by high resolution melting analysis (HRMA) was developed, tested on 25 reference Candida collection strains and validated on an additional 143 clinical isolates in this study. All reference strains and clinical isolates inconclusive when using phenotypic methods and/or HRMA were analysed using ITS2 sequencing. Considering reference and clinical strains together, 23 out of 27 Candida species could be clearly distinguished by HRMA, while the remaining 4 species were grouped in 2 pairs, when applying the mean Tm ± 3 SD values, the shape of the derivative melting curve (dMelt curve) and, in some cases, the normalized and temperature—shifted difference plot against C. krusei. HRMA as a simple, rapid and inexpensive tool was shown to be useful in identifying a wide spectrum of clinically important Candida species. It may complement the current clinical diagnostic approach based on commercially available biochemical kits. PMID:25689781

  8. First screening for Brachyspira hampsonii in Swiss pigs applying a new high resolution melting assay.

    PubMed

    Scherrer, Simone; Borgström, Anna; Frei, Daniel; Wittenbrink, Max M

    2016-09-25

    A new High Resolution Melting (HRM) assay was developed for the rapid detection of Brachyspira (B.) hampsonii. B. hampsonii occurs in different European countries, however, until today it has not been encountered in Switzerland. Four B. hampsonii reference strains were used to develop the HRM assay: B. hampsonii clade I ATCC BAA2463 and clade II ATCC BAA2464 strain, as well as two isolated strains P280/1 from the UK and the German isolate 5369-1x/12. A conserved region of the nox gene was used to design B. hampsonii-specific primers. The HRM melting curves for the four reference strains showed reproducible difference graphs with distinct differences between the four strains based on a slight variation between the four amplicon sequences. In addition, DNA from 22 B. hampsonii strains representing four genetic B. hampsonii groups was used to validate the method. Melting temperatures in the interval between 73.1 and 74°C were obtained for all B. hampsonii strains and allow differentiating B. hampsonii from other Brachyspira species. In total 897 Swiss porcine fecal Brachyspira isolates, cultured between 2009 and 2015, were analysed by the HRM protocol. B. hampsonii was not detected among these Swiss Brachyspira isolates. In conclusion, the rapid and low-cost HRM approach allows a sensitive and specific identification of B. hampsonii. PMID:27599925

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

    PubMed

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

    2014-12-01

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

  10. Genotyping of classical swine fever virus using high-resolution melt analysis.

    PubMed

    Titov, Ilya; Tsybanov, Sodnom; Malogolovkin, Alexander

    2015-11-01

    Discrimination between different field and vaccine strains of classical swine fever virus (CSFV) is crucial for meaningful disease diagnosis and epidemiological investigation. In this study, a rapid method for differentiating vaccine strains and outbreak CSFV isolates by combined RT-PCR and high-resolution melt (HRM) analysis has been developed. The assay is based on PCR amplification of short fragments from the most variable region of CSFVgene E2, followed by HRM analysis of amplicons. Real-Time PCR/HRM for CSFV detection and differentiation analysis has sensitivity comparable to RT-qPCR and genotyping resolution comparable to E2 nucleotide sequencing. This assay in one step enables rapid and sensitive identification and genotype discrimination of CSFV in field samples, and thus will be valuable for CSF outbreak response and disease control. PMID:26300371

  11. Use of a New High Resolution Melting Method for Genotyping Pathogenic Leptospira spp.

    PubMed Central

    Naze, Florence; Desvars, Amélie; Picardeau, Mathieu; Bourhy, Pascale; Michault, Alain

    2015-01-01

    Background Leptospirosis is a worldwide zoonosis that is endemic in tropical areas, such as Reunion Island. The species Leptospira interrogans is the primary agent in human infections, but other pathogenic species, such as L. kirschner and L. borgpetersenii, are also associated with human leptospirosis. Methods and Findings In this study, a melting curve analysis of the products that were amplified with the primer pairs lfb1 F/R and G1/G2 facilitated an accurate species classification of Leptospira reference strains. Next, we combined an unsupervised high resolution melting (HRM) method with a new statistical approach using primers to amplify a two variable-number tandem-repeat (VNTR) for typing at the subspecies level. The HRM analysis, which was performed with ScreenClust Software, enabled the identification of genotypes at the serovar level with high resolution power (Hunter-Gaston index 0.984). This method was also applied to Leptospira DNA from blood samples that were obtained from Reunion Island after 1998. We were able to identify a unique genotype that is identical to that of the L. interrogans serovars Copenhageni and Icterohaemorrhagiae, suggesting that this genotype is the major cause of leptospirosis on Reunion Island. Conclusions Our simple, rapid, and robust genotyping method enables the identification of Leptospira strains at the species and subspecies levels and supports the direct genotyping of Leptospira in biological samples without requiring cultures. PMID:26154161

  12. Automated Microfluidic Platform for Serial Polymerase Chain Reaction and High-Resolution Melting Analysis.

    PubMed

    Cao, Weidong; Bean, Brian; Corey, Scott; Coursey, Johnathan S; Hasson, Kenton C; Inoue, Hiroshi; Isano, Taisuke; Kanderian, Sami; Lane, Ben; Liang, Hongye; Murphy, Brian; Owen, Greg; Shinoda, Nobuhiko; Zeng, Shulin; Knight, Ivor T

    2016-06-01

    We report the development of an automated genetic analyzer for human sample testing based on microfluidic rapid polymerase chain reaction (PCR) with high-resolution melting analysis (HRMA). The integrated DNA microfluidic cartridge was used on a platform designed with a robotic pipettor system that works by sequentially picking up different test solutions from a 384-well plate, mixing them in the tips, and delivering mixed fluids to the DNA cartridge. A novel image feedback flow control system based on a Canon 5D Mark II digital camera was developed for controlling fluid movement through a complex microfluidic branching network without the use of valves. The same camera was used for measuring the high-resolution melt curve of DNA amplicons that were generated in the microfluidic chip. Owing to fast heating and cooling as well as sensitive temperature measurement in the microfluidic channels, the time frame for PCR and HRMA was dramatically reduced from hours to minutes. Preliminary testing results demonstrated that rapid serial PCR and HRMA are possible while still achieving high data quality that is suitable for human sample testing. PMID:25827436

  13. High resolution laser micro sintering / melting using q-switched and high brilliant laser radiation

    NASA Astrophysics Data System (ADS)

    Exner, H.; Streek, A.

    2015-03-01

    Since the discovery of selective laser sintering/melting, numerous modifications have been made to upgrade or customize this technology for industrial purposes. Laser micro sintering (LMS) is one of those modifications: Powders with particles in the range of a few micrometers are used to obtain products with highly resolved structures. Pulses of a q-switched laser had been considered necessary in order to generate sinter layers from the micrometer scaled metal powders. LMS has been applied with powders from metals as well as from ceramic and cermet feedstock's to generate micro parts. Recent technological progress and the application of high brilliant continuous laser radiation have now allowed an efficient laser sintering/melting of micrometer scaled metal powders. Thereby it is remarkable that thin sinter layers are generated using high continuous laser power. The principles of the process, the state of the art in LMS concerning its advantages and limitations and furthermore the latest results of the recent development of this technology will be presented. Laser Micro Sintering / Laser Micro Melting (LMM) offer a vision for a new dimension of additive fabrication of miniature and precise parts also with application potential in all engineering fields.

  14. DNA-based identification of Peucedanum ostruthium specimens and detection of common adulterants by high-resolution melting curve analysis.

    PubMed

    Schmiderer, Corinna; Ruzicka, Joana; Novak, Johannes

    2015-12-01

    Masterwort (Peucedanum ostruthium, syn. Imperatoria ostruthium, Apiaceae) is an old economic plant in Alpine countries cultivated as ornamental plant and used for spirits and in folk medicine. P. ostruthium is a species that has often been confused with related Apiaceae species or morphologically similar roots or tubers resulting in products of minor quality. Masterwort can be distinguished from other Apiaceae species by nrDNA (ITS1 and ITS2). The analysed chloroplast markers (trnK 5' intron, trnT-trnL, and psbA-trnH), however, showed no species-specific mutations. With the application of two primer pairs amplifying parts of ITS and developed for high-resolution melting curve analysis (HRM) the target species was distinguishable from the other Peucedanum and Apiaceae species of our reference set. A multiplex PCR/HRM was developed to detect adulterations with Gentiana spp., Aconitum napellus and Veratrum album. PMID:26493217

  15. Review of selective laser melting: Materials and applications

    NASA Astrophysics Data System (ADS)

    Yap, C. Y.; Chua, C. K.; Dong, Z. L.; Liu, Z. H.; Zhang, D. Q.; Loh, L. E.; Sing, S. L.

    2015-12-01

    Selective Laser Melting (SLM) is a particular rapid prototyping, 3D printing, or Additive Manufacturing (AM) technique designed to use high power-density laser to melt and fuse metallic powders. A component is built by selectively melting and fusing powders within and between layers. The SLM technique is also commonly known as direct selective laser sintering, LaserCusing, and direct metal laser sintering, and this technique has been proven to produce near net-shape parts up to 99.9% relative density. This enables the process to build near full density functional parts and has viable economic benefits. Recent developments of fibre optics and high-power laser have also enabled SLM to process different metallic materials, such as copper, aluminium, and tungsten. Similarly, this has also opened up research opportunities in SLM of ceramic and composite materials. The review presents the SLM process and some of the common physical phenomena associated with this AM technology. It then focuses on the following areas: (a) applications of SLM materials and (b) mechanical properties of SLM parts achieved in research publications. The review is not meant to put a ceiling on the capabilities of the SLM process but to enable readers to have an overview on the material properties achieved by the SLM process so far. Trends in research of SLM are also elaborated in the last section.

  16. Review of selective laser melting: Materials and applications

    SciTech Connect

    Yap, C. Y.; Chua, C. K. Liu, Z. H. Zhang, D. Q. Loh, L. E. Sing, S. L.; Dong, Z. L.

    2015-12-15

    Selective Laser Melting (SLM) is a particular rapid prototyping, 3D printing, or Additive Manufacturing (AM) technique designed to use high power-density laser to melt and fuse metallic powders. A component is built by selectively melting and fusing powders within and between layers. The SLM technique is also commonly known as direct selective laser sintering, LaserCusing, and direct metal laser sintering, and this technique has been proven to produce near net-shape parts up to 99.9% relative density. This enables the process to build near full density functional parts and has viable economic benefits. Recent developments of fibre optics and high-power laser have also enabled SLM to process different metallic materials, such as copper, aluminium, and tungsten. Similarly, this has also opened up research opportunities in SLM of ceramic and composite materials. The review presents the SLM process and some of the common physical phenomena associated with this AM technology. It then focuses on the following areas: (a) applications of SLM materials and (b) mechanical properties of SLM parts achieved in research publications. The review is not meant to put a ceiling on the capabilities of the SLM process but to enable readers to have an overview on the material properties achieved by the SLM process so far. Trends in research of SLM are also elaborated in the last section.

  17. Rapid detection of fungal pathogens in bronchoalveolar lavage samples using panfungal PCR combined with high resolution melting analysis.

    PubMed

    Bezdicek, Matej; Lengerova, Martina; Ricna, Dita; Weinbergerova, Barbora; Kocmanova, Iva; Volfova, Pavlina; Drgona, Lubos; Poczova, Miroslava; Mayer, Jiri; Racil, Zdenek

    2016-10-01

    Despite advances in the treatment of invasive fungal diseases (IFD), mortality rates remain high. Moreover, due to the expanding spectrum of causative agents, fast and accurate pathogen identification is necessary. We designed a panfungal polymerase chain reaction (PCR), which targets the highly variable ITS2 region of rDNA genes and uses high resolution melting analysis (HRM) for subsequent species identification. The sensitivity and specificity of this method was tested on a broad spectrum of the most clinically important fungal pathogens including Aspergillus spp., Candida spp. and mucormycetes. Despite the fact that fluid from bronchoalveolar lavage (BAL) is one of the most frequently tested materials there is a lack of literature sources aimed at panfungal PCR as an IFD diagnostic tool from BAL samples. The applicability of this method in routine practice was evaluated on 104 BAL samples from immunocompromised patients. Due to high ITS region variability, we obtained divergent melting peaks for different fungal species. Thirteen out of 18 patients with proven or probable IFD were positive. Therefore, the sensitivity, specificity, positive predictive value and negative predictive value of our method were 67%, 100%, 100%, and 94%, respectively. In our assay, fungal pathogens identification is based on HRM, therefore omitting the expensive and time consuming sequencing step. With the high specificity, positive and negative predictive values, short time needed to obtain a result, and low price, the presented assay is intended to be used as a quick screening method for patients at risk of IFD. PMID:27161789

  18. Cancer mutation screening: Comparison of high-resolution melt analysis between two platforms.

    PubMed

    Ebili, Henry O; Ilyas, Mohammad

    2015-01-01

    High-resolution melt analysis (HRMA) is a cheap and reliable post-polymerase chain reaction (PCR) cancer mutation screening technique, which is fast gaining clinical relevance. The HRMA capabilities of the LightScanner (Idaho Technology) have been severally studied. However, the ABI 7500 HRM has not been tested against the purpose-built HRM instrument such as the LightScanner. DNA from formalin-fixed, paraffin-embedded gastric cancer, colorectal cancer, and normal tissue as well as from colorectal cancer cell lines were amplified at exons 2, 3, and 4 of KRAS, and at exons 11 and 15 of BRAF in the ABI 7500 fast real-time PCR machine and subjected to melting both on the ABI and on the LightScanner. HRMA data were analysed with the ABI HRM software v2.0.1 and the LightScanner Call-IT 2.5. We tested the ABI 7500 HRM for internal precision, accuracy, sensitivity, and specificity at mutation screening relative to the LightScanner, using crude percentage concordance, kappa statistics, and the area under the receiver operator characteristics (AUROC) curve on SPSS version 19. The results show that the ABI 7500 HRMA has a high internal precision, and excellent concordance, sensitivity, and specificity at mutation screening compared with the LightScanner. However, in contrast to the LightScanner HRM software analysis, the ABI HRM software v.2.0.1, cannot distinguish real from certain pseudovariations in PCR amplicons that are sometimes brought about by the artefacts of the melting process. In conclusion, the ABI HRM has a comparable performance level with the LightScanner, although in certain respects mentioned previously, the LightScanner has an edge over the ABI. PMID:25932046

  19. Exploring crystallization kinetics in natural rhyolitic melts using high resolution CT imagery of spherulites

    NASA Astrophysics Data System (ADS)

    Clow, T. W.; Befus, K. S.; Gardner, J. E.

    2014-12-01

    Little of our understanding of crystallization kinetics has been directly derived from studies of natural samples. We examine crystallization of rhyolitic melts by quantifying spherulite sizes and number densities in obsidian collected from Yellowstone caldera using high-resolution x-ray computed tomography (CT) imagery. Spherulites are spherical to ellipsoidal masses of intergrown alkali feldspar and quartz in a radiating, fibrous structure. They are thought to form in response to relatively rapid crystallization of melt in response to large amounts of undercooling. Recent research using compositional gradients that form outside of spherulites has suggested that they nucleate at 700 to 500 ˚C and their growth slows exponentially until it eventually ceases at ~400 ˚C. By quantifying spherulite textures, and using those temperature constraints, we derive new kinetic information regarding crystallization in natural rhyolitic systems. We find that spherulites range from 0.2 to 12.3 mm in diameter, and are 0.004 to 49.5 mm3 in volume. Such values generate number densities of 70 to 185 spherulites cm-3. Histograms of size display positively skewed distributions indicating small spherulites are far more abundant than larger ones. Those distributions imply nucleation rates change as a function of temperature. At higher temperatures where the melt is undercooled by 400-500 ˚C, nucleation is rare and growth is favored. With decreasing temperature, nucleation rates increase rapidly until cold enough temperatures are reached that diffusion limits crystallization and causes it to cease (undercoolings of ~650 ˚C). Assuming a cooling rate for the host obsidian of 10-5 ˚C s-1, then overall spherulite nucleation rates are 0.01 to 0.03 spherulites cm-3 hour-1.

  20. Sex Determination in Highly Fragmented Human DNA by High-Resolution Melting (HRM) Analysis

    PubMed Central

    Álvarez-Sandoval, Brenda A.; Manzanilla, Linda R.; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design. PMID:25098828

  1. Sex determination in highly fragmented human DNA by high-resolution melting (HRM) analysis.

    PubMed

    Álvarez-Sandoval, Brenda A; Manzanilla, Linda R; Montiel, Rafael

    2014-01-01

    Sex identification in ancient human remains is a common problem especially if the skeletons are sub-adult, incomplete or damaged. In this paper we propose a new method to identify sex, based on real-time PCR amplification of small fragments (61 and 64 bp) of the third exon within the amelogenin gene covering a 3-bp deletion on the AMELX-allele, followed by a High Resolution Melting analysis (HRM). HRM is based on the melting curves of amplified fragments. The amelogenin gene is located on both chromosomes X and Y, showing dimorphism in length. This molecular tool is rapid, sensitive and reduces the risk of contamination from exogenous genetic material when used for ancient DNA studies. The accuracy of the new method described here has been corroborated by using control samples of known sex and by contrasting our results with those obtained with other methods. Our method has proven to be useful even in heavily degraded samples, where other previously published methods failed. Stochastic problems such as the random allele drop-out phenomenon are expected to occur in a less severe form, due to the smaller fragment size to be amplified. Thus, their negative effect could be easier to overcome by a proper experimental design. PMID:25098828

  2. High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities.

    PubMed

    Miller, Manuel; Zorn, Julia; Brielmeier, Markus

    2015-01-01

    Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice. PMID:26556281

  3. Reliable Discrimination of 10 Ungulate Species Using High Resolution Melting Analysis of Faecal DNA

    PubMed Central

    Ramón-Laca, Ana; Gleeson, Dianne; Yockney, Ivor; Perry, Michael; Nugent, Graham; Forsyth, David M.

    2014-01-01

    Identifying species occupying an area is essential for many ecological and conservation studies. Faecal DNA is a potentially powerful method for identifying cryptic mammalian species. In New Zealand, 10 species of ungulate (Order: Artiodactyla) have established wild populations and are managed as pests because of their impacts on native ecosystems. However, identifying the ungulate species present within a management area based on pellet morphology is unreliable. We present a method that enables reliable identification of 10 ungulate species (red deer, sika deer, rusa deer, fallow deer, sambar deer, white-tailed deer, Himalayan tahr, Alpine chamois, feral sheep, and feral goat) from swabs of faecal pellets. A high resolution melting (HRM) assay, targeting a fragment of the 12S rRNA gene, was developed. Species-specific primers were designed and combined in a multiplex PCR resulting in fragments of different length and therefore different melting behaviour for each species. The method was developed using tissue from each of the 10 species, and was validated in blind trials. Our protocol enabled species to be determined for 94% of faecal pellet swabs collected during routine monitoring by the New Zealand Department of Conservation. Our HRM method enables high-throughput and cost-effective species identification from low DNA template samples, and could readily be adapted to discriminate other mammalian species from faecal DNA. PMID:24637802

  4. High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities

    PubMed Central

    Miller, Manuel; Zorn, Julia; Brielmeier, Markus

    2015-01-01

    Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes “Jawetz” and “Heyl”, Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice. PMID:26556281

  5. Rapid genotyping of beak and feather disease virus using high-resolution DNA melt curve analysis.

    PubMed

    Sarker, Subir; Ghorashi, Seyed A; Forwood, Jade K; Raidal, Shane R

    2014-11-01

    Beak and feather disease virus (BFDV) is a significant pathogen both for wild and captive psittacine birds globally. Genotypic differentiation of BFDV isolates is crucial to establish effective control strategies for the conservation of endangered species and epidemiological investigations of disease outbreaks. The technique developed in this study is a simple, rapid and inexpensive genotyping method for BFDV using PCR and subsequent high-resolution melt (HRM) curve analysis. This was achieved using PCR amplification of the conserved Rep gene in the presence of a fluorescent DNA intercalating dye (SYTO9). HRM curve analysis of the resultant amplicon could readily differentiate between reference strain (92-SR14) and 18 other BFDV isolates used in this study. Analysis of the nucleotide sequences of the amplicon from each isolate revealed that each melt curve profile was related to a unique DNA sequence. The potential of the PCR-HRM curve analysis to differentiate inter-host genetic variation among critically endangered orange-bellied parrots, lorikeets and cockatoos was also evaluated. Phylogenetic tree topology based on partial Rep gene sequences used in this study showed that BFDV Rep gene sequence patterns were correlated with the results of HRM curve analysis. The results presented in this study indicate that this technique could be used in both clinical research and differentiation of BFDV isolates in a fraction of time without further nucleotide sequencing and provides a novel approach for the genetic screening of BFDV in clinical virology laboratories. PMID:25102431

  6. Reliable discrimination of 10 ungulate species using high resolution melting analysis of faecal DNA.

    PubMed

    Ramón-Laca, Ana; Gleeson, Dianne; Yockney, Ivor; Perry, Michael; Nugent, Graham; Forsyth, David M

    2014-01-01

    Identifying species occupying an area is essential for many ecological and conservation studies. Faecal DNA is a potentially powerful method for identifying cryptic mammalian species. In New Zealand, 10 species of ungulate (Order: Artiodactyla) have established wild populations and are managed as pests because of their impacts on native ecosystems. However, identifying the ungulate species present within a management area based on pellet morphology is unreliable. We present a method that enables reliable identification of 10 ungulate species (red deer, sika deer, rusa deer, fallow deer, sambar deer, white-tailed deer, Himalayan tahr, Alpine chamois, feral sheep, and feral goat) from swabs of faecal pellets. A high resolution melting (HRM) assay, targeting a fragment of the 12S rRNA gene, was developed. Species-specific primers were designed and combined in a multiplex PCR resulting in fragments of different length and therefore different melting behaviour for each species. The method was developed using tissue from each of the 10 species, and was validated in blind trials. Our protocol enabled species to be determined for 94% of faecal pellet swabs collected during routine monitoring by the New Zealand Department of Conservation. Our HRM method enables high-throughput and cost-effective species identification from low DNA template samples, and could readily be adapted to discriminate other mammalian species from faecal DNA. PMID:24637802

  7. Glacier surface melt characterization and trend analysis (1992-2011) in the Russian High Arctic from combined resolution-enhanced scatterometer and passive microwave data

    NASA Astrophysics Data System (ADS)

    Zhao, M.; Ramage, J. M.; Semmens, K. A.

    2012-12-01

    Global warming has been pronounced in the remote glacierized archipelagoes (Severnaya Zemlya, Novaya Zemlya and Franz Josef Land) of the Russian High Arctic (RHA) and its effect on the low altitude, high latitude small ice caps needs examination. The timing and spatial variability of snow melt onset, duration and intensity are key factors influencing mass balance and the ice marginal hydrological system as well as important indicators of glacial response to anthropogenic and natural forcings. Characterization and trend analysis of RHA glacier melt behaviors provide insight about assessing the mass loss rate under recent Arctic climate change. However, due to the harsh environment, long term records of glaciological data for RHA are limited, necessitating the application of remotely sensed data to accomplish the research. The high sensitivity to liquid water and the ability to penetrate non-precipitating clouds enables microwave remote sensing to detect glacier surface melt. The appearance of melt water in snow dramatically decreases the returned scatterometer radar signal from active microwave sensors and sharply augments passive microwave emission. Based on this feature, we combined resolution-enhanced ERS-1/2 C-band (1992-2000), QuickSCAT Ku-band (2000-2009), ASCAT C-band (2009-2011) scatterometer data and SSMI 37 GHz (1995-2007) vertically polarized passive microwave products from Brigham Young University and analyzed glacier surface melt trends from 1992 to 2011 with a spatial resolution downscaled to 4.45km. We concatenated scatterometer derived melt behaviors by overlapping years and refined the results based on passive microwave data. Cross-validation shows that melt timing to be consistent between the active and passive sensors. Trend analysis (α < 0.005) reveals that the average glacier surface melt onset date occurs earlier by approximately 0.85 days/year in Severnaya Zemlya which outpaced the mean advancing rate in the pan-Arctic. Surrounded by ocean

  8. Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria.

    PubMed

    Daniels, Rachel; Hamilton, Elizabeth J; Durfee, Katelyn; Ndiaye, Daouda; Wirth, Dyann F; Hartl, Daniel L; Volkman, Sarah K

    2015-01-01

    Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs. One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures. SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status. While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele. In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs

  9. Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

    PubMed Central

    Daniels, Rachel; Hamilton, Elizabeth J.; Durfee, Katelyn; Ndiaye, Daouda; Wirth, Dyann F.; Hartl, Daniel L.; Volkman, Sarah K.

    2015-01-01

    Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs. One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures. SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status. While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele. In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs

  10. Performance and applications of quench melt-growth bulk magnets

    NASA Astrophysics Data System (ADS)

    Nariki, S.; Teshima, H.; Morita, M.

    2016-03-01

    This paper describes the progress in quench melt-growth (QMG) bulk magnets, developed by the Nippon Steel & Sumitomo Metal Corporation, which consist of single crystalline RE123 phase and finely dispersed RE211 particles. QMG bulks can trap high magnetic fields. The field-trapping ability of QMG bulks is largely increased with an improvement in its J c and size, promising the realization of various applications such as flywheel energy-storage systems, ship motors, NMR/MRI spectrometers, wind-power generators and so on. Intensive research has revealed that the optimal RE element is different depending on application requirements. Gd-QMG bulk is the most promising material for several high-field engineering applications. The trapped magnetic field of Gd-QMG bulk 60 mm in diameter at 77 K is twice as large as that of Y-QMG bulk with a similar size due to its excellent J c properties. The large Gd-based QMG bulks up to 150 mm in diameter are fabricated by incorporating the RE compositional gradient method. Compact NMR/MRI spectrometers are one of the promising applications of bulk superconductors. Eu-QMG bulks are suitable for NMR magnets. NMR applications require extremely homogeneous magnetic fields. In the Eu-system, the small paramagnetic moment of a Eu ion compared to a Gd ion improves the field homogeneity in the bulk. For the application of current leads, Dy-based QMG is available by utilizing a low thermal conductivity.

  11. A Rapid Multiplex Real-Time PCR High-Resolution Melt Curve Assay for the Simultaneous Detection of Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus in Food.

    PubMed

    Forghani, Fereidoun; Wei, Shuai; Oh, Deog-Hwan

    2016-05-01

    Three important foodborne pathogens, Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, are of great concern for food safety. They may also coexist in food matrices and, in the case of B. cereus and S. aureus, the resulting illnesses can resemble each other owing to similar symptoms. Therefore, their simultaneous detection may have advantages in terms of cost savings and rapidity. Given this context, a rapid multiplex real-time PCR high-resolution melt curve assay for the simultaneous detection of these three pathogens in food was developed. The assay successfully detected B. cereus (gyrB), L. monocytogenes (hly), and S. aureus (nuc) in a single reaction, and the average melting temperatures were 76.23, 80.19, and 74.01°C, respectively. The application of SYTO9 dye and a slow melt curve analysis ramp rate (0.1°C/s) enabled the production of sharp, high-resolution melt curve peaks that were easily distinguishable from each other. The detection limit in food (milk, rice, and lettuce) was 3.7 × 10(3) CFU/g without an enrichment step and 3.7 × 10(1) CFU/g following the 10-h enrichment. Hence, the assay developed here is specific and sensitive, providing an efficient tool for implementation in food for the simultaneous detection of B. cereus, L. monocytogenes, and S. aureus . PMID:27296430

  12. Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species

    PubMed Central

    Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Ounjai, Sarawut; Rora, Jantarika A.; Madesis, Panagiotis; de Boer, Hugo

    2015-01-01

    DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability. PMID:26406615

  13. Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis

    PubMed Central

    Ngui, Romano; Lim, Yvonne A. L.; Chua, Kek Heng

    2012-01-01

    Background Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. Methods Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. Conclusion The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species. PMID:22844538

  14. Rapid Diagnosis of Old World Leishmaniasis by High-Resolution Melting Analysis of the 7SL RNA Gene▿ †

    PubMed Central

    Nasereddin, Abedelmajeed; Jaffe, Charles L.

    2010-01-01

    High-resolution melt analysis PCR (HRM PCR) for diagnosis of Old World Leishmania was developed using the 7SL RNA gene. Cutaneous leishmaniasis samples were analyzed. Sensitivity and specificity of HRM PCR were significantly better (P < 0.001) than those of internal transcribed spacer 1 PCR and similar to those of kinetoplast DNA PCR. PMID:20392923

  15. A PCR–High-Resolution Melt Assay for Rapid Differentiation of Nontypeable Haemophilus influenzae and Haemophilus haemolyticus

    PubMed Central

    Binks, Michael J.; Beissbarth, Jemima; Hare, Kim M.; Kirkham, Lea-Ann S.; Smith-Vaughan, Heidi

    2014-01-01

    We have developed a PCR–high-resolution melt (PCR-HRM) assay to discriminate nontypeable Haemophilus influenzae (NTHi) colonies from Haemophilus haemolyticus. This method is rapid and robust, with 96% sensitivity and 92% specificity compared to the hpd#3 assay. PCR-HRM is ideal for high-throughput screening for NTHi surveillance and clinical trials. PMID:24478508

  16. Silicate-melt inclusions in magmatic rocks: applications to petrology

    NASA Astrophysics Data System (ADS)

    Frezzotti, Maria-Luce

    2001-01-01

    Silicate-melt inclusions in igneous rocks provide important information on the composition and evolution of magmatic systems. Such inclusions represent accidentally trapped silicate melt (±immiscible H 2O and/or CO 2 fluids) that allow one to follow the evolution of magmas through snapshots, corresponding to specific evolution steps. This information is available on condition that they remained isolated from the enclosing magma after their entrapment. The following steps of investigation are discussed: (a) detailed petrographic studies to characterise silicate-melt inclusion primary characters and posttrapping evolution, including melt crystallisation; (b) high temperature studies to rehomogenise the inclusion content and select chemically representative inclusions: chemical compositions should be compared to relevant phase diagrams. Silicate-melt inclusion studies allow us to concentrate on specific topics; inclusion studies in early crystallising phases allow the characterisation of primary magmas, while in more differentiated rocks, they unravel the subsequent chemical evolution. The distribution of volatile species (i.e., H 2O, CO 2, S, Cl) in inclusion glass can provide information on the degassing processes and on recycling of subducted material. In intrusive rocks, silicate melt inclusions may preserve direct evidence of magmatic stage evolution (e.g., immiscibility phenomena). Melt inclusions in mantle xenoliths indicate that high-silica melts can coexist with mantle peridotites and give information on the presence of carbonate melt within the upper mantle. Thus, combining silicate-melt inclusion data with conventional petrological and geochemical information and experimental petrology can increase our ability to model magmatic processes.

  17. Applications of nonequilibrium melting concept to damage-accumulation processes

    SciTech Connect

    Lam, N.Q.; Okamoto, P.R.

    1998-01-01

    The authors recent study of crystalline-to-amorphous transformation led to the successful development of a unified thermodynamic description of disorder-induced amorphization and heat-induced melting, based on a generalized version of the Lindemann melting criterion. The generalized criterion requires that the melting temperature of a defective crystal decreases with increasing static atomic disorder. Hence, any crystal can melt at temperatures below the melting point of its perfect crystalline state when driven far from equilibrium by introducing critical amounts of misfitting solute atoms and lattice imperfections, radiation damage, and/or tensile stresses. This conceptual approach to nonequilibrium melting provides new insight into long-standing materials problems such as brittle fracture, embrittlement, and environmentally-induced cracking, for example irradiation-assisted stress corrosion cracking.

  18. High-resolution melting system to perform multilocus sequence typing of Campylobacter jejuni.

    PubMed

    Lévesque, Simon; Michaud, Sophie; Arbeit, Robert D; Frost, Eric H

    2011-01-01

    Multi-locus sequence typing (MLST) has emerged as the state-of-the-art method for resolving bacterial population genetics but it is expensive and time consuming. We evaluated the potential of high resolution melting (HRM) to identify known MLST alleles of Campylobacter jejuni at reduced cost and time. Each MLST locus was amplified in two or three sub fragments, which were analyzed by HRM. The approach was investigated using 47 C. jejuni isolates, previously characterized by classical MLST, representing isolates from diverse environmental, animal and clinical sources and including the six most prevalent sequence types (ST) and the most frequent alleles. HRM was then applied to a validation set of 84 additional C. jejuni isolates from chickens; 92% of the alleles were resolved in 35 hours of laboratory time and the cost of reagents per isolate was $20 compared with $100 for sequence-based typing. HRM has the potential to complement sequence-based methods for resolving SNPs and to facilitate a wide range of genotyping studies. PMID:21297862

  19. High resolution melting: improvements in the genetic diagnosis of hypertrophic cardiomyopathy in a Portuguese cohort

    PubMed Central

    2012-01-01

    Background Hypertrophic Cardiomyopathy (HCM) is a complex myocardial disorder with a recognized genetic heterogeneity. The elevated number of genes and mutations involved in HCM limits a gene-based diagnosis that should be considered of most importance for basic research and clinical medicine. Methodology In this report, we evaluated High Resolution Melting (HRM) robustness, regarding HCM genetic testing, by means of analyzing 28 HCM-associated genes, including the most frequent 4 HCM-associated sarcomere genes, as well as 24 genes with lower reported HCM-phenotype association. We analyzed 80 Portuguese individuals with clinical phenotype of HCM allowing simultaneously a better characterization of this disease in the Portuguese population. Results HRM technology allowed us to identify 60 mutated alleles in 72 HCM patients: 49 missense mutations, 3 nonsense mutations, one 1-bp deletion, one 5-bp deletion, one in frame 3-bp deletion, one insertion/deletion, 3 splice mutations, one 5'UTR mutation in MYH7, MYBPC3, TNNT2, TNNI3, CSRP3, MYH6 and MYL2 genes. Significantly 22 are novel gene mutations. Conclusions HRM was proven to be a technique with high sensitivity and a low false positive ratio allowing a rapid, innovative and low cost genotyping of HCM. In a short return, HRM as a gene scanning technique could be a cost-effective gene-based diagnosis for an accurate HCM genetic diagnosis and hopefully providing new insights into genotype/phenotype correlations. PMID:22429680

  20. [Molecular identification of hairy antler by analysis of high resolution melting].

    PubMed

    Chen, Kang; Jiang, Chao; Yuan, Yuan; Huang, Lu-qi; Jin, Yan

    2015-02-01

    High resolution melting (HRM) , an important technology for genotyping and mutation scanning, has broad prospects in the authenticity of traditional Chinese medicine. This paper selected universal CO I primers and used HRM to establish a new method for authenticity of Hairy Antler. PCR was conducted at the annealing temperature of 60 °C and 45 cycles. The range of the DNA template concentration, the primer concentration and the Mg2+ ion concentration were further optimized. The results showed that the Tm values of Cervus nippon were (81.96 ± 0.07), (84.51 ± 0.03) °C and Cervus elaphus was(82.58 ± 0.13), (85.95 ± 0.05) °C with 10-100 mg · L(-1) DNA template, 0.2 µLmol · L(-1) primer, 2.0 mmol · L(-1) Mg2+. This method can authenticate of hairy antler and is simple, fast, high-throughput, visualization. PMID:26137679

  1. Identification of Uvaria sp by barcoding coupled with high-resolution melting analysis (Bar-HRM).

    PubMed

    Osathanunkul, M; Madesis, P; Ounjai, S; Pumiputavon, K; Somboonchai, R; Lithanatudom, P; Chaowasku, T; Wipasa, J; Suwannapoom, C

    2016-01-01

    DNA barcoding, which was developed about a decade ago, relies on short, standardized regions of the genome to identify plant and animal species. This method can be used to not only identify known species but also to discover novel ones. Numerous sequences are stored in online databases worldwide. One of the ways to save cost and time (by omitting the sequencing step) in species identification is to use available barcode data to design optimized primers for further analysis, such as high-resolution melting analysis (HRM). This study aimed to determine the effectiveness of the hybrid method Bar-HRM (DNA barcoding combined with HRM) to identify species that share similar external morphological features, rather than conduct traditional taxonomic identification that require major parts (leaf, flower, fruit) of the specimens. The specimens used for testing were those, which could not be identified at the species level and could either be Uvaria longipes or Uvaria wrayias, indicated by morphological identification. Primer pairs derived from chloroplast regions (matK, psbA-trnH, rbcL, and trnL) were used in the Bar-HRM. The results obtained from psbA-trnH primers were good enough to help in identifying the specimen while the rest were not. Bar-HRM analysis was proven to be a fast and cost-effective method for plant species identification. PMID:26909907

  2. High Resolution Melting Analysis for JAK2 Exon 14 and Exon 12 Mutations

    PubMed Central

    Rapado, Inmaculada; Grande, Silvia; Albizua, Enriqueta; Ayala, Rosa; Hernández, José-Angel; Gallardo, Miguel; Gilsanz, Florinda; Martinez-Lopez, Joaquin

    2009-01-01

    JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations. PMID:19225136

  3. Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria

    PubMed Central

    Chua, Kek Heng; Lim, Siew Chee; Ng, Ching Ching; Lee, Ping Chin; Lim, Yvonne Ai Lian; Lau, Tze Pheng; Chai, Hwa Chia

    2015-01-01

    Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNexTM, a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1–100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNexTM. This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNexTM. The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis. PMID:26507008

  4. Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria.

    PubMed

    Chua, Kek Heng; Lim, Siew Chee; Ng, Ching Ching; Lee, Ping Chin; Lim, Yvonne Ai Lian; Lau, Tze Pheng; Chai, Hwa Chia

    2015-01-01

    Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNex(TM), a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1-100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNex(TM). This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNex(TM). The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis. PMID:26507008

  5. Hot-melt extrusion technology and pharmaceutical application.

    PubMed

    Wilson, Matthew; Williams, Marcia A; Jones, David S; Andrews, Gavin P

    2012-06-01

    The use of hot-melt extrusion (HME) within the pharmaceutical industry is steadily increasing, due to its proven ability to efficiently manufacture novel products. The process has been utilized readily in the plastics industry for over a century and has been used to manufacture medical devices for several decades. The development of novel drugs with poor solubility and bioavailability brought the application of HME into the realm of drug-delivery systems. This has specifically been shown in the development of drug-delivery systems of both solid dosage forms and transdermal patches. HME involves the application of heat, pressure and agitation through an extrusion channel to mix materials together, and subsequently forcing them out through a die. Twin-screw extruders are most popular in solid dosage form development as it imparts both dispersive and distributive mixing. It blends materials while also imparting high shear to break-up particles and disperse them. HME extrusion has been shown to molecularly disperse poorly soluble drugs in a polymer carrier, increasing dissolution rates and bioavailability. The most common difficulty encountered in producing such dispersions is stabilization of amorphous drugs, which prevents them from recrystallization during storage. Pharmaceutical industrial suppliers, of both materials and equipment, have increased their development of equipment and chemicals for specific use with HME. Clearly, HME has been identified as an important and significant process to further enhance drug solubility and solid-dispersion production. PMID:22838073

  6. Development of a fast, simple profiling method for sample screening using high resolution melting (HRM) of STRs.

    PubMed

    Nicklas, Janice A; Noreault-Conti, Trisha; Buel, Eric

    2012-03-01

    A screening assay has been developed to provide preliminary individualization of crime scene samples thus eliminating expensive, time-consuming short tandem repeat (STR) profiling of nonprobative samples. High resolution melting performed in a real-time PCR instrument is used to detect the slight melting differences between the length and sequence variations of 22 forensic STRs. Three STRs (vWA, D18S51, THO1) were chosen to develop an assay which was optimized for Mg++ concentration, annealing/extension time/temperature, assay volume, and bovine serum albumin addition. The assay was tested for reproducibility, uniformity for genotype, melting profile consistency, effects of inhibitors, and mixture effects. The assay could be used to determine DNA concentration when a standard curve is run simultaneously. Calculations of costs show that the assay can save significant time and money for a crime with many samples or suspects. PMID:22150300

  7. A novel method for the rapid and prospective identification of Beijing Mycobacterium tuberculosis strains by high-resolution melting analysis.

    PubMed

    Alonso, M; Navarro, Y; Barletta, F; Martínez Lirola, M; Gotuzzo, E; Bouza, E; García de Viedma, D

    2011-03-01

    Genotypic analysis of Mycobacterium tuberculosis (MTB) has enabled the definition of several lineages. The Beijing family, which is considered highly virulent and transmissible, has been associated with resistance in certain settings and involved in severe outbreaks, making it one of the most closely-monitored lineages. Therefore, rapid prospective identification of Beijing MTB strains could be relevant. In the present study, we evaluate a real-time PCR followed by high-resolution melting (HRM) based on the identification of a single nucleotide polymorphism (SNP) in the Rv2629 gene which defines Beijing lineage (A191C for Beijing genotype and A191A for non-Beijing genotype). This combined methodology efficiently differentiated Beijing and non-Beijing strains in 100% of the isolates from a collection of reference strains without requiring specific DNA probes. Additionally, HRM was able to assign a Beijing/non-Beijing genotype in 90.9% of the respiratory specimens assayed. Its applicability was tested on a Peruvian sample of circulating MTB strains, in which it identified 10.7% as belonging to the Beijing genotype; this proportion reached 20% in the North Lima area. HRM analysis of the A191C SNP is a rapid, reliable, and sensitive method for the efficient prospective survey of high-risk Beijing MTB strains, even in developing settings where MTB culture is often not available. PMID:20384709

  8. Rapid detection of HLA-B*51 by real-time polymerase chain reaction and high-resolution melting analysis.

    PubMed

    Imperiali, C; Alía-Ramos, P; Padró-Miquel, A

    2015-08-01

    HLA-B*51, a class I human leukocyte antigen (HLA) molecule, is the strongest known genetic risk factor for Behçet disease. However, there are only few articles reporting methods to determine the presence or absence of HLA-B51. For this reason, we designed and developed an easy, fast, and inexpensive real-time high-resolution melting (HRM) assay to detect HLA-B*51. We genotyped 61 samples by our HRM assay and by conventional polymerase chain reaction, and no discrepancies were found between results. Besides, a subgroup of 25 samples was also genotyped in a different laboratory, and another subgroup of 16 samples was obtained from the International Histocompatibility Working Group DNA Bank, and a full concordance of results was observed with those obtained by HRM. Regarding the identifying system evaluated, we obtained 100% of specificity, sensibility, and repeatability, and 0% of false positive and false negative rates. Therefore, this HRM analysis is easily applicable to the rapid detection of HLA-B*51, exhibits a high speed, and requires a very low budget. PMID:26176813

  9. Quantitative high-resolution melting PCR analysis for monitoring of fermentation microbiota in sourdough.

    PubMed

    Lin, Xiaoxi B; Gänzle, Michael G

    2014-09-01

    Current methods of monitoring the microbial ecology of food fermentation system are generally labor intensive and/or time consuming. This study developed two methods based on high-resolution melting curves (HRM) to monitor sourdough microbiota during fermentation and to investigate the effect of cereal substrate on microbial ecology. A strain cocktail of Lactobacillus fermentum FUA3165, Lactobacillus plantarum FUA3309, Lactobacillus paracasei FUA3166 and Lactobacillus reuteri FUA3168 was used to ferment red (Town and PAN8609) and white (commercial and Segaolane) sorghum sourdough, and wheat sourdough. The microbial composition of sourdoughs was determined by plate count and HRM-qPCR to differentiate at the species level. The resistance of each species to sorghum phenolic extract was measured. There was no difference in microbial composition among the four sorghum sourdoughs, with L. fermentum FUA3165 in all sourdoughs. The competiveness of the strains in sorghum sourdoughs corresponded to their resistance to sorghum phenolic extract. In a second experiment, five L. reuteri strains, the human-lineage strains FUA3400 and 3401 isolated from wheat sourdough, the rodent-lineage strain FUA5448 isolated from rye sourdough and the sorghum isolates FUA3168 and 3324, were used to ferment wheat, rye and sorghum sourdoughs. The microbial composition of sourdoughs was determined by plate counts and HRM-qPCR to different L. reuteri strains representing different host-adapted lineages. No difference among different substrates was observed; indicating cereal type had no selective effect on sourdough microbial ecology. In conclusion, HRM-qPCR assays were established as rapid and highly specific tool for monitoring of sourdough microbiota. The ability to distinguish highly similar microbes in samples containing only few genotypes makes HRM-qPCR suitable for quality control in other food fermentation systems. The presence of phenolic compounds in sorghum sourdough favored organisms

  10. Detection of a G>C single nucleotide polymorphism within a repetitive DNA sequence by high-resolution DNA melting.

    PubMed

    Schmidt, Ulrike; Hulkkonen, Johannes; Naue, Jana

    2016-09-01

    In standard forensic DNA analysis, single base mutations within short tandem repeats (STR) mostly escape detection. In this study, high-resolution DNA melting (HRM) is compared to minisequencing and Sanger sequencing as to determine the most suitable method for detection of a G to C mutation within a repetitive DNA sequence, the STR system DXS10161. It shows an ATG/ATC polymorphism surrounded by a variable number of (TATC) and (ATCT) motifs. Neutral base changes like G:C to C:G result in very low differences in the melting temperature (T m) of the PCR amplicons. By enhanced resolution of fluorescence vs. temperature in HRM, the technique showed to be suitable for detecting a G to C transversion in this repetitive DNA sequence context. Compared to minisequencing, HRM is more time- and cost-effective. Results were confirmed by Sanger sequencing. PMID:26972692

  11. A Melting Layer Model for Passive/Active Microwave Remote Sensing Applications. Part 1; Model Formulation and Comparison with Observations

    NASA Technical Reports Server (NTRS)

    Olson, William S.; Bauer, Peter; Viltard, Nicolas F.; Johnson, Daniel E.; Tao, Wei-Kuo

    2000-01-01

    In this study, a 1-D steady-state microphysical model which describes the vertical distribution of melting precipitation particles is developed. The model is driven by the ice-phase precipitation distributions just above the freezing level at applicable gridpoints of "parent" 3-D cloud-resolving model (CRM) simulations. It extends these simulations by providing the number density and meltwater fraction of each particle in finely separated size categories through the melting layer. The depth of the modeled melting layer is primarily determined by the initial material density of the ice-phase precipitation. The radiative properties of melting precipitation at microwave frequencies are calculated based upon different methods for describing the dielectric properties of mixed phase particles. Particle absorption and scattering efficiencies at the Tropical Rainfall Measuring Mission Microwave Imager frequencies (10.65 to 85.5 GHz) are enhanced greatly for relatively small (approx. 0.1) meltwater fractions. The relatively large number of partially-melted particles just below the freezing level in stratiform regions leads to significant microwave absorption, well-exceeding the absorption by rain at the base of the melting layer. Calculated precipitation backscatter efficiencies at the Precipitation Radar frequency (13.8 GHz) increase in proportion to the particle meltwater fraction, leading to a "bright-band" of enhanced radar reflectivities in agreement with previous studies. The radiative properties of the melting layer are determined by the choice of dielectric models and the initial water contents and material densities of the "seeding" ice-phase precipitation particles. Simulated melting layer profiles based upon snow described by the Fabry-Szyrmer core-shell dielectric model and graupel described by the Maxwell-Garnett water matrix dielectric model lead to reasonable agreement with radar-derived melting layer optical depth distributions. Moreover, control profiles

  12. A Melting Layer Model for Passive/Active Microwave Remote Sensing Applications. Part 2; Simulation of TRMM Observations

    NASA Technical Reports Server (NTRS)

    Olson, William S.; Bauer, Peter; Kummerow, Christian D.; Tao, Wei-Kuo

    2000-01-01

    The one-dimensional, steady-state melting layer model developed in Part I of this study is used to calculate both the microphysical and radiative properties of melting precipitation, based upon the computed concentrations of snow and graupel just above the freezing level at applicable horizontal gridpoints of 3-dimensional cloud resolving model simulations. The modified 3-dimensional distributions of precipitation properties serve as input to radiative transfer calculations of upwelling radiances and radar extinction/reflectivities at the TRMM Microwave Imager (TMI) and Precipitation Radar (PR) frequencies, respectively. At the resolution of the cloud resolving model grids (approx. 1 km), upwelling radiances generally increase if mixed-phase precipitation is included in the model atmosphere. The magnitude of the increase depends upon the optical thickness of the cloud and precipitation, as well as the scattering characteristics of ice-phase precipitation aloft. Over the set of cloud resolving model simulations utilized in this study, maximum radiance increases of 43, 28, 18, and 10 K are simulated at 10.65, 19.35 GHz, 37.0, and 85.5 GHz, respectively. The impact of melting on TMI-measured radiances is determined not only by the physics of the melting particles but also by the horizontal extent of the melting precipitation, since the lower-frequency channels have footprints that extend over 10''s of kilometers. At TMI resolution, the maximum radiance increases are 16, 15, 12, and 9 K at the same frequencies. Simulated PR extinction and reflectivities in the melting layer can increase dramatically if mixed-phase precipitation is included, a result consistent with previous studies. Maximum increases of 0.46 (-2 dB) in extinction optical depth and 5 dBZ in reflectivity are simulated based upon the set of cloud resolving model simulations.

  13. Co-evolution of tidewater glacier calving front morphology and submarine melt rates in a high resolution ocean model

    NASA Astrophysics Data System (ADS)

    Slater, D. A.; Nienow, P. W.; Goldberg, D. N.; Cowton, T. R.; Sole, A. J.

    2015-12-01

    Rapid dynamic changes at the margins of the Greenland Ice Sheet, synchronous with ocean warming, have raised concern that tidewater glaciers can respond rapidly and sensitively to ocean forcing. One way in which ocean forcing would manifest is through the melting of the submerged parts of tidewater glacier calving fronts, with the spatial distribution of submarine melt a control on their morphology. Calving front morphology has thus far received little attention and yet has the potential to significantly impact calving rates and therefore tidewater glacier dynamics. Here we present a model which allows us to study the evolution of calving front morphology in two dimensions. We outline a new routine for calculating submarine melt rates from ocean models at calving fronts of arbitrary geometry, and for adjusting this geometry according to the calculated melt rates. This routine is applied to a high resolution (~1m) non-hydrostatic ocean model (MITgcm) with a glacier boundary (calving front) which evolves in time according to the simulated submarine melt rates. The model shows, consistent with recent observations, that submarine melting leads to undercutting of tidewater glacier calving fronts. We examine how undercut magnitude, undercut depth and potential steady states respond to variation in subglacial discharge, ice velocity, and fjord depth, temperature and stratification. In addition to this analysis we use a diagnostic full-Stokes flow-line ice model to examine how these geometries affect ice internal stress and potential for calving. In undertaking this work we aim to elucidate a process which - supposing tidewater glaciers are sensitive to ocean forcing - must provide a fundamental link between the ocean and the ice.

  14. Development of a high-resolution melting method for the screening of TNFAIP3 gene mutations.

    PubMed

    Chang, Yuli Christine; Chang, Ya-Sian; Chang, Chun-Chi; Liu, Ta-Chih; Ko, Ying-Chin; Lee, Chien-Chin; Chang, Shun-Jen; Chang, Jan-Gowth

    2016-05-01

    Tumor necrosis factor, α-induced protein 3 (TNFAIP3) which encodes a ubiquitin-modifying enzyme (A20), acts as a negative regulator of the NF-κB pathway, and in lymphoma and autoimmune diseases it is frequently inactivated by mutations and/or deletions. We investigated the prevalence of the inactivation of TNFAIP3 in oral squamous cell carcinoma (OSCC). DNA was extracted from 81 cases of OSCC and 50 peripheral blood samples from normal controls. A high-resolution melting (HRM) analysis was used to characterize TNFAIP3 mutations, and the results were confirmed by direct DNA sequencing. Three mutations and three single-nucleotide polymorphisms (SNPs) were found to be associated with OSCC; the TNFAIP3 mutation occurred in 3.7% (3/81) of the OSCC cases examined. All mutations were in exon 7 [c.1081G>A (p.E361K), c.1398C>G (p.S466R) (rs200878487) and c.1760C>T (p.P587L) (rs150056192)], and p.E361K was identified as a novel mutation. We further used SIFT and PolyPhen-2 software to assess potentially functional mutations. Two SNPs, c.296‑20_296-18delCTC (rs71670547) and c.380T>G (p.F127C) (rs2230926), were located in exon 3, and c.2140C>T (p.P714S) was located in exon 9. A novel SNP, p.P714S differed from the one reported previously (p.P714A) (rs369155845) at that site. We also identified five SNPs in 50 normal Taiwanese individuals, and two of them [c.296‑15C>T (rs377482653) and c.305A>G (p.N102S) (rs146534657)] were not found in our OSCC tissue. HRM facilitated the screening of genetic changes. In addition, our results indicate that the prevalence of the TNFAIP3 mutation is low in OSCC. PMID:26986245

  15. Refining lunar impact chronology through high spatial resolution (40)Ar/(39)Ar dating of impact melts.

    PubMed

    Mercer, Cameron M; Young, Kelsey E; Weirich, John R; Hodges, Kip V; Jolliff, Bradley L; Wartho, Jo-Anne; van Soest, Matthijs C

    2015-02-01

    Quantitative constraints on the ages of melt-forming impact events on the Moon are based primarily on isotope geochronology of returned samples. However, interpreting the results of such studies can often be difficult because the provenance region of any sample returned from the lunar surface may have experienced multiple impact events over the course of billions of years of bombardment. We illustrate this problem with new laser microprobe (40)Ar/(39)Ar data for two Apollo 17 impact melt breccias. Whereas one sample yields a straightforward result, indicating a single melt-forming event at ca. 3.83 Ga, data from the other sample document multiple impact melt-forming events between ca. 3.81 Ga and at least as young as ca. 3.27 Ga. Notably, published zircon U/Pb data indicate the existence of even older melt products in the same sample. The revelation of multiple impact events through (40)Ar/(39)Ar geochronology is likely not to have been possible using standard incremental heating methods alone, demonstrating the complementarity of the laser microprobe technique. Evidence for 3.83 Ga to 3.81 Ga melt components in these samples reinforces emerging interpretations that Apollo 17 impact breccia samples include a significant component of ejecta from the Imbrium basin impact. Collectively, our results underscore the need to quantitatively resolve the ages of different melt generations from multiple samples to improve our current understanding of the lunar impact record, and to establish the absolute ages of important impact structures encountered during future exploration missions in the inner Solar System. PMID:26601128

  16. High-Resolution Melting Analysis as a Powerful Tool to Discriminate and Genotype Pseudomonas savastanoi Pathovars and Strains

    PubMed Central

    Gori, Andrea; Cerboneschi, Matteo; Tegli, Stefania

    2012-01-01

    Pseudomonas savastanoi is a serious pathogen of Olive, Oleander, Ash, and several other Oleaceae. Its epiphytic or endophytic presence in asymptomatic plants is crucial for the spread of Olive and Oleander knot disease, as already ascertained for P. savastanoi pv. savastanoi (Psv) on Olive and for pv. nerii (Psn) on Oleander, while no information is available for pv. fraxini (Psf) on Ash. Nothing is known yet about the distribution on the different host plants and the real host range of these pathovars in nature, although cross-infections were observed following artificial inoculations. A multiplex Real-Time PCR assay was recently developed to simultaneously and quantitatively discriminate in vitro and in planta these P. savastanoi pathovars, for routine culture confirmation and for epidemiological and diagnostical studies. Here an innovative High-Resolution Melting Analysis (HRMA)-based assay was set up to unequivocally discriminate Psv, Psn and Psf, according to several single nucleotide polymorphisms found in their Type Three Secretion System clusters. The genetic distances among 56 P. savastanoi strains belonging to these pathovars were also evaluated, confirming and refining data previously obtained by fAFLP. To our knowledge, this is the first time that HRMA is applied to a bacterial plant pathogen, and one of the few multiplex HRMA-based assays developed so far. This protocol provides a rapid, sensitive, specific tool to differentiate and detect Psv, Psn and Psf strains, also in vivo and against other related bacteria, with lower costs than conventional multiplex Real-Time PCR. Its application is particularly suitable for sanitary certification programs for P. savastanoi, aimed at avoiding the spreading of this phytopathogen through asymptomatic plants. PMID:22295075

  17. Refining lunar impact chronology through high spatial resolution 40Ar/39Ar dating of impact melts

    PubMed Central

    Mercer, Cameron M.; Young, Kelsey E.; Weirich, John R.; Hodges, Kip V.; Jolliff, Bradley L.; Wartho, Jo-Anne; van Soest, Matthijs C.

    2015-01-01

    Quantitative constraints on the ages of melt-forming impact events on the Moon are based primarily on isotope geochronology of returned samples. However, interpreting the results of such studies can often be difficult because the provenance region of any sample returned from the lunar surface may have experienced multiple impact events over the course of billions of years of bombardment. We illustrate this problem with new laser microprobe 40Ar/39Ar data for two Apollo 17 impact melt breccias. Whereas one sample yields a straightforward result, indicating a single melt-forming event at ca. 3.83 Ga, data from the other sample document multiple impact melt–forming events between ca. 3.81 Ga and at least as young as ca. 3.27 Ga. Notably, published zircon U/Pb data indicate the existence of even older melt products in the same sample. The revelation of multiple impact events through 40Ar/39Ar geochronology is likely not to have been possible using standard incremental heating methods alone, demonstrating the complementarity of the laser microprobe technique. Evidence for 3.83 Ga to 3.81 Ga melt components in these samples reinforces emerging interpretations that Apollo 17 impact breccia samples include a significant component of ejecta from the Imbrium basin impact. Collectively, our results underscore the need to quantitatively resolve the ages of different melt generations from multiple samples to improve our current understanding of the lunar impact record, and to establish the absolute ages of important impact structures encountered during future exploration missions in the inner Solar System. PMID:26601128

  18. Antimicrobial thermoplastic materials for biomedical applications prepared by melt processing

    NASA Astrophysics Data System (ADS)

    Botta, L.; Scaffaro, R.; Ceraulo, M.; Gallo, G.

    2014-05-01

    In this work thermoplastic polymers with antimicrobial properties were prepared by incorporating an antibiotic, i.e., ciprofloxacin (CFX), by melt processing. Two different polymers were used as matrices, i.e., polypropylene (PP) and poly(lactid acid) (PLA) and different concentrations of CFX have been incorporated. The antimicrobial properties, the release kinetic and the mechanical performances of the prepared materials were evaluated.

  19. Rapid real-time PCR and high resolution melt analysis in a self-filling thermoplastic chip.

    PubMed

    Sposito, A; Hoang, V; DeVoe, D L

    2016-09-21

    A microfluidic platform designed for point-of-care PCR-based nucleic acid diagnostics is described. Compared to established microfluidic PCR technologies, the system is unique in its ability to achieve exceptionally rapid PCR amplification in a low cost thermoplastic format, together with high temperature accuracy enabling effective validation of reaction product by high resolution melt analysis performed in the same chamber as PCR. In addition, the system employs capillary pumping for automated loading of sample into the reaction chamber, combined with an integrated hydrophilic valve for precise self-metering of sample volumes into the device. Using the microfluidic system to target a mutation in the G6PC gene, efficient PCR from human genomic DNA template is achieved with cycle times as low as 14 s, full amplification in 8.5 min, and final melt analysis accurately identifying the desired amplicon. PMID:27460504

  20. Rapid Discrimination between Anopheles gambiae s.s. and Anopheles arabiensis by High-Resolution Melt (HRM) Analysis

    PubMed Central

    Zianni, Michael R.; Nikbakhtzadeh, Mahmood R.; Jackson, Bryan T.; Panescu, Jenny; Foster, Woodbridge A.

    2013-01-01

    There is a need for more cost-effective options to more accurately discriminate among members of the Anopheles gambiae complex, particularly An. gambiae and Anopheles arabiensis. These species are morphologically indistinguishable in the adult stage, have overlapping distributions, but are behaviorally and ecologically different, yet both are efficient vectors of malaria in equatorial Africa. The method described here, High-Resolution Melt (HRM) analysis, takes advantage of minute differences in DNA melting characteristics, depending on the number of incongruent single nucleotide polymorphisms in an intragenic spacer region of the X-chromosome-based ribosomal DNA. The two species in question differ by an average of 13 single-nucleotide polymorphisms giving widely divergent melting curves. A real-time PCR system, Bio-Rad CFX96, was used in combination with a dsDNA-specific dye, EvaGreen, to detect and measure the melting properties of the amplicon generated from leg-extracted DNA of selected mosquitoes. Results with seven individuals from pure colonies of known species, as well as 10 field-captured individuals unambiguously identified by DNA sequencing, demonstrated that the method provided a high level of accuracy. The method was used to identify 86 field mosquitoes through the assignment of each to the two common clusters with a high degree of certainty. Each cluster was defined by individuals from pure colonies. HRM analysis is simpler to use than most other methods and provides comparable or more accurate discrimination between the two sibling species but requires a specialized melt-analysis instrument and software. PMID:23543777

  1. Rapid and efficient differentiation of Yersinia species using high-resolution melting analysis.

    PubMed

    Souza, Roberto A; Frazão, Miliane R; Almeida, Alzira M P; Falcão, Juliana P

    2015-08-01

    The primary goal of clinical microbiology is the accurate identification of the causative agent of the disease. Here, we describe a method for differentiation between Yersinia species using PCR-HRMA. The results revealed species-specific melting profiles. The herein developed assay can be used as an effective method to differentiate Yersinia species. PMID:25980404

  2. Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis.

    PubMed

    Mohamed Zahidi, Jama'ayah; Bee Yong, Tay; Hashim, Rohaidah; Mohd Noor, Azura; Hamzah, Siti Hawa; Ahmad, Norazah

    2015-04-01

    Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level. PMID:25641125

  3. Differentiation of infectious bursal disease virus strains using real-time RT-PCR and high resolution melt curve analysis.

    PubMed

    Ghorashi, Seyed A; O'Rourke, Denise; Ignjatovic, Jagoda; Noormohammadi, Amir H

    2011-01-01

    Differentiation of infectious bursal disease virus (IBDV) strains is crucial for effective vaccination programs and epidemiological investigations. In this study, a combination of real-time RT-PCR and high resolution melt (HRM) curve analysis was developed for simultaneous detection and differentiation of IBDV strains/isolates. The hypervariable region of VP2 gene was amplified from several IBDV strains and subjected to HRM curve analysis. The method could readily differentiate between classical vaccines/isolates and variants. Analysis of the nucleotide sequence of the amplicons from each strain revealed that each melt curve profile was related to a unique DNA sequence. The real-time RT-PCR HRM curve analysis was also able to differentiate IBDV strains/isolates directly in bursal tissues from field submissions and from vaccinated commercial flocks. The differences between melting peaks generated from IBDV strains were significantly different (P<0.0001) demonstrating the high discriminatory power of this technique. The results presented in this study indicated that real-time RT-PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping IBDV isolates/strains and can contribute to effective control of IBDV outbreaks. PMID:21111004

  4. Precise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data

    PubMed Central

    Hatae, Ryusuke; Yoshimoto, Koji; Kuga, Daisuke; Akagi, Yojiro; Murata, Hideki; Suzuki, Satoshi O.; Mizoguchi, Masahiro; Iihara, Koji

    2016-01-01

    High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for IDH1R132 and IDH2R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAFV600E mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all IDH1 mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of IDH2R172 and BRAFV600E mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments. PMID:27529619

  5. Precise Detection of IDH1/2 and BRAF Hotspot Mutations in Clinical Glioma Tissues by a Differential Calculus Analysis of High-Resolution Melting Data.

    PubMed

    Hatae, Ryusuke; Hata, Nobuhiro; Yoshimoto, Koji; Kuga, Daisuke; Akagi, Yojiro; Murata, Hideki; Suzuki, Satoshi O; Mizoguchi, Masahiro; Iihara, Koji

    2016-01-01

    High resolution melting (HRM) is a simple and rapid method for screening mutations. It offers various advantages for clinical diagnostic applications. Conventional HRM analysis often yields equivocal results, especially for surgically obtained tissues. We attempted to improve HRM analyses for more effective applications to clinical diagnostics. HRM analyses were performed for IDH1R132 and IDH2R172 mutations in 192 clinical glioma samples in duplicate and these results were compared with sequencing results. BRAFV600E mutations were analyzed in 52 additional brain tumor samples. The melting profiles were used for differential calculus analyses. Negative second derivative plots revealed additional peaks derived from heteroduplexes in PCR products that contained mutations; this enabled unequivocal visual discrimination of the mutations. We further developed a numerical expression, the HRM-mutation index (MI), to quantify the heteroduplex-derived peak of the mutational curves. Using this expression, all IDH1 mutation statuses matched those ascertained by sequencing, with the exception of three samples. These discordant results were all derived from the misinterpretation of sequencing data. The effectiveness of our approach was further validated by analyses of IDH2R172 and BRAFV600E mutations. The present analytical method enabled an unequivocal and objective HRM analysis and is suitable for reliable mutation scanning in surgically obtained glioma tissues. This approach could facilitate molecular diagnostics in clinical environments. PMID:27529619

  6. Development of Novel High-Resolution Melting-Based Assays for Genotyping Two Alu Insertion Polymorphisms (FXIIIB and PV92).

    PubMed

    González-Giraldo, Yeimy; Rodríguez-Dueñas, Marisol; Forero, Diego A

    2016-03-01

    Insertion/Deletion polymorphisms (InDels) are a common type of genetic variation, with a growing role in population genetics and applied genomics. There is the need for the development of novel cost-effective assays for genotyping InDels of high importance. The main objective of this study was to develop high-resolution melting-based assays for genotyping two commonly studied Alu insertion polymorphisms: FXIIIB and PV92 (rs70942849 and rs3138523). Three primers (two forward and one reverse) were designed for each marker, and high-resolution melting (HRM) analyses in a qPCR platform were performed, using EvaGreen fluorescent dye. For each one of the two Alu insertion polymorphisms, HRM analyses identified distinguishable peaks for the three genotypes, allowing a robust genotyping. Results were validated using 96 DNA samples previously genotyped and the assays worked with different DNA concentrations. In this study, we developed novel cost-effective assays, using qPCR, for genotyping two Alu insertion polymorphisms (widely used as ancestry markers). Our results highlight the feasibility of using HRM analyses for genotyping InDel polymorphisms of medical and biotechnological importance. PMID:26843017

  7. High-resolution melt analysis for species identification of coagulase-negative staphylococci derived from bovine milk.

    PubMed

    Ajitkumar, Praseeda; Barkema, Herman W; Zadoks, Ruth N; Morck, Douglas W; van der Meer, Frank J U M; De Buck, Jeroen

    2013-03-01

    Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens isolated from bovine milk. In this study, we report a rapid assay for species identification of CNS using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time polymerase chain reaction amplification of 16S rRNA gene fragment, spanning the variable region V1 and V2, was performed with a resulting amplicon of 215 bp. A library of distinct melt curves of reference strains of 13 common CNS species was created using HRMA. Sequencing of 16S rRNA and rpoB genes, and, when needed, tuf gene, of 100 CNS isolates obtained from Canadian Bovine Mastitis Research Network was done to determine their species identity, allowing for subsequent evaluation of the performance of HRMA for field isolates of bovine CNS. A combination of HRMA and sequencing revealed that Staphylococcus chromogenes, S. xylosus, S. simulans, and S. sciuri had multiple genotypes, complicating their resolution by HRMA. As the 3 genotypes of S. chromogenes had distinct melt curves, the 3 distinct genotypes were employed as reference strains in a blinded trial of 156 CNS isolates to identify S. chromogenes. HRMA correctly identified all S. chromogenes isolates which were later confirmed by sequencing. Staphylococcus chromogenes (68%) was most frequently found among the CNS isolates, followed by S. haemolyticus (10%) and S. xylosus (6%). The present study revealed that HRMA of 16S rRNA gene (V1-V2) could be used as a rapid, efficient, low-cost, and minimally cumbersome technique for S. chromogenes identification, the most common CNS derived from bovine milk. PMID:23273337

  8. Viscosity of Carbonate-Silicate Melts Using Ultra-High Resolution Falling Sphere Viscometry

    NASA Astrophysics Data System (ADS)

    Hummer, D. R.; Kavner, A.; Manning, C. E.; Park, C.; Kono, Y.; Kenney-Benson, C.

    2014-12-01

    Carbonatites are carbon-rich magmas that participate in the deep-Earth carbon cycle, and may be precursors to many types of terrestrial magmatism. [1] Viscosity is a crucial parameter in determining migration rates and behavior of melts from the upper mantle to Earth's surface, but very little is known about the viscosity of carbonate-silicate liquids at conditions relevant to Earth's interior. To examine the viscosity of carbonate-silicate liquids as a function of composition, we performed in situ falling sphere viscosity experiments using a high speed X-ray camera and Paris-Edinburgh press at the HPCAT beamline (Advanced Photon Source, Argonne National Laboratory). Mixtures from the CaCO3-CaSiO3 (calcite-wollastonite) binary system were used to simulate mantle silicate carbonatites. Samples were loaded using the experimental setup of Yamada et al [2], held at 3 GPa, and heated until the sample was fully molten (between 1350-1650 oC). The high speed camera recorded the falling rate of a platinum sphere placed near the top of the sample chamber, enabling the calculation of terminal velocity and hence viscosity. Results indicate that pure CaCO3 at upper mantle conditions has a very low viscosity of ~0.006 Pa-s, only a little higher than that of water. This viscosity is 2-6 times lower than that of potassium carbonates at similar pressures (2.5-4.0 GPa) but at somewhat lower temperatures (800-1200 oC). [3] Our measured viscosity as a function of increasing silicate content increases along a log-linear trend, reaching 0.256 Pa-s for CaSiO3 liquid. This heavy dependence of viscosity on composition has implications for melt migration processes at different depths, suggesting either viscosity-driven or porosity-driven migration depending on both extent of melting and carbonate content. [1] Dasgupta, R. et al. (2013) Nature 493, 211-215. [2] Yamada, A. et al. (2011) Rev. Sci. Instr. 82, 015103. [3] Dobson, D. et al. (1996) Earth Plan. Sci. Lett. 143, 207-215.

  9. High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains.

    PubMed

    Bingga, Gali; Liu, Zhicheng; Zhang, Jianfeng; Zhu, Yujun; Lin, Lifeng; Ding, Shuangyang; Guo, Pengju

    2014-01-01

    A high resolution melting (HRM) curve method was developed to identify canine parvovirus type 2 (CPV-2) strains by nested PCR. Two sets of primers, CPV-426F/426R and CPV-87R/87F, were designed that amplified a 52 bp and 53 bp product from the viral VP2 capsid gene. The region amplified by CPV-426F/426R included the A4062G and T4064A mutations in CPV-2a, CPV-2b and CPV-2c. The region amplified by CPV-87F/87R included the A3045T mutation in the vaccine strains of CPV-2 and CPV-2a, CPV-2b and CPV-2c. Faecal samples were obtained from 30 dogs that were CPV antigen-positive. The DNA was isolated from the faecal samples and PCR-amplified using the two sets of primers, and genotyped by HRM curve analysis. The PCR-HRM assay was able to distinguish single nucleotide polymorphisms between CPV-2a, CPV-2b and CPV-2c using CPV-426F/426R. CPV-2a was distinguished from CPV-2b and CPV-2c by differences in the melting temperature. CPV-2b and CPV-2c could be distinguished based on the shape of the melting curve after generating heteroduplexes using a CPV-2b reference sample. The vaccine strains of CPV-2 were identified using CPV-87F/87R. Conventional methods for genotyping CPV strains are labor intensive, expensive or time consuming; the present PCR-based HRM assay might be an attractive alternative. PMID:25159576

  10. Melt Infiltrated Ceramic Composites (Hipercomp) for Gas Turbine Engine Applications

    SciTech Connect

    Gregory Corman; Krishan Luthra

    2005-09-30

    This report covers work performed under the Continuous Fiber Ceramic Composites (CFCC) program by GE Global Research and its partners from 1994 through 2005. The processing of prepreg-derived, melt infiltrated (MI) composite systems based on monofilament and multifilament tow SiC fibers is described. Extensive mechanical and environmental exposure characterizations were performed on these systems, as well as on competing Ceramic Matrix Composite (CMC) systems. Although current monofilament SiC fibers have inherent oxidative stability limitations due to their carbon surface coatings, the MI CMC system based on multifilament tow (Hi-Nicalon ) proved to have excellent mechanical, thermal and time-dependent properties. The materials database generated from the material testing was used to design turbine hot gas path components, namely the shroud and combustor liner, utilizing the CMC materials. The feasibility of using such MI CMC materials in gas turbine engines was demonstrated via combustion rig testing of turbine shrouds and combustor liners, and through field engine tests of shrouds in a 2MW engine for >1000 hours. A unique combustion test facility was also developed that allowed coupons of the CMC materials to be exposed to high-pressure, high-velocity combustion gas environments for times up to {approx}4000 hours.

  11. Ecological applications of high resolution spectrometry

    NASA Technical Reports Server (NTRS)

    Lawrence, William T.

    1989-01-01

    Future directions of NASA's space program plans include a significant effort at studying the Earth as a system of interrelated ecosystems. As part of NASA's Earth Observing System (Eos) Program a series of space platforms will be launched and operated to study the Earth with a variety of active and passive instruments. Several of the Eos instruments will be capable of imaging the planet's surface reflectance on a large number of very narrow portions of the solar spectrum. After the development of appropriate algorithms, this reflectance information will be used to determine key parameters about the structure and function of terrestrial and aquatic ecosystems and the pattern and processes of those systems across large areas of the globe. Algorithm development applicable to terrestrial systems will permit the inference of ecological processes from high resolution spectrometry data, similar to that to be forthcoming from the Eos mission. The first summer was spent working with tropical soils and relating their reflectance characteristics to particle size, iron content, and color. This summer the emphasis is on vegetation and work was begun with the Forest Ecosystems Dynamics Project in the Earth Resources Branch where both optical and radar characteristics of a mixed conifer/hardwood forest in Maine are being studied for use in a ecological modeling effort. A major series of aircraft overflights will take place throughout the summer. Laboratory and field spectrometers are used to measure the spectral reflectance of a hierarchy of vegetation from individual leaves to whole canopies for eventual modeling of their nutrient content using reflectance data. Key leaf/canopy parameters are being approximated including chlorophyll, nitrogen, phosphorus, water content, and leaf specific weight using high resolution spectrometry alone. Measurements are made of carbon exchange across the landscape for input to a spatial modeling effort to gauge production within the forest. A

  12. A high-resolution melting (HRM) assay for the differentiation between Israeli field and Neethling vaccine lumpy skin disease viruses.

    PubMed

    Menasherow, Sophia; Erster, Oran; Rubinstein-Giuni, Marisol; Kovtunenko, Anita; Eyngor, Evgeny; Gelman, Boris; Khinich, Evgeny; Stram, Yehuda

    2016-06-01

    Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ∼0.5 °C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable. PMID:26902159

  13. Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis

    PubMed Central

    Banowary, Banya; Dang, Van Tuan; Sarker, Subir; Connolly, Joanne H.; Chenu, Jeremy; Groves, Peter; Ayton, Michelle; Raidal, Shane; Devi, Aruna; Vanniasinkam, Thiru; Ghorashi, Seyed A.

    2015-01-01

    Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates. PMID:26394042

  14. Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

    PubMed Central

    Cousins, Matthew M.; Donnell, Deborah; Eshleman, Susan H.

    2013-01-01

    We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology. PMID:23178437

  15. High Resolution Melting Analysis for Rapid Mutation Screening in Gyrase and Topoisomerase IV Genes in Quinolone-Resistant Salmonella enterica

    PubMed Central

    Thong, Kwai Lin

    2014-01-01

    The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes. PMID:25371903

  16. High resolution melting analysis for rapid mutation screening in gyrase and Topoisomerase IV genes in quinolone-resistant Salmonella enterica.

    PubMed

    Ngoi, Soo Tein; Thong, Kwai Lin

    2014-01-01

    The increased Salmonella resistance to quinolones and fluoroquinolones is a public health concern in the Southeast Asian region. The objective of this study is to develop a high resolution melt curve (HRM) assay to rapidly screen for mutations in quinolone-resistant determining region (QRDR) of gyrase and topoisomerase IV genes. DNA sequencing was performed on 62 Salmonella strains to identify mutations in the QRDR of gyrA, gyrB, parC, and parE genes. Mutations were detected in QRDR of gyrA (n = 52; S83F, S83Y, S83I, D87G, D87Y, and D87N) and parE (n = 1; M438I). Salmonella strains with mutations within QRDR of gyrA are generally more resistant to nalidixic acid (MIC 16 > 256 μg/mL). Mutations were uncommon within the QRDR of gyrB, parC, and parE genes. In the HRM assay, mutants can be distinguished from the wild-type strains based on the transition of melt curves, which is more prominent when the profiles are displayed in difference plot. In conclusion, HRM analysis allows for rapid screening for mutations at the QRDRs of gyrase and topoisomerase IV genes in Salmonella. This assay markedly reduced the sequencing effort involved in mutational studies of quinolone-resistance genes. PMID:25371903

  17. A method to distinguish morphologically similar Peromyscus species using extracellular RNA and high-resolution melt analysis.

    PubMed

    Seifert, Veronica A; Clarke, Benjamin L; Crossland, Janet P; Bemis, Lynne T

    2016-09-01

    A method applying high-resolution melt (HRM) analysis to PCR products copied and amplified from extracellular RNA (exRNA) has been developed to distinguish two morphologically similar Peromyscus species: Peromyscus leucopus and Peromyscus maniculatus. P. leucopus is considered the primary reservoir host of Borrelia burgdorferi, the causative agent for Lyme disease in North America. In northern Minnesota the habitat ranges of P. leucopus overlaps with that of P. maniculatus. Serum samples from live mice of both species were collected from cheek bleeds, total extracellular RNA (exRNA) was extracted, copied using reverse transcription and amplified by PCR followed by HRM analysis. A circulating ribosomal RNA (rRNA) was identified which differed at seven nucleotides between the two species and a method of HRM analysis was developed allowing rapid species confirmation. In the future, this HRM based method may be adapted for additional species. PMID:27349513

  18. Melt spreading code assessment, modifications, and application to the EPR core catcher design.

    SciTech Connect

    Farmer, M. T .; Nuclear Engineering Division

    2009-03-30

    The Evolutionary Power Reactor (EPR) is under consideration by various utilities in the United States to provide base load electrical production, and as a result the design is undergoing a certification review by the U.S. Nuclear Regulatory Commission (NRC). The severe accident design philosophy for this reactor is based upon the fact that the projected power rating results in a narrow margin for in-vessel melt retention by external cooling of the reactor vessel. As a result, the design addresses ex-vessel core melt stabilization using a mitigation strategy that includes: (1) an external core melt retention system to temporarily hold core melt released from the vessel; (2) a layer of 'sacrificial' material that is admixed with the melt while in the core melt retention system; (3) a melt plug in the lower part of the retention system that, when failed, provides a pathway for the mixture to spread to a large core spreading chamber; and finally, (4) cooling and stabilization of the spread melt by controlled top and bottom flooding. The overall concept is illustrated in Figure 1.1. The melt spreading process relies heavily on inertial flow of a low-viscosity admixed melt to a segmented spreading chamber, and assumes that the melt mass will be distributed to a uniform height in the chamber. The spreading phenomenon thus needs to be modeled properly in order to adequately assess the EPR design. The MELTSPREAD code, developed at Argonne National Laboratory, can model segmented, and both uniform and nonuniform spreading. The NRC is thus utilizing MELTSPREAD to evaluate melt spreading in the EPR design. MELTSPREAD was originally developed to support resolution of the Mark I containment shell vulnerability issue. Following closure of this issue, development of MELTSPREAD ceased in the early 1990's, at which time the melt spreading database upon which the code had been validated was rather limited. In particular, the database that was utilized for initial validation consisted

  19. Detection of Sequence Polymorphism in Rubus Occidentalis L. Monomorphic Microsatellite Markers by High Resolution Melting

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsatellite, or simple sequence repeat (SSR) markers, are valuable as co-dominant genetic markers with a variety of applications such as DNA fingerprinting, linkage mapping, and population structure analysis. Development of microsatellite primers through the identification of appropriate repeate...

  20. High resolution melting detects sequence polymorphism in rubus occidentalis L. monomorphic microsatellite markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microsatellite, or simple sequence repeat (SSR) markers, are valuable as co-dominant genetic markers with a variety of applications such as DNA fingerprinting, linkage mapping, and population structure analysis. However, primer pairs designed from the regions that flank SSRs often generate fragment...

  1. High resolution SAR applications and instrument design

    NASA Technical Reports Server (NTRS)

    Dionisio, C.; Torre, A.

    1993-01-01

    The Synthetic Aperture Radar (SAR) has viewed, in the last two years, a huge increment of interest from many preset and potential users. The good spatial resolution associated to the all weather capability lead to considering SAR not only a scientific instrument but a tool for verifying and controlling the daily human relationships with the Earth Environment. New missions were identified for SAR as spatial resolution became lower than three meters: disasters, pollution, ships traffic, volcanic eruptions, earthquake effect are only a few of the possible objects which can be effectively detected, controlled and monitored by SAR mounted on satellites. High resolution radar design constraints and dimensioning are discussed.

  2. Genotyping single-nucleotide polymorphisms of human genes involved in organophosphate detoxification by high-resolution melting.

    PubMed

    Kurdyukov, Ivan; Rodionov, Gennady; Radilov, Andrey; Babakov, Vladimir

    2014-08-01

    Paraoxonase-1 (PON1) and butyrylcholinesterase (BCHE) are natural bioscavengers of organophosphate acetylcholinesterase inhibitors in the human body, which can determine individual sensitivity to organophosphate toxicity. Interindividual differences in activity of PON1 (catalytic bioscavenger) and substrate specificity are strongly associated with the substitution of two amino acids: Leu/Met (L/M) at position 55 (rs854560) and Gln/Arg (Q/R) at position 192 (rs662). In the case of BCHE (stoichiometric bioscavenger) substitution, Ala/Thr (A/T) at position 539 produces the so-called "K-variant" of the enzyme (rs1803274). Threonine allele is often co-inherited with an atypical BCHE allele (rs1799807). The atypical variant of BCHE displays a lower affinity for cholinesterase inhibitors. Genotyping rs662 and rs1803274 single-nucleotide polymorphisms (SNP) by high-resolution melting (HRM) is facilitated by the nucleotide substitution A>G (G>A), which resulted in a changed number of hydrogen bonds in the PCR product and, consequently, shifted T m. In the case of rs854560, genotyping is complicated by the nucleotide substitution T>A, which has no significant effect on the T m of the PCR product. An addition of a small quantity of LL homozygote DNA into the reaction mixture before PCR discriminates the three genotypes by the melt curves due to different amounts of heteroduplexes formed in the LM and MM samples. HRM analysis can be applied for genotyping human rs854560, rs662, and rs1803274 SNPs. PMID:24705954

  3. High Resolution Melting Analysis: A Rapid Screening and Typing Tool for Common β-Thalassemia Mutation in Chinese Population

    PubMed Central

    Lin, Min; Jiao, Ji-Wei; Zhan, Xiu-Hui; Zhan, Xiao-Fen; Pan, Mei-Chen; Wang, Jun-Li; Wang, Chun-Fang; Zhong, Tian-Yu; Zhang, Qin; Yu, Xia; Wu, Jiao-Ren; Yang, Hui-Tian; Lin, Fen; Tong, Xin; Yang, Hui; Zha, Guang-Cai; Wang, Qian; Zheng, Lei; Wen, Ying-Fang; Yang, Li-Ye

    2014-01-01

    β-thalassemia is a common inherited disorder worldwide including southern China, and at least 45 distinct β-thalassemia mutations have been identified in China. High-resolution melting (HRM) assay was recently introduced as a rapid, inexpensive and effective method for genotyping. However, there was no systemic study on the diagnostic capability of HRM to identify β-thalassemia. Here, we used an improved HRM method to screen and type 12 common β-thalassemia mutations in Chinese, and the rapidity and reliability of this method was investigated. The whole PCR and HRM procedure could be completed in 40 min. The heterozygous mutations and 4 kinds of homozygous mutations could be readily differentiated from the melting curve except c.-78A>G heterozygote and c.-79A>G heterozygote. The diagnostic reliability of this HRM assay was evaluated on 756 pre-typed genomic DNA samples and 50 cases of blood spots on filter paper, which were collected from seven high prevalent provinces in southern China. If c.-78A>G heterozygote and c.-79A>G heterozygote were classified into the same group (c.-78&79 A>G heterozygote), the HRM method was in complete concordance with the reference method (reverse dot blot/DNA-sequencing). In a conclusion, the HRM method appears to be an accurate and sensitive method for the rapid screening and identification of β-thalassemia mutations. In the future, we suggest this technology to be used in neonatal blood spot screening program. It could enlarge the coverage of β-thalassemia screening program in China. At the same time, its value should be confirmed in prospectively clinical and epidemiological studies. PMID:25089872

  4. Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis.

    PubMed

    Sady, Hany; Al-Mekhlafi, Hesham M; Ngui, Romano; Atroosh, Wahib M; Al-Delaimy, Ahmed K; Nasr, Nabil A; Dawaki, Salwa; Abdulsalam, Awatif M; Ithoi, Init; Lim, Yvonne A L; Chua, Kek Heng; Surin, Johari

    2015-01-01

    The present study describes a real-time PCR approach with high resolution melting-curve (HRM) assay developed for the detection and differentiation of Schistosoma mansoni and S. haematobium in fecal and urine samples collected from rural Yemen. The samples were screened by microscopy and PCR for the Schistosoma species infection. A pair of degenerate primers were designed targeting partial regions in the cytochrome oxidase subunit I (cox1) gene of S. mansoni and S. haematobium using real-time PCR-HRM assay. The overall prevalence of schistosomiasis was 31.8%; 23.8% of the participants were infected with S. haematobium and 9.3% were infected with S. mansoni. With regards to the intensity of infections, 22.1% and 77.9% of S. haematobium infections were of heavy and light intensities, respectively. Likewise, 8.1%, 40.5% and 51.4% of S. mansoni infections were of heavy, moderate and light intensities, respectively. The melting points were distinctive for S. mansoni and S. haematobium, categorized by peaks of 76.49 ± 0.25 °C and 75.43 ± 0.26 °C, respectively. HRM analysis showed high detection capability through the amplification of Schistosoma DNA with as low as 0.0001 ng/µL. Significant negative correlations were reported between the real-time PCR-HRM cycle threshold (Ct) values and microscopic egg counts for both S. mansoni in stool and S. haematobium in urine (p < 0.01). In conclusion, this closed-tube HRM protocol provides a potentially powerful screening molecular tool for the detection of S. mansoni and S. haematobium. It is a simple, rapid, accurate, and cost-effective method. Hence, this method is a good alternative approach to probe-based PCR assays. PMID:26193254

  5. Detection of Schistosoma mansoni and Schistosoma haematobium by Real-Time PCR with High Resolution Melting Analysis

    PubMed Central

    Sady, Hany; Al-Mekhlafi, Hesham M.; Ngui, Romano; Atroosh, Wahib M.; Al-Delaimy, Ahmed K.; Nasr, Nabil A.; Dawaki, Salwa; Abdulsalam, Awatif M.; Ithoi, Init; Lim, Yvonne A. L.; Chua, Kek Heng; Surin, Johari

    2015-01-01

    The present study describes a real-time PCR approach with high resolution melting-curve (HRM) assay developed for the detection and differentiation of Schistosoma mansoni and S. haematobium in fecal and urine samples collected from rural Yemen. The samples were screened by microscopy and PCR for the Schistosoma species infection. A pair of degenerate primers were designed targeting partial regions in the cytochrome oxidase subunit I (cox1) gene of S. mansoni and S. haematobium using real-time PCR-HRM assay. The overall prevalence of schistosomiasis was 31.8%; 23.8% of the participants were infected with S. haematobium and 9.3% were infected with S. mansoni. With regards to the intensity of infections, 22.1% and 77.9% of S. haematobium infections were of heavy and light intensities, respectively. Likewise, 8.1%, 40.5% and 51.4% of S. mansoni infections were of heavy, moderate and light intensities, respectively. The melting points were distinctive for S. mansoni and S. haematobium, categorized by peaks of 76.49 ± 0.25 °C and 75.43 ± 0.26 °C, respectively. HRM analysis showed high detection capability through the amplification of Schistosoma DNA with as low as 0.0001 ng/µL. Significant negative correlations were reported between the real-time PCR-HRM cycle threshold (Ct) values and microscopic egg counts for both S. mansoni in stool and S. haematobium in urine (p < 0.01). In conclusion, this closed-tube HRM protocol provides a potentially powerful screening molecular tool for the detection of S. mansoni and S. haematobium. It is a simple, rapid, accurate, and cost-effective method. Hence, this method is a good alternative approach to probe-based PCR assays. PMID:26193254

  6. Evaluation of low melting halide systems for battery applications

    NASA Astrophysics Data System (ADS)

    Mamantov, G.; Perrovic, C.

    1981-03-01

    This three year program involves evaluation of selected low temperature molten salt solvent systems containing inorganic and/or organic chlorides and bromides for battery applications. The research involves determination of the liquidus temperatures, the specific electrical conductivity, and the electrochemical span of selected halide systems. Characterization of the solvent species by Raman spectroscopy, vapor pressure measurements, and the electrochemical study of a few cathode and anode systems will be undertaken for the most promising solvent systems. The research during the second year of this project involved the determination of liquidus temperatures and/or specific electrical conductivities for a number of binary and ternary molten salt systems containing AlCl3, AlBr3, SbCl3, FeCl3, and GaCl3.

  7. Molecular simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus by real-time RT-PCR and high resolution melting analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, real-time RT-PCR assays were combined with high resolution melting (HRM) analysis for the simultaneous detection of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) infection in sweet cherry trees. Detection of CNRMV and CGRMV was performed using a...

  8. Recovery of zinc and lead from fly ash from ash-melting and gasification-melting processes of MSW--comparison and applicability of chemical leaching methods.

    PubMed

    Okada, T; Tojo, Y; Tanaka, N; Matsuto, T

    2007-01-01

    Fly ash generated from MSW ash-melting and gasification-melting plants, known as Melting Furnace Fly Ash (MFA), contains considerable amounts of heavy metals such as Pb and Zn. These metals can be recovered using a smelting furnace after "pre-treatment" for removal of unnecessary elements such as Cl, Sn and Si. Chemical methods have been studied for pretreatment in the past. However, they have been discussed only with regard to treatment cost and the concentration of Pb and Zn recovered, but neither applicability to various types of MFA nor the environmental impact have been considered. In this study, acid, alkaline and ammonia/chloride leaching methods were compared from the standpoints of: (1) applicability to MFA, (2) concentration of Pb and Zn recovered, (3) treatment cost, and (4) environmental impact. Twenty-three samples of MFAs were collected and classified into 4 types based on element contents. A Pb and Zn recovery experiment was conducted for the representative MFA of those types. The results showed: (1) MFA from gasification-melting plants cannot be treated by chemical methods; (2) the other MFA can be treated to an acceptable quality by existing smelting furnaces; (3) only MFA from electric resistance ash-melting plants can be treated easily by the water washing method; and (4) alkaline and ammonia/chloride leaching methods were more effective than acid leaching. PMID:16488594

  9. Identification of Molecular Markers Associated with Verticillium Wilt Resistance in Alfalfa (Medicago Sativa L.) Using High-Resolution Melting

    PubMed Central

    Zhang, Tiejun; Yu, Long-Xi; McCord, Per; Miller, David; Bhamidimarri, Suresh; Johnson, David; Monteros, Maria J.; Ho, Julie; Reisen, Peter; Samac, Deborah A.

    2014-01-01

    Verticillium wilt, caused by the soilborne fungus, Verticillium alfalfae, is one of the most serious diseases of alfalfa (Medicago sativa L.) worldwide. To identify loci associated with resistance to Verticillium wilt, a bulk segregant analysis was conducted in susceptible or resistant pools constructed from 13 synthetic alfalfa populations, followed by association mapping in two F1 populations consisted of 352 individuals. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers were used for genotyping. Phenotyping was done by manual inoculation of the pathogen to replicated cloned plants of each individual and disease severity was scored using a standard scale. Marker-trait association was analyzed by TASSEL. Seventeen SNP markers significantly associated with Verticillium wilt resistance were identified and they were located on chromosomes 1, 2, 4, 7 and 8. SNP markers identified on chromosomes 2, 4 and 7 co-locate with regions of Verticillium wilt resistance loci reported in M. truncatula. Additional markers identified on chromosomes 1 and 8 located the regions where no Verticillium resistance locus has been reported. This study highlights the value of SNP genotyping by high resolution melting to identify the disease resistance loci in tetraploid alfalfa. With further validation, the markers identified in this study could be used for improving resistance to Verticillium wilt in alfalfa breeding programs. PMID:25536106

  10. High-resolution melting curve analysis for genotyping of common SNP in MTHFR gene using fixed-cell suspension.

    PubMed

    Sinthuwiwat, Thivaratana; Poowasanpetch, Phanasit; Wongngamrungroj, Angsana; Promso, Somying; Auewarakul, Chirayu; Mooney, Sean; Tocharoentanaphol, Chintana

    2008-01-01

    Genetic variation in MTHFR might explain the interindividual differences in both therapeutic and toxic responses to the treatment of cancer and rheumatoid arthritis with methotrexate, and can be involved in the sensitivity of developing diseases like cancer and congenital anomalies. We investigated the common sequence variation, C677T, in the MTHFR gene in fixed-cell specimens archived after chromosomal analysis using a novel gene scanning method based on post PCR analysis of high-resolution melting curves (HRM). These fixed specimens were stored after routine chromosomal analysis for 1 year at -20 degrees C in a 3:1 methanol:acetic acid solution. The method revealed a distinct pattern between homozygous and heterozygous alleles. Sensitivity and specificity of the HRM based method were comparable to that obtained by a hybridization probe. While the success rate for genotyping of a common SNP in MTHFR was similar to the hybridization probe approach, the HRM based method was more cost-effective and had a shorter turnaround time. PMID:18725286

  11. Probe-free and sensitive detection of diarrhea-causing pathogens using RT-PCR combined high resolution melting analysis.

    PubMed

    Wang, Hai-Bo; Mo, Qiu-Hua; Wang, Qi; Wu, Bi-Mei; Feng, Zi-Li; Lin, Ji-Can; Yang, Ze

    2016-09-01

    Rapid and sensitive diagnostic methods are needed to help physicians make faster and better treatment decision for patients suffered from diarrhea. In the present study, a probe-free and sensitive RT-PCR combined high resolution melting analysis (HRMA) assay was established successfully for the detection of four major diarrhea-causing pathogens. The lower limit of detection of the assay were 10(0), 10(2), 10(0) and 10(3) copies/reaction for rotaviruses group A, astroviruses serotype 1, noroviruses genogroup II, and sapoviruses genegroup I, respectively, which were 1000-fold, 10-fold, 1000-fold and 10-fold more sensitive than conventional RT-PCR assay developed in parallel and comparable to or higher than commercially available real-time RT-PCR assay. Blinded sample evaluation showed that the assay was 100% concordant to both conventional RT-PCR and commercial real-time RT-PCR, indicating high reliability of the new assay. Therefore, the assay could provide a valuable platform for the probe-free and sensitive diagnosis of these pathogens. PMID:27461241

  12. Combined Use of Molecular Markers and High-Resolution Melting (HRM) to Assess Chromosome Dosage in Potato Hybrids.

    PubMed

    Villano, Clizia; Miraglia, Valeria; Iorizzo, Massimo; Aversano, Riccardo; Carputo, Domenico

    2016-03-01

    In plants, the most widely used cytological techniques to assess parental genome contributions are based on in situ hybridization (FISH and GISH), but they are time-consuming and need specific expertise and equipment. Recent advances in genomics and molecular biology have made PCR-based markers a straightforward, affordable technique for chromosome typing. Here, we describe the development of a molecular assay that uses single-copy conserved ortholog set II (COSII)-based single nucleotide polymorphisms (SNPs) and the high-resolution melting (HRM) technique to assess the chromosome dosage of interspecific hybrids between a Solanum phureja-S. tuberosum diploid (2n = 2x = 24) hybrid and its wild relative S. commersonii. Screening and analysis of 45 COSII marker sequences allowed S. commersonii-specific SNPs to be identified for all 12 chromosomes. Combining the HRM technique with the establishment of synthetic DNA hybrids, SNP markers were successfully used to predict the expected parental chromosome ratio of 5 interspecific triploid hybrids. These results demonstrate the ability of this strategy to distinguish diverged genomes from each other, and to estimate chromosome dosage. The method could potentially be applied to any species as a tool to assess paternal to maternal ratios in the framework of a breeding program or following transformation techniques. PMID:26663623

  13. High Resolution Melting Analysis Targeting hsp70 as a Fast and Efficient Method for the Discrimination of Leishmania Species

    PubMed Central

    Zampieri, Ricardo Andrade; Laranjeira-Silva, Maria Fernanda; Muxel, Sandra Marcia; Stocco de Lima, Ana Carolina; Shaw, Jeffrey Jon; Floeter-Winter, Lucile Maria

    2016-01-01

    Background Protozoan parasites of the genus Leishmania cause a large spectrum of clinical manifestations known as Leishmaniases. These diseases are increasingly important public health problems in many countries both within and outside endemic regions. Thus, an accurate differential diagnosis is extremely relevant for understanding epidemiological profiles and for the administration of the best therapeutic protocol. Methods/Principal Findings Exploring the High Resolution Melting (HRM) dissociation profiles of two amplicons using real time polymerase chain reaction (real-time PCR) targeting heat-shock protein 70 coding gene (hsp70) revealed differences that allowed the discrimination of genomic DNA samples of eight Leishmania species found in the Americas, including Leishmania (Leishmania) infantum chagasi, L. (L.) amazonensis, L. (L.) mexicana, L. (Viannia) lainsoni, L. (V.) braziliensis, L. (V.) guyanensis, L. (V.) naiffi and L. (V.) shawi, and three species found in Eurasia and Africa, including L. (L.) tropica, L. (L.) donovani and L. (L.) major. In addition, we tested DNA samples obtained from standard promastigote culture, naturally infected phlebotomines, experimentally infected mice and clinical human samples to validate the proposed protocol. Conclusions/Significance HRM analysis of hsp70 amplicons is a fast and robust strategy that allowed for the detection and discrimination of all Leishmania species responsible for the Leishmaniases in Brazil and Eurasia/Africa with high sensitivity and accuracy. This method could detect less than one parasite per reaction, even in the presence of host DNA. PMID:26928050

  14. Genotyping of the C742T mutation of the FGFR3 gene causing type 1 thanatophoric dysplasia by high-resolution melting analysis.

    PubMed

    Liu, Ying-Na; Li, Ru; Li, Dong-Zhi

    2011-01-01

    Type 1 thanatophoric dysplasia (TD) is typically a lethal dwarfism. It is not always possible to distinguish fetuses with TD from other skeletal dysplasia in utero by ultrasonography. A definite diagnosis should be established by molecular genetic analysis to find out the abnormal mutations in the fibroblast growth factor receptor 3 (FGFR 3) gene. Among the known mutations of this gene, the C742T (R248C) mutation is the most common one associated with type 1 TD. Exon 7 of the FGFR3 gene was analyzed in 10 prenatal samples with type 1 TD, as well as in 30 control individuals for the presence of the c.742 C > T variant using melting curve analysis with a high-resolution melting instrument. The high-resolution melting curve analysis successfully genotyped this mutation in all 10 samples with type 1 TD without the need of further assays. The technique had a sensitivity and specificity of 100%. This study suggest that high-resolution melting analysis is a simple, rapid, and sensitive one tube assay for genotyping the FGFR3 gene. PMID:20569165

  15. Detection and characterization of Leishmania (Leishmania) and Leishmania (Viannia) by SYBR green-based real-time PCR and high resolution melt analysis targeting kinetoplast minicircle DNA.

    PubMed

    Ceccarelli, Marcello; Galluzzi, Luca; Migliazzo, Antonella; Magnani, Mauro

    2014-01-01

    Leishmaniasis is a neglected disease with a broad clinical spectrum which includes asymptomatic infection. A thorough diagnosis, able to distinguish and quantify Leishmania parasites in a clinical sample, constitutes a key step in choosing an appropriate therapy, making an accurate prognosis and performing epidemiological studies. Several molecular techniques have been shown to be effective in the diagnosis of leishmaniasis. In particular, a number of PCR methods have been developed on various target DNA sequences including kinetoplast minicircle constant regions. The first aim of this study was to develop a SYBR green-based qPCR assay for Leishmania (Leishmania) infantum detection and quantification, using kinetoplast minicircle constant region as target. To this end, two assays were compared: the first used previously published primer pairs (qPCR1), whereas the second used a nested primer pairs generating a shorter PCR product (qPCR2). The second aim of this study was to evaluate the possibility to discriminate among subgenera Leishmania (Leishmania) and Leishmania (Viannia) using the qPCR2 assay followed by melting or High Resolution Melt (HRM) analysis. Both assays used in this study showed good sensitivity and specificity, and a good correlation with standard IFAT methods in 62 canine clinical samples. However, the qPCR2 assay allowed to discriminate between Leishmania (Leishmania) and Leishmania (Viannia) subgenera through melting or HRM analysis. In addition to developing assays, we investigated the number and genetic variability of kinetoplast minicircles in the Leishmania (L.) infantum WHO international reference strain (MHOM/TN/80/IPT1), highlighting the presence of minicircle subclasses and sequence heterogeneity. Specifically, the kinetoplast minicircle number per cell was estimated to be 26,566±1,192, while the subclass of minicircles amplifiable by qPCR2 was estimated to be 1,263±115. This heterogeneity, also observed in canine clinical samples

  16. High time-resolution photodetectors for PET applications

    SciTech Connect

    Ronzhin, Anatoly

    2015-11-27

    This paper describes recent developments aiming at the improvement of the time resolution of photodetectors used in positron emission tomography (PET). Promising photodetector candidates for future PET-time-of-flight (TOF) applications are also discussed.

  17. High time-resolution photodetectors for PET applications

    DOE PAGESBeta

    Ronzhin, Anatoly

    2016-02-01

    This paper describes recent developments aiming at the improvement of the time resolution of photodetectors used in positron emission tomography (PET). Promising photodetector candidates for future PET-time-of-flight (TOF) applications are also discussed.

  18. Applications of high-resolution remote sensing image data

    NASA Technical Reports Server (NTRS)

    Strome, W. M.; Leckie, D.; Miller, J.; Buxton, R.

    1990-01-01

    There are many situations in which the image resolution of satellite data is insufficient to provide the detail required for resource management and environmental monitoring. This paper will focus on applications of high-resolution (0.4 to 10 m) airborne multispectral and imaging spectrometer data acquired in Canada using the MEIS II multispectral line imager and the PMI imaging spectrometer. Applications discussed will include forestry, mapping, and geobotany.

  19. Multiplex Real-Time PCR Assay with High-Resolution Melting Analysis for Characterization of Antimicrobial Resistance in Neisseria gonorrhoeae.

    PubMed

    Donà, Valentina; Kasraian, Sara; Lupo, Agnese; Guilarte, Yuvia N; Hauser, Christoph; Furrer, Hansjakob; Unemo, Magnus; Low, Nicola; Endimiani, Andrea

    2016-08-01

    Resistance to antibiotics used against Neisseria gonorrhoeae infections is a major public health concern. Antimicrobial resistance (AMR) testing relies on time-consuming culture-based methods. Development of rapid molecular tests for detection of AMR determinants could provide valuable tools for surveillance and epidemiological studies and for informing individual case management. We developed a fast (<1.5-h) SYBR green-based real-time PCR method with high-resolution melting (HRM) analysis. One triplex and three duplex reactions included two sequences for N. gonorrhoeae identification and seven determinants of resistance to extended-spectrum cephalosporins (ESCs), azithromycin, ciprofloxacin, and spectinomycin. The method was validated by testing 39 previously fully characterized N. gonorrhoeae strains, 19 commensal Neisseria species strains, and an additional panel of 193 gonococcal isolates. Results were compared with results of culture-based AMR determination. The assay correctly identified N. gonorrhoeae and the presence or absence of the seven AMR determinants. There was some cross-reactivity with nongonococcal Neisseria species, and the detection limit was 10(3) to 10(4) genomic DNA (gDNA) copies/reaction. Overall, the platform accurately detected resistance to ciprofloxacin (sensitivity and specificity, 100%), ceftriaxone (sensitivity, 100%; specificity, 90%), cefixime (sensitivity, 92%; specificity, 94%), azithromycin (sensitivity and specificity, 100%), and spectinomycin (sensitivity and specificity, 100%). In conclusion, our methodology accurately detects mutations that generate resistance to antibiotics used to treat gonorrhea. Low assay sensitivity prevents direct diagnostic testing of clinical specimens, but this method can be used to screen collections of gonococcal isolates for AMR more quickly than current culture-based AMR testing. PMID:27225407

  20. Comparison of a High-Resolution Melting Assay to Next-Generation Sequencing for Analysis of HIV Diversity

    PubMed Central

    Cousins, Matthew M.; Ou, San-San; Wawer, Maria J.; Munshaw, Supriya; Swan, David; Magaret, Craig A.; Mullis, Caroline E.; Serwadda, David; Porcella, Stephen F.; Gray, Ronald H.; Quinn, Thomas C.; Donnell, Deborah; Eshleman, Susan H.

    2012-01-01

    Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution. PMID:22785188

  1. High resolution melting analysis of the NR1I3 genetic variants: Is there an association with neonatal hyperbilirubinemia?

    PubMed

    Cheung, Tian Pei; Van Rostenberghe, Hans; Ismail, Rosliza; Nawawi, Noor Namirah; Abdullah, Nurul Amierah; Ramli, Noraida; Ibrahim, Nor Rosidah; Hj Abd Majid, Noorizan; Mohd Yusoff, Narazah; Nishio, Hisahide; Yusoff, Surini

    2015-12-01

    Constitutive androstane receptor (CAR) encoded by the nuclear receptor subfamily 1, group I, member 3 (NR1I3) gene regulates the elimination of bilirubin through activating the components of the bilirubin clearance pathway. Hence, NR1I3 genetic variants may affect bilirubin metabolism and result in neonatal hyperbilirubinemia. Thus far, research which investigates the association between NR1I3 variants and neonatal hyperbilirubinemia has not been undertaken in any population. The present study aimed to evaluate the influence of MPJ6_1I3008 (rs10157822), IVS8+116T>G (rs4073054) and 540A>G (rs2307424) on neonatal hyperbilirubinemia development in the Malay population. Buccal swabs were collected from 232 hyperbilirubinemia and 277 control term newborns with gestational age ≥37weeks and birth weight ≥2500g. The NR1I3 variants were genotyped by using high resolution melting (HRM) assays and verified by DNA sequencing. Gender, mode of delivery and birth weight did not differ between hyperbilirubinemia and control groups. The genotypic and allelic frequencies of MPJ6_1I3008, IVS8+116T>G and 540A>G were not significantly different between the groups. However, stratification by gender revealed a significant inverse association between homozygous variant genotype of MPJ6_1I3008 and risk of neonatal hyperbilirubinemia in the females (OR, 0.44; 95% CI, 0.20-0.95; p=0.034). This study demonstrates that the homozygous variant genotype of MPJ6_1I3008 was associated with a significant reduced risk of neonatal hyperbilirubinemia in the females. PMID:26188155

  2. Identifying the last bloodmeal of questing sheep tick nymphs (Ixodes ricinus L.) using high resolution melting analysis.

    PubMed

    Collini, Margherita; Albonico, Francesca; Hauffe, Heidi C; Mortarino, Michele

    2015-06-15

    The sheep tick, Ixodes ricinus L., is an important hematophagous vector of zoonotic disease of both veterinary and public health importance in Europe. Risk models for tick-borne diseases can be improved by identifying the main hosts of this species in any given area. However, this generalist tick stays on a host for only a few days a year over its life cycle, making the study of its feeding ecology difficult. In contrast, ticks can easily be collected from vegetation when they are questing. Molecular methods have proved to be a reliable alternative to field observation, but most current methods have low sensitivity and/or low identification success (i.e. hosts are only identified to taxonomic levels higher than species). In this study we use Real-time PCR coupled with High Resolution Melting Analysis (HRMA) to identify the source of the last bloodmeal in questing tick nymphs. Twenty of the most important tick hosts were grouped taxonomically and six group-specific primer sets, targeting short mitochondrial DNA regions, were designed de novo. Firstly, we show that these primers successfully amplify target host DNA (from host tissue or engorged ticks), and that HRMA can be used to reliably identify hosts to species (or genera in the case of Sorex and Apodemus). Secondly, the new protocol was tested on field-collected questing nymphs. Bloodmeal source was identified in 65.4% of 52 individuals. In 83.3% of these, the host was identified to species or genera using HRMA alone. Moreover, the primer sets designed here can unequivocally identify mixed bloodmeals. The combination of sensitivity and identification success together with the closed-tube and single step approach that minimizes contamination, make Real-time HRMA a good alternative to current methods for bloodmeal identification. PMID:25941127

  3. Application of Markov Chain Monte Carlo Method to Mantle Melting: An Example from REE Abundances in Abyssal Peridotites

    NASA Astrophysics Data System (ADS)

    LIU, B.; Liang, Y.

    2015-12-01

    Markov chain Monte Carlo (MCMC) simulation is a powerful statistical method in solving inverse problems that arise from a wide range of applications, such as nuclear physics, computational biology, financial engineering, among others. In Earth sciences applications of MCMC are primarily in the field of geophysics [1]. The purpose of this study is to introduce MCMC to geochemical inverse problems related to trace element fractionation during concurrent melting, melt transport and melt-rock reaction in the mantle. MCMC method has several advantages over linearized least squares methods in inverting trace element patterns in basalts and mantle rocks. First, MCMC can handle equations that have no explicit analytical solutions which are required by linearized least squares methods for gradient calculation. Second, MCMC converges to global minimum while linearized least squares methods may be stuck at a local minimum or converge slowly due to nonlinearity. Furthermore, MCMC can provide insight into uncertainties of model parameters with non-normal trade-off. We use MCMC to invert for extent of melting, amount of trapped melt, and extent of chemical disequilibrium between the melt and residual solid from REE data in abyssal peridotites from Central Indian Ridge and Mid-Atlantic Ridge. In the first step, we conduct forward calculation of REE evolution with melting models in a reasonable model space. We then build up a chain of melting models according to Metropolis-Hastings algorithm to represent the probability of specific model. We show that chemical disequilibrium is likely to play an important role in fractionating LREE in residual peridotites. In the future, MCMC will be applied to more realistic but also more complicated melting models in which partition coefficients, diffusion coefficients, as well as melting and melt suction rates vary as functions of temperature, pressure and mineral compositions. [1]. Sambridge & Mosegarrd [2002] Rev. Geophys.

  4. Applications of high resolution inverse Raman spectroscopy

    SciTech Connect

    Owyoung, A.; Esherick, P.

    1980-01-01

    The use of high-power, narrow-band lasers has significantly improved the resolving power and sensitivity of inverse Raman spectroscopy of gases. In this paper we shall describe this technique, illustrate its capabilities by showing some Q-branch spectra of heavy spherical tops, and survey some possible future applications.

  5. High-resolution melting analysis of the spa locus reveals significant diversity within sequence type 93 methicillin-resistant Staphylococcus aureus from northern Australia.

    PubMed

    Tong, S Y C; Lilliebridge, R A; Holt, D C; McDonald, M I; Currie, B J; Giffard, P M

    2009-12-01

    High-resolution melting analysis is an inherently robust, easy and inexpensive approach to the examination of genomic regions containing single-nucleotide polymorphisms and hypervariable loci. Staphylococcus aureus sequence type (ST) 93 is a singleton, Panton-Valentine leukocidin-positive clone unique to Australia. A high-resolution melting-based method for the identification of ST93 was developed, and a similar approach was used to reveal diversity within the spa locus of this lineage. Statistical and graphical methods that account for instrumental and operator-dependent variation in high-resolution melting curves were developed, to allow greater confidence and reproducibility in deciding whether another curve is truly different from the baseline curve of an amplicon with known sequence. The data support a very early acquisition, or multiple independent acquisitions, of SCCmec by ST93 methicillin-susceptible S. aureus (MSSA), and the coexistence of MSSA and methicillin-resistant S. aureus versions of the same lineage within northern Australia. PMID:19392885

  6. Sensitive Detection and Serovar Differentiation of Typhoidal and Nontyphoidal Salmonella enterica Species Using 16S rRNA Gene PCR Coupled with High-Resolution Melt Analysis

    PubMed Central

    Masek, Billie J.; Hardick, Justin; Won, Helen; Yang, Samuel; Hsieh, Yu-Hsiang; Rothman, Richard E.; Gaydos, Charlotte A.

    2015-01-01

    Salmonella enterica species infections are a significant public health problem causing high morbidity rates worldwide and high mortality rates in the developing world. These infections are not always rapidly diagnosed as a cause of bloodstream infections because of the limitations of blood culture, which greatly affects clinical care as a result of treatment delays. A molecular diagnostic assay that could rapidly detect and identify S. enterica species infections as a cause of sepsis is needed. Nine typhoidal and nontyphoidal S. enterica serovars were used to establish the limit of detection (LOD) of a previously published 16S rRNA gene PCR (16S PCR) in mock whole blood specimens. In addition, 16 typhoidal and nontyphoidal S. enterica serovars were used to evaluate the serovar differentiation capability of 16S PCR coupled with high-resolution melt analysis. The overall LOD of 16S PCR for the nine typhoidal and nontyphoidal S. enterica serovars analyzed was <10 colony-forming units per milliliter (CFU/mL) in mock whole blood specimens, with the lowest and highest LOD at <1 CFU/mL and 9 CFU/mL, respectively. By high-resolution melt analysis, the typhoidal and nontyphoidal S. enterica serovar groups analyzed each generated a unique grouping code, allowing for serovar-level identification. 16S PCR coupled with high-resolution melt analysis could be a useful molecular diagnostic that could enhance the current diagnostic, treatment, and surveillance methods of S. enterica bloodstream infections. PMID:24365382

  7. Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis.

    PubMed

    Hong, Yanbin; Pandey, Manish K; Liu, Ying; Chen, Xiaoping; Liu, Hong; Varshney, Rajeev K; Liang, Xuanqiang; Huang, Shangzhi

    2015-01-01

    The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut. PMID:26697032

  8. Towards a Pathogenic Escherichia coli Detection Platform Using Multiplex SYBR®Green Real-Time PCR Methods and High Resolution Melting Analysis

    PubMed Central

    Kagkli, Dafni-Maria; Folloni, Silvia; Barbau-Piednoir, Elodie; Van den Eede, Guy; Van den Bulcke, Marc

    2012-01-01

    Escherichia coli is a group of bacteria which has raised a lot of safety concerns in recent years. Five major intestinal pathogenic groups have been recognized amongst which the verocytotoxin or shiga-toxin (stx1 and/or stx2) producing E. coli (VTEC or STEC respectively) have received a lot of attention recently. Indeed, due to the high number of outbreaks related to VTEC strains, the European Food Safety Authority (EFSA) has requested the monitoring of the “top-five” serogroups (O26, O103, O111, O145 and O157) most often encountered in food borne diseases and addressed the need for validated VTEC detection methods. Here we report the development of a set of intercalating dye Real-time PCR methods capable of rapidly detecting the presence of the toxin genes together with intimin (eae) in the case of VTEC, or aggregative protein (aggR), in the case of the O104:H4 strain responsible for the outbreak in Germany in 2011. All reactions were optimized to perform at the same annealing temperature permitting the multiplex application in order to minimize the need of material and to allow for high-throughput analysis. In addition, High Resolution Melting (HRM) analysis allowing the discrimination among strains possessing similar virulence traits was established. The development, application to food samples and the flexibility in use of the methods are thoroughly discussed. Together, these Real-time PCR methods facilitate the detection of VTEC in a new highly efficient way and could represent the basis for developing a simple pathogenic E. coli platform. PMID:22761753

  9. Identification and Evaluation of Single-Nucleotide Polymorphisms in Allotetraploid Peanut (Arachis hypogaea L.) Based on Amplicon Sequencing Combined with High Resolution Melting (HRM) Analysis

    PubMed Central

    Hong, Yanbin; Pandey, Manish K.; Liu, Ying; Chen, Xiaoping; Liu, Hong; Varshney, Rajeev K.; Liang, Xuanqiang; Huang, Shangzhi

    2015-01-01

    The cultivated peanut (Arachis hypogaea L.) is an allotetraploid (AABB) species derived from the A-genome (Arachis duranensis) and B-genome (Arachis ipaensis) progenitors. Presence of two versions of a DNA sequence based on the two progenitor genomes poses a serious technical and analytical problem during single nucleotide polymorphism (SNP) marker identification and analysis. In this context, we have analyzed 200 amplicons derived from expressed sequence tags (ESTs) and genome survey sequences (GSS) to identify SNPs in a panel of genotypes consisting of 12 cultivated peanut varieties and two diploid progenitors representing the ancestral genomes. A total of 18 EST-SNPs and 44 genomic-SNPs were identified in 12 peanut varieties by aligning the sequence of A. hypogaea with diploid progenitors. The average frequency of sequence polymorphism was higher for genomic-SNPs than the EST-SNPs with one genomic-SNP every 1011 bp as compared to one EST-SNP every 2557 bp. In order to estimate the potential and further applicability of these identified SNPs, 96 peanut varieties were genotyped using high resolution melting (HRM) method. Polymorphism information content (PIC) values for EST-SNPs ranged between 0.021 and 0.413 with a mean of 0.172 in the set of peanut varieties, while genomic-SNPs ranged between 0.080 and 0.478 with a mean of 0.249. Total 33 SNPs were used for polymorphism detection among the parents and 10 selected lines from mapping population Y13Zh (Zhenzhuhei × Yueyou13). Of the total 33 SNPs, nine SNPs showed polymorphism in the mapping population Y13Zh, and seven SNPs were successfully mapped into five linkage groups. Our results showed that SNPs can be identified in allotetraploid peanut with high accuracy through amplicon sequencing and HRM assay. The identified SNPs were very informative and can be used for different genetic and breeding applications in peanut. PMID:26697032

  10. Strong thermodynamic coupling between sub-ice-shelf melting and sea ice in a high-resolution global sea-ice-ocean isopycnal model

    NASA Astrophysics Data System (ADS)

    Sergienko, O. V.

    2015-12-01

    Sub-ice-shelf melting(freezing) of the Antarctic ice shelves acts as a source(sink) of freshwater, therefore, affects ocean water properties and circulation. In its turn, sub-ice-shelf melting/freezing is controlled by the ocean water properties that reach the sub-ice-shelf cavities. The properties of these water masses are determined by heat and fresh-water exchange with sea ice and atmosphere. Simulations of a high-resolution (1/8 deg) global sea-ice-ocean isopycnal model capable to resolve the ocean circulation in sub-ice-shelf cavities of Antarctic ice shelves and account for the thermodynamic interaction of the circulation with ice shelves show that melting/freezing rates have a strong seasonal cycle with highest melting rates observed in the Austral Fall. On the continental shelf, subsurface ocean temperatures (100-300 m) have a similar seasonal cycle which is lagged with respect to the surface. Shelf temperatures peak in the summertime, followed by rapid cooling towards the freezing point as seasonal ice cover increases. The lagged warming in the subsurface is attributable to reduced heat loss to the atmosphere in the presence of seasonal sea ice. This suggests that the seasonal cycle in melt rates is controlled by the phasing of subsurface temperatures on the continental shelf, which is in turn dominated by sea ice. The outflowing fresh, cold and light meltwater formed in sub-ice-shelf cavities remains in the mixed layer and promotes formation of sea ice and its longer persistence into the Austral Summer. These processes suggest the presence of strong mutual feedbacks between sub-ice-shelf melting and sea ice formation around Antarctic ice shelves.

  11. High-resolution melting analysis (HRM) for differentiation of four major Taeniidae species in dogs Taenia hydatigena, Taenia multiceps, Taenia ovis, and Echinococcus granulosus sensu stricto.

    PubMed

    Dehghani, Mansoureh; Mohammadi, Mohammad Ali; Rostami, Sima; Shamsaddini, Saeedeh; Mirbadie, Seyed Reza; Harandi, Majid Fasihi

    2016-07-01

    Tapeworms of the genus Taenia include several species of important parasites with considerable medical and veterinary significance. Accurate identification of these species in dogs is the prerequisite of any prevention and control program. Here, we have applied an efficient method for differentiating four major Taeniid species in dogs, i.e., Taenia hydatigena, T. multiceps, T. ovis, and Echinococcus granulosus sensu stricto. High-resolution melting (HRM) analysis is simpler, less expensive, and faster technique than conventional DNA-based assays and enables us to detect PCR amplicons in a closed system. Metacestode samples were collected from local abattoirs from sheep. All the isolates had already been identified by PCR-sequencing, and their sequence data were deposited in the GenBank. Real-time PCR coupled with HRM analysis targeting mitochondrial cox1 and ITS1 genes was used to differentiate taeniid species. Distinct melting curves were obtained from ITS1 region enabling accurate differentiation of three Taenia species and E. granulosus in dogs. The HRM curves of Taenia species and E .granulosus were clearly separated at Tm of 85 to 87 °C. In addition, double-pick melting curves were produced in mixed infections. Cox1 melting curves were not decisive enough to distinguish four taeniids. In this work, the efficiency of HRM analysis to differentiate four major taeniid species in dogs has been demonstrated using ITS1 gene. PMID:27008188

  12. Detection of plant oil DNA using high resolution melting (HRM) post PCR analysis: a tool for disclosure of olive oil adulteration.

    PubMed

    Vietina, Michelangelo; Agrimonti, Caterina; Marmiroli, Nelson

    2013-12-15

    Extra virgin olive oil is frequently subjected to adulterations with addition of oils obtained from plants other than olive. DNA analysis is a fast and economic tool to identify plant components in oils. Extraction and amplification of DNA by PCR was tested in olives, in milled seeds and in oils, to investigate its use in olive oil traceability. DNA was extracted from different oils made of hazelnut, maize, sunflower, peanut, sesame, soybean, rice and pumpkin. Comparing the DNA melting profiles in reference plant materials and in the oils, it was possible to identify any plant components in oils and mixtures of oils. Real-Time PCR (RT-PCR) platform has been added of the new methodology of high resolution melting (HRM), both were used to analyse olive oils mixed with different percentage of other oils. Results showed HRM a cost effective method for efficient detection of adulterations in olive oils. PMID:23993554

  13. The utility of high-resolution melting analysis of SNP nucleated PCR amplicons--an MLST based Staphylococcus aureus typing scheme.

    PubMed

    Lilliebridge, Rachael A; Tong, Steven Y C; Giffard, Philip M; Holt, Deborah C

    2011-01-01

    High resolution melting (HRM) analysis is gaining prominence as a method for discriminating DNA sequence variants. Its advantage is that it is performed in a real-time PCR device, and the PCR amplification and HRM analysis are closed tube, and effectively single step. We have developed an HRM-based method for Staphylococcus aureus genotyping. Eight single nucleotide polymorphisms (SNPs) were derived from the S. aureus multi-locus sequence typing (MLST) database on the basis of maximized Simpson's Index of Diversity. Only G↔A, G↔T, C↔A, C↔T SNPs were considered for inclusion, to facilitate allele discrimination by HRM. In silico experiments revealed that DNA fragments incorporating the SNPs give much higher resolving power than randomly selected fragments. It was shown that the predicted optimum fragment size for HRM analysis was 200 bp, and that other SNPs within the fragments contribute to the resolving power. Six DNA fragments ranging from 83 bp to 219 bp, incorporating the resolution optimized SNPs were designed. HRM analysis of these fragments using 94 diverse S. aureus isolates of known sequence type or clonal complex (CC) revealed that sequence variants are resolved largely in accordance with G+C content. A combination of experimental results and in silico prediction indicates that HRM analysis resolves S. aureus into 268 "melt types" (MelTs), and provides a Simpson's Index of Diversity of 0.978 with respect to MLST. There is a high concordance between HRM analysis and the MLST defined CCs. We have generated a Microsoft Excel key which facilitates data interpretation and translation between MelT and MLST data. The potential of this approach for genotyping other bacterial pathogens was investigated using a computerized approach to estimate the densities of SNPs with unlinked allelic states. The MLST databases for all species tested contained abundant unlinked SNPs, thus suggesting that high resolving power is not dependent upon large numbers of SNPs

  14. Hybrid analysis (barcode-high resolution melting) for authentication of Thai herbal products, Andrographis paniculata (Burm.f.) Wall.ex Nees

    PubMed Central

    Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Khamyong, Nuttaluck; Pintakum, Danupol; Lamphun, Santisuk Na; Triwitayakorn, Kanokporn; Osathanunkul, Kitisak; Madesis, Panagiotis

    2016-01-01

    Background: Andrographis paniculata Nees is a medicinal plant with multiple pharmacological properties. It has been used over many centuries as a household remedy. A. paniculata products sold on the markets are in processed forms so it is difficult to authenticate. Therefore buying the herbal products poses a high-risk of acquiring counterfeited, substituted and/or adulterated products. Due to these issues, a reliable method to authenticate products is needed. Materials and Methods: High resolution melting analysis coupled with DNA barcoding (Bar-HRM) was applied to detect adulteration in commercial herbal products. The rbcL barcode was selected to use in primers design for HRM analysis to produce standard melting profile of A. paniculata species. DNA of the tested commercial products was isolated and their melting profiles were then generated and compared with the standard A. paniculata. Results: The melting profiles of the rbcL amplicons of the three closely related herbal species (A. paniculata, Acanthus ebracteatus and Rhinacanthus nasutus) are clearly separated so that they can be distinguished by the developed method. The method was then used to authenticate commercial herbal products. HRM curves of all 10 samples tested are similar to A. paniculata which indicated that all tested products were contained the correct species as labeled. Conclusion: The method described in this study has been proved to be useful in aiding identification and/or authenticating A. paniculata. This Bar-HRM analysis has allowed us easily to determine the A. paniculata species in herbal products on the markets even they are in processed forms. SUMMARY We propose the use of DNA barcoding combined with High Resolution Melting analysis for authenticating of Andrographis paniculata products.The developed method can be used regardless of the type of the DNA template (fresh or dried tissue, leaf, and stem).rbcL region was chosen for the analysis and work well with our samplesWe can easily

  15. Hot-melt extrusion of polyvinyl alcohol for oral immediate release applications.

    PubMed

    De Jaeghere, W; De Beer, T; Van Bocxlaer, J; Remon, J P; Vervaet, C

    2015-08-15

    The primary purpose of this study was to process partially hydrolyzed PVOH grades (degree of hydroxylation (DH): 33-88%) via HME and to evaluate them as carrier for oral immediate release dosage forms in order to improve the release rate of poorly water soluble drugs (i.e., HCT and CEL) via the formulation of solid dispersions. PVOH grades (DH >70%) were able to solubilize HCT and CEL up to 15%, but required higher extrusion temperature, due to the crystalline nature of PVOH. The highest drug release rate was observed from hot-melt extruded PVOH samples with a high DH. While drug release from extrudates consisting of PVOH with a low DH was affected by ionic strength, there was no influence of pH and ionic strength on HCT release from PVOH samples with a higher DH. However, PVOH (DH >70%) required higher extrusion temperatures, which could hamper its application for thermosensitive drugs. Therefore, the secondary purpose was to investigate the effect of sorbitol, a water-soluble plasticizer, on the thermal properties of hot-melt extruded PVOH (DH >70%). The melting of PVOH/sorbitol mixture was required to establish molecular interactions between PVOH and sorbitol. These molecular interactions were reflected in the HME behavior: whereas an extrusion temperature of 180 °C was necessary to process physical mixtures of PVOH (DH >70%) and sorbitol, only 140 °C was necessary during re-extrusion (after quench cooling and cryomilling) of the PVOH/sorbitol mixture. In addition, the in vitro and in vivo dug release of plasticized PVOH was examined; whereas the CEL/PVO/sorbitol system was able to maintain supersaturation during in vitro dissolution (0.1N HCl) compared to Celebrex(®), the in vivo pharmacokinetic parameters (AUC0-24h, Cmax and Tmax) were highly comparable. PMID:26160667

  16. Hot-Melt Extrusion: from Theory to Application in Pharmaceutical Formulation.

    PubMed

    Patil, Hemlata; Tiwari, Roshan V; Repka, Michael A

    2016-02-01

    Hot-melt extrusion (HME) is a promising technology for the production of new chemical entities in the developmental pipeline and for improving products already on the market. In drug discovery and development, industry estimates that more than 50% of active pharmaceutical ingredients currently used belong to the biopharmaceutical classification system II (BCS class II), which are characterized as poorly water-soluble compounds and result in formulations with low bioavailability. Therefore, there is a critical need for the pharmaceutical industry to develop formulations that will enhance the solubility and ultimately the bioavailability of these compounds. HME technology also offers an opportunity to earn intellectual property, which is evident from an increasing number of patents and publications that have included it as a novel pharmaceutical formulation technology over the past decades. This review had a threefold objective. First, it sought to provide an overview of HME principles and present detailed engineered extrusion equipment designs. Second, it included a number of published reports on the application of HME techniques that covered the fields of solid dispersions, microencapsulation, taste masking, targeted drug delivery systems, sustained release, films, nanotechnology, floating drug delivery systems, implants, and continuous manufacturing using the wet granulation process. Lastly, this review discussed the importance of using the quality by design approach in drug development, evaluated the process analytical technology used in pharmaceutical HME monitoring and control, discussed techniques used in HME, and emphasized the potential for monitoring and controlling hot-melt technology. PMID:26159653

  17. Applications of super-resolution and deblurring to practical sensors

    NASA Astrophysics Data System (ADS)

    Young, S. Susan; Sims, Richard; Kraples, Keith; Waterman, James; Smith, Leslie; Jacobs, Eddie; Corbin, Ted; Larsen, Louis; Driggers, Ronald G.

    2008-04-01

    In image formation and recording process, there are many factors that affect sensor performance and image quality that result in loss of high-frequency information. Two of these common factors are undersampled sensors and sensor's blurring function. Two image processing algorithms, including super-resolution image reconstruction and deblur filtering, have been developed based on characterizing the sources of image degradation from image formation and recording process. In this paper, we discuss the applications of these two algorithms to three practical thermal imaging systems. First, super-resolution and deblurring are applied to a longwave uncooled sensor in a missile seeker. Target resolution is improved in the flight phase of the seeker operation. Second, these two algorithms are applied to a midwave target acquisition sensor for use in long-range target identification. Third, the two algorithms are applied to a naval midwave distributed aperture sensor (DAS) for infrared search and track (IRST) system that is dual use in missile detection and force protection/anti-terrorism applications. In this case, super-resolution and deblurring are used to improve the resolution of on-deck activity discrimination.

  18. Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR.

    PubMed

    Thanchomnang, Tongjit; Intapan, Pewpan M; Tantrawatpan, Chairat; Lulitanond, Viraphong; Chungpivat, Sudchit; Taweethavonsawat, Piyanan; Kaewkong, Worasak; Sanpool, Oranuch; Janwan, Penchom; Choochote, Wej; Maleewong, Wanchai

    2013-12-01

    A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors. PMID:24516268

  19. Allele-specific extension allows base-pair neutral homozygotes to be discriminated by high-resolution melting of small amplicons.

    PubMed

    Cai, Yanning; Yuan, Yanpeng; Lin, Qingling; Chan, Piu

    2010-11-01

    Not all single-nucleotide polymorphisms (SNPs) can be determined using high-resolution melting (HRM) of small amplicons, especially class 3 and 4 SNPs. This is due mainly to the small shift in the melting temperature (Tm) between two types of homozygote. Choosing rs1869458 (a class 4 SNP) as a sample, we developed a modified small amplicon HRM assay. An allele-specific extension (ASE) primer, which ended at an SNP site and matched only one of the alleles, was added to the reaction as well as additional thermal steps for ASE. Following asymmetric polymerase chain reaction and melting curve analysis, heterozygotes were easily identified. Two types of homozygote were also distinguishable, indicating that extension primers 11 to 13 bases in length worked efficiently in an allele-specific way. Modification of the limiting amplification primer with locked nucleic acid increased the Tm difference between extension and amplification peaks and facilitated subsequent genotyping. In addition, 194 human genomic DNA samples were genotyped with the developed assay and by direct sequencing, with the different methods providing identical genotyping results. In conclusion, ASE-HRM is a simple, inexpensive, closed-tube genotyping method that can be used to examine all types of SNP. PMID:20599636

  20. Rapid Detection and Identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in Mosquito Vectors and Blood Samples by High Resolution Melting Real-Time PCR

    PubMed Central

    Thanchomnang, Tongjit; Intapan, Pewpan M.; Tantrawatpan, Chairat; Lulitanond, Viraphong; Chungpivat, Sudchit; Taweethavonsawat, Piyanan; Kaewkong, Worasak; Sanpool, Oranuch; Janwan, Penchom; Choochote, Wej

    2013-01-01

    A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors. PMID:24516268

  1. Polymeric formulations for drug release prepared by hot melt extrusion: application and characterization.

    PubMed

    Stanković, Milica; Frijlink, Henderik W; Hinrichs, Wouter L J

    2015-07-01

    Over the past few decades hot melt extrusion (HME) has emerged as a powerful processing technology for the production of pharmaceutical solid dosage forms in which an active pharmaceutical ingredient (API) is dispersed into polymer matrices. It has been shown that formulations using HME can provide time-controlled, sustained and targeted drug delivery, and improved bioavailability of poorly soluble drugs. In this review, the basic principles of the HME process are described together with an overview of some of the most common biodegradable and nonbiodegradable polymers used for the preparation of different formulations using this method. Further, the applications of HME in drug delivery and analytical techniques employed to characterize HME products are addressed. PMID:25660507

  2. Application of hot melt extrusion for poorly water-soluble drugs: limitations, advances and future prospects.

    PubMed

    Lu, Ming; Guo, Zhefei; Li, Yongcheng; Pang, Huishi; Lin, Ling; Liu, Xu; Pan, Xin; Wu, Chuanbin

    2014-01-01

    Hot melt extrusion (HME) is a powerful technology to enhance the solubility and bioavailability of poorly water-soluble drugs by producing amorphous solid dispersions. Although the number of articles and patents about HME increased dramatically in the past twenty years, there are very few commercial products by far. The three main obstacles limiting the commercial application of HME are summarized as thermal degradation of heat-sensitive drugs at high process temperature, recrystallization of amorphous drugs during storage and dissolving process, and difficulty to obtain products with reproducible physicochemical properties. Many efforts have been taken in recent years to understand the basic mechanism underlying these obstacles and then to overcome them. This article reviewed and summarized the limitations, recent advances, and future prospects of HME. PMID:23651401

  3. Rapid Detection and Statistical Differentiation of KPC Gene Variants in Gram-Negative Pathogens by Use of High-Resolution Melting and ScreenClust Analyses

    PubMed Central

    Roth, Amanda L.

    2013-01-01

    In the United States, the production of the Klebsiella pneumoniae carbapenemase (KPC) is an important mechanism of carbapenem resistance in Gram-negative pathogens. Infections with KPC-producing organisms are associated with increased morbidity and mortality; therefore, the rapid detection of KPC-producing pathogens is critical in patient care and infection control. We developed a real-time PCR assay complemented with traditional high-resolution melting (HRM) analysis, as well as statistically based genotyping, using the Rotor-Gene ScreenClust HRM software to both detect the presence of blaKPC and differentiate between KPC-2-like and KPC-3-like alleles. A total of 166 clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii with various β-lactamase susceptibility patterns were tested in the validation of this assay; 66 of these organisms were known to produce the KPC β-lactamase. The real-time PCR assay was able to detect the presence of blaKPC in all 66 of these clinical isolates (100% sensitivity and specificity). HRM analysis demonstrated that 26 had KPC-2-like melting peak temperatures, while 40 had KPC-3-like melting peak temperatures. Sequencing of 21 amplified products confirmed the melting peak results, with 9 isolates carrying blaKPC-2 and 12 isolates carrying blaKPC-3. This PCR/HRM assay can identify KPC-producing Gram-negative pathogens in as little as 3 h after isolation of pure colonies and does not require post-PCR sample manipulation for HRM analysis, and ScreenClust analysis easily distinguishes blaKPC-2-like and blaKPC-3-like alleles. Therefore, this assay is a rapid method to identify the presence of blaKPC enzymes in Gram-negative pathogens that can be easily integrated into busy clinical microbiology laboratories. PMID:23077125

  4. A novel gap-PCR with high resolution melting analysis for the detection of α-thalassaemia Southeast Asian and Filipino β0-thalassaemia deletion

    PubMed Central

    Leng Kho, Siew; Heng Chua, Kek; George, Elizabeth; Tan, Jin Ai Mary Anne

    2015-01-01

    Homozygosity for the α-thalassaemia Southeast Asian (α-SEA) and Filipino β0-thalassaemia (β-FIL) deletions can cause serious complications leading to foetal death or life-long blood transfusions. A rapid and accurate molecular detection assay is essential in populations where the deletions are common. In this study, gap-polymerase chain reaction (PCR) with high resolution melting (HRM) analysis was developed to detect both the large deletions. Melting curves at 86.9 ± 0.1 °C were generated by normal individuals without the α-SEA deletion, 84.7 ± 0.1 °C by homozygous α-SEA deletion individuals and two melting curves at 84.7 ± 0.1 °C and 86.9 ± 0.1 °C by α-SEA deletion carriers. Normal individuals without the β-FIL deletion produce amplicons with a melting temperature (Tm) at 74.6 ± 0.1 °C, homozygous β-FIL individuals produce amplicons with Tm at 73.6 ± 0.1 °C and heterozygous β-FIL individuals generate two amplicons with Tm at 73.6 ± 0.1 °C and 74.6 ± 0.1 °C. Evaluation using blinded tests on 220 DNA samples showed 100% sensitivity and specificity. The developed assays are sensitive and specific for rapid molecular and prenatal diagnosis for the α-SEA and β-FIL deletions. PMID:26365497

  5. A novel gap-PCR with high resolution melting analysis for the detection of α-thalassaemia Southeast Asian and Filipino β°-thalassaemia deletion.

    PubMed

    Kho, Siew Leng; Chua, Kek Heng; George, Elizabeth; Tan, Jin Ai Mary Anne

    2015-01-01

    Homozygosity for the α-thalassaemia Southeast Asian (α-SEA) and Filipino β°-thalassaemia (β-FIL) deletions can cause serious complications leading to foetal death or life-long blood transfusions. A rapid and accurate molecular detection assay is essential in populations where the deletions are common. In this study, gap-polymerase chain reaction (PCR) with high resolution melting (HRM) analysis was developed to detect both the large deletions. Melting curves at 86.9 ± 0.1 °C were generated by normal individuals without the α-SEA deletion, 84.7 ± 0.1 °C by homozygous α-SEA deletion individuals and two melting curves at 84.7 ± 0.1 °C and 86.9 ± 0.1 °C by α-SEA deletion carriers. Normal individuals without the β-FIL deletion produce amplicons with a melting temperature (Tm) at 74.6 ± 0.1 °C, homozygous β-FIL individuals produce amplicons with Tm at 73.6 ± 0.1 °C and heterozygous β-FIL individuals generate two amplicons with Tm at 73.6 ± 0.1 °C and 74.6 ± 0.1 °C. Evaluation using blinded tests on 220 DNA samples showed 100% sensitivity and specificity. The developed assays are sensitive and specific for rapid molecular and prenatal diagnosis for the α-SEA and β-FIL deletions. PMID:26365497

  6. Melt-growth bulk superconductors and application to an axial-gap-type rotating machine

    NASA Astrophysics Data System (ADS)

    Zhang, Yufeng; Zhou, Difan; Ida, Tetsuya; Miki, Motohiro; Izumi, Mitsuru

    2016-04-01

    The present manuscript addresses key issues in the course of our study of materials processing of bulk high-temperature superconductors, trapped flux and its application to a prototype axial-gap-type rotating machine. The TUMSAT group has conducted a series of studies since 2003 on the growth of GdBa2Cu3O7-δ bulk material and its application in a compact low-speed high-torque rotating machine. In the stage of material growth, gaining the advantage of a large motive torque density requires large integrated flux in the motor/generators. A large grain surface might be required with sophisticated techniques for the melt-growth texture in the bulk with optimal flux pinning. In the second stage, the in situ magnetization procedure for bulk superconductors in the applied machine is a crucial part of the technology. Pulsed current excitation by using an armature copper winding has magnetized field pole bulks on the rotor. The axial-gap flux synchronous machine studied in the past decade is a condensed technology and indicates that further scientific development is required for a future compact machine to be superior to conventional ones in accordance with the cryogenic periphery and flux stabilization.

  7. Amundsen Sea sector ice shelf thickness, melt rates, and inland response from annual high-resolution DEM mosaics

    NASA Astrophysics Data System (ADS)

    Shean, D. E.; Joughin, I. R.; Smith, B. E.; Alexandrov, O.; Moratto, Z.; Porter, C. C.; Morin, P. J.

    2014-12-01

    Significant grounding line retreat, acceleration, and thinning have occurred along the Amundsen Sea sector of West Antarctica in recent decades. These changes are driven primarily by ice-ocean interaction beneath ice shelves, but existing observations of the spatial distribution, timing, and magnitude of ice shelf melt are limited. Using the NASA Ames Stereo Pipeline, we generated digital elevation models (DEMs) with ~2 m posting from all ~450 available WorldView-1/2 along-track stereopairs for the Amundsen Sea sector. A novel iterative closest point algorithm was used to coregister DEMs to filtered Operation IceBridge ATM/LVIS data and ICESat-1 GLAS data, offering optimal sub-meter horizontal/vertical accuracy. The corrected DEMs were used to produce annual mosaics for the entire ~500x700 km region with focused, sub-annual products for ice shelves and grounding zones. These mosaics provide spatially-continuous measurements of ice shelf topography with unprecedented detail. Using these data, we derive estimates of ice shelf thickness for regions in hydrostatic equilibrium and map networks of sub-shelf melt channels for the Pine Island (PIG), Thwaites, Crosson, and Dotson ice shelves. We also document the break-up of the Thwaites ice shelf and PIG rift evolution leading up to the 2013 calving event. Eulerian difference maps document 2010-2014 thinning over fast-flowing ice streams and adjacent grounded ice. These data reveal the greatest thinning rates over the Smith Glacier ice plain and slopes beyond the margins of the fast-flowing PIG trunk. Difference maps also highlight the filling of at least two subglacial lakes ~30 km upstream of the PIG grounding line in 2011. Lagrangian difference maps reveal the spatial distribution of ice shelf thinning, which can primarily be attributed to basal melt. Preliminary results show focused ice shelf thinning within troughs and large basal channels, especially along the western margin of the Dotson ice shelf. These new data

  8. Impact of melt segregation on chemical composition with application to deep crustal hot zones

    NASA Astrophysics Data System (ADS)

    Solano, J.; Jackson, M.; Sparks, R. S.; Blundy, J. D.

    2010-12-01

    Models of heat transfer during the emplacement of mantle-derived basaltic sills in the mid- to lower crust demonstrate that large volumes of evolved melt may be generated in deep crustal hot zones (DCHZ). These models consider only the thermal evolution of a DCHZ, yet melt must also segregate from along the grain boundaries where it initially resides to form a magma which leaves the DCHZ. However, models which include melt migration describe phase change using simple melt fraction-temperature relations, which do not capture the impact of melt segregation on the chemical evolution of melt and residual solid. We present a model of melting and buoyancy-driven melt segregation in which phase change is described using a phase diagram and the chemical evolution of the melt and residual solid is properly captured. Melt migration is assumed to occur along grain boundaries so local thermodynamic equilibrium is maintained. We begin by using a simple binary phase diagram and model a 1-D column with several different initial compositions and thermal boundary conditions. We investigate this simple case because it could be closely replicated in the laboratory, and allows aspects of the physics which hitherto have been poorly understood to be clearly observed and explained. It is trivial to extend our model to more complex systems. For an initially homogenous column, in which the fraction of component A is less than the eutectic composition, we find that the melt fraction at the base decreases and the bulk composition becomes enriched in component A, while the melt fraction at the top increases and the bulk composition tends towards the eutectic composition. Melt segregation provides a mechanism for accumulating melt of (or close to) the eutectic composition, but at much higher melt fractions than predicted by purely thermal models; for example, static melting to 10% may yield the eutectic composition, but melt segregation allows that composition to accumulate to 100%. For a

  9. High-resolution adaptive holographic interferometer for biomedical applications

    NASA Astrophysics Data System (ADS)

    Dovgalenko, George; Dagdanova, Ayuna

    2007-07-01

    We realized new adaptive holographic sensor and interferometer, which allows to visualize high-resolution 3D images of diffuse reflected objects in Continue Hologram Registration Regime- CHRR. The coupled laser wave nonlinear theory was applied for optimization of hologram recording in crystals symmetry 23 and optimized experimental set up. Experimentally demonstrated dynamical holographic image sensors on doped 23 symmetry photosensitive crystals, with resolution 7900-lines/mm at 632 nm and 11641 lines/mm at 440 nm for 15 mW CW HeNe and He-Cd lasers. The results are presented for holographic visualization of Cryogenic and Ultrasonic near field images of Surgical Medical Instrument. Application of CHRR interferometer for hologram registration of moving biological object in "vivo" is illustrated.

  10. Clinical applications of high-resolution ocular magnetic resonance imaging.

    PubMed

    Tanitame, Keizo; Sone, Takashi; Kiuchi, Yoshiaki; Awai, Kazuo

    2012-11-01

    Magnetic resonance imaging (MRI) using fast sequences with subjects staring at a target can provide motion-free ocular images, and small receiver surface coils make it possible to produce ocular images with high spatial resolution. MRI using half-Fourier single-shot rapid acquisition with a relaxation enhancement sequence as a fast T2-weighted imaging yields useful images for the morphologic diagnosis of ocular diseases, and MRI using a fast spoiled gradient-recalled-echo sequence as a T1-weighted imaging yields additional information by the administration of gadolinium-based contrast material for assessing the vascularity of intraocular tumors. These ocular imaging techniques are useful for the evaluation of patients with angle closure glaucoma, congenital abnormality of ocular globes, intraocular tumors and several types of detachments, as well as patients after ocular surgery. In this pictorial essay, we demonstrate the clinical applications of fast high-resolution ocular MRI with fixation of the subjects' visual foci. PMID:22923185

  11. Application of the Billet Casting Method to Determine the Onset of Incipient Melting of 319 Al Alloy Engine Blocks

    NASA Astrophysics Data System (ADS)

    Lombardi, A.; Ravindran, C.; MacKay, R.

    2015-06-01

    The increased use of Al for automotive applications has resulted from the need to improve vehicle fuel efficiency. Aluminum alloy engine blocks fulfil the need of lightweighting. However, there are many challenges associated with thermo-mechanical mismatch between Al and the gray cast iron cylinder liners, which result in large tensile residual stress along the cylinder bores. This requires improced mechanical properties in this region to prevent premature engine failure. In this study, replicating billet castings were used to simulate the engine block solution heat treatment process and determine the onset of incipient melting. Microstructural changes during heat treatment were assessed with SEM and EDX, while thermal analysis was carried out using differential scanning calorimetry. The results suggest that solution heat treatment at 500 °C was effective in dissolving secondary phase particles, while solutionizing at 515 or 530 °C caused incipient melting of Al2Cu and Al5Mg8Cu2Si6. Incipient melting caused the formation ultra-fine eutectic clusters consisting of Al, Al2Cu, and Al5Mg8Cu2Si6 on quenching. In addition, DSC analysis found that incipient melting initiated at 507 °C for all billets, although the quantity of local melting reduced with microstructural refinement as evidenced by smaller endothermic peaks and energy absorption. The results from this study will assist in improving engine block casting integrity and process efficiency.

  12. Identification and Differentiation of Monilinia Species Causing Brown Rot of Pome and Stone Fruit using High-Resolution Melting (HRM) Analysis.

    PubMed

    Papavasileiou, Antonios; Madesis, Panagiotis B; Karaoglanidis, George S

    2016-09-01

    Brown rot is a devastating disease of stone fruit caused by Monilinia spp. Among these species, Monilinia fructicola is a quarantine pathogen in Europe but has recently been detected in several European countries. Identification of brown rot agents relies on morphological differences or use of molecular methods requiring fungal isolation. The current study was initiated to develop and validate a high-resolution melting (HRM) method for the identification of the Monilinia spp. and for the detection of M. fructicola among other brown rot pathogens. Based on the sequence of the cytb intron from M. laxa, M. fructicola, M. fructigena, M. mumecola, M. linhartiana, and M. yunnanensis isolates originating from several countries, a pair of universal primers for species identification and a pair of primers specific to M. fructicola were designed. The specificity of the primers was verified to ensure against cross-reaction with other fungal species. The melting curve analysis using the universal primers generated six different HRM curve profiles, each one specific for each species. Τhe HRM analysis primers specific to M. fructicola amplified a 120-bp region with a distinct melt profile corresponding to the presence of M. fructicola, regardless of the presence of other species. HRM analysis can be a useful tool for rapid identification and differentiation of the six Monilinia spp. using a single primer pair. This novel assay has the potential for simultaneous identification and differentiation of the closely related Monilinia spp. as well as for the differentiation of M. fructicola from other common pathogens or saprophytes that may occur on the diseased fruit. PMID:27247082

  13. Application of MODIS snow cover products: wildfire impacts on snow and melt in the Sierra Nevada

    NASA Astrophysics Data System (ADS)

    Micheletty, P. D.; Kinoshita, A. M.; Hogue, T. S.

    2014-07-01

    The current work evaluates the spatial and temporal variability in snow after a large forest fire in northern California with Moderate Resolution Imaging Spectroradiometer (MODIS) snow covered area and grain size (MODSCAG) algorithm. MODIS MOD10A1 fractional snow covered area and MODSCAG fractional snow cover products are utilized to detect spatial and temporal changes in snowpack after the 2007 Moonlight Fire and an unburned basin, Grizzly Ridge, for water years (WY) 2002-2012. Estimates of canopy adjusted and non-adjusted MODSCAG fractional snow covered area (fSCA) are smoothed and interpolated to provide a continuous timeseries of daily basin average snow extent over the two basins. The removal of overstory canopy by wildfire exposes more snow cover; however, elemental pixel comparisons and statistical analysis show that the MOD10A1 product has a tendency to overestimate snow coverage pre-fire, muting the effects of wildfire. The MODSCAG algorithm better distinguishes sub-pixel snow coverage in forested areas and is highly correlated to soil burn severity after the fire. Annual MODSCAG fSCA estimates show statistically significant increased fSCA in the Moonlight Fire study area after the fire (WY 2008-2011; P < 0.01) compared to pre-fire averages and the control basin. After the fire, the number of days exceeding a pre-fire high snow cover threshold increased by 81%. Canopy reduction increases exposed viewable snow area and the amount of solar radiation that reaches the snowpack leading to earlier basin average melt-out dates compared to the nearby unburned basin. There is also a significant increase in MODSCAG fSCA post-fire regardless of slope or burn severity. Alteration of regional snow cover has significant implications for both short and long-term water supplies for downstream communities and resource managers.

  14. Application of MODIS snow cover products: wildfire impacts on snow and melt in the Sierra Nevada

    NASA Astrophysics Data System (ADS)

    Micheletty, P. D.; Kinoshita, A. M.; Hogue, T. S.

    2014-11-01

    The current work evaluates the spatial and temporal variability in snow after a large forest fire in northern California using Moderate Resolution Imaging Spectroradiometer (MODIS) snow-covered area and grain size (MODSCAG). MODIS MOD10A1 fractional snow-covered area and MODSCAG fractional snow cover products are utilized to detect spatial and temporal changes in snowpack after the 2007 Moonlight Fire and an unburned basin, Grizzly Ridge, for water years (WY) 2002-2012. Estimates of canopy-adjusted and non-adjusted MODSCAG fractional snow-covered area (fSCA) are smoothed and interpolated to provide a continuous time series of average daily snow extent over the two basins. The removal of overstory canopy by wildfire exposes more snow cover; however, elemental pixel comparisons and statistical analysis show that the MOD10A1 product has a tendency to overestimate snow coverage pre-fire, muting the observed effects of wildfire. The MODSCAG algorithm better distinguishes subpixel snow coverage in forested areas and is highly correlated to soil burn severity after the fire. Annual MODSCAG fSCA estimates show statistically significant increased fSCA in the Moonlight Fire study area after the fire (P < 0.01 for WY 2008-2011) compared to pre-fire averages and the control basin. After the fire, the number of days exceeding a pre-fire high snow-cover threshold increased by 81%. Canopy reduction increases exposed viewable snow area and the amount of solar radiation that reaches the snowpack, leading to earlier basin average melt-out dates compared to the nearby unburned basin. There is also a significant increase in MODSCAG fSCA post-fire regardless of slope or burn severity. Regional snow cover change has significant implications for both short- and long-term water supply for impacted ecosystems, downstream communities, and resource managers.

  15. Rapid and Reliable Detection of Glucose-6-Phosphate Dehydrogenase (G6PD) Gene Mutations in Han Chinese Using High-Resolution Melting Analysis

    PubMed Central

    Yan, Jing-bin; Xu, Hong-ping; Xiong, Can; Ren, Zhao-rui; Tian, Guo-li; Zeng, Fanyi; Huang, Shu-zhen

    2010-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked inherited disease, is one of the most common enzymopathies and affects over 400 million people worldwide. In China at least 21 distinct point mutations have been identified so far. In this study high-resolution melting (HRM) analysis was used to screen for G6PD mutations in 260 unrelated Han Chinese individuals, and the rapidity and reliability of this method was investigated. The mutants were readily differentiated by using HRM analysis, which produced distinct melting curves for each tested mutation. Interestingly, G1388A and G1376T, the two most common variants accounting for 50% to 60% of G6PD deficiency mutations in the Chinese population, could be differentiated in a single reaction. Further, two G6PD mutations not previously reported in the Chinese population were identified in this study. One of these mutations, designated “G6PD Jiangxi G1340T,” involved a G1340T substitution in exon 11, predicting a Gly447Val change in the protein. The other mutation involved a C406T substitution in exon 5. The frequencies of the common polymorphism site C1311T/IVS (intervening sequence) XI t93c between patients with G6PD and healthy volunteers were not significantly different. Thus, HRM analysis will be a useful alternative for screening G6PD mutations. PMID:20203002

  16. Rapid and reliable detection of glucose-6-phosphate dehydrogenase (G6PD) gene mutations in Han Chinese using high-resolution melting analysis.

    PubMed

    Yan, Jing-bin; Xu, Hong-ping; Xiong, Can; Ren, Zhao-rui; Tian, Guo-li; Zeng, Fanyi; Huang, Shu-zhen

    2010-05-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked inherited disease, is one of the most common enzymopathies and affects over 400 million people worldwide. In China at least 21 distinct point mutations have been identified so far. In this study high-resolution melting (HRM) analysis was used to screen for G6PD mutations in 260 unrelated Han Chinese individuals, and the rapidity and reliability of this method was investigated. The mutants were readily differentiated by using HRM analysis, which produced distinct melting curves for each tested mutation. Interestingly, G1388A and G1376T, the two most common variants accounting for 50% to 60% of G6PD deficiency mutations in the Chinese population, could be differentiated in a single reaction. Further, two G6PD mutations not previously reported in the Chinese population were identified in this study. One of these mutations, designated "G6PD Jiangxi G1340T," involved a G1340T substitution in exon 11, predicting a Gly447Val change in the protein. The other mutation involved a C406T substitution in exon 5. The frequencies of the common polymorphism site C1311T/IVS (intervening sequence) XI t93c between patients with G6PD and healthy volunteers were not significantly different. Thus, HRM analysis will be a useful alternative for screening G6PD mutations. PMID:20203002

  17. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations.

    PubMed

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease's high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics' assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions' setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning. PMID:27351925

  18. Development of a high-resolution melting analysis assay for rapid and high-throughput identification of clinically important dermatophyte species.

    PubMed

    Didehdar, M; Khansarinejad, B; Amirrajab, N; Shokohi, T

    2016-07-01

    Accurate identification of dermatophyte species is important both for epidemiological studies and for implementing antifungal treatment strategies. Although nucleic acid amplification-based assays have several advantages over conventional mycological methods, a major disadvantage is their high cost. The aim of this study was to develop a rapid and accurate real-time PCR-based high-resolution melting (HRM) assay for differentiation of the most common dermatophyte species. The oligonucleotide primers were designed to amplify highly conserved regions of the dermatophyte ribosomal DNA. Analysis of a panel containing potentially interfering fungi demonstrated no cross reactivity with the assay. To evaluate the performance characteristics of the method, a total of 250 clinical isolates were tested in comparison with the long-established PCR-RFLP method and the results were reassessed using DNA sequencing, as the reference standard method. The assay is able to type dermatophytes using normalised melting peak, difference plot analysis or electrophoresis on agarose gel methods. The results showed that, in comparison to PCR-RFLP, the developed HRM assay was able to differentiate at least 10 common dermatophytes species with a higher speed, throughput and accuracy. These results indicate that the HRM assay will be a useful sensitive, high throughput and cost-effective method for differentiating the most common dermatophyte species. PMID:26991756

  19. Duplex High-Resolution Melting Assay for the Simultaneous Genotyping of IL28B rs12979860 and PNPLA3 rs738409 Polymorphisms in Chronic Hepatitis C Patients

    PubMed Central

    Enache, Elena L.; Sin, Anca; Bancu, Ligia; Ramière, Christophe; Diaz, Olivier; André, Patrice; Enache, Liviu S.

    2015-01-01

    Chronic hepatitis C (CHC) is a major burden for public health worldwide. Although newer direct-acting antivirals show good efficacy, their cost precludes their wide adoption in resource-limited regions. Thus, strategies are being developed to help identify patients with high susceptibility to response to classic PEG-interferon + ribavirin therapy. IL28B polymorphism rs12979860 C/T is an important predictor for an efficient response to interferon-based therapy. A genetic variant in adiponutrin (PNPLA3) gene, rs738409 C/G, is associated with steatosis, severity, and progression of liver fibrosis in CHC patients, and predicts treatment outcome in difficult-to-cure HCV-infected patients with advanced fibrosis. We developed a rapid and inexpensive assay based on duplex high-resolution melting (HRM) for the simultaneous genotyping of these two polymorphisms. The assay validation was performed on synthetic DNA templates and 132 clinical samples from CHC patients. When compared with allele-specific PCR and sequencing, our assay showed 100% (95% CI: 0.9724–1) accuracy, with 100% sensitivity and specificity. Our assay was robust against concentration and quality of DNA samples, melting curve normalization intervals, HRM analysis algorithm, and sequence variations near the targeted SNPs (single nucleotide polymorphism). This duplex assay should provide useful information for patient-oriented management and clinical decision-making in CHC. PMID:26389885

  20. Development of a High-Resolution Melting Approach for Scanning Beta Globin Gene Point Mutations in the Greek and Other Mediterranean Populations

    PubMed Central

    Chassanidis, Christos; Boutou, Effrossyni; Voskaridou, Ersi; Balassopoulou, Angeliki

    2016-01-01

    Beta-thalassaemia is one of the most common autosomal recessive disorders worldwide. The disease’s high incidence, which is observed in the broader Mediterranean area has led to the establishment of molecular diagnostics’ assays to prevent affected births. Therefore, the development of a reliable, cost-effective and rapid scanning method for β globin gene point mutations, easily adapted to a routine laboratory, is absolutely essential. Here, we describe, for the first time, the development of a High-Resolution Melting Analysis (HRMA) approach, suitable for scanning the particularly heterogeneous beta globin gene mutations present in the Greek population, and thus adaptable to the Mediterranean and other areas where these mutations have been identified. Within this context, β globin gene regions containing mutations frequently identified in the Greek population were divided in ten overlapping amplicons. Our reactions’ setup allowed for the simultaneous amplification of multiple primer sets and partial multiplexing, thereby resulting in significant reduction of the experimental time. DNA samples from β-thalassaemia patients/carriers with defined genotypes were tested. Distinct genotypes displayed distinguishable melting curves, enabling accurate detection of mutations. The described HRMA can be adapted to a high-throughput level. It represents a rapid, simple, cost-effective, reliable, highly feasible and sensitive method for β-thalassaemia gene scanning. PMID:27351925

  1. Duplex High-Resolution Melting Assay for the Simultaneous Genotyping of IL28B rs12979860 and PNPLA3 rs738409 Polymorphisms in Chronic Hepatitis C Patients.

    PubMed

    Enache, Elena L; Sin, Anca; Bancu, Ligia; Ramière, Christophe; Diaz, Olivier; André, Patrice; Enache, Liviu S

    2015-01-01

    Chronic hepatitis C (CHC) is a major burden for public health worldwide. Although newer direct-acting antivirals show good efficacy, their cost precludes their wide adoption in resource-limited regions. Thus, strategies are being developed to help identify patients with high susceptibility to response to classic PEG-interferon + ribavirin therapy. IL28B polymorphism rs12979860 C/T is an important predictor for an efficient response to interferon-based therapy. A genetic variant in adiponutrin (PNPLA3) gene, rs738409 C/G, is associated with steatosis, severity, and progression of liver fibrosis in CHC patients, and predicts treatment outcome in difficult-to-cure HCV-infected patients with advanced fibrosis. We developed a rapid and inexpensive assay based on duplex high-resolution melting (HRM) for the simultaneous genotyping of these two polymorphisms. The assay validation was performed on synthetic DNA templates and 132 clinical samples from CHC patients. When compared with allele-specific PCR and sequencing, our assay showed 100% (95% CI: 0.9724-1) accuracy, with 100% sensitivity and specificity. Our assay was robust against concentration and quality of DNA samples, melting curve normalization intervals, HRM analysis algorithm, and sequence variations near the targeted SNPs (single nucleotide polymorphism). This duplex assay should provide useful information for patient-oriented management and clinical decision-making in CHC. PMID:26389885

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

    PubMed

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

    2014-02-01

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

  3. A new identification method for five species of oysters in genus Crassostrea from China based on high-resolution melting analysis

    NASA Astrophysics Data System (ADS)

    Wang, Jiafeng; Xu, Fei; Li, Li; Zhang, Guofan

    2014-03-01

    The high phenotypic plasticity in the shell of oysters presents a challenge during taxonomic and phylogenetic studies of these economically important bivalves. However, because DNA can exhibit marked differences among morphologically similar species, DNA barcoding offers a potential means for oyster identification. We analyzed the complete sequences of the cytochrome oxidase subunit I (COI) of five common Crassostrea species in China (including Hong Kong oyster C. hongkongensis, Jinjiang oyster C. ariakensis, Portuguese oyster C. angulata, Kumamoto oyster C. sikamea, and Pacific oyster C. gigas) and screened for distinct fragments. Using these distinct fragments on a high-resolution melting analysis platform, we developed an identification method that does not rely on species-specific PCR or fragment length polymorphism and is efficient, reliable, and easy to visualize. Using a single pair of primers (Oyster-COI-1), we were able to successfully distinguish among the five oyster species. This new method provides a simple and powerful tool for the identification of oyster species.

  4. Specific heat treatment of selective laser melted Ti-6Al-4V for biomedical applications

    NASA Astrophysics Data System (ADS)

    Huang, Qianli; Liu, Xujie; Yang, Xing; Zhang, Ranran; Shen, Zhijian; Feng, Qingling

    2015-12-01

    The ductility of as-fabricated Ti-6Al-4V falls far short of the requirements for biomedical titanium alloy implants and the heat treatment remains the only applicable option for improvement of their mechanical properties. In the present study, the decomposition of as-fabricated martensite was investigated to provide a general understanding on the kinetics of its phase transformation. The decomposition of asfabricated martensite was found to be slower than that of water-quenched martensite. It indicates that specific heat treatment strategy is needed to be explored for as-fabricated Ti-6Al-4V. Three strategies of heat treatment were proposed based on different phase transformation mechanisms and classified as subtransus treatment, supersolvus treatment and mixed treatment. These specific heat treatments were conducted on selective laser melted samples to investigate the evolutions of microstructure and mechanical properties. The subtransus treatment leaded to a basket-weave structure without changing the morphology of columnar prior β grains. The supersolvus treatment resulted in a lamellar structure and equiaxed β grains. The mixed treatment yielded a microstructure that combines both features of the subtransus treatment and supersolvus treatment. The subtransus treatment is found to be the best choice among these three strategies for as-fabricated Ti-6Al-4V to be used as biomedical implants.

  5. Unraveling Host-Vector-Arbovirus Interactions by Two-Gene High Resolution Melting Mosquito Bloodmeal Analysis in a Kenyan Wildlife-Livestock Interface

    PubMed Central

    Omondi, David; Masiga, Daniel K.; Ajamma, Yvonne Ukamaka; Fielding, Burtram C.; Njoroge, Laban; Villinger, Jandouwe

    2015-01-01

    The blood-feeding patterns of mosquitoes are directly linked to the spread of pathogens that they transmit. Efficient identification of arthropod vector bloodmeal hosts can identify the diversity of vertebrate species potentially involved in disease transmission cycles. While molecular bloodmeal analyses rely on sequencing of cytochrome b (cyt b) or cytochrome oxidase 1 gene PCR products, recently developed bloodmeal host identification based on high resolution melting (HRM) analyses of cyt b PCR products is more cost-effective. To resolve the diverse vertebrate hosts that mosquitoes may potentially feed on in sub-Saharan Africa, we utilized HRM profiles of both cyt b and 16S ribosomal RNA genes. Among 445 blood-fed Aedeomyia, Aedes, Anopheles, Culex, Mansonia, and Mimomyia mosquitoes from Kenya’s Lake Victoria and Lake Baringo regions where many mosquito-transmitted pathogens are endemic, we identified 33 bloodmeal hosts including humans, eight domestic animal species, six peridomestic animal species and 18 wildlife species. This resolution of vertebrate host species was only possible by comparing profiles of both cyt b and 16S markers, as melting profiles of some pairs of species were similar for either marker but not both. We identified mixed bloodmeals in a Culex pipiens from Mbita that had fed on a goat and a human and in two Mansonia africana mosquitoes from Baringo that each had fed on a rodent (Arvicanthis niloticus) in addition to a human or baboon. We further detected Sindbis and Bunyamwera viruses in blood-fed mosquito homogenates by Vero cell culture and RT-PCR in Culex, Aedeomyia, Anopheles and Mansonia mosquitoes from Baringo that had fed on humans and livestock. The observed mosquito feeding on both arbovirus amplifying hosts (including sheep and goats) and possible arbovirus reservoirs (birds, porcupine, baboons, rodents) informs arbovirus disease epidemiology and vector control strategies. PMID:26230507

  6. Application of the high resolution return beam vidicon

    NASA Technical Reports Server (NTRS)

    Cantella, M. J.

    1977-01-01

    The Return Beam Vidicon (RBV) is a high-performance electronic image sensor and electrical storage component. It can accept continuous or discrete exposures. Information can be read out with a single scan or with many repetitive scans for either signal processing or display. Resolution capability is 10,000 TV lines/height, and at 100 lp/mm, performance matches or exceeds that of film, particularly with low-contrast imagery. Electronic zoom can be employed effectively for image magnification and data compression. The high performance and flexibility of the RBV permit wide application in systems for reconnaissance, scan conversion, information storage and retrieval, and automatic inspection and test. This paper summarizes the characteristics and performance parameters of the RBV and cites examples of feasible applications.

  7. Enhancing the resolution of gpr spectra for pavement engineering applications

    NASA Astrophysics Data System (ADS)

    Benedetto, F.; Benedetto, A.

    2012-04-01

    Ground Penetrating Radar (GPR) is a geophysical method that uses radar pulses to image the subsurface. This non-destructive method uses electromagnetic radiation and detects the reflected signals from subsurface structures. It can detect objects, changes in material, and voids and cracks. GPR has many applications in a number of fields. In the field of civil engineering one of the most advanced technologies used for road pavement monitoring is based on the deployment of advanced GPR systems. One of the most relevant causes of road pavement damage is often referable to water intrusion in structural layers. In this context, GPR has been recently proposed as a method to estimate moisture content in a porous medium without preventive calibration. Hence, the development of methods to obtain an estimate of the moisture content is a crucial research field involving economic, social and strategic aspects in road safety for a great number of public and private Agencies. In particular, a recent new approach was proposed to estimate moisture content in a porous medium basing on the theory of Rayleigh scattering, showing a shift of the frequency peak of the GPR spectrum towards lower frequencies as the moisture content increases in the soil. The weakness characterizing this approach is represented by the needs of high resolution signals, whereas GPR spectra are affected by low resolution. Hence, the rising requirement for high resolution leads to specific demands for improved prediction methods. Recently, a new technique combining the response of the conventional fast Fourier transform (FFT, well known for its high-precision receiving signal level) with that of the MUSIC (multiple signal classification) algorithm, well known for its super-resolution capacity has been proposed. This combined method has been proved to obtain a high precision level in quantifying the shift of the frequency peak of the GPR spectrum. This combined method can perform a reliable coarse estimate of

  8. Analysis of impact melt and vapor production in CTH for planetary applications

    DOE PAGESBeta

    Quintana, S. N.; Crawford, D. A.; Schultz, P. H.

    2015-05-19

    This study explores impact melt and vapor generation for a variety of impact speeds and materials using the shock physics code CTH. The study first compares the results of two common methods of impact melt and vapor generation to demonstrate that both the peak pressure method and final temperature method are appropriate for high-speed impact models (speeds greater than 10 km/s). However, for low-speed impact models (speeds less than 10 km/s), only the final temperature method is consistent with laboratory analyses to yield melting and vaporization. Finally, a constitutive model for material strength is important for low-speed impacts because strengthmore » can cause an increase in melting and vaporization.« less

  9. Analysis of impact melt and vapor production in CTH for planetary applications

    SciTech Connect

    Quintana, S. N.; Crawford, D. A.; Schultz, P. H.

    2015-05-19

    This study explores impact melt and vapor generation for a variety of impact speeds and materials using the shock physics code CTH. The study first compares the results of two common methods of impact melt and vapor generation to demonstrate that both the peak pressure method and final temperature method are appropriate for high-speed impact models (speeds greater than 10 km/s). However, for low-speed impact models (speeds less than 10 km/s), only the final temperature method is consistent with laboratory analyses to yield melting and vaporization. Finally, a constitutive model for material strength is important for low-speed impacts because strength can cause an increase in melting and vaporization.

  10. Machine learning with systematic density-functional theory calculations: Application to melting temperatures of single- and binary-component solids

    NASA Astrophysics Data System (ADS)

    Seko, Atsuto; Maekawa, Tomoya; Tsuda, Koji; Tanaka, Isao

    2014-02-01

    A combination of systematic density-functional theory (DFT) calculations and machine learning techniques has a wide range of potential applications. This study presents an application of the combination of systematic DFT calculations and regression techniques to the prediction of the melting temperature for single and binary compounds. Here we adopt the ordinary least-squares regression, partial least-squares regression, support vector regression, and Gaussian process regression. Among the four kinds of regression techniques, SVR provides the best prediction. The inclusion of physical properties computed by the DFT calculation to a set of predictor variables makes the prediction better. In addition, limitation of the predictive power is shown when extrapolation from the training dataset is required. Finally, a simulation to find the highest melting temperature toward the efficient materials design using kriging is demonstrated. The kriging design finds the compound with the highest melting temperature much faster than random designs. This result may stimulate the application of kriging to efficient materials design for a broad range of applications.

  11. Wide genetic variations at 18S ribosomal RNA locus of Cyclospora cayetanensis isolated from Egyptian patients using high resolution melting curve.

    PubMed

    Hussein, Eman M; El-Moamly, Amal A; Mahmoud, Moushira A; Ateek, Nayera S

    2016-07-01

    A variable clinical picture of cyclosporiasis including gastrointestinal tract (GIT) symptomatic or asymptomatic beside extraintestinal consequences suggests a possibility of heterogenicity of Cyclospora cayetanensis. The present work aimed to explore the possibility of genetic variation of C. cayetanensis using high-resolution melting (HRM) curve of polymerase chain reaction (PCR) amplified 18S rRNA genes. DNAs extracted from the stool samples of 70 cyclosporiasis patients were amplified and scanned by PCR/HRM curve. The results showed that there are four different genotypic profiles of C. cayetanensis with presence of mixed ones. Although Tm of all profiles was within the same range, they were discerned by plotting of the temperature-shifted florescence difference between normalized melting curves (dF/dT). Genotypic profile I was found alone in 40 % of patients and mixed with genotypic profile II and/or III in 25.7 % of patients, followed by genotypic profile II in 14.3 % then genotypic profile III and IV (10 % each). A significant relation was found between genotypic profiles and GIT symptomatic status as profile I and profile II were mostly detected in patients with acute GIT symptoms without or with chronic illness, respectively, while profile IV cases only were GIT asymptomatic. Statistical significance relations between genotypic profiles and age, gender, residence and oocyst shape index were determined. In conclusion, PCR/HRM proved a wide variation on C. cayetanensis genes that could be reflected on its pathogenic effects and explaining the variability of the clinical manifestations presented by cyclosporiasis patients. PMID:27041342

  12. Shear melting and high temperature embrittlement: theory and application to machining titanium.

    PubMed

    Healy, Con; Koch, Sascha; Siemers, Carsten; Mukherji, Debashis; Ackland, Graeme J

    2015-04-24

    We describe a dynamical phase transition occurring within a shear band at high temperature and under extremely high shear rates. With increasing temperature, dislocation deformation and grain boundary sliding are supplanted by amorphization in a highly localized nanoscale band, which allows for massive strain and fracture. The mechanism is similar to shear melting and leads to liquid metal embrittlement at high temperature. From simulation, we find that the necessary conditions are lack of dislocation slip systems, low thermal conduction, and temperature near the melting point. The first two are exhibited by bcc titanium alloys, and we show that the final one can be achieved experimentally by adding low-melting-point elements: specifically, we use insoluble rare earth metals (REMs). Under high shear, the REM becomes mixed with the titanium, lowering the melting point within the shear band and triggering the shear-melting transition. This in turn generates heat which remains localized in the shear band due to poor heat conduction. The material fractures along the shear band. We show how to utilize this transition in the creation of new titanium-based alloys with improved machinability. PMID:25955055

  13. A review of melt and vapor growth techniques for polydiacetylene thin films for nonlinear optical applications

    NASA Technical Reports Server (NTRS)

    Penn, B. G.; Shields, A.; Frazier, D. O.

    1988-01-01

    Methods for the growth of polydiacetylene thin films by melt and vapor growth and their subsequent polymerization are summarized. Films with random orientations were obtained when glass or quartz were used as substrates in the vapor growth process. Oriented polydiacetylene films were fabricated by the vapor deposition of diacetylene monomer onto oriented polydiacetylene on a glass substrate and its subsequent polymerization by UV light. A method for the growth of oriented thin films by a melt-shear growth process as well as a method of film growth by seeded recrstallization from the melt between glass plates, that may be applied to the growth of polydiacetylene films, are described. Moreover, a method is presented for the fabrication of single crystal thin films of polyacetylenes by irradiation of the surface of diacetylene single crystals to a depth between 100 and 2000 angstroms.

  14. An electrochemical series of redox couples in silicate melts - A review and applications to geochemistry

    NASA Technical Reports Server (NTRS)

    Schreiber, Henry D.

    1987-01-01

    An electrochemical series for redox couples in a glass-forming oxide melt is developed. This series is a quantitative numerical scale of reference reduction potentials of the redox couples in a silicate melt that is a model for basaltic magmas. The redox couples are ordered in terms of their reference reduction potentials; the order appears to be relatively independent of the exact melt composition and temperature. Thus, upon calibration to a desired composition, oxygen fugacity, and temperature, this electrochemical series can provide estimates of redox state proportions in basaltic magmas on different planetary bodies. The geochemical electrochemical series can also be used to understand the interrelationship of the redox state of the magma and the presence of volatile species such as oxygen, water, sulfur gases, and carbon gases.

  15. Application of rock melting to construction of storage holes for nuclear waste

    SciTech Connect

    Neudecker, J.W. Jr.

    1988-12-31

    Rock melting technology can provide in-situ glass liners in nuclear waste package emplacement holes to reduce permeability and increase borehole stability. Reduction of permeability would reduce the time and probability of groundwater contacting the waste packages. Increasing the stability of the storage boreholes would enhance the retrievability of the nuclear waste packages. The rock melting hole forming technology has already been tested in volcanic tuff similar to the geology at the proposed nuclear waste repository at Yucca Mountain, Nevada. 6 refs., 5 figs., 2 tabs.

  16. Predicting Melting Points of Organic Molecules: Applications to Aqueous Solubility Prediction Using the General Solubility Equation.

    PubMed

    McDonagh, J L; van Mourik, T; Mitchell, J B O

    2015-11-01

    In this work we make predictions of several important molecular properties of academic and industrial importance to seek answers to two questions: 1) Can we apply efficient machine learning techniques, using inexpensive descriptors, to predict melting points to a reasonable level of accuracy? 2) Can values of this level of accuracy be usefully applied to predicting aqueous solubility? We present predictions of melting points made by several novel machine learning models, previously applied to solubility prediction. Additionally, we make predictions of solubility via the General Solubility Equation (GSE) and monitor the impact of varying the logP prediction model (AlogP and XlogP) on the GSE. We note that the machine learning models presented, using a modest number of 2D descriptors, can make melting point predictions in line with the current state of the art prediction methods (RMSE≥40 °C). We also find that predicted melting points, with an RMSE of tens of degrees Celsius, can be usefully applied to the GSE to yield accurate solubility predictions (log10 S RMSE<1) over a small dataset of drug-like molecules. PMID:27491032

  17. Nickel and Cobalt Partitioning Between Spinel and Basaltic Melt: Applications to Planetary Basalt Suites

    NASA Technical Reports Server (NTRS)

    Righter, K.

    2002-01-01

    New experimental spinel/melt partition coefficients for Ni and Co have been measured in basalt samples with natural levels of Ni and Co, are lower than previous high doping experiments, and are applied to several planetary basalt suites. Additional information is contained in the original extended abstract.

  18. Thermodynamics of Oligonucleotide Duplex Melting

    ERIC Educational Resources Information Center

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

  19. Macroscopic coupling of deformation and melt migration at continental interiors, with applications to the Colorado Plateau

    NASA Astrophysics Data System (ADS)

    Roy, Mousumi; Gold, Stav; Johnson, Alex; Osuna Orozco, Rodrigo; Holtzman, Benjamin K.; Gaherty, James

    2016-05-01

    Within continental interiors far from plate boundaries, we hypothesize that the architecture of the lithosphere will influence dynamic pressure gradients produced as the lithosphere moves relative to the underlying asthenosphere. We investigate how these dynamic pressure gradients affect melt migration by percolative flow within the mantle beneath continental interiors. Our models show that for a lithospheric keel that protrudes into the "mantle wind," the behavior of the system can be classified into three categories: (1) small-scale convective instabilities modify both the geometry of the lithosphere-asthenophere boundary (LAB) and associated dynamic pressure gradients so that the surface flux of melt is largest above a narrow downwelling at the center of the keel; (2) weak convection does not substantially modify the keel and persistent gradients at the LAB at margins of the keel control dynamic pressure gradients so that surface melt flux is elevated within the keel relative to its surroundings; and (3) when convective instabilities are suppressed due to sufficiently high viscosity in the asthenosphere, asymmetric dynamic pressure gradients due to mantle shear beneath the keel focus melt streamlines toward the upwind flank of the keel and direct melt away from the downwind flank. We interpret surface patterns of Cenozoic magmatism at the Colorado Plateau, particularly the "upwind-downwind" asymmetry relative to the direction of motion of North America over the underlying asthenosphere, in terms of category (3) above. Our models quantitatively assess the viability of spatially variable dynamic pressure gradients as a physical mechanism to explain disparate observations at the Colorado Plateau, such as surface magmatism, heat flow, xenolith data, and seismic structure.

  20. Taxonomic identification of mediterranean pines and their hybrids based on the high resolution melting (HRM) and trnL approaches: from cytoplasmic inheritance to timber tracing.

    PubMed

    Ganopoulos, Ioannis; Aravanopoulos, Filippos; Madesis, Panagiotis; Pasentsis, Konstantinos; Bosmali, Irene; Ouzounis, Christos; Tsaftaris, Athanasios

    2013-01-01

    Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples. PMID:23577179

  1. Detection of NPM1 exon 12 mutations and FLT3 – internal tandem duplications by high resolution melting analysis in normal karyotype acute myeloid leukemia

    PubMed Central

    Tan, Angela YC; Westerman, David A; Carney, Dennis A; Seymour, John F; Juneja, Surender; Dobrovic, Alexander

    2008-01-01

    Background Molecular characterisation of normal karyotype acute myeloid leukemia (NK-AML) allows prognostic stratification and potentially can alter treatment choices and pathways. Approximately 45–60% of patients with NK-AML carry NPM1 gene mutations and are associated with a favourable clinical outcome when FLT3-internal tandem duplications (ITD) are absent. High resolution melting (HRM) is a novel screening method that enables rapid identification of mutation positive DNA samples. Results We developed HRM assays to detect NPM1 mutations and FLT3-ITD and tested diagnostic samples from 44 NK-AML patients. Eight were NPM1 mutation positive only, 4 were both NPM1 mutation and FLT3-ITD positive and 4 were FLT3-ITD positive only. A novel point mutation Y572C (c.1715A>G) in exon 14 of FLT3 was also detected. In the group with de novo NK-AML, 40% (12/29) were NPM1 mutation positive whereas NPM1 mutations were observed in 20% (3/15) of secondary NK-AML cases. Sequencing was performed and demonstrated 100% concordance with the HRM results. Conclusion HRM is a rapid and efficient method of screening NK-AML samples for both novel and known NPM1 and FLT3 mutations. NPM1 mutations can be observed in both primary and secondary NK-AML cases. PMID:18664261

  2. Rapid Detection and Identification of Mucormycetes in Bronchoalveolar Lavage Samples from Immunocompromised Patients with Pulmonary Infiltrates by Use of High-Resolution Melt Analysis

    PubMed Central

    Racil, Zdenek; Hrncirova, Kristyna; Kocmanova, Iva; Volfova, Pavlina; Ricna, Dita; Bejdak, Petr; Moulis, Mojmir; Pavlovsky, Zdenek; Weinbergerova, Barbora; Toskova, Martina; Mayer, Jiri

    2014-01-01

    Rapid differential diagnostics of pulmonary infiltrates suspected of invasive fungal disease in an immunocompromised host and early initiation of effective antifungal therapy are crucial for patient outcomes. There are no serological tests available to detect mucormycetes; therefore, PCR-based methods are highly suitable. We validated our previously published PCR followed by high-resolution melt analysis (PCR/HRMA) to detect Rhizopus spp., Rhizomucor pusillus, Lichtheimia corymbifera, and Mucor spp. in bronchoalveolar lavage (BAL) samples from immunocompromised patients who were at risk of invasive fungal disease. All PCR/HRMA-positive samples were retested using novel real-time quantitative PCR (RQ PCR) assays specific to the species identified. In total, between January 2009 and December 2012 we analyzed 99 BAL samples from 86 patients with pulmonary abnormalities using PCR/HRMA. Ninety (91%) BAL samples were negative, and 9 (9%) samples were positive. The sensitivity and specificity of PCR/HRMA were 100% and 93%, respectively. By combining the positive results of PCR/HRMA with positive RQ PCR results, the specificity was raised to 98%. PCR/HRMA, due to its high negative predictive value (99%), represents a fast and reliable tool for routine BAL sample screening for the differential diagnosis of pulmonary infiltrates in immunocompromised patients for the four most clinically important mucormycetes. PMID:24850354

  3. Rapid detection and identification of mucormycetes in bronchoalveolar lavage samples from immunocompromised patients with pulmonary infiltrates by use of high-resolution melt analysis.

    PubMed

    Lengerova, Martina; Racil, Zdenek; Hrncirova, Kristyna; Kocmanova, Iva; Volfova, Pavlina; Ricna, Dita; Bejdak, Petr; Moulis, Mojmir; Pavlovsky, Zdenek; Weinbergerova, Barbora; Toskova, Martina; Mayer, Jiri

    2014-08-01

    Rapid differential diagnostics of pulmonary infiltrates suspected of invasive fungal disease in an immunocompromised host and early initiation of effective antifungal therapy are crucial for patient outcomes. There are no serological tests available to detect mucormycetes; therefore, PCR-based methods are highly suitable. We validated our previously published PCR followed by high-resolution melt analysis (PCR/HRMA) to detect Rhizopus spp., Rhizomucor pusillus, Lichtheimia corymbifera, and Mucor spp. in bronchoalveolar lavage (BAL) samples from immunocompromised patients who were at risk of invasive fungal disease. All PCR/HRMA-positive samples were retested using novel real-time quantitative PCR (RQ PCR) assays specific to the species identified. In total, between January 2009 and December 2012 we analyzed 99 BAL samples from 86 patients with pulmonary abnormalities using PCR/HRMA. Ninety (91%) BAL samples were negative, and 9 (9%) samples were positive. The sensitivity and specificity of PCR/HRMA were 100% and 93%, respectively. By combining the positive results of PCR/HRMA with positive RQ PCR results, the specificity was raised to 98%. PCR/HRMA, due to its high negative predictive value (99%), represents a fast and reliable tool for routine BAL sample screening for the differential diagnosis of pulmonary infiltrates in immunocompromised patients for the four most clinically important mucormycetes. PMID:24850354

  4. Taxonomic Identification of Mediterranean Pines and Their Hybrids Based on the High Resolution Melting (HRM) and trnL Approaches: From Cytoplasmic Inheritance to Timber Tracing

    PubMed Central

    Ganopoulos, Ioannis; Aravanopoulos, Filippos; Madesis, Panagiotis; Pasentsis, Konstantinos; Bosmali, Irene; Ouzounis, Christos; Tsaftaris, Athanasios

    2013-01-01

    Fast and accurate detection of plant species and their hybrids using molecular tools will facilitate the assessment and monitoring of local biodiversity in an era of climate and environmental change. Herein, we evaluate the utility of the plastid trnL marker for species identification applied to Mediterranean pines (Pinus spp.). Our results indicate that trnL is a very sensitive marker for delimiting species biodiversity. Furthermore, High Resolution Melting (HRM) analysis was exploited as a molecular fingerprint for fast and accurate discrimination of Pinus spp. DNA sequence variants. The trnL approach and the HRM analyses were extended to wood samples of two species (Pinus nigra and Pinus sylvestris) with excellent results, congruent to those obtained using leaf tissue. Both analyses demonstrate that hybrids from the P. brutia (maternal parent) × P. halepensis (paternal parent) cross, exhibit the P. halepensis profile, confirming paternal plastid inheritance in Group Halepensis pines. Our study indicates that a single one-step reaction method and DNA marker are sufficient for the identification of Mediterranean pines, their hybrids and the origin of pine wood. Furthermore, our results underline the potential for certain DNA regions to be used as novel biological information markers combined with existing morphological characters and suggest a relatively reliable and open taxonomic system that can link DNA variation to phenotype-based species or hybrid assignment status and direct taxa identification from recalcitrant tissues such as wood samples. PMID:23577179

  5. Characterization of Genetic Diversity of Bacillus anthracis in France by Using High-Resolution Melting Assays and Multilocus Variable-Number Tandem-Repeat Analysis ▿ †

    PubMed Central

    Derzelle, S.; Laroche, S.; Le Flèche, P.; Hauck, Y.; Thierry, S.; Vergnaud, G.; Madani, N.

    2011-01-01

    Using high-resolution melting (HRM) analysis, we developed a cost-effective method to genotype a set of 13 phylogenetically informative single-nucleotide polymorphisms (SNPs) within the genome of Bacillus anthracis. SNP discrimination assays were performed in monoplex or duplex and applied to 100 B. anthracis isolates collected in France from 1953 to 2009 and a few reference strains. HRM provided a reliable and cheap alternative to subtype B. anthracis into one of the 12 major sublineages or subgroups. All strains could be correctly positioned on the canonical SNP (canSNP) phylogenetic tree, except the divergent Pasteur vaccine strain ATCC 4229. We detected the cooccurrence of three canSNP subgroups in France. The dominant B.Br.CNEVA sublineage was found to be prevalent in the Alps, the Pyrenees, the Auvergne region, and the Saône-et-Loire department. Strains affiliated with the A.Br.008/009 subgroup were observed throughout most of the country. The minor A.Br.001/002 subgroup was restricted to northeastern France. Multiple-locus variable-number tandem-repeat analysis using 24 markers further resolved French strains into 60 unique profiles and identified some regional patterns. Diversity found within the A.Br.008/009 and B.Br.CNEVA subgroups suggests that these represent old, ecologically established clades in France. Phylogenetic relationships with strains from other parts of the world are discussed. PMID:21998431

  6. Mutation scanning in a single and a stacked genetically modified (GM) event by real-time PCR and high resolution melting (HRM) analysis.

    PubMed

    Ben Ali, Sina-Elisabeth; Madi, Zita Erika; Hochegger, Rupert; Quist, David; Prewein, Bernhard; Haslberger, Alexander G; Brandes, Christian

    2014-01-01

    Genetic mutations must be avoided during the production and use of seeds. In the European Union (EU), Directive 2001/18/EC requires any DNA construct introduced via transformation to be stable. Establishing genetic stability is critical for the approval of genetically modified organisms (GMOs). In this study, genetic stability of two GMOs was examined using high resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) employing Scorpion primers for amplification. The genetic variability of the transgenic insert and that of the flanking regions in a single oilseed rape variety (GT73) and a stacked maize (MON88017×MON810) was studied. The GT73 and the 5' region of MON810 showed no instabilities in the examined regions. However; two out of 100 analyzed samples carried a heterozygous point mutation in the 3' region of MON810 in the stacked variety. These results were verified by direct sequencing of the amplified PCR products as well as by sequencing of cloned PCR fragments. The occurrence of the mutation suggests that the 5' region is more suitable than the 3' region for the quantification of MON810. The identification of the single nucleotide polymorphism (SNP) in a stacked event is in contrast to the results of earlier studies of the same MON810 region in a single event where no DNA polymorphism was found. PMID:25365178

  7. Differentiation of four strains of Chinese soft-shelled turtle (Pelodiscus sinensis) based on high-resolution melting analysis of single nucleotide polymorphism sites in mitochondrial DNA.

    PubMed

    Zhang, H Q; Zhang, C; Xu, X J; Zhu, J J; He, Z Y; Shao, J Z

    2015-01-01

    The Chinese soft-shelled turtle (Pelodiscus sinensis) has been one of the most economically important aquatic animals in China for thousands of years, and several breeding strains have been formed. Since the morphological characteristics of some strains are similar, a rapid and accurate molecular method to differentiate between strains is required. In this study, partial sequences of mitochondrial DNA from four turtle strains, Taihu Lake Strain, Taiwan Strain, Japanese Strain, and Yellow River Strain, were amplified and sequenced based on selected strain-specific single nucleotide polymorphism (SNP) sites. The corresponding primers were designed and a high-resolution melting (HRM) technique was employed for genotyping these SNPs. The results indicated that a total of seven SNPs can be detected by HRM. Among these SNPs, one can be used for identifying the Taihu Lake Strain, one for the Japanese Strain, two for the Taiwan Strain, and three for the Yellow River Strain. This method is rapid and convenient, which offers technical support for strain identification and selective breeding in Chinese soft-shelled turtles. PMID:26535627

  8. Screening of the BRCA1 gene in Brazilian patients with breast and/or ovarian cancer via high-resolution melting reaction analysis.

    PubMed

    de Oliveira, Eneida Santos; Soares, Bárbara Luisa; Lemos, Sara; Rosa, Reginaldo Cruz Alves; Rodrigues, Angélica Nogueira; Barbosa, Leandro Augusto; de Oliveira Lopes, Débora; dos Santos, Luciana Lara

    2016-04-01

    The aim of this study was to evaluate the profile of BRCA1 mutations among cancer-affected Brazilian women from the Midwest region of Minas Gerais state with clearly defined risk factors for hereditary breast and ovarian cancer (HBOC) syndrome. In this Brazilian region, the first Center for Hereditary Cancer Control began operation in 2011, and 90% of patients receive assistance from the public health service. Eighteen patients at high risk for HBOC were subjected to molecular analysis. Primers were designed for 22 coding exons of the gene; DNA was extracted; and real-time PCR followed by high-resolution melting reaction was performed. The amplicons were sequenced to confirm the identified profiles. Only exon 11 was directly sequenced due its length. Multiplex ligation-dependent probe amplification (MLPA) was performed for those patients in whom no pathogenic mutations were found. Among the 14 alterations identified in this study, the c.5263_5264insC pathogenic mutation was present in two patients (11.1%). Four alterations showed no clinical relevance; one exhibited inconclusive clinical relevance according to the examined databases; and eight alterations presented a divergent classification between the databases. No deletions or duplications were found using the MLPA technique. The HRM methodology was highly sensitive in identifying variants in the BRCA1 gene and can dramatically reduce the amount of sequencing required to identify germline mutations in BRCA genes, enabling cheaper tests and increasing their availability to Brazilian women assisted by the public health service. PMID:26666763

  9. Rapid Identification of Echinococcus granulosus and E. canadensis Using High-Resolution Melting (HRM) Analysis by Focusing on a Single Nucleotide Polymorphism.

    PubMed

    Safa, Ahmad Hosseini; Harandi, Majid Fasihi; Tajaddini, Mohammadhasan; Rostami-Nejad, Mohammad; Mohtashami-Pour, Mehdi; Pestehchian, Nader

    2016-07-22

    High-resolution melting (HRM) is a reliable and sensitive scanning method to detect variation in DNA sequences. We used this method to better understand the epidemiology and transmission of Echinococcus granulosus. We tested the use of HRM to discriminate the genotypes of E. granulosus and E. canadensis. One hundred forty-one hydatid cysts were collected from slaughtered animals in different parts of Isfahan-Iran in 2013. After DNA extraction, the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was amplified using PCR coupled with the HRM curve. The result of HRM analysis using partial the sequences of cox1 gene revealed that 93, 35, and 2 isolates were identified as G1, G3, and G6 genotypes, respectively. A single nucleotide polymorphism (SNP) was found in locus 9867 of the cox1 gene. This is a critical locus for the differentiation between the G6 and G7 genotypes. In the phylogenic tree, the sample with a SNP was located between the G6 and G7 genotypes, which suggest that this isolate has a G6/G7 genotype. The HRM analysis developed in the present study provides a powerful technique for molecular and epidemiological studies on echinococcosis in humans and animals. PMID:26567833

  10. Use of a High Resolution Melting (HRM) Assay to Compare Gag, Pol, and Env Diversity in Adults with Different Stages of HIV Infection

    PubMed Central

    Cousins, Matthew M.; Laeyendecker, Oliver; Beauchamp, Geetha; Brookmeyer, Ronald; Towler, William I.; Hudelson, Sarah E.; Khaki, Leila; Koblin, Beryl; Chesney, Margaret; Moore, Richard D.; Kelen, Gabor D.; Coates, Thomas; Celum, Connie; Buchbinder, Susan P.; Seage, George R.; Quinn, Thomas C.; Donnell, Deborah; Eshleman, Susan H.

    2011-01-01

    Background Cross-sectional assessment of HIV incidence relies on laboratory methods to discriminate between recent and non-recent HIV infection. Because HIV diversifies over time in infected individuals, HIV diversity may serve as a biomarker for assessing HIV incidence. We used a high resolution melting (HRM) diversity assay to compare HIV diversity in adults with different stages of HIV infection. This assay provides a single numeric HRM score that reflects the level of genetic diversity of HIV in a sample from an infected individual. Methods HIV diversity was measured in 203 adults: 20 with acute HIV infection (RNA positive, antibody negative), 116 with recent HIV infection (tested a median of 189 days after a previous negative HIV test, range 14–540 days), and 67 with non-recent HIV infection (HIV infected >2 years). HRM scores were generated for two regions in gag, one region in pol, and three regions in env. Results Median HRM scores were higher in non-recent infection than in recent infection for all six regions tested. In multivariate models, higher HRM scores in three of the six regions were independently associated with non-recent HIV infection. Conclusions The HRM diversity assay provides a simple, scalable method for measuring HIV diversity. HRM scores, which reflect the genetic diversity in a viral population, may be useful biomarkers for evaluation of HIV incidence, particularly if multiple regions of the HIV genome are examined. PMID:22073290

  11. rpoB gene high-resolution melt curve analysis: a rapid approach for diagnosis and screening of drug resistance in tuberculous meningitis.

    PubMed

    Sharma, Kusum; Modi, Manish; Kaur, Harsimran; Sharma, Aman; Ray, Pallab; Varma, Subhash

    2015-10-01

    Timely and rapid diagnosis of multidrug resistance in tuberculous meningitis (TBM) is a challenge both for a microbiologist and neurologist. The present study was conducted to evaluate role of real-time polymerase chain reaction (PCR) using rpoB, IS6110, and MPB64 as targets in diagnosis of TBM in 110 patients and subsequent high-resolution melt (HRM) curve analysis of rpoB gene amplicons for screening of drug resistance. The sensitivity of smear, culture, and real-time PCR was 1.8%, 10.9%, and 83.63%, respectively. All 120 control patients showed negative results. With HRM rpoB analysis, rifampicin resistance was detected in 3 out of 110 cases of TBM (3.33%). Subsequently, results of HRM analysis were confirmed by rpoB gene sequencing, and mutations were observed at 516 (2 patients) and 531 (1 patient) codons, respectively. rpoB HRM analysis can be a promising tool for rapid diagnosis and screening of drug resistance in TBM patients in 90minutes. PMID:26254140

  12. Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes

    PubMed Central

    Pholwat, Suporn; Liu, Jie; Stroup, Suzanne; Gratz, Jean; Banu, Sayera; Rahman, S. M. Mazidur; Ferdous, Sara Sabrina; Foongladda, Suporn; Boonlert, Duangjai; Ogarkov, Oleg; Zhdanova, Svetlana; Kibiki, Gibson; Heysell, Scott

    2015-01-01

    ABSTRACT Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes. PMID:25714709

  13. Mutation Scanning in a Single and a Stacked Genetically Modified (GM) Event by Real-Time PCR and High Resolution Melting (HRM) Analysis

    PubMed Central

    Ben Ali, Sina-Elisabeth; Madi, Zita Erika; Hochegger, Rupert; Quist, David; Prewein, Bernhard; Haslberger, Alexander G.; Brandes, Christian

    2014-01-01

    Genetic mutations must be avoided during the production and use of seeds. In the European Union (EU), Directive 2001/18/EC requires any DNA construct introduced via transformation to be stable. Establishing genetic stability is critical for the approval of genetically modified organisms (GMOs). In this study, genetic stability of two GMOs was examined using high resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) employing Scorpion primers for amplification. The genetic variability of the transgenic insert and that of the flanking regions in a single oilseed rape variety (GT73) and a stacked maize (MON88017 × MON810) was studied. The GT73 and the 5' region of MON810 showed no instabilities in the examined regions. However; two out of 100 analyzed samples carried a heterozygous point mutation in the 3' region of MON810 in the stacked variety. These results were verified by direct sequencing of the amplified PCR products as well as by sequencing of cloned PCR fragments. The occurrence of the mutation suggests that the 5' region is more suitable than the 3' region for the quantification of MON810. The identification of the single nucleotide polymorphism (SNP) in a stacked event is in contrast to the results of earlier studies of the same MON810 region in a single event where no DNA polymorphism was found. PMID:25365178

  14. High Resolution Reconstruction of the Ionosphere for SAR Applications

    NASA Astrophysics Data System (ADS)

    Minkwitz, David; Gerzen, Tatjana; Hoque, Mainul

    2014-05-01

    Caused by ionosphere's strong impact on radio signal propagation, high resolution and highly accurate reconstructions of the ionosphere's electron density distribution are demanded for a large number of applications, e.g. to contribute to the mitigation of ionospheric effects on Synthetic Aperture Radar (SAR) measurements. As a new generation of remote sensing satellites the TanDEM-L radar mission is planned to improve the understanding and modelling ability of global environmental processes and ecosystem change. TanDEM-L will operate in L-band with a wavelength of approximately 24 cm enabling a stronger penetration capability compared to X-band (3 cm) or C-band (5 cm). But accompanied by the lower frequency of the TanDEM-L signals the influence of the ionosphere will increase. In particular small scale irregularities of the ionosphere might lead to electron density variations within the synthetic aperture length of the TanDEM-L satellite and in turn might result into blurring and azimuth pixel shifts. Hence the quality of the radar image worsens if the ionospheric effects are not mitigated. The Helmholtz Alliance project "Remote Sensing and Earth System Dynamics" (EDA) aims in the preparation of the HGF centres and the science community for the utilisation and integration of the TanDEM-L products into the study of the Earth's system. One significant point thereby is to cope with the mentioned ionospheric effects. Therefore different strategies towards achieving this objective are pursued: the mitigation of the ionospheric effects based on the radar data itself, the mitigation based on external information like global Total Electron Content (TEC) maps or reconstructions of the ionosphere and the combination of external information and radar data. In this presentation we describe the geostatistical approach chosen to analyse the behaviour of the ionosphere and to provide a high resolution 3D electron density reconstruction. As first step the horizontal structure of

  15. E-beam GIDC resolution enhancement technology in practical applications

    NASA Astrophysics Data System (ADS)

    Martens, S.; Butschke, J.; Galler, R.; Krüger, M.; Sailer, H.; Sülzle, M.

    2013-09-01

    For nearly all relevant applications of e-beam lithography the resolution and pattern quality requirements are approaching or exceeding the limits of the available process. On one hand, for shrinking feature dimensions, the e-beam proximity effect and process effects such as photo acid diffusion limit the pattern contrast and process window. On the other hand, e-beam process related parasitic effects such as shot noise, fogging, developer loading, heating, charging, and inhomogeneous bake introduce some significant errors. Even though e-beam tool and process tool suppliers continue to implement new or improve current strategies to avoid or correct these effects, the amount of residual errors requires some reasonable e-beam process window, in particular for high end applications. For some patterns the undersize-overdose approach (SIZE) improves the pattern fidelity and process window. However, for patterns with high fill factors this approach increases the overall deposited electron dose, which due to the increased backscattering diminishes or even eliminates the advantages. The geometrically induced dose correction (GIDC) method overcomes this issue by combining the SIZE concept with a short range framing technique, which reduces the deposited dose in large filled pattern areas. This paper provides a comparison of the standard, SIZE, and GIDC correction approaches for 1D test patterns as well as production patterns. For a broad comparison, patterns were printed onto negative and positive chemically amplified resists and on wafer and mask substrates using a Vistec SB352HR variable shape e-beam writer. Both wafers were also etched. The outcome of the study is that the SIZE and GIDC approaches often outperform the standard proximity effect correction. For dense patterns, GIDC still provides a better pattern quality and process window, while the SIZE approach suffers from the increased overall deposited electron dose and clearly falls behind GIDC in terms of process

  16. High spatial resolution geochemistry and textural characteristics of 'microtektite' glass spherules in proximal Cretaceous-Paleogene sections: Insights into glass alteration patterns and precursor melt lithologies

    NASA Astrophysics Data System (ADS)

    Belza, Joke; Goderis, Steven; Smit, Jan; Vanhaecke, Frank; Baert, Kitty; Terryn, Herman; Claeys, Philippe

    2015-03-01

    Using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), we have conducted spatially resolved trace element analysis on fresh, unaltered microtektite glasses linked to the Cretaceous-Paleogene (K-Pg) boundary Chicxulub crater and on their surrounding alteration phases. This unique approach offers the opportunity to study in situ and at high spatial resolution both the mixing of different target lithologies and the variation of the major and trace element budget during the alteration process. In addition, two-dimensional element distribution maps reveal important geochemical information beyond the capabilities of single spot laser drilling. Glasses from two localities in opposite quadrants from the source crater were studied. At the Beloc locality (Haiti), the glass population is dominated by the presence of yellow high-Ca glass and black andesitic glass formed by admixture of carbonate/dolomite/anhydrite platform lithologies with crystalline basement. These glasses alter according to the well-established hydration-palagonitization model postulated for mafic volcanic glasses. REEs become progressively leached from the glass to below the detection limit for the applied spot size, while immobile Zr, Hf, Nb, and Ta passively accumulate in the process exhibiting both inter-element ratios and absolute concentrations similar to those for the original glass. In contrast, The Arroyo El Mimbral locality (NE Mexico) is characterized by abundant green glass fragments high in Si, Al and alkalis, and low in Mg, Ca, Fe. Low Si black glass is less abundant though similar in composition to the black glass variety at Beloc. The alteration pattern of high-Si, Al green glass at the Mimbral locality is more complex, including numerous competing reaction processes (ion-exchange, hydration, dissolution, and secondary mineral precipitation) generally controlled by the pH and composition of the surrounding fluid. All green, high-Si, Al glasses are hydrated and

  17. Melting-point measurements at high static pressures from laser heating methods: Application to uranium

    SciTech Connect

    Sitaud, B.; Thevenin, T.

    1999-07-01

    Two experimental approaches dealing with the determination of melting at high static pressures are described and analyzed. With the sample squeezed inside a diamond anvil cell, high temperatures up to the solid-liquid transition are obtained using Nd:YAG laser heating. Two methods have been investigated. In the first technique, the heating is accomplished with a pulsed laser and the brief radiation variations (t {lt} 10 ms) emitted from the sample are recorded with two high-speed infrared detectors. The melting location is defined by a plateau or changes of slope of the signals, and the temperatures are calculated by assuming a constant value of emissivity factor at the end of the transition over the studied pressure range. The second system employs a continuous laser and a two-dimensional CCD detector to measure temperatures using multispectral pyrometry. Melting is detected from criteria related either to textural change in the sample involving interference contrast under a laser illumination or to the specific variations of temperatures and emissivity as a function of laser power. Thermal radiation is fitted to Planck's law with temperature and emissivity as the free parameters. Advantages and drawbacks are presented from results obtained on pure uranium.

  18. Application of a hot-melt granulation process to enhance fenofibrate solid dose manufacturing.

    PubMed

    Chaudhary, Rakesh Singh; Amankwaa, Edward; Kumar, Sandeep; Hu, Tom; Chan, Mohamed; Sanghvi, Pradeep

    2016-07-01

    Evaluation of hot-melt granulation of fenofibrate and croscarmellose sodium and its cooling time for the molten mass in a ratio of 55:45 was conducted to assess the manufacturing process capability to produce an acceptable granulation which flows well on Korsch PH300 tablet compression machine. The formation of the drug-polymer eutectic mixture was investigated by differential scanning calorimetry, scanning electron microscopy and X-ray powder diffraction. The physical properties of the hot-melt was determined by examining the milled blocks after solidification and milling after cooling periods of 10, 20 and 30 d. The milled material was assessed for the effect of hold time of the blend on the solid dose compression characteristics. The impact of cooling on the processing of the blocks was assessed after 10, 20 and 30 d of cooling. The study suggests that after the hot-melt formed the fenofibrate crystallized independently and a solid solution with croscarmellose sodium was not formed. The age of the blocks determined the hardness of the crystals, changing the processing nature of the granules with respect to compression and powder flow characteristics. The blocks processed after 20 d and beyond produced granules with a characteristic suitable for holding the blend for 14 d in the bin with no impact on flow properties and compressibility of the blend. There was no chipping, capping, sticking or picking observed and a higher compression speed was achieved. PMID:26552938

  19. Application of Aerosondes to melt-pond observation over Arctic sea ice

    NASA Astrophysics Data System (ADS)

    Inoue, J.; Curry, J. A.

    2006-12-01

    Continuous observation of sea ice using a small robotic aircraft called the Aerosonde was made over the Arctic Ocean from Barrow, Alaska, on 20-21 July 2003. Over a region of 350 km off the coast of Barrow, ice concentration data that consists of ice and ponded areas was made using images taken by the aircraft. Melt- pond fraction (A_p) increased northward from 15 % to 30 % as the ice fraction (A_i) increased. However, the fraction of the ice that is ponded (A_p/(A_i+A_p)) was over 30 % in the perennial ice zone while about 25 % in the first year ice zone, suggesting the different stages of melt-pond evolution in each regions. AMSRE-derived ice concentration was underestimated 7% as compared with the observed one (A_i+A_p). These analyses demonstrate new technology for monitoring the spatial variations in the sea ice with melt ponds that would be very difficult to measure using satellites.

  20. Thermal Assessment of a Latent-Heat Energy Storage Module During Melting and Freezing for Solar Energy Applications

    NASA Astrophysics Data System (ADS)

    Ramos Archibold, Antonio

    Capital investment reduction, exergetic efficiency improvement and material compatibility issues have been identified as the primary techno-economic challenges associated, with the near-term development and deployment of thermal energy storage (TES) in commercial-scale concentrating solar power plants. Three TES techniques have gained attention in the solar energy research community as possible candidates to reduce the cost of solar-generated electricity, namely (1) sensible heat storage, (2) latent heat (tank filled with phase change materials (PCMs) or encapsulated PCMs packed in a vessel) and (3) thermochemical storage. Among these the PCM macro-encapsulation approach seems to be one of the most-promising methods because of its potential to develop more effective energy exchange, reduce the cost associated with the tank and increase the exergetic efficiency. However, the technological barriers to this approach arise from the encapsulation techniques used to create a durable capsule, as well as an assessment of the fundamental thermal energy transport mechanisms during the phase change. A comprehensive study of the energy exchange interactions and induced fluid flow during melting and solidification of a confined storage medium is reported in this investigation from a theoretical perspective. Emphasis has been placed on the thermal characterization of a single constituent storage module rather than an entire storage system, in order to, precisely capture the energy exchange contributions of all the fundamental heat transfer mechanisms during the phase change processes. Two-dimensional, axisymmetric, transient equations for mass, momentum and energy conservation have been solved numerically by the finite volume scheme. Initially, the interaction between conduction and natural convection energy transport modes, in the absence of thermal radiation, is investigated for solar power applications at temperatures (300--400°C). Later, participating thermal radiation

  1. Applications of web produced by hot air assisted melt differential electrospinning method

    NASA Astrophysics Data System (ADS)

    Bubakir, Mahmoud M.; Li, Haoyi; Wu, Weifeng; Li, Xiaohu; Ma, Shuai; Yang, Weimin

    2014-08-01

    Melt electrospinning, a technique that has gained increasing attention since it easily can generate continuous ultrafine fibers directly from polymer melts without the use of any solvent. Therefore, it is considered as a safe, cost effective, and environmental friendly technique. However, with all those great advantages, the technique still suffers some drawbacks such as: large fiber diameter and low throughput. The hot air assisted melt differential electrospinning (MDES) is a new technique invented by our research team that can solve or eliminate those drawbacks. The most important features of our used apparatus are: Needleless nozzle that could generate multiple Taylor cones around the bottom edge of the nozzle, which can result in a high throughput. The stretching force acting on the jets can be further strengthened by an air current provided by an air pressure gun. Interference between the high voltage supply and temperature sensors could be prevented through the grounding of the nozzle. The ultrafine pp webs produced using the same apparatus was in the micro/nano scale with a diameter of 600nm-6um and a smooth surface. Porosity of the webs ranges from 86.5%-99.4% when different collecting devices are used. The resultant ultrafine webs were applied in three areas: oil sorption, water treatment, and hydrophilic PP membrane. The results were very promising as for oil the sorption capacity was 129.0g/g; for water treatment, the rejection rate for 3um particles was 95%. And for the hydrophilic PP membrane, the water sorption capacity was 12.3 g/g.

  2. Hypervariable pili and flagella genes provide suitable new targets for DNA high-resolution melt-based genotyping of dairy Geobacillus spp.

    PubMed

    Chauhan, Kanika; Seale, R Brent; Deeth, Hilton C; Turner, Mark S

    2014-10-01

    Although nonpathogenic in nature, spores of Geobacillus are able to attach to surfaces, germinate, and form biofilms, allowing rapid multiplication and persistence within milk powder processing plants, causing final product contamination, and eventually leading to a loss of revenue in terms of downgraded product quality. As a result, Geobacillus spp. have been found to be common contaminants of milk powder worldwide. Genotyping methods can help in gaining insight into the ecology and transmission of these thermophilic bacteria within and between dairy processing plants. The objective of this study was to use the assembled draft genomes of two Geobacillus spp. to identify and test new hypervariable genotyping targets for differentiating closely related dairy Geobacillus isolates. The two Geobacillus spp. strains obtained from high spore count powders were obtained in 2010 (isolate 7E) and in 1995 (isolate 126) and were previously shown to be of same genotype based on a variable number tandem repeat genotyping method. Significant nucleotide sequence variation was found in genes encoding pili and flagella, which were further investigated as suitable loci for a new high-resolution melt analysis (HRMA)-based genotyping method. Three genes encoding pulG (containing prepilin-type N-terminal cleavage domain), pilT (pili retraction protein), and fliW (flagellar assembly protein) were selected as targets for the new pili/flagella gene (PilFla) HRMA genotyping method. The three-gene-based PilFla-HRMA genotyping method differentiated 35 milk powder Geobacillus spp. isolates into 19 different genotype groups (D = 0.93), which compared favorably to the previous method (which used four variable number tandem repeat loci) that generated 16 different genotype groups (D = 0.90). In conclusion, through comparative genomics of two closely related dairy Geobacillus strains, we have identified new hypervariable regions that prove to be useful targets for highly discriminatory genotyping

  3. Evaluation of the Capacity of PCR and High-Resolution Melt Curve Analysis for Identification of Mixed Infection with Mycoplasma gallisepticum Strains.

    PubMed

    Ghorashi, Seyed A; Kanci, Anna; Noormohammadi, Amir H

    2015-01-01

    Pathogenicity and presentation of Mycoplasma gallisepticum (MG) infection may differ from one strain to another and this may have implications on control measures. Infection of individual birds with more than one MG strain has been reported. A PCR followed by high resolution melt (HRM) curve analysis has been developed in our laboratory and routinely used for detection and differentiation of MG strains. However the potential of this test for identification of MG strains in a mixed specimen has not been evaluated. In the present study, the capability of PCR-HRM curve analysis technique, targeting vlhA and pvpA genes was assessed for identification of individual MG strains in a mixed population. Different DNA ratios of two MG strains from 1 to 10(-4) ng were tested with some generated conventional and normalized curves distinct from those of individual strains alone. Using genotype confidence percentages (GCP) generated from HRM curve analysis, it was found that vlhA PCR-HRM was more consistent than pvpA PCR-HRM for the detection of MG ts-11 vaccine strain mixed with any of the MG strains 6/85, F, S6 or a field isolate. The potential of vlhA PCR-HRM to detect mixed MG strains in a specimen was found to be primarily dependent on quantity and proportion of the target DNAs in the mixture. This is the first study examining the capacity of PCR-HRM technique for identification of individual MG strains in a mixed strain population. PMID:25970590

  4. Evaluation of the Capacity of PCR and High-Resolution Melt Curve Analysis for Identification of Mixed Infection with Mycoplasma gallisepticum Strains

    PubMed Central

    Ghorashi, Seyed A.; Kanci, Anna; Noormohammadi, Amir H.

    2015-01-01

    Pathogenicity and presentation of Mycoplasma gallisepticum (MG) infection may differ from one strain to another and this may have implications on control measures. Infection of individual birds with more than one MG strain has been reported. A PCR followed by high resolution melt (HRM) curve analysis has been developed in our laboratory and routinely used for detection and differentiation of MG strains. However the potential of this test for identification of MG strains in a mixed specimen has not been evaluated. In the present study, the capability of PCR-HRM curve analysis technique, targeting vlhA and pvpA genes was assessed for identification of individual MG strains in a mixed population. Different DNA ratios of two MG strains from 1 to 10-4 ng were tested with some generated conventional and normalized curves distinct from those of individual strains alone. Using genotype confidence percentages (GCP) generated from HRM curve analysis, it was found that vlhA PCR-HRM was more consistent than pvpA PCR-HRM for the detection of MG ts-11 vaccine strain mixed with any of the MG strains 6/85, F, S6 or a field isolate. The potential of vlhA PCR-HRM to detect mixed MG strains in a specimen was found to be primarily dependent on quantity and proportion of the target DNAs in the mixture. This is the first study examining the capacity of PCR-HRM technique for identification of individual MG strains in a mixed strain population. PMID:25970590

  5. Rapid detection of multidrug-resistant Mycobacterium tuberculosis by use of real-time PCR and high-resolution melt analysis.

    PubMed

    Ramirez, Melissa V; Cowart, Kelley C; Campbell, Patricia J; Morlock, Glenn P; Sikes, David; Winchell, Jonas M; Posey, James E

    2010-11-01

    The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis. The rifampicin resistance determinant region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTC) strains from nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High-resolution melting (HRM) analysis following real-time PCR was used to identify M. tuberculosis strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific single-nucleotide polymorphism (SNP) transversion mutations. This method was used to screen 252 M. tuberculosis clinical isolates, including 154 RIF-resistant strains and 174 INH-resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF-resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in M. tuberculosis strains and offers several advantages over current molecular and culture-based techniques. PMID:20810777

  6. Real-Time PCR and High-Resolution Melt Analysis for Rapid Detection of Mycobacterium leprae Drug Resistance Mutations and Strain Types

    PubMed Central

    Li, Wei; Matsuoka, Masanori; Kai, Masanori; Thapa, Pratibha; Khadge, Saraswoti; Hagge, Deanna A.; Brennan, Patrick J.

    2012-01-01

    Drug resistance surveillance and strain typing of Mycobacterium leprae are necessary to investigate ongoing transmission of leprosy in regions of endemicity. To enable wider implementation of these molecular analyses, novel real-time PCR–high-resolution melt (RT-PCR-HRM) assays without allele-specific primers or probes and post-PCR sample handling were developed. For the detection of mutations within drug resistance-determining regions (DRDRs) of folP1, rpoB, and gyrA, targets for dapsone, rifampin, and fluoroquinolones, real-time PCR-HRM assays were developed. Wild-type and drug-resistant mouse footpad-derived strains that included three folP1, two rpoB, and one gyrA mutation types in a reference panel were tested. RT-PCR-HRM correctly distinguished the wild type from the mutant strains. In addition, RT-PCR-HRM analyses aided in recognizing samples with mixed or minor alleles and also a mislabeled sample. When tested in 121 sequence-characterized clinical strains, HRM identified all the folP1 mutants representing two mutation types, including one not within the reference panel. The false positives (<5%) could be attributed to low DNA concentration or PCR inhibition. A second set of RT-PCR-HRM assays for identification of three previously reported single nucleotide polymorphisms (SNPs) that have been used for strain typing were developed and validated in 22 reference and 25 clinical strains. Real-time PCR-HRM is a sensitive, simple, rapid, and high-throughput tool for routine screening known DRDR mutants in new and relapsed cases, SNP typing, and detection of minor mutant alleles in the wild-type background at lower costs than current methods and with the potential for quality control in leprosy investigations. PMID:22170923

  7. Quantitation of CDH1 promoter methylation in formalin-fixed paraffin-embedded tissues of breast cancer patients using differential high resolution melting analysis

    PubMed Central

    Naghitorabi, Mojgan; Mohammadi-Asl, Javad; Sadeghi, Hamid Mir Mohammad; Rabbani, Mohammad; Jafarian-Dehkordi, Abbas; Javanmard, Shaghayegh Haghjooy

    2016-01-01

    Background: E-cadherin (CDH1) plays an important role in cell–cell adhesion of epithelial tissues. Loss of E-cadherin expression can lead to loss of tissue integrity, metastasis, and cancer progression. Also loss of E-cadherin expression might be related to aberrant promoter methylation of the CDH1 gene. Many studies have been performed on CDH1 promoter methylation, especially in breast cancer. Although most of the studies have used qualitative methods for methylation analysis, this study is designed to quantitatively investigate CDH1 promoter methylation in breast cancer and its correlation with patients’ clinicopathological features. Materials and Methods: Using differential high resolution melting analysis (D-HRMA), the methylation level of the CDH1 gene promoter was quantified in 98 breast cancer formalin-fixed paraffin-embedded (FFPE) tissues and also 10 fresh frozen normal breast tissues. Results: All samples were detected to be methylated at the CDH1 promoter region. About 74.5% of the breast cancer samples were hypermethylated with an average methylation level of around 60%, while 25.5% of the patients were methylated with the mean methylation level of about 33%, and 90% of the normal samples had a mean methylation level of about 18%. Statistical analyses represented a significant correlation between CDH1 promoter methylation and cancer progression hallmarks, such as, clinical stage, nodal involvement, tumor size, and histological grade. Conclusion: In summary, quantitation of CDH1 promoter methylation can serve as a diagnostic and prognostic tool in breast cancer. Also D-HRMA can be used as a fast and reliable method for quantitation of promoter methylation. PMID:27308263

  8. Al-Fe-Zr alloys for high temperature applications produced by rapid quenching from the melt

    SciTech Connect

    Okazaki, K.; Skinner, D.J.

    1984-09-01

    The melt-spinning technique to produce microstructure for high temperature-high strength Al-Fe-Zr alloys, meeting aerospace structural requirements, is presented, including the investigations for the improvement of the thermal stability of iron aluminide dispersoids by addition of Zr. The alloys containing about 27 to 30 percent volume fraction of dispersoids would be the optimum to meet the requirements from the strength and ductility viewpoint. The thermal stability of the alloys was determined by measuring the microvickers hardness on the isochronally annealed ribbons. 9 references.

  9. Sulfur in vacuum - Sublimation effects on frozen melts, and applications to Io's surface and torus

    NASA Technical Reports Server (NTRS)

    Nash, Douglas B.

    1987-01-01

    Vacuum sublimation effects on solid sulfur yield a form of the element that is white at room temperature, is fluffy in texture, and forms on frozen sulfur in vacuum through differential evaporation of molecular species in the solid. This vacuum sulfur should exist in large quantity on Io, if the solid free sulfur there has solidified from a melt; a sulfur volcanism model for Io is accordingly developed on this basis which implies that the color and spectra of different sulfur regions of Io could indicate their relative crystallization ages and cooling histories. The flux of sublimating hotspot sulfur appears consistent with estimated turnover rates of the Io surface.

  10. Ground state of two-dimensional Yukawa bosons: Applications to vortex melting

    NASA Astrophysics Data System (ADS)

    Magro, W. R.; Ceperley, D. M.

    1993-07-01

    Using variational and diffusion Monte Carlo techniques, we investigate the ground state of bosons interacting in the continuum through a repulsive modified-Bessel-function potential, ɛK0(r/σ), in two dimensions. This is a simplified model for flux lines in high-Tc superconductors. A pair-product trial function is first optimized so that its variational energy is very near the ground-state energy, then the diffusion Monte Carlo technqiue is used to calculate the exact ground-state energy. As a function of mass and density, we calculate the region of stability of the solid for densities greater than 0.01/σ2. The quantum crystal melts at high density, due to the potential's soft core, and at low density, due to the exponentially weak interaction. Bosons with ħ2/2mσ2ɛ>0.09 do not crystallize at any density. Within the flux model, we compute the flux-line phase diagram for Bi2Sr2CaCu2O8. Pair-correlation functions, structure factors, and Lindemann ratios at melting are also comptued.

  11. Application of mixtures of polymeric carriers for dissolution enhancement of fenofibrate using hot-melt extrusion.

    PubMed

    Kalivoda, Adela; Fischbach, Matthias; Kleinebudde, Peter

    2012-06-15

    Hot-melt extrusion was applied to improve dissolution behavior of poorly soluble model drug fenofibrate. Blends of polymers were used as carrier: copovidone (COP), polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol copolymer (PVCL-PVAc-PEG) and hypromellose 2910/5 (HPMC). The ratio of fenofibrate to COP remained constantly 1+3 (weighted parts) with varying amounts of PVCL-PVAc-PEG and HPMC. Solid state of fenofibrate was characterized by X-ray diffractometry and differential scanning calorimetry. Dissolution performance was compared to marketed formulations Lipidil and Lipidil-Ter. Stability studies were conducted at 25°C/60%rH. The dissolution rate from extrudates was significantly increased when compared to pure fenofibrate powder or physical mixture of the components. A supersaturation of 7.6-12.1 was reached with the pelletized extrudates. All extrudates were superior to marketed formulations. No recrystallization was observed after 26 weeks of storage for fenofibrate-COP extrudates 1+3 (weighted parts) with or without polymeric additives. Even so, both degree and duration of supersaturation decreased with increasing storage periods with the exception of fenofibrate-HPMC extrudates. Of particular interest is the finding that by adding polymers with differing release characteristics to the drug-carrier mixture, the dissolution performance of hot-melt extruded solid dosage forms can be readily adapted to meet specific requirements. PMID:22440149

  12. Full Spatial Resolution Infrared Sounding Application in the Preconvection Environment

    NASA Astrophysics Data System (ADS)

    Liu, C.; Liu, G.; Lin, T.

    2013-12-01

    Advanced infrared (IR) sounders such as the Atmospheric Infrared Sounder (AIRS) and Infrared Atmospheric Sounding Interferometer (IASI) provide atmospheric temperature and moisture profiles with high vertical resolution and high accuracy in preconvection environments. The derived atmospheric stability indices such as convective available potential energy (CAPE) and lifted index (LI) from advanced IR soundings can provide critical information 1 ; 6 h before the development of severe convective storms. Three convective storms are selected for the evaluation of applying AIRS full spatial resolution soundings and the derived products on providing warning information in the preconvection environments. In the first case, the AIRS full spatial resolution soundings revealed local extremely high atmospheric instability 3 h ahead of the convection on the leading edge of a frontal system, while the second case demonstrates that the extremely high atmospheric instability is associated with the local development of severe thunderstorm in the following hours. The third case is a local severe storm that occurred on 7-8 August 2010 in Zhou Qu, China, which caused more than 1400 deaths and left another 300 or more people missing. The AIRS full spatial resolution LI product shows the atmospheric instability 3.5 h before the storm genesis. The CAPE and LI from AIRS full spatial resolution and operational AIRS/AMSU soundings along with Geostationary Operational Environmental Satellite (GOES) Sounder derived product image (DPI) products were analyzed and compared. Case studies show that full spatial resolution AIRS retrievals provide more useful warning information in the preconvection environments for determining favorable locations for convective initiation (CI) than do the coarser spatial resolution operational soundings and lower spectral resolution GOES Sounder retrievals. The retrieved soundings are also tested in a regional data assimilation WRF 3D-var system to evaluate the

  13. A new perturbation theory of solids and fluids and its applications to high-pressure melting problems

    SciTech Connect

    Ree, F.H.

    1990-05-01

    A statistical mechanical theory that can describe both solids and fluids in a self-consistent way is described. This theory utilizes a optimized reference potential whose repulsive range shrinks with density. A unique feature of the new theory is that solid- and fluid-phase thermodynamic properties are both computed within a single theoretical framework. Hence, it allows us to study melting phenomena in a self-consistent manner. For solids, the new theory treats both harmonic and anharmonic effects in thermodynamic properties on equal footing. Applications to several model and rare gas systems show that the new theory can accurately predict fluid, solid, and fluid-solid transition properties. Effective pair potentials inferred from the analysis of krypton and xenon isotherms contain short- and long-range modifications to the Aziz-Slaman pair potential. The long-range correction is repulsive and originates from the Axilrod-Teller three-body force, while the short-range correction probably originates from many-body forces. Using the computed melting curves of krypton and neon, we discuss the range of validity of the corresponding states principle for rare gas systems. 68 refs., 8 figs., 6 tabs.

  14. Miniature high-resolution thermometer for low-temperature applications

    NASA Astrophysics Data System (ADS)

    Welander, Paul B.; Hahn, Inseob

    2001-09-01

    We report on a new miniature, high-resolution, susceptibility thermometer that employs LaxGd1-xCl3 as the paramagnetic material for temperature measurement below 4 K. The thermometer utilizes small permanent disk magnets to provide the required magnetic field, and has a total mass of only 2 g. The thermometer shows a resolution of 0.2 nK in a 1 Hz bandwidth and an absolute drift rate less than 50 fK/s at 2.2 K. In this article, we describe the thermometer design, assembly procedure, and experimental test results, including its thermal time constant and susceptibility to external magnetic fields.

  15. Identification of BRCA1/2 Founder Mutations in Southern Chinese Breast Cancer Patients Using Gene Sequencing and High Resolution DNA Melting Analysis

    PubMed Central

    Kwong, Ava; Ng, Enders Kai On; Wong, Chris Lei Po; Law, Fian Bic Fai; Au, Tommy; Wong, Hong Nei; Kurian, Allison W.; West, Dee W.; Ford, James M.; Ma, Edmond Siu Kwan

    2012-01-01

    Background Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of BRCA1 and BRCA2 mutations in different populations. Knowledge of BRCA mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of BRCA mutations in Chinese breast and ovarian cancer patients from Southern China. Methodology/Principal Findings A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive BRCA1 and BRCA2 mutation screening was performed using bi-directional sequencing of all coding exons of BRCA1 and BRCA2. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM) analysis. Among the 451 probands analyzed, 69 (15.3%) deleterious BRCA mutations were identified, comprising 29 in BRCA1 and 40 in BRCA2. The four recurrent BRCA1 mutations (c.470_471delCT, c.3342_3345delAGAA, c.5406+1_5406+3delGTA and c.981_982delAT) accounted for 34.5% (10/29) of all BRCA1 mutations in this cohort. The four recurrent BRCA2 mutations (c.2808_2811delACAA, c.3109C>T, c.7436_7805del370 and c.9097_9098insA) accounted for 40% (16/40) of all BRCA2 mutations. Haplotype analysis was performed to confirm 1 BRCA1 and 3 BRCA2 mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated. Conclusion In this study, our findings suggest that BRCA mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment. PMID:22970155

  16. Kawai-type apparatus equipped with sintered diamond and its application to melting of mantle materials

    NASA Astrophysics Data System (ADS)

    Ito, E.; Kubo, A.; Katsura, T.; Walter, M. J.

    2003-12-01

    Kawai-type (the 6-8 type) of multi-anvil apparatus has been widely used in the mineral physics because of its versatile abilities such as large volume and pressure environment of high hydrostacity. However, it has been realized for last two decades that the maximum attainable pressure is limited to ca. 27 GPa when using tungsten carbide (WC) as anvil material. We have tried to extend capability of Kawai-type apparatus by adopting sintered diamond (SD) cubes of 14 mm edge length with 1.5 or 2.0 mm truncations together with an octahedral magnesia pressure medium. Recently generated pressures of 54 GPa and 40 GPa were confirmed for 1.5 and 2.0 mm truncations, respectively, at room temperature based on the MgO pressure scale. Following above technical innovation, we have carried out melting experiments on peridotite and CI model mantle material up to 35 GPa to examine the hypothesis for crystal fractionation in deep magma ocean in early stage of the Earth's history. Powdered starting material was put directly into a small cylindrical Re heater, which was set in the octahedron with a LaCrO3 sleeve. The sample was heated to ca. 2500° C for 2-3 min at the prescribed load. The quenched products were made to polished sections, which were examined by electron microscopy and then analyzed by the electron probe micro analyzer. In peridotite, ferropericlase (Fp) is the liquidus phase up to about 30 GPa. Both Fp and Mg-perovskite (Mg-Pv), however, coexist on the liquidus at 31 GPa, indicating multiple saturation of these phases. At higher than 32 GPa the front of Fp grains moves back from the liquidus to the slightly lower temperature region and Mg-Pv becomes the liquidus phase. Ca-perovskite (Ca-Pv) crystallizes at a fairly lower temperature than Fp and Mg-Pv at pressures up to ca. 29 GPa. However the crystallization temperatures of Fp and Ca-Pv become closer with increasing pressure, and the former might be only a few degrees higher than the latter at 33 GPa. In CI mantle, on

  17. Astrophysical applications of high angular resolution array-telescopes

    NASA Technical Reports Server (NTRS)

    Linsley, J.

    1985-01-01

    The air shower array-telescopes which are currently being used to search for and study point sources of UHE gamma-rays have angular resolution similar to 1 deg, limited by either the small total area of particle detectors or poor timing resolution. As the signal to noise ratio depends sensitively on the angular resolution, it seems certain that this figure will quickly be surpassed when second generation instruments come into operation. Since the trajectories of galactic cosmic rays with E 100,000 GeV are practically straight lines on scales of 1 A.U. or less, these new instruments will be able to observe a shadow cast by the Moon (angular diameter 0.5 deg). Although the angular diameter of the Sun is practically the same, its shadow will be more complex because of its magnetic field. Thus, high angular resolution observations of the Sun afford a means of investigating the solar magnetic field, and also the charge composition of cosmic rays, including the ratio of antiprotons to protons.

  18. Application of Classification Models to Pharyngeal High-Resolution Manometry

    ERIC Educational Resources Information Center

    Mielens, Jason D.; Hoffman, Matthew R.; Ciucci, Michelle R.; McCulloch, Timothy M.; Jiang, Jack J.

    2012-01-01

    Purpose: The authors present 3 methods of performing pattern recognition on spatiotemporal plots produced by pharyngeal high-resolution manometry (HRM). Method: Classification models, including the artificial neural networks (ANNs) multilayer perceptron (MLP) and learning vector quantization (LVQ), as well as support vector machines (SVM), were…

  19. Experiments on flow focusing in soluble porous media, with applications to melt extraction from the mantle

    SciTech Connect

    Kelemen, P.B.; Whitehead, J.A.; Aharonov, E.; Jordahl, K.A.

    1995-01-01

    We demonstrate finite strucutres formed as a consequence of the `reactive infiltration instability` in a series of laboratory and numerical experiments with growth of solution channels parallel to the fluid flow direction. Our experiments demonstrate channel growth in the presence of an initial solution front and without an initial solution front where there is a gradient in the solubility of the solid matrix. In the gradient case, diffuse flow is unstable everywhere, channels can form and grow at any point, and channels may extend over the length scale of the gradient. As a consequence of the gradient results, we suggest that the reactive infiltration instability is important in the Earth`s mantle, where partial melts in the mantle ascend adiabatically. This hypothesis represents an important alternative to mid-oceanic ridge basalts (MORB) extraction in fractures, since fractures may not form in weak, viscously deforming asthenospheric mantle. We also briefly consider the effects of crystallization, rather than dissolution reactions, on the morphology of porous flow via a second set of experiments where fluid becomes supersaturated in a solid phase. This process may produce a series of walled conduits, as in our experiments. Development of a low-porosity cap overlying high porosity conduits may create hydrostatic overpressure sufficient to cause fracture and magma transport to the surface in dikes.

  20. Collimator application for microchannel plate image intensifier resolution improvement

    DOEpatents

    Thomas, S.W.

    1996-02-27

    A collimator is included in a microchannel plate image intensifier (MCPI). Collimators can be useful in improving resolution of MCPIs by eliminating the scattered electron problem and by limiting the transverse energy of electrons reaching the screen. Due to its optical absorption, a collimator will also increase the extinction ratio of an intensifier by approximately an order of magnitude. Additionally, the smooth surface of the collimator will permit a higher focusing field to be employed in the MCP-to-collimator region than is currently permitted in the MCP-to-screen region by the relatively rough and fragile aluminum layer covering the screen. Coating the MCP and collimator surfaces with aluminum oxide appears to permit additional significant increases in the field strength, resulting in better resolution. 2 figs.

  1. Collimator application for microchannel plate image intensifier resolution improvement

    DOEpatents

    Thomas, Stanley W.

    1996-02-27

    A collimator is included in a microchannel plate image intensifier (MCPI). Collimators can be useful in improving resolution of MCPIs by eliminating the scattered electron problem and by limiting the transverse energy of electrons reaching the screen. Due to its optical absorption, a collimator will also increase the extinction ratio of an intensifier by approximately an order of magnitude. Additionally, the smooth surface of the collimator will permit a higher focusing field to be employed in the MCP-to-collimator region than is currently permitted in the MCP-to-screen region by the relatively rough and fragile aluminum layer covering the screen. Coating the MCP and collimator surfaces with aluminum oxide appears to permit additional significant increases in the field strength, resulting in better resolution.

  2. High-resolution electron microscopy and its applications.

    PubMed

    Li, F H

    1987-12-01

    A review of research on high-resolution electron microscopy (HREM) carried out at the Institute of Physics, the Chinese Academy of Sciences, is presented. Apart from the direct observation of crystal and quasicrystal defects for some alloys, oxides, minerals, etc., and the structure determination for some minute crystals, an approximate image-contrast theory named pseudo-weak-phase object approximation (PWPOA), which shows the image contrast change with crystal thickness, is described. Within the framework of PWPOA, the image contrast of lithium ions in the crystal of R-Li2Ti3O7 has been observed. The usefulness of diffraction analysis techniques such as the direct method and Patterson method in HREM is discussed. Image deconvolution and resolution enhancement for weak-phase objects by use of the direct method are illustrated. In addition, preliminary results of image restoration for thick crystals are given. PMID:3505590

  3. Vacuum Shear Force Microscopy Application to High Resolution Work

    NASA Astrophysics Data System (ADS)

    Polonski, Vitali; Yamamoto, Yoh; White, Jonathon; Kourogi, Motonobu; Ohtsu, Motoichi

    1999-07-01

    A new technique—Vacuum Shear Force Microscopy (VSFM)—is introduced as a reliable method for maintaining a constant separation between a probe and sample. Elimination of many of the instabilities observed when applying the shear force mechanism to imaging under ambient conditions, allows for routine nanometer lateral and sub-nanometer normal resolution. In this paper this technique is applied, firstly, to the imaging of microtubules (biology) and, secondly, to the patterning and subsequent imaging of nanoscale metal lines (nanofabrication).

  4. High resolution frequency analysis techniques with application to the redshift experiment

    NASA Technical Reports Server (NTRS)

    Decher, R.; Teuber, D.

    1975-01-01

    High resolution frequency analysis methods, with application to the gravitational probe redshift experiment, are discussed. For this experiment a resolution of .00001 Hz is required to measure a slowly varying, low frequency signal of approximately 1 Hz. Major building blocks include fast Fourier transform, discrete Fourier transform, Lagrange interpolation, golden section search, and adaptive matched filter technique. Accuracy, resolution, and computer effort of these methods are investigated, including test runs on an IBM 360/65 computer.

  5. High-Resolution Melting Is a Sensitive, Cost-Effective, Time-Saving Technique for BRAF V600E Detection in Thyroid FNAB Washing Liquid: A Prospective Cohort Study

    PubMed Central

    Marino, Marco; Monzani, Maria Laura; Brigante, Giulia; Cioni, Katia; Madeo, Bruno; Santi, Daniele; Maiorana, Antonino; Bettelli, Stefania; Moriondo, Valeria; Pignatti, Elisa; Bonacini, Lara; Carani, Cesare; Rochira, Vincenzo; Simoni, Manuela

    2015-01-01

    Objective The diagnostic accuracy of thyroid fine needle aspiration biopsy (FNAB) can be improved by the combination of cytological and molecular analysis. In this study, washing liquids of FNAB (wFNAB) were tested for the BRAF V600E mutation, using the sensitive and cost-effective technique called high-resolution melting (HRM). The aim was to demonstrate the feasibility of BRAF analysis in wFNAB and its diagnostic utility, combined with cytology. Design Prospective cohort study. Methods 481 patients, corresponding to 648 FNAB samples, were subjected to both cytological (on cells smeared onto a glass slide) and molecular analysis (on fluids obtained washing the FNAB needle with 1 ml of saline) of the same aspiration. BRAF V600E analysis was performed by HRM after methodological validation for application to wFNAB (technique sensitivity: 5.4%). Results The cytological results of the FNAB were: 136 (21%) nondiagnostic (THY1); 415 (64%) benign (THY2); 80 (12.4%) indeterminate (THY3); 9 (1.4%) suspicious for malignancy (THY4); 8 (1.2%) diagnostic of malignancy (THY5). The BRAF V600E mutation was found in 5 THY2, 2 THY3, 6 THY4 and 6 THY5 samples. Papillary carcinoma diagnosis was histologically confirmed in all BRAF+ thyroidectomized patients. BRAF combined with cytology improved the diagnostic value compared to cytology alone in a subgroup of 74 operated patients. Conclusions HRM was demonstrated to be a feasible method for BRAF analysis in wFNAB. Thanks to its sensitivity and cost-effectiveness, it might be routinely used on a large scale in clinical practice. In perspective, standby wFNAB samples could be analyzed a posteriori in case of indeterminate cytology and/or suspicious findings on ultrasound. PMID:26279992

  6. Application and evaluation of high-resolution WRF-CMAQ with simple urban parameterization

    EPA Science Inventory

    The 2-way coupled WRF-CMAQ meteorology and air quality modeling system is evaluated for high-resolution applications by comparing to a regional air quality field study (Discover-AQ). The model was modified to better account for the effects of urban environments. High-resolution...

  7. Application and evaluation of high-resolution WRF-CMAQ with simple urban parameterization.

    EPA Science Inventory

    The 2-way coupled WRF-CMAQ meteorology and air quality modeling system is evaluated for high-resolution applications by comparing to a regional air quality field study (Discover-AQ). The model was modified to better account for the effects of urban environments. High-resolution...

  8. High Spatiotemporal-Resolution Magnetic Tweezers: Calibration and Applications for DNA Dynamics.

    PubMed

    Dulin, David; Cui, Tao Ju; Cnossen, Jelmer; Docter, Margreet W; Lipfert, Jan; Dekker, Nynke H

    2015-11-17

    The observation of biological processes at the molecular scale in real time requires high spatial and temporal resolution. Magnetic tweezers are straightforward to implement, free of radiation or photodamage, and provide ample multiplexing capability, but their spatiotemporal resolution has lagged behind that of other single-molecule manipulation techniques, notably optical tweezers and AFM. Here, we present, to our knowledge, a new high-resolution magnetic tweezers apparatus. We systematically characterize the achievable spatiotemporal resolution for both incoherent and coherent light sources, different types and sizes of beads, and different types and lengths of tethered molecules. Using a bright coherent laser source for illumination and tracking at 6 kHz, we resolve 3 Å steps with a 1 s period for surface-melted beads and 5 Å steps with a 0.5 s period for double-stranded-dsDNA-tethered beads, in good agreement with a model of stochastic bead motion in the magnetic tweezers. We demonstrate how this instrument can be used to monitor the opening and closing of a DNA hairpin on millisecond timescales in real time, together with attendant changes in the hairpin dynamics upon the addition of deoxythymidine triphosphate. Our approach opens up the possibility of observing biological events at submillisecond timescales with subnanometer resolution using camera-based detection. PMID:26588570

  9. 77 FR 70433 - Resolute Marine Energy, Inc.; Notice of Preliminary Permit Application Accepted for Filing and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-11-26

    ... Alaska Wave Energy Project No. 14438. Resolute supplemented its application on September 19, 2012. The...) Between 7 and 15 Surge Wave Energy Converters for a maximum total installed capacity of 750 kilowatts;...

  10. High Resolution Atmospheric Modeling for Wind Energy Applications

    SciTech Connect

    Simpson, M; Bulaevskaya, V; Glascoe, L; Singer, M

    2010-03-18

    The ability of the WRF atmospheric model to forecast wind speed over the Nysted wind park was investigated as a function of time. It was found that in the time period we considered (August 1-19, 2008), the model is able to predict wind speeds reasonably accurately for 48 hours ahead, but that its forecast skill deteriorates rapidly after 48 hours. In addition, a preliminary analysis was carried out to investigate the impact of vertical grid resolution on the forecast skill. Our preliminary finding is that increasing vertical grid resolution does not have a significant impact on the forecast skill of the WRF model over Nysted wind park during the period we considered. Additional simulations during this period, as well as during other time periods, will be run in order to validate the results presented here. Wind speed is a difficult parameter to forecast due the interaction of large and small length scale forcing. To accurately forecast the wind speed at a given location, the model must correctly forecast the movement and strength of synoptic systems, as well as the local influence of topography / land use on the wind speed. For example, small deviations in the forecast track or strength of a large-scale low pressure system can result in significant forecast errors for local wind speeds. The purpose of this study is to provide a preliminary baseline of a high-resolution limited area model forecast performance against observations from the Nysted wind park. Validating the numerical weather prediction model performance for past forecasts will give a reasonable measure of expected forecast skill over the Nysted wind park. Also, since the Nysted Wind Park is over water and some distance from the influence of terrain, the impact of high vertical grid spacing for wind speed forecast skill will also be investigated.

  11. Application of the Plagioclase-Liquid Hygrometer to the Bishop Tuff: Consistency with Melt Inclusion H2O Contents

    NASA Astrophysics Data System (ADS)

    Jolles, J.; Lange, R. A.

    2015-12-01

    High-silica (74-77 wt% SiO2) rhyolites are the most evolved magmas on Earth and constitute some of the largest eruptions (1000s of km3). Of these, one classic example is the Bishop Tuff, a 760 ka eruption of >670 km3 of high-silica rhyolite erupted from Long Valley caldera, CA. Documenting dissolved H2O contents is crucial for understanding its origin and evolution. Analyses of water contents measured in quartz-hosted melt inclusions from the Bishop Tuff (Wallace et al. 1999; Anderson et al. 2000) show that the Early and Middle Bishop Tuff (Ig1Eb, Ig2Ea) have higher water contents (≤ 6.3 wt% H2O) than the Late Bishop Tuff (Ig2NWa; ≤ 5.2 wt%). Our work utilizes the revised plagioclase-liquid hygrometer (Waters & Lange, 2015), which is applicable to rhyolite, to evaluate internal consistency between Fe-Ti oxide temperatures, the plagioclase hygrometer, and melt inclusion H2O analyses. Two-oxide thermometry (Ghiorso & Evans, 2008), using all possible Fe-Ti oxide pairs (between 56 and 1500 pairs for individual samples), was carried out on 2-3 pumice clasts for each sampled eruptive unit. Resulting temperatures (°C ± 1σ) for individual clasts are: 705 ± 12, 728 ± 10 for unit Ig1Eb; 710 ± 12, 728 ± 11 for unit Ig2Ea; 752 ±10, 776 ± 8, 778 ± 7 for unit Ig2NWa; 791 ± 7, 795 ± 8 for unit Ig2Nb. The compositions of the most calcic plagioclase phenocrysts in the Early and Middle units are An17-19, whereas in the Late units they are An29-30. When the Fe-Ti oxide temperatures, whole rock analyses, and plagioclase compositions are incorporated into the plagioclase hygrometer, they give water contents at the onset of plagioclase crystallization of 6.6-6.9 wt% for the Early and Middle units and 4.8-4.9 wt % for the Late units. These results show internal consistency between melt inclusion analyses of water, Fe-Ti oxide thermometry, and the plagioclase-liquid hygrometer; they further support a temperature gradient across the Early, Middle, and Late Bishop Tuff units.

  12. Real-time RT-PCR high-resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated virus 3 variant groups I, II, III and VI

    PubMed Central

    2012-01-01

    Background Grapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. Methods In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. Results A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM. Conclusion The real-time RT-PCR HRM

  13. Dynamics in Polymer Melts and Nanocomposites

    NASA Astrophysics Data System (ADS)

    Schneider, Gerald

    Intense research has led to substantial progress in the field of polymer melts and nanocomposites, both regarding the fundamental understanding and the relationship to applications. From a fundamental point of view, knowing the microscopic single chain dynamics is important. It may even lead to optimized materials ranging from the classical car tire to battery or fuel cell applications. In polymer melts, different processes, such as diffusion, reptation, contour length fluctuations, etc. occur and determine the macroscopic results, e.g. obtained by rheology. In nanocomposites confinement effects and interactions of chains with surfaces play an important role. High resolution techniques, such as small-angle neutron scattering or neutron spin echo spectroscopy are suited to explore the structure and dynamics of chains. The presentation illuminates the fundamental relationship between the microscopic dynamics and the mesoscopic properties, exploiting different experimental techniques, such as dielectric spectroscopy, rheology, neutron scattering and neutron spin echo spectroscopy.

  14. Application of high-resolution EFTEM SI in an AEM.

    PubMed

    Schaffer, Bernhard; Grogger, Werner; Kothleitner, Gerald; Hofer, Ferdinand

    2008-03-01

    In this work we show how energy-filtered imaging can be used to obtain spectrum images of electron energy-loss spectrometric data. Focus is placed on improved energy resolution within these data sets. Using two multilayer samples (GaN/AlN and InP/InAs), we demonstrate the advantages of spectrum-imaging and its extended mapping capabilities. Plasmon-ratio maps are used to quickly create high-contrast material maps with high signal-to-noise ratio, ratio-contrast plots are used to gain optimum settings for the ratio maps, and plasmon-position maps are used to map small shifts of the energy position of bulk plasmon peaks. PMID:17874077

  15. Monitoring the melt-extrusion transesterification of ethylene-vinylacetate copolymer by self-modeling curve resolution analysis of on-line near-infrared spectra.

    PubMed

    Sasić, S; Kita, Y; Furukawa, T; Watari, M; Siesler, H W; Ozaki, Y

    2000-12-01

    The transesterification of molten ethylene-vinylacetate (EVA) copolymers by octanol with sodium methoxide as catalyst in an extruder has been monitored by on-line near infrared (NIR) spectroscopy. A total of 60 NIR spectra were acquired for 37 min with the last spectrum recorded 31 min after the addition of octanol and catalyst was stopped. The experimental spectra show strong baseline fluctuations which are corrected for by multiplicative scatter correction (MSC). The chemometric methods of the orthogonal projection approach (OPA) and multivariate curve resolution (MCR) were used to resolve the spectra and to derive concentration profiles of the species. The detailed analysis reveals the absence of completely pure variables which leads to small errors in the calculation of pure spectra. The initial estimation of a concentration that is necessary as an input parameter for MCR also presents a non-trivial task. We obtained results that were not ideal but applicable for practical concentration control. They enable a fast monitoring of the process in real-time and resolve the spectra of the EVA copolymer and the ethylene-vinyl alcohol (EVAL) copolymer to be very close to the reference spectra. The chemometric methods used and the decomposed spectra are discussed in detail. PMID:11219074

  16. High Spatial Resolution 40Ar/39Ar Geochronology of Impact Melt Breccias from Apollo 17 Boulders at Stations 2, 6, and 7

    NASA Astrophysics Data System (ADS)

    Mercer, C. M.; Hodges, K. V.; Jolliff, B. L.; Van Soest, M. C.; Wartho, J. A.; Weirich, J. R.

    2015-12-01

    Several boulders located at the bases of the North and South Massifs were among the primary field targets of the Apollo 17 mission to the Taurus-Littrow Valley on the Moon [1]. Some boulders are polylithologic, including Boulder 1 at Station 2 and the boulders at Stations 6 and 7. These boulders were the subjects of consortium studies [2, 3] that included 40Ar/39Ar geochronology to determine the ages of distinct lithologies within each boulder [e.g., 4-6]. We report new 40Ar/39Ar data for the impact melt breccias 72255, 76315, 77075, and 77135 obtained using the UV laser ablation microprobe (UVLAMP) methods of [7]. For 72255, we obtained a preliminary isochron date ca. 3814 Ma from 22 melt analyses, which is younger than published plateau dates (e.g., 3951-3835 Ma [4, 8]). Fifteen melt analyses of 76315 yield a preliminary isochron date ca. 3850 Ma, younger than the 3900 ± 16 Ma date reported by [8]. Melt analyses of 77075 yield preliminary dates between ca. 3797-3584 Ma, possibly reflecting partial loss of 40Ar. In this case, the oldest date may provide a minimum age for the formation of melt in 77075. Finally, the UVLAMP dates for the 77135 melt range from 3810-3361 Ma and corresponding Ca/K ratios range from ca. 100-6. Electron microprobe analyses of small (ca. 10s of microns wide) pockets of K-rich materials show that both K-rich glass and K-feldspar are present. The UVLAMP dates for 77135 likely reflect spatially variable 40Ar loss, consistent with published step heating results [e.g., 6]. References: [1] Schmitt (1973) Science, 182, 681-690. [2] Ryder (1993). Catalog of Apollo 17 Rocks: Volume 1 - Stations 2 and 3 (South Massif). LPI. [3] Ryder (1993). Catalog of Apollo 17 Rocks: Volume 4 - North Massif. LPI. [4] Leich et al. (1975) The Moon, 14, 407-444. [5] Cadogan & Turner (1976). LPSC, 7, 2267-2285. [6] Stettler et al. (1978). LPSC, 9, 1113-1115. [7] Mercer et al. (2015) Sci. Adv., 1, e1400050. [8] Dalrymple & Ryder (1996). JGR, 101, 26069-26084.

  17. Application of Metabolomics for High Resolution Phenotype Analysis

    PubMed Central

    Fukusaki, Eiichiro

    2014-01-01

    Metabolome, a total profile of whole metabolites, is placed on downstream of proteome. Metabolome is thought to be results of implementation of genomic information. In other words, metabolome can be called as high resolution phenotype. The easiest operation of metabolomics is the integration to the upstream ome information including transcriptome and/or proteome. Those trials have been reported at a certain scientific level. In addition, metabolomics can be operated in stand-alone mode without any other ome information. Among metabolomics tactics, the author’s group is particularly focusing on metabolic fingerprinting, in which metabolome information is employed as explanatory variant to evaluate response variant. Metabolic fingerprinting technique is expected not only for analyzing slight difference depending on genotype difference but also for expressing dynamic variation of living organisms. The author introduces several good examples which he performed. Those are useful for easy understanding of the power of metabolomics. In addition, the author mentions the latest technology for analysis of metabolic dynamism. The author’s group developed a facile analytical method for semi-quantitative metabolic dynamism. The author introduces the novel method that uses time dependent variation of isotope distribution based on stable isotope dilution. PMID:26819889

  18. Applications of High-Resolution Observations at Millimeter Wavelengths

    NASA Astrophysics Data System (ADS)

    Rosenfeld, Katherine

    Interferometric observations at millimeter wavelengths provide a precious, detailed view of certain astrophysical objects. This thesis is composed of studies that both rely on and enable this technique to study the structure of planet-forming disks and soon image the closest regions around super-massive black holes. Young stars form out of a cloud of gas and dust that, before its eventual dissipation, flattens to a disk. However the disk population is diverse and recent high-resolution images have revealed a wide variety of interesting features. To understand these observations we use detailed radiative transfer models to motivate various physical scenarios. First we identify a set of traits in the disk around V4046 Sgr that marks the coupled progression of the gas and dust distributions in the presence of at least one embedded companion. Next, we investigate how the vertical temperature structure of a disk can be spatially resolved and apply our framework to observations of the disk around HD163296. Lastly, we show how large-scale radial flows of gas may be observable and question how this phenomenon might be distinguished from other scenarios such as warps or outflows. The last chapter summarizes the APHIDS project which changes the sampling rate of data taken at the SMA so that it may be used for VLBI campaigns.

  19. Multivariate curve resolution: a review of advanced and tailored applications and challenges.

    PubMed

    Ruckebusch, C; Blanchet, L

    2013-02-26

    Multivariate curve resolution (MCR) is a widespread methodology for the analysis of process data in many different application fields. This article intends to propose a critical review of the recently published works. Particular attention will be paid to situations requiring advanced and tailored applications of multivariate curve resolution, dealing with improvements in preprocessing methods, multi-set data arrangements, tailored constraints, issues related to non-ideal noise structure and deviation to linearity. These analytical issues are tackling the limits of applicability of MCR methods and, therefore, they can be considered as the most challenging ones. PMID:23410623

  20. Ultra high resolution stepper motors design, development, performance and application

    NASA Technical Reports Server (NTRS)

    Moll, H.; Roeckl, G.

    1979-01-01

    The design and development of stepper motors with steps in the 10 arc sec to 2 arc min range is described. Some of the problem areas, e.g. rotor suspension, tribology aspects and environmental conditions are covered. A summary of achieved test results and the employment in different mechanisms already developed and tested is presented to give some examples of the possible use of this interesting device. Adaptations to military and commercial requirements are proposed and show the wide range of possible applications.

  1. Microstructures and mechanical properties of Co-29Cr-6Mo alloy fabricated by selective laser melting process for dental applications.

    PubMed

    Takaichi, Atsushi; Suyalatu; Nakamoto, Takayuki; Joko, Natsuka; Nomura, Naoyuki; Tsutsumi, Yusuke; Migita, Satoshi; Doi, Hisashi; Kurosu, Shingo; Chiba, Akihiko; Wakabayashi, Noriyuki; Igarashi, Yoshimasa; Hanawa, Takao

    2013-05-01

    The selective laser melting (SLM) process was applied to a Co-29Cr-6Mo alloy, and its microstructure, mechanical properties, and metal elution were investigated to determine whether the fabrication process is suitable for dental applications. The microstructure was evaluated using scanning electron microscopy with energy-dispersed X-ray spectroscopy (SEM-EDS), X-ray diffractometry (XRD), and electron back-scattered diffraction pattern analysis. The mechanical properties were evaluated using a tensile test. Dense builds were obtained when the input energy of the laser scan was higher than 400 J mm⁻³, whereas porous builds were formed when the input energy was lower than 150 J mm⁻³. The microstructure obtained was unique with fine cellular dendrites in the elongated grains parallel to the building direction. The γ phase was dominant in the build and its preferential <001> orientation was confirmed along the building direction, which was clearly observed for the builds fabricated at lower input energy. Although the mechanical anisotropy was confirmed in the SLM builds due to the unique microstructure, the yield strength, UTS, and elongation were higher than those of the as-cast alloy and satisfied the type 5 criteria in ISO22764. Metal elution from the SLM build was smaller than that of the as-cast alloy, and thus, the SLM process for the Co-29Cr-6Mo alloy is a promising candidate for fabricating dental devices. PMID:23500549

  2. New melt-processable thermoplastic polyimides for opto-electronic applications

    NASA Astrophysics Data System (ADS)

    Narayanan, Aditya; Haralur, Gurulingamurthy

    2012-10-01

    The rapid development and adoption of digital technology is leading to an increase in demand for smaller, faster digital data devices and faster digital telecommunication networks. This trend requires increased network bandwidth to handle large amounts of data and seamless integration of network devices with compatible end-user devices. This need is being met by using fiber-optic and photonics technology, infra-red (IR) signals to transmit information, and is fundamental changing the communication industry, thereby creating a need for new polymeric materials. New ULTEM* polyetherimide (PEI) and EXTEM* thermoplastic polyimide (TPI) resins meet the material requirements for the optoelectronics industry. These resins have building blocks enabling IR light transmission without degrading signal quality. They can be injection-molded into thin, precision optical lenses and connectors. ULTEM* resins are been widely used in this industry as fiber-optic components in trans-receivers. EXTEM* resins are amenable to lead-free soldering (LFS), a greener industrial assembly process. While still being IR-transparent, EXTEM* resin is an ideal material for LFS capable substrates, connectors and lenses. An optical product portfolio has been developed and is being presented as a solution to the opto-electronics component industry and some of the successful applications therein.

  3. Melt pool dynamics during selective electron beam melting

    NASA Astrophysics Data System (ADS)

    Scharowsky, T.; Osmanlic, F.; Singer, R. F.; Körner, C.

    2014-03-01

    Electron beam melting is a promising additive manufacturing technique for metal parts. Nevertheless, the process is still poorly understood making further investigations indispensable to allow a prediction of the part's quality. To improve the understanding of the process especially the beam powder interaction, process observation at the relevant time scale is necessary. Due to the difficult accessibility of the building area, the high temperatures, radiation and the very high scanning speeds during the melting process the observation requires an augmented effort in the observation equipment. A high speed camera in combination with an illumination laser, band pass filter and mirror system is suitable for the observation of the electron beam melting process. The equipment allows to observe the melting process with a high spatial and temporal resolution. In this paper the adjustment of the equipment and results of the lifetime and the oscillation frequencies of the melt pool for a simple geometry are presented.

  4. A Survey on Large High-Resolution Display Technologies, Techniques, and Applications

    SciTech Connect

    Ni, Tao; Schmidt, Greg S.; Staadt, Oliver G.; Livingston, Mark A.; Ball, Robert; May, Richard A.

    2006-03-27

    Continued advances in display hardware, computing power, networking, and rendering algorithms have all converged to dramatically improve large high-resolution display capabilities. We present a survey on prior research with large high-resolution displays. In the hardware configurations section we examine systems including multi-monitor workstations, recon*gurable projector arrays, and others. Rendering and the data pipeline are addressed with an overview of current technologies. We discuss many applications for large high-resolution displays such as automotive design, scientific visualization, control centers, and others. Quantifying the effect of large high-resolution displays on human performance and other aspects is important as we look toward future advances in display technology and how it is applied in different situations. Interacting with these displays brings a different set of challenges for HCI professionals, so an overview of some of this work is provided. Finally, we present our view of the top ten greatest challenges in large high-resolution displays.

  5. Minimizing the grid-resolution dependence of flow-routing algorithms for geomorphic applications

    NASA Astrophysics Data System (ADS)

    Pelletier, Jon D.

    2010-10-01

    The results of flow-routing methods currently used in the geomorphic literature depend on grid resolution. This poses a problem for landscape evolution models, which must be independent of grid resolution to the greatest extent possible. Here I illustrate a refinement of currently-used flow-routing algorithms that yields unit contributing areas (i.e. contributing areas per unit width of flow) with minimal grid-resolution effects. I illustrate the application of this method in idealized topography, in high-resolution Digital Elevation Models (DEMs) of real-world topography, and by integration into a landscape evolution model for ridge-and-valley topography. The landscape evolution model produces grid-resolution-independent results in a more straightforward way than previous models for this type of landscape.

  6. Advances in super-resolution technology and application in biomedical research

    NASA Astrophysics Data System (ADS)

    O'Connell, Christopher B.; Ross, Stephen T.

    2012-02-01

    The diffraction limit of the conventional light microscope establishes a barrier that limits resolution and prevents observation of fine structural details within biological specimens. A number of commercially available systems now enable researchers to beat diffraction and achieve up to ten-fold improvements in resolution. These super resolution systems generally rely on one of two strategies. They either add optical elements in order to overcome the diffraction limit or they implement computational power and fitting algorithms to circumvent it. We have now entered the next phase in the development of super resolution systems, where probes, hardware, and software are gaining the refinements necessary to facilitate their application to a range of biological problems. Here, we highlight the recent developments in these areas for two types of super resolution imaging systems, structured illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM). These improvements are promoting the fast acquisition and processing speeds needed for live cell imaging beyond the diffraction limit.

  7. High resolution mesospheric sodium properties for adaptive optics applications

    NASA Astrophysics Data System (ADS)

    Pfrommer, T.; Hickson, P.

    2014-05-01

    Context. The performance of laser guide star adaptive optics (AO) systems for large optical and infrared telescopes is affected by variability of the sodium layer, located at altitudes between 80 and 120 km in the upper mesosphere and lower thermosphere. The abundance and density structure of the atomic sodium found in this region is subject to local and global weather effects, planetary and gravity waves and magnetic storms, and is variable on time scales down to tens of milliseconds, a range relevant to AO. Aims: It is therefore important to characterize the structure and dynamical evolution of the sodium region on small, as well as large spatial and temporal scales. Parameters of particular importance for AO are the mean sodium altitude, sodium layer width and the temporal power spectrum of the centroid altitude. Methods: We have conducted a three-year campaign employing a high-resolution lidar system installed on the 6-m Large Zenith Telescope (LZT) located near Vancouver, Canada. During this period, 112 nights of useful data were obtained. Results: The vertical density profile of atomic sodium shows remarkable structure and variability. Smooth Gaussian-shaped profiles rarely occur. Multiple internal layers are frequently found. These layers often have sharp lower edges, with scale heights of just a few hundred meters, and tend to drift downwards at a typical rate of one kilometer every two to three hours. Individual layers can persist for many hours, but their density and internal structure can be highly variable. Sporadic layers are seen reaching peak densities several times the average, often in just a few minutes. Coherent vertical oscillations are often found, typically extending over tens of kilometers in altitude. Regions of turbulence are evident and Kelvin-Helmholtz instability are sometimes seen. The mean value of the centroid altitude is found to be 90.8 ± 0.1 km. The sodium layer width was determined by computing the altitude range that contains a

  8. Calculating Freshwater Input from Iceberg Melt in Greenlandic Fjords by Combining In Situ Observations of Iceberg Movement with High Resolution Satellite Imagery

    NASA Astrophysics Data System (ADS)

    Sulak, D. J.; Sutherland, D.; Stearns, L. A.; Hamilton, G. S.

    2015-12-01

    Understanding fjord circulation in Greenland's outlet glacial fjords is crucial to explaining recent temporal and spatial variability in glacier dynamics, as well as freshwater transport on the continental shelf. The fjords are commonly assumed to exhibit a plume driven circulation that draws in warmer and saltier Atlantic-origin water toward the glacier at depth. Freshwater input at glacier termini directly drives this circulation and significantly influences water column stratification, which indirectly feeds back on the plume driven circulation. Previous work has focused on freshwater inputs from surface runoff and submarine melting, but the contribution from iceberg melt, a potentially important freshwater source, has not been quantified. Here, we develop a new technique combining in situ observations of movement from iceberg-mounted GPS units with multispectral satellite imagery from Landsat 8. The combination of datasets allows us to examine the details of iceberg movement and quantify mean residence times in a given fjord. We then use common melt rate parameterizations to estimate freshwater input for a given iceberg, utilizing novel satellite-derived iceberg distributions to scale up to a fjord-wide freshwater contribution. We apply this technique to Rink Isbræ and Kangerlussuup Sermia in west Greenland, and Helheim Glacier in southeast Greenland. The analysis can be rapidly expanded to look at other systems as well as seasonal and interannual changes in how icebergs affect the circulation and stratification of Greenland's outlet glacial fjords. Ultimately, this work will lead to a more complete understanding of the wide range of factors that control the observed regional variability in Greenland's glaciers.

  9. Chloride-bearing liquids and partial melting of mantle eclogites: experimental study and application to the diamond-forming processes.

    NASA Astrophysics Data System (ADS)

    Safonov, Oleg

    2010-05-01

    Recent studies prove that the partial melting in some eclogite xenoliths in kimberlites is closely related to formation of diamonds in these rocks at 4-6 GPa and 1150-12500C [e.g. 1, 2]. Along with specific mineral assemblages, the products of the eclogite partial melting commonly include relics of potassium-rich silicic melts (45-65 wt. % of SiO2, 4-14 wt. % of K2O and K2O/Na2O > 1.0) [1, 2]. Available experimental data, however, demonstrate that such melts can not be produced by 'dry' or hydrous melting of a common eclogite. It implies that partial melting and conjugate diamond formation in mantle eclogites was triggered by infiltration of potassic fluids/melts. Assemblages of Cl-bearing phases and carbonates in eclogite xenoliths [1], and eclogitic diamonds [3-6] suggest that these agents were chloride-carbonate-H2O melts or/and chloride-H2O-CO2 fluids. In order to characterize interaction of both types of liquids with eclogites and their minerals, experiments in the eclogite-related systems with participation of CaCO3-Na2CO3-KCl-H2O or H2O-CO2-KCl are reviewed. Melting relations in the system eclogite-CaCO3-Na2CO3-KCl-H2O follow the general scheme proposed earlier for chloride-carbonate-silicate systems [7]. Below 12000C, Grt, Cpx and phlogopite (Phl) coexist with LCC only. Formation of Phl and Ca-rich Grt after Cpx indicate active reactions of Cpx with LCC accompanied by CO2 degassing and depletion of the clinopyroxene in jadeite. Subsequent dissolution of silicates in LCC at >1200OC results in formation of potassic silica-undersaturated carbonate and Cl-bearing melt (LCS) (37-40 wt. % of SiO2, 10-12 wt. % of K2O, ~3.5 wt. % of Cl) immiscible with the LCC. Compositional feature of this melt is very comparable to those of low-Mg carbonate-silicate melt inclusions in diamonds [6]. However, it is not relevant to the melt relics preserved in the partially molten eclogite xenoliths. Melting of eclogites with participation of the H2O-CO2-KCl fluid at 5 GPa at 1200

  10. Experimental investigation of the electrical behavior of olivine during partial melting under pressure and application to the lunar mantle

    NASA Astrophysics Data System (ADS)

    Pommier, A.; Leinenweber, K.; Tasaka, M.

    2015-09-01

    Electrical conductivity measurements were performed during melting experiments of olivine compacts (dry and hydrous Fo77 and Fo90) at 4 and 6 GPa in order to investigate melt transport properties and quantify the effect of partial melting on electrical properties. Experiments were performed in the multi-anvil apparatus and electrical measurements were conducted using the impedance spectroscopy technique with the two-electrode method. Changes in impedance spectra were used to identify the transition from an electrical response controlled by the solid matrix to an electrical response controlled by the melt phase. This transition occurs slightly above the solidus temperature and lasts until Tsolidus + 75 °C (±25). At higher temperature, a significant increase in conductivity (corresponding to an increase in conductivity values by a factor ranging from ∼30 to 100) is observed, consistent with the transition from a tube-dominated network to a structure in which melt films and pools become prominent features. This increase in conductivity corresponds to an abrupt jump for all dry samples and to a smoother increase for the hydrous sample. It is followed by a plateau at higher temperature, suggesting that the electrical response of the investigated samples lacks sensitivity to temperature at an advanced stage of partial melting. Electron microprobe analyses on quenched products indicated an increase in Mg# (molar Mg / (Mg +Fe)) of olivine during experiments (∼77-93 in the quenched samples with an initial Fo77 composition and ∼92-97 in the quenched samples with an initial Fo90 composition) due to the partitioning of iron to the melt phase. Assuming a respective melt fraction of 0.10 and 0.20 before and after the phase of significant increase in conductivity, in agreement with previous electrical and permeability studies, our results can be reproduced satisfactorily by two-phase electrical models (the Hashin and Shtrikman bounds and the modified brick layer model