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Sample records for high-resolution melting curve

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

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    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.

  2. Evaluation of PCR and high-resolution melt curve analysis for differentiation of Salmonella isolates.

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    Saeidabadi, Mohammad Sadegh; Nili, Hassan; Dadras, Habibollah; Sharifiyazdi, Hassan; Connolly, Joanne; Valcanis, Mary; Raidal, Shane; Ghorashi, Seyed Ali

    2017-06-01

    Consumption of poultry products contaminated with Salmonella is one of the major causes of foodborne diseases worldwide and therefore detection and differentiation of Salmonella spp. in poultry is important. In this study, oligonucleotide primers were designed from hemD gene and a PCR followed by high-resolution melt (HRM) curve analysis was developed for rapid differentiation of Salmonella isolates. Amplicons of 228 bp were generated from 16 different Salmonella reference strains and from 65 clinical field isolates mainly from poultry farms. HRM curve analysis of the amplicons differentiated Salmonella isolates and analysis of the nucleotide sequence of the amplicons from selected isolates revealed that each melting curve profile was related to a unique DNA sequence. The relationship between reference strains and tested specimens was also evaluated using a mathematical model without visual interpretation of HRM curves. In addition, the potential of the PCR-HRM curve analysis was evaluated for genotyping of additional Salmonella isolates from different avian species. The findings indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of Salmonella isolates to determine the serovar/serotype.

  3. A Strategy to Setup Codominant Microsatellite Analysis for High-Resolution-Melting-Curve-Analysis (HRM)

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    Mader, Eduard; Lukas, Brigitte; Novak, Johannes

    2008-01-01

    Background High resolution melting curve analysis (HRM) is a technique that measures exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double stranded DNA. The measurement is immediately following PCR in a one-step, closed-tube method. The shape of the melting curve depends on the GC content, length and sequence of the amplicon. Hence it is a powerful, fast and cheap method to detect Single Nucleotide Polymorphisms (SNPs) and other mutations. Results Here we present a strategy to set up microsatellite analysis for HRM including the correct assignment of heterozygous samples by comparative analysis and artificial mixtures of samples. The approach is demonstrated on two Simple Sequence Repeat (SSR) loci of different complexity in the genus Origanum. Following this strategy all alleles of our sample sets could be classified correctly. Conclusion HRM can be used in microsatellite analysis and other codominant marker systems implementing a protocol of comparative melting curve assignment with artificial mixtures of samples to overcome difficulties in correctly assigning heterozygous samples. The method is faster, more sensitive and cheaper than standard protocols for microsatellite analysis. PMID:18980665

  4. A Strategy to Setup Codominant Microsatellite Analysis for High-Resolution-Melting-Curve-Analysis (HRM

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    Novak Johannes

    2008-11-01

    Full Text Available Abstract Background High resolution melting curve analysis (HRM is a technique that measures exactly the decreasing fluorescence of intercalating dye in the process of dissociation of double stranded DNA. The measurement is immediately following PCR in a one-step, closed-tube method. The shape of the melting curve depends on the GC content, length and sequence of the amplicon. Hence it is a powerful, fast and cheap method to detect Single Nucleotide Polymorphisms (SNPs and other mutations. Results Here we present a strategy to set up microsatellite analysis for HRM including the correct assignment of heterozygous samples by comparative analysis and artificial mixtures of samples. The approach is demonstrated on two Simple Sequence Repeat (SSR loci of different complexity in the genus Origanum. Following this strategy all alleles of our sample sets could be classified correctly. Conclusion HRM can be used in microsatellite analysis and other codominant marker systems implementing a protocol of comparative melting curve assignment with artificial mixtures of samples to overcome difficulties in correctly assigning heterozygous samples. The method is faster, more sensitive and cheaper than standard protocols for microsatellite analysis.

  5. Differentiation of five enterohepatic Helicobacter species by nested PCR with high-resolution melting curve analysis.

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    Wu, Miaoli; Rao, Dan; Zhu, Yujun; Wang, Jing; Yuan, Wen; Zhang, Yu; Huang, Ren; Guo, Pengju

    2017-04-01

    Enterohepatic Helicobacter species (EHS) are widespread in rodent species around the world. Several studies have demonstrated that infection with EHS can interfere with the outcomes of animal experiments in cancer research and significantly influence the study results. Therefore, it is essential to establish a rapid detection and identification of EHS for biomedical research using laboratory rodents. Our study aimed to develop a rapid and sensitive method to detect and distinguish five enterohepatic Helicobacter species. Nested PCR followed by high-resolution melting curve analysis (HRM) was developed for identification of H. bilis, H. rodentium, H. muridarum, H. typhlonius, as well as H. hepaticus. To validate the accuracy of nested PCR-HRM analysis, quantitative real-time PCR methods for five different enterohepatic Helicobacter species were developed. A total of 50 cecal samples were tested using both nested PCR-HRM analysis and qPCR method. The nested PCR-HRM method could distinguish five enterohepatic Helicobacter species by different melting temperatures. The melting curve were characterized by peaks of 78.7 ± 0.12°C for H. rodentium, 80.51 ± 0.09°C for H. bilis, 81.6 ± 0.1°C for H. typhlonius, 82.11 ± 0.18°C for H. muridarum, and 82.95 ± 0.09°C for H. hepaticus. The nested PCR-HRM assay is a simple, rapid, and cost-effective assay. This assay could be a useful tool for molecular epidemiology study of enterohepatic Helicobacter infection and an attractive alternative for genotyping of enterohepatic Helicobacter species. © 2016 John Wiley & Sons Ltd.

  6. Identification and differentiation of Campylobacter species by high-resolution melting curve analysis.

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    Hoseinpour, Fatemeh; Foroughi, Azadeh; Nomanpour, Bizhan; Nasab, Rezvan Sobhani

    2017-07-01

    Campylobacter jejuni and Campylobacter coli are the important food-born zoonotic pathogen, also are leading causes of human food borne illnesses worldwide. cadF gene is expressed in all C. jejuni and C. coli strains and mediates cell binding to the cell matrix protein, Fibronectin. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The goal of this study was to apply HRM analysis to identification of C. jejuni and C. coli. A total of 100 samples were obtained from chicken in Kermanshah, Iran. HRM analysis based on cadF gene and Eva Green was developed to the identification of Campylobacter to the species level. Fifty-five of 100 samples were positive as campylobacter (7 C. jejuni, 29 C. coli, 16 mixes and 3 unknown). Minor variations were observed in melting point temperatures of C. coli or C. jejuni isolates and this variation can be used to the differentiation between C. coli or C. jejuni isolates. The results of this study indicated that HRM curve analysis can be a suitable technique and rapid technique for distinguishing between C. jejuni and C. coli isolates. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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    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.

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

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    Manuel Miller

    Full Text Available 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.

  9. High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis.

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    Thomas, Holly R; Percival, Stefanie M; Yoder, Bradley K; Parant, John M

    2014-01-01

    With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high

  10. High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis.

    Directory of Open Access Journals (Sweden)

    Holly R Thomas

    Full Text Available With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs; we observed nuclease generated HRM positive targeting in 1 of 6 (16% open pool derived ZFNs, 14 of 23 (60% TALENs, and 58 of 77 (75% CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases; yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will

  11. Assessing contamination of microalgal astaxanthin producer Haematococcus cultures with high-resolution melting curve analysis.

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    Dawidziuk, Adam; Popiel, Delfina; Luboinska, Magda; Grzebyk, Michal; Wisniewski, Maciej; Koczyk, Grzegorz

    2017-05-01

    Due to its superior antioxidant capabilities and higher activity than other carotenoids, astaxanthin is used widely in the nutraceutical and medicine industries. The most prolific natural producer of astaxanthin is the unicellular green microalga Haematococcus pluvialis. The correct identification of any contaminants in H. pluvialis cultures is both essential and nontrivial for several reasons. Firstly, while it is possible to distinguish the main microalgal contaminant Coelastrella sp. (in H. pluvialis cultures), in practice, it is frequently a daunting and error-prone task for personnel without extensive experience in the microscopic identification of algal species. Secondly, the undetected contaminants may decrease or stop production of astaxanthin. Lastly, the presence of other contaminants such as fungi can eventually infect and destroy the whole algae collection. In this study, high-resolution melting (HRM) analysis was developed to detect microalgal and fungal contamination. The developed diagnostic procedure allowed to distinguish pure H. pluvialis samples from cultures contaminated with low amounts (1.25 ng/ml) of microalgal DNA and fungal DNA (2.5 ng/ml). Such discrimination is not possible with the use of microscopy observations and allows fast and efficient collection testing.

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

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    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.

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

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    Banya Banowary

    Full Text Available 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.

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

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    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Dynamic time warping assessment of high-resolution melt curves provides a robust metric for fungal identification

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    Phatak, Sayali S.; Li, Dongmei; Luka, Janos; Calderone, Richard A.

    2017-01-01

    Fungal infections are a global problem imposing considerable disease burden. One of the unmet needs in addressing these infections is rapid, sensitive diagnostics. A promising molecular diagnostic approach is high-resolution melt analysis (HRM). However, there has been little effort in leveraging HRM data for automated, objective identification of fungal species. The purpose of these studies was to assess the utility of distance methods developed for comparison of time series data to classify HRM curves as a means of fungal species identification. Dynamic time warping (DTW), first introduced in the context of speech recognition to identify temporal distortion of similar sounds, is an elastic distance measure that has been successfully applied to a wide range of time series data. Comparison of HRM curves of the rDNA internal transcribed spacer (ITS) region from 51 strains of 18 fungal species using DTW distances allowed accurate classification and clustering of all 51 strains. The utility of DTW distances for species identification was demonstrated by matching HRM curves from 243 previously identified clinical isolates against a database of curves from standard reference strains. The results revealed a number of prior misclassifications, discriminated species that are not resolved by routine phenotypic tests, and accurately identified all 243 test strains. In addition to DTW, several other distance functions, Edit Distance on Real sequence (EDR) and Shape-based Distance (SBD), showed promise. It is concluded that DTW-based distances provide a useful metric for the automated identification of fungi based on HRM curves of the ITS region and that this provides the foundation for a robust and automatable method applicable to the clinical setting. PMID:28264030

  16. Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

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    Anthwal, Divya; Gupta, Rakesh Kumar; Bhalla, Manpreet; Bhatnagar, Shinjini

    2017-01-01

    ABSTRACT Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB, katG, and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH-katG, and INH-inhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases. PMID:28330890

  17. High resolution melting curve analysis, a rapid and affordable method for mutation analysis in childhood acute myeloid leukemia

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    Yin eLiu

    2014-09-01

    Full Text Available Background: Molecular genetic alterations with prognostic significance have been described in childhood acute myeloid leukemia (AML. The aim of this study was to establish cost-effective techniques to detect mutations of FMS-like tyrosine kinase 3 (FLT3, Nucleophosmin 1 (NPM1, and a partial tandem duplication within the mixed lineage leukemia (MLL-PTD genes in childhood AML. Procedure: Ninety-nine children with newly diagnosed AML were included in this study. We developed a fluoresent dye SYTO-82 based high resolution melting curve (HRM anaylsis to detect FLT3 internal tandem duplication (FLT3-ITD, FLT3 tyrosine kinase domain (FLT3-TKD and NPM1 mutations. MLL-PTD was screened by real-time quantitative PCR. Results: The HRM methodology correlated well with gold standard Sanger sequencing with less cost. Among the 99 patients studied, the FLT3-ITD mutation was associated with significantly worse event free survival (EFS. Patients with the NPM1 mutation had significantly better EFS and overall survival. However, HRM was not sensitive enough for minimal residual disease monitoring. Conclusions: HRM was a rapid and efficient method for screening of FLT3 and NPM1 gene mutations. It was both affordable and accurate, especially in resource underprivileged regions. Our results indicated that HRM could be a useful clinical tool for rapid and cost effective screening of the FLT3 and NPM1 mutations in AML patients.

  18. Gateway-Compatible CRISPR-Cas9 Vectors and a Rapid Detection by High-Resolution Melting Curve Analysis

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    Cynthia J. Denbow

    2017-07-01

    Full Text Available CRISPR-Cas9 system rapidly became an indispensable tool in plant biology to perform targeted mutagenesis. A CRISPR-Cas9-mediated double strand break followed by non-homologous end joining (NHEJ repair most frequently results in a single base pair deletion or insertions (indels, which is hard to detect using methods based on enzymes that detect heteroduplex DNA. In addition, somatic tissues of the T1 generation inevitably contain a mosaic population, in which the portion of cells carrying the mutation can be too small to be detected by the enzyme-based methods. Here we report an optimized experimental protocol for detecting Arabidopsis mutants carrying a CRISPR-Cas9 mediated mutation, using high-resolution melting (HRM curve analysis. Single-base pair insertion or deletion (indel can be easily detected using this method. We have also examined the detection limit for the template containing a one bp indel compared to the WT genome. Our results show that <5% of mutant DNA containing one bp indel can be detected using this method. The vector developed in this study can be used with a Gateway technology-compatible derivative of pCUT vectors, with which off-target mutations could not be detected even by a whole genome sequencing.

  19. Gateway-Compatible CRISPR-Cas9 Vectors and a Rapid Detection by High-Resolution Melting Curve Analysis.

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    Denbow, Cynthia J; Lapins, Samantha; Dietz, Nick; Scherer, Raelynn; Nimchuk, Zachary L; Okumoto, Sakiko

    2017-01-01

    CRISPR-Cas9 system rapidly became an indispensable tool in plant biology to perform targeted mutagenesis. A CRISPR-Cas9-mediated double strand break followed by non-homologous end joining (NHEJ) repair most frequently results in a single base pair deletion or insertions (indels), which is hard to detect using methods based on enzymes that detect heteroduplex DNA. In addition, somatic tissues of the T1 generation inevitably contain a mosaic population, in which the portion of cells carrying the mutation can be too small to be detected by the enzyme-based methods. Here we report an optimized experimental protocol for detecting Arabidopsis mutants carrying a CRISPR-Cas9 mediated mutation, using high-resolution melting (HRM) curve analysis. Single-base pair insertion or deletion (indel) can be easily detected using this method. We have also examined the detection limit for the template containing a one bp indel compared to the WT genome. Our results show that <5% of mutant DNA containing one bp indel can be detected using this method. The vector developed in this study can be used with a Gateway technology-compatible derivative of pCUT vectors, with which off-target mutations could not be detected even by a whole genome sequencing.

  20. Genetic diversity of Dientamoeba fragilis isolates of irritable bowel syndrome patients by high-resolution melting-curve (HRM) analysis.

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    Hussein, Eman M; Al-Mohammed, Hamdan I; Hussein, Abdalla M

    2009-10-01

    Dientamoeba fragilis is a parasite that has been recognized as a causative agent of gastrointestinal symptoms. The search for genetic variation in D. fragilis based on the small-subunit (SSU) rRNA gene using restriction fragment length polymorphism was found not useful for molecular epidemiology. In this study, genetic variability of different clinical isolates of D. fragilis was explored by high-resolution melting curve (HRM) following polymerase chain reaction (PCR) in a one-step closed-tube method. Thirty fecal samples from irritable bowel syndrome (IBS) patients having D. fragilis trophozoites and negative for other organisms were involved in this study. According to the type of diarrhea, eight patients had acute, 14 patients had chronic intermittent, and eight patients had diarrhea alternating with constipation. HRM proved that four profiles (subtypes) were present as detecting by scanning mutation. One of these profiles (profile 1) was predominant (50%). Profile 2 was present on 20%. Profiles 3 and 4 were present on 16.7% and 13.4%, respectively. No mixed profiles were detected among the samples. The melting curves characterized by T(m)1=77.17+/-0.29 degrees C in profile 1, T(m)1=77.37+/-1.45 degrees C in profile 2, T(m)1=74.24+/-0.08 degrees C and T(m)2=79.64+/-0.09 degrees C in profile 3, and T(m)1=75.51 +/- 0.09 degrees C and T(m)=79.42 +/- 0.09 degrees C in profile 4. The relation between these profiles and types of diarrhea proved that the majority of patients having profile 1 (73.4%) and profile 4 (75%) had chronic intermittent diarrhea. All of the patients having profile 2 had acute diarrhea while all of the patients having profile 3 had diarrhea alternating with constipation. Although profile 1 was detected among all types of diarrhea, it was corresponding to 11/14 of patients with chronic intermittent diarrhea. All the differences were clinically and statistically significant. In conclusion, HRM following PCR was proved as a wide variation on D

  1. High-resolution DNA melt-curve analysis for cost-effective mass screening of pairwise species interactions.

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    McCarthy, James K; Didham, Raphael K; Brockerhoff, Eckehard G; van Bysterveldt, Katherine A; Varsani, Arvind

    2013-09-01

    Ecological studies of pairwise interactions are constrained by the methods available for rapid species identification of the interacting organisms. The resolution of data required to characterize species interaction networks at multiple spatio-temporal scales can be intensive, and therefore laborious and costly to collect. We explore the utility of high-resolution DNA melt-curve analysis (HRM) as a rapid species identification method. An approach was developed to identify organisms at the pairwise interaction level, with particular application to cryptic species interactions that are traditionally difficult to study. Here, we selected a challenging application; to identify the presence/absence of pathogenic fungi (Sporothrix inflata, Ophiostoma nigrocarpum and Ophiostoma galeiforme) transported by bark beetle vectors (Hylastes ater and Hylurgus ligniperda). The technique was able to distinguish between different species of DNA within a single, pooled sample. In test applications, HRM was effective in the mass screening and identification of pathogenic fungal species carried by many individual bark beetle vectors (n = 455 beetles screened) across large geographic scales. For two of the fungal species, there was no difference in the frequency of association with either of their vectors, but for the third fungal species there was a shift in vector-pathogen associations across locations. This technique allows rapid, mass screening and characterization of species interactions at a fraction of the time and cost of traditional methods. It is anticipated that this method can be readily applied to explore other cryptic species interactions, or other studies requiring rapid generation of large data sets and/or high-throughput efficiency. © 2013 John Wiley & Sons Ltd.

  2. Detection of monoclonal immunoglobulin heavy chain gene rearrangement (FR3 in Thai malignant lymphoma by High Resolution Melting curve analysis

    Directory of Open Access Journals (Sweden)

    Pongpruttipan Tawatchai

    2010-05-01

    Full Text Available Abstract Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Detection of antigen receptor gene rearrangement including T cell receptor (TCR and immunoglobulin heavy chain (IgH by polymerase chain reaction followed by heteroduplex has currently become standard whereas fluorescent fragment analysis (GeneScan has been used for confirmation test. In this study, three techniques had been compared: thermocycler polymerase chain reaction (PCR followed by heteroduplex and polyacrylamide gel electrophoresis, GeneScan analysis, and real time PCR with High Resolution Melting curve analysis (HRM. The comparison was carried out with DNA extracted from paraffin embedded tissues diagnosed as B- cell non-Hodgkin lymphoma. Specific PCR primers sequences for IgH gene variable region 3, including fluorescence labeled IgH primers were used and results were compared with HRM. In conclusion, the detection IgH gene rearrangement by HRM in the LightCycler System showed potential for distinguishing monoclonality from polyclonality in B-cell non-Hodgkin lymphoma. Introduction Malignant lymphoma, especially non-Hodgkin lymphoma, is one of the most common hematologic malignancies in Thailand. The incidence rate as reported by Ministry of Public Health is 3.1 per 100,000 population in female whereas the rate in male is 4.5 per 100,000 population 1. At Siriraj Hospital, the new cases diagnosed as malignant lymphoma were 214.6 cases/year 2. The diagnosis of malignant lymphoma is often problematic, especially in early stages of the disease. Therefore, detection of antigen receptor gene rearrangement including T cell receptor (TCR and immunoglobulin heavy chain (IgH by polymerase chain reaction (PCR assay has recently become a standard laboratory test for discrimination of reactive from malignant clonal

  3. High-resolution melt-curve analysis of random-amplified-polymorphic-DNA markers, for the characterisation of pathogenic Leptospira

    DEFF Research Database (Denmark)

    Tulsiani, Suhella; Craig, S B; Graham, G C

    2010-01-01

    A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used...... to define serovars or detect genotypes. Ten serovars, of leptospires from the species Leptospira interrogans (serovars Australis, Robinsoni, Hardjo, Pomona, Zanoni, Copenhageni and Szwajizak), L. borgpetersenii (serovar Arborea), L. kirschneri (serovar Cynopteri) and L. weilii (serovar Celledoni), were...... of Leptospira serotypes using a DNA-based methodology is now possible. As an objective and relatively inexpensive and rapid method of serovar identification, at least for cultured isolates, RAPD-HRM assays show convincing potential....

  4. ‘‘Blind'' mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis

    DEFF Research Database (Denmark)

    Studer, Bruno; Jensen, Louise Bach; Fiil, Alice

    2009-01-01

    curves. In this study, HRM was used for simultaneous screening and genotyping of genic DNA sequence polymorphisms identified in the Lolium perenne F2 mapping population VrnA. Melting profiles of PCR products amplified from previously published gene loci and from a novel gene putatively involved...... knowledge of allelic sequences in the key grassland species perennial ryegrass (Lolium perenne L.)....

  5. 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.

    Science.gov (United States)

    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 .

  6. Optimizing a multiplex high resolution melting curve to diagnose G6PD deficiency based on viangchan and canton mutations

    Directory of Open Access Journals (Sweden)

    Nghia Le Tri

    2016-08-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD deficiency which is caused by mutation on G6PD gene is the most common enzyme disorder in human. There have been 184 discovered mutations among which Viangchan [Val291Met] and Canton mutation [Arg459Leu] are the most common variants in Vietnamese. Due to the severity of this disease, several methods have been devised for diagnostics. However, time-consuming, low sensitivity and expensiveness are major problems of those techniques. Recently, High Resolution Melting (HRM has been developed and proven to be an effective method for DNA genotyping, mutation scanning and sequence matching. Hence, in this study a multiplex HRM has been developed aiming at detecting these two mutations concurrently. At first, a singleplex HRM was designed for each mutation. Then, conditions for these singleplex assay were combined and optimized again in order to get the optimal condition for multiplex HRM. Although this method showed a promising potential with a high accuracy, sensitivity, and specificity, it is impractial to continue developing this method because of the lack of controls. However, the optimized singleplex PCR-HRM in this study still can be used for single mutation detection and serve as the background to develop PCR-HRM for other G6PD mutations in Vietnamese-Kinh population. [Biomed Res Ther 2016; 3(8.000: 757-769

  7. Assessment of genetic mutations in the XRCC2 coding region by high resolution melting curve analysis and the risk of differentiated thyroid carcinoma in Iran

    Directory of Open Access Journals (Sweden)

    Shima Fayaz

    2012-01-01

    Full Text Available Homologous recombination (HR is the major pathway for repairing double strand breaks (DSBs in eukaryotes and XRCC2 is an essential component of the HR repair machinery. To evaluate the potential role of mutations in gene repair by HR in individuals susceptible to differentiated thyroid carcinoma (DTC we used high resolution melting (HRM analysis, a recently introduced method for detecting mutations, to examine the entire XRCC2 coding region in an Iranian population. HRM analysis was used to screen for mutations in three XRCC2 coding regions in 50 patients and 50 controls. There was no variation in the HRM curves obtained from the analysis of exons 1 and 2 in the case and control groups. In exon 3, an Arg188His polymorphism (rs3218536 was detected as a new melting curve group (OR: 1.46; 95%CI: 0.432-4.969; p = 0.38 compared with the normal melting curve. We also found a new Ser150Arg polymorphism in exon 3 of the control group. These findings suggest that genetic variations in the XRCC2 coding region have no potential effects on susceptibility to DTC. However, further studies with larger populations are required to confirm this conclusion.

  8. High-Resolution Melting Curve Analysis of the 16S Ribosomal Gene to Detect and Identify Pathogenic and SaprophyticLeptospiraSpecies in Colombian Isolates.

    Science.gov (United States)

    Peláez Sánchez, Ronald G; Quintero, Juan Álvaro López; Pereira, Martha María; Agudelo-Flórez, Piedad

    2017-05-01

    AbstractIt is important to identify the circulating Leptospira agent to enhance the performance of serodiagnostic tests by incorporating specific antigens of native species, develop vaccines that take into account the species/serovars circulating in different regions, and optimize prevention and control strategies. The objectives of this study were to develop a polymerase chain reaction (PCR)-high-resolution melting (HRM) assay for differentiating between species of the genus Leptospira and to verify its usefulness in identifying unknown samples to species level. A set of primers from the initial region of the 16S ribosomal gene was designed to detect and differentiate the 22 species of Leptospira . Eleven reference strains were used as controls to establish the reference species and differential melting curves. Twenty-five Colombian Leptospira isolates were studied to evaluate the usefulness of the PCR-HRM assay in identifying unknown samples to species level. This identification was confirmed by sequencing and phylogenetic analysis of the 16S ribosomal gene. Eleven Leptospira species were successfully identified, except for Leptospira meyeri / Leptospira yanagawae because the sequences were 100% identical. The 25 isolates from humans, animals, and environmental water sources were identified as Leptospira santarosai (twelve), Leptospira interrogans (nine), and L. meyeri / L. yanagawae (four). The species verification was 100% concordant between PCR-HRM and phylogenetic analysis of the 16S ribosomal gene. The PCR-HRM assay designed in this study is a useful tool for identifying Leptospira species from isolates.

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

    Directory of Open Access Journals (Sweden)

    Seyed A Ghorashi

    Full Text Available 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.

  10. Classification of fowl adenoviruses by use of phylogenetic analysis and high-resolution melting-curve analysis of the hexon L1 gene region.

    Science.gov (United States)

    Marek, Ana; Günes, Ayse; Schulz, Evelyn; Hess, Michael

    2010-12-01

    A total of 44 fowl adenovirus (FAdV) samples from 6 European countries, Pakistan, India, Kuwait, Mexico, Peru and Ecuador were used in this study and the phylogenetic analyses based on the loop 1 (L1) region of hexon gene were performed. For comparison, available hexon sequences of representatives of different FAdV species were also used. At least 12 genotypes within the five FAdV species (A-E) were revealed and the existence of these genotypes was supported by high bootstrap values. Furthermore, three primer pairs binding to the conserved pedestal regions (HexL1s/HexL1as and HexA/HexB) and pedestal (P1) region and loop 2 (L2) region (HexF1/HexR1) of the FAdV hexon gene were used for high-resolution melting (HRM)-curve analysis and results were compared with those of phylogenetic analyses. HRM-curve analysis based on the HexL1s/HexL1as region grouped all tested field isolates and reference strains into 22 subgroups, consistently with phylogenetic analysis. This method is a rapid and cost-effective alternative to existing serotype identification methods and offers a possibility to classify FAdV isolates more precisely. However, it has limitations such as need for extensive interpretation of results and potential for indeterminate results. Gaining of hexon sequences of further field isolates offers the potential for novel and additional information in analysis of the molecular epidemiology of FAdV. Copyright © 2010 Elsevier B.V. All rights reserved.

  11. Real-time PCR followed by high-resolution melting curve analysis: A rapid and pragmatic approach for screening of multidrug-resistant extrapulmonary tuberculosis.

    Science.gov (United States)

    Sharma, Kusum; Sharma, Megha; Singh, Shreya; Modi, Manish; Sharma, Aman; Ray, Pallab; Varma, Subhash

    2017-09-01

    Multidrug resistance (MDR) in extrapulmonary tuberculosis (EPTB) is a diagnostic challenge in an endemic country like India. Timely detection of MDR-TB can contribute to a better patient outcome. To perform real-time PCR (qPCR) using rpoB, mpb64 and IS6110 gene on a variety of EPTB samples and to compare the performance of different gene targets. All qPCR positive samples were subjected to high resolution melt-curve analysis (HRM analysis) for rpoB and katG gene to evaluate its potential for MDR screening among different sample types. Real-time PCR using rpoB, mpb64 and IS6110 genes was carried out on 200 cases of study group and 100 cases of non-TB control group. The study group consisted of 100 culture-confirmed and 100 clinically suspected cases of EPTB. Phenotypic drug susceptibility testing (DST) for culture isolates was performed by the 1% indirect agar proportion method. DNA extracted from all qPCR positive samples was subjected to rpoB and katG HRM analysis for screening of MDR. Sequencing was used to confirm the results of HRM analysis and the results were also compared with phenotypic DST in all culture positive cases. The sensitivity of qPCR using rpoB, mpb64 and IS6110 was 86.5%, 86.5% and 76.5%, respectively. All isolates from the control group were negative by all the three targets, giving a specificity of 100%. HRM analysis detected MDR in 22/200 (11%) isolates. 3/200 (1.5%) had mono-rifampicin resistance while 8/200 (4%) had mono-isoniazid resistance. HRM analysis identified an additional 4 MDR cases directly from the samples which were negative by culture. On sequencing, mutations were observed at codon 531 (60%); 533 (16%); 516 (12%) and 526 (12%) of the rpoB gene and at codon 315 (100%) of the katG gene. There was 100% concordance in the results of phenotypic DST, HRM analysis and sequencing. The HRM analysis can play a promising role in the reliable and rapid screening of EPTB samples for detection of MDR. Copyright © 2017 Elsevier Ltd. All

  12. High Resolution Melting Analysis for fast and cheap polymorphism screening of marine populations

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Anne-Leila Meistertzheim, Isabelle Calves, Sébastien Artigaud, Carolyn S. Friedman, Christine Paillard, Jean Laroche & Claude Ferec ### Abstract This protocol permits the mutation scanning of PCR products by high-resolution DNA melting analysis requiring the inclusion of a saturating intercalating dye in the PCR mix without labelled probe. During a scanning process, fluorescent melting curves of PCR amplicons are analyzed. Mutations modifying melting curve shapes, are allowed...

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Comparison of multiple genes and 16S-23S rRNA intergenic space region for their capacity in high resolution melt curve analysis to differentiate Mycoplasma gallisepticum vaccine strain ts-11 from field strains.

    Science.gov (United States)

    Ghorashi, Seyed A; Bradbury, Janet M; Ferguson-Noel, Naola M; Noormohammadi, Amir H

    2013-12-27

    Mycoplasma gallisepticum (MG) is an important avian pathogen causing significant economic losses in the global poultry industry. In an attempt to compare and evaluate existing genotyping methods for differentiation of MG strains/isolates, high resolution melt (HRM) curve analysis was applied to 5 different PCR methods targeting vlhA, pvpA, gapA, mgc2 genes and 16S-23S rRNA intergenic space region (IGSR). To assess the discriminatory power of PCR-HRM of examined genes and IGSR, MG strains ts-11, F, 6/85 and S6, and, initially, 8 field isolates were tested. All MG strains/isolates were differentiated using PCR-HRM curve analysis and genotype confidence percentage (GCP) values of vlhA and pvpA genes, while only 0, 3 and 4 out of 12 MG strains/isolates were differentiated using gapA, mgc2 genes and IGSR, respectively. The HRM curve analysis of vlhA and pvpA genes was found to be highly correlated with the genetic diversity of the targeted genes confirmed by sequence analysis of amplicons generated from MG strains. The potential of the vlhA and pvpA genes was also demonstrated for genotyping of 12 additional MG strains from Europe and the USA. Results from this study provide a direct comparison between genes previously used in sequencing-based genotyping methods for MG strain identification and highlight the usefulness of vlhA and pvpA HRM curve analyses as rapid and reliable tools specially for diagnosis and differentiation of MG strains used here. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Amplification of real-time high resolution melting analysis PCR ...

    African Journals Online (AJOL)

    In this study, we assessed the usefulness of eight common primers amplifying the respective genes in real-time high resolution melting analysis PCR (real-time HRMA PCR) in terms of time, cost and sensitivity with respect to PCR-SSCP method. We found that case sample can easily be differentiated from control sample by ...

  16. Application of high-resolution melting for variant scanning in ...

    African Journals Online (AJOL)

    High-resolution melting (HRM) analysis is a rapid and sensitive method for single nucleotide polymorphism (SNP) analysis. In this study, a novel HRM assay was carried out to detect SNPs in the chloroplast gene atpB which encodes the beta subunit of the ATP synthase and atpB upstream intergenic region.

  17. [High resolution melting and its application in identity study of traditional Chinese medicine].

    Science.gov (United States)

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

    2015-12-01

    High resolution melting (HRM), based on melting curve analysis, requires not only saturating dyes that fluoresce in the presence of double-stranded DNA, but also higher resolution detection equipment. The melting curve is a novel method for sequence matching, genotyping and mutation scanning. The technology is simple, accurate, rapid, closed-tube, low-cost, and high-throughput, which make it gain more and more applications. This review article presents the basic principles, key factors and both the advantage and limitations of HRM. The potential application is discussed in the study of molecular identity of traditional Chinese medicine.

  18. High resolution melting curve analysis targeting the HBB gene mutational hot-spot offers a reliable screening approach for all common as well as most of the rare beta-globin gene mutations in Bangladesh.

    Science.gov (United States)

    Islam, Md Tarikul; Sarkar, Suprovath Kumar; Sultana, Nusrat; Begum, Mst Noorjahan; Bhuyan, Golam Sarower; Talukder, Shezote; Muraduzzaman, A K M; Alauddin, Md; Islam, Mohammad Sazzadul; Biswas, Pritha Promita; Biswas, Aparna; Qadri, Syeda Kashfi; Shirin, Tahmina; Banu, Bilquis; Sadya, Salma; Hussain, Manzoor; Sarwardi, Golam; Khan, Waqar Ahmed; Mannan, Mohammad Abdul; Shekhar, Hossain Uddin; Chowdhury, Emran Kabir; Sajib, Abu Ashfaqur; Akhteruzzaman, Sharif; Qadri, Syed Saleheen; Qadri, Firdausi; Mannoor, Kaiissar

    2018-01-02

    Bangladesh lies in the global thalassemia belt, which has a defined mutational hot-spot in the beta-globin gene. The high carrier frequencies of beta-thalassemia trait and hemoglobin E-trait in Bangladesh necessitate a reliable DNA-based carrier screening approach that could supplement the use of hematological and electrophoretic indices to overcome the barriers of carrier screening. With this view in mind, the study aimed to establish a high resolution melting (HRM) curve-based rapid and reliable mutation screening method targeting the mutational hot-spot of South Asian and Southeast Asian countries that encompasses exon-1 (c.1 - c.92), intron-1 (c.92 + 1 - c.92 + 130) and a portion of exon-2 (c.93 - c.217) of the HBB gene which harbors more than 95% of mutant alleles responsible for beta-thalassemia in Bangladesh. Our HRM approach could successfully differentiate ten beta-globin gene mutations, namely c.79G > A, c.92 + 5G > C, c.126_129delCTTT, c.27_28insG, c.46delT, c.47G > A, c.92G > C, c.92 + 130G > C, c.126delC and c.135delC in heterozygous states from the wild type alleles, implying the significance of the approach for carrier screening as the first three of these mutations account for ~85% of total mutant alleles in Bangladesh. Moreover, different combinations of compound heterozygous mutations were found to generate melt curves that were distinct from the wild type alleles and from one another. Based on the findings, sixteen reference samples were run in parallel to 41 unknown specimens to perform direct genotyping of the beta-thalassemia specimens using HRM. The HRM-based genotyping of the unknown specimens showed 100% consistency with the sequencing result. Targeting the mutational hot-spot, the HRM approach could be successfully applied for screening of beta-thalassemia carriers in Bangladesh as well as in other countries of South Asia and Southeast Asia. The approach could be a useful supplement of hematological and

  19. Identification of a new genetic marker in Mycoplasma synoviae vaccine strain MS-H and development of a strategy using polymerase chain reaction and high-resolution melting curve analysis for differentiating MS-H from field strains.

    Science.gov (United States)

    Zhu, Ling; Konsak, Barbara M; Olaogun, Olusola M; Agnew-Crumptona, Rebecca; Kanci, Anna; Marenda, Marc S; Browning, Glenn F; Noormohammadi, Amir H

    2017-10-01

    Mycoplasma synoviae (MS) is an economically important avian pathogen worldwide, causing subclinical respiratory tract infection and infectious synovitis in chickens and turkeys. A temperature-sensitive (ts(+)) live attenuated vaccine MS-H, derived from the Australian field strain 86079/7NS, is now widely used in many countries to control the disease induced by MS. Differentiation of MS-H vaccine from field strains is crucial for monitoring vaccination programs in commercial poultry. Comparison of genomic sequences of MS-H and its parent strain revealed an adenine deletion at nucleotide position 468 of the MS-H oppF-1 gene. This mutation was shown to be unique to MS-H in further comparative analyses of oppF-1 genes of MS-H re-isolates and field strains from Australia and other countries. Based on this single nucleotide, a combination of nested PCR and high-resolution melting (HRM) curve analysis was used to evaluate its potential for use in differentiation of MS-H from field strains. The mean genotype confidence percentages of 99.27 and 48.20 for MS-H and field strains, respectively, demonstrated the high discriminative power of the newly developed assay (oppF PCR-HRM). A set of 13 tracheal swab samples collected from MS-H vaccinated specific pathogen free birds and commercial chicken flocks infected with MS were tested using the oppF PCR-HRM test and results were totally consistent with those obtained using vlhA genotyping. The nested-PCR HRM method established in this study proved to be a rapid, simple and cost effective tool for discriminating the MS-H vaccine strain from Australian and international strains in pure cultures and on tracheal swabs. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Differentiation of Staphylococcus spp. by high-resolution melting analysis.

    Science.gov (United States)

    Slany, Michal; Vanerkova, Martina; Nemcova, Eva; Zaloudikova, Barbora; Ruzicka, Filip; Freiberger, Tomas

    2010-12-01

    High-resolution melting analysis (HRMA) is a fast (post-PCR) high-throughput method to scan for sequence variations in a target gene. The aim of this study was to test the potential of HRMA to distinguish particular bacterial species of the Staphylococcus genus even when using a broad-range PCR within the 16S rRNA gene where sequence differences are minimal. Genomic DNA samples isolated from 12 reference staphylococcal strains (Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus intermedius, Staphylococcus saprophyticus, Staphylococcus sciuri, Staphylococcus simulans, Staphylococcus warneri, and Staphylococcus xylosus) were subjected to a real-time PCR amplification of the 16S rRNA gene in the presence of fluorescent dye EvaGreen™, followed by HRMA. Melting profiles were used as molecular fingerprints for bacterial species differentiation. HRMA of S. saprophyticus and S. xylosus resulted in undistinguishable profiles because of their identical sequences in the analyzed 16S rRNA region. The remaining reference strains were fully differentiated either directly or via high-resolution plots obtained by heteroduplex formation between coamplified PCR products of the tested staphylococcal strain and phylogenetically unrelated strain.

  1. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons.

    Science.gov (United States)

    Liew, Michael; Pryor, Robert; Palais, Robert; Meadows, Cindy; Erali, Maria; Lyon, Elaine; Wittwer, Carl

    2004-07-01

    High-resolution melting of PCR amplicons with the DNA dye LCGreen I was recently introduced as a homogeneous, closed-tube method of genotyping that does not require probes or real-time PCR. We adapted this system to genotype single-nucleotide polymorphisms (SNPs) after rapid-cycle PCR (12 min) of small amplicons (SNP base changes. In addition, clinical protocols for factor V (Leiden) 1691G>A, prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 1298A>C, hemochromatosis (HFE) 187C>G, and beta-globin (hemoglobin S) 17A>T were developed. LCGreen I was included in the reaction mixture before PCR, and high-resolution melting was obtained within 2 min after amplification. In all cases, heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves. Approximately 84% of human SNPs involve a base exchange between A::T and G::C base pairs, and the homozygotes are easily genotyped by melting temperatures (T(m)s) that differ by 0.8-1.4 degrees C. However, in approximately 16% of SNPs, the bases only switch strands and preserve the base pair, producing very small T(m) differences between homozygotes (G protocol, but, as predicted from the sequence changes, was not needed for the other four clinical protocols. SNP genotyping by high-resolution melting analysis is simple, rapid, and inexpensive, requiring only PCR, a DNA dye, and melting instrumentation. The method is closed-tube, performed without probes or real-time PCR, and can be completed in less than 2 min after completion of PCR.

  2. High-Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science.

    Science.gov (United States)

    Dos Santos Rocha, Alípio; de Amorim, Isis Salviano Soares; Simão, Tatiana de Almeida; da Fonseca, Adenilson de Souza; Garrido, Rodrigo Grazinoli; Mencalha, Andre Luiz

    2017-08-23

    Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high-resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual-dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics. © 2017 American Academy of Forensic Sciences.

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

    Science.gov (United States)

    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. © 2015 Society for Laboratory Automation and Screening.

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

    Directory of Open Access Journals (Sweden)

    Florence Naze

    Full Text Available 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.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.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.

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

    Science.gov (United States)

    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

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

    National Research Council Canada - National Science Library

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

    2016-01-01

    .... 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...

  7. Assessment of high resolution melting analysis as a potential SNP genotyping technique in forensic casework.

    Science.gov (United States)

    Venables, Samantha J; Mehta, Bhavik; Daniel, Runa; Walsh, Simon J; van Oorschot, Roland A H; McNevin, Dennis

    2014-11-01

    High resolution melting (HRM) analysis is a simple, cost effective, closed tube SNP genotyping technique with high throughput potential. The effectiveness of HRM for forensic SNP genotyping was assessed with five commercially available HRM kits evaluated on the ViiA™ 7 Real Time PCR instrument. Four kits performed satisfactorily against forensically relevant criteria. One was further assessed to determine the sensitivity, reproducibility, and accuracy of HRM SNP genotyping. The manufacturer's protocol using 0.5 ng input DNA and 45 PCR cycles produced accurate and reproducible results for 17 of the 19 SNPs examined. Problematic SNPs had GC rich flanking regions which introduced additional melting domains into the melting curve (rs1800407) or included homozygotes that were difficult to distinguish reliably (rs16891982; a G to C SNP). A proof of concept multiplexing experiment revealed that multiplexing a small number of SNPs may be possible after further investigation. HRM enables genotyping of a number of SNPs in a large number of samples without extensive optimization. However, it requires more genomic DNA as template in comparison to SNaPshot®. Furthermore, suitably modifying pre-existing forensic intelligence SNP panels for HRM analysis may pose difficulties due to the properties of some SNPs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.).

    Science.gov (United States)

    Han, Yuanhong; Khu, Dong-Man; Monteros, Maria J

    2012-02-01

    Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic polymorphism in plant genomes. SNP markers are valuable tools for genetic analysis of complex traits of agronomic importance, linkage and association mapping, genome-wide selection, map-based cloning, and marker-assisted selection. Current challenges for SNP genotyping in polyploid outcrossing species include multiple alleles per loci and lack of high-throughput methods suitable for variant detection. In this study, we report on a high-resolution melting (HRM) analysis system for SNP genotyping and mapping in outcrossing tetraploid genotypes. The sensitivity and utility of this technology is demonstrated by identification of the parental genotypes and segregating progeny in six alfalfa populations based on unique melting curve profiles due to differences in allelic composition at one or multiple loci. HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes. Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9566-x) contains supplementary material, which is available to authorized users.

  9. Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis.

    Science.gov (United States)

    Li, Juan; Zhao, Guang-Hui; Lin, RuiQing; Blair, David; Sugiyama, Hiromu; Zhu, Xing-Quan

    2015-11-01

    Schistosomiasis, caused by blood flukes belonging to several species of the genus Schistosoma, is a serious and widespread parasitic disease. Accurate and rapid differentiation of these etiological agents of animal and human schistosomiasis to species level can be difficult. We report a real-time PCR assay coupled with a high-resolution melt (HRM) assay targeting a portion of the nuclear 18S rDNA to detect, identify, and distinguish between four major blood fluke species (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma mekongi). Using this system, the Schistosoma spp. was accurately identified and could also be distinguished from all other trematode species with which they were compared. As little as 10(-5) ng genomic DNA from a Schistosoma sp. could be detected. This process is inexpensive, easy, and can be completed within 3 h. Examination of 21 representative Schistosoma samples from 15 geographical localities in seven endemic countries validated the value of the HRM detection assay and proved its reliability. The melting curves were characterized by peaks of 83.65 °C for S. japonicum and S. mekongi, 85.65 °C for S. mansoni, and 85.85 °C for S. haematobium. The present study developed a real-time PCR coupled with HRM analysis assay for detection and differential identification of S. mansoni, S. haematobium, S. japonicum, and S. mekongi. This method is rapid, sensitive, and inexpensive. It has important implications for epidemiological studies of Schistosoma.

  10. Identification of gene mutation in patients with osteogenesis imperfect using high resolution melting analysis.

    Science.gov (United States)

    Wang, Jianhai; Ren, Xiuzhi; Bai, Xue; Zhang, Tianke; Wang, Yi; Li, Keqiu; Li, Guang

    2015-08-26

    Osteogenesis imperfecta (OI), a congenital bone disorder, is caused by mutations in COL1A1 and COL1A2 genes, leading to deficiency of type I collagen. The high resolution melting (HRM) analysis has been used for detecting mutations, polymorphisms and epigenetic alteration in double-stranded DNAs. This study was to evaluate the potential application of HRM analysis for identifying gene mutations in patients with OI. This study included four children with OI and their parents and fifty normal people as controls. Blood samples were collected for HRM analysis of PCR-amplified exons and flanking DNA sequences of COL1A1 and COL1A2 genes. Direct gene sequencing was performed to validate HRM-identified gene mutations. As compared to controls, HRM analysis of samples form children with OI showed abnormal melting curves in exons 11 and 33-34 of the COL1A1 gene and exons 19 and 48 of the COL1A2 gene, which indicates the presence of heterozygous mutations in COL1A1 and COL1A2 genes. In addition to two known mutations in the COL1A2 gene, c.982G > A and c.3197G > T, sequencing analysis identified two novel mutations in the COL1A1 gene, c.2321delC and c.768dupC mutations, which function as premature stop codons. These results support future studies of applying HRM analysis as a diagnostic approach for OI.

  11. Rapid detection and identification of human hookworm infections through high resolution melting (HRM analysis.

    Directory of Open Access Journals (Sweden)

    Romano Ngui

    Full Text Available 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.

  12. Rapid detection and identification of human hookworm infections through high resolution melting (HRM) analysis.

    Science.gov (United States)

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

    2012-01-01

    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. 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. 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.

  13. Detection of somatic mutations by high-resolution DNA melting (HRM analysis in multiple cancers.

    Directory of Open Access Journals (Sweden)

    Jesus Gonzalez-Bosquet

    Full Text Available Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each. HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

  14. Differentiation Borrelia Species in Environmental Samples with High-Resolution DNA Melting Analysis.

    Science.gov (United States)

    Wodecka, Beata; Skotarczak, Bogumiła

    2015-01-01

    Borrelia burgdorferi sensu lato includes at least 20 species in the world, and half of these are found in Europe. The usefulness of high resolution melting (HRM) analysis of DNA denaturation curves has been assessed for differentiation of Borrelia species. HRM protocol for Borrelia species was used to examine the 77 DNA extracts selected from earlier studies with the use of three different molecular markers: flaB, rplL, and groEL. The studies revealed that the best marker is the groEL gene, which enables identification of 8 Borrelia species, including B. miyamotoi from the relapsing fever borreliae group and 7 of B. burgdorferi s.l. complex (B. garinii, B, afzelii, B. burgdorferi s.s., B. valaisiana, B. lusitaniae, B. bissetii, B. spielmanii). The HRM method, when compared with other PCR variants with regard to the reduced time of analysis, is an alternative for the procedures used in the molecular diagnostics of borreliosis including testing of blood samples or saved Ixodes ticks for the presence and genotyping of Borrelia burgdorferi after biting a patient.

  15. Development of a high-resolution melting method for the detection of hemoglobin alpha variants.

    Science.gov (United States)

    Shih, Hung-Chang; Er, Tze-Kiong; Chang, Tien-Jye; Chang, Ya-Sian; Liu, Ta-Chih; Chang, Jan-Gowth

    2010-05-01

    The present study was aimed at identifying hemoglobin (Hb) alpha variants. To identify Hb variants, a high-resolution melting (HRM) method was performed. The diagnostic strategy was found to be successful in identifying Hb alpha variants including HBA1:c.27G>T, (Hb Hekinan) HBA1:c.46G>C (Hb Ottawa), HBA2:c.31_33AG (Hb alpha2-globin gene codon del AG), HBA1:c.223G>C (Hb G-Taichung), HBA1:p.Phe118_Thr119insIle (Hb Phnom Penh), HBA2:c.369C>G (Hb Westmead), HBA2:c.364G>A (or HBA1) (Hb Owari), HBA2:c.377T>C (Hb Quong Sze), and HBA2:c.427T>C (Hb Constant Spring). Each Hb variant could be readily and easily identified through the difference in plotted curves. In addition, the Hb variants could be distinguished to be located at either HBA1 or HBA2 gene. The HRM analysis is found to be a good tool for identifying Hb variants in alpha globin genes. 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  16. ScreenClust: Advanced statistical software for supervised and unsupervised high resolution melting (HRM) analysis.

    Science.gov (United States)

    Reja, Valin; Kwok, Alister; Stone, Glenn; Yang, Linsong; Missel, Andreas; Menzel, Christoph; Bassam, Brant

    2010-04-01

    High resolution melting (HRM) is an emerging new method for interrogating and characterizing DNA samples. An important aspect of this technology is data analysis. Traditional HRM curves can be difficult to interpret and the method has been criticized for lack of statistical interrogation and arbitrary interpretation of results. Here we report the basic principles and first applications of a new statistical approach to HRM analysis addressing these concerns. Our method allows automated genotyping of unknown samples coupled with formal statistical information on the likelihood, if an unknown sample is of a known genotype (by discriminant analysis or "supervised learning"). It can also determine the assortment of alleles present (by cluster analysis or "unsupervised learning") without a priori knowledge of the genotypes present. The new algorithms provide highly sensitive and specific auto-calling of genotypes from HRM data in both supervised an unsupervised analysis mode. The method is based on pure statistical interrogation of the data set with a high degree of standardization. The hypothesis-free unsupervised mode offers various possibilities for de novo HRM applications such as mutation discovery. Copyright 2010. Published by Elsevier Inc.

  17. Differentiation of BHV-1 isolates from vaccine virus by high-resolution melting analysis.

    Science.gov (United States)

    Ostertag-Hill, Claire; Fang, Liang; Izume, Satoko; Lee, Megan; Reed, Aimee; Jin, Ling

    2015-02-16

    An efficacious bovine herpesvirus type-1 (BHV-1) vaccine has been used for many years. However, in the past few years, abortion and respiratory diseases have occurred after administration of the modified live vaccine. To investigate whether BHV-1 isolates from disease outbreaks are identical to those of the vaccines used, selected regions of the BHV-1 genome were investigated by high-resolution melting (HRM) analysis and PCR-DNA sequencing. When a target region within the thymidine kinase (TK) gene was examined by HRM analysis, 6 out of the 11 isolates from abortion cases and 22 out of the 25 isolates from bovine respiratory disease (BRD) cases had different melting curves compared to the vaccine virus. Surprisingly, when a conserved region within the US6 gene that encodes glycoprotein D (gD) was examined by HRM analysis, 5 out of the 11 abortion isolates and 18 out of the 23 BRD isolates had different melting curves from the vaccine virus. To determine whether SNPs within the coding regions of glycoprotein E (gE) and TK genes can be used to differentiate the isolates from the vaccine virus, PCR-DNA sequencing was used to examine these SNPs in all the isolates. This revealed that only 1 out of 11 of the abortion isolates and 4 out of 24 of the BRD isolates are different in the target region of gE from the vaccine virus, while 5 out of 11 abortion isolates and 4 out of 22 BRD isolates are different in the target region of TK from the vaccine virus. No DNA sequence differences were observed in glycoprotein G (gG) region between disease and vaccine isolates. Our study demonstrated that many disease isolates had genetic differences from the vaccine virus in regions examined by HRM and PCR-DNA sequencing analysis. In addition, many isolates contained more than one type of mutation and were composed of mixed variants. Our study suggests that a mixture of variants were present in isolates collected post-vaccination. HRM is a rapid diagnostic method that can be used for

  18. Primate genotyping via high resolution melt analysis: rapid and reliable identification of color vision status in wild lemurs.

    Science.gov (United States)

    Jacobs, Rachel L; Spriggs, Amanda N; MacFie, Tammie S; Baden, Andrea L; Irwin, Mitchell T; Wright, Patricia C; Louis, Edward E; Lawler, Richard R; Mundy, Nicholas I; Bradley, Brenda J

    2016-10-01

    Analyses of genetic polymorphisms can aid our understanding of intra- and interspecific variation in primate sociality, ecology, and behavior. Studies of primate opsin genes are prime examples of this, as single nucleotide variants (SNVs) in the X-linked opsin gene underlie variation in color vision. For primate species with polymorphic trichromacy, genotyping opsin SNVs can generally indicate whether individual primates are red-green color-blind (denoted homozygous M or homozygous L) or have full trichromatic color vision (heterozygous ML). Given the potential influence of color vision on behavior and fitness, characterizing the color vision status of study subjects is becoming commonplace for many primate field projects. Such studies traditionally involve a multi-step sequencing-based method that can be costly and time-consuming. Here we present a new reliable, rapid, and relatively inexpensive method for characterizing color vision in primate populations using high resolution melt analysis (HRMA). Using lemurs as a case study, we characterized variation at exons 3 and/or 5 of the X-linked opsin gene for 87 individuals representing nine species. We scored opsin genotypes and color vision status using both traditional sequencing-based methods as well as our novel melting-curve based HRMA protocol. For each species, the melting curves of varying genotypes (homozygous M, homozygous L, heterozygous ML) differed in melting temperature and/or shape. Melting curves for each sample were consistent across replicates, and genotype-specific melting curves were consistent across DNA sources (blood vs. feces). We show that opsin genotypes can be quickly and reliably scored using HRMA once lab-specific reference curves have been developed based on known genotypes. Although the protocol presented here focuses on genotyping lemur opsin loci, we also consider the larger potential for applying this approach to various types of genetic studies of primate populations.

  19. Evaluation of DNA barcoding coupled high resolution melting for discrimination of closely related species in phytopharmaceuticals.

    Science.gov (United States)

    Osathanunkul, Maslin; Suwannapoom, Chatmongkon; Osathanunkul, Kitisak; Madesis, Panagiotis; de Boer, Hugo

    2016-02-15

    Phytopharmaceuticals are increasingly popular as alternative medicines, but poorly regulated in many countries. The manufacturers of these products should be subject to strict controls regarding each product's quality and constituents. Routine testing and identification of raw materials should be performed to ensure that the raw materials used in pharmaceutical products are suitable for their intended use. We have applied DNA Barcoding - High Resolution Melting (Bar-HRM), an emerging method for identifying of medicinal plant species based on DNA dissociation kinetics and DNA barcoding, for the authentication of medicinal plant species. Commonly commercialized Thai medicinal plants that are widely used for medicinal purposes were used in this study. Publicly available sequences of four plastid markers were used for universal primer design. Species discrimination efficiency of the designed primers was evaluated as single and multi-locus analyses by using the primers sets. HRM analysis was performed in triplicate on each of the 26 taxa to establish the Tm for each primer set (matK, rbcLA, rbcLB, rbcLC, rpoC1, and trnL). The shapes of the melting curves were analyzed to distinguish the different plant species. Bar-HRM species identification success rates were assessed for each single-locus as well as for multi-locus combinations to establish the optimal combination of primer sets. In single locus analysis the rpoC1 primer set gave the highest discrimination (58%), and in multi locus analysis this could be increased from 87% to 99% depending on the total number of regions included. Different combinations proved to be more or less effective at discrimination, depending on the genus or family examined. Bar-HRM has proven to be a cost-effective and reliable method for the identification of species in this study of Thai medicinal plants, and results show an identification success rate of 99% among species in the test set. Copyright © 2016 Elsevier GmbH. All rights reserved.

  20. Evaluation of spa-typing of methicillin-resistant Staphylococcus aureus using high-resolution melting analysis.

    Science.gov (United States)

    Mazi, Waleed; Sangal, Vartul; Sandstrom, Gunnar; Saeed, Amir; Yu, Jun

    2015-09-01

    spa-typing of methicillin-resistant Staphylococcus aureus (MRSA) has been used widely in clinical diagnostics and epidemiological studies. The aim of this study was to evaluate high-resolution melting (HRM) as a rapid and cost-effective method, to replace DNA-sequencing, for spa-typing in a global collection of 50 MRSA isolates. The polymorphic X region of the spa gene was amplified by colony PCR using the SensiMix HRM kit, and the melting temperature (Tm) and melting curves of the amplicons were analyzed in close tubes using a Rotor-Gene 6000 instrument. Fifteen out of 19 spa-types each had a distinct Tm, which was sufficient to unambiguously type each of these spa-types. The remaining four spa-types could not be separated by Tm alone: t008 and t2770 shared a Tm (80.3°C) and t021 and t311 shared a Tm (80.0°C). However, they could be separated based on the shapes of their melting curves. There are discrepancies between the findings of the present study and those of previous studies, suggesting that standardization remains a challenge for cross-referencing. HRM-based spa-typing is reproducible, simple, rapid, and cost-effective. t037 is prevalent in Brazil and Sudan, while diverse spa-types are found in Scotland and Saudi Arabia. Standardization is required for cross-referencing between laboratories globally. Copyright © 2015. Published by Elsevier Ltd.

  1. Study of the role of tumor necrosis factor-α (-308 G/A) and interleukin-10 (-1082 G/A) polymorphisms as potential risk factors to acute kidney injury in patients with severe sepsis using high-resolution melting curve analysis.

    Science.gov (United States)

    Hashad, Doaa I; Elsayed, Eman T; Helmy, Tamer A; Elawady, Samier M

    2017-11-01

    Septic acute kidney injury (AKI) is a prevalent complication in intensive care units with an increased incidence of complications. The aim of the present study was to assess the use of high-resolution melting curve (HRM) analysis in investigating whether the genetic polymorphisms; -308 G/A of tumor necrosis factor-α (TNF-α), and -1082 G /A of Interleukin-10 (IL-10) genes may predispose patients diagnosed with severe sepsis to the development of AKI. One hundred and fifty patients with severe sepsis participated in the present study; only sixty-six developed AKI. Both polymorphisms were studied using HRM analysis. The low producer genotype of both studied polymorphism of TNF-α and IL-10 genes was associated with AKI. Using logistic regression analysis, the low producer genotypes remained an independent risk factor for AKI. A statistically significant difference was detected between both studied groups as regards the low producer genotype in both TNF-α (-308 G/A) and interleukin-10 (IL-10) (-1082 G/A) polymorphisms being prevalent in patients developing AKI. Principle conclusions: The low producer genotypes of both TNF-α (-308 G/A) and IL-10 (-1082 G/A) polymorphisms could be considered a risk factor for the development of AKI in critically ill patients with severe sepsis, thus management technique implemented for this category should be modulated rescuing this sector of patients from the grave deterioration to acute kidney injury. Using HRM for genotyping proved to be a highly efficient, simple, cost-effective genotyping technique that is most appropriate for the routine study of large-scale samples.

  2. High-resolution melting analysis (HRMA): a highly sensitive inexpensive genotyping alternative for population studies.

    Science.gov (United States)

    Smith, B L; Lu, C-P; Alvarado Bremer, J R

    2010-01-01

    High-resolution melting analysis (HRMA) is a highly sensitive closed-tube genotyping method used primarily in clinical studies. As the method is rapid, inexpensive and amenable to high throughput, we decided to investigate its applicability to population studies. Small amplicons and unlabelled probes were used to genotype the nuclear genes, lactate dehydrogenase-A (ldh-A), myosin light chain-2 (mlc-2), acidic ribosomal phosphoprotein P0 (ARP) and calmodulin (CaM) in populations of swordfish, Xiphias gladius. Results indicate that HRMA is a powerful genotyping tool to study wild populations. © 2009 Blackwell Publishing Ltd.

  3. Direct genotyping of single nucleotide polymorphisms in methyl metabolism genes using probe-free high-resolution melting analysis.

    Science.gov (United States)

    Kristensen, Lasse S; Dobrovic, Alexander

    2008-05-01

    High-resolution melting (HRM) shows great promise for high-throughput, rapid genotyping of individual polymorphic loci. We have developed HRM assays for genotyping single nucleotide polymorphisms (SNP) in several key genes that are involved in methyl metabolism and may directly or indirectly affect the methylation status of the DNA. The SNPs are in the 5,10-methylenetetrahydrofolate reductase (MTHFR; C677T and A1298C), methionine synthetase (MTR; 5-methyltetrahydrofolate-homocysteine methyltransferase; A2756G), and DNA methyltransferase 3b (DNMT3b; C46359T and C31721T) loci. The choice of short amplicons led to greater melting temperature (Tm) differences between the two homozygous genotypes, which allowed accurate genotyping without the use of probes or spiking with control DNA. In the case of MTHFR, there is a second rarer SNP (rs4846051) close to the A1298C SNP that may result in inaccurate genotyping. We masked this second SNP by placing the primer over it and choosing a base at the polymorphic position that was equally mismatched to both alleles. The HRM assays were done on HRM capable real-time PCR machines rather than stand-alone HRM machines. Monitoring the amplification allows ready identification of samples that may give rise to aberrant melting curves because of PCR abnormalities. We show that samples amplifying markedly late can give rise to shifted melting curves without alteration of shapes and potentially lead to misclassification of genotypes. In conclusion, rapid and high-throughput SNP analysis can be done with probe-free HRM if sufficient attention is paid to amplicon design and quality control to omit aberrantly amplifying samples.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Rapid identification of HBB gene mutations by high-resolution melting analysis.

    Science.gov (United States)

    Shih, Hung-Chang; Er, Tze-Kiong; Chang, Tien-Jye; Chang, Ya-Sian; Liu, Ta-Chih; Chang, Jan-Gowth

    2009-11-01

    This study was undertaken to identify HBB gene mutation. Herein we evaluated high-resolution melting analysis in the identification of HBB mutations. We have successfully established a diagnostic strategy for identifying HBB gene mutations including c.-78A>G, c.-79A>G, c.2T>G, c.79_80insT, c.84_85insC, c.123_124insT, c.125_128delTCTT, c.130 G>T, c.170G>A, c.216_217ins A and c.316-197 C>T from wild-type DNA using HRM analysis. The results of HRM analysis were confirmed by direct DNA sequencing. In summary, we report that HRM analysis is an appealing technique for the identification of HBB mutations. We also believe that HRM can be used as a method for prenatal diagnosis of beta-thalassemia.

  6. The spa typing of methicillin-resistant Staphylococcus aureus isolates by High Resolution Melting (HRM) analysis.

    Science.gov (United States)

    Fasihi, Yasser; Fooladi, Saba; Mohammadi, Mohammad Ali; Emaneini, Mohammad; Kalantar-Neyestanaki, Davood

    2017-09-06

    Molecular typing is an important tool for control and prevention of infection. A suitable molecular typing method for epidemiological investigation must be easy to perform, highly reproducible, inexpensive, rapid and easy to interpret. In this study, two molecular typing methods including the conventional PCR-sequencing method and high resolution melting (HRM) analysis were used for staphylococcal protein A (spa) typing of 30 Methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from clinical samples. Based on PCR-sequencing method results, 16 different spa types were identified among the 30 MRSA isolates. Among the 16 different spa types, 14 spa types separated by HRM method. Two spa types including t4718 and t2894 were not separated from each other. According to our results, spa typing based on HRM analysis method is very rapid, easy to perform and cost-effective, but this method must be standardized for different regions, spa types, and real-time machinery.

  7. High-resolution melting facilitates mutation screening of PYGM in patients with McArdle disease

    DEFF Research Database (Denmark)

    Duno, M.; Quinlivan, R.; Vissing, J.

    2009-01-01

    Mutations in PYGM, encoding the muscle-specific glycogen phosphorylase (myophosphorylase), are responsible for McArdle disease. Among Caucasians, a large proportion of patients are homozygous for the R50X mutation, but other mutations can affect all the 20 exons of PYGM, making mutation detection...... variations. Thirteen of these are pathogenic, and three were classified as polymorphisms. Nine variations had not previously been described. One of the novel mutations, c.2430C > T, was initially predicted to result in a silent G810G change, but cDNA analysis demonstrated that the mutation led to abnormal m...... laborious. We have developed a high-resolution melting (HRM) assay for mutation detection in PYGM. Twelve McArdle patients were investigated, in whom pre-screening had ruled out homozygosity or compound heterozygosity for the two common G205S and R50X mutations. In total, we identified 16 different...

  8. Genome-wide SNP discovery in tetraploid alfalfa using 454 sequencing and high resolution melting analysis

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    Zhao Patrick X

    2011-07-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs are the most common type of sequence variation among plants and are often functionally important. We describe the use of 454 technology and high resolution melting analysis (HRM for high throughput SNP discovery in tetraploid alfalfa (Medicago sativa L., a species with high economic value but limited genomic resources. Results The alfalfa genotypes selected from M. sativa subsp. sativa var. 'Chilean' and M. sativa subsp. falcata var. 'Wisfal', which differ in water stress sensitivity, were used to prepare cDNA from tissue of clonally-propagated plants grown under either well-watered or water-stressed conditions, and then pooled for 454 sequencing. Based on 125.2 Mb of raw sequence, a total of 54,216 unique sequences were obtained including 24,144 tentative consensus (TCs sequences and 30,072 singletons, ranging from 100 bp to 6,662 bp in length, with an average length of 541 bp. We identified 40,661 candidate SNPs distributed throughout the genome. A sample of candidate SNPs were evaluated and validated using high resolution melting (HRM analysis. A total of 3,491 TCs harboring 20,270 candidate SNPs were located on the M. truncatula (MT 3.5.1 chromosomes. Gene Ontology assignments indicate that sequences obtained cover a broad range of GO categories. Conclusions We describe an efficient method to identify thousands of SNPs distributed throughout the alfalfa genome covering a broad range of GO categories. Validated SNPs represent valuable molecular marker resources that can be used to enhance marker density in linkage maps, identify potential factors involved in heterosis and genetic variation, and as tools for association mapping and genomic selection in alfalfa.

  9. A novel typing method for Listeria monocytogenes using high-resolution melting analysis (HRMA) of tandem repeat regions.

    Science.gov (United States)

    Ohshima, Chihiro; Takahashi, Hajime; Iwakawa, Ai; Kuda, Takashi; Kimura, Bon

    2017-07-17

    Listeria monocytogenes, which is responsible for causing food poisoning known as listeriosis, infects humans and animals. Widely distributed in the environment, this bacterium is known to contaminate food products after being transmitted to factories via raw materials. To minimize the contamination of products by food pathogens, it is critical to identify and eliminate factory entry routes and pathways for the causative bacteria. High resolution melting analysis (HRMA) is a method that takes advantage of differences in DNA sequences and PCR product lengths that are reflected by the disassociation temperature. Through our research, we have developed a multiple locus variable-number tandem repeat analysis (MLVA) using HRMA as a simple and rapid method to differentiate L. monocytogenes isolates. While evaluating our developed method, the ability of MLVA-HRMA, MLVA using capillary electrophoresis, and multilocus sequence typing (MLST) was compared for their ability to discriminate between strains. The MLVA-HRMA method displayed greater discriminatory ability than MLST and MLVA using capillary electrophoresis, suggesting that the variation in the number of repeat units, along with mutations within the DNA sequence, was accurately reflected by the melting curve of HRMA. Rather than relying on DNA sequence analysis or high-resolution electrophoresis, the MLVA-HRMA method employs the same process as PCR until the analysis step, suggesting a combination of speed and simplicity. The result of MLVA-HRMA method is able to be shared between different laboratories. There are high expectations that this method will be adopted for regular inspections at food processing facilities in the near future. Copyright © 2017. Published by Elsevier B.V.

  10. High-resolution melting analysis for detection of MYH9 mutations.

    Science.gov (United States)

    Provaznikova, Dana; Kumstyrova, Tereza; Kotlin, Roman; Salaj, Peter; Matoska, Vaclav; Hrachovinova, Ingrid; Rittich, Simon

    2008-09-01

    May-Hegglin anomaly (MHA), Sebastian (SBS), Fechtner (FTNS) and Epstein (EPS) syndromes are rare autosomal dominant disorders with giant platelets and thrombocytopenia. Other manifestations of these disorders are combinations of the presence of granulocyte inclusions and deafness, cataracts and renal failure. Currently, MHA, SBS, FTNS and EPS are considered to be distinct clinical manifestation of a single illness caused by mutations of the MYH9 gene encoding the heavy chain of non-muscle myosin IIA (NMMHC-IIA). As the MYH9 gene has a high number of exons, it takes much time and material to use this method for the detection of MYH9 mutations. Recently, a new method has been introduced for scanning DNA mutations without the need for direct sequencing: high-resolution melting analysis (HRMA). Mutation detection with HRMA relies on the intercalation of the specific dye (LC Green plus) in double-strand DNA and fluorescence monitoring of PCR product melting profiles. In our study, we optimized the conditions and used HRMA for rapid screening of mutations in all MYH9 exons in seven affected individuals from four unrelated families with suspected MYH9 disorders. Samples identified by HRMA as positive for the mutation were analysed by direct sequencing. HRMA saved us over 85% of redundant sequencing.

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

    Directory of Open Access Journals (Sweden)

    Ana Ramón-Laca

    Full Text Available 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.

  12. The use of high-resolution melting analysis for rapid spa typing on methicillin-resistant Staphylococcus aureus clinical isolates.

    Science.gov (United States)

    Chen, Jonathan Hon-Kwan; Cheng, Vincent Chi-Chung; Chan, Jasper Fuk-Woo; She, Kevin Kin-Kwan; Yan, Mei-Kum; Yau, Miranda Chong-Yee; Kwan, Grace See-Wai; Yam, Wing-Cheong; Yuen, Kwok-Yung

    2013-02-15

    Methicillin-resistant Staphylococcus aureus (MRSA) has been endemic in Hong Kong for three decades. This study evaluated the practical use of high-resolution melting (HRM) real-time PCR analysis on MRSA staphylococcal Protein A (spa) typing on local MRSA isolates. Among 55 clinical MRSA isolates collected in 2011, 12 different spa types were observed by the conventional PCR-sequencing method including the locally predominant spa type t1081 and two locally predominant community acquired MRSA spa types t019 and t437. By using the HRM method, it could differentiate all 12 spa genotypes by distinct melting curves and HRM difference plot analysis. These two methods demonstrated 100% concordance whereas the HRM method required only 3h of turnaround time and one-fifth of reagent cost compared to the conventional method. Our study confirmed that the cost effective and rapid HRM typing approach is practically useful for MRSA community transmission monitoring and nosocomial outbreak control in Hong Kong. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. A curved image-plate detector system for high-resolution synchrotron X-ray diffraction.

    Science.gov (United States)

    Sarin, P; Haggerty, R P; Yoon, W; Knapp, M; Berghaeuser, A; Zschack, P; Karapetrova, E; Yang, N; Kriven, W M

    2009-03-01

    The developed curved image plate (CIP) is a one-dimensional detector which simultaneously records high-resolution X-ray diffraction (XRD) patterns over a 38.7 degrees 2theta range. In addition, an on-site reader enables rapid extraction, transfer and storage of X-ray intensity information in response is not compromised in the unsaturated regions of the image plate, regardless of saturation in another region. The speed of XRD data acquisition together with excellent resolution afforded by the CIP detector is unique and opens up wide possibilities in materials research accessible through X-ray diffraction. This article presents details of the basic features, operation and performance of the CIP detector along with some examples of applications, including high-temperature XRD.

  14. High-resolution fiber optic temperature sensors using nonlinear spectral curve fitting technique.

    Science.gov (United States)

    Su, Z H; Gan, J; Yu, Q K; Zhang, Q H; Liu, Z H; Bao, J M

    2013-04-01

    A generic new data processing method is developed to accurately calculate the absolute optical path difference of a low-finesse Fabry-Perot cavity from its broadband interference fringes. The method combines Fast Fourier Transformation with nonlinear curve fitting of the entire spectrum. Modular functions of LabVIEW are employed for fast implementation of the data processing algorithm. The advantages of this technique are demonstrated through high performance fiber optic temperature sensors consisting of an infrared superluminescent diode and an infrared spectrometer. A high resolution of 0.01 °C is achieved over a large dynamic range from room temperature to 800 °C, limited only by the silica fiber used for the sensor.

  15. DNA Barcoding Coupled with High Resolution Melting Analysis Enables Rapid and Accurate Distinction of Aspergillus species.

    Science.gov (United States)

    Fidler, Gabor; Kocsube, Sandor; Leiter, Eva; Biro, Sandor; Paholcsek, Melinda

    2017-08-01

    We describe a high-resolution melting (HRM) analysis method that is rapid, reproducible, and able to identify reference strains and further 40 clinical isolates of Aspergillus fumigatus (14), A. lentulus (3), A. terreus (7), A. flavus (8), A. niger (2), A. welwitschiae (4), and A. tubingensis (2). Asp1 and Asp2 primer sets were designed to amplify partial sequences of the Aspergillus benA (beta-tubulin) genes in a closed-, single-tube system. Human placenta DNA, further Aspergillus (3), Candida (9), Fusarium (6), and Scedosporium (2) nucleic acids from type strains and clinical isolates were also included in this study to evaluate cross reactivity with other relevant pathogens causing invasive fungal infections. The barcoding capacity of this method proved to be 100% providing distinctive binomial scores; 14, 34, 36, 35, 25, 15, 26 when tested among species, while the within-species distinction capacity of the assay proved to be 0% based on the aligned thermodynamic profiles of the Asp1, Asp2 melting clusters allowing accurate species delimitation of all tested clinical isolates. The identification limit of this HRM assay was also estimated on Aspergillus reference gDNA panels where it proved to be 10-102 genomic equivalents (GE) except the A. fumigatus panel where it was 103 only. Furthermore, misidentification was not detected with human genomic DNA or with Candida, Fusarium, and Scedosporium strains. Our DNA barcoding assay introduced here provides results within a few hours, and it may possess further diagnostic utility when analyzing standard cultures supporting adequate therapeutic decisions. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

    Science.gov (United States)

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

    2016-01-13

    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.

  17. Application of barcode high-resolution melting for rapid authentication of the medicinal plant Psammosilene tunicoides

    Directory of Open Access Journals (Sweden)

    Jingjian Li

    2016-07-01

    Full Text Available Psammosilene tunicoides is an important herb used in traditional Chinese medicine. It has been proved to benefit the stomach and treat rheumatism and gout. On the market, this herbal medicine is frequently adulterated by the related species Silene viscidula. Correct identification of P. tunicoides is important to ensure herbal quality, safety, authenticity and health for consumers. However, the identification of the P. tunicoides and S. viscidula species is complicated because of their morphological similarities. Therefore, a reliable method to authenticate these two medicinal plants is needed. In this study, the ITS2 barcode region coupled with high-resolution melting (Bar-HRM was evaluated as a novel approach for differentiating P. tunicoides from its adulterant S. viscidula. Our findings showed that Bar-HRM not only detected the adulteration, but also quantified the most common admixture. Bar-HRM sensitivity in adulterant detection was assessed by analysing samples mixed with different proportions of S. viscidula and P. tunicoides control. The results are presented as a linear regression with R2 = 0.9852, which implied the capability of the method to detect adulteration. This study is significant to verify the authenticity for better quality control of this herbal species.

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

    Science.gov (United States)

    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.

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

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    Santos Susana

    2012-03-01

    Full Text Available Abstract 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.

  20. The high resolution melting analysis (HRM) as a molecular tool for monitoring parasites of the wildlife.

    Science.gov (United States)

    Héritier, Laurent; Verneau, Olivier; Breuil, Gregory; Meistertzheim, Anne-Leila

    2017-04-01

    In an interconnected world, the international pet trade on wild animals is becoming increasingly important. As a consequence, non-native parasite species are introduced, which affect the health of wildlife and contribute to the loss of biodiversity. Because the investigation of parasite diversity within vulnerable host species implies the molecular identification of large samples of parasite eggs, the sequencing of DNA barcodes is time-consuming and costly. Thereby, the objectives of our study were to apply the high resolution melting (HRM) approach for species determination from pools of parasite eggs. Molecular assays were validated on flatworm parasites (polystomes) infecting the Mediterranean pond turtle Mauremys leprosa and the invasive red-eared slider Trachemys scripta elegans in French natural environments. HRM analysis results indicated that double or multiple parasitic infections could be detected from wild animal populations. They also showed that the cycle of parasite eggs production was not regular over time and may depend on several factors, among which the ecological niche and the target species. Thereby, monitoring parasites from wild endangered animals implies periodic parasitological surveys to avoid false negative diagnostics, based solely on eggs production.

  1. High Resolution Melt analysis for mutation screening in PKD1 and PKD2

    Directory of Open Access Journals (Sweden)

    Fontes Michel

    2011-10-01

    Full Text Available Abstract Background Autosomal dominant polycystic kidney disease (ADPKD is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. Methods We aim to prove that high resolution melt analysis (HRM, a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. Results We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. Conclusion HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes.

  2. High Resolution Melt analysis for mutation screening in PKD1 and PKD2.

    Science.gov (United States)

    Bataille, Stanislas; Berland, Yvon; Fontes, Michel; Burtey, Stéphane

    2011-10-18

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 ) within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes.

  3. Differential Identification of Mycobacterial Species Using High-Resolution Melting Analysis

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    Azar D. Khosravi

    2017-10-01

    Full Text Available Infections caused by non-tuberculous mycobacteria (NTM is increasing wordwide. Due to the difference in treatment of NTM infections and tuberculosis, rapid species identification of mycobacterial clinical isolates is necessary for the effective management of mycobacterial diseases treatment and their control strategy. In this study, a cost-effective technique, real-time PCR coupled with high-resolution melting (HRM analysis, was developed for the differentiation of Mycobacterial species using a novel rpoBC sequence. A total of 107 mycobacterial isolates (nine references and 98 clinical isolates were subjected to differentiation using rpoBC locus sequence in a real-time PCR-HRM assay scheme. From 98 Mycobacterium clinical isolates, 88 species (89.7%, were identified at the species level by rpoBC locus sequence analysis as a gold standard method. M. simiae was the most frequently encountered species (41 isolates, followed by M. fortuitum (20 isolates, M. tuberculosis (15 isolates, M. kansassi (10 isolates, M. abscessus group (5 isolates, M. avium (5 isolates, and M. chelonae and M. intracellulare one isolate each. The HRM analysis generated six unique specific groups representing M. tuberculosis complex, M. kansasii, M. simiae, M. fortuitum, M. abscessus–M. chelonae group, and M. avium complex. In conclusion, this study showed that the rpoBC-based real-time PCR followed by HRM analysis could differentiate the majority of mycobacterial species that are commonly encountered in clinical specimens.

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

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    Maslin Osathanunkul

    Full Text Available 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.

  5. Traceability of PDO Olive Oil “Terra di Bari” Using High Resolution Melting

    Directory of Open Access Journals (Sweden)

    Cinzia Montemurro

    2015-01-01

    Full Text Available The aim of the research was to verify the applicability of microsatellite (SSR markers in High Resolution Melting (HRM analysis for the identification of the olive cultivars used in the “Terra di Bari” PDO extra virgin olive oil. A panel of nine cultivars, widespread in Apulia region, was tested with seventeen SSR primer pairs and the PCR products were at first analysed with a Genetic Analyzer automatic sequencer. An identification key was obtained for the nine cultivars, which showed an unambiguous discrimination among the varieties constituting the “Terra di Bari” PDO extra virgin olive oil: Cima di Bitonto, Coratina, and Ogliarola. Subsequently, an SSR based method was set up with the DCA18 marker, coupled with HRM analysis for the distinction of the Terra di Bari olive oil from non-Terra di Bari olive oil using different mixtures. Thus, this analysis enabled the distinction and identification of the PDO mixtures. Hence, this assay provided a flexible, cost-effective, and closed-tube microsatellite genotyping method, well suited to varietal identification and authentication analysis in olive oil.

  6. High-resolution melting (HRM) for genotyping bovine ephemeral fever virus (BEFV).

    Science.gov (United States)

    Erster, Oran; Stram, Rotem; Menasherow, Shopia; Rubistein-Giuni, Marisol; Sharir, Binyamin; Kchinich, Evgeni; Stram, Yehuda

    2017-02-02

    In recent years there have been several major outbreaks of bovine ephemeral disease in the Middle East, including Israel. Such occurrences raise the need for quick identification of the viruses responsible for the outbreaks, in order to rapidly identify the entry of viruses that do not belong to the Middle-East BEFV lineage. This challenge was met by the development of a high-resolution melt (HRM) assay. The assay is based on the viral G gene sequence and generation of an algorithm that calculates and evaluates the GC content of various fragments. The algorithm was designed to scan 50- to 200-base-long segments in a sliding-window manner, compare and rank them using an Order of Technique of Preference by Similarity to Ideal Solution (TOPSIS) the technique for order preference by similarity to ideal solution technique, according to the differences in GC content of homologous fragments. Two fragments were selected, based on a match to the analysis criteria, in terms of size and GC content. These fragments were successfully used in the analysis to differentiate between different virus lineages, thus facilitating assignment of the viruses' geographical origins. Moreover, the assay could be used for differentiating infected from vaccinated animales (DIVA). The new algorithm may therefore be useful for development of improved genotyping studies for other viruses and possibly other microorganisms. Copyright © 2016. Published by Elsevier B.V.

  7. Application of high-resolution DNA melting for genotyping in lepidopteran non-model species: Ostrinia furnacalis (Crambidae.

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    FengBo Li

    Full Text Available Development of an ideal marker system facilitates a better understanding of the genetic diversity in lepidopteran non-model organisms, which have abundant species, but relatively limited genomic resources. Single nucleotide polymorphisms (SNPs discovered within single-copy genes have proved to be desired markers, but SNP genotyping by current techniques remain laborious and expensive. High resolution melting (HRM curve analysis represents a simple, rapid and inexpensive genotyping method that is primarily confined to clinical and diagnostic studies. In this study, we evaluated the potential of HRM analysis for SNP genotyping in the lepidopteran non-model species Ostrinia furnacalis (Crambidae. Small amplicon and unlabeled probe assays were developed for the SNPs, which were identified in 30 females of O. furnacalis from 3 different populations by our direct sequencing. Both assays were then applied to genotype 90 unknown female DNA by prior mixing with known wild-type DNA. The genotyping results were compared with those that were obtained using bi-directional sequencing analysis. Our results demonstrated the efficiency and reliability of the HRM assays. HRM has the potential to provide simple, cost-effective genotyping assays and facilitates genotyping studies in any non-model lepidopteran species of interest.

  8. Genotyping of the protozoan pathogen Toxoplasma gondii using high-resolution melting analysis of the repeated B1 gene.

    Science.gov (United States)

    Costa, Jean-Marc; Cabaret, Odile; Moukoury, Sandrine; Bretagne, Stéphane

    2011-09-01

    Genetic studies of the protozoan parasite Toxoplasma gondii have identified three main distinct types according to virulence in some hosts. Several methods have been developed to differentiate genotypes currently dominated by microsatellite markers targeting single-copy loci. We analyzed the possibility of using the 35-fold repetitive B1 gene via high-resolution melting (HRM) curve analysis. Sequencing of the B1 gene of 14 reference strains (four Type I, six Type II, and four Type III strains) identified 18 single nucleotide polymorphisms (SNP). Primers were designed to amplify eight of them for HRM analysis and for relative quantification of each nucleotide variation using SNaPshot mini-sequencing. Genotyping with five microsatellite markers was performed for comparison. Two to four HRM profiles were obtained depending on the SNP tested. The differences observed relied on the different ratios of nucleotides at the SNP locus as evidenced via SNaPshot mini-sequencing. The three main lineages could be distinguished by using several HRM profiles. Some HRM profiles proved more informative than the analysis based on five microsatellite markers, showing additional differences in Type I and Type II strains. Using HRM analysis, we obtained at least an equally good discrimination of the main lineages than that based on five microsatellite markers. Copyright © 2011 Elsevier B.V. All rights reserved.

  9. First report of Tasmanian sheep strain (G2) genotype isolated from Iranian goat using the high resolution melting (HRM) analysis.

    Science.gov (United States)

    Hosseini-Safa, Ahmad; Mohag Hegh, Mohammad Ali; Pestechian, Nader; Ganji, Maryam; Mohammadi, Rasoul; Mahmoudi Lamouki, Reza; Rostami-Nejad, Mohammad

    2016-12-01

    The present study was aimed to evaluate E. granulosus genotypes isolated from goats using HRM analysis in Isfahan province. Cystic echincoccosis, so-called hydatidosis, is widespread infection caused by the larval stage of Echinococcus granulosus . This is an important zoonotic disease worldwide, especially in the developing countries such as Iran. To date, molecular studies mainly based on the mitochondrial DNA sequences have identified distinct genotypes termed G1-G10 which can differ in some characteristics such as the growth and infectivity to different intermediate hosts or the survival rate in the definitive hosts that are important for the development of control strategies. From August to December 2014, 1341 goats were investigated and hydatid cysts were collected from the liver and lungs of 43 infected goats in Isfahan province abattoirs, Isfahan, Iran. Total genomic DNA was extracted from each sample, amplified for the presence of polymorphism of mitochondrial gene coding for cytochrome c oxidase subunit 1 (CO1), using high resolution melting curve (HRM) method. the results of HRM analysis using the sequence of CO1 gene for 43 Echinococcus granulosus isolates from goats showed 31, 2 and 10 isolates were identified as G1, G2, and G3 genotypes, respectively. G1 is the predominant genotype in the isolated goat samples in Isfahan province, and the presence of G2 strain was reported for the first time in goat in Iran.

  10. Methodological streamlining of SNP discovery and genotyping via high-resolution melting analysis (HRMA) in non-model species.

    Science.gov (United States)

    Smith, Brad L; Lu, Ching-Ping; Alvarado Bremer, Jaime R

    2013-03-01

    The exponential growth of genetic resources is fueled by continued advances in genomic technologies and the adoption of single nucleotide polymorphisms (SNPs) for population studies. Concomitant to these developments, there is growing need for rapid screening and subsequent genotyping of SNPs in non-model organisms. Here we provide a rapid and low-cost workflow utilizing high-resolution melting analysis (HRMA) for nuclear marker development and genotyping of 774 Atlantic and Mediterranean swordfish (Xiphias gladius) that is amendable to other species. Preliminary HRMA screening of amplicons (>290bp) for 10 nuclear loci revealed the presence of nucleotide polymorphisms, however, length and variability precluded diagnostic genotyping. Two variants of HRMA were therefore utilized to provide diagnostic genotyping assays. Short-amplicon HRMA (SA-HRMA), in which primers flank closely a SNP of interest, was identified as a low cost, rapid, closed-tube diagnostic genotyping assay that could distinguish between homozygous genotypes by ΔTm, and heterozygous genotypes by heteroduplex melting curve profiles. When the patterns of sequence variation were not suitable for SA-HRMA, unlabeled probe (UP)-HRMA was utilized. UP-HRMA has the advantage of being capable of genotyping multiple linked SNPs in a single closed-tube assay without Bayesian haplotype reconstruction, and can identify new SNPs while genotyping populations. Almost 37% of the SNPs genotyped via UP-HRMA were discovered while genotyping populations and not from preliminary screening. Analysis of swordfish in the North Atlantic (NA, n=419), South Atlantic (SA, n=296), and Mediterranean (MED, n=59) found no significant linkage disequilibrium. To assess whether deviations in HWE could be the result of genotyping error rather than population admixture only swordfish from reported spawning areas in the NA (n=49), MED (n=59), and SA (n=42) were analyzed and all loci were in Hardy-Weinberg equilibrium. Significant genetic

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

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    Hany Sady

    2015-07-01

    Full Text Available 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.

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

    Science.gov (United States)

    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 PCR assays. PMID:26193254

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

    Science.gov (United States)

    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

    2015-07-16

    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.

  14. Rapid multiplex high resolution melting method to analyze inflammatory related SNPs in preterm birth

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    Pereyra Silvana

    2012-01-01

    Full Text Available Abstract Background Complex traits like cancer, diabetes, obesity or schizophrenia arise from an intricate interaction between genetic and environmental factors. Complex disorders often cluster in families without a clear-cut pattern of inheritance. Genomic wide association studies focus on the detection of tens or hundreds individual markers contributing to complex diseases. In order to test if a subset of single nucleotide polymorphisms (SNPs from candidate genes are associated to a condition of interest in a particular individual or group of people, new techniques are needed. High-resolution melting (HRM analysis is a new method in which polymerase chain reaction (PCR and mutations scanning are carried out simultaneously in a closed tube, making the procedure fast, inexpensive and easy. Preterm birth (PTB is considered a complex disease, where genetic and environmental factors interact to carry out the delivery of a newborn before 37 weeks of gestation. It is accepted that inflammation plays an important role in pregnancy and PTB. Methods Here, we used real time-PCR followed by HRM analysis to simultaneously identify several gene variations involved in inflammatory pathways on preterm labor. SNPs from TLR4, IL6, IL1 beta and IL12RB genes were analyzed in a case-control study. The results were confirmed either by sequencing or by PCR followed by restriction fragment length polymorphism. Results We were able to simultaneously recognize the variations of four genes with similar accuracy than other methods. In order to obtain non-overlapping melting temperatures, the key step in this strategy was primer design. Genotypic frequencies found for each SNP are in concordance with those previously described in similar populations. None of the studied SNPs were associated with PTB. Conclusions Several gene variations related to the same inflammatory pathway were screened through a new flexible, fast and non expensive method with the purpose of analyzing

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

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    Ying Wang

    Full Text Available 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.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.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%.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.

  16. DMSO increases mutation-scanning detection sensitivity in clinical samples using high resolution melting

    Science.gov (United States)

    Song, Chen; Castellanos-Rizaldos, Elena; Bejar, Rafael; Ebert, Benjamin L.; Makrigiorgos, G. Mike

    2016-01-01

    BACKGROUND Mutation scanning provides the simplest, lowest cost method for identifying DNA variations on single PCR amplicons, and it may be performed prior to sequencing to avoid screening of non-informative wild type samples. High resolution melting (HRM) is the most commonly used method for mutation scanning. However, by using PCR-HRM mutations below ≈ 3–10% that can still be clinically significant may often be missed. Therefore, enhancing HRM detection sensitivity is important for mutation scanning and its clinical application. METHODS We used serial dilution of TP53 exon 8 mutation containing cell lines to demonstrate the improvement in detection sensitivity for conventional-PCR-HRM in the presence of DMSO. We also conducted full-COLD-PCR to further enrich low-level mutations prior to HRM±DMSO and employed droplet-digital PCR to derive the optimal conditions for mutation enrichment. Both conventional-PCR-HRM and full-COLD-PCR-HRM ±DMSO were used for mutation scanning in TP53 exon 8 in cancer samples containing known mutations and in myelodysplastic syndrome samples with unknown mutations. Mutations in other genes were also examined. RESULTS The detection sensitivity of PCR-HRM-scanning increases 2–5-fold in the presence of DMSO, depending also on mutation type and sequence context, and can typically detect mutation abundance of about 1%. When mutation enrichment is applied during amplification using full-COLD-PCR and followed by HRM in the presence of DMSO, mutations with 0.2–0.3% mutation abundance in TP53 exon 8 can be detected. CONCLUSIONS DMSO improves HRM mutation scanning sensitivity. When full-COLD-PCR is employed, followed by DMSO-HRM, the overall improvement is about 20-fold as compared to conventional PCR-HRM. PMID:26432802

  17. High-resolution melting analysis of MED12 mutations in uterine leiomyomas in Chinese patients.

    Science.gov (United States)

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

    2015-03-01

    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. 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. 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 (χ(2)=0.011, p=0.916). In addition, MED12 mutations were identified in 1/157 (4.17%) case of other endometrial pathologies by both methods. 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.

  18. Molecular identification of broomrape species from a single seed by High Resolution Melting analysis

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    Mathieu Rolland

    2016-12-01

    Full Text Available Broomrapes are holoparasitic plants spreading through seeds. Each plant produces hundreds of thousands of seeds which remain viable in the soils for decades. To limit their spread, drastic measures are being taken and the contamination of a commercial seed lot by a single broomrape seed can lead to its rejection. Considering that broomrapes species identification from a single seed is extremely difficult even for trained botanists and that among all the described species, only a few are really noxious for the crops, numerous seed lots are rejected because of the contamination by seeds of non-noxious broomrape species. The aim of this study was to develop and evaluate a High Resolution Melting assay identifying the eight most noxious and common broomrape species (P. aegyptiaca, O. cernua, O. crenata, O. cumana, O. foetida, O. hederae, O. minor, and P. ramosa from a single seed. Based on trnL and rbcL plastidial genes amplification, the designed assay successfully identifies O. cumana, O. cernua, O. crenata, O. minor, O. hederae, and O. foetida; P. ramosa and P. aegyptiaca can be differentiated from other species but not from each other. Tested on 50 seed lots, obtained results perfectly matched identifications performed by sequencing. Through the analysis of common seed lots by different analysts, the reproducibility of the assay was evaluated at 90 %. Despite an original sample preparation process it was not possible to extract enough DNA from some seeds (10% of the samples. The described assay fulfils its objectives and allows an accurate identification of the targeted broomrape species. It can be used to identify contaminants in commercial seed lots or for any other purpose. The assay might be extended to vegetative material.

  19. High-Resolution Melting Analysis of the TPMT Gene: A Study in the Polish Population

    Science.gov (United States)

    Borun, Pawel; Milanowska, Katarzyna; Jakubowska-Burek, Ludwika; Zakerska, Oliwia; Dobrowolska-Zachwieja, Agnieszka; Plawski, Andrzej; Froster, Ursula G.; Szalata, Marlena; Slomski, Ryszard

    2013-01-01

    The thiopurine S-methyltransferase (TPMT) gene encoding thiopurine methyltransferase is a crucial enzyme in metabolism of thiopurine drugs: azathioprine and 6-mercoptopurine, which are used in the treatment of leukemia or inflammatory bowel diseases. Genetic polymorphism of the TPMT gene correlates with activity of this enzyme, individual reaction, and dosing of thiopurines. Thirty-one variants of the TPMT gene with low enzymatic activity have been described with three major alleles: TPMT*2 (c.238G>C), *3A (c.460 G>A, c.719A>G), and *3C (c.719A>G), accounting for 80% to 95% of inherited TPMT deficiency in different populations in the world. The aim of the study was to establish a rapid and highly sensitive method of analysis for the complete coding sequence of the TPMT gene and to determine the spectrum and prevalence of the TPMT gene sequence variations in the Polish population. Recently, high-resolution melting analysis (HRMA) has become a highly sensitive, automated, and economical technique for mutation screening or genotyping. We applied HRMA for the first time to TPMT gene scanning. In total, we analyzed 548 alleles of the Polish population. We found 11 different sequence variations, where two are novel changes: c.200T>C (p.P67S, TPMT*30) and c.595G>A (p.V199I, TPMT*31). Detection of these new rare alleles TPMT*30 and *31 in the Polish population suggests the need to analyze the whole TPMT gene and maybe also the extension of routinely used tests containing three major alleles, TPMT*2, *3A, and *3C. Identification of sequence variants using HRMA is highly sensitive and less time consuming compared to standard sequencing. We conclude that HRMA can be easy integrated into genetic testing of the TPMT gene in patients treated with thiopurines. PMID:23252704

  20. Cross-Validation of High-Resolution Melting Analysis-Based Genotyping Platform.

    Science.gov (United States)

    Langaee, Taimour; Stauffer, Lynda; Galloway, Cheryl; Solayman, Mohamed H; Cavallari, Larisa

    2017-04-01

    Developing genetic and pharmacogenetic panels enhances genetic testing in clinical molecular diagnostics and precision medicine. This study was designed to cross-validate the performance of Canon's multiplex high-resolution DNA melting analysis platform with the Applied Biosystems TaqMan®-based Quant Studio Real-Time PCR System and Pyrosequencing® genotyping platforms for common genetic polymorphisms of the vitamin K epoxide reductase complex 1 (VKORC1) and CYP2C9. Genomic DNA isolated from 240 blood and saliva samples was used to genotype the VKORC1-1639 G/A (rs9923231), CYP2C9*2 (430C>T, rs28371674), and CYP2C9*3 (1075A>C, rs1057910) single-nucleotide polymorphisms (SNPs) on the three above-mentioned genotyping platforms. There was 99.2%, 100%, and 100% concordance among the Canon DNA analyzer, the TaqMan-based QuantStudio, and the Pyrosequencing genotyping results for the VKORC1 (rs9923231), CYP2C9*2, and CYP2C9*3 SNPs, respectively, in DNA samples isolated from blood. The DNA samples isolated from saliva showed 100% concordance among the three test platforms for the three tested SNPs. These results show that, the DNA analyzer performed very well when compared with two commonly used genotyping platforms. The reliability, multiple genetic variant testing capability, and short turnaround time for up to eight samples make the DNA analyzer an ideal genotyping platform for genetic testing in the clinical practice setting, where efficient genotyping is important to prevent delays in optimizing drug therapy.

  1. Putative hybrids between two Anisakis cryptic species: molecular genotyping using High Resolution Melting.

    Science.gov (United States)

    Cavallero, S; Costa, A; Caracappa, S; Gambetta, B; D'Amelio, S

    2014-11-01

    The genus Anisakis includes nine recognized species and the complex of cryptic species Anisakis simplex s. l. is often associated with the human disease known as anisakiasis. During the last decades the use of nuclear ribosomal ITS allowed the identification and description of numerous anisakid nematodes and the discovery of recombinant genotypes or putative hybrids even in other parasitic helminths, such as those between A. simplex sensu stricto and A. pegreffii. The existence of pure hybrids of the two sibling species has been long debated due to the large recovery of larval forms from sympatric areas and the rare observation of adult hybrids. The aims of the present report were to identify anisakid nematodes collected from Stenella coeruleoalba using PCR-RFLP of ITS and to focus the interest on hybrid forms using a High Resolution Melting (HRM) and direct sequencing analyses, since the new record of putative hybrid at adult stage. The PCR-RFLP analysis enabled to identify A. simplex s.s., A. pegreffii, the heterozygous genotype of the two species and A. physeteris. The use of the genotyping approach based on HRM confirmed the profiles of the two species A. simplex s.s. and A. pegreffii, and of the hybrid individuals. The new record of adult hybrids in definitive hosts rekindles the long debate about their existence and their evolutionary meaning. Since the reproductive isolation between A. simplex s.s. and A. pegreffii is the assumption for their existence as separated species, the use of alternative molecular markers and population genetic studies on adult anisakids are recommended. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. High-resolution Laue-type DuMond curved crystal spectrometer.

    Science.gov (United States)

    Szlachetko, M; Berset, M; Dousse, J-Cl; Hoszowska, J; Szlachetko, J

    2013-09-01

    We report on a high-resolution transmission-type curved crystal spectrometer based on the modified DuMond slit geometry. The spectrometer was developed at the University of Fribourg for the study of photoinduced X-ray spectra. K and L X-ray transitions with energies above about 10 keV can be measured with an instrumental resolution comparable to their natural linewidths. Construction details and operational characteristics of the spectrometer are presented. The variation of the energy resolution as a function of the focal distance and diffraction order is discussed. The high sensitivity of the spectrometer is demonstrated via the 2s-1s dipole-forbidden X-ray transition of Gd which could be observed despite its extremely low intensity. The precision of the instrument is illustrated by comparing the sum of the energies of the Au K-L2 and L2-M3 cascading transitions with the energy of the crossover K-M3 transition as well as by considering the energy differences of the Gd Kα1 X-ray line measured at five different diffraction orders. Finally, to demonstrate the versatility of the spectrometer, it is shown that the latter can also be used for in-house extended X-ray absorption fine structure measurements.

  3. Automated curve matching techniques for reproducible, high-resolution palaeomagnetic dating

    Science.gov (United States)

    Lurcock, Pontus; Channell, James

    2016-04-01

    High-resolution relative palaeointensity (RPI) and palaeosecular variation (PSV) data are increasingly important for accurate dating of sedimentary sequences, often in combination with oxygen isotope (δ18O) measurements. A chronology is established by matching a measured downcore signal to a dated reference curve, but there is no standard methodology for performing this correlation. Traditionally, matching is done by eye, but this becomes difficult when two parameters (e.g. RPI and δ18O) are being matched simultaneously, and cannot be done entirely objectively or repeatably. More recently, various automated techniques have appeared for matching one or more signals. We present Scoter, a user-friendly program for dating by signal matching and for comparing different matching techniques. Scoter is a cross-platform application implemented in Python, and consists of a general-purpose signal processing and correlation library linked to a graphical desktop front-end. RPI, PSV, and other records can be opened, pre-processed, and automatically matched with reference curves. A Scoter project can be exported as a self-contained bundle, encapsulating the input data, pre-processing steps, and correlation parameters, as well as the program itself. The analysis can be automatically replicated by anyone using only the resources in the bundle, ensuring full reproducibility. The current version of Scoter incorporates an experimental signal-matching algorithm based on simulated annealing, as well as an interface to the well-established Match program of Lisiecki and Lisiecki (2002), enabling results of the two approaches to be compared directly.

  4. Transmission and selection of macrolide resistant Mycoplasma genitalium infections detected by rapid high resolution melt analysis.

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    Jimmy Twin

    Full Text Available BACKGROUND: Mycoplasma genitalium (MG causes urethritis, cervicitis and pelvic inflammatory disease. The MG treatment failure rate using 1 g azithromycin at an Australian Sexual Health clinic in 2007-9 was 31% (95%CI 23-40%. We developed a rapid high resolution melt analysis (HRMA assay targeting resistance mutations in the MG 23S rRNA gene, and validated it against DNA sequencing by examining pre- and post-treatment archived samples from MG-infected patients. METHODOLOGY/PRINCIPAL FINDINGS: Available MG-positive pre-treatment (n = 82 and post-treatment samples from individuals with clinical treatment failure (n = 20 were screened for 23S rRNA gene mutations. Sixteen (20% pre-treatment samples possessed resistance mutations (A2058G, A2059G, A2059C, which were significantly more common in patients with symptomatic azithromycin-treatment failure (12/26; 44% than in those clinically cured (4/56; 7%, p<0.001. All 20 patients experiencing azithromycin-failure had detectable mutations in their post-treatment samples. In 9 of these cases, the same mutational types were present in both pre- and post-treatment samples indicating transmitted resistance, whilst in 11 of these cases (55%, mutations were absent in pre-treatment samples indicating likely selection of resistant isolates have occurred. HRMA was able to detect all mutational changes determined in this study by DNA sequencing. An additional HRMA assay incorporating an unlabelled probe was also developed to detect type 4 single-nucleotide polymorphisms found in other populations, with a slightly lower sensitivity of 90%. CONCLUSIONS/SIGNIFICANCE: Treatment failure is associated with the detection of macrolide resistance mutations, which appear to be almost equally due to selection of resistant isolates following exposure to 1 g azithromycin and pre-existing transmitted resistance. The application of a rapid molecular assay to detect resistance at the time of initial detection of infection allows

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

    Science.gov (United States)

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

    2016-01-01

    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. 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. 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. 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. 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 determine the A. paniculata species in herbal products tested

  6. High Resolution Melting Analysis for Detecting p53 Gene Mutations in Patients with Non-small Cell Lung Cancer

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    Zhihong CHEN

    2011-10-01

    Full Text Available Background and objective It has been proven that p53 gene was related to many human cancers. The mutations in p53 gene play an important role in carcinogensis and mostly happened in exon 5-8. The aim of this study is to establish a high resolution melting (HRM assay to detect p53 mutations from patients with non-small cell lung cancer (NSCLC, to investigate the characteristics of p53 gene mutations, and to analyze the relationship between p53 mutations and evolution regularity of pathogenesis. Methods p53 mutations in exon 5-8 were detected by HRM assay on DNA insolated from 264 NSCLC samples derived from tumor tissues and 54 control samples from pericancerous pulmonary tissues. The mutation samples by the HRM assay were confirmed by sequencing technique. Samples which were positive by HRM but wild type by sequencing were further confirmed by sub-clone and sequencing. Results No mutation was found in 54 pericancerous pulmonary samples by HRM assay. 104 of the 264 tumor tissues demonstrated mutation curves by HRM assay, 102 samples were confirmed by sequencing, including 95 point mutations and 7 frame shift mutations by insertion or deletion. The mutation rate of p53 gene was 39.4%. The mutation rate from exon 5-8 were 11.7%, 8%, 12.5% and 10.6%, respectively and there was no statistically significant difference between them (P=0.35. p53 mutations were significantly more frequent in males than that in females, but not related to the other clinicopathologic characteristics. Conclusion The results indicate that HRM is a sensitive in-tube methodology to detect for mutations in clinical samples. The results suggest that the arising p53 mutations in NSCLC may be due to spontaneous error in DNA synthesis and repair.

  7. Using high resolution tritium profiles to quantify the effects of melt on two Spitsbergen ice cores

    NARCIS (Netherlands)

    van der Wel, L.G.; Streurman, H.J.; Isaksson, E.; Helsen, M.M.|info:eu-repo/dai/nl/325802459; van de Wal, R.S.W.|info:eu-repo/dai/nl/101899556; Martma, T.; Pohjola, V.A.; Moore, J.C.; Meijer, H.A.J.

    2011-01-01

    Ice cores from small ice caps provide valuable climatic information, additional to that of Greenland and Antarctica. However, their integrity is usually compromised by summer meltwater percolation. To determine to what extent this can affect such ice cores, we performed high-resolution tritium

  8. High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

    DEFF Research Database (Denmark)

    Hjelmsø, Mathis Hjort; Hansen, Lars Hestbjerg; Bælum, Jacob

    2014-01-01

    -resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized...... that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide...

  9. Molecular differentiation of Schistosoma japonicum and Schistosoma mekongi by real-time PCR with high resolution melting analysis.

    Science.gov (United States)

    Kongklieng, Amornmas; Kaewkong, Worasak; Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Lulitanond, Viraphong; Sri-Aroon, Pusadee; Limpanont, Yanin; Maleewong, Wanchai

    2013-12-01

    Human schistosomiasis caused by Schistosoma japonicum and Schistosoma mekongi is a chronic and debilitating helminthic disease still prevalent in several countries of Asia. Due to morphological similarities of cercariae and eggs of these 2 species, microscopic differentiation is difficult. High resolution melting (HRM) real-time PCR is developed as an alternative tool for the detection and differentiation of these 2 species. A primer pair was designed for targeting the 18S ribosomal RNA gene to generate PCR products of 156 base pairs for both species. The melting points of S. japonicum and S. mekongi PCR products were 84.5±0.07℃ and 85.7±0.07℃, respectively. The method permits amplification from a single cercaria or an egg. The HRM real-time PCR is a rapid and simple tool for differentiation of S. japonicum and S. mekongi in the intermediate and final hosts.

  10. Rapid detection and identification of Brugia malayi, B. pahangi, and Dirofilaria immitis by high-resolution melting assay.

    Science.gov (United States)

    Wongkamchai, Sirichit; Monkong, Nuntiya; Mahannol, Pakpimom; Taweethavonsawat, Piyanan; Loymak, Sumat; Foongladda, Suporn

    2013-01-01

    Human lymphatic filariasis is caused by filarial worms such as Brugia malayi for which the major reservoir is domestic cats. However, domestic cats or dogs also carry nonhuman filaria such as Brugia pahangi and Dirofilaria immitis. We have developed a single-tube, real-time PCR with a high-resolution melting (HRM) analysis assay for detection and identification of B. malayi, B. pahangi, and D. immitis in blood samples. The designated primer pair in the PCR can amplify a 114-bp region of mitochondrial 12S rRNA genes of these filarial worms. Subsequently, the HRM assay showed a specific melting temperature for each species. The assay showed the highest sensitivity and specificity in comparison with DNA sequences after assessment with 34 cat and 14 dog blood samples. This assay could be helpful for epidemiological studies of reservoirs and vectors.

  11. Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies

    National Research Council Canada - National Science Library

    Do, Hongdo; Dobrovic, Alexander

    2009-01-01

    .... High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true...

  12. Rapid detection and simultaneous genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in powdered infant formula using real-time PCR and high resolution melting (HRM) analysis

    National Research Council Canada - National Science Library

    Cai, Xian-Quan; Yu, Hai-Qiong; Ruan, Zhou-Xi; Yang, Lei-Liang; Bai, Jian-Shan; Qiu, De-Yi; Jian, Zhi-Hua; Xiao, Yi-Qian; Yang, Jie-Yang; Le, Thanh Hoa; Zhu, Xing-Quan

    2013-01-01

    .... The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp...

  13. Rapid Detection and Simultaneous Genotyping of Cronobacter spp. (formerly Enterobacter sakazakii) in Powdered Infant Formula Using Real-time PCR and High Resolution Melting (HRM) Analysis: e67082

    National Research Council Canada - National Science Library

    Xian-Quan Cai; Hai-Qiong Yu; Zhou-Xi Ruan; Lei-Liang Yang; Jian-Shan Bai; De-Yi Qiu; Zhi-Hua Jian; Yi-Qian Xiao; Jie-Yang Yang; Thanh Hoa Le; Xing-Quan Zhu

    2013-01-01

    .... The present study developed an assay integrating real-time PCR and high resolution melting (HRM) analysis targeting the OmpA gene for the specific detection and rapid identification of Cronobacter spp...

  14. Identification of TNIP1 Polymorphisms by High Resolution Melting Analysis with Unlabelled Probe: Association with Systemic Lupus Erythematosus

    Science.gov (United States)

    Zhang, Jie; Chen, Yuewen; Shao, Yong; Wu, Qi; Guan, Ming; Zhang, Wei; Wan, Jun; Yu, Bo

    2012-01-01

    Background. TNFα-induced protein 3 (TNFAIP3) interacting with protein 1 (TNIP1) acts as a negative regulator of NF-κB and plays an important role in maintaining the homeostasis of immune system. A recent genome-wide association study (GWAS) showed that the polymorphism of TNIP1 was associated with the disease risk of SLE in Caucasian. In this study, we investigated whether the association of TNIP1 with SLE was replicated in Chinese population. Methods. The association of TNIP1 SNP rs7708392 (G/C) was determined by high resolution melting (HRM) analysis with unlabeled probe in 285 SLE patients and 336 healthy controls. Results. A new SNP rs79937737 located on 5 bp upstream of rs7708392 was discovered during the HRM analysis. No association of rs7708392 or rs79937737 with the disease risk of SLE was found. Furthermore, rs7708392 and rs79937737 were in weak linkage disequilibrium (LD). Hypotypes analysis of the two SNPs also showed no association with SLE in Chinese population. Conclusions. High resolution melting analysis with unlabeled probes proves to be a powerful and efficient genotyping method for identifying and screening SNPs. No association of rs7708392 or rs79937737 with the disease risk of SLE was observed in Chinese population. PMID:22852072

  15. Identification of TNIP1 Polymorphisms by High Resolution Melting Analysis with Unlabelled Probe: Association with Systemic Lupus Erythematosus

    Directory of Open Access Journals (Sweden)

    Jie Zhang

    2012-01-01

    Full Text Available Background. TNFα-induced protein 3 (TNFAIP3 interacting with protein 1 (TNIP1 acts as a negative regulator of NF-κB and plays an important role in maintaining the homeostasis of immune system. A recent genome-wide association study (GWAS showed that the polymorphism of TNIP1 was associated with the disease risk of SLE in Caucasian. In this study, we investigated whether the association of TNIP1 with SLE was replicated in Chinese population. Methods. The association of TNIP1 SNP rs7708392 (G/C was determined by high resolution melting (HRM analysis with unlabeled probe in 285 SLE patients and 336 healthy controls. Results. A new SNP rs79937737 located on 5 bp upstream of rs7708392 was discovered during the HRM analysis. No association of rs7708392 or rs79937737 with the disease risk of SLE was found. Furthermore, rs7708392 and rs79937737 were in weak linkage disequilibrium (LD. Hypotypes analysis of the two SNPs also showed no association with SLE in Chinese population. Conclusions. High resolution melting analysis with unlabeled probes proves to be a powerful and efficient genotyping method for identifying and screening SNPs. No association of rs7708392 or rs79937737 with the disease risk of SLE was observed in Chinese population.

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

    Science.gov (United States)

    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.

  17. High-resolution melting analysis for detection of a single-nucleotide polymorphism and the genotype of the myostatin gene in warmblood horses.

    Science.gov (United States)

    Serpa, Priscila B S; Garbade, Petra; Natalini, Cláudio C; Pires, Ananda R; Tisotti, Tainor M

    2017-01-01

    OBJECTIVE To develop a high-resolution melting (HRM) assay to detect the g.66493737C>T polymorphism in the myostatin gene (MSTN) and determine the frequency of 3 previously defined g.66493737 genotypes (T/T, T/C, and C/C) in warmblood horses. SAMPLES Blood samples from 23 horses. PROCEDURES From each blood sample, DNA was extracted and analyzed by standard PCR methods and an HRM assay to determine the MSTN genotype. Three protocols (standard protocol, protocol in which a high-salt solution was added to the reaction mixture before the first melting cycle, and protocol in which an unlabeled probe was added to the reaction mixture before analysis) for the HRM assay were designed and compared. Genotype results determined by the HRM protocol that generated the most consistent melting curves were compared with those determined by sequencing. RESULTS The HRM protocol in which an unlabeled probe was added to the reaction mixture generated the most consistent melting curves. The genotypes of the g.66493737C>T polymorphism were determined for 22 horses (16 by HRM analysis and 20 by sequencing); 14, 7, and 1 had the T/T, T/C, and C/C genotypes, respectively. The genotype determined by HRM analysis agreed with that determined by sequencing for 14 of 16 horses. The frequency of alleles T and C was 79.5% and 20.5%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HRM analysis may be a faster and more economical alternative than PCR methods for genotyping. Genotyping results might be useful as predictors of athletic performance for horses.

  18. High resolution melting analysis is a more sensitive and effective alternative to gel-based platforms in analysis of SSR--an example in citrus.

    Directory of Open Access Journals (Sweden)

    Gaetano Distefano

    Full Text Available High resolution melting curve analysis (HRM has been used as an efficient, accurate and cost-effective tool to detect single nucleotide polymorphisms (SNPs or insertions or deletions (INDELs. However, its efficiency, accuracy and applicability to discriminate microsatellite polymorphism have not been extensively assessed. The traditional protocols used for SSR genotyping include PCR amplification of the DNA fragment and the separation of the fragments on electrophoresis-based platform. However, post-PCR handling processes are laborious and costly. Furthermore, SNPs present in the sequences flanking repeat motif cannot be detected by polyacrylamide-gel-electrophoresis based methods. In the present study, we compared the discriminating power of HRM with the traditional electrophoresis-based methods and provided a panel of primers for HRM genotyping in Citrus. The results showed that sixteen SSR markers produced distinct polymorphic melting curves among the Citrus spp investigated through HRM analysis. Among those, 10 showed more genotypes by HRM analysis than capillary electrophoresis owing to the presence of SNPs in the amplicons. For the SSR markers without SNPs present in the flanking region, HRM also gave distinct melting curves which detected same genotypes as were shown in capillary electrophoresis (CE analysis. Moreover, HRM analysis allowed the discrimination of most of the 15 citrus genotypes and the resulting genetic distance analysis clustered them into three main branches. In conclusion, it has been approved that HRM is not only an efficient and cost-effective alternative of electrophoresis-based method for SSR markers, but also a method to uncover more polymorphisms contributed by SNPs present in SSRs. It was therefore suggested that the panel of SSR markers could be used in a variety of applications in the citrus biodiversity and breeding programs using HRM analysis. Furthermore, we speculate that the HRM analysis can be employed to

  19. Curved crystals for high-resolution focusing of X and gamma rays through a Laue lens

    Science.gov (United States)

    Guidi, Vincenzo; Bellucci, Valerio; Camattari, Riccardo; Neri, Ilaria

    2013-08-01

    Crystals with curved diffracting planes have been investigated as high-efficiency optical components for the realization of a Laue lens for satellite-borne experiments in astrophysics. At Sensor and Semiconductor Laboratory (Ferrara, Italy) a research and development plan to implement Si and Ge curved crystals by surface grooving technique has been undertaken. The method of surface grooving allows obtaining Si and Ge curved crystals with self-standing curvature, i.e., with no need for external bending device, which is a mandatory issue in satellite-borne experiments. Si and Ge grooved crystals have been characterized by X-ray diffraction at ESRF and ILL to prove their functionality for a high-reflectivity Laue lens.

  20. Sparse SVD Method for High-Resolution Extraction of the Dispersion Curves of Ultrasonic Guided Waves.

    Science.gov (United States)

    Xu, Kailiang; Minonzio, Jean-Gabriel; Ta, Dean; Hu, Bo; Wang, Weiqi; Laugier, Pascal

    2016-10-01

    The 2-D Fourier transform analysis of multichannel signals is a straightforward method to extract the dispersion curves of guided modes. Basically, the time signals recorded at several positions along the waveguide are converted to the wavenumber-frequency space, so that the dispersion curves (i.e., the frequency-dependent wavenumbers) of the guided modes can be extracted by detecting peaks of energy trajectories. In order to improve the dispersion curve extraction of low-amplitude modes propagating in a cortical bone, a multiemitter and multireceiver transducer array has been developed together with an effective singular vector decomposition (SVD)-based signal processing method. However, in practice, the limited number of positions where these signals are recorded results in a much lower resolution in the wavenumber axis than in the frequency axis. This prevents a clear identification of overlapping dispersion curves. In this paper, a sparse SVD (S-SVD) method, which combines the signal-to-noise ratio improvement of the SVD-based approach with the high wavenumber resolution advantage of the sparse optimization, is presented to overcome the above-mentioned limitation. Different penalty constraints, i.e., l1 -norm, Frobenius norm, and revised Cauchy norm, are compared with the sparse characteristics. The regularization parameters are investigated with respect to the convergence property and wavenumber resolution. The proposed S-SVD method is investigated using synthetic wideband signals and experimental data obtained from a bone-mimicking phantom and from an ex-vivo human radius. The analysis of the results suggests that the S-SVD method has the potential to significantly enhance the wavenumber resolution and to improve the extraction of the dispersion curves.

  1. High-resolution melting analysis using unlabeled probe and amplicon scanning simultaneously detects several lactase persistence variants

    DEFF Research Database (Denmark)

    Janukonyté, Jurgita; Vestergaard, Else M; Ladefoged, Søren A

    2010-01-01

    persistence can be associated with other variants nearby the -13910C>T variant, limiting the use of the -13910C>T-based SNP analysis, e.g. TaqMan assays for the diagnosis of lactose intolerance. Using high-resolution melting analysis, we identified five samples that were heterozygous for the -13915T>G variant...... the -13910C>T and -13915T>G variants in addition to rarer variants surrounding the -13910 site. This new method may contribute to improve the diagnostic performance of the genetic analysis for lactose intolerance.......Lactase persistence and thereby tolerance to lactose is a common trait in people of Northern European descent. It is linked to the LCT -13910C>T variant located in intron 13 of the MCM6 gene 13.9 kb upstream of the lactase (LCT) gene. In people of African and Middle Eastern descent, lactase...

  2. Mutation analysis of SLC26A4 for Pendred syndrome and nonsyndromic hearing loss by high-resolution melting

    DEFF Research Database (Denmark)

    Chen, Neng; Tranebjærg, Lisbeth; Rendtorff, Nanna Dahl

    2011-01-01

    to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed...... for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed...... a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously...

  3. Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis.

    Science.gov (United States)

    Kristensen, Lasse S; Andersen, Gitte B; Hager, Henrik; Hansen, Lise Lotte

    2012-01-01

    Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis. © 2011 Wiley Periodicals, Inc.

  4. MUSE stares into the shadows: the high-resolution dust attenuation curve of NGC 5626

    Science.gov (United States)

    Viaene, S.; Sarzi, M.; Baes, M.; Fritz, J.; Puerari, I.

    2017-12-01

    The newest generation of integral field unit spectrographs brings three-dimensional mapping of nearby galaxies one step closer. While the focus up to this point was mostly on stars and ionized gas, it is also possible to look at dust in a new, more complete way. Using MUSE science verification observations of NGC 5626, we map the interstellar matter in this dusty lenticular. We use the resolving power of MUSE to measure the optical attenuation with a spectral resolution of 6.25 Å, at physical scales of 0.1-1 kpc. The integrated attenuation curve of NGC 5626 shows a smooth, slightly steeper than Milky Way and SMC attenuation curves. Several sharp features are superimposed: we measure lower attenuation at spectral emission lines and higher attenuation for the sodium line doublet. No correlation was observed between sodium line strength and reddening by dust on spatially resolved scales. Additionally, the continuum attenuation was found to be independent from the Balmer decrement (tracing ionized gas attenuation). We model and interpret the variations in the attenuation curves of each spatial resolution element of NGC 5626. We find that the amount and distribution of dust along the line of sight is highly degenerate with any variation in the intrinsic extinction law. Our analysis shows that the interstellar matter in NGC 5626 resides in a regular and well-settled disc. Our results preach caution in the application of simple recipes to de-redden global galaxy spectra and underlines the need for more realistic dust geometries when constructing such correction formulas.

  5. Causes of bimodal melting curve:Asymmetric guaninecytosine (GC ...

    African Journals Online (AJOL)

    The aim of this study was to present a new situation in which a relatively single short PCR-product might show two separate peaks with sequence specific shapes at the dissociation curve. SYBR-Green I real-time RT-PCR was performed on Lhcgr-gene transcripts in rats. Different programs were used for melting curve ...

  6. High-resolution melting of 12S rRNA and cytochrome b DNA sequences for discrimination of species within distinct European animal families.

    Directory of Open Access Journals (Sweden)

    Jana Naue

    Full Text Available The cheap and easy identification of species is necessary within multiple fields of molecular biology. The use of high-resolution melting (HRM of DNA provides a fast closed-tube method for analysis of the sequence composition of the mitochondrial genes 12S rRNA and cytochrome b. We investigated the potential use of HRM for species identification within eleven different animal groups commonly found in Europe by animal-group-specific DNA amplification followed by DNA melting. Influence factors as DNA amount, additional single base alterations, and the existence of mixed samples were taken into consideration. Visual inspection combined with mathematical evaluation of the curve shapes did resolve nearly all species within an animal group. The assay can therefore not only be used for identification of animal groups and mixture analysis but also for species identification within the respective groups. The use of a universal 12S rRNA system additionally revealed a possible approach for species discrimination, mostly by exclusion. The use of the HRM assay showed to be a reliable, fast, and cheap method for species discrimination within a broad range of different animal species and can be used in a flexible "modular" manner depending on the question to be solved.

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

    Science.gov (United States)

    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

  8. Application of the High Resolution Melting analysis for genetic mapping of Sequence Tagged Site markers in narrow-leafed lupin (Lupinus angustifolius L.).

    Science.gov (United States)

    Kamel, Katarzyna A; Kroc, Magdalena; Święcicki, Wojciech

    2015-01-01

    Sequence tagged site (STS) markers are valuable tools for genetic and physical mapping that can be successfully used in comparative analyses among related species. Current challenges for molecular markers genotyping in plants include the lack of fast, sensitive and inexpensive methods suitable for sequence variant detection. In contrast, high resolution melting (HRM) is a simple and high-throughput assay, which has been widely applied in sequence polymorphism identification as well as in the studies of genetic variability and genotyping. The present study is the first attempt to use the HRM analysis to genotype STS markers in narrow-leafed lupin (Lupinus angustifolius L.). The sensitivity and utility of this method was confirmed by the sequence polymorphism detection based on melting curve profiles in the parental genotypes and progeny of the narrow-leafed lupin mapping population. Application of different approaches, including amplicon size and a simulated heterozygote analysis, has allowed for successful genetic mapping of 16 new STS markers in the narrow-leafed lupin genome.

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

    Directory of Open Access Journals (Sweden)

    Elena L. Enache

    2015-09-01

    Full Text Available 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.

  10. Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor®

    Directory of Open Access Journals (Sweden)

    Vincent Kate

    2009-12-01

    Full Text Available Abstract Background TILLING (Targeting Induced Local Lesions IN Genomes is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor® software, aimed at simultaneous detection of mutations in three homoeologous genes. Results We demonstrate that High Resolution Melting (HRM analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor® is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. Conclusion Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence

  11. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis.

    Science.gov (United States)

    Hanson, Erin K; Ballantyne, Jack

    2013-01-01

    Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive

  12. Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting

    Science.gov (United States)

    Chen, Neng; Tranebjærg, Lisbeth; Rendtorff, Nanna Dahl; Schrijver, Iris

    2011-01-01

    Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss and mutations in the SLC26A4 gene. Extensive allelic heterogeneity, however, necessitates analysis of all exons and splice sites to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously identified by sequencing. Subsequently, a 384-well plate format was designed for up to three patient samples per run. Blinded HRM testing on these plates of patient samples collected over 1 year in a clinical diagnostic laboratory accurately detected all variants identified by sequencing. In conclusion, HRM with targeted sequencing is a reliable, simple, and cost-effective method for SLC26A4 mutation screening and detection. PMID:21704276

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

    Science.gov (United States)

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

    2016-02-01

    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. 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. 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.

  14. High resolution melting analytical platform for rapid prenatal and postnatal diagnosis of β-thalassemia common among Southeast Asian population.

    Science.gov (United States)

    Prajantasen, Thanet; Fucharoen, Supan; Fucharoen, Goonnapa

    2015-02-20

    High resolution melting (HRM) analysis is a powerful technology for scanning sequence alteration. We have applied this HRM assay to screen common β-thalassemia mutations found among Southeast Asian population. Known DNA samples with 8 common mutations were used in initial development of the methods including -28 A-G, codon 17 A-T, IVSI-1G-T, IVSI-5G-C, codon 26G-A (Hb E), codons 41/42 -TTCT, codons 71/72+A and IVSII-654 C-T. Further validation was done on 60 postnatal and 6 prenatal diagnoses of β-thalassemia. Each mutation has specific HRM profile which could be used in rapid screening. Apart from those with DNA deletions, the results of HRM assay matched 100% with those of routine diagnosis made by routine allele specific PCR. In addition, the HRM assay could initially recognize three unknown mutations including a hitherto un-described one in Thai population. The established HRM assay should prove useful for rapid and high throughput platform for screening and prenatal diagnosis of β-thalassemia common in the region. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Barcode High Resolution Melting (Bar-HRM) analysis for detection and quantification of PDO "Fava Santorinis" (Lathyrus clymenum) adulterants.

    Science.gov (United States)

    Ganopoulos, Ioannis; Madesis, Panagiotis; Darzentas, Nikos; Argiriou, Anagnostis; Tsaftaris, Athanasios

    2012-07-15

    Legumes considered as one of the most important crops worldwide. Due to high price as a PDO product, commercial products of "Fava Santorinis" are often subjected to adulterations from other legume products coming from other Lathyrus or Vicia and Pisum species. Using plant DNA barcoding regions (trnL and rpoC) coupled with High Resolution Melting (Bar-HRM) we have developed a method allowing us to detect and authenticate PDO "Fava Santorinis". Bar-HRM proved to be a very sensitive tool able to genotype Lathyrus and its closed relative species and to detect admixtures, being sensitive enough to as low as 1:100 of non-"Fava Santorinis" in "Fava Santorinis" commercial products. In conclusion, Bar-HRM analysis can be a faster, with higher resolution and cost effectiveness alternative method to authenticate PDO "Fava Santorinis" and to quantitatively detect adulterations in "Fava Santorinis" with other relative commercial "Fava" food products. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. Rapid Detection and Differentiation of Clonorchis sinensis and Opisthorchis viverrini Using Real-Time PCR and High Resolution Melting Analysis

    Directory of Open Access Journals (Sweden)

    Xian-Quan Cai

    2014-01-01

    Full Text Available Clonorchis sinensis and Opisthorchis viverrini are both important fish-borne pathogens, causing serious public health problem in Asia. The present study developed an assay integrating real-time PCR and high resolution melting (HRM analysis for the specific detection and rapid identification of C. sinensis and O. viverrini. Primers targeting COX1 gene were highly specific for these liver flukes, as evidenced by the negative amplification of closely related trematodes. Assays using genomic DNA extracted from the two flukes yielded specific amplification and their identity was confirmed by sequencing, having the accuracy of 100% in reference to conventional methods. The assay was proved to be highly sensitive with a detection limit below 1 pg of purified genomic DNA, 5 EPG, or 1 metacercaria of C. sinensis. Moreover, C. sinensis and O. viverrini were able to be differentiated by their HRM profiles. The method can reduce labor of microscopic examination and the contamination of agarose electrophoresis. Moreover, it can differentiate these two flukes which are difficult to be distinguished using other methods. The established method provides an alternative tool for rapid, simple, and duplex detection of C. sinensis and O. viverrini.

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

    Science.gov (United States)

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

    2015-02-28

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

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

    Directory of Open Access Journals (Sweden)

    Ricardo Andrade Zampieri

    2016-02-01

    Full Text Available 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.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.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.

  19. Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: identification of novel mutations.

    Science.gov (United States)

    Raymond, Laure; Diebold, Bertrand; Leroux, Céline; Maurey, Hélène; Drouin-Garraud, Valérie; Delahaye, Andre; Dulac, Olivier; Metreau, Julia; Melikishvili, Gia; Toutain, Annick; Rivier, François; Bahi-Buisson, Nadia; Bienvenu, Thierry

    2013-01-01

    Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Development of a high-resolution melting-based approach for efficient differentiation among Bacillus cereus group isolates.

    Science.gov (United States)

    Antolinos, Vera; Fernández, Pablo S; Ros-Chumillas, María; Periago, Paula M; Weiss, Julia

    2012-09-01

    Strains belonging to Bacillus cereus Group include six different species, among which are Bacillus thuringiensis, Bacillus weihenstephanensis, and Bacillus cereus sensu stricto, a causative agent of food poisoning. Sequence of the panC-housekeeping gene is used for B. cereus Group affiliation to seven major phylogenetic groups (I-VII) with different ecological niches and variations in thermal growth range and spore heat resistance of B. cereus Group microorganisms varies among phylogenetic groups. We assigned a selection of B. cereus sensu stricto strains related to food poisoning from the Spanish cultivar Collection (Valencia) to Group IV strains based on panC gene sequence. Thermal inactivation assays revealed variability of spore heat resistance within these Group IV strains. Adequate food sanitizing treatments therefore require fast and reliable identification of particular strains. In the present study, feasibility of genotyping via high-resolution melting (HRM) analysis was examined. HRM analysis of amplified polymorphic 16S-23 intergenic spacer region (ISR) region proved to be discriminatory for B. cereus sensu stricto strain typing, while two other polymorphic regions within the bacterial rRNA operon allowed differentiation between Bacillus species, demonstrating its applicability for discrimination on the species and strain level within B. cereus Group.

  1. Development of a high-resolution melting marker for selecting Fusarium crown and root rot resistance in tomato.

    Science.gov (United States)

    Kim, Bichseam; Kim, Nahui; Kim, Jun Young; Kim, Byung Sup; Jung, Hee-Jeong; Hwang, Indoek; Noua, Ill-Sup; Sim, Sung-Chur; Park, Younghoon

    2016-03-01

    Fusarium crown and root rot is a severe fungal disease of tomato caused by Fusarium oxysporum f. sp. radicis-lycopersici (FORL). In this study, the genomic location of the FORL-resistance locus was determined using a set of molecular markers on chromosome 9 and an F2 population derived from FORL-resistant inbred 'AV107-4' (Solanum lycopersicum) × susceptible 'L3708' (Solanum pimpinellifolium). Bioassay performed using Korean FORL strain KACC 40031 showed single dominant inheritance of FORL resistance in the F2 population. In all, 13 polymerase chain reaction-based markers encompassing approximately 3.6-72.0 Mb of chromosome 9 were developed based on the Tomato-EXPEN 2000 map and SolCAP Tomato single nucleotide polymorphism array analysis. These markers were genotyped on 345 F2 plants, and the FORL-resistance locus was found to be present on a pericentromeric region of suppressed chromosomal recombination in chromosome 9. The location of the FORL-resistance locus was further confirmed by testing these markers against diverse commercial tomato and stock cultivars resistant to FORL. A restriction fragment length polymorphism marker, PNU-D4, located at approximately 6.1 Mb of chromosome 9 showed the highest match with the resistance locus and was used for conducting high-resolution melting analysis for marker-assisted selection of FORL resistance.

  2. Identification of molecular markers associated with Verticillium wilt resistance in alfalfa (Medicago sativa L. using high-resolution melting.

    Directory of Open Access Journals (Sweden)

    Tiejun Zhang

    Full Text Available 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.

  3. Identification of molecular markers associated with Verticillium wilt resistance in alfalfa (Medicago sativa L.) using high-resolution melting.

    Science.gov (United States)

    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.

  4. Melting of cross-linked DNA. III. Calculation of differential melting curves.

    Science.gov (United States)

    Lando, D Y; Fridman, A S; Krot, V I; Akhrem, A A

    1998-08-01

    In our previous papers I and II (D. Y. Lando et al, J. Biomol. Struct. Dynam. (1997) v. 15, N1, p. 129-140, p. 141-150), two methods were developed for calculation of melting curves of cross-linked DNA. One of them is based on Poland's and another on the Fixman-Freire approach. In the present communication, III, a new theoretical method is developed for computation of differential melting curves of DNAs cross-linked by anticancer drugs and their inactive analogs. As Poland's approach, the method allows study of the influence of the loop entropy factor, delta(n), on melting behavior (n is the length of a loop in base pairs). However the method is much faster and requires computer time that inherent for the most rapid Fixman-Freire calculation approach. In contrast to the computation procedures described before in communications I and II, the method is suitable for computation of differential melting curves in the case of long DNA chains, arbitrary loop entropy factors of melted regions and arbitrary degree of cross-linking including very low values that occur in vivo after administration of antitumor drugs. The method is also appropriate for DNAs without cross-links. The results of calculation demonstrate that even very low degree of cross-linking alters the DNA differential melting curve. Cross-linking also markedly strengthens the influence of particular function delta(n) upon melting behavior.

  5. Numerical prediction of the melting curve of n-octane

    NARCIS (Netherlands)

    Polson, J.M.; Frenkel, D.

    1999-01-01

    We compute the melting curve of n-octane using Molecular Dynamics simulations with a realistic all-atom molecular model. Thermodynamic integration methods are used to calculate the free energy of the system in both the crystalline solid and isotropic liquid phases. The Gibbs–Duhem integration

  6. Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species.

    Science.gov (United States)

    Gopaul, Krishna K; Sells, Jessica; Lee, Robin; Beckstrom-Sternberg, Stephen M; Foster, Jeffrey T; Whatmore, Adrian M

    2014-12-11

    The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays. Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel. The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material.

  7. High Resolution Melting (HRM) analysis for mutation screening of RGSL1,RGS16 and RGS8 in breast cancer

    DEFF Research Database (Denmark)

    Wiechec, Emilia; Wiuf, Carsten Henrik; Overgaard, Jens

    2011-01-01

    Abstract BACKGROUND: Identification of specific mutation targets in cancer may lead to discovery of the genes modulating cancer susceptibility and/or prognosis. The RGSL1, RGS16, and RGS8 genes within the 1q25.3 region belong to the novel family of regulators of G protein signaling (RGS) genes...... coding exons of RGSL1, RGS16, and RGS8 in tumors from 200 breast cancer patients. All sequence variants detected by HRM resulted in abnormal shape of the melting curves. The identified mutations and known single nucleotide polymorphisms (SNP) were subsequently confirmed by sequencing, and distribution...... cancer patients. In addition, a total of seven known SNPs were identified in this study. Genotype distributions were not significantly different between breast cancer patients and controls. CONCLUSIONS AND IMPACT: Identification of novel mutations within RGSL1 provides a new insight...

  8. Genotype-specific real-time PCR combined with high-resolution melting analysis for rapid identification of red-spotted grouper nervous necrosis virus.

    Science.gov (United States)

    Toubanaki, Dimitra K; Karagouni, Evdokia

    2017-08-01

    A real-time genotype-specific polymerase chain reaction (PCR) assay combined with high-resolution melting (HRM) analysis was developed to assess the most common genotypes of nervous necrosis viruses or nodaviruses. Nodaviruses are the causal agents of viral nervous necrosis infections, which have been wreaking havoc in the aquaculture industry worldwide, with fish mortality up to 100%. The four different genotypes of nodaviruses correlate with differences in viral pathogenicity. Therefore, rational development of effective vaccines and diagnostics requires analysis of genetic variation among viruses. The aim of the present study was to develop a real-time tetra-primer genotype-specific PCR assay for genotype identification. Four primers were utilized for simultaneous amplification of nodavirus genotype-specific products in a single closed-tube PCR after a reverse-transcription reaction using RNA isolated from fish samples. For high-throughput sample analysis, SYBR Green-based real-time PCR was used in combination with HRM analysis. The assay was evaluated in terms of specificity and sensitivity. The analysis resulted in melting curves that were indicative of each genotype. The detection limit when using reference plasmids was 100 ag/µL for both genotypes, while the sensitivity of the assays when testing a complex mixture was 10 fg/µL for red-spotted grouper nervous necrosis virus (RGNNV) and 100 fg/µL for striped jack nervous necrosis virus (SJNNV). To test the capability of this method under real-world conditions, 58 samples were examined. All samples belonged to the RGNNV genotype, which was fully validated. The results were in full agreement with genotyping by reference methods. The proposed methodology provides a rapid, sensitive, specific, robust and automatable assay for nodavirus genotyping, making it a useful tool for diagnosis and screening for epidemiological studies.

  9. High-resolution time series of Pseudomonas aeruginosa gene expression and rhamnolipid secretion through growth curve synchronization

    Directory of Open Access Journals (Sweden)

    Xavier João B

    2011-06-01

    Full Text Available Abstract Background Online spectrophotometric measurements allow monitoring dynamic biological processes with high-time resolution. Contrastingly, numerous other methods require laborious treatment of samples and can only be carried out offline. Integrating both types of measurement would allow analyzing biological processes more comprehensively. A typical example of this problem is acquiring quantitative data on rhamnolipid secretion by the opportunistic pathogen Pseudomonas aeruginosa. P. aeruginosa cell growth can be measured by optical density (OD600 and gene expression can be measured using reporter fusions with a fluorescent protein, allowing high time resolution monitoring. However, measuring the secreted rhamnolipid biosurfactants requires laborious sample processing, which makes this an offline measurement. Results Here, we propose a method to integrate growth curve data with endpoint measurements of secreted metabolites that is inspired by a model of exponential cell growth. If serial diluting an inoculum gives reproducible time series shifted in time, then time series of endpoint measurements can be reconstructed using calculated time shifts between dilutions. We illustrate the method using measured rhamnolipid secretion by P. aeruginosa as endpoint measurements and we integrate these measurements with high-resolution growth curves measured by OD600 and expression of rhamnolipid synthesis genes monitored using a reporter fusion. Two-fold serial dilution allowed integrating rhamnolipid measurements at a ~0.4 h-1 frequency with high-time resolved data measured at a 6 h-1 frequency. We show how this simple method can be used in combination with mutants lacking specific genes in the rhamnolipid synthesis or quorum sensing regulation to acquire rich dynamic data on P. aeruginosa virulence regulation. Additionally, the linear relation between the ratio of inocula and the time-shift between curves produces high-precision measurements of

  10. High Resolution Melt (HRM analysis is an efficient tool to genotype EMS mutants in complex crop genomes

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    Lochlainn Seosamh Ó

    2011-12-01

    Full Text Available Abstract Background Targeted Induced Loci Lesions IN Genomes (TILLING is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs and insertion/deletions (IN/DELs enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. Results We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Conclusions Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service.

  11. Sensitive High-Resolution Melting Analysis for Screening of KRAS and BRAF Mutations in Iranian Human Metastatic Colorectal Cancers

    Science.gov (United States)

    Niya, Mohammad Hadi Karbalaie; Basi, Ali; Koochak, Aghigh; Tameshkel, Fahimeh Safarnezhad; Rakhshani, Nasser; Zamani, Farhad; Imanzade, Farid; Rezvani, Hamid; sereshki, Mohammad Mahdi Adib; Sohrabi, Masoud Reza

    2016-01-01

    Background: Investigations of methods for detection of mutations have uncovered major weaknesses of direct sequencing and pyrosequencing, with their high costs and low sensitivity in screening for both known and unknown mutations. High resolution melting (HRM) analysis is an alternative tool for the rapid detection of mutations. Here we describe the accuracy of HRM in screening for KRAS and BRAF mutations in metastatic colorectal cancer (mCRCs) samples. Materials and Methods: A total of 1000 mCRC patients in Mehr Hospital, Tehran, Iran, from Feb 2008 to May 2012 were examined for KRAS mutations and 242 of them were selected for further assessment of BRAF mutations by HRM analysis. In order to calculate the sensitivity and specificity, HRM results were checked by pyrosequencing as the golden standard and Dxs Therascreen as a further method. Results: In the total of 1,000 participants, there were 664 (66.4%) with wild type and 336 (33.6%) with mutant codons 12 and/or 13 of the KRAS gene. Among 242 samples randomly checked for the BRAF gene, all were wild type by HRM. Pyrosequencing and Dxs Therascreen results were in line with those of the HRM. In this regard, the sensitivity and specificity of HRM were evaluated as 100%. Conclusion: The findings suggest that the HRM, in comparison with DNA sequencing, is a more appropriate method for precise scanning of KRAS and BRAF mutations. It is also possible to state that HRM may be an attractive technique for the detection of known or unknown somatic mutations in other genes. PMID:28122448

  12. Rapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting.

    Science.gov (United States)

    Wojdacz, Tomasz K; Dobrovic, Alexander; Algar, Elizabeth M

    2008-10-01

    Beckwith Wiedemann syndrome (BWS) and Russell Silver syndrome (RS) are growth disorders with opposing epimutations affecting the H19/IGF2 imprinting center at 11p15.5. Overgrowth and tumor risk in BWS is caused by aberrant expression of the paternally expressed, imprinted IGF2 gene, occurring as a consequence of mosaic hypermethylation within the imprinting center, or to mosaic paternal uniparental disomy (UPD). RS is characterized by severe intrauterine growth retardation (IUGR). A subset of RS cases were recently shown to have mosaic hypomethylation within the H19/IGF2 imprinting center, predicted to silence paternally expressed IGF2 in early development. Molecular diagnosis for BWS and RS involves methylation analysis of the H19 locus, enabling discrimination of allelic methylation patterns. In this study, methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyze methylation within the intergenic region of the H19 locus. A total of 36 samples comprising normal control (11), BWS (19), and RS (six) DNA were analyzed in a blinded study and scored as hypermethylated, normal, or hypomethylated. Results were compared with those derived by methylation-sensitive Southern blotting using the restriction enzymes Rsa I and Hpa II. A total of 100% concordance was obtained for the Southern blotting and MS-HRM scores. A total of three samples with paternal duplication affecting the H19/IGF2 region were scored as equivocal by both methods; however, 33 out of 36 (92%) the samples were unambiguously scored as being hypermethylated, hypomethylated, or normally methylated using MS-HRM. We conclude that MS-HRM is a rapid, cost-effective, and sensitive method for screening mosaic methylation changes at the H19 locus in BWS and RS.

  13. High resolution melting analysis of the MMAB gene in cblB patients and in those with undiagnosed methylmalonic aciduria.

    Science.gov (United States)

    Illson, Margaret L; Dempsey-Nunez, Laura; Kent, Jana; Huang, Qiuying; Brebner, Alison; Raff, Michael L; Watkins, David; Gilfix, Brian M; Wittwer, Carl T; Rosenblatt, David S

    2013-01-01

    Isolated methylmalonic aciduria (MMA) results either from a defect in the mitochondrial enzyme methylmalonylCoA mutase (MCM), or in the intracellular conversion of vitamin B12 (cobalamin) into its active coenzyme adenosylcobalamin (AdoCbl). Mutations in the MMAB gene affect the function of the enzyme ATP:cob(I)alamin adenosyltransferase (ATR) and the production of AdoCbl. Measurement of MCM function in cultured patient fibroblasts, followed by somatic cell complementation analysis in cases where MCM function is decreased, has classically been used to diagnose the cblB cobalamin disorder. A patient with persistent MMA, who could not be diagnosed using traditional somatic cell studies, was subsequently shown by sequencing in a clinical laboratory to contain two variants in the MMAB gene. This observation brings into question whether somatic cell studies have failed to diagnose other cblB patients with mild cellular phenotypes. A high resolution melting analysis (HRMA) assay was developed for the MMAB gene. It was used to scan 96 reference samples and two cohorts of patients: 42 patients diagnosed with cblB by complementation studies; and 181 patients with undiagnosed MMA. MMAB mutations, including one novel nonsense mutation (c.12 C>A [p.C4X]), were identified in all members of the cblB cohort. Four patients with undiagnosed MMA, including the index case described above, were found to contain variants in the MMAB gene: c.185C>T (p.T62M), c.394T>C (p.C132R), c.398C>T (p.S133F), c.521C>T (p.S174L), c.572G>A (p.R191Q). Only the index case was found to have two variants, suggesting that somatic cell studies diagnose almost all cblB patients. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. High-resolution melting analysis as a powerful tool to discriminate and genotype Pseudomonas savastanoi pathovars and strains.

    Directory of Open Access Journals (Sweden)

    Andrea Gori

    Full Text Available 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.

  15. Rapid and high throughput molecular identification of diverse mosquito species by high resolution melting analysis [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Yvonne Ukamaka Ajamma

    2016-08-01

    Full Text Available Mosquitoes are a diverse group of invertebrates, with members that are among the most important vectors of diseases. The correct identification of mosquitoes is paramount to the control of the diseases that they transmit. However, morphological techniques depend on the quality of the specimen and often unavailable taxonomic expertise, which may still not be able to distinguish mosquitoes among species complexes (sibling and cryptic species. High resolution melting (HRM analyses, a closed-tube, post-polymerase chain reaction (PCR method used to identify variations in nucleic acid sequences, has been used to differentiate species within the Anopheles gambiae and Culex pipiens complexes. We validated the use of PCR-HRM analyses to differentiate species within Anopheles and within each of six genera of culicine mosquitoes, comparing primers targeting cytochrome b (cyt b, NADH dehydrogenase subunit 1 (ND1, intergenic spacer region (IGS and cytochrome c oxidase subunit 1 (COI gene regions. HRM analyses of amplicons from all the six primer pairs successfully differentiated two or more mosquito species within one or more genera (Aedes (Ae. vittatus from Ae. metallicus, Culex (Cx. tenagius from Cx. antennatus, Cx. neavei from Cx. duttoni, cryptic Cx. pipiens species, Anopheles (An. gambiae s.s. from An. arabiensis and Mansonia (Ma. africana from Ma. uniformis based on their HRM profiles. However, PCR-HRM could not distinguish between species within Aedeomyia (Ad. africana and Ad. furfurea, Mimomyia (Mi. hispida and Mi. splendens and Coquillettidia (Cq. aurites, Cq. chrysosoma, Cq. fuscopennata, Cq. metallica, Cq. microannulatus, Cq. pseudoconopas and Cq. versicolor genera using any of the primers. The IGS and COI barcode region primers gave the best and most definitive separation of mosquito species among anopheline and culicine mosquito genera, respectively, while the other markers may serve to confirm identifications of closely related sub

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

    Science.gov (United States)

    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. Diagnosis of ABCB11 gene mutations in children with intrahepatic cholestasis using high resolution melting analysis and direct sequencing.

    Science.gov (United States)

    Hu, Guorui; He, Ping; Liu, Zhifeng; Chen, Qian; Zheng, Bixia; Zhang, Qihua

    2014-09-01

    Intrahepatic cholestasis represents a heterogeneous group of disorders that begin during childhood, most commonly manifesting as neonatal cholestasis, and lead to ongoing liver dysfunction in children and adults. For children, inherited pathogenic factors of cholestasis have gained increasing attention owing to the rapid development of molecular biology technology. However, these methods have their advantages and disadvantages in terms of simplicity, sensitivity, specificity, time required and expense. In the present study, an effective, sensitive and economical method is recommended, termed high-resolution melting (HRM) analysis and direct sequencing, based on general polymerase chain reaction, to detect mutations in disease‑causing genes. As one type of inherited intrahepatic cholestasis, progressive familial intrahepatic cholestasis type 2 (PFIC2) is caused by pathogenic mutations in the ABCB11 gene, HRM was used to detect mutations in the ABCB11 gene in the present study, and the diagnosis for PFIC2 was made by comprehensive analysis of genetic findings and clinical features. Furthermore, the characteristics of mutations and single nucleotide polymorphisms (SNPs) in the ABCB11 gene were elucidated. A total of 14 types of mutations/polymorphisms were identified in 20 patients from mainland China, including six missense mutations (p.Y337H, p.Y472C, p.R696W, p.Q931P, p.D1131V and p.H1198R), one nonsense mutation (p.R928X) and seven SNPs (p.D36D/rs3815675, p.F90F/rs4148777, p.Y269Y/rs2287616, p.I416I/rs183390670, p.V444A/rs2287622, p.A865V/rs118109635 and p.A1028A/rs497692). Five mutations were novel. The majority of the mutations were different from those detected in other population groups. A total of 4/20 patients (1/5) were diagnosed to be PFIC2 by combining genetic findings with the clinical features. Polymorphisms V444A and A1028A, with an allele frequency of 74.5 and 67.2%, respectively, were highly prevalent in the mainland Chinese subjects. No

  18. Comparative study of IDH1 mutations in gliomas by high resolution melting analysis, immunohistochemistry and direct DNA sequencing.

    Science.gov (United States)

    Li, Juan; Zhang, Haiyan; Wang, Li; Yang, Chuanhong; Lai, Huangwen; Zhang, Wei; Chen, Xiaodong; Wang, Jie

    2015-09-01

    Patients with glioblastomas with a specific mutation in the isocitrate dehydrogenase 1 (IDH1) gene have a better prognosis than those with gliomas with wild‑type IDH1. IDH1 analysis has become part of the standard diagnostic procedure and a promising tool used for stratification in clinical trials. The present study aimed to compare high resolution melting (HRM) analysis, immunohistochemistry (IHC) and direct DNA sequencing for the detection of IDH mutations in gliomas. Fifty‑one formalin‑fixed paraffin‑embedded tumor samples were selected. For the HRM analysis and direct DNA sequencing, DNA was extracted from the tissues. For IHC, sections were stained with an anti‑IDH1‑R132H specific antibody. The HRM analysis method identified 33 cases of IDH1 gene mutations, and all mutations occurred at the R132H site. There were 33 cases of IDH1 gene mutations found by IHC, which was consistent with that identified using the HRM analysis method. However, only 30 IDH1 samples were confirmed by sequencing, in which mutations occurred at the IDH1 exon 4 R132H site. No mutation was detected in the other three of these 33 cases (two grade II oligodendroglioma and one grade II diffuse astrocytoma) by sequencing, while IHC was positive for IDH1‑R132H. The results showed that the mutation detection rate was not identified to be significantly different (P=0.250) when determined by the HRM analysis method or by direct DNA sequencing, as the concordant rate between the two methods was high (κ=0.866). The HRM analysis method in glioma IDH1 gene mutation detection has advantages of high sensitivity, good repeatability, simple operation and accurate results. It provides a novel method for detecting mutations of the IDH1 gene in paraffin embedded tissue samples of clinical glioma. Related to a small amount of sample, there was no evidence showing that HRM analysis method is superior to IHC. Direct DNA sequencing, HRM analysis and IHC results were consistent; however, HRM and

  19. Simulating sub-daily Intensity-Frequency-Duration curves in Australia using a dynamical high-resolution regional climate model

    Science.gov (United States)

    Mantegna, Gabriel A.; White, Christopher J.; Remenyi, Tomas A.; Corney, Stuart P.; Fox-Hughes, Paul

    2017-11-01

    Climate change has the potential to significantly alter the characteristics of high-intensity, short-duration rainfall events, potentially leading to more severe and more frequent flash floods. Research has shown that future changes to such events could far exceed expectations based on temperature scaling and basic physical principles alone, but that computationally expensive convection-permitting models are required to accurately simulate sub-daily extreme rainfall events. It is therefore crucial to be able to model future changes to sub-daily duration extreme rainfall events as cost effectively as possible, especially in Australia where such information is scarce. In this study, we seek to determine what the shortest duration of extreme rainfall is that can be simulated by a less computationally expensive convection-parametrizing Regional Climate Model (RCM). We examine the ability of the Conformal Cubic Atmospheric Model (CCAM), a ∼10 km high-resolution convection-parametrizing RCM, to reproduce sub-daily Intensity-Frequency-Duration (IFD) curves corresponding to two long-term observational stations in the Australian island state of Tasmania, and examine the future model projections. We find that CCAM simulates observed extreme rainfall statistics well for 3-h durations and longer, challenging the current understanding that convection-permitting models are needed to accurately model sub-daily extreme rainfall events. Further, future projections from CCAM for the end of this Century show that extreme sub-daily rainfall intensities could increase by more than 15% per °C, far exceeding the 7% scaling estimate predicted by the Clausius-Clapeyron vapour pressure relationship and the 5% scaling estimate recommended by the Australian Rainfall and Runoff guide.

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

    OpenAIRE

    Roth, Amanda L.; Hanson, Nancy D.

    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 statisticall...

  1. High-resolution melting PCR assay, applicable for diagnostics and screening studies, allowing detection and differentiation of several Babesia spp. infecting humans and animals.

    Science.gov (United States)

    Rozej-Bielicka, Wioletta; Masny, Aleksander; Golab, Elzbieta

    2017-10-01

    The goal of the study was to design a single tube PCR test for detection and differentiation of Babesia species in DNA samples obtained from diverse biological materials. A multiplex, single tube PCR test was designed for amplification of approximately 400 bp region of the Babesia 18S rRNA gene. Universal primers were designed to match DNA of multiple Babesia spp. and to have low levels of similarity to DNA sequences of other intracellular protozoa and Babesia hosts. The PCR products amplified from Babesia DNA isolated from human, dog, rodent, deer, and tick samples were subjected to high-resolution melting analysis for Babesia species identification. The designed test allowed detection and differentiation of four Babesia species, three zoonotic (B. microti, B. divergens, B. venatorum) and one that is generally not considered zoonotic-Babesia canis. Both detection and identification of all four species were possible based on the HRM curves of the PCR products in samples obtained from the following: humans, dogs, rodents, and ticks. No cross-reactivity with DNA of Babesia hosts or Plasmodium falciparum and Toxoplasma gondii was observed. The lack of cross-reactivity with P. falciparum DNA might allow using the assay in endemic malaria areas. The designed assay is the first PCR-based test for detection and differentiation of several Babesia spp. of medical and veterinary importance, in a single tube reaction. The results of the study show that the designed assay for Babesia detection and identification could be a practical and inexpensive tool for diagnostics and screening studies of diverse biological materials.

  2. Establishment of a simple and rapid identification method for Listeria spp. by using high-resolution melting analysis, and its application in food industry.

    Science.gov (United States)

    Ohshima, Chihiro; Takahashi, Hajime; Phraephaisarn, Chirapiphat; Vesaratchavest, Mongkol; Keeratipibul, Suwimon; Kuda, Takashi; Kimura, Bon

    2014-01-01

    Listeria monocytogenes is the causative bacteria of listeriosis, which has a higher mortality rate than that of other causes of food poisoning. Listeria spp., of which L. monocytogenes is a member, have been isolated from food and manufacturing environments. Several methods have been published for identifying Listeria spp.; however, many of the methods cannot identify newly categorized Listeria spp. Additionally, they are often not suitable for the food industry, owing to their complexity, cost, or time consumption. Recently, high-resolution melting analysis (HRMA), which exploits DNA-sequence differences, has received attention as a simple and quick genomic typing method. In the present study, a new method for the simple, rapid, and low-cost identification of Listeria spp. has been presented using the genes rarA and ldh as targets for HRMA. DNA sequences of 9 Listeria species were first compared, and polymorphisms were identified for each species for primer design. Species specificity of each HRM curve pattern was estimated using type strains of all the species. Among the 9 species, 7 were identified by HRMA using rarA gene, including 3 new species. The remaining 2 species were identified by HRMA of ldh gene. The newly developed HRMA method was then used to assess Listeria isolates from the food industry, and the method efficiency was compared to that of identification by 16S rDNA sequence analysis. The 2 methods were in coherence for 92.6% of the samples, demonstrating the high accuracy of HRMA. The time required for identifying Listeria spp. was substantially low, and the process was considerably simplified, providing a useful and precise method for processing multiple samples per day. Our newly developed method for identifying Listeria spp. is highly valuable; its use is not limited to the food industry, and it can be used for the isolates from the natural environment.

  3. Using high-resolution tritium profiles to quantify the effects of melt on two Spitsbergen ice cores

    NARCIS (Netherlands)

    Wel, L.G. van der; Streurman, H.J.; Isaksson, E.; Helsen, M.M.; Wal, R.S.W. van de; Martma, T.; Pohjola, V.A.; Moore, J.C.; Meijer, H.A.J.

    2011-01-01

    Ice cores from small ice caps provide valuable climatic information, additional to that of Greenland and Antarctica. However, their integrity is usually compromised by summer meltwater percolation. To determine to what extent this can affect such ice cores, we performed high-resolution tritium

  4. A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis.

    Science.gov (United States)

    Marin, M S; Quintana, S; Leunda, M R; Recavarren, M; Pagnuco, I; Späth, E; Pérez, S; Odeón, A

    2016-01-01

    Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) are antigenically and genetically similar. The aim of this study was to develop a simple and reliable one-step real time PCR assay with high resolution melting (HRM) analysis for the simultaneous detection and differentiation of BoHV-1 and BoHV-5. Optimization of assay conditions was performed with DNA from reference strains. Then, DNA from field isolates, clinical samples and tissue samples of experimentally infected animals were studied by real time PCR-HRM. An efficient amplification of real time PCR products was obtained, and a clear melting curve and appropriate melting peaks for both viruses were achieved in the HRM curve analysis for BoHV type identification. BoHV was identified in all of the isolates and clinical samples, and BoHV types were properly differentiated. Furthermore, viral DNA was detected in 12/18 and 7/18 samples from BoHV-1- and BoHV-5-infected calves, respectively. Real time PCR-HRM achieved a higher sensitivity compared with virus isolation or conventional PCR. In this study, HRM was used as a novel procedure. This method provides rapid, sensitive, specific and simultaneous detection of bovine alpha-herpesviruses DNA. Thus, this technique is an excellent tool for diagnosis, research and epidemiological studies of these viruses in cattle. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Snapback primer genotyping of the Gilbert syndrome UGT1A1 (TA)(n) promoter polymorphism by high-resolution melting.

    Science.gov (United States)

    Farrar, Jared S; Palais, Robert A; Wittwer, Carl T

    2011-09-01

    Gilbert syndrome, a chronic nonhemolytic unconjugated hyperbilirubinemia, is associated with thymine-adenine (TA) insertions in the UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1) promoter. The UGT1A1 promoter genotype also correlates with toxicity induced by the chemotherapeutic drug irinotecan. Current closed-tube assays for genotyping the UGT1A1 (TA)(n) promoter polymorphism require multiple labeled probes and/or have difficulty classifying the (TA)(5) and (TA)(8) alleles. An unlabeled 5' extension on one primer that creates a hairpin after asymmetric PCR was used to develop a snapback primer high-resolution melting assay for the (TA)(n) polymorphism. A new method that plots the local deviation from exponential decay to improve genotype clustering was used to remove background fluorescence and to analyze the data. The snapback assay was compared with small-amplicon melting and fragment length analyses in a blinded study of DNA samples from 100 African Americans. Genotyping results obtained by small-amplicon melting and snapback primer melting were 83% and 99% concordant, respectively, with results obtained by fragment analysis. Reanalysis of the single discordant sample in the results of the snapback genotyping assay and the fragment analysis revealed an error in the fragment analysis. High-resolution melting was required for accurate snapback genotyping of the UGT1A1 (TA)(n) polymorphism. The 100% accuracy obtained with a capillary-based instrument fell to ≤81% with plate-based instruments. In contrast to small-amplicon genotyping, snapback primer genotyping can distinguish all UGT1A1 promoter genotypes. Rapid-cycle PCR combined with snapback primer analysis with only 2 unlabeled PCR primers (one with a 5' extension) and a saturating DNA dye can genotype loci with several alleles in <30 min.

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

    Science.gov (United States)

    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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Applications of the method of high resolution melting analysis for diagnosis of Leber's disease and the three primary mutation spectrum of LHON in the Han Chinese population.

    Science.gov (United States)

    Cui, Guanglin; Ding, Hu; Xu, Yujun; Li, Bin; Wang, Dao Wen

    2013-01-01

    Current screening methods, such as single strand conformational polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC) and direct DNA sequencing that are used for detecting mutation in Leber's hereditary optic neuropathy (LHON) subjects are time consuming and costly. Here we tested high-resolution melt (HRM) analysis for mtDNA primary mutations in LHON patients. In this study, we applied the high resolution melting (HRM) technology to screen mtDNA primary mutations in 50 LHON patients from their peripheral blood. In order to evaluate the reliability of this technique, we compared the results obtained by HRM and direct mtDNA sequencing. We also investigated the spectrum of three most common mtDNA mutations implicated in LHON in the Han Chinese population. The results showed HRM analysis differentiated all of the mtDNA primary mutations and identified 4 additional mtDNA mutations from 50 patients in the blind study. The prevalence of three primary mutations were 11778G>A (87.9%), 14484T>C (6.5%) and 3460G>A (1.7%) in the Han Chinese population. In conclusion, HRM analysis is a rapid, reliable, and low-cost tool for detecting mtDNA primary mutations and has practical applications in molecular genetics. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves.

    Science.gov (United States)

    Lando, Dmitri Y; Fridman, Alexander S; Chang, Chun-Ling; Grigoryan, Inessa E; Galyuk, Elena N; Murashko, Oleg N; Chen, Chun-Chung; Hu, Chin-Kun

    2015-06-15

    Many factors that change the temperature position and interval of the DNA helix-coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, Tm, and temperature melting width, ΔT, of the entire DNA transition. Changes in Tm and ΔT can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining Tm and ΔT. Indeed, some of the methods give unreasonable "jagged" Tm and ΔT dependences on varying relative concentration of DNA chemical modifications (rb), [Na(+)], and GC content. At the same time, Tm determined as the helix-coil transition average temperature, and ΔT, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of Tm and ΔT on rb, [Na(+)], and GC content. For multi-peak DMCs, Tm value determined in this way is the closest to the thermodynamic melting temperature (the helix-coil transition enthalpy/entropy ratio). Copyright © 2015. Published by Elsevier Inc.

  9. High-Resolution, High-Efficiency, Curved Diffraction Gratings Fabricated by Conformable, Maskless,100-nm Lithography Project

    Data.gov (United States)

    National Aeronautics and Space Administration — In this program, we will develop a novel process for fabricating large-area ultraviolet diffraction gratings on curved surfaces. This process is based on a unique...

  10. Development of a two-step high-resolution melting (HRM) analysis for screening sequence variants associated with resistance to the QoIs, benzimidazoles and dicarboximides in airborne inoculum of Botrytis cinerea.

    Science.gov (United States)

    Chatzidimopoulos, Michael; Ganopoulos, Ioannis; Vellios, Evangelos; Madesis, Panagiotis; Tsaftaris, Athanasios; Pappas, Athanassios C

    2014-11-01

    A rapid, high-resolution melting (HRM) analysis protocol was developed to detect sequence variations associated with resistance to the QoIs, benzimidazoles and dicarboximides in Botrytis cinerea airborne inoculum. HRM analysis was applied directly in fungal DNA collected from air samplers with selective medium. Three and five different genotypes were detected and classified according to their melting profiles in BenA and bos1 genes associated with resistance to benzimidazoles and dicarboximides, respectively. The sensitivity of the methodology was evident in the case of the QoIs, where genotypes varying either by a single nucleotide polymorphism or an additional 1205-bp intron were separated accurately with a single pair of primers. The developed two-step protocol was completed in 82 min and showed reduced variation in the melting curves' formation. HRM analysis rapidly detected the major mutations found in greenhouse strains providing accurate data for successfully controlling grey mould. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  11. 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.

    Science.gov (United States)

    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.

  12. Rapid detection and statistical differentiation of KPC gene variants in Gram-negative pathogens by use of high-resolution melting and ScreenClust analyses.

    Science.gov (United States)

    Roth, Amanda L; Hanson, Nancy D

    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 bla(KPC) 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 bla(KPC) 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 bla(KPC-2) and 12 isolates carrying bla(KPC-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 bla(KPC-2-like) and bla(KPC-3-like) alleles. Therefore, this assay is a rapid method to identify the presence of bla(KPC) enzymes in Gram-negative pathogens that can be easily integrated into busy clinical microbiology laboratories.

  13. High pressure melting curves of silver, gold and copper

    Directory of Open Access Journals (Sweden)

    Ho Khac Hieu

    2013-11-01

    Full Text Available In this work, based on the Lindemann's formula of melting and the pressure-dependent Grüneisen parameter, we have investigated the pressure effect on melting temperature of silver, gold and copper metals. The analytical expression of melting temperature as a function of volume compression has been derived. Our results are compared with available experimental data as well as with previous theoretical studies and the good and reasonable agreements are found. We also proposed the potential of this approach on predicting melting of copper at very high pressure.

  14. High pressure melting curves of silver, gold and copper

    Energy Technology Data Exchange (ETDEWEB)

    Hieu, Ho Khac, E-mail: hieuhk@duytan.edu.vn [Research and Development Center for Science and Technology, Duy Tan University, K7/25 Quang Trung, Danang (Viet Nam); Ha, Nguyen Ngoc [VNU-Hanoi University of Science, 334 Nguyen Trai, Hanoi (Viet Nam)

    2013-11-15

    In this work, based on the Lindemann's formula of melting and the pressure-dependent Grüneisen parameter, we have investigated the pressure effect on melting temperature of silver, gold and copper metals. The analytical expression of melting temperature as a function of volume compression has been derived. Our results are compared with available experimental data as well as with previous theoretical studies and the good and reasonable agreements are found. We also proposed the potential of this approach on predicting melting of copper at very high pressure.

  15. Molecular dynamics simulations of the melting curve of NiAl alloy under pressure

    Directory of Open Access Journals (Sweden)

    Wenjin Zhang

    2014-05-01

    Full Text Available The melting curve of B2-NiAl alloy under pressure has been investigated using molecular dynamics technique and the embedded atom method (EAM potential. The melting temperatures were determined with two approaches, the one-phase and the two-phase methods. The first one simulates a homogeneous melting, while the second one involves a heterogeneous melting of materials. Both approaches reduce the superheating effectively and their results are close to each other at the applied pressures. By fitting the well-known Simon equation to our melting data, we yielded the melting curves for NiAl: 1783(1 + P/9.8010.298 (one-phase approach, 1850(1 + P/12.8060.357 (two-phase approach. The good agreement of the resulting equation of states and the zero-pressure melting point (calc., 1850 ± 25 K, exp., 1911 K with experiment proved the correctness of these results. These melting data complemented the absence of experimental high-pressure melting of NiAl. To check the transferability of this EAM potential, we have also predicted the melting curves of pure nickel and pure aluminum. Results show the calculated melting point of Nickel agrees well with experiment at zero pressure, while the melting point of aluminum is slightly higher than experiment.

  16. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.

    Science.gov (United States)

    Ririe, K M; Rasmussen, R P; Wittwer, C T

    1997-02-15

    A microvolume fluorometer integrated with a thermal cycler was used to acquire DNA melting curves during polymerase chain reaction by fluorescence monitoring of the double-stranded DNA specific dye SYBR Green I. Plotting fluorescence as a function of temperature as the thermal cycler heats through the dissociation temperature of the product gives a DNA melting curve. The shape and position of this DNA melting curve are functions of the GC/AT ratio, length, and sequence and can be used to differentiate amplification products separated by less than 2 degrees C in melting temperature. Desired products can be distinguished from undesirable products, in many cases eliminating the need for gel electrophoresis. Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes. Complete amplification and analysis of products can be performed in less than 15 min.

  17. MEASUREMENT OF THE SHOCK-HEATED MELT CURVE OF LEAD USING PYROMETRY AND REFLECTOMETRY

    Energy Technology Data Exchange (ETDEWEB)

    D. Partouche-Sebban and J. L. Pelissier, Commissariat a` l' Energie Atomique, F. G. Abeyta, Los Alamos National Laboratory; W. W. Anderson, Los Alamos National Laboratory; M. E. Byers, Los Alamos National Laboratory; D. Dennis-Koller, Los Alamos National Laboratory; J. S. Esparza, Los Alamos National Laboratory; S. D. Borror, Bechtel Nevada; C. A. Kruschwitz, Bechtel Nevada

    2004-01-01

    Data on the high-pressure melting temperatures of metals is of great interest in several fields of physics including geophysics. Measuring melt curves is difficult but can be performed in static experiments (with laser-heated diamond-anvil cells for instance) or dynamically (i.e., using shock experiments). However, at the present time, both experimental and theoretical results for the melt curve of lead are at too much variance to be considered definitive. As a result, we decided to perform a series of shock experiments designed to provide a measurement of the melt curve of lead up to about 50 GPa in pressure. At the same time, we developed and fielded a new reflectivity diagnostic, using it to make measurements on tin. The results show that the melt curve of lead is somewhat higher than the one previously obtained with static compression and heating techniques.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    David Omondi

    Full Text Available 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.

  20. Development of a quantitation approach for total human and male DNA based on real time PCR followed by high resolution melting analysis.

    Science.gov (United States)

    Ginart, Santiago; Caputo, Mariela; Alechine, Evguenia; Corach, Daniel; Sala, Andrea

    2016-10-01

    We developed and validated a total human DNA quantitation technique that simultaneously allows male DNA detection. This assay, called Amel-Y, is a duplex Real Time PCR followed by HRM (high resolution melting) analysis using the intercalating dye SYTO9. Amel-Y duplex produces two amplicons, one for the amelogenin gene (106/112 bp, female/male) and another (84 bp) corresponding to human Y chromosome-specific fragment to detect male DNA. After HRM analysis, two melting peaks differing in 5.3°C-5.5°C are detected if both male and female DNA are present and only one if only female DNA is present. For specificity assessment, the inclusion of high concentrations of bacterial and fungal DNA in the quantitation reactions allowed discarding species cross-reactivity. A set of crime scene evidence from forensic casework has been quantified with commercial kits and compared with Amel-Y duplex. Our method detected male DNA from a concentration of 18 pg/μL and supports autosomal/Y DNA detection ratio up to 200:1. A limitation of the technique is its inability to quantify male and female donnors in a mixed sample. The Amel-Y duplex demonstrated to be an efficient system for quantifying total human DNA being a specific, rapid, sensitive, and cost-effective method suitable for mixed DNA samples and applicable to any field where human DNA quantification is required, such as molecular diagnosis, population genetics, and forensic identification. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. High-resolution melt and morphological analyses of mealybugs (Hemiptera: Pseudococcidae) from cacao: tools for the control of Cacao swollen shoot virus spread.

    Science.gov (United States)

    Wetten, Andy; Campbell, Colin; Allainguillaume, Joël

    2016-03-01

    Mealybugs (Hemiptera: Coccoidea: Pseudococcidae) are key vectors of badnaviruses, including Cacao swollen shoot virus (CSSV), the most damaging virus affecting cacao (Theobroma cacao L.). The effectiveness of mealybugs as virus vectors is species dependent, and it is therefore vital that CSSV resistance breeding programmes in cacao incorporate accurate mealybug identification. In this work, the efficacy of a CO1-based DNA barcoding approach to species identification was evaluated by screening a range of mealybugs collected from cacao in seven countries. Morphologically similar adult females were characterised by scanning electron microscopy, and then, following DNA extraction, were screened with CO1 barcoding markers. A high degree of CO1 sequence homology was observed for all 11 individual haplotypes, including those accessions from distinct geographical regions. This has allowed the design of a high-resolution melt (HRM) assay capable of rapid identification of the commonly encountered mealybug pests of cacao. HRM analysis readily differentiated between mealybug pests of cacao that cannot necessarily be identified by conventional morphological analysis. This new approach, therefore, has potential to facilitate breeding for resistance to CSSV and other mealybug-transmitted diseases. © 2015 Society of Chemical Industry.

  2. ITS2 barcoding DNA region combined with high resolution melting (HRM) analysis of Hyoscyami Semen, the mature seed of Hyoscyamus niger.

    Science.gov (United States)

    Xiong, Chao; Hu, Zhi-Gang; Tu, Yuan; Liu, He-Gang; Wang, Ping; Zhao, Ming-Ming; SHIi, Yu-Hua; Wu, Lan; Sun, Wei; Chen, Shi-Lin

    2016-12-01

    Hyoscyami Semen, the mature dried seed of Hyoscyamus niger L., has long been used as a traditional Chinese medicine to treat human diseases. Hyoscyami Semen is found in local markets in China. In markets, sellers and buyers commonly inadvertently mix the seeds of H. niger with the seeds of related species such as Hygrophila salicifolia (Vahl) Nees, Astragalus complanatus R. Br., Cuscuta australis R. Br., Cuscuta chinensis Lam., and Impatiens balsamina L. because of their similar morphologies or similar names. Thus, developing a reliable method for discriminating H. niger seeds from its adulterants is necessary to reduce confusion and ensure the safe use of Hyoscyami Semen. The present study was designed to evaluate the efficiency of high-resolution melting analysis combined with DNA barcoding (Bar-HRM) with internal transcribed spacer 2 to discriminate H. niger. Our results show that Bar-HRM successfully identified the adulterants and detected the proportion of H. niger DNA extract within an admixture. In particular, HRM detected H. niger DNA extract in A. complanatus DNA extract at concentrations as low as 1%. In conclusion, the Bar-HRM method developed in the present study for authenticating H. niger is rapid and cost-effective. It can be used in the future to guarantee the purity of Hyoscyami Semen for the clinical use. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  3. High-resolution melt analysis to identify and map sequence-tagged site anchor points onto linkage maps: a white lupin (Lupinus albus) map as an exemplar.

    Science.gov (United States)

    Croxford, Adam E; Rogers, Tom; Caligari, Peter D S; Wilkinson, Michael J

    2008-01-01

    * The provision of sequence-tagged site (STS) anchor points allows meaningful comparisons between mapping studies but can be a time-consuming process for nonmodel species or orphan crops. * Here, the first use of high-resolution melt analysis (HRM) to generate STS markers for use in linkage mapping is described. This strategy is rapid and low-cost, and circumvents the need for labelled primers or amplicon fractionation. * Using white lupin (Lupinus albus, x = 25) as a case study, HRM analysis was applied to identify 91 polymorphic markers from expressed sequence tag (EST)-derived and genomic libraries. Of these, 77 generated STS anchor points in the first fully resolved linkage map of the species. The map also included 230 amplified fragment length polymorphisms (AFLP) loci, spanned 1916 cM (84.2% coverage) and divided into the expected 25 linkage groups. * Quantitative trait loci (QTL) analyses performed on the population revealed genomic regions associated with several traits, including the agronomically important time to flowering (tf), alkaloid synthesis and stem height (Ph). Use of HRM-STS markers also allowed us to make direct comparisons between our map and that of the related crop, Lupinus angustifolius, based on the conversion of RFLP, microsatellite and single nucleotide polymorphism (SNP) markers into HRM markers.

  4. A high resolution melting (HRM) technology-based assay for cost-efficient clinical detection and genotyping of herpes simplex virus (HSV)-1 and HSV-2.

    Science.gov (United States)

    Lieveld, M; Carregosa, A; Benoy, I; Redzic, N; Berth, M; Vanden Broeck, D

    2017-10-01

    Genital herpes can be caused by two very similar viruses, herpes simplex virus (HSV)-1 or HSV-2. These two HSV types cannot be distinguished clinically, but genotyping is recommended in the first-episodes of genital herpes to guide counselling and management. Quantitative polymerase chain reaction (qPCR) is the preferred diagnostic method for HSV typing. However, commercial qPCR methods use expensive fluorescent labeled probes for detection. Furthermore, most low-cost methods are not able to differentiate between HSV-1 and -2. The aim of this study was to develop a high resolution melting (HRM) technology-based assay for sensitive HSV-1 and HSV-2 detection and genotyping. Using a panel of 46 clinical specimens, the performance of the HRM assay was compared to two commercial HSV tests: the HRM assay detected HSV in all 23 positive samples, with no false positive results (100% concordance with HSV I/II Real-TM assay). Additionally, the HRM assay correctly genotyped both HSV types in a subset of these clinical samples, as determined by the Realstar HSV PCR Kit. The HSV HRM assay provides a cost-effective alternative method to conventional more expensive assays and can be used in routine clinical specimens, in cases where it is particularly necessary to detect and distinguish HSV-1 from -2. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. FADS gene polymorphisms confer the risk of coronary artery disease in a Chinese Han population through the altered desaturase activities: based on high-resolution melting analysis.

    Directory of Open Access Journals (Sweden)

    Si-Wei Li

    Full Text Available OBJECTIVE: We explored the desaturase activities and the correlation of fatty acid desaturases (FADS gene single nucleotide polymorphisms (SNPs with plasma fatty acid in coronary artery disease (CAD patients in a Chinese Han population. METHODS: Plasma fatty acids were measured by gas chromatography in CAD patients (n = 505 and a control group (n = 510. Five SNPs in the FADS gene were genotyped with high-resolution melting (HRM methods. RESULTS: After adjustment, D6D activity, assessed as arachidonic acid (AA, C20:4n-6/linoleic acid (LA, C18:2n-6, was higher in CAD patients (pT and rs174460 C>T were different between the two groups. The rs174537 T allele was associated with a lower risk of CAD [OR 0.743, 95% CI (0.624, 0.884, p = 0.001]. Carriers of the rs174460 C allele were associated with a higher risk of CAD [OR 1.357, 95% CI (1.106, 1.665, p = 0.003]. CONCLUSIONS: We firstly report that the rs174460 C allele is associated with a higher risk of CAD, and confirm that the rs174537 T allele is associated with a lower risk of CAD. Our results indicate that FADS gene polymorphisms are likely to influence plasma fatty acid concentrations and desaturase activities.

  6. FADS gene polymorphisms confer the risk of coronary artery disease in a Chinese Han population through the altered desaturase activities: based on high-resolution melting analysis.

    Science.gov (United States)

    Li, Si-Wei; Lin, Kun; Ma, Pei; Zhang, Zhen-Lu; Zhou, Yi-Dan; Lu, Shuang-Yan; Zhou, Xin; Liu, Song-Mei

    2013-01-01

    We explored the desaturase activities and the correlation of fatty acid desaturases (FADS) gene single nucleotide polymorphisms (SNPs) with plasma fatty acid in coronary artery disease (CAD) patients in a Chinese Han population. Plasma fatty acids were measured by gas chromatography in CAD patients (n = 505) and a control group (n = 510). Five SNPs in the FADS gene were genotyped with high-resolution melting (HRM) methods. After adjustment, D6D activity, assessed as arachidonic acid (AA, C20:4n-6)/linoleic acid (LA, C18:2n-6), was higher in CAD patients (pT and rs174460 C>T were different between the two groups. The rs174537 T allele was associated with a lower risk of CAD [OR 0.743, 95% CI (0.624, 0.884), p = 0.001]. Carriers of the rs174460 C allele were associated with a higher risk of CAD [OR 1.357, 95% CI (1.106, 1.665), p = 0.003]. We firstly report that the rs174460 C allele is associated with a higher risk of CAD, and confirm that the rs174537 T allele is associated with a lower risk of CAD. Our results indicate that FADS gene polymorphisms are likely to influence plasma fatty acid concentrations and desaturase activities.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Ioannis Ganopoulos

    Full Text Available 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.

  9. A novel closed-tube method based on high resolution melting (HRM) analysis for authenticity testing and quantitative detection in Greek PDO Feta cheese.

    Science.gov (United States)

    Ganopoulos, Ioannis; Sakaridis, Ioannis; Argiriou, Anagnostis; Madesis, Panagiotis; Tsaftaris, Athanasios

    2013-11-15

    Animal species identification of milk and dairy products has received increasing attention concerning food composition, traceability, allergic pathologies and accurate consumer information. Here we sought to develop an easy to use and robust method for species identification in cheese with emphasis on an authenticity control of PDO Feta cheese products. We used specific mitochondrial DNA regions coupled with high resolution melting (HRM) a closed-tube method allowing us to detect bovine, ovine and caprine species and authenticate Greek PDO Feta cheese. The primers successfully amplified DNA isolated from milk and cheese and showed a high degree of specificity. HRM was proven capable of accurately identifying the presence of bovine milk (not allowed in Feta) down to 0.1% and also of quantifying the ratio of sheep to goat milk mixture in different Feta cheese commercial products. In conclusion, HRM analysis can be a faster, with higher resolution and a more cost effective alternative method to authenticate milk and dairy products including PDO Feta cheese and to quantitatively detect its sheep milk adulterations. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Rapid and inexpensive detection of common HBB gene mutations in Tunisian population by high-resolution melting analysis: implication for molecular diagnosis.

    Science.gov (United States)

    Ouragini, Houyem; Haddad, Faten; Darragi, Imen; Abbes, Salem

    2014-03-01

    In Tunisia, β-thalassemia is a common hereditary disease with a carrying rate of 2.21%. Up to now, detection of responsible mutations was made by laborious, expensive, and/or time consuming methods. The aim of this study is to develop and validate a specific assay for detection of the two most frequent mutations in Tunisian population, the IVS-I-110 (G → A) and Cd39 (C → T) mutations. In this study, we optimize high resolution melting analysis (HRMA) conditions for these mutations, using control DNAs. Then, we evaluate the strength of this methodology by screening a cohort of patients with β-thalassemia. All examined reference DNA samples were unambiguously distinguished from each other. For the blinded test, the results were completely compatible with direct sequencing, performed after the HRMA. As HRMA represents a highly sensitive and high-throughput gene scanning method, it can provide timely diagnosis at low cost for effective clinical management of β-thalassemia.

  11. Simultaneous detection of Cherry necrotic rusty mottle virus and Cherry green ring mottle virus using real-time PCR and high resolution melting analysis.

    Science.gov (United States)

    Komorowska, Beata; Fiore, Nicola; Zamorano, Alan; Li, Ruhui

    2014-08-01

    In this study, the real-time 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 in a real-time PCR using a primer set for both of them or duplex real-time PCR that included one specific primer set for each virus. These two strategies allowed us to confirmed virus infection in all tested samples. In 17 field samples the technique revealed samples positive for CNRMV or CGRMV as well as positive for both viruses. In addition, the HRM analysis made it possible to differentiate clearly between CNRMV and CGRMV. Sequence variations among CNRMV and CGRMV isolates observed from the HRM peaks were confirmed by sequencing. To test the capability to use this method in field, forty one sweet cherry samples were examined by HRM analysis. The HRM data showed that seven samples were positive for CNRMV and three were infected with CGRMV. The results presented in this study indicated that real-time PCR followed by HRM analysis provides sensitive, automated and rapid tool to detect and differentiate between CNRMV and CGRMV isolates. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Sina-Elisabeth Ben Ali

    2014-10-01

    Full Text Available 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.

  13. Rapid differentiation of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood by real-time PCR coupled to high resolution melting analysis.

    Science.gov (United States)

    Albonico, Francesca; Loiacono, Monica; Gioia, Gloria; Genchi, Claudio; Genchi, Marco; Mortarino, Michele

    2014-02-24

    Dirofilaria immitis and D. repens are the principal causative agents of canine filariosis and, although the number of dogs subjected to specific prevention is increasing, the prevalence of these parasites remains high in many areas of the world. The discrimination between the two Dirofilaria species using the classical diagnostic methods can be difficult and may lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. Over the last years, several molecular methods with higher sensitivity and specificity compared to classical microscopy and ELISA assays were designed. Nevertheless, a need for simple, rapid, and cost-effective molecular protocols to accurately discriminate between D. immitis and D. repens still remains. High resolution melting analysis coupled to real-time PCR (real-time PCR-HRMA) is a widely used technique to target sequence polymorphisms of the same gene in different species without the need to perform DNA sequencing or to use species-specific probes. In this work, a fast and cost-effective real-time PCR-HRMA protocol to detect and differentiate simultaneously and unequivocally D. immitis and D. repens microfilarial DNA extracted from peripheral dog blood samples is described. The present method is simpler to use than most other DNA-based methods and provides comparable discrimination between the two sibling species. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. 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.

    Science.gov (United States)

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

    2015-10-27

    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.

  15. High resolution melt-curve analysis to fine map a locus controlling the paternal sorting of mitochondria

    Science.gov (United States)

    Mitochondria are required for normal growth and development and play an important role in programmed cell death and aging. The mitochondrial DNA is maternally transmitted in the vast majority of eukaryotes. One exception is cucumber (Cucumis sativus), whose mitochondrial DNA is paternally transmit...

  16. Whole-genome amplified DNA from stored dried blood spots is reliable in high resolution melting curve and sequencing analysis

    DEFF Research Database (Denmark)

    Winkel, Bo G; Hollegaard, Mads Vilhelm; Olesen, Morten S

    2011-01-01

    The use of dried blood spots (DBS) samples in genomic workup has been limited by the relative low amounts of genomic DNA (gDNA) they contain. It remains to be proven that whole genome amplified DNA (wgaDNA) from stored DBS samples, constitutes a reliable alternative to gDNA.We wanted to compare...

  17. Dynamic time warping assessment of high-resolution melt curves provides a robust metric for fungal identification

    National Research Council Canada - National Science Library

    Sha Lu; Gordana Mirchevska; Sayali S Phatak; Dongmei Li; Janos Luka; Richard A Calderone; William A Fonzi

    2017-01-01

    .... Dynamic time warping (DTW), first introduced in the context of speech recognition to identify temporal distortion of similar sounds, is an elastic distance measure that has been successfully applied to a wide range of time series data...

  18. 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.

    Directory of Open Access Journals (Sweden)

    Marcello Ceccarelli

    Full Text Available 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

  19. Vitis vinifera L. Single-Nucleotide Polymorphism Detection with High-Resolution Melting Analysis Based on the UDP-Glucose:Flavonoid 3-O-Glucosyltransferase Gene.

    Science.gov (United States)

    Pereira, Leonor; Martins-Lopes, Paula

    2015-10-21

    Vitis vinifera L. is a species with a large number of varieties, which differ in terms of anthocyanin content. The genes involved in the anthocyanin biosynthesis pathway have a direct effect in the anthocyanin profile of each variety, being potentially interesting for varietal identification. The current study aimed at the design of an assay suitable for the discrimination of the largest number of grapevine varieties. Two genes of the anthocyanin pathway, chalcone isomerase (CHI) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT), were sequenced in 22 grapevine varieties. The CHI gene presented 5 SNPs within the sequence. A total of 58 SNPs and 1 INDEL were found among the UFGT gene, allowing the discrimination of 18 different genotypes within the 22 grapevine varieties. A HRM assay designed for UFGT, containing 704 bp, produced differentiated melting curves for each of the 18 haplotypes. The developed HRM assay is efficient in grapevine varietal discrimination.

  20. Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR).

    Science.gov (United States)

    Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

    2016-10-19

    Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.

  1. Combining COLD-PCR and high-resolution melt analysis for rapid detection of low-level, rifampin-resistant mutations in Mycobacterium tuberculosis.

    Science.gov (United States)

    Pang, Yu; Liu, Guan; Wang, Yufeng; Zheng, Suhua; Zhao, Yan-Lin

    2013-04-01

    Multidrug-resistant Mycobacterium tuberculosis (M. tuberculosis) remains a serious threat to public health. Mutational analysis of the gene encoding the beta subunit of RNA polymerase (rpoB) is an established and widely used surrogate marker for multidrug-resistant tuberculosis (MDR-TB). The rpoB-based drug-resistant assay requires relatively less time to detect drug resistance in M. tuberculosis, yet it fails to detect low-level mutations in wild-type DNA. Here, we describe a low-level mutation detection method that combines co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR) with high-resolution melting (HRM) analysis, aimed at detecting low-level, rifampin-resistant mutations in M. tuberculosis. Compared to conventional polymerase chain reaction (PCR), dilution experiments demonstrated a four- to eightfold improvement in selectivity using COLD-PCR/HRM to detect low-level, rifampin-resistant mutations. The mutation detection limit of conventional PCR/HRM was approximately 20%, whereas COLD-PCR/HRM had a mutation detection limit of 2.5%. Using traditional PCR/HRM and DNA sequencing, we found rpoB mutation in 110 rifampin-resistant isolates. The use of COLD-PCR/HRM allowed us to detect 10 low-level, rifampin-resistant mutations in 16 additional drug-resistant isolates. The sensitivity of COLD-PCR/HRM (95.2%) is significantly higher than that of PCR/HRM (87.3%). Our findings demonstrate that combined use of COLD-PCR with HRM can provide a sensitivity of at least 5% in detecting rpoB-mutated populations in a wild-type background, decreasing the delay in drug-resistant TB diagnosis and leading to faster, cheaper, more efficient, and more personalized antibiotic treatment, especially for low-level drug resistance mutations among the excess wild-type DNA. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. High resolution melting analysis of the MMAA gene in patients with cblA and in those with undiagnosed methylmalonic aciduria.

    Science.gov (United States)

    Dempsey-Nunez, Laura; Illson, Margaret L; Kent, Jana; Huang, Qiuying; Brebner, Alison; Watkins, David; Gilfix, Brian M; Wittwer, Carl T; Rosenblatt, David S

    2012-11-01

    The gene product of MMAA is required for the intracellular metabolism of cobalamin (Cbl). Mutations in this gene lead to the cblA class of disorders, characterized by isolated methylmalonic aciduria. We have been concerned that somatic cell methods of diagnosis may miss patients with mild cellular phenotypes. A high resolution melting analysis (HRMA) assay was developed to rapidly scan the coding exons and flanking intronic regions of the MMAA gene for variants. DNA was scanned by HRMA from 96 unaffected reference individuals, 72 cblA patients confirmed by complementation, and 181 patients with isolated elevated methylmalonic acid, who could not be diagnosed using complementation analysis. Suspected variants were confirmed by Sanger sequencing. In the cblA cohort, HRMA correctly identified all previously known mutations as well as an additional 22 variants, 10 of which had not been previously reported. Novel variants included one duplication (c.551dupG, p.C187LfsX3), one deletion (c.387delC, p.Y129YfsX13), one splice site mutation (c.440-2A>G, splice site), 4 missense mutations (c.748G>A, p.E520K; c.820G>A, p.G274S; c.627G>T, p.R209S; c.826A>G, p.K276E), and 3 nonsense mutations (c.960G>A, p.W320X; c.1075C>T, p.E359X; c.1084C>T, p.Q362X). All novel missense variants affect highly conserved residues and are predicted to be damaging. Scanning of MMAA in the 181 undiagnosed samples revealed a single novel heterozygous missense change (c.821G>A, p.G274D). Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Towards a pathogenic Escherichia coli detection platform using multiplex SYBR®Green Real-time PCR methods and high resolution melting analysis.

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    Dafni-Maria Kagkli

    Full Text Available 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.

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

    Science.gov (United States)

    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 (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

  5. A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC libraries using High Resolution Melt analysis

    Directory of Open Access Journals (Sweden)

    Caligari Peter DS

    2010-05-01

    Full Text Available Abstract Background The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR. Results Here, we report the first combined use of Multiplex Tandem PCR (MT-PCR and High Resolution Melt (HRM analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker. Conclusions A pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes.

  6. Quantitative assessment of AKAP12 promoter methylation in colorectal cancer using methylation-sensitive high resolution melting: Correlation with Duke's stage.

    Science.gov (United States)

    Liu, Weiwei; Guan, Ming; Su, Bing; Ye, Chuanzhong; Li, Ji; Zhang, Xinju; Liu, Chunfang; Li, Min; Lin, Yong; Lu, Yuan

    2010-06-01

    The expression of AKAP12 (A Kinase anchoring protein 12) is markedly reduced in a variety of cancers. The purpose of this study was to establish a methylation-sensitive high resolution melting (MS-HRM) assay for the quantitative detection of AKAP12 promoter methylation and expression and the association with clinicopathological variables in human colorectal cancer. We also assessed the effect of AKAP12 re-expression on cell growth and colony formation. Downregulation or loss of AKAP12 mRNA expression was detected in 31 of 45 tissue samples (68.9%). No significant correlation was observed between the reduced expression levels and patient age, gender, Duke's stage or tumor differentiation. Methylation (>1%) of the AKAP12 promoter region was present in 35 of 45 (77.8%) carcinoma tissue samples and 6 of 45 (13.3%) adjacent tissue samples. AKAP12 methylation was significantly higher in the colorectal cancer tissues exhibiting advanced Duke's stages. Treatment of the three colorectal carcinoma cell lines (LoVo, COLO320 and SW480) with completely methylated AKAP12 with inhibitors of DNA methyltransferase (5-Aza-2'-deoxycytidine) markedly increased expression of AKAP12 and decreased methylation levels. Ectopic expression of AKAP12 in the LoVo cell line suppressed cell growth and inhibited colony formation. The AKAP12 gene was examined by quantitative RT-PCR, MS-HRM analysis and bisulfite sequencing in 45 paired tissue samples obtained from primary colorectal carcinomas and the corresponding adjacent tissues. In addition, five colorectal carcinoma cell lines (LoVo, COLO205, SW480, LS174T and COLO320) were investigated and western blot analysis was used to investigate changes in protein expression. A proliferation assay and soft agar assay were performed after overexpression of AKAP12. Our study demonstrated that MS-HRM is a robust, fast and sensitive method for AKAP12 methylation analysis. AKAP12 methylation represents a potential molecular biomarker for predicting the

  7. Entropy and transport properties of liquid metals along the melting curve

    Science.gov (United States)

    Cao, Qi-Long; Wang, Pan-Pan; Shao, Ju-Xiang; Wang, Fan-Hou

    2017-02-01

    Molecular dynamics simulations are performed for several monatomic metals and Fe0.9Ni0.1 metallic alloy to study the transport properties and entropy of liquids along melting curve. Our results show that the self-diffusion coefficients and viscosity of liquids increase with increasing pressure along the melting curves. Analysis suggests that, at high pressure conditions, the pair correlation entropy S2 of liquids along melting curve is bout -3.71kB, independent of the pressure and variety of liquids, which indicates that there is no obvious change in liquid structure along the melting curve. The Rosenfeld entropy-scaling laws with S2 = -3.71kB and the special values of scaling parameters can give reasonable estimates for the self-diffusion coefficients and viscosity of liquid metals along melting curves. The effect of pressure on transport coefficients can be quantified through its corresponding effect on the melting temperature and number density, and this result is in consistent with the Andrade's model. In addition, the variation of S2 provides a useful, experimentally accessible, structure-based criterion for freezing of liquid metals.

  8. Identification of BRCA1/2 founder mutations in Southern Chinese breast cancer patients using gene sequencing and high resolution DNA melting analysis.

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    Ava Kwong

    Full Text Available 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.

  9. Identification of BRCA1/2 founder mutations in Southern Chinese breast cancer patients using gene sequencing and high resolution DNA melting analysis.

    Science.gov (United States)

    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

    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. 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. 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.

  10. qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients.

    Directory of Open Access Journals (Sweden)

    Sergio Araujo

    2017-06-01

    Full Text Available Real-Time PCR-High Resolution Melting (qPCR-HRM analysis has been recently described for rapid drug susceptibility testing (DST of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens.The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR, rifampin (rpoB DRDR and ofloxacin (gyrA DRDR was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB and 55 paucibacillary (PB cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA were obtained from both MB (154/156; 98.7% and PB (35/55; 63.3% patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172, formalin-fixed paraffin embedded (FFPE tissues (n = 36 or archival Fite's stained slides (n = 3 were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able

  11. High resolution melting: a useful field-deployable method to measure dhfr and dhps drug resistance in both highly and lowly endemic Plasmodium populations.

    Science.gov (United States)

    Ndiaye, Yaye Dié; Diédhiou, Cyrille K; Bei, Amy K; Dieye, Baba; Mbaye, Aminata; Mze, Nasserdine Papa; Daniels, Rachel F; Ndiaye, Ibrahima M; Déme, Awa B; Gaye, Amy; Sy, Mouhamad; Ndiaye, Tolla; Badiane, Aida S; Ndiaye, Mouhamadou; Premji, Zul; Wirth, Dyann F; Mboup, Souleymane; Krogstad, Donald; Volkman, Sarah K; Ahouidi, Ambroise D; Ndiaye, Daouda

    2017-04-19

    Emergence and spread of drug resistance to every anti-malarial used to date, creates an urgent need for development of sensitive, specific and field-deployable molecular tools for detection and surveillance of validated drug resistance markers. Such tools would allow early detection of mutations in resistance loci. The aim of this study was to compare common population signatures and drug resistance marker frequencies between two populations with different levels of malaria endemicity and history of anti-malarial drug use: Tanzania and Sénégal. This was accomplished by implementing a high resolution melting assay to study molecular markers of drug resistance as compared to polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP) methodology. Fifty blood samples were collected each from a lowly malaria endemic site (Sénégal), and a highly malaria endemic site (Tanzania) from patients presenting with uncomplicated Plasmodium falciparum malaria at clinic. Data representing the DHFR were derived using both PCR-RFLP and HRM assay; while genotyping data representing the DHPS were evaluated in Senegal and Tanzania using HRM. Msp genotyping analysis was used to characterize the multiplicity of infection in both countries. A high prevalence of samples harbouring mutant DHFR alleles was observed in both population using both genotyping techniques. HRM was better able to detect mixed alleles compared to PCR/RFLP for DHFR codon 51 in Tanzania; and only HRM was able to detect mixed infections from Senegal. A high prevalence of mutant alleles in DHFR (codons 51, 59, 108) and DHPS (codon 437) were found among samples from Sénégal while no mutations were observed at DHPS codons 540 and 581, from both countries. Overall, the frequency of samples harbouring either a single DHFR mutation (S108N) or double mutation in DHFR (C59R/S108N) was greater in Sénégal compared to Tanzania. Here the results demonstrate that HRM is a rapid, sensitive, and field

  12. Quantitative evaluation of interstitial pneumonia using 3D-curved high-resolution CT imaging parallel to the chest wall: A pilot study.

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    Hiroyasu Umakoshi

    Full Text Available To quantify the imaging findings of patients with interstitial pneumonia (IP and emphysema using three-dimensional curved high-resolution computed tomography (3D-cHRCT at a constant depth from the chest wall, and compare the results to visual assessment of IP and each patient's diffusing capacity of the lungs for carbon monoxide (DLco.We retrospectively reviewed the axial CT findings and pulmonary function test results of 95 patients with lung cancer (72 men and 23 women, aged 45-84 years with or without IP, as follows: non-IP (n = 47, mild IP (n = 31, and moderate IP (n = 17. The 3D-cHRCT images of the lung at a 1-cm depth from the chest wall were reconstructed automatically using original software; total area (TA, high-attenuation area (HAA >-500 HU, and low-attenuation area (LAA <-950 HU were calculated on a workstation. The %HAA and %LAA were calculated as follows: [Formula: see text], and [Formula: see text].The %HAA and %LAA respective values were 3.2±0.9 and 27.7±8.2, 3.9±1.2 and 27.6±5.9, and 6.9±2.2 and 25.4±8.7 in non-IP, mild IP, and moderate IP patients, respectively. There were significant differences in %HAA between the 3 groups of patients (P<0.001, but no differences in %LAA (P = 0.558. Multiple linear regression analysis revealed that %HAA and %LAA were negatively correlated with predicted DLco (standard partial regression coefficient [b*] = -0.453, P<0.001; b* = -0.447, P<0.001, respectively.The %HAA and %LAA values computed using 3D-cHRCT were significantly correlated with DLco and may be important quantitative parameters for both IP and emphysema.

  13. Absence of low temperature anomaly on the melting curve of $^4$He

    OpenAIRE

    Todoshchenko, I. A.; Alles, H.; Junes, H. J.; Parshin, A. Ya.; Tsepelin, V.

    2007-01-01

    We have measured the melting pressure and pressure in the liquid at constant density of ultra-pure $^4$He (0.3 ppb of $^3$He impurities) with the accuracy of about 0.5 $\\mu$bar in the temperature range from 10 to 320 mK. Our measurements show that the anomaly on the melting curve below 80 mK which we have recently observed is entirely due to an anomaly in the elastic modulus of Be-Cu from which our pressure gauge is made of. We thus conclude that the melting pressure of $^4$He follows the $T^...

  14. COLD-PCR enhanced melting curve analysis improves diagnostic accuracy for KRAS mutations in colorectal carcinoma.

    Science.gov (United States)

    Pritchard, Colin C; Akagi, Laura; Reddy, Poluru L; Joseph, Loren; Tait, Jonathan F

    2010-11-26

    KRAS mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer. Sensitive methods are required to reliably detect KRAS mutations in tumor samples due to admixture with non-mutated cells. Many laboratories have implemented sensitive tests for KRAS mutations, but the methods often require expensive instrumentation and reagents, parallel reactions, multiple steps, or opening PCR tubes. We developed a highly sensitive, single-reaction, closed-tube strategy to detect all clinically significant mutations in KRAS codons 12 and 13 using the Roche LightCycler® instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (Tc) of 81°C increased the sensitivity of the assay >10-fold for the majority of KRAS mutations. We compared the COLD-PCR enhanced melting curve method to melting curve analysis without COLD-PCR and to traditional Sanger sequencing. In a cohort of 61 formalin-fixed paraffin-embedded colorectal cancer specimens, 29/61 were classified as mutant and 28/61 as wild type across all methods. Importantly, 4/61 (6%) were re-classified from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significant KRAS mutations by COLD-PCR DNA sequencing. Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background. We have developed and validated an inexpensive, rapid, and highly sensitive clinical assay for KRAS mutations that is the first report of COLD-PCR combined with probe-based melting curve analysis. This assay significantly improved diagnostic

  15. COLD-PCR enhanced melting curve analysis improves diagnostic accuracy for KRAS mutations in colorectal carcinoma

    Directory of Open Access Journals (Sweden)

    Joseph Loren

    2010-11-01

    Full Text Available Abstract Background KRAS mutational analysis is the standard of care prior to initiation of treatments targeting the epidermal growth factor receptor (EGFR in patients with metastatic colorectal cancer. Sensitive methods are required to reliably detect KRAS mutations in tumor samples due to admixture with non-mutated cells. Many laboratories have implemented sensitive tests for KRAS mutations, but the methods often require expensive instrumentation and reagents, parallel reactions, multiple steps, or opening PCR tubes. Methods We developed a highly sensitive, single-reaction, closed-tube strategy to detect all clinically significant mutations in KRAS codons 12 and 13 using the Roche LightCycler® instrument. The assay detects mutations via PCR-melting curve analysis with a Cy5.5-labeled sensor probe that straddles codons 12 and 13. Incorporating a fast COLD-PCR cycling program with a critical denaturation temperature (Tc of 81°C increased the sensitivity of the assay >10-fold for the majority of KRAS mutations. Results We compared the COLD-PCR enhanced melting curve method to melting curve analysis without COLD-PCR and to traditional Sanger sequencing. In a cohort of 61 formalin-fixed paraffin-embedded colorectal cancer specimens, 29/61 were classified as mutant and 28/61 as wild type across all methods. Importantly, 4/61 (6% were re-classified from wild type to mutant by the more sensitive COLD-PCR melting curve method. These 4 samples were confirmed to harbor clinically-significant KRAS mutations by COLD-PCR DNA sequencing. Five independent mixing studies using mutation-discordant pairs of cell lines and patient specimens demonstrated that the COLD-PCR enhanced melting curve assay could consistently detect down to 1% mutant DNA in a wild type background. Conclusions We have developed and validated an inexpensive, rapid, and highly sensitive clinical assay for KRAS mutations that is the first report of COLD-PCR combined with probe

  16. Screening of WT1 mutations in exon 8 and 9 in children with steroid resistant nephrotic syndrome from a single centre and establishment of a rapid screening assay using high-resolution melting analysis in a clinical setting

    OpenAIRE

    Siji, Annes; Pardeshi, Varsha Chhotusing; Ravindran, Shilpa; Vasudevan, Ambily; Vasudevan, Anil

    2017-01-01

    Background Mutations in Wilm?s tumor 1 (WT1) gene is one of the commonly reported genetic mutations in children with steroid resistant nephrotic syndrome (SRNS). We report the results of direct sequencing of exons 8 and 9 of WT1 gene in 100 children with SRNS from a single centre. We standardized and validated High Resolution Melt (HRM) as a rapid and cost effective screening step to identify individuals with normal sequence and distinguish it from those with a potential mutation. Since only ...

  17. Rapid and accurate identification of isolates of Candida species by melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA)

    NARCIS (Netherlands)

    Decat, E.; van Mechelen, E.; Saerens, B.; Vermeulen, S.J.T.; Boekhout, T.; de Blaiser, S.; Vaneechoutte, M.; Deschaght, P.

    2013-01-01

    Rapid identification of clinically important yeasts can facilitate the initiation of anti-fungal therapy, since susceptibility is largely species-dependent. We evaluated melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA) as an identification

  18. On-chip magnetic bead-based DNA melting curve analysis using a magnetoresistive sensor

    Energy Technology Data Exchange (ETDEWEB)

    Rizzi, Giovanni, E-mail: giori@nanotech.dtu.dk; Østerberg, Frederik W.; Henriksen, Anders D.; Dufva, Martin; Hansen, Mikkel F., E-mail: mikkel.hansen@nanotech.dtu.dk

    2015-04-15

    We present real-time measurements of DNA melting curves in a chip-based system that detects the amount of surface-bound magnetic beads using magnetoresistive magnetic field sensors. The sensors detect the difference between the amount of beads bound to the top and bottom sensor branches of the differential sensor geometry. The sensor surfaces are functionalized with wild type (WT) and mutant type (MT) capture probes, differing by a single base insertion (a single nucleotide polymorphism, SNP). Complementary biotinylated targets in suspension couple streptavidin magnetic beads to the sensor surface. The beads are magnetized by the field arising from the bias current passed through the sensors. We demonstrate the first on-chip measurements of the melting of DNA hybrids upon a ramping of the temperature. This overcomes the limitation of using a single washing condition at constant temperature. Moreover, we demonstrate that a single sensor bridge can be used to genotype a SNP. - Highlights: • We apply magnetoresistive sensors to study solid-surface hybridization kinetics of DNA. • We measure DNA melting profiles for perfectly matching DNA duplexes and for a single base mismatch. • We present a procedure to correct for temperature dependencies of the sensor output. • We reliably extract melting temperatures for the DNA hybrids. • We demonstrate direct measurement of differential binding signal for two probes on a single sensor.

  19. On-chip magnetic bead-based DNA melting curve analysis using a magnetoresistive sensor

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Østerberg, Frederik Westergaard; Henriksen, Anders Dahl

    2014-01-01

    We present real-time measurements of DNA melting curves in a chip-based system that detects the amount of surface-bound magnetic beads using magnetoresistive magnetic field sensors. The sensors detect the difference between the amount of beads bound to the top and bottom sensor branches...... of the differential sensor geometry. The sensor surfaces are functionalized with wild type (WT) and mutant type (MT) capture probes, differing by a single base insertion (a single nucleotide polymorphism, SNP). Complementary biotinylated targets in suspension couple streptavidin magnetic beads to the sensor surface....... The beads are magnetized by the field arising from the bias current passed through the sensors. We demonstrate the first on-chip measurements of the melting of DNA hybrids upon a ramping of the temperature. This overcomes the limitation of using a single washing condition at constant temperature. Moreover...

  20. Identification of squid species by melting temperature shifts on fluorescence melting curve analysis (FMCA) using single dual-labeled probe

    Science.gov (United States)

    Koh, Eunjung; Song, Ha Jeong; Kwon, Na Young; Kim, Gi Won; Lee, Kwang Ho; Jo, Soyeon; Park, Sujin; Park, Jihyun; Park, Eun Kyeong; Hwang, Seung Yong

    2017-06-01

    Real time PCR is a standard method for identification of species. One of limitations of the qPCR is that there would be false-positive result due to mismatched hybridization between target sequence and probe depending on the annealing temperature in the PCR condition. As an alternative, fluorescence melting curve analysis (FMCA) could be applied for species identification. FMCA is based on a dual-labeled probe. Even with subtle difference of target sequence, there are visible melting temperature (Tm) shift. One of FMCA applications is distinguishing organisms distributed and consumed globally as popular food ingredients. Their prices are set by species or country of origin. However, counterfeiting or distributing them without any verification procedure are becoming social problems and threatening food safety. Besides distinguishing them in naked eye is very difficult and almost impossible in any processed form. Therefore, it is necessary to identify species in molecular level. In this research three species of squids which have 1-2 base pair differences each are selected as samples since they have the same issue. We designed a probe which perfectly matches with one species and the others mismatches 2 and 1 base pair respectively and labeled with fluorophore and quencher. In an experiment with a single probe, we successfully distinguished them by Tm shift depending on the difference of base pair. By combining FMCA and qPCR chip, smaller-scale assay with higher sensitivity and resolution could be possible, andc furthermore, enabling results analysis with smart phone would realize point-of-care testing (POCT).

  1. Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR

    Directory of Open Access Journals (Sweden)

    Lázcoz Paula

    2008-02-01

    Full Text Available Abstract Background We present two melting curve analysis (MCA-based semiquantitative real time PCR techniques to detect the promoter methylation status of genes. The first, MCA-MSP, follows the same principle as standard MSP but it is performed in a real time thermalcycler with results being visualized in a melting curve. The second, MCA-Meth, uses a single pair of primers designed with no CpGs in its sequence. These primers amplify both unmethylated and methylated sequences. In clinical applications the MSP technique has revolutionized methylation detection by simplifying the analysis to a PCR-based protocol. MCA-analysis based techniques may be able to further improve and simplify methylation analyses by reducing starting DNA amounts, by introducing an all-in-one tube reaction and by eliminating a final gel stage for visualization of the result. The current study aimed at investigating the feasibility of both MCA-MSP and MCA-Meth in the analysis of promoter methylation, and at defining potential advantages and shortcomings in comparison to currently implemented techniques, i.e. bisulfite sequencing and standard MSP. Methods The promoters of the RASSF1A (3p21.3, BLU (3p21.3 and MGMT (10q26 genes were analyzed by MCA-MSP and MCA-Meth in 13 astrocytoma samples, 6 high grade glioma cell lines and 4 neuroblastoma cell lines. The data were compared with standard MSP and validated by bisulfite sequencing. Results Both, MCA-MSP and MCA-Meth, successfully determined promoter methylation. MCA-MSP provided information similar to standard MSP analyses. However the analysis was possible in a single tube and avoided the gel stage. MCA-Meth proved to be useful in samples with intermediate methylation status, reflected by a melting curve position shift in dependence on methylation extent. Conclusion We propose MCA-MSP and MCA-Meth as alternative or supplementary techniques to MSP or bisulfite sequencing.

  2. NMR system for imaging [sup 3]He-[sup 4]He mixtures on the melting curve

    Energy Technology Data Exchange (ETDEWEB)

    Swirski, Y. (Dept. of Physis, Technion-Israel Inst. of Tech., Haifa (Israel)); Shuster, I. (Dept. of Physis, Technion-Israel Inst. of Tech., Haifa (Israel)); Schmidt, E.J. (Dept. of Physis, Technion-Israel Inst. of Tech., Haifa (Israel)); Polturak, E. (Dept. of Physis, Technion-Israel Inst. of Tech., Haifa (Israel)); Lipson, S.G. (Dept. of Physis, Technion-Israel Inst. of Tech., Haifa (Israel))

    1994-02-01

    A pulsed 10 MHz NMR system for imaging [sup 3]He-[sup 4]He mixtures on the melting curve between 0.4K to 1.5K is described. The spatial distribution of [sup 3]He, T[sub 1] and T[sub 2] relaxation times were mapped along the vertical direction, perpendicular to the liquid-solid interface. Multi-exponential fits for the relaxation times were made. These are useful to follow the relaxation of structural inhomogeneities in the crystal. (orig.)

  3. High-resolution heat-transfer-coefficient maps applicable to compound-curve surfaces using liquid crystals in a transient wind tunnel

    Science.gov (United States)

    Jones, Terry V.; Hippensteele, Steven A.

    1988-01-01

    Tests were performed in a transient heat transfer tunnel in which the model under test was preheated prior to allowing room temperature air to be suddenly drawn over the model. The resulting movement of isothermal contours on the model is revealed using a surface coating of thermochromic liquid crystals that display distinctive colors at particular temperatures. A video record is obtained of a temperature and time data pair for all points on the model during a single test. Experiments on a duct model are reported in which the model was preheated using a hot air stream. A manner in which initial model temperature nonuniformities could be taken into account was investigated. The duct model was also tested with a steady-state measurement technique and results were compared with the transient measurements, but recognizing that differences existed between the upstream thermal boundary conditions. The steady-state and transient measurements were shown to be consistent with predicted values. The main advantage of this transient heat transfer technique using liquid crystals is that since the test model need not be actively heated, high-resolution measurements on surfaces with complex shapes may be obtained.

  4. High-resolution melting analysis of gyrA codon 84 and grlA codon 80 mutations conferring resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples.

    Science.gov (United States)

    Loiacono, Monica; Martino, Piera A; Albonico, Francesca; Dell'Orco, Francesca; Ferretti, Manuela; Zanzani, Sergio; Mortarino, Michele

    2017-09-01

    Staphylococcus pseudintermedius is an opportunistic pathogen of dogs and cats. A high-resolution melting analysis (HRMA) protocol was designed and tested on 42 clinical isolates with known fluoroquinolone (FQ) susceptibility and gyrA codon 84 and grlA codon 80 mutation status. The HRMA approach was able to discriminate between FQ-sensitive and FQ-resistant strains and confirmed previous reports that the main mutation site associated with FQ resistance in S. pseudintermedius is located at position 251 (Ser84Leu) of gyrA. Routine, HRMA-based FQ susceptibility profiles may be a valuable tool to guide therapy. The FQ resistance-predictive power of the assay should be tested in a significantly larger number of isolates.

  5. The Melting Point of Palladium Using Miniature Fixed Points of Different Ceramic Materials: Part II—Analysis of Melting Curves and Long-Term Investigation

    Science.gov (United States)

    Edler, F.; Huang, K.

    2016-12-01

    Fifteen miniature fixed-point cells made of three different ceramic crucible materials (Al2O3, ZrO2, and Al2O3(86 %)+ZrO2(14 %)) were filled with pure palladium and used to calibrate type B thermocouples (Pt30 %Rh/Pt6 %Rh). A critical point by using miniature fixed points with small amounts of fixed-point material is the analysis of the melting curves, which are characterized by significant slopes during the melting process compared to flat melting plateaus obtainable using conventional fixed-point cells. The method of the extrapolated starting point temperature using straight line approximation of the melting plateau was applied to analyze the melting curves. This method allowed an unambiguous determination of an electromotive force (emf) assignable as melting temperature. The strict consideration of two constraints resulted in a unique, repeatable and objective method to determine the emf at the melting temperature within an uncertainty of about 0.1 μ V. The lifetime and long-term stability of the miniature fixed points was investigated by performing more than 100 melt/freeze cycles for each crucible of the different ceramic materials. No failure of the crucibles occurred indicating an excellent mechanical stability of the investigated miniature cells. The consequent limitation of heating rates to values below {± }3.5 K min^{-1} above 1100° C and the carefully and completely filled crucibles (the liquid palladium occupies the whole volume of the crucible) are the reasons for successfully preventing the crucibles from breaking. The thermal stability of the melting temperature of palladium was excellent when using the crucibles made of Al2O3(86 %)+ZrO2(14 %) and ZrO2. Emf drifts over the total duration of the long-term investigation were below a temperature equivalent of about 0.1 K-0.2 K.

  6. Competitive PCR-High Resolution Melting Analysis (C-PCR-HRMA) for large genomic rearrangements (LGRs) detection: A new approach to assess quantitative status of BRCA1 gene in a reference laboratory.

    Science.gov (United States)

    Minucci, Angelo; De Paolis, Elisa; Concolino, Paola; De Bonis, Maria; Rizza, Roberta; Canu, Giulia; Scaglione, Giovanni Luca; Mignone, Flavio; Scambia, Giovanni; Zuppi, Cecilia; Capoluongo, Ettore

    2017-07-01

    Evaluation of copy number variation (CNV) in BRCA1/2 genes, due to large genomic rearrangements (LGRs), is a mandatory analysis in hereditary breast and ovarian cancers families, if no pathogenic variants are found by sequencing. LGRs cannot be detected by conventional methods and several alternative methods have been developed. Since these approaches are expensive and time consuming, identification of alternative screening methods for LGRs detection is needed in order to reduce and optimize the diagnostic procedure. The aim of this study was to investigate a Competitive PCR-High Resolution Melting Analysis (C-PCR-HRMA) as molecular tool to detect recurrent BRCA1 LGRs. C-PCR-HRMA was performed on exons 3, 14, 18, 19, 20 and 21 of the BRCA1 gene; exons 4, 6 and 7 of the ALB gene were used as reference fragments. This study showed that it is possible to identify recurrent BRCA1 LGRs, by melting peak height ratio between target (BRCA1) and reference (ALB) fragments. Furthermore, we underline that a peculiar amplicon-melting profile is associated to a specific BRCA1 LGR. All C-PCR-HRMA results were confirmed by Multiplex ligation-dependent probe amplification. C-PCR-HRMA has proved to be an innovative, efficient and fast method for BRCA1 LGRs detection. Given the sensitivity, specificity and ease of use, c-PCR-HRMA can be considered an attractive and powerful alternative to other methods for BRCA1 CNVs screening, improving molecular strategies for BRCA testing in the context of Massive Parallel Sequencing. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. High Resolution Elevation Contours

    Data.gov (United States)

    Minnesota Department of Natural Resources — This dataset contains contours generated from high resolution data sources such as LiDAR. Generally speaking this data is 2 foot or less contour interval.

  8. Multiplex fluorescence melting curve analysis for mutation detection with dual-labeled, self-quenched probes.

    Directory of Open Access Journals (Sweden)

    Qiuying Huang

    2011-04-01

    Full Text Available Probe-based fluorescence melting curve analysis (FMCA is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.

  9. A novel real time PCR assay using melt curve analysis for ivory identification.

    Science.gov (United States)

    Kitpipit, Thitika; Penchart, Kitichaya; Ouithavon, Kanita; Satasook, Chutamas; Linacre, Adrian; Thanakiatkrai, Phuvadol

    2016-10-01

    Demand for ivory and expansion of human settlements have resulted in a rapid decline in the number of elephants. Enforcement of local and international laws and regulations requires identification of the species from which any ivory, or ivory products, originated. Further geographical assignment of the dead elephant from which the ivory was taken can assist in forensic investigations. In this study, a real-time PCR assay using melt curve analysis was developed and fully validated for forensic use. The presence or absence of three Elephantidae-specific and elephant species-specific melting peaks was used to identify the elephant species. Using 141 blood and ivory samples from the three extant elephant species, the assay demonstrated very high reproducibility and accuracy. The limit of detection was as low as 0.031ng of input DNA for conventional amplification and 0.002ng for nested amplification. Both DNA concentrations are typically encountered in forensic casework, especially for degraded samples. No cross-reactivity was observed for non-target species. Evaluation of direct amplification and nested amplification demonstrated the assay's flexibility and capability of analyzing low-template DNA samples and aged samples. Additionally, blind trial testing showed the assay's suitability application in real casework. In conclusion, wildlife forensic laboratories could use this novel, quick, and low-cost assay to help combat the continuing poaching crises leading to the collapse of elephant numbers in the wild. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  10. [Restriction endonuclease digest - melting curve analysis: a new SNP genotyping and its application in traditional Chinese medicine authentication].

    Science.gov (United States)

    Jiang, Chao; Huang, Lu-Qi; Yuan, Yuan; Chen, Min; Hou, Jing-Yi; Wu, Zhi-Gang; Lin, Shu-Fang

    2014-04-01

    Single nucleotide polymorphisms (SNP) is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp (cggcgggagggcgg) to 5' end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes macrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.

  11. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM analysis [v1; ref status: indexed, http://f1000r.es/2hj

    Directory of Open Access Journals (Sweden)

    Erin K. Hanson

    2013-12-01

    Full Text Available Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE or quantitative RT-PCR (qRT-PCR platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions, IL1F7 (skin, ALAS2 (blood, MMP10 (menstrual blood, HTN3 (saliva and TGM4 (semen.  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green. Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively

  12. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM analysis [v2; ref status: indexed, http://f1000r.es/314

    Directory of Open Access Journals (Sweden)

    Erin K. Hanson

    2014-02-01

    Full Text Available Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE or quantitative RT-PCR (qRT-PCR platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions, IL1F7 (skin, ALAS2 (blood, MMP10 (menstrual blood, HTN3 (saliva and TGM4 (semen.  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green. Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively

  13. A mitochondrial species identification assay for Australian blacktip sharks (Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides) using real-time PCR and high-resolution melt analysis.

    Science.gov (United States)

    Morgan, Jess A T; Welch, David J; Harry, Alistair V; Street, Raewyn; Broderick, Damien; Ovenden, Jennifer R

    2011-09-01

    Tropical Australian shark fisheries target two morphologically indistinguishable blacktip sharks, the Australian blacktip (Carcharhinus tilstoni) and the common blacktip (C. limbatus). Their relative contributions to northern and eastern Australian coastal fisheries are unclear because of species identification difficulties. The two species differ in their number of precaudal vertebrae, which is difficult and time consuming to obtain in the field. But, the two species can be distinguished genetically with diagnostic mutations in their mitochondrial DNA ND4 gene. A third closely related sister species, the graceful shark C. amblyrhynchoides, can also be distinguished by species-specific mutations in this gene. DNA sequencing is an effective diagnostic tool, but is relatively expensive and time consuming. In contrast, real-time high-resolution melt (HRM) PCR assays are rapid and relatively inexpensive. These assays amplify regions of DNA with species-specific genetic mutations that result in PCR products with unique melt profiles. A real-time HRM PCR species-diagnostic assay (RT-HRM-PCR) has been developed based on the mtDNA ND4 gene for rapid typing of C. tilstoni, C. limbatus and C. amblyrhynchoides. The assay was developed using ND4 sequences from 66 C. tilstoni, 33. C. limbatus and five C. amblyrhynchoides collected from Indonesia and Australian states and territories; Western Australia, the Northern Territory, Queensland and New South Wales. The assay was shown to be 100% accurate on 160 unknown blacktip shark tissue samples by full mtDNA ND4 sequencing. © 2011 Blackwell Publishing Ltd.

  14. High resolution melting analysis (HRM) as a new tool for the identification of species belonging to the Lactobacillus casei group and comparison with species-specific PCRs and multiplex PCR.

    Science.gov (United States)

    Iacumin, Lucilla; Ginaldi, Federica; Manzano, Marisa; Anastasi, Veronica; Reale, Anna; Zotta, Teresa; Rossi, Franca; Coppola, Raffaele; Comi, Giuseppe

    2015-04-01

    The correct identification and characterisation of bacteria is essential for several reasons: the classification of lactic acid bacteria (LAB) has changed significantly over the years, and it is important to distinguish and define them correctly, according to the current nomenclature, avoiding problems in the interpretation of literature, as well as mislabelling when probiotic are used in food products. In this study, species-specific PCR and HRM (high-resolution melting) analysis were developed to identify strains belonging to the Lactobacillus casei group and to classify them into L. casei, Lactobacillus paracasei and Lactobacillus rhamnosus. HRM analysis confirmed to be a potent, simple, fast and economic tool for microbial identification. In particular, 201 strains, collected from International collections and attributed to the L. casei group, were examined using these techniques and the results were compared with consolidated molecular methods, already published. Seven of the tested strains don't belong to the L. casei group. Among the remaining 194 strains, 6 showed inconsistent results, leaving identification undetermined. All the applied techniques were congruent for the identification of the vast majority of the tested strains (188). Notably, for 46 of the strains, the identification differed from the previous attribution. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Screening of WT1 mutations in exon 8 and 9 in children with steroid resistant nephrotic syndrome from a single centre and establishment of a rapid screening assay using high-resolution melting analysis in a clinical setting.

    Science.gov (United States)

    Siji, Annes; Pardeshi, Varsha Chhotusing; Ravindran, Shilpa; Vasudevan, Ambily; Vasudevan, Anil

    2017-01-10

    Mutations in Wilm's tumor 1 (WT1) gene is one of the commonly reported genetic mutations in children with steroid resistant nephrotic syndrome (SRNS). We report the results of direct sequencing of exons 8 and 9 of WT1 gene in 100 children with SRNS from a single centre. We standardized and validated High Resolution Melt (HRM) as a rapid and cost effective screening step to identify individuals with normal sequence and distinguish it from those with a potential mutation. Since only mutation positive samples identified by HRM will be further processed for sequencing it will help in reducing the sequencing burden and speed up the screening process. One hundred SRNS children were screened for WT1 mutations in Exon 8 and 9 using Sanger sequencing. HRM assay was standardized and validated by performing analysis for exon 8 and 9 on 3 healthy control and 5 abnormal variants created by site directed mutagenesis and verified by sequencing. To further test the clinical applicability of the assay, we screened additional 91 samples for HRM testing and performed a blinded assessment. WT1 mutations were not observed in the cohort of children with SRNS. The results of HRM analysis were concordant with the sequencing results. The WT1 gene mutations were not observed in the SRNS cohort indicating it has a low prevalence. We propose applying this simple, rapid and cost effective assay using HRM technique as the first step for screening the WT1 gene hot spot region in a clinical setting.

  16. Comparison of high-resolution melting analysis with direct sequencing for the detection of recurrent mutations in DNA methyltransferase 3A and isocitrate dehydrogenase 1 and 2 genes in acute myeloid leukemia patients.

    Science.gov (United States)

    Gorniak, Patryk; Ejduk, Anna; Borg, Katarzyna; Makuch-Lasica, Hanna; Nowak, Grazyna; Lech-Maranda, Ewa; Prochorec-Sobieszek, Monika; Warzocha, Krzysztof; Juszczynski, Przemyslaw

    2016-02-01

    Acute myeloid leukemia (AML) cells harbor frequent mutations in genes responsible for epigenetic modifications. Increasing evidence of clinical role of DNMT3A and IDH1/2 mutations highlights the need for a robust and inexpensive test to identify these mutations in routine diagnostic work-up. Herein, we compared routinely used direct sequencing method with high-resolution melting (HRM) assay for screening DNMT3A and IDH1/2 mutations in patients with AML. We show very high concordance between HRM and Sanger sequencing (100% samples for IDH2-R140 and DNMT3-R882 mutations, 99% samples for IDH1-R132 and IDH2-R172 mutations). HRM method reported no false-negative results, suggesting that it can be used for mutations screening. Moreover, HRM displayed much higher sensitivity in comparison with DNA sequencing in all assessed loci. With Sanger sequencing, robust calls were observed when the sample contained 50% of mutant DNA in the background of wild-type DNA. In marked contrast, the detection limit of HRM improved down to 10% of mutated DNA. Given the ubiquitous presence of wild-type DNA background in bone marrow aspirates and clonal variations regarding mutant allele burden, these results favor HRM as a sensitive, specific, labor-, and cost-effective tool for screening and detection of mutations in IDH1/2 and DNMT3A genes in patients with AML. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. MECP2 mutations in Czech patients with Rett syndrome and Rett-like phenotypes: novel mutations, genotype-phenotype correlations and validation of high-resolution melting analysis for mutation scanning.

    Science.gov (United States)

    Zahorakova, Daniela; Lelkova, Petra; Gregor, Vladimir; Magner, Martin; Zeman, Jiri; Martasek, Pavel

    2016-07-01

    Rett syndrome (RTT) is an X-linked neurodevelopmental disorder characterized by developmental regression with loss of motor, communication and social skills, onset of stereotypic hand movements and often seizures. RTT is primarily caused by de novo mutations in the methyl-CpG-binding protein 2 gene (MECP2). We established a high-resolution melting (HRM) technique for mutation scanning of the MECP2 gene and performed analyses in Czech patients with RTT, autism spectrum conditions and intellectual disability with Rett-like features. In the cases with confirmed MECP2 mutations, we determined X-chromosome inactivation (XCI), examined the relationships between genotype and clinical severity and evaluated the modifying influence of XCI. Our results demonstrate that HRM analysis is a reliable method for the detection of point mutations, small deletions and duplications in the MECP2 gene. We identified 29 pathogenic mutations in 75 girls, including four novel mutations: c.155_1189del1035;909_932inv;insC, c.573delC, c.857_858dupAA and c.1163_1200del38. Skewed XCI (ratio >75%) was found in 19.3% of the girls, but no gross divergence in clinical severity was observed. Our findings confirm a high mutation frequency in classic RTT (92%) and a correlation between the MECP2 mutation type and clinical severity. We also demonstrate limitations of XCI in explaining all of the phenotypic differences in RTT.

  18. High-throughput gender identification of penguin species using melting curve analysis.

    Science.gov (United States)

    Tseng, Chao-Neng; Chang, Yung-Ting; Chiu, Hui-Tzu; Chou, Yii-Cheng; Huang, Hurng-Wern; Cheng, Chien-Chung; Liao, Ming-Hui; Chang, Hsueh-Wei

    2014-04-03

    Most species of penguins are sexual monomorphic and therefore it is difficult to visually identify their genders for monitoring population stability in terms of sex ratio analysis. In this study, we evaluated the suitability using melting curve analysis (MCA) for high-throughput gender identification of penguins. Preliminary test indicated that the Griffiths's P2/P8 primers were not suitable for MCA analysis. Based on sequence alignment of Chromo-Helicase-DNA binding protein (CHD)-W and CHD-Z genes from four species of penguins (Pygoscelis papua, Aptenodytes patagonicus, Spheniscus magellanicus, and Eudyptes chrysocome), we redesigned forward primers for the CHD-W/CHD-Z-common region (PGU-ZW2) and the CHD-W-specific region (PGU-W2) to be used in combination with the reverse Griffiths's P2 primer. When tested with P. papua samples, PCR using P2/PGU-ZW2 and P2/PGU-W2 primer sets generated two amplicons of 148- and 356-bp, respectively, which were easily resolved in 1.5% agarose gels. MCA analysis indicated the melting temperature (Tm) values for P2/PGU-ZW2 and P2/PGU-W2 amplicons of P. papua samples were 79.75°C-80.5°C and 81.0°C-81.5°C, respectively. Females displayed both ZW-common and W-specific Tm peaks, whereas male was positive only for ZW-common peak. Taken together, our redesigned primers coupled with MCA analysis allows precise high throughput gender identification for P. papua, and potentially for other penguin species such as A. patagonicus, S. magellanicus, and E. chrysocome as well.

  19. High-Resolution Melting Curve Analysis for High-Throughput Genotyping of NOD2/CARD15 Mutations and Distribution of These Mutations in Slovenian Inflammatory Bowel Diseases Patients

    Directory of Open Access Journals (Sweden)

    Mitja Mitrovič

    2011-01-01

    in CD patients, 25/197 (12.69% in UC patients and in 26/256 (10.15% in healthy controls. We have successfully implemented NOD2/CARD15 HRMA assays, which may contribute to the development of genetic profiles for risk prediction of developing CD and for differential diagnosis of CD vs. UC.

  20. High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires

    DEFF Research Database (Denmark)

    Tulsiani, Suhella; Craig, S B; Graham, G C

    2010-01-01

    could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild...

  1. Ultra high resolution tomography

    Energy Technology Data Exchange (ETDEWEB)

    Haddad, W.S.

    1994-11-15

    Recent work and results on ultra high resolution three dimensional imaging with soft x-rays will be presented. This work is aimed at determining microscopic three dimensional structure of biological and material specimens. Three dimensional reconstructed images of a microscopic test object will be presented; the reconstruction has a resolution on the order of 1000 A in all three dimensions. Preliminary work with biological samples will also be shown, and the experimental and numerical methods used will be discussed.

  2. Validation of High Resolution Melting Analysis (HRM) of the Amplified ITS2 Region for the Detection and Identification of Yeasts from Clinical Samples: Comparison with Culture and MALDI-TOF Based Identification.

    Science.gov (United States)

    Duyvejonck, Hans; Cools, Piet; Decruyenaere, Johan; Roelens, Kristien; Noens, Lucien; Vermeulen, Stefan; Claeys, Geert; Decat, Ellen; Van Mechelen, Els; Vaneechoutte, Mario

    2015-01-01

    Candida species are known as opportunistic pathogens, and a possible cause of invasive infections. Because of their species-specific antimycotic resistance patterns, reliable techniques for their detection, quantification and identification are needed. We validated a DNA amplification method for direct detection of Candida spp. from clinical samples, namely the ITS2-High Resolution Melting Analysis (direct method), by comparing it with a culture and MALDI-TOF Mass Spectrometry based method (indirect method) to establish the presence of Candida species in three different types of clinical samples. A total of 347 clinical samples, i.e. throat swabs, rectal swabs and vaginal swabs, were collected from the gynaecology/obstetrics, intensive care and haematology wards at the Ghent University Hospital, Belgium. For the direct method, ITS2-HRM was preceded by NucliSENS easyMAG DNA extraction, directly on the clinical samples. For the indirect method, clinical samples were cultured on Candida ID and individual colonies were identified by MALDI-TOF. For 83.9% of the samples there was complete concordance between both techniques, i.e. the same Candida species were detected in 31.1% of the samples or no Candida species were detected in 52.8% of the samples. In 16.1% of the clinical samples, discrepant results were obtained, of which only 6.01% were considered as major discrepancies. Discrepancies occurred mostly when overall numbers of Candida cells in the samples were low and/or when multiple species were present in the sample. Most of the discrepancies could be decided in the advantage of the direct method. This is due to samples in which no yeast could be cultured whereas low amounts could be detected by the direct method and to samples in which high quantities of Candida robusta according to ITS2-HRM were missed by culture on Candida ID agar. It remains to be decided whether the diagnostic advantages of the direct method compensate for its disadvantages.

  3. Development of a novel real-time PCR assay with high-resolution melt analysis to detect and differentiate OXA-48-Like β-lactamases in carbapenem-resistant Enterobacteriaceae.

    Science.gov (United States)

    Hemarajata, Peera; Yang, Shangxin; Hindler, Janet A; Humphries, Romney M

    2015-09-01

    The rapid global spread of carbapenem-resistant Enterobacteriaceae (CRE) poses an urgent threat to public health. More than 250 class D β-lactamases (OXAs) have been described in recent years, with variations in hydrolytic activity for β-lactams. The plasmid-borne OXA-48 β-lactamase and its variants are identified only sporadically in the United States but are common in Europe. Recognition of these OXA-48-like carbapenemases is vital in order to control their dissemination. We developed a real-time PCR assay based on high-resolution melt analysis, using bla OXA-48-like-specific primers coupled with an unlabeled 3'-phosphorylated oligonucleotide probe (LunaProbe) homologous to OXA-48-like carbapenemase genes. The assay was validated using genomic DNA from 48 clinical isolates carrying a variety of carbapenemase genes, including bla KPC, bla SME, bla IMP, bla NDM-1, bla VIM, bla OXA-48, bla OXA-162, bla OXA-181, bla OXA-204, bla OXA-244, bla OXA-245, and bla OXA-232. Our assay identified the presence of bla OXA-48-like β-lactamase genes and clearly distinguished between bla OXA-48 and its variants in control strains, including between bla OXA-181 and bla OXA-232, which differ by only a single base pair in the assay target region. This approach has potential for use in epidemiological investigations and continuous surveillance to help control the spread of CRE strains producing OXA-48-like enzymes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    Science.gov (United States)

    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

    2015-06-01

    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. Prospective cohort study. 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%). 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. 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.

  5. Melting curve analysis of a groEL PCR fragment for the rapid genotyping of strains belonging to the Lactobacillus casei group of species.

    Science.gov (United States)

    Koirala, Ranjan; Taverniti, Valentina; Balzaretti, Silvia; Ricci, Giovanni; Fortina, Maria Grazia; Guglielmetti, Simone

    2015-04-01

    Lactobacillus casei group (Lcs) consists of three phylogenetically closely related species (L. casei, L. paracasei, and L. rhamnosus), which are widely used in the dairy and probiotic industrial sectors. Strategies to easily and rapidly characterize Lcs are therefore of interest. To this aim, we developed a method according to a technique known as high resolution melting analysis (HRMa), which was applied to a 150 bp groEL gene fragment. The analysis was performed on 53 Lcs strains and 29 strains representatives of species that are commonly present in dairy and probiotic products and can be most probably co-isolated with Lcs strains. DNA amplification was obtained only from Lcs strains, demonstrating the specificity of the groEL primers designed in this study. The HRMa clustered Lcs strains in three groups that exactly corresponded to the species of the L. casei group. A following HRMa separated the 39 L. paracasei strains in two well distinct intraspecific groups, indicating the possible existence of at least two distinct genotypes inside the species. Nonetheless, the phenotypic characterization demonstrated that the genotypes do not correspond to the two L. paracasei subspecies, namely paracasei and tolerans. In conclusion, the melting curve analysis developed in this study is demonstrably a simple, labor-saving, and rapid strategy obtain the genotyping of a bacterial isolate and simultaneously potentially confirm its affiliation to the L. casei group of species. The application of this method to a larger collection of strains may validate the possibility to use the proposed HRMa protocol for the taxonomic discrimination of L. casei group of species. In general, this study suggests that HRMa can be a suitable technique for the genetic typization of Lactobacillus strains. Copyright © 2015 Elsevier GmbH. All rights reserved.

  6. Silver ion-mediated killing of a food pathogen: Melting curve analysis data of silver resistance genes and growth curve data

    Directory of Open Access Journals (Sweden)

    Kuppan Gokulan

    2017-04-01

    Full Text Available Limited antibacterial activity of silver ions leached from silver-impregnated food contact materials could be due to: 1 the presence of silver resistance genes in tested bacteria; or 2 lack of susceptibility to silver ion-mediated killing in the bacterial strain (K. Williams, L. Valencia, K. Gokulan, R. Trbojevich, S. Khare, 2016 [1]. This study contains data to address the specificity of silver resistance genes in Salmonella Typhimurium during the real time PCR using melting curve analysis and an assessment of the minimum inhibitory concentration of silver ions for Salmonella.

  7. Validation of High Resolution Melting Analysis (HRM of the Amplified ITS2 Region for the Detection and Identification of Yeasts from Clinical Samples: Comparison with Culture and MALDI-TOF Based Identification.

    Directory of Open Access Journals (Sweden)

    Hans Duyvejonck

    Full Text Available Candida species are known as opportunistic pathogens, and a possible cause of invasive infections. Because of their species-specific antimycotic resistance patterns, reliable techniques for their detection, quantification and identification are needed. We validated a DNA amplification method for direct detection of Candida spp. from clinical samples, namely the ITS2-High Resolution Melting Analysis (direct method, by comparing it with a culture and MALDI-TOF Mass Spectrometry based method (indirect method to establish the presence of Candida species in three different types of clinical samples.A total of 347 clinical samples, i.e. throat swabs, rectal swabs and vaginal swabs, were collected from the gynaecology/obstetrics, intensive care and haematology wards at the Ghent University Hospital, Belgium. For the direct method, ITS2-HRM was preceded by NucliSENS easyMAG DNA extraction, directly on the clinical samples. For the indirect method, clinical samples were cultured on Candida ID and individual colonies were identified by MALDI-TOF.For 83.9% of the samples there was complete concordance between both techniques, i.e. the same Candida species were detected in 31.1% of the samples or no Candida species were detected in 52.8% of the samples. In 16.1% of the clinical samples, discrepant results were obtained, of which only 6.01% were considered as major discrepancies. Discrepancies occurred mostly when overall numbers of Candida cells in the samples were low and/or when multiple species were present in the sample.Most of the discrepancies could be decided in the advantage of the direct method. This is due to samples in which no yeast could be cultured whereas low amounts could be detected by the direct method and to samples in which high quantities of Candida robusta according to ITS2-HRM were missed by culture on Candida ID agar. It remains to be decided whether the diagnostic advantages of the direct method compensate for its disadvantages.

  8. Barcoding melting curve analysis for rapid, sensitive, and discriminating authentication of saffron (Crocus sativus L.) from its adulterants.

    Science.gov (United States)

    Jiang, Chao; Cao, Liang; Yuan, Yuan; Chen, Min; Jin, Yan; Huang, Luqi

    2014-01-01

    Saffron (Crocus sativus L.) is one of the most important and expensive medicinal spice products in the world. Because of its high market value and premium price, saffron is often adulterated through the incorporation of other materials, such as Carthamus tinctorius L. and Calendula officinalis L. flowers, Hemerocallis L. petals, Daucus carota L. fleshy root, Curcuma longa L. rhizomes, Zea may L., and Nelumbo nucifera Gaertn. stigmas. To develop a straightforward, nonsequencing method for rapid, sensitive, and discriminating detection of these adulterants in traded saffron, we report here the application of a barcoding melting curve analysis method (Bar-MCA) that uses the universal chloroplast plant DNA barcoding region trnH-psbA to identify adulterants. When amplified at DNA concentrations and annealing temperatures optimized for the curve analysis, peaks were formed at specific locations for saffron (81.92°C) and the adulterants: D. carota (81.60°C), C. tinctorius (80.10°C), C. officinalis (79.92°C), Dendranthema morifolium (Ramat.) Tzvel. (79.62°C), N. nucifera (80.58°C), Hemerocallis fulva (L.) L. (84.78°C), and Z. mays (84.33°C). The constructed melting curves for saffron and its adulterants have significantly different peak locations or shapes. In conclusion, Bar-MCA could be a faster and more cost-effective method to authenticate saffron and detect its adulterants.

  9. Real-time RT-PCR and SYBR Green I melting curve analysis for the identification of Plum pox virus strains C, EA, and W: effect of amplicon size, melt rate, and dye translocation.

    Science.gov (United States)

    Varga, Aniko; James, Delano

    2006-03-01

    Real-time RT-PCR and SYBR green I melt curve analysis of a 74 bp amplicon enabled identification of Plum pox virus strains C, EA, and W, with distinct T(m)'s associated with each strain. This test is a useful supplement to a real-time RT-PCR test described earlier that was used to distinguish PPV strains D and M. A longer fragment of 155 bp was not effective for strain identification. A simplified one-tube protocol, with dithiothreitol eliminated from the reaction, showed similar sensitivity when compared to a two-tube protocol. For melt curve analysis, a slower melt rate of 0.1 degrees C/s, compared to 0.4 degrees C/s, was effective for detecting weak amplicons, and improved resolution of the T(m) of amplicons amplified simultaneously. SYBR green I was useful for duplex melt curve analysis. In repeated melt run treatments (total of 14) of a single sample containing co-amplified targets, complete translocation of SYBR green I was observed, going from a 74 bp fragment to a 114 bp fragment. The duration of the melt run may be a critical factor affecting SYBR green I binding and translocation, and its manipulation may facilitate improved resolution and simultaneous detection of multiple targets. This phenomenon may explain inconsistent SYBR green I fluorescence patterns associated with melt curve analysis of some amplicon complexes.

  10. A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β-Thalassemia Mutations

    Science.gov (United States)

    Xiong, Fu; Huang, Qiuying; Chen, Xiaoyun; Zhou, Yuqiu; Zhang, Xinhua; Cai, Ren; Chen, Yajun; Xie, Jiansheng; Feng, Shanwei; Wei, Xiaofeng; Xiao, Qizhi; Zhang, Tianlang; Luo, Shiqiang; Yang, Xuehuang; Hao, Ying; Qu, Yanxia; Li, Qingge; Xu, Xiangmin

    2011-01-01

    The increasing number of disease-causing mutations demands a simple, direct, and cost-effective diagnostic genotyping technique capable of detecting multiple mutations. This study validated the efficacy of a novel melting curve analysis–based genotyping assay (MeltPro HBB assay) for 24 β-thalassemia mutations in the Chinese population. The diagnostic potential of this assay was evaluated in 1022 pretyped genomic DNA samples, including 909 clinical cases of β-thalassemia minor or major, using a double-blind analysis in a multicenter validation study. Reproducibility of the assay was 100%, and the limit of detection was 10 pg per reaction. All 24 β-thalassemia mutations were accurately genotyped, and β-thalassemia genotypes were correctly determined in all 1022 samples, yielding overall sensitivity and specificity of 100%. The concordance rate was 99.4% between this assay and the reference method. It was concluded that the MeltPro HBB assay is useful for reliable genotyping of multiple β-thalassemia mutations in clinical settings and may have potential as a versatile method for rapid genotyping of known mutations because of its high throughput, accuracy, ease of use, and low cost. PMID:21704277

  11. Simultaneous detection of Fusarium culmorum and F. graminearum in plant material by duplex PCR with melting curve analysis

    Directory of Open Access Journals (Sweden)

    Karlovsky Petr

    2006-01-01

    Full Text Available Abstract Background Fusarium head blight (FHB is a disease of cereal crops, which has a severe impact on wheat and barley production worldwide. Apart from reducing the yield and impairing grain quality, FHB leads to contamination of grain with toxic secondary metabolites (mycotoxins, which pose a health risk to humans and livestock. The Fusarium species primarily involved in FHB are F. graminearum and F. culmorum. A key prerequisite for a reduction in the incidence of FHB is an understanding of its epidemiology. Results We describe a duplex-PCR-based method for the simultaneous detection of F. culmorum and F. graminearum in plant material. Species-specific PCR products are identified by melting curve analysis performed in a real-time thermocycler in the presence of the fluorescent dye SYBR Green I. In contrast to multiplex real-time PCR assays, the method does not use doubly labeled hybridization probes. Conclusion PCR with product differentiation by melting curve analysis offers a cost-effective means of qualitative analysis for the presence of F. culmorum and F. graminearum in plant material. This method is particularly suitable for epidemiological studies involving a large number of samples.

  12. Noise Pollution Status in a Metal Melting Industry and the Map of Its Isosonic Curve

    Directory of Open Access Journals (Sweden)

    Forouharmajd

    2015-10-01

    Full Text Available Background Steel industry is one of the most important industries of each country and noise pollution is one of the very annoying factors in this industry that causes various diseases; above all, the damage to the auditory system of the work force is high. Therefore, the assessment of noise pollution and the identification of the sources and causes of its creation are inevitable for controlling and teaching the prevention programs and hearing protection. Objectives This study aimed to draw the noise map of isosonic curves and assess the noise pollution using Surfer modeling software. Materials and Methods In the present study, first, preliminary maps and stationing were drawn, and then, the measurement of the sound pressure level and the octave band analysis were performed by a noise measurement device called Casella-CEL 445; then, after recording the data in software of Surfer version 8, they were used for drawing the isosonic curves. Results The results of the peripheral measurements showed that the maximum sound pressure of level 109 dBA was related to the electric arc furnace. A map of the graphic isosonic curves showed most of the noise pollution in the following areas: furnace, casting, tundishes, and the cooling beds. Conclusions According to the factory noise map, except for the control rooms with 81 dBA noise, other cases of measuring stations were higher than the standard exposure of 85 dBA noise. As a result, the implementation of control strategies, such as inspection and performance of control systems and insulation of the control room to prevent unnecessary exposure noise, are necessary.

  13. Viscosity jump in the lower mantle inferred from melting curves of ferropericlase.

    Science.gov (United States)

    Deng, Jie; Lee, Kanani K M

    2017-12-08

    Convection provides the mechanism behind plate tectonics, which allows oceanic lithosphere to be subducted into the mantle as "slabs" and new rock to be generated by volcanism. Stagnation of subducting slabs and deflection of rising plumes in Earth's shallow lower mantle have been suggested to result from a viscosity increase at those depths. However, the mechanism for this increase remains elusive. Here, we examine the melting behavior in the MgO-FeO binary system at high pressures using the laser-heated diamond-anvil cell and show that the liquidus and solidus of (Mg x Fe 1-x )O ferropericlase (x = ~0.52-0.98), exhibit a local maximum at ~40 GPa, likely caused by the spin transition of iron. We calculate the relative viscosity profiles of ferropericlase using homologous temperature scaling and find that viscosity increases 10-100 times from ~750 km to ~1000-1250 km, with a smaller decrease at deeper depths, pointing to a single mechanism for slab stagnation and plume deflection.

  14. Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR.

    Science.gov (United States)

    Guion, Chase E; Ochoa, Theresa J; Walker, Christopher M; Barletta, Francesca; Cleary, Thomas G

    2008-05-01

    Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx(1) and stx(2) for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.

  15. Detection of Diarrheagenic Escherichia coli by Use of Melting-Curve Analysis and Real-Time Multiplex PCR ▿

    Science.gov (United States)

    Guion, Chase E.; Ochoa, Theresa J.; Walker, Christopher M.; Barletta, Francesca; Cleary, Thomas G.

    2008-01-01

    Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx1 and stx2 for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli. PMID:18322059

  16. The effect of changing wind forcing on Antarctic ice shelf melting in high-resolution, global sea ice-ocean simulations with the Accelerated Climate Model for Energy (ACME)

    Science.gov (United States)

    Asay-Davis, Xylar; Price, Stephen; Petersen, Mark; Wolfe, Jonathan

    2017-04-01

    The capability for simulating sub-ice shelf circulation and submarine melting and freezing has recently been added to the U.S. Department of Energy's Accelerated Climate Model for Energy (ACME). With this new capability, we use an eddy permitting ocean model to conduct two sets of simulations in the spirit of Spence et al. (GRL, 41, 2014), who demonstrate increased warm water upwelling along the Antarctic coast in response to poleward shifting and strengthening of Southern Ocean westerly winds. These characteristics, symptomatic of a positive Southern Annular Mode (SAM), are projected to continue into the 21st century under anthropogenic climate change (Fyfe et al., J. Clim., 20, 2007). In our first simulation, we force the climate model using the standard CORE interannual forcing dataset (Large and Yeager; Clim. Dyn., 33, 2009). In our second simulation, we force our climate model using an altered version of CORE interannual forcing, based on the latter half of the full time series, which we take as a proxy for a future climate state biased towards a positive SAM. We compare ocean model states and sub-ice shelf melt rates with observations, exploring sources of model biases as well as the effects of the two forcing scenarios.

  17. A practical approach to obtain the soil freezing characteristic curve and the freezing/melting point of a soil-water system

    NARCIS (Netherlands)

    Aukenthaler, Manuel; Brinkgreve, R.B.J.; Haxaire, A

    2016-01-01

    Knowing that extensive field tests and laboratory tests are time-consuming and expensive, this paper describes a practical approach to obtain crucial properties of frozen soil such as the soil freezing characteristic curve (SFCC), the freezing/melting point of a soil-water system and its hydraulic

  18. Genotyping of a tri-allelic polymorphism by a novel melting curve assay in MTHFD1L: an association study of nonsyndromic Cleft in Ireland

    LENUS (Irish Health Repository)

    Minguzzi, Stefano

    2012-04-20

    AbstractBackgroundPolymorphisms within the MTHFD1L gene were previously associated with risk of neural tube defects in Ireland. We sought to test the most significant MTHFD1L polymorphisms for an association with risk of cleft in an Irish cohort. This required the development of a new melting curve assay to genotype the technically challenging MTHFD1L triallelic deletion\\/insertion polymorphism (rs3832406).MethodsMelting curve analysis was used to genotype the MTHFD1L triallelic deletion\\/insertion polymorphism (rs3832406) and a Single Nucleotide Polymorphism rs17080476 in an Irish cohort consisting of 981 Irish case-parent trios and 1,008 controls. Tests for association with nonsyndromic cleft lip with or without cleft palate and cleft palate included case\\/control analysis, mother\\/control analysis and Transmission Disequilibrium Tests of case-parent trios.ResultsA successful melting curve genotyping assay was developed for the deletion\\/insertion polymorphism (rs3832406). The TDT analysis initially showed that the rs3832406 polymorphism was associated with isolated cleft lip with or without cleft palate. However, corrected p-values indicated that this association was not significant.ConclusionsMelting Curve Analysis can be employed to successfully genotype challenging polymorphisms such as the MTHFD1L triallelic deletion\\/insertion polymorphism (DIP) reported here (rs3832406) and is a viable alternative to capillary electrophoresis. Corrected p-values indicate no association between MTHFD1L and risk of cleft in an Irish cohort.

  19. Genotyping of a tri-allelic polymorphism by a novel melting curve assay in MTHFD1L: an association study of nonsyndromic Cleft in Ireland

    Directory of Open Access Journals (Sweden)

    Minguzzi Stefano

    2012-04-01

    Full Text Available Abstract Background Polymorphisms within the MTHFD1L gene were previously associated with risk of neural tube defects in Ireland. We sought to test the most significant MTHFD1L polymorphisms for an association with risk of cleft in an Irish cohort. This required the development of a new melting curve assay to genotype the technically challenging MTHFD1L triallelic deletion/insertion polymorphism (rs3832406. Methods Melting curve analysis was used to genotype the MTHFD1L triallelic deletion/insertion polymorphism (rs3832406 and a Single Nucleotide Polymorphism rs17080476 in an Irish cohort consisting of 981 Irish case-parent trios and 1,008 controls. Tests for association with nonsyndromic cleft lip with or without cleft palate and cleft palate included case/control analysis, mother/control analysis and Transmission Disequilibrium Tests of case-parent trios. Results A successful melting curve genotyping assay was developed for the deletion/insertion polymorphism (rs3832406. The TDT analysis initially showed that the rs3832406 polymorphism was associated with isolated cleft lip with or without cleft palate. However, corrected p-values indicated that this association was not significant. Conclusions Melting Curve Analysis can be employed to successfully genotype challenging polymorphisms such as the MTHFD1L triallelic deletion/insertion polymorphism (DIP reported here (rs3832406 and is a viable alternative to capillary electrophoresis. Corrected p-values indicate no association between MTHFD1L and risk of cleft in an Irish cohort.

  20. A single nucleotide polymorphism melt curve assay employing an intercalating dye probe fluorescence resonance energy transfer for forensic analysis.

    Science.gov (United States)

    Halpern, Micah D; Ballantyne, Jack

    2009-08-01

    The characterization and use of DNA sequence polymorphisms are an important aspect of forensic analysis. A number of approaches are being explored for single nucleotide polymorphism (SNP) genotyping, but current detection methods are subject to limitations that adversely impact their utility for forensic analysis. We have developed a novel method for genotyping both single and multiple SNPs that uses an intercalating dye and a probe labeled with a single fluorophore to affect a fluorescence energy transfer. Melting curve analysis is then used to distinguish true alleles from mismatched alleles. We term the new method dye probe fluorescence resonance energy transfer (dpFRET). In the current work, development proceeded at first with synthetic DNA template testing to establish proof of concept for the chemistry involved, followed by the design of polymerase chain reaction (PCR)-based genomic DNA assays to demonstrate potential forensic applications. The loci chosen for testing included both nuclear (MHC DRB) and mitochondrial DNA (cytochrome b) genes. A preliminary assessment of the sensitivity limits of the technology indicated that dpFRET was capable of accurately genotyping DNA from one single diploid cell equivalent. This technology could also potentially impact a wide range of nonforensic disciplines to aid in discovery, screening, and association of DNA sequence polymorphisms.

  1. A novel method of multiple nucleic acid detection: Real-time RT-PCR coupled with probe-melting curve analysis.

    Science.gov (United States)

    Han, Yang; Hou, Shao-Yang; Ji, Shang-Zhi; Cheng, Juan; Zhang, Meng-Yue; He, Li-Juan; Ye, Xiang-Zhong; Li, Yi-Min; Zhang, Yi-Xuan

    2017-11-15

    A novel method, real-time reverse transcription PCR (real-time RT-PCR) coupled with probe-melting curve analysis, has been established to detect two kinds of samples within one fluorescence channel. Besides a conventional TaqMan probe, this method employs another specially designed melting-probe with a 5' terminus modification which meets the same label with the same fluorescent group. By using an asymmetric PCR method, the melting-probe is able to detect an extra sample in the melting stage effectively while it almost has little influence on the amplification detection. Thus, this method allows the availability of united employment of both amplification stage and melting stage for detecting samples in one reaction. The further demonstration by simultaneous detection of human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in one channel as a model system is presented in this essay. The sensitivity of detection by real-time RT-PCR coupled with probe-melting analysis was proved to be equal to that detected by conventional real-time RT-PCR. Because real-time RT-PCR coupled with probe-melting analysis can double the detection throughputs within one fluorescence channel, it is expected to be a good solution for the problem of low-throughput in current real-time PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. High-resolution multiphoton cryomicroscopy.

    Science.gov (United States)

    König, Karsten; Uchugonova, Aisada; Breunig, Hans Georg

    2014-03-15

    An ultracompact high-resolution multiphoton cryomicroscope with a femtosecond near infrared fiber laser has been utilized to study the cellular autofluorescence during freezing and thawing of cells. Cooling resulted in an increase of the intracellular fluorescence intensity followed by morphological modifications at temperatures below -10 °C, depending on the application of the cryoprotectant DMSO and the cooling rate. Furthermore, fluorescence lifetime imaging revealed an increase of the mean lifetime with a decrease in temperature. Non-destructive, label-free optical biopsies of biomaterial in ice can be obtained with sub-20 mW mean powers. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Principles of high resolution NMR in solids

    CERN Document Server

    Mehring, Michael

    1983-01-01

    The field of Nuclear Magnetic Resonance (NMR) has developed at a fascinating pace during the last decade. It always has been an extremely valuable tool to the organic chemist by supplying molecular "finger print" spectra at the atomic level. Unfortunately the high resolution achievable in liquid solutions could not be obtained in solids and physicists and physical chemists had to live with unresolved lines open to a wealth of curve fitting procedures and a vast amount of speculations. High resolution NMR in solids seemed to be a paradoxon. Broad structure­ less lines are usually encountered when dealing with NMR in solids. Only with the recent advent of mUltiple pulse, magic angle, cross-polarization, two-dimen­ sional and multiple-quantum spectroscopy and other techniques during the last decade it became possible to resolve finer details of nuclear spin interactions in solids. I have felt that graduate students, researchers and others beginning to get involved with these techniques needed a book which trea...

  4. Single nucleotide polymorphism genotyping by two colour melting curve analysis using the MGB Eclipse Probe System in challenging sequence environment.

    Science.gov (United States)

    Belousov, Yevgeniy S; Welch, Robert A; Sanders, Silvia; Mills, Alan; Kulchenko, Alena; Dempcy, Robert; Afonina, Irina A; Walburger, David K; Glaser, Cynthia L; Yadavalli, Sunita; Vermeulen, Nicolaas M J; Mahoney, Walt

    2004-03-01

    Probe and primer design for single nucleotide polymorphism (SNP) detection can be very challenging for A-T DNA-rich targets, requiring long sequences with lower specificity and stability, while G-C-rich DNA targets present limited design options to lower GC-content sequences only. We have developed the MGB Eclipse Probe System, which is composed of the following elements: MGB Eclipse probes and primers, specially developed software for the design of probes and primers, a unique set of modified bases and a Microsoft Excel macro for automated genotyping, which ably solves, in large part, this challenge. Fluorogenic MGB Eclipse probes are modified oligonucleotides containing covalently attached duplex-stabilising dihydrocyclopyrroloindole tripeptide (DPI3), the MGB ligand (MGB is a trademark of Epoch Biosciences, Bothell, WA), which has the combined properties of allowing the use of short sequences and providing great mismatch discrimination. The MGB moiety prevents probe degradation during polymerase chain reaction (PCR), allowing the researcher to use real time data; alternatively, hybridisation can be accurately measured by a post-PCR two-colour melt curve analysis. Using MGB Eclipse probes and primers containing modified bases further enhances the analysis of difficult SNP targets. G- or C-rich sequences can be refractory to analysis due to Hoogsteen base pairing. Substitution of normal G with Epoch's modified G prevents Hoogsteen base pairing, allowing both superior PCR and probe-based analysis of GC-rich targets. The use of modified A and T bases allows better stabilisation by significantly increasing the Tm of the oligonucleotides. Modified A creates A-T base pairs that have a stability slightly lower than a G-C base pair, and modified T creates T-A base pairs that have a stability about 30 per cent higher than the unmodified base pair. Together, the modified bases permit the use of short probes, providing good mismatch discrimination and primers that allow PCR

  5. Single nucleotide polymorphism genotyping by two colour melting curve analysis using the MGB Eclipse™ Probe System in challenging sequence environment

    Directory of Open Access Journals (Sweden)

    Belousov Yevgeniy S

    2004-03-01

    Full Text Available Abstract Probe and primer design for single nucleotide polymorphism (SNP detection can be very challenging for A-T DNA-rich targets, requiring long sequences with lower specificity and stability, while G-C-rich DNA targets present limited design options to lower GC-content sequences only. We have developed the MGB Eclipsee™ Probe System, which is composed of the following elements: MGB Eclipse probes and primers, specially developed software for the design of probes and primers, a unique set of modified bases and a Microsoft Excel macro for automated genotyping, which ably solves, in large part, this challenge. Fluorogenic MGB Eclipse probes are modified oligo-nucleotides containing covalently attached duplex-stabilising dihydrocyclopyrroloindole tripeptide (DPI3, the MGB ligand (MGB™ is a trademark of Epoch Biosciences, Bothell, WA, which has the combined properties of allowing the use of short sequences and providing great mismatch discrimination. The MGB moiety prevents probe degradation during polymerase chain reaction (PCR, allowing the researcher to use real time data; alternatively, hybridisation can be accurately measured by a post-PCR two-colour melt curve analysis. Using MGB Eclipse probes and primers containing modified bases further enhances the analysis of difficult SNP targets. G- or C-rich sequences can be refractory to analysis due to Hoogsteen base pairing. Substitution of normal G with Epoch's modified G prevents Hoogsteen base pairing, allowing both superior PCR and probe-based analysis of GC-rich targets. The use of modified A and T bases allows better stabilisation by significantly increasing the Tm of the oligonucleotides. Modified A creates A-T base pairs that have a stability slightly lower than a G-C base pair, and modified T creates T-A base pairs that have a stability about 30 per cent higher than the unmodified base pair. Together, the modified bases permit the use of short probes, providing good mismatch

  6. High resolution X-ray diffraction studies on unirradiated and ...

    Indian Academy of Sciences (India)

    High-resolution X-ray diffraction technique, employing a three-crystal monochromator–collimator combination is used to study the irradiation induced defects in flux grown Sr-hexaferrite crystals irradiated with 50 MeV Li3+ ion beams at room temperature with a fluence value of 1 × 1014 ions/cm2. The diffraction curves of the ...

  7. Enhanced High Resolution RBS System

    Science.gov (United States)

    Pollock, Thomas J.; Hass, James A.; Klody, George M.

    2011-06-01

    Improvements in full spectrum resolution with the second NEC high resolution RBS system are summarized. Results for 50 Å TiN/HfO films on Si yielding energy resolution on the order of 1 keV are also presented. Detector enhancements include improved pulse processing electronics, upgraded shielding for the MCP/RAE detector, and reduced noise generated from pumping. Energy resolution measurements on spectra front edge coupled with calculations using 0.4mStr solid angle show that beam energy spread at 400 KeV from the Pelletron® accelerator is less than 100 eV. To improve user throughput, magnet control has been added to the automatic data collection. Depth profiles derived from experimental data are discussed. For the thin films profiled, depth resolutions were on the Angstrom level with the non-linear energy/channel conversions ranging from 100 to 200 eV.

  8. High resolution tomographic instrument development

    Energy Technology Data Exchange (ETDEWEB)

    1992-01-01

    Our recent work has concentrated on the development of high-resolution PET instrumentation reflecting in part the growing importance of PET in nuclear medicine imaging. We have developed a number of positron imaging instruments and have the distinction that every instrument has been placed in operation and has had an extensive history of application for basic research and clinical study. The present program is a logical continuation of these earlier successes. PCR-I, a single ring positron tomograph was the first demonstration of analog coding using BGO. It employed 4 mm detectors and is currently being used for a wide range of biological studies. These are of immense importance in guiding the direction for future instruments. In particular, PCR-II, a volume sensitive positron tomograph with 3 mm spatial resolution has benefited greatly from the studies using PCR-I. PCR-II is currently in the final stages of assembly and testing and will shortly be placed in operation for imaging phantoms, animals and ultimately humans. Perhaps the most important finding resulting from our previous study is that resolution and sensitivity must be carefully balanced to achieve a practical high resolution system. PCR-II has been designed to have the detection characteristics required to achieve 3 mm resolution in human brain under practical imaging situations. The development of algorithms by the group headed by Dr. Chesler is based on a long history of prior study including his joint work with Drs. Pelc and Reiderer and Stearns. This body of expertise will be applied to the processing of data from PCR-II when it becomes operational.

  9. High resolution tomographic instrument development

    Energy Technology Data Exchange (ETDEWEB)

    1992-08-01

    Our recent work has concentrated on the development of high-resolution PET instrumentation reflecting in part the growing importance of PET in nuclear medicine imaging. We have developed a number of positron imaging instruments and have the distinction that every instrument has been placed in operation and has had an extensive history of application for basic research and clinical study. The present program is a logical continuation of these earlier successes. PCR-I, a single ring positron tomograph was the first demonstration of analog coding using BGO. It employed 4 mm detectors and is currently being used for a wide range of biological studies. These are of immense importance in guiding the direction for future instruments. In particular, PCR-II, a volume sensitive positron tomograph with 3 mm spatial resolution has benefited greatly from the studies using PCR-I. PCR-II is currently in the final stages of assembly and testing and will shortly be placed in operation for imaging phantoms, animals and ultimately humans. Perhaps the most important finding resulting from our previous study is that resolution and sensitivity must be carefully balanced to achieve a practical high resolution system. PCR-II has been designed to have the detection characteristics required to achieve 3 mm resolution in human brain under practical imaging situations. The development of algorithms by the group headed by Dr. Chesler is based on a long history of prior study including his joint work with Drs. Pelc and Reiderer and Stearns. This body of expertise will be applied to the processing of data from PCR-II when it becomes operational.

  10. Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

    Directory of Open Access Journals (Sweden)

    Zhiyuan Wu

    Full Text Available BACKGROUND: JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. METHODOLOGY/PRINCIPAL FINDINGS: Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. CONCLUSIONS: With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

  11. High-resolution infrared imaging

    Science.gov (United States)

    Falco, Charles M.

    2010-08-01

    The hands and mind of an artist are intimately involved in the creative process of image formation, intrinsically making paintings significantly more complex than photographs to analyze. In spite of this difficulty, several years ago the artist David Hockney and I identified optical evidence within a number of paintings that demonstrated artists began using optical projections as early as c1425 - nearly 175 years before Galileo - as aids for producing portions of their images. In the course of our work, Hockney and I developed insights that I have been applying to a new approach to computerized image analysis. Recently I developed and characterized a portable high resolution infrared for capturing additional information from paintings. Because many pigments are semi-transparent in the IR, in a number of cases IR photographs ("reflectograms") have revealed marks made by the artists that had been hidden under paint ever since they were made. I have used this IR camera to capture photographs ("reflectograms") of hundreds of paintings in over a dozen museums on three continents and, in some cases, these reflectograms have provided new insights into decisions the artists made in creating the final images that we see in the visible.

  12. Detection and discrimination of two Brucella species by multiplex real-time PCR and high-resolution melt analysis curve from human blood and comparison of results using RFLP

    Directory of Open Access Journals (Sweden)

    Vahhab Piranfar

    2015-09-01

    Conclusion: The present study showed that this technique, which scans gene segments and creates an analysis pattern for detection of clinical samples, is useful and more dominant compared with PCR-RFLP. Thus, this method can be used for brucellosis detection, and clinical and epidemiological research.

  13. High Resolution Tactile Sensors for Curved Robotic Fingertips

    DEFF Research Database (Denmark)

    Drimus, Alin; Jankovics, Vince; Gorsic, Matija

    2014-01-01

    ]. For humans, extracting high level information from touch provides a better understanding of the objects manipulated while for insects it is essential for locomotion[3]. While robot designers have been using vision systems to provide the robot with information about its surroundings, this is not always...

  14. Evaluation of Advanced Bionics high resolution mode.

    Science.gov (United States)

    Buechner, Andreas; Frohne-Buechner, Carolin; Gaertner, Lutz; Lesinski-Schiedat, Anke; Battmer, Rolf-Dieter; Lenarz, Thomas

    2006-07-01

    The objective of this paper is to evaluate the advantages of the Advanced Bionic high resolution mode for speech perception, through a retrospective analysis. Forty-five adult subjects were selected who had a minimum experience of three months' standard mode (mean of 10 months) before switching to high resolution mode. Speech perception was tested in standard mode immediately before fitting with high resolution mode, and again after a maximum of six months high resolution mode usage (mean of two months). A significant improvement was found, between 11 and 17%, depending on the test material. The standard mode preference does not give any indication about the improvement when switching to high resolution. Users who are converted within any study achieve a higher performance improvement than those converted in the clinical routine. This analysis proves the significant benefits of high resolution mode for users, and also indicates the need for guidelines for individual optimization of parameter settings in a high resolution mode program.

  15. Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood.

    Directory of Open Access Journals (Sweden)

    Chia-Hsiang Chen

    Full Text Available Genetic polymorphisms of apolipoprotein E (APOE are associated with various health conditions and diseases, such as Alzheimer's disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories.

  16. Detection and differentiation of Plum pox virus using real-time multiplex PCR with SYBR Green and melting curve analysis: a rapid method for strain typing.

    Science.gov (United States)

    Varga, Aniko; James, Delano

    2005-02-01

    A real-time multiplex PCR procedure with melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid and reliable identification of Plum pox virus (PPV) isolates of strains D and M. Members of the different strains were identified by their distinctive melting temperatures (T(m)s); 84.3-84.43 degrees C for D isolates, and 85.34-86.11 degrees C for M isolates. The associated amplicon sizes were 114 and 380 bp, respectively. The procedure was used for detection and identification of PPV in both herbaceous and woody hosts. The Tm for members of a particular strain was very similar, with a host effect that did not hinder strain identification. Universal primers included in the study detected all isolates of PPV tested, amplifying a 74 bp fragment. The Tm of this fragment varied from 80.12 to 81.52 degrees C and may have supplementary value for PPV identification. SYBR Green-based detection was compared to detection using a hybridization LUX fluorogenic primer. Better resolution of the melting peaks was observed with SYBR Green I, than with the LUX primers, hence strain identification with SYBR Green I was more reliable. This is a simple approach to PPV strain identification with the relatively inexpensive dye SYBR Green I, and eliminates any need for electrophoretic analysis of amplicons or RFLP patterns using ethidium bromide.

  17. Melting curve analysis for the screening of hepatitis B virus genotypes A, D and F in patients from a general hospital in southern Brazil.

    Science.gov (United States)

    Becker, Carlos Eduardo; Kretzmann, Nelson Alexandre; Mattos, Angelo Alves de; Veiga, Ana Beatriz Gorini da

    2013-01-01

    Hepatitis B virus (HBV) can cause fulminant hepatitis, cirrhosis and hepatocellular carcinoma, and is one of the most common causes of acute and chronic liver failure. The genetic variants of HBV can be decisive for the evolution of these diseases as well as for the election of therapy. The aim of this study was to evaluate and standardize an in house methodology based on the analysis of the melting curve polymerase chain reaction (PCR) of real-time (qPCR) to screen for genotypes A, D and F of HBV in patients from a hospital in Rio Grande do Sul, Brazil. We evaluated 104 patients presumably with HBV chronic infection. Viral DNA was extracted from plasma and viral genotypes and different mutations were determined using PCR-based protocols. A PCR-based methodology was standardized for the analysis of genotypes A, D and F of HBV. The technique was based in a nested PCR with the final step consisting of a multiplex real-time PCR, using the melting curve as a tool for the differentiation of fragments. A higher frequency of genotype D (44.4%), followed by genotype A (22.2%) and genotype F (3.7%) was observed. The standardized assay, a nested PCR-multiplex qPCR using specific primers, provides a rapid and accurate method for the differentiation of HBV genotypes that are more frequent in Southern Brazil - A, D and F. This method can be applied in the clinical practice.

  18. Section on High Resolution Optical Imaging (HROI)

    Data.gov (United States)

    Federal Laboratory Consortium — The Section on High Resolution Optical Imaging (HROI) develops novel technologies for studying biological processes at unprecedented speed and resolution. Research...

  19. Genotyping of velvet antlers for identification of country of origin using mitochondrial DNA and fluorescence melting curve analysis with locked nucleic acid probes.

    Science.gov (United States)

    Ahn, Jeong Jin; Kim, Youngjoo; Hong, Ji Young; Kim, Gi Won; Hwang, Seung Yong

    2016-07-01

    Velvet antlers are used medicinally in Asia and possess various therapeutic effects. Prices are set according to the country of origin, which is unidentifiable to the naked eye, and therefore counterfeiting is prevalent. Additionally, antlers of the Canadian elk, which can generate chronic wasting disease, are prevalently smuggled and distributed in the market. Thus, a method for identifying the country of origin of velvet antlers was developed, using polymorphisms in mitochondrial DNA, fluorescence melting curve analysis and analysis of locked nucleic acids (LNA). This combined method is capable of identifying five genotypes of velvet antlers in a single experiment using two probes. It also has advantages in multiplexing, simplicity and efficiency in genotyping, when compared to real-time PCR or microarrays. The developed method can be used to improve identification rates in the velvet antler market and, by extension, research based on polymorphisms in DNA sequences.

  20. Development of a simultaneous identification method for 13 animal species using two multiplex real-time PCR assays and melting curve analysis.

    Science.gov (United States)

    Ishida, Natsumi; Sakurada, Makoto; Kusunoki, Hiroshi; Ueno, Yasuhiro

    2017-11-26

    We developed a simple and rapid method for animal species identification in the forensic science field based on mitochondrial DNA using two multiplex real-time PCRs and analysis of the resultant SYBR Green I melting curves. This method was designed to identify nine domestic animals simultaneously (dog, cat, rabbit, cattle, pig, chicken, goat, sheep and horse) and four wild animals (deer, raccoon-dog, monkey and bear) by comparing the different melting temperatures of the amplicons produced from samples originating from each species. For this analysis, we targeted various mitochondrial genes, including those encoding cytochrome b (cytb), NADH dehydrogenase 5 (ND5), cytochrome c oxidase 3 (COX3), tRNA-ND5, and tRNA-ATP synthase 8 (ATP8). For practical applications, this study presents a validation of this assay including its specificity, sensitivity and robustness. The limits of detection in the multiplex reactions were 10 pg for eight of the nine animals, excluding horse (1 pg for horse). The method was able to correctly identify the animal species from artificial forensic samples including blood stains, saliva, hair and bone, and samples digested in artificial gastric fluid, and for 17 forensic casework samples. The data from the multiplex real-time PCR assays are obtainable only 30 min after DNA extraction of the samples, making the assays useful for screening samples containing DNA from unknown animal origin in the forensic field. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Diagnosis and identification of Leishmania spp. from Giemsa-stained slides, by real-time PCR and melting curve analysis in south-west of Iran.

    Science.gov (United States)

    Khademvatan, S; Neisi, N; Maraghi, S; Saki, J

    2011-12-01

    The aim of present study was describing a real-time PCR assay for the diagnosis and direct identification of Leishmania species on Giemsa-stained slides in south-west of Iran. Altogether, 102 Giemsa-stained slides were collected from different part of south-west of Iran between 2008 and 2011. All the Giemsa-stained slides were examined under light microscope. After DNA extraction, real-time PCR amplification and detection were conducted with fluorescent SYBR Green I. For identification, PCR products were analysed with melting curve analysis. One hundred and two archived slides from suspected lesion examined by microscopy and real-time PCR. The sensitivity of the real-time PCR on Giemsa-stained slid was 98% (96/102). The melting curve analysis (T(m)) were 88·3±0·2°C for L. tropica (MHOM/IR/02/Mash10), 86·5±0·2°C for L. major (MHOM/IR/75/ER) and 89·4±0·3°C for L. infantum (MCAN/IR/97/LON 49), respectively. This study is first report in use of real-time PCR for diagnosis and identification of Leishmania spp. in Iran. Up to now, in Iran, the majority of identification of Leishmania species is restriction fragment length polymorphism (PCR-RFLP) of ITS1 and kinetoplast DNA. Our data showed that Giemsa-stained slides that were stored more than 3 years, can be use for Leishmania DNA extraction and amplification by real-time PCR. Compared to conventional PCR-based methods, the real-time PCR is extremely rapid with results and more samples can be processed at one time.

  2. Comparison of Single Nucleotide Polymorphism genotyping of CYP2C19 by Loop-mediated isothermal amplification and real-time PCR melting curve analysis.

    Science.gov (United States)

    Kwong, K M; Tam, C C; Chan, Raymond; Lee, Stephen W L; Ip, P; Kwok, Janette

    2017-12-13

    CYP2C19, a member of cytochrome P450 enzymes, is involved in various drug metabolisms, such as Clopidogrel. Common Single Nucleotide Polymorphisms (SNPs) of CYP2C19 gene, CYP2C19*2 and CYP2C19*3, are liable for the poor metabolism of Clopidogrel. It is crucial to identify poor metabolizers for alternative treatment as poor metabolism of Clopidogrel has been shown to correlate with worse clinical outcome in acute coronary syndrome (ACS) patients. A genotyping method, Loop-mediated isothermal amplification (LAMP) was employed in this study. CYP2C19*2 and CYP2C19*3 were adapted from Iwasaki M. et al. with modifications in the reaction mixtures and end-point detection method where simpler visual detection using SYBR® Safe was employed instead of a more technical and equipment demanding real-time PCR. Real-time PCR melting curve analysis is a common method for SNPs analysis and hence chosen as a reference for results obtained from the LAMP assay. The CYP2C19-LAMP assay successfully detected CYP2C19*2 and CYPC19*3 mutants. The typing results of CYP2C19-LAMP assay, performed in triplicates, were concordant with the real-time PCR melting curve analysis results. CYP2C19-LAMP assay using SYBR® Safe dye for visual detection of end-point result is a simple, rapid and cost-effective method for CYP2C19 genotyping. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Melting curve analysis after T allele enrichment (MelcaTle as a highly sensitive and reliable method for detecting the JAK2V617F mutation.

    Directory of Open Access Journals (Sweden)

    Soji Morishita

    Full Text Available Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs. However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle. MelcaTle comprises three steps: 1 two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2 selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA probe, and 3 a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.

  4. Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

    Science.gov (United States)

    Morishita, Soji; Takahashi, Kochi; Araki, Marito; Hironaka, Yumi; Sunami, Yoshitaka; Edahiro, Yoko; Tsutsui, Miyuki; Ohsaka, Akimichi; Tsuneda, Satoshi; Komatsu, Norio

    2015-01-01

    Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs. PMID:25794279

  5. VT Hydrography Dataset - High Resolution NHD

    Data.gov (United States)

    Vermont Center for Geographic Information — (Link to Metadata) The Vermont Hydrography Dataset (VHD) is compliant with the local resolution (also known as High Resolution) National Hydrography Dataset (NHD)...

  6. High Resolution Silicon Deformable Mirrors Project

    Data.gov (United States)

    National Aeronautics and Space Administration — In this proposal we describe a plan to build a deformable mirror suitable for space-based operation in systems for high-resolution imaging. The prototype DM will be...

  7. High Resolution Silicon Deformable Mirrors Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This proposal describes a plan to build a prototype small stroke, high precision deformable mirror suitable for space-based operation in systems for high-resolution...

  8. Structure of high-resolution NMR spectra

    CERN Document Server

    Corio, PL

    2012-01-01

    Structure of High-Resolution NMR Spectra provides the principles, theories, and mathematical and physical concepts of high-resolution nuclear magnetic resonance spectra.The book presents the elementary theory of magnetic resonance; the quantum mechanical theory of angular momentum; the general theory of steady state spectra; and multiple quantum transitions, double resonance and spin echo experiments.Physicists, chemists, and researchers will find the book a valuable reference text.

  9. Differential detection of Trichinella papuae, T. spiralis and T. pseudospiralis by real-time fluorescence resonance energy transfer PCR and melting curve analysis.

    Science.gov (United States)

    Tantrawatpan, Chairat; Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Boonmars, Thidarut; Wu, Zhiliang; Morakote, Nimit; Maleewong, Wanchai

    2012-04-30

    Trichinellosis caused by nematodes of Trichinella spp. is a zoonotic foodborne disease. Three Trichinella species of the parasite including Trichinella spiralis, Trichinella papuae and Trichinella pseudospiralis, have been etiologic agents of human trichinellosis in Thailand. Definite diagnosis of this helminthiasis is based on a finding of the Trichinella larva (e) in a muscle biopsy. The parasite species or genotype can be determined using molecular methods, e.g., polymerase chain reaction (PCR). This study has utilized real-time fluorescence resonance energy transfer PCR (real-time FRET PCR) and a melting curve analysis for the differential diagnosis of trichinellosis. Three common Trichinella species in Thailand were studied using one set of primers and fluorophore-labeled hybridization probes specific for the small subunit of the mitochondrial ribosomal RNA gene. Using fewer than 35 cycles as the cut-off for positivity and using different melting temperatures (T(m)), this assay detected T. spiralis, T. papuae and T. pseudospiralis in muscle tissue and found the mean T(m) ± SD values to be 51.79 ± 0.06, 66.09 ± 0.46 and 51.46 ± 0.09, respectively. The analytical sensitivity of the technique enabled the detection of a single Trichinella larva of each species, and the detection limit for the target DNA sequence was 16 copies of positive control plasmid. A test of the technique's analytical specificity showed no fluorescence signal for a panel of 19 non-Trichinella parasites or for human and mouse genomic DNA. Due to the sensitivity and specificity of the detection of these Trichinella species, as well as the fast and high-throughput nature of these tools, this method has application potential in differentiating non-encapsulated larvae of T. papuae from T. spiralis and T. pseudospiralis in tissues of infected humans and animals. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Qualitative interpretation of high resolution aeromagnetic (HRAM ...

    African Journals Online (AJOL)

    Qualitative interpretation of high resolution aeromagnetic (HRAM) data from some parts of offshore Niger delta, Nigeria. ... Open Access DOWNLOAD FULL TEXT ... The original raster map, obtained from the Nigeria Geological Survey Agency (NGSA) in half degree sheet, was subjected to qualitative data analysis using the ...

  11. A High-Resolution Stopwatch for Cents

    Science.gov (United States)

    Gingl, Z.; Kopasz, K.

    2011-01-01

    A very low-cost, easy-to-make stopwatch is presented to support various experiments in mechanics. The high-resolution stopwatch is based on two photodetectors connected directly to the microphone input of a sound card. Dedicated free open-source software has been developed and made available to download. The efficiency is demonstrated by a free…

  12. Compressive sensing for high resolution radar imaging

    NARCIS (Netherlands)

    Anitori, L.; Otten, M.P.G.; Hoogeboom, P.

    2010-01-01

    In this paper we present some preliminary results on the application of Compressive Sensing (CS) to high resolution radar imaging. CS is a recently developed theory which allows reconstruction of sparse signals with a number of measurements much lower than what is required by the Shannon sampling

  13. Compact high-resolution spectral phase shaper

    NARCIS (Netherlands)

    Postma, S.; van der Walle, P.; Offerhaus, Herman L.; van Hulst, N.F.

    2005-01-01

    The design and operation of a high-resolution spectral phase shaper with a footprint of only 7×10 cm2 is presented. The liquid-crystal modulator has 4096 elements. More than 600 independent degrees of freedom can be positioned with a relative accuracy of 1 pixel. The spectral shaping of pulses from

  14. High resolution analysis of interphase chromosome domains

    NARCIS (Netherlands)

    Visser, A. E.; Jaunin, F.; Fakan, S.; Aten, J. A.

    2000-01-01

    Chromosome territories need to be well defined at high resolution before functional aspects of chromosome organization in interphase can be explored. To visualize chromosomes by electron microscopy (EM), the DNA of Chinese hamster fibroblasts was labeled in vivo with thymidine analogue BrdU. Labeled

  15. Rapid detection and grouping of porcine bocaviruses by an EvaGreen(®) based multiplex real-time PCR assay using melting curve analysis.

    Science.gov (United States)

    Zheng, Xiaowen; Liu, Gaopeng; Opriessnig, Tanja; Wang, Zining; Yang, Zongqi; Jiang, Yonghou

    2016-08-01

    Several novel porcine bocaviruses (PBoVs) have been identified in pigs in recent years and association of these viruses with respiratory signs or diarrhea has been suggested. In this study, an EvaGreen(®)-based multiplex real-time PCR (EG-mPCR) with melting curve analysis was developed for simultaneous detection and grouping of novel PBoVs into the same genogroups G1, G2 and G3. Each target produced a specific amplicon with a melting peak of 81.3 ± 0.34 °C for PBoV G1, 78.2 ± 0.37 °C for PBoV G2, and 85.0 ± 0.29 °C for PBoV G3. Non-specific reactions were not observed when other pig viruses were used to assess the EG-mPCR assay. The sensitivity of the EG-mPCR assay using purified plasmid constructs containing the specific viral target fragments was 100 copies for PBoV G1, 50 for PBoV G2 and 100 for PBoV G3. The assay is able to detect and distinguish three PBoV groups with intra-assay and inter-assay variations ranging from 0.13 to 1.59%. The newly established EG-mPCR assay was validated with 227 field samples from pigs. PBoV G1, G2 and G3 was detected in 15.0%, 25.1% and 41.9% of the investigated samples and coinfections of two or three PBoV groups were also detected in 25.1% of the cases, indicating that all PBoV groups are prevalent in Chinese pigs. The agreement of the EG-mPCR assay with an EvaGreen-based singleplex real-time PCR (EG-sPCR) assay was 99.1%. This EG-mPCR will serve as a rapid, sensitive, reliable and cost effective alternative for routine surveillance testing of multiple PBoVs in pigs and will enhance our understanding of the epidemiological features and possible also pathogenetic changes associated with these viruses in pigs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. High resolution technology for FPD lithography tools

    Science.gov (United States)

    Yabu, Nobuhiko; Nagai, Yoshiyuki; Tomura, Satoshi; Yoshikawa, Tomohiro

    2013-06-01

    As the resolution of LCD panels adapted for Smartphone and Tablet PC rapidly becomes higher, the performance needed for lithography tools to produce them also becomes higher than ever. To respond to such needs, we have developed new lithography tools for mass production of high resolution LCD panels. We have executed various exposure tests to evaluate their performance. In this paper, we present the results of these tests. By employing higher NA projection optics, high resolution (2.0μm and under) has been achieved. We also present the effect of special illumination and the difference in profile between kinds of photoresist. Furthermore, we also refer what will be needed for masks and blanks in the next generation. To achieve even higher resolution, it is necessary for masks and blanks to have high flatness, low level of defects and small linewidth error.

  17. Radiation length imaging with high resolution telescopes

    OpenAIRE

    Stolzenberg, U.; Frey, A.; Schwenker, B; Wieduwilt, P.; Marinas, C; Lütticke, F.

    2016-01-01

    The construction of low mass vertex detectors with a high level of system integration is of great interest for next generation collider experiments. Radiation length images with a sufficient spatial resolution can be used to measure and disentangle complex radiation length $X$/$X_0$ profiles and contribute to the understanding of vertex detector systems. Test beam experiments with multi GeV particle beams and high-resolution tracking telescopes provide an opportunity to obtain precise 2D imag...

  18. High Resolution Spectra of HE Detonations

    Science.gov (United States)

    1980-07-07

    region. We shall assume for present purposes that the emissivity of the detonation products of a 50 to 100 lb HE explosion is also in the viciity of... speed . Incorporated in the emulsion layers are dye forming coup- lers which react simultaneously during I , developmentto produce a separate dye S...Best Available Cop 1~EV~ AFTAC-TR-80-24 HIGH RESOLUTION SPECTRA OF HE DETONATIONS HSS Inc 2 Alfred Circle Bedford, MA 01730 7 JULY 1980 AUG 4 9D

  19. High resolution NMR theory and chemical applications

    CERN Document Server

    Becker, Edwin D

    1969-01-01

    High Resolution NMR: Theory and Chemical Applications focuses on the applications of nuclear magnetic resonance (NMR), as well as chemical shifts, lattices, and couplings. The book first offers information on the theory of NMR, including nuclear spin and magnetic moment, spin lattice relaxation, line widths, saturation, quantum mechanical description of NMR, and ringing. The text then ponders on instrumentation and techniques and chemical shifts. Discussions focus on the origin of chemical shifts, reference compounds, empirical correlations of chemical shifts, modulation and phase detection,

  20. Multiplex real-time PCR assays for detection of eight Shiga toxin-producing Escherichia coli in food samples by melting curve analysis.

    Science.gov (United States)

    Singh, Prashant; Mustapha, Azlin

    2015-12-23

    Shiga toxin-producing Escherichia coli (STEC) are pathogenic strains of E. coli that can cause bloody diarrhea and kidney failure. Seven STEC serogroups, O157, O26, O45, O103, O111, O121 and O145 are responsible for more than 71% of the total infections caused by this group of pathogens. All seven serogroups are currently considered as adulterants in non-intact beef products in the U.S. In this study, two multiplex melt curve real-time PCR assays with internal amplification controls (IACs) were standardized for the detection of eight STEC serogroups. The first multiplex assay targeted E. coli serogroups O145, O121, O104, and O157; while the second set detected E. coli serogroups O26, O45, O103 and O111. The applicability of the assays was tested using 11 different meat and produce samples. For food samples spiked with a cocktail of four STEC serogroups with a combined count of 10 CFU/25 g food, all targets of the multiplex assays were detected after an enrichment period of 6h. The assays also worked efficiently when 325 g of food samples were spiked with 10 CFU of STECs. The assays are not dependent on fluorescent-labeled probes or immunomagnetic beads, and can be used for the detection of eight STEC serogroups in less than 11h. Routine preliminary screening of STECs in food samples is performed by testing for the presence of STEC virulence genes. The assays developed in this study can be useful as a first- or second-tier test for the identification of the eight O serogroup-specific genes in suspected food samples. Copyright © 2015. Published by Elsevier B.V.

  1. Quantitative PCR Coupled with Melt Curve Analysis for Detection of Selected Pseudo-nitzschia spp. (Bacillariophyceae) from the Northwestern Mediterranean Sea▿

    Science.gov (United States)

    Andree, Karl B.; Fernández-Tejedor, Margarita; Elandaloussi, Laurence M.; Quijano-Scheggia, Sonia; Sampedro, Nagore; Garcés, Esther; Camp, Jordi; Diogène, Jorge

    2011-01-01

    The frequency and intensity of Pseudo-nitzschia spp. blooms along the coast of Catalonia have been increasing over the past 20 years. As species from this genus that are documented as toxigenic have been found in local waters, with both toxic and nontoxic species cooccurring in the same bloom, there is a need to develop management tools for discriminating the difference. Currently, differentiation of toxic and nontoxic species requires time-consuming electron microscopy to distinguish taxonomic features that would allow identification as to species, and cryptic species can still remain misidentified. In this study, cells of Pseudo-nitzschia from clonal cultures isolated from seawater were characterized to their species identity using scanning electron microscopy, and subsamples of each culture were used to create an internal transcribed spacer 1 (ITS-1), 5.8S, and ITS-2 ribosomal DNA database for development of species-specific quantitative PCR (qPCR) assays. Once developed, these qPCR assays were applied to field samples collected over a 2-year period in Alfaques Bay in the northwestern Mediterranean Sea to evaluate the possibility of a comprehensive surveillance for all Pseudo-nitzschia spp. using molecular methods to supplement optical microscopy, which can discern taxonomy only to the genus level within this taxon. Total Pseudo-nitzschia cell density was determined by optical microscopy from water samples collected weekly and compared to results obtained from the sum of eight Pseudo-nitzschia species-specific qPCR assays using duplicate samples. Species-specific qPCR followed by melt curve analysis allowed differentiation of amplicons and identification of false positives, and results correlated well with the total Pseudo-nitzschia cell counts from optical microscopy. PMID:21193668

  2. Detection of clonal antigen receptor gene rearrangement in dogs with lymphoma by real-time polymerase chain reaction and melting curve analysis.

    Science.gov (United States)

    Langner, Kathrin F A; Joetzke, Alexa E; Nerschbach, Verena; Eberle, Nina; Schuberth, Hans-Joachim; Koy, Mirja; Nolte, Ingo; Betz, Daniela

    2014-01-03

    Molecular techniques that detect canine lymphoma cells by their clonal antigen receptor gene rearrangement play an increasing role for diagnosis as well as for monitoring minimal residual disease during and after cytostatic therapy. However, the methods currently available are time-consuming and/or cost-intensive thus impeding the use in clinical routine. The aim of the present study was to develop and evaluate a real-time polymerase chain reaction (PCR) with subsequent melting curve analysis (MCA) for the detection of clonally rearranged antigen receptor genes in dogs with B and T cell lymphoma on non formalin-fixed and paraffin-embedded lymph node samples. In lymph node aspirates from 30 dogs with multicentric B cell lymphoma, real-time PCR with MCA detected clonal rearrangement in 100% and conventional PCR with polyacrylamide gel electrophoresis (PAGE) in 93% of samples. Both methods correctly identified clonality in 80% of lymph node aspirates of 10 dogs with T cell lymphoma. None of the two PCR systems detected clonal rearrangement in samples from 9 dogs with lymph node hyperplasia. Using a dilutional series with regular lymphoid desoxyribonucleic acid (DNA), detection limits of lymphoma DNA were as low as 0.8% and 6.25% for B and T cell clonal rearrangement with real-time PCR and MCA and at 3.13% and 12.5% with the conventional system. Median absolute detection limits of lymphoma DNA were shown to be at 0.1 ng and 1 ng for the B and T cell immunophenotype with the real-time PCR system and at 10 ng each with conventional PCR and PAGE. Real-time PCR with MCA is a convenient and reliable method with a good analytical sensitivity. Thus, the method may assist the detection of clonal antigen receptor gene rearrangement in canine lymphoma patients in a clinical setting also in the presence of small amounts of neoplastic cells.

  3. Ultra-high resolution coded wavefront sensor

    KAUST Repository

    Wang, Congli

    2017-06-08

    Wavefront sensors and more general phase retrieval methods have recently attracted a lot of attention in a host of application domains, ranging from astronomy to scientific imaging and microscopy. In this paper, we introduce a new class of sensor, the Coded Wavefront Sensor, which provides high spatio-temporal resolution using a simple masked sensor under white light illumination. Specifically, we demonstrate megapixel spatial resolution and phase accuracy better than 0.1 wavelengths at reconstruction rates of 50 Hz or more, thus opening up many new applications from high-resolution adaptive optics to real-time phase retrieval in microscopy.

  4. High resolution extremity CT for biomechanics modeling

    Energy Technology Data Exchange (ETDEWEB)

    Ashby, A.E.; Brand, H.; Hollerbach, K.; Logan, C.M.; Martz, H.E.

    1995-09-23

    With the advent of ever more powerful computing and finite element analysis (FEA) capabilities, the bone and joint geometry detail available from either commercial surface definitions or from medical CT scans is inadequate. For dynamic FEA modeling of joints, precise articular contours are necessary to get appropriate contact definition. In this project, a fresh cadaver extremity was suspended in parafin in a lucite cylinder and then scanned with an industrial CT system to generate a high resolution data set for use in biomechanics modeling.

  5. Detailed mitochondrial phenotyping by high resolution metabolomics.

    Directory of Open Access Journals (Sweden)

    James R Roede

    Full Text Available Mitochondrial phenotype is complex and difficult to define at the level of individual cell types. Newer metabolic profiling methods provide information on dozens of metabolic pathways from a relatively small sample. This pilot study used "top-down" metabolic profiling to determine the spectrum of metabolites present in liver mitochondria. High resolution mass spectral analyses and multivariate statistical tests provided global metabolic information about mitochondria and showed that liver mitochondria possess a significant phenotype based on gender and genotype. The data also show that mitochondria contain a large number of unidentified chemicals.

  6. A Portable, High Resolution, Surface Measurement Device

    Science.gov (United States)

    Ihlefeld, Curtis M.; Burns, Bradley M.; Youngquist, Robert C.

    2012-01-01

    A high resolution, portable, surface measurement device has been demonstrated to provide micron-resolution topographical plots. This device was specifically developed to allow in-situ measurements of defects on the Space Shuttle Orbiter windows, but is versatile enough to be used on a wide variety of surfaces. This paper discusses the choice of an optical sensor and then the decisions required to convert a lab bench optical measurement device into an ergonomic portable system. The necessary trade-offs between performance and portability are presented along with a description of the device developed to measure Orbiter window defects.

  7. High-Resolution PET Detector. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Karp, Joel

    2014-03-26

    The objective of this project was to develop an understanding of the limits of performance for a high resolution PET detector using an approach based on continuous scintillation crystals rather than pixelated crystals. The overall goal was to design a high-resolution detector, which requires both high spatial resolution and high sensitivity for 511 keV gammas. Continuous scintillation detectors (Anger cameras) have been used extensively for both single-photon and PET scanners, however, these instruments were based on NaI(Tl) scintillators using relatively large, individual photo-multipliers. In this project we investigated the potential of this type of detector technology to achieve higher spatial resolution through the use of improved scintillator materials and photo-sensors, and modification of the detector surface to optimize the light response function.We achieved an average spatial resolution of 3-mm for a 25-mm thick, LYSO continuous detector using a maximum likelihood position algorithm and shallow slots cut into the entrance surface.

  8. Limiting liability via high resolution image processing

    Energy Technology Data Exchange (ETDEWEB)

    Greenwade, L.E.; Overlin, T.K.

    1996-12-31

    The utilization of high resolution image processing allows forensic analysts and visualization scientists to assist detectives by enhancing field photographs, and by providing the tools and training to increase the quality and usability of field photos. Through the use of digitized photographs and computerized enhancement software, field evidence can be obtained and processed as `evidence ready`, even in poor lighting and shadowed conditions or darkened rooms. These images, which are most often unusable when taken with standard camera equipment, can be shot in the worst of photographic condition and be processed as usable evidence. Visualization scientists have taken the use of digital photographic image processing and moved the process of crime scene photos into the technology age. The use of high resolution technology will assist law enforcement in making better use of crime scene photography and positive identification of prints. Valuable court room and investigation time can be saved and better served by this accurate, performance based process. Inconclusive evidence does not lead to convictions. Enhancement of the photographic capability helps solve one major problem with crime scene photos, that if taken with standard equipment and without the benefit of enhancement software would be inconclusive, thus allowing guilty parties to be set free due to lack of evidence.

  9. High-Resolution Scintimammography: A Pilot Study

    Energy Technology Data Exchange (ETDEWEB)

    Rachel F. Brem; Joelle M. Schoonjans; Douglas A. Kieper; Stan Majewski; Steven Goodman; Cahid Civelek

    2002-07-01

    This study evaluated a novel high-resolution breast-specific gamma camera (HRBGC) for the detection of suggestive breast lesions. Methods: Fifty patients (with 58 breast lesions) for whom a scintimammogram was clinically indicated were prospectively evaluated with a general-purpose gamma camera and a novel HRBGC prototype. The results of conventional and high-resolution nuclear studies were prospectively classified as negative (normal or benign) or positive (suggestive or malignant) by 2 radiologists who were unaware of the mammographic and histologic results. All of the included lesions were confirmed by pathology. Results: There were 30 benign and 28 malignant lesions. The sensitivity for detection of breast cancer was 64.3% (18/28) with the conventional camera and 78.6% (22/28) with the HRBGC. The specificity with both systems was 93.3% (28/30). For the 18 nonpalpable lesions, sensitivity was 55.5% (10/18) and 72.2% (13/18) with the general-purpose camera and the HRBGC, respectively. For lesions 1 cm, 7 of 15 were detected with the general-purpose camera and 10 of 15 with the HRBGC. Four lesions (median size, 8.5 mm) were detected only with the HRBGC and were missed by the conventional camera. Conclusion: Evaluation of indeterminate breast lesions with an HRBGC results in improved sensitivity for the detection of cancer, with greater improvement shown for nonpalpable and 1-cm lesions.

  10. Multilayer Patterning of High Resolution Intrinsically Stretchable Electronics

    Science.gov (United States)

    Tybrandt, Klas; Stauffer, Flurin; Vörös, Janos

    2016-05-01

    Stretchable electronics can bridge the gap between hard planar electronic circuits and the curved, soft and elastic objects of nature. This has led to applications like conformal displays, electronic skin and soft neuroprosthetics. A remaining challenge, however, is to match the dimensions of the interfaced systems, as all require feature sizes well below 100 μm. Intrinsically stretchable nanocomposites are attractive in this context as the mechanical deformations occur on the nanoscale, although methods for patterning high performance materials have been lacking. Here we address these issues by reporting on a multilayer additive patterning approach for high resolution fabrication of stretchable electronic devices. The method yields highly conductive 30 μm tracks with similar performance to their macroscopic counterparts. Further, we demonstrate a three layer micropatterned stretchable electroluminescent display with pixel sizes down to 70 μm. These presented findings pave the way towards future developments of high definition displays, electronic skins and dense multielectrode arrays.

  11. A high resolution X-ray crystal spectrometer to study electron and ...

    Indian Academy of Sciences (India)

    We have studied fast ion–atom and electron–atom collision processes using a reconditioned high resolution X-ray spectrometer. The X-rays, generated by the collisions, are dispersed by a curved ADP crystal (Johansson geometry) and detected by a gas proportional counter. A self-written LabVIEW based program has ...

  12. High-Resolution Broadband Spectral Interferometry

    Energy Technology Data Exchange (ETDEWEB)

    Erskine, D J; Edelstein, J

    2002-08-09

    We demonstrate solar spectra from a novel interferometric method for compact broadband high-resolution spectroscopy. The spectral interferometer (SI) is a hybrid instrument that uses a spectrometer to externally disperse the output of a fixed-delay interferometer. It also has been called an externally dispersed interferometer (EDI). The interferometer can be used with linear spectrometers for imaging spectroscopy or with echelle spectrometers for very broad-band coverage. EDI's heterodyning technique enhances the spectrometer's response to high spectral-density features, increasing the effective resolution by factors of several while retaining its bandwidth. The method is extremely robust to instrumental insults such as focal spot size or displacement. The EDI uses no moving parts, such as purely interferometric FTS spectrometers, and can cover a much wider simultaneous bandpass than other internally dispersed interferometers (e.g. HHS or SHS).

  13. Development of New High Resolution Neutron Detector

    Science.gov (United States)

    Mostella, L. D., III; Rajabali, M.; Loureiro, D. P.; Grzywacz, R.

    2017-09-01

    Beta-delayed neutron emission is a prevalent form of decay for neutron-rich nuclei. This occurs when an unstable nucleus undergoes beta decay, but produces a daughter nucleus in an excited state above the neutron separation energy. The daughter nucleus then de-excites by ejecting one or more neutrons. We wish to map the states from which these nuclei decay via neutron spectroscopy using NEXT, a new high resolution neutron detector. NEXT utilizes silicon photomultipliers and 6 mm thick pulse-shape discriminating plastic scintillators, allowing for smaller and more compact modular geometries in the NEXT array. Timing measurements for the detector were performed and a resolution of 893 ps (FWHM) has been achieved so far. Aspects of the detector that were investigated and will be presented here include scintillator geometry, wrapping materials, fitting functions for the digitized signals, and electronic components coupled to the silicon photomultipliers for signal shaping.

  14. High Resolution, High Frame Rate Video Technology

    Science.gov (United States)

    1990-01-01

    Papers and working group summaries presented at the High Resolution, High Frame Rate Video (HHV) Workshop are compiled. HHV system is intended for future use on the Space Shuttle and Space Station Freedom. The Workshop was held for the dual purpose of: (1) allowing potential scientific users to assess the utility of the proposed system for monitoring microgravity science experiments; and (2) letting technical experts from industry recommend improvements to the proposed near-term HHV system. The following topics are covered: (1) State of the art in the video system performance; (2) Development plan for the HHV system; (3) Advanced technology for image gathering, coding, and processing; (4) Data compression applied to HHV; (5) Data transmission networks; and (6) Results of the users' requirements survey conducted by NASA.

  15. High Resolution Regional Climate Simulations over Alaska

    Science.gov (United States)

    Monaghan, A. J.; Clark, M. P.; Arnold, J.; Newman, A. J.; Musselman, K. N.; Barlage, M. J.; Xue, L.; Liu, C.; Gutmann, E. D.; Rasmussen, R.

    2016-12-01

    In order to appropriately plan future projects to build and maintain infrastructure (e.g., dams, dikes, highways, airports), a number of U.S. federal agencies seek to better understand how hydrologic regimes may shift across the country due to climate change. Building on the successful completion of a series of high-resolution WRF simulations over the Colorado River Headwaters and contiguous USA, our team is now extending these simulations over the challenging U.S. States of Alaska and Hawaii. In this presentation we summarize results from a newly completed 4-km resolution WRF simulation over Alaska spanning 2002-2016 at 4-km spatial resolution. Our aim is to gain insight into the thermodynamics that drive key precipitation processes, particularly the extremes that are most damaging to infrastructure.

  16. Constructing a WISE High Resolution Galaxy Atlas

    Science.gov (United States)

    Jarrett, T. H.; Masci, F.; Tsai, C. W.; Petty, S.; Cluver, M.; Assef, Roberto J.; Benford, D.; Blain, A.; Bridge, C.; Donoso, E.; hide

    2012-01-01

    After eight months of continuous observations, the Wide-field Infrared Survey Explorer (WISE) mapped the entire sky at 3.4 micron, 4.6 micron, 12 micron, and 22 micron. We have begun a dedicated WISE High Resolution Galaxy Atlas project to fully characterize large, nearby galaxies and produce a legacy image atlas and source catalog. Here we summarize the deconvolution techniques used to significantly improve the spatial resolution of WISE imaging, specifically designed to study the internal anatomy of nearby galaxies. As a case study, we present results for the galaxy NGC 1566, comparing the WISE enhanced-resolution image processing to that of Spitzer, Galaxy Evolution Explorer, and ground-based imaging. This is the first paper in a two-part series; results for a larger sample of nearby galaxies are presented in the second paper.

  17. High-Resolution Movement EEG Classification

    Directory of Open Access Journals (Sweden)

    Jakub Štastný

    2007-01-01

    Full Text Available The aim of the contribution is to analyze possibilities of high-resolution movement classification using human EEG. For this purpose, a database of the EEG recorded during right-thumb and little-finger fast flexion movements of the experimental subjects was created. The statistical analysis of the EEG was done on the subject's basis instead of the commonly used grand averaging. Statistically significant differences between the EEG accompanying movements of both fingers were found, extending the results of other so far published works. The classifier based on hidden Markov models was able to distinguish between movement and resting states (classification score of 94–100%, but it was unable to recognize the type of the movement. This is caused by the large fraction of other (nonmovement related EEG activities in the recorded signals. A classification method based on advanced EEG signal denoising is being currently developed to overcome this problem.

  18. Improved methods for high resolution electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, J.R.

    1987-04-01

    Existing methods of making support films for high resolution transmission electron microscopy are investigated and novel methods are developed. Existing methods of fabricating fenestrated, metal reinforced specimen supports (microgrids) are evaluated for their potential to reduce beam induced movement of monolamellar crystals of C/sub 44/H/sub 90/ paraffin supported on thin carbon films. Improved methods of producing hydrophobic carbon films by vacuum evaporation, and improved methods of depositing well ordered monolamellar paraffin crystals on carbon films are developed. A novel technique for vacuum evaporation of metals is described which is used to reinforce microgrids. A technique is also developed to bond thin carbon films to microgrids with a polymer bonding agent. Unique biochemical methods are described to accomplish site specific covalent modification of membrane proteins. Protocols are given which covalently convert the carboxy terminus of papain cleaved bacteriorhodopsin to a free thiol. 53 refs., 19 figs., 1 tab.

  19. High resolution multimodal clinical ophthalmic imaging system.

    Science.gov (United States)

    Mujat, Mircea; Ferguson, R Daniel; Patel, Ankit H; Iftimia, Nicusor; Lue, Niyom; Hammer, Daniel X

    2010-05-24

    We developed a multimodal adaptive optics (AO) retinal imager which is the first to combine high performance AO-corrected scanning laser ophthalmoscopy (SLO) and swept source Fourier domain optical coherence tomography (SSOCT) imaging modes in a single compact clinical prototype platform. Such systems are becoming ever more essential to vision research and are expected to prove their clinical value for diagnosis of retinal diseases, including glaucoma, diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinitis pigmentosa. The SSOCT channel operates at a wavelength of 1 microm for increased penetration and visualization of the choriocapillaris and choroid, sites of major disease activity for DR and wet AMD. This AO system is designed for use in clinical populations; a dual deformable mirror (DM) configuration allows simultaneous low- and high-order aberration correction over a large range of refractions and ocular media quality. The system also includes a wide field (33 deg.) line scanning ophthalmoscope (LSO) for initial screening, target identification, and global orientation, an integrated retinal tracker (RT) to stabilize the SLO, OCT, and LSO imaging fields in the presence of lateral eye motion, and a high-resolution LCD-based fixation target for presentation of visual cues. The system was tested in human subjects without retinal disease for performance optimization and validation. We were able to resolve and quantify cone photoreceptors across the macula to within approximately 0.5 deg (approximately 100-150 microm) of the fovea, image and delineate ten retinal layers, and penetrate to resolve features deep into the choroid. The prototype presented here is the first of a new class of powerful flexible imaging platforms that will provide clinicians and researchers with high-resolution, high performance adaptive optics imaging to help guide therapies, develop new drugs, and improve patient outcomes.

  20. High-resolution transcriptome of human macrophages.

    Directory of Open Access Journals (Sweden)

    Marc Beyer

    Full Text Available Macrophages are dynamic cells integrating signals from their microenvironment to develop specific functional responses. Although, microarray-based transcriptional profiling has established transcriptional reprogramming as an important mechanism for signal integration and cell function of macrophages, current knowledge on transcriptional regulation of human macrophages is far from complete. To discover novel marker genes, an area of great need particularly in human macrophage biology but also to generate a much more thorough transcriptome of human M1- and M1-like macrophages, we performed RNA sequencing (RNA-seq of human macrophages. Using this approach we can now provide a high-resolution transcriptome profile of human macrophages under classical (M1-like and alternative (M2-like polarization conditions and demonstrate a dynamic range exceeding observations obtained by previous technologies, resulting in a more comprehensive understanding of the transcriptome of human macrophages. Using this approach, we identify important gene clusters so far not appreciated by standard microarray techniques. In addition, we were able to detect differential promoter usage, alternative transcription start sites, and different coding sequences for 57 gene loci in human macrophages. Moreover, this approach led to the identification of novel M1-associated (CD120b, TLR2, SLAMF7 as well as M2-associated (CD1a, CD1b, CD93, CD226 cell surface markers. Taken together, these data support that high-resolution transcriptome profiling of human macrophages by RNA-seq leads to a better understanding of macrophage function and will form the basis for a better characterization of macrophages in human health and disease.

  1. High-Resolution Transcriptome of Human Macrophages

    Science.gov (United States)

    Xue, Jia; Staratschek-Jox, Andrea; Vorholt, Daniela; Krebs, Wolfgang; Sommer, Daniel; Sander, Jil; Mertens, Christina; Nino-Castro, Andrea; Schmidt, Susanne V.; Schultze, Joachim L.

    2012-01-01

    Macrophages are dynamic cells integrating signals from their microenvironment to develop specific functional responses. Although, microarray-based transcriptional profiling has established transcriptional reprogramming as an important mechanism for signal integration and cell function of macrophages, current knowledge on transcriptional regulation of human macrophages is far from complete. To discover novel marker genes, an area of great need particularly in human macrophage biology but also to generate a much more thorough transcriptome of human M1- and M1-like macrophages, we performed RNA sequencing (RNA-seq) of human macrophages. Using this approach we can now provide a high-resolution transcriptome profile of human macrophages under classical (M1-like) and alternative (M2-like) polarization conditions and demonstrate a dynamic range exceeding observations obtained by previous technologies, resulting in a more comprehensive understanding of the transcriptome of human macrophages. Using this approach, we identify important gene clusters so far not appreciated by standard microarray techniques. In addition, we were able to detect differential promoter usage, alternative transcription start sites, and different coding sequences for 57 gene loci in human macrophages. Moreover, this approach led to the identification of novel M1-associated (CD120b, TLR2, SLAMF7) as well as M2-associated (CD1a, CD1b, CD93, CD226) cell surface markers. Taken together, these data support that high-resolution transcriptome profiling of human macrophages by RNA-seq leads to a better understanding of macrophage function and will form the basis for a better characterization of macrophages in human health and disease. PMID:23029029

  2. Integrated High Resolution Monitoring of Mediterranean vegetation

    Science.gov (United States)

    Cesaraccio, Carla; Piga, Alessandra; Ventura, Andrea; Arca, Angelo; Duce, Pierpaolo; Mereu, Simone

    2017-04-01

    The study of the vegetation features in a complex and highly vulnerable ecosystems, such as Mediterranean maquis, leads to the need of using continuous monitoring systems at high spatial and temporal resolution, for a better interpretation of the mechanisms of phenological and eco-physiological processes. Near-surface remote sensing techniques are used to quantify, at high temporal resolution, and with a certain degree of spatial integration, the seasonal variations of the surface optical and radiometric properties. In recent decades, the design and implementation of global monitoring networks involved the use of non-destructive and/or cheaper approaches such as (i) continuous surface fluxes measurement stations, (ii) phenological observation networks, and (iii) measurement of temporal and spatial variations of the vegetation spectral properties. In this work preliminary results from the ECO-SCALE (Integrated High Resolution Monitoring of Mediterranean vegetation) project are reported. The project was manly aimed to develop an integrated system for environmental monitoring based on digital photography, hyperspectral radiometry , and micrometeorological techniques during three years of experimentation (2013-2016) in a Mediterranean site of Italy (Capo Caccia, Alghero). The main results concerned the analysis of chromatic coordinates indices from digital images, to characterized the phenological patterns for typical shrubland species, determining start and duration of the growing season, and the physiological status in relation to different environmental drought conditions; then the seasonal patterns of canopy phenology, was compared to NEE (Net Ecosystem Exchange) patterns, showing similarities. However, maximum values of NEE and ER (Ecosystem respiration), and short term variation, seemed mainly tuned by inter annual pattern of meteorological variables, in particular of temperature recorded in the months preceding the vegetation green-up. Finally, green signals

  3. Superconducting High Resolution Fast-Neutron Spectrometers

    Energy Technology Data Exchange (ETDEWEB)

    Hau, Ionel Dragos [Univ. of California, Berkeley, CA (United States)

    2006-01-01

    Superconducting high resolution fast-neutron calorimetric spectrometers based on 6LiF and TiB{sub 2} absorbers have been developed. These novel cryogenic spectrometers measure the temperature rise produced in exothermal (n, α) reactions with fast neutrons in 6Li and 10B-loaded materials with heat capacity C operating at temperatures T close to 0.1 K. Temperature variations on the order of 0.5 mK are measured with a Mo/Cu thin film multilayer operated in the transition region between its superconducting and its normal state. The advantage of calorimetry for high resolution spectroscopy is due to the small phonon excitation energies kBT on the order of μeV that serve as signal carriers, resulting in an energy resolution ΔE ~ (kBT2C)1/2, which can be well below 10 keV. An energy resolution of 5.5 keV has been obtained with a Mo/Cu superconducting sensor and a TiB2 absorber using thermal neutrons from a 252Cf neutron source. This resolution is sufficient to observe the effect of recoil nuclei broadening in neutron spectra, which has been related to the lifetime of the first excited state in 7Li. Fast-neutron spectra obtained with a 6Li-enriched LiF absorber show an energy resolution of 16 keV FWHM, and a response in agreement with the 6Li(n, α)3H reaction cross section and Monte Carlo simulations for energies up to several MeV. The energy resolution of order of a few keV makes this novel instrument applicable to fast-neutron transmission spectroscopy based on the unique elemental signature provided by the neutron absorption and scattering resonances. The optimization of the energy resolution based on analytical and numerical models of the detector response is discussed in the context of these applications.

  4. High resolution measurement of the glycolytic rate

    Directory of Open Access Journals (Sweden)

    Carla X Bittner

    2010-09-01

    Full Text Available The glycolytic rate is sensitive to physiological activity, hormones, stress, aging and malignant transformation. Standard techniques to measure the glycolytic rate are based on radioactive isotopes, are not able to resolve single cells and have poor temporal resolution, limitations that hamper the study of energy metabolism in the brain and other organs. A new method is described in this article, which makes use of a recently-developed FRET glucose nanosensor to measure the rate of glycolysis in single cells with high temporal resolution. Used in cultured astrocytes, the method showed for the first time that glycolysis can be activated within seconds by a combination of glutamate and K+, supporting a role for astrocytes in neurometabolic and neurovascular coupling in the brain. It was also possible to make a direct comparison of metabolism in neurons and astrocytes lying in close proximity, paving the way to a high-resolution characterization of brain energy metabolism. Single-cell glycolytic rates were also measured in fibroblasts, adipocytes, myoblasts and tumor cells, showing higher rates for undifferentiated cells and significant metabolic heterogeneity within cell types. This method should facilitate the investigation of tissue metabolism at the single-cell level and is readily adaptable for high-throughput analysis.

  5. High resolution quantum metrology via quantum interpolation

    Science.gov (United States)

    Ajoy, Ashok; Liu, Yixiang; Saha, Kasturi; Marseglia, Luca; Jaskula, Jean-Christophe; Cappellaro, Paola

    2016-05-01

    Nitrogen Vacancy (NV) centers in diamond are a promising platform for quantum metrology - in particular for nanoscale magnetic resonance imaging to determine high resolution structures of single molecules placed outside the diamond. The conventional technique for sensing of external nuclear spins involves monitoring the effects of the target nuclear spins on the NV center coherence under dynamical decoupling (the CPMG/XY8 pulse sequence). However, the nuclear spin affects the NV coherence only at precise free evolution times - and finite timing resolution set by hardware often severely limits the sensitivity and resolution of the method. In this work, we overcome this timing resolution barrier by developing a technique to supersample the metrology signal by effectively implementing a quantum interpolation of the spin system dynamics. This method will enable spin sensing at high magnetic fields and high repetition rate, allowing significant improvements in sensitivity and spectral resolution. We experimentally demonstrate a resolution boost by over a factor of 100 for spin sensing and AC magnetometry. The method is shown to be robust, versatile to sensing normal and spurious signal harmonics, and ultimately limited in resolution only by the number of pulses that can be applied.

  6. AIRBORNE HIGH-RESOLUTION DIGITAL IMAGING SYSTEM

    Directory of Open Access Journals (Sweden)

    Prado-Molina, J.

    2006-04-01

    Full Text Available A low-cost airborne digital imaging system capable to perform aerial surveys with small-format cameras isintroduced. The equipment is intended to obtain high-resolution multispectral digital photographs constituting so aviable alternative to conventional aerial photography and satellite imagery. Monitoring software handles all theprocedures involved in image acquisition, including flight planning, real-time graphics for aircraft position updatingin a mobile map, and supervises the main variables engaged in the imaging process. This software also creates fileswith the geographical position of the central point of every image, and the flight path followed by the aircraftduring the entire survey. The cameras are mounted on a three-axis stabilized platform. A set of inertial sensorsdetermines platform's deviations independently from the aircraft and an automatic control system keeps thecameras at a continuous nadir pointing and heading, with a precision better than ± 1 arc-degree in three-axis. Thecontrol system is also in charge of saving the platform’s orientation angles when the monitoring software triggersthe camera. These external orientation parameters, together with a procedure for camera calibration give theessential elements for image orthocorrection. Orthomosaics are constructed using commercial GIS software.This system demonstrates the feasibility of large area coverage in a practical and economical way using smallformatcameras. Monitoring and automatization reduce the work while increasing the quality and the amount ofuseful images.

  7. Fundamental constants and high-resolution spectroscopy

    Science.gov (United States)

    Bonifacio, P.; Rahmani, H.; Whitmore, J. B.; Wendt, M.; Centurion, M.; Molaro, P.; Srianand, R.; Murphy, M. T.; Petitjean, P.; Agafonova, I. I.; D'Odorico, S.; Evans, T. M.; Levshakov, S. A.; Lopez, S.; Martins, C. J. A. P.; Reimers, D.; Vladilo, G.

    2014-01-01

    Absorption-line systems detected in high resolution quasar spectra can be used to compare the value of dimensionless fundamental constants such as the fine-structure constant, α, and the proton-to-electron mass ratio, μ = m_p/m_e, as measured in remote regions of the Universe to their value today on Earth. In recent years, some evidence has emerged of small temporal and also spatial variations in α on cosmological scales which may reach a fractional level of ≈ 10 ppm (parts per million). We are conducting a Large Programme of observations with the Very Large Telescope's Ultraviolet and Visual Echelle Spectrograph (UVES), and are obtaining high-resolution ({R ≈ 60 000}) and high signal-to-noise ratio (S/N ≈ 100) spectra calibrated specifically to study the variations of the fundamental constants. We here provide a general overview of the Large Programme and report on the first results for these two constants, discussed in detail in Molaro et al. (2013) and Rahmani et al. (2013). A stringent bound for Δα/α is obtained for the absorber at z_abs = 1.6919 towards HE 2217-2818. The absorption profile is complex with several very narrow features, and is modeled with 32 velocity components. The relative variation in α in this system is +1.3± 2.4_stat ± 1.0_sys ppm if Al II λ 1670 Å and three Fe II transitions are used, and +1.1 ± 2.6_stat ppm in a slightly different analysis with only Fe II transitions used. This is one of the tightest bounds on α-variation from an individual absorber and reveals no evidence for variation in α at the 3-ppm precision level (1σ confidence). The expectation at this sky position of the recently-reported dipolar variation of α is (3.2-5.4)±1.7 ppm depending on dipole model used and this constraint of Δα/α at face value is not supporting this expectation but not inconsistent with it at the 3σ level. For the proton-to-electron mass ratio the analysis of the H_2 absorption lines of the z_abs ≈ 2.4018 damped Lyα system

  8. High-resolution phylogenetic microbial community profiling

    Energy Technology Data Exchange (ETDEWEB)

    Singer, Esther; Coleman-Derr, Devin; Bowman, Brett; Schwientek, Patrick; Clum, Alicia; Copeland, Alex; Ciobanu, Doina; Cheng, Jan-Fang; Gies, Esther; Hallam, Steve; Tringe, Susannah; Woyke, Tanja

    2014-03-17

    The representation of bacterial and archaeal genome sequences is strongly biased towards cultivated organisms, which belong to merely four phylogenetic groups. Functional information and inter-phylum level relationships are still largely underexplored for candidate phyla, which are often referred to as microbial dark matter. Furthermore, a large portion of the 16S rRNA gene records in the GenBank database are labeled as environmental samples and unclassified, which is in part due to low read accuracy, potential chimeric sequences produced during PCR amplifications and the low resolution of short amplicons. In order to improve the phylogenetic classification of novel species and advance our knowledge of the ecosystem function of uncultivated microorganisms, high-throughput full length 16S rRNA gene sequencing methodologies with reduced biases are needed. We evaluated the performance of PacBio single-molecule real-time (SMRT) sequencing in high-resolution phylogenetic microbial community profiling. For this purpose, we compared PacBio and Illumina metagenomic shotgun and 16S rRNA gene sequencing of a mock community as well as of an environmental sample from Sakinaw Lake, British Columbia. Sakinaw Lake is known to contain a large age of microbial species from candidate phyla. Sequencing results show that community structure based on PacBio shotgun and 16S rRNA gene sequences is highly similar in both the mock and the environmental communities. Resolution power and community representation accuracy from SMRT sequencing data appeared to be independent of GC content of microbial genomes and was higher when compared to Illumina-based metagenome shotgun and 16S rRNA gene (iTag) sequences, e.g. full-length sequencing resolved all 23 OTUs in the mock community, while iTags did not resolve closely related species. SMRT sequencing hence offers various potential benefits when characterizing uncharted microbial communities.

  9. High resolution CT findings of pseudoalveolar sarcoidosis

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Ji Eun; Park, Jun Gyun; Choe, Kyu Ok; Kim, Sang Jin [Yonsei University College of Medicine, Seoul (Korea, Republic of); Ryu, Young Hoon; Im, Jung Gi [Seoul National University College of Medicine, Seoul (Korea, Republic of); Lee, Kyoung Soo [Sungkunkwan University College of Medicine, Seoul (Korea, Republic of); Song, Koun Sik [University of Ulsan College of Medicine, Seoul (Korea, Republic of); Kim, Hyae Young [National Cancer Centar, Seoul (Korea, Republic of)

    2002-08-01

    To determine the specific high-resolution CT features of sarcoidosis in which the observed pattern is predominantly pseudoalveolar. We retrospectively reviewed the HRCT findings in 15 cases in which chest radiography demonstrated pseudoalveolar consolidation. In all 15, sarcoidosis was pathologically proven. The distribution and characterization of the following CT features was meticulously scrutinized: distribution and characterization of pseudoalveolar lesions, air-bronchograms, micronodules, thickening of bronchovascular bundles and interlobular septa, lung distortion, ground-glass opacities and combined hilar and mediastinal lymphadenopathy. Follow-up CT scans were available in three cases after corticosteroid administration. Between one and 12 (mean, 5.6) pseudoalveolar lesions appeared as dense homogeneous or inhomogeneous opacities 1-4.5 cm in diameter and with an irregular margin located either at the lung periphery adjacent to the pleural surface or along the bronchovascular bundles, with mainly bilateral distribution (n=14, 93%). An air-bronchogram was observed in ten cases. Micronodules were observed at the periphery of the lesion or surrounding lung, which along with a thickened bronchovascular bundle was a consistent feature in all cases. Additional CT features included hilar and mediastinal lymphadenopathy (n=14, 93%), thickened interlobular septa (n=12, 80%), and ground-glass opacity (n=10, 67%). Lung distortion was noted in only one case (7%). After steroid administration pseudoalveolar lesions decreased in number and size in all three cases in which follow-up CT was available. The consistent HRCT features of pseudoalveolar sarcoidosis are bilateral multifocal dense homogenous or inhomogenous opacity and an irregular margin located either at the lung periphery adjacent to the pleural surface or along the bronchovascular bundles. Micronodules are present at the periphery of the lesion or surrounding lung. The features are reversible administration.

  10. High-resolution downscaling for hydrological management

    Science.gov (United States)

    Ulbrich, Uwe; Rust, Henning; Meredith, Edmund; Kpogo-Nuwoklo, Komlan; Vagenas, Christos

    2017-04-01

    Hydrological modellers and water managers require high-resolution climate data to model regional hydrologies and how these may respond to future changes in the large-scale climate. The ability to successfully model such changes and, by extension, critical infrastructure planning is often impeded by a lack of suitable climate data. This typically takes the form of too-coarse data from climate models, which are not sufficiently detailed in either space or time to be able to support water management decisions and hydrological research. BINGO (Bringing INnovation in onGOing water management; ) aims to bridge the gap between the needs of hydrological modellers and planners, and the currently available range of climate data, with the overarching aim of providing adaptation strategies for climate change-related challenges. Producing the kilometre- and sub-daily-scale climate data needed by hydrologists through continuous simulations is generally computationally infeasible. To circumvent this hurdle, we adopt a two-pronged approach involving (1) selective dynamical downscaling and (2) conditional stochastic weather generators, with the former presented here. We take an event-based approach to downscaling in order to achieve the kilometre-scale input needed by hydrological modellers. Computational expenses are minimized by identifying extremal weather patterns for each BINGO research site in lower-resolution simulations and then only downscaling to the kilometre-scale (convection permitting) those events during which such patterns occur. Here we (1) outline the methodology behind the selection of the events, and (2) compare the modelled precipitation distribution and variability (preconditioned on the extremal weather patterns) with that found in observations.

  11. High Resolution Global View of Io

    Science.gov (United States)

    1997-01-01

    Io, the most volcanic body in the solar system is seen in the highest resolution obtained to date by NASA's Galileo spacecraft. The smallest features that can be discerned are 2.5 kilometers in size. There are rugged mountains several kilometers high, layered materials forming plateaus, and many irregular depressions called volcanic calderas. Several of the dark, flow-like features correspond to hot spots, and may be active lava flows. There are no landforms resembling impact craters, as the volcanism covers the surface with new deposits much more rapidly than the flux of comets and asteroids can create large impact craters. The picture is centered on the side of Io that always faces away from Jupiter; north is to the top.Color images acquired on September 7, 1996 have been merged with higher resolution images acquired on November 6, 1996 by the Solid State Imaging (CCD) system aboard NASA's Galileo spacecraft. The color is composed of data taken, at a range of 487,000 kilometers, in the near-infrared, green, and violet filters and has been enhanced to emphasize the extraordinary variations in color and brightness that characterize Io's face. The high resolution images were obtained at ranges which varied from 245,719 kilometers to 403,100 kilometers.Launched in October 1989, Galileo entered orbit around Jupiter on December 7, 1995. The spacecraft's mission is to conduct detailed studies of the giant planet, its largest moons and the Jovian magnetic environment. The Jet Propulsion Laboratory, Pasadena, CA manages the mission for NASA's Office of Space Science, Washington, DC.This image and other images and data received from Galileo are posted on the World Wide Web, on the Galileo mission home page at URL http://galileo.jpl.nasa.gov. Background information and educational context for the images can be found at URL http://www.jpl.nasa.gov/galileo/sepo

  12. Caracterização da fusão peritética do cloreto de sódio di-hidratado por curvas de aquecimento Sodium chloride dihydrate peritetic melting characterization through heating curves

    Directory of Open Access Journals (Sweden)

    Edward de Souza

    2009-01-01

    Full Text Available Binary mixture phase diagrams are normally obtained from thermal analysis involving freezing point curves. However, that approach is not always reliable and easy to follow to all kinds of mixtures in any proportion. In fact, even for a simple system, such as NaCl-H2O, this freezing methodology gives mixed results when one starts from a solid-solution system, due mostly to the formation of the NaCl.2H2O, which has an incongruent melting point, and the dependence of its solubility with the temperature. In this work we report a trustworthy, simple and cheap method involving heating curves to drawn the NaCl-H2O phase diagram.

  13. A new high-resolution climatology for the Nordic Seas

    Science.gov (United States)

    Korablev, A.; Johannessen, O. M.; Pnyushkov, A.; Smirnov, A.

    2009-04-01

    Constantly growing interests in high-resolution oceanographic fields stimulate compilation of comprehensive initial datasets and advanced methodology of the objective analysis. Observed level database for the Nordic Seas and the North Atlantic compiled from large amount of initial sources was recently considerably updated by adding historical and modern measurements. Improved database allows evaluating ocean climate variability in the area for 1900-2007. Applied quality control algorithms on observed data were specifically designed to preserve regional variability and to produce the oceanographic fields with enhanced spatial resolution. Objectively analyzed (OA) temperature, salinity and dissolved oxygen monthly fields at the standard levels for the 1900-2007 on 0.25 x 0.5 gr. latitude-longitude grid were computed by means of block variant of ordinary kriging system. The OA monthly fields comprise climatology and can be used for studying of temporal and spatial parameters variation. A number of stable regimes and periods with abrupt changes of the water masses thermohaline properties were identified and will be discussed. Monthly mean fields were compared with available high-resolution (better then 0.5 gr.) climatology fields, including Generalized Digital Environmental Model (GDEM) version 3.0 and NODC/NOAA products based on data from World Ocean Databases 2001 and 2005. Results show considerable discrepancies originated from differences in initial datasets, quality control algorithms and methods of objective analysis. Allocation of the collected oceanographic stations for more than one century and derived climatological fields over the Nordic Seas reveals a disproportion in data coverage. Repeated standard stations, sections and polygons are most important for uniform long-term time series compositing and ocean climate variation study. Luck of observations over the west and northern parts of the region do not allows reliable climatology fields compilation

  14. Novel high resolution tactile robotic fingertips

    DEFF Research Database (Denmark)

    Drimus, Alin; Jankovics, Vince; Gorsic, Matija

    2014-01-01

    This paper describes a novel robotic fingertip based on piezoresistive rubber that can sense pressure tactile stimuli with a high spatial resolution over curved surfaces. The working principle is based on a three-layer sandwich structure (conductive electrodes on top and bottom and piezoresistive...... rubber in the middle). For the conductive layers we use ring patterns of silver epoxy and flex PCB electrode arrays. The proposed sensorised fingertip has 60 sensitive regions (taxels) arranged in 5 rings and 12 columns that have a smooth pressure to resistance characteristic. Using the sensor...

  15. HIGH RESOLUTION AIRBORNE SHALLOW WATER MAPPING

    Directory of Open Access Journals (Sweden)

    F. Steinbacher

    2012-07-01

    Full Text Available In order to meet the requirements of the European Water Framework Directive (EU-WFD, authorities face the problem of repeatedly performing area-wide surveying of all kinds of inland waters. Especially for mid-sized or small rivers this is a considerable challenge imposing insurmountable logistical efforts and costs. It is therefore investigated if large-scale surveying of a river system on an operational basis is feasible by employing airborne hydrographic laser scanning. In cooperation with the Bavarian Water Authority (WWA Weilheim a pilot project was initiated by the Unit of Hydraulic Engineering at the University of Innsbruck and RIEGL Laser Measurement Systems exploiting the possibilities of a new LIDAR measurement system with high spatial resolution and high measurement rate to capture about 70 km of riverbed and foreland for the river Loisach in Bavaria/Germany and the estuary and parts of the shoreline (about 40km in length of lake Ammersee. The entire area surveyed was referenced to classic terrestrial cross-section surveys with the aim to derive products for the monitoring and managing needs of the inland water bodies forced by the EU-WFD. The survey was performed in July 2011 by helicopter and airplane and took 3 days in total. In addition, high resolution areal images were taken to provide an optical reference, offering a wide range of possibilities on further research, monitoring, and managing responsibilities. The operating altitude was about 500 m to maintain eye-safety, even for the aided eye, the airspeed was about 55 kts for the helicopter and 75 kts for the aircraft. The helicopter was used in the alpine regions while the fixed wing aircraft was used in the plains and the urban area, using appropriate scan rates to receive evenly distributed point clouds. The resulting point density ranged from 10 to 25 points per square meter. By carefully selecting days with optimum water quality, satisfactory penetration down to the river

  16. High Resolution Airborne Shallow Water Mapping

    Science.gov (United States)

    Steinbacher, F.; Pfennigbauer, M.; Aufleger, M.; Ullrich, A.

    2012-07-01

    In order to meet the requirements of the European Water Framework Directive (EU-WFD), authorities face the problem of repeatedly performing area-wide surveying of all kinds of inland waters. Especially for mid-sized or small rivers this is a considerable challenge imposing insurmountable logistical efforts and costs. It is therefore investigated if large-scale surveying of a river system on an operational basis is feasible by employing airborne hydrographic laser scanning. In cooperation with the Bavarian Water Authority (WWA Weilheim) a pilot project was initiated by the Unit of Hydraulic Engineering at the University of Innsbruck and RIEGL Laser Measurement Systems exploiting the possibilities of a new LIDAR measurement system with high spatial resolution and high measurement rate to capture about 70 km of riverbed and foreland for the river Loisach in Bavaria/Germany and the estuary and parts of the shoreline (about 40km in length) of lake Ammersee. The entire area surveyed was referenced to classic terrestrial cross-section surveys with the aim to derive products for the monitoring and managing needs of the inland water bodies forced by the EU-WFD. The survey was performed in July 2011 by helicopter and airplane and took 3 days in total. In addition, high resolution areal images were taken to provide an optical reference, offering a wide range of possibilities on further research, monitoring, and managing responsibilities. The operating altitude was about 500 m to maintain eye-safety, even for the aided eye, the airspeed was about 55 kts for the helicopter and 75 kts for the aircraft. The helicopter was used in the alpine regions while the fixed wing aircraft was used in the plains and the urban area, using appropriate scan rates to receive evenly distributed point clouds. The resulting point density ranged from 10 to 25 points per square meter. By carefully selecting days with optimum water quality, satisfactory penetration down to the river bed was achieved

  17. High-Resolution Ion Cyclotron Mobility Spectrometry

    Science.gov (United States)

    Merenbloom, Samuel I.; Glaskin, Rebecca S.; Henson, Zachary B.; Clemmer, David E.

    2009-01-01

    A novel ion mobility spectrometry instrument incorporating a cyclotron geometry drift tube is presented. The drift tube consists of eight regions, four curved drift tubes and four ion funnels. Packets of ions are propagated around the drift tube by changing the drift field at a frequency that is resonant with the ion’s drift time through each region. The approach trims each packet of ions as it leaves and enters each new region. An electrostatic gate allows ions to be kept in the drift tube for numerous cycles, increasing the ability to resolve specified ions. We demonstrate the approach by isolating the [M+2H]2+ or [M+3H]3+ charge state of substance P as well as individual trisaccharide isomers from a mixture of melezitose and raffinose. Resolving powers in excess of 300 are obtainable with this approach. PMID:19143495

  18. High resolution X-ray diffraction studies on unirradiated and ...

    Indian Academy of Sciences (India)

    High-resolution X-ray diffraction technique, employing a three-crystal monochromator–collimator ... observed by high-resolution electron microscopy in both ..... 1988 Nucl. Instrum. Meth. B34 228. Kato N 1992 J. Acta Crystallogr. A48 834. Kaur B, Bhat M, Licci F, Kumar R, Kotru P N and Bamzai K K. 2004 Nucl. Instrum. Meth ...

  19. Scalable Algorithms for Large High-Resolution Terrain Data

    DEFF Research Database (Denmark)

    Mølhave, Thomas; Agarwal, Pankaj K.; Arge, Lars Allan

    2010-01-01

    In this paper we demonstrate that the technology required to perform typical GIS computations on very large high-resolution terrain models has matured enough to be ready for use by practitioners. We also demonstrate the impact that high-resolution data has on common problems. To our knowledge, so...

  20. Achieving sensitive, high-resolution laser spectroscopy at CRIS

    Energy Technology Data Exchange (ETDEWEB)

    Groote, R. P. de [Instituut voor Kern- en Stralingsfysica, KU Leuven (Belgium); Lynch, K. M., E-mail: kara.marie.lynch@cern.ch [EP Department, CERN, ISOLDE (Switzerland); Wilkins, S. G. [The University of Manchester, School of Physics and Astronomy (United Kingdom); Collaboration: the CRIS collaboration

    2017-11-15

    The Collinear Resonance Ionization Spectroscopy (CRIS) experiment, located at the ISOLDE facility, has recently performed high-resolution laser spectroscopy, with linewidths down to 20 MHz. In this article, we present the modifications to the beam line and the newly-installed laser systems that have made sensitive, high-resolution measurements possible. Highlights of recent experimental campaigns are presented.

  1. High-resolution X-ray diffraction studies of multilayers

    DEFF Research Database (Denmark)

    Christensen, Finn Erland; Hornstrup, Allan; Schnopper, H. W.

    1988-01-01

    High-resolution X-ray diffraction studies of the perfection of state-of-the-art multilayers are presented. Data were obtained using a triple-axis perfect-crystal X-ray diffractometer. Measurements reveal large-scale figure errors in the substrate. A high-resolution triple-axis set up is required...

  2. High resolution UV spectroscopy and laser-focused nanofabrication

    NARCIS (Netherlands)

    Myszkiewicz, G.

    2005-01-01

    This thesis combines two at first glance different techniques: High Resolution Laser Induced Fluorescence Spectroscopy (LIF) of small aromatic molecules and Laser Focusing of atoms for Nanofabrication. The thesis starts with the introduction to the high resolution LIF technique of small aromatic

  3. Achieving sensitive, high-resolution laser spectroscopy at CRIS

    Science.gov (United States)

    de Groote, R. P.; Lynch, K. M.; Wilkins, S. G.

    2017-11-01

    The Collinear Resonance Ionization Spectroscopy (CRIS) experiment, located at the ISOLDE facility, has recently performed high-resolution laser spectroscopy, with linewidths down to 20 MHz. In this article, we present the modifications to the beam line and the newly-installed laser systems that have made sensitive, high-resolution measurements possible. Highlights of recent experimental campaigns are presented.

  4. On the Design of High Resolution Imaging Systems

    Science.gov (United States)

    Eckardt, A.; Reulke, R.

    2017-05-01

    The design of high-resolution systems is always a consideration of many parameters. Technological parameter of the imaging system, e.g. diameter of the imaging system, mass and power, as well as storage and data transfer, have an direct impact on spacecraft size and design. The paper describes the essential design parameters for the description of high-resolution systems.

  5. Ultrafast real-time PCR with integrated melting curve analysis and duplex capacities using a low-cost polymer lab-on-a-chip system

    Science.gov (United States)

    Gransee, Rainer; Schneider, Tristan; Elyorgun, Deniz; Strobach, Xenia; Schunck, Tobias; Gatscha, Theresia; Winkler, Christian; Höth, Julian

    2015-05-01

    Nucleic amplification using quantitative polymeric chain reaction (qPCR) has become the gold standard of molecular testing. These systems offer both amplification and simultaneous fluorescence detection. An ultrafast microfluidic module (allowing 30 PCR cycles in 6 minutes) based on the oscillating fluid plug concept was previously developed [1,2] allowing the amplification of native genomic deoxyribonucleic acid (DNA) molecules. This abstract presents the actual status of the advanced system. The upgraded system generates high quality qPCR amplification plots and additional sensitive melting point analysis comparable to data obtained from commercial real-time cyclers. These features provide the user with all information needed to analyze PCR products. The system uses light emitting diodes (LED) for illumination and a low cost Charge-coupled Device (CCD) camera for optical detection. Image data processing allows the automated process control of the overall system components. The system enables the performance of rapid and robust nucleic acid amplifications together with the integration of real time measurement technology. This allows the amplification and simultaneous quantification of the targeted pathogens. The integration of duplex amplification performance allows the incorporation of the necessary controls into the device to validate the PCR performance. This demonstrator can be run either as fully autonomously working device or as OEM part of a sample-to-answer platform.

  6. High-resolution dynamic CE-MRA of the thorax enabled by iterative TWIST reconstruction.

    Science.gov (United States)

    Wetzl, Jens; Forman, Christoph; Wintersperger, Bernd J; D'Errico, Luigia; Schmidt, Michaela; Mailhe, Boris; Maier, Andreas; Stalder, Aurélien F

    2017-02-01

    To evaluate the clinical benefit of using a new iterative reconstruction technique fully integrated on a standard clinical scanner and reconstruction system using a TWIST acquisition for high-resolution dynamic three-dimensional contrast-enhanced MR angiography (CE-MRA). Low-dose, high-resolution TWIST datasets of 11 patients were reconstructed using both standard GRAPPA-based reconstruction for reference and iterative reconstruction, which reduces the temporal footprint of reconstructed images. Image quality of both techniques was assessed by two experienced readers, as well as quantitatively evaluated using a time-signal curve analysis. Image quality scores consistently and significantly improved by using iterative reconstruction compared with the standard approach. Most notably, the delineation of small to mid-size vasculature improved from a mean Likert score between "nondiagnostic" and "poor" for standard to between "good" and "excellent" for iterative reconstruction. The full width at half maximum of the contrast agent bolus computed from the time-signal curve was also reduced by iterative reconstruction, allowing for more precise bolus timing. Iterative reconstruction can substantially improve high-resolution dynamic CE-MRA image quality, most notably in small to mid-size vasculature. Dynamic CE-MRA with iterative reconstruction could become an alternative to conventional static 3D CE-MRA, thus simplifying the clinical workflow. Magn Reson Med 77:833-840, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

  7. High Resolution, Range/Range-Rate Imager Project

    Data.gov (United States)

    National Aeronautics and Space Administration — Visidyne proposes to develop a design for a small, lightweight, high resolution, in x, y, and z Doppler imager to assist in the guidance, navigation and control...

  8. Hurricane Satellite (HURSAT) from Advanced Very High Resolution Radiometer (AVHRR)

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Huricane Satellite (HURSAT)-Advanced Very High Resolution Radiometer (AVHRR) is used to extend the HURSAT data set such that appling the Objective Dvorak technique...

  9. Methodology of high-resolution photography for mural condition database

    Science.gov (United States)

    Higuchi, R.; Suzuki, T.; Shibata, M.; Taniguchi, Y.

    2015-08-01

    Digital documentation is one of the most useful techniques to record the condition of cultural heritage. Recently, high-resolution images become increasingly useful because it is possible to show general views of mural paintings and also detailed mural conditions in a single image. As mural paintings are damaged by environmental stresses, it is necessary to record the details of painting condition on high-resolution base maps. Unfortunately, the cost of high-resolution photography and the difficulty of operating its instruments and software have commonly been an impediment for researchers and conservators. However, the recent development of graphic software makes its operation simpler and less expensive. In this paper, we suggest a new approach to make digital heritage inventories without special instruments, based on our recent our research project in Üzümlü church in Cappadocia, Turkey. This method enables us to achieve a high-resolution image database with low costs, short time, and limited human resources.

  10. A Forward-Looking High-Resolution GPR System

    National Research Council Canada - National Science Library

    Kositsky, Joel; Milanfar, Peyman

    1999-01-01

    A high-resolution ground penetrating radar (GPR) system was designed to help define the optimal radar parameters needed for the efficient standoff detection of buried and surface-laid antitank mines...

  11. High Resolution Orthoimagery = Orthorectified Metro Areas: 2000 - Present

    Data.gov (United States)

    U.S. Geological Survey, Department of the Interior — High resolution orthorectified images combine the image characteristics of an aerial photograph with the geometric qualities of a map. An orthoimage is a...

  12. Topological Data Analysis of High-Resolution Temporal Rainfall

    Science.gov (United States)

    Carsteanu, Alin Andrei; Fernández Méndez, Félix; Vásquez Aguilar, Raciel

    2017-04-01

    This study applies topological data analysis (TDA) to the state space representations of high-resolution temporal rainfall intensity data from Iowa City (IIHR, U of Iowa). Using a sufficient embedding dimension, topological properties of the underlying manifold are depicted.

  13. NOAA High-Resolution Sea Surface Temperature (SST) Analysis Products

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This archive covers two high resolution sea surface temperature (SST) analysis products developed using an optimum interpolation (OI) technique. The analyses have a...

  14. Developing Visual Editors for High-Resolution Haptic Patterns

    DEFF Research Database (Denmark)

    Cuartielles, David; Göransson, Andreas; Olsson, Tony

    2012-01-01

    In this article we give an overview of our iterative work in developing visual editors for creating high resolution haptic patterns to be used in wearable, haptic feedback devices. During the past four years we have found the need to address the question of how to represent, construct and edit high...... resolution haptic patterns so that they translate naturally to the user’s haptic experience. To solve this question we have developed and tested several visual editors...

  15. Assessment for Melting Temperature Measurement of Nucleic Acid by HRM

    National Research Council Canada - National Science Library

    Jing Wang; Xiaoming Pan; Xingguo Liang

    2016-01-01

    High resolution melting (HRM), with a high sensitivity to distinguish the nucleic acid species with small variations, has been widely applied in the mutation scanning, methylation analysis, and genotyping...

  16. High resolution laser beam induced current focusing for photoactive surface characterization

    Energy Technology Data Exchange (ETDEWEB)

    Fernandez-Lorenzo, C. [Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Cadiz, Apartado de Correos 40, 11510 Puerto Real, Cadiz (Spain)]. E-mail: concha.fernandez@uca.es; Poce-Fatou, J.A. [Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Cadiz, Apartado de Correos 40, 11510 Puerto Real, Cadiz (Spain); Alcantara, R. [Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Cadiz, Apartado de Correos 40, 11510 Puerto Real, Cadiz (Spain); Navas, J. [Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Cadiz, Apartado de Correos 40, 11510 Puerto Real, Cadiz (Spain); Martin, J. [Departamento de Quimica Fisica, Facultad de Ciencias, Universidad de Cadiz, Apartado de Correos 40, 11510 Puerto Real, Cadiz (Spain)

    2006-12-15

    The micro-characterization of several surface properties of the solar cells can be accomplished using high-resolution laser beam induced current images. For obtaining these images, a very precise laser beam focusing on the photoactive surface is required. For this purpose, a methodology for obtaining the best focalization associated to the maximum of a peak curve has been developed. In this paper, a data set, obtained from the inner photoconversion properties of the system, has been evaluated with three different numerical analysis techniques: (a) derivative (b) length and (c) Fourier Transform, in order to get the finest possible peak distribution. Then, an amount of 13 analytical peak curves using the Levenberg Marquardt algorithm to find the best curve that adjusts the data distribution have been analyzed.

  17. Evaluation of high-resolution melting and other molecular methods in discrimination of Lactobacillus isolates.

    Science.gov (United States)

    Szén, O P; Kiss, A; Naár, Z; Pál, K

    2014-10-01

    Fermented and probiotic products are mainly based on the activity of diverse Lactobacillus species. Distinguishing of these species, especially the closely related ones might be problematic. Our aim was to compare and evaluate molecular methods that have the potential of discrimination and might serve as alternatives of traditional microbiological techniques. In our experiments, PCR methods using general and species-specific bacterial primers, RFLP, sequencing and HRM assays were tested and their efficiency compared. A new universal primer pair was designed for amplification of short fragments of the 16S rDNA of six Lactobacillus, a Lactococcus and a Streptococcus species; furthermore, successful HRM analysis was performed on them that resulted in the separation of each species, including the almost indistinguishable Lact. paracasei ssp. paracasei and Lact. paracasei ssp. tolerans subspecies. The results showed that HRM might be a useful, time- and cost-saving one-step tool for preliminary classification of isolates, although the use of additional techniques, like species-specific PCR, analysis of RFLP patterns and sequencing, might be necessary for confirmation of the results. The newly developed HRM primers offer a quick and efficient tool for discrimination of lactobacilli, including very closely related Lactobacillus subspecies. © 2014 The Society for Applied Microbiology.

  18. High resolution melt analysis (HRMA); a viable alternative to agarose gel electrophoresis for mouse genotyping

    National Research Council Canada - National Science Library

    Thomsen, Nicole; Ali, Radiya G; Ahmed, Jehangir N; Arkell, Ruth M

    2012-01-01

    .... In contrast to the range of new technologies for mouse mutagenesis, genotyping methods have remained relatively static with alleles typically discriminated by agarose gel electrophoresis of PCR products...

  19. Identification of rare and frequent variants of the CASR gene by high-resolution melting

    DEFF Research Database (Denmark)

    Nissen, Peter H; Christensen, Signe E; Ladefoged, Søren A

    2012-01-01

    BACKGROUND: Calcium metabolic disorders like familial hypocalciuric hypercalcemia (FHH) and autosomal dominant familial isolated hypoparathyroidism (FIH) can be caused by rare variants of the calcium sensing receptor gene (CASR). Molecular genetic screening of the CASR is often based on DNA seque...

  20. Evaluation of Resistance in Pepper Germplasm to Cucumber mosaic virus by High Resolution Melting Analysis

    Directory of Open Access Journals (Sweden)

    Na Young Ro

    2012-12-01

    Full Text Available In this study, total number of 1941 Capsicum accessions conserved at RDA Genebank was evaluated for theirresponse to Cucumber mosaic virus (CMV. These accessions were composed with 9 species originated from89 countries, included 839 Capsicum annuum, 277 C. baccatum, 395 C. chinense, 343 C. frutescens, 49 C.pubescens, and other 38 wild pepper species (C. chacoense, C. galapagoense, etc.. Resistant to CMV wasscreened with the 240H02SP6 SNP marker related to the Cmr1 (Cucumber mosaic resistance 1. Eighty nineaccessions of pepper germplasm were resistant to CMV based on the marker. One hundred sixty twoaccessions showed heterozygosity. One thousand two hundred seventy accessions were susceptible to CMV.Four hundred twenty accessions did not show distinction by 240H02SP6 marker. These 89 resistant peppergermplasm can be used in a pepper breeding program against CMV.

  1. EMODnet High Resolution Seabed Mapping - further developing a high resolution digital bathymetry for European seas

    Science.gov (United States)

    Schaap, Dick M. A.; Schmitt, Thierry

    2017-04-01

    Access to marine data is a key issue for the EU Marine Strategy Framework Directive and the EU Marine Knowledge 2020 agenda and includes the European Marine Observation and Data Network (EMODnet) initiative. EMODnet aims at assembling European marine data, data products and metadata from diverse sources in a uniform way. The EMODnet data infrastructure is developed through a stepwise approach in three major phases. Currently EMODnet is entering its 3rd phase with operational portals providing access to marine data for bathymetry, geology, physics, chemistry, biology, seabed habitats and human activities, complemented by checkpoint projects, analysing the fitness for purpose of data provision. The EMODnet Bathymetry project has developed Digital Terrain Models (DTM) for the European seas. These have been produced from survey and aggregated data sets that are indexed with metadata by adopting the SeaDataNet Catalogue services. SeaDataNet is a network of major oceanographic data centres around the European seas that manage, operate and further develop a pan-European infrastructure for marine and ocean data management. The latest EMODnet Bathymetry DTM release has a resolution of 1/8 arcminute * 1/8 arcminute and covers all European sea regions. Use has been made of circa 7800 gathered survey datasets and composite DTMs from 27 European data providers from 15 countries. For areas without coverage use has been made of the latest GEBCO DTM. The catalogue services and the generated EMODnet DTM have been published at the dedicated EMODnet Bathymetry portal which includes a versatile DTM viewing service that also supports downloading in various formats. End December 2016 the Bathymetry project has been succeeded by EMODnet High Resolution Seabed Mapping (HRSM) as part of the third phase of EMODnet. This new project will continue gathering of bathymetric in-situ data sets with extra efforts for near coastal waters and coastal zones. In addition Satellite Derived Bathymetry

  2. A high-resolution vehicle emission inventory for China

    Science.gov (United States)

    Zheng, B.; Zhang, Q.; He, K.; Huo, H.; Yao, Z.; Wang, X.

    2012-12-01

    Developing high resolution emission inventory is an essential task for air quality modeling and management. However, current vehicle emission inventories in China are usually developed at provincial level and then allocated to grids based on various spatial surrogates, which is difficult to get high spatial resolution. In this work, we developed a new approach to construct a high-resolution vehicle emission inventory for China. First, vehicle population at county level were estimated by using the relationship between per-capita GDP and vehicle ownership. Then the Weather Research and Forecasting (WRF) model were used to drive the International Vehicle Emission (IVE) model to get monthly emission factors for each county. Finally, vehicle emissions by county were allocated to grids with 5-km horizon resolution by using high-resolution road network data. This work provides a better understanding of spatial representation of vehicle emissions in China and can benefit both air quality modeling and management with improved spatial accuracy.

  3. Single sensor processing to obtain high resolution color component signals

    Science.gov (United States)

    Glenn, William E. (Inventor)

    2010-01-01

    A method for generating color video signals representative of color images of a scene includes the following steps: focusing light from the scene on an electronic image sensor via a filter having a tri-color filter pattern; producing, from outputs of the sensor, first and second relatively low resolution luminance signals; producing, from outputs of the sensor, a relatively high resolution luminance signal; producing, from a ratio of the relatively high resolution luminance signal to the first relatively low resolution luminance signal, a high band luminance component signal; producing, from outputs of the sensor, relatively low resolution color component signals; and combining each of the relatively low resolution color component signals with the high band luminance component signal to obtain relatively high resolution color component signals.

  4. Compact and high-resolution optical orbital angular momentum sorter

    Directory of Open Access Journals (Sweden)

    Chenhao Wan

    2017-03-01

    Full Text Available A compact and high-resolution optical orbital angular momentum (OAM sorter is proposed and demonstrated. The sorter comprises a quadratic fan-out mapper and a dual-phase corrector positioned in the pupil plane and the Fourier plane, respectively. The optical system is greatly simplified compared to previous demonstrations of OAM sorting, and the performance in resolution and efficiency is maintained. A folded configuration is set up using a single reflective spatial light modulator (SLM to demonstrate the validity of the scheme. The two phase elements are implemented on the left and right halves of the SLM and connected by a right-angle prism. Experimental results demonstrate the high resolution of the compact OAM sorter, and the current limit in efficiency can be overcome by replacing with transmissive SLMs and removing the beam splitters. This novel scheme paves the way for the miniaturization and integration of high-resolution OAM sorters.

  5. High-resolution interference with programmable classical incoherent light.

    Science.gov (United States)

    Zhang, Er-Feng; Liu, Wei-Tao; Chen, Ping-Xing

    2015-07-01

    A scheme of high-resolution interference with classical incoherent light is proposed. In this scheme, the classical incoherent light is programmable in the amplitude distribution and wavefront, and with the programmable classical incoherent light we improve the resolution of the interference pattern by a factor of 2 compared with the scheme by Erkmen [J. Opt. Soc. Am. A29, 782 (2012)JOAOD60740-323210.1364/JOSAA.29.000782]. Compared with other schemes for observing interference patterns, only single-pixel detection is needed in our proposal. Moreover, the high-resolution interference pattern can be inverted to obtain an image with better resolution compared with that of the scheme proposed by Erkmen. Furthermore, this scheme of high-resolution interference is verified in detail by theoretical analysis and numerical simulations.

  6. O-space with high resolution readouts outperforms radial imaging.

    Science.gov (United States)

    Wang, Haifeng; Tam, Leo; Kopanoglu, Emre; Peters, Dana C; Constable, R Todd; Galiana, Gigi

    2017-04-01

    While O-Space imaging is well known to accelerate image acquisition beyond traditional Cartesian sampling, its advantages compared to undersampled radial imaging, the linear trajectory most akin to O-Space imaging, have not been detailed. In addition, previous studies have focused on ultrafast imaging with very high acceleration factors and relatively low resolution. The purpose of this work is to directly compare O-Space and radial imaging in their potential to deliver highly undersampled images of high resolution and minimal artifacts, as needed for diagnostic applications. We report that the greatest advantages to O-Space imaging are observed with extended data acquisition readouts. A sampling strategy that uses high resolution readouts is presented and applied to compare the potential of radial and O-Space sequences to generate high resolution images at high undersampling factors. Simulations and phantom studies were performed to investigate whether use of extended readout windows in O-Space imaging would increase k-space sampling and improve image quality, compared to radial imaging. Experimental O-Space images acquired with high resolution readouts show fewer artifacts and greater sharpness than radial imaging with equivalent scan parameters. Radial images taken with longer readouts show stronger undersampling artifacts, which can cause small or subtle image features to disappear. These features are preserved in a comparable O-Space image. High resolution O-Space imaging yields highly undersampled images of high resolution and minimal artifacts. The additional nonlinear gradient field improves image quality beyond conventional radial imaging. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. High-resolution low-dose scanning transmission electron microscopy.

    Science.gov (United States)

    Buban, James P; Ramasse, Quentin; Gipson, Bryant; Browning, Nigel D; Stahlberg, Henning

    2010-01-01

    During the past two decades instrumentation in scanning transmission electron microscopy (STEM) has pushed toward higher intensity electron probes to increase the signal-to-noise ratio of recorded images. While this is suitable for robust specimens, biological specimens require a much reduced electron dose for high-resolution imaging. We describe here protocols for low-dose STEM image recording with a conventional field-emission gun STEM, while maintaining the high-resolution capability of the instrument. Our findings show that a combination of reduced pixel dwell time and reduced gun current can achieve radiation doses comparable to low-dose TEM.

  8. Theoretical Problems in High Resolution Solar Physics, 2

    Science.gov (United States)

    Athay, G. (Editor); Spicer, D. S. (Editor)

    1987-01-01

    The Science Working Group for the High Resolution Solar Observatory (HRSO) laid plans beginning in 1984 for a series of workshops designed to stimulate a broadbased input from the scientific community to the HRSO mission. These workshops have the dual objectives of encouraging an early start on the difficult theoretical problems in radiative transfer, magnetohydrodynamics, and plasma physics that will be posed by the HRSO data, and maintaining current discussions of results in high resolution solar studies. This workshop was the second in the series. The workshop format presented invited review papers during the formal sessions and contributed poster papers for discussions during open periods. Both are presented.

  9. In situ visualization of tears on contact lens using ultra high resolution optical coherence tomography.

    Science.gov (United States)

    Wang, Jianhua; Jiao, Shuliang; Ruggeri, Marco; Shousha, Mohamed Abou; Shousha, Mohammed Abou; Chen, Qi

    2009-03-01

    To demonstrate the capability of directly visualizing the tear film on contact lenses using optical coherence tomography (OCT). Six eyes of three healthy subjects wearing PureVision and ACUVUE Advance soft and Boston RGP hard contact lenses were imaged with a custom built, high speed, ultra-high resolution spectral domain optical coherence tomograph. Refresh Liquigel was used to demonstrate the effect of artificial tears on the tear film. Ultra high resolution images of the pre- and post-lens films were directly visualized when each lens was inserted onto the eye. After the instillation of artificial tears during lens wear, the tear film was thicker. The post-lens tear film underneath the lens edge was clearly shown. Interactions between the lens edges and the ocular surface were obtained for each of the lens types and base curves. With a contrast enhancement agent, tear menisci on the contact lenses around the upper and lower eyelids were highlighted. With hard contact lenses, the tear film was visualized clearly and changed after a blink when the lens was pulled up by the lid. Ultra-high resolution OCT is a potentially promising technique for imaging tears around contact lenses. This successful demonstration of in situ post-lens tear film imaging suggests that OCT could open a new era in studying tear dynamics during contact lens wear. The novel method may lead to new ways of evaluating contact lens fitting.

  10. Application of a real-time fluorescence resonance energy transfer polymerase chain reaction assay with melting curve analysis for the detection of Paragonimus heterotremus eggs in the feces of experimentally infected cats.

    Science.gov (United States)

    Tantrawatpan, Chairat; Intapan, Pewpan M; Thanchomnang, Tongjit; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Anamnart, Witthaya; Maleewong, Wanchai

    2013-09-01

    Paragonimus heterotremus is a medically important lung fluke that causes human and animal paragonimiasis in Southeast Asia, including Thailand. In the current study, a real-time fluorescence resonance energy transfer polymerase chain reaction (real-time FRET PCR) with melting curve analysis was developed and evaluated to detect P. heterotremus eggs in the feces of experimentally infected cats. The detection limit of this method for the P. heterotremus DNA sequence was 3 × 10(2) copies of the positive control plasmid and 10(-3) ng of P. heterotremus genomic DNA. The assay system could detect 10 eggs of P. heterotremus per gram of cat feces. No fluorescence signal was observed when DNA purified from 16 other organisms or genomic DNA from cats and human beings were tested. Real-time FRET PCR yielded positive results for all fecal samples from 17 P. heterotremus-infected cats and showed a negative relationship (r = -0.852, P analysis. This assay can be useful for the detection of, and epidemiological studies on, P. heterotremus infection in endemic areas.

  11. Hydrologic Simulation in Mediterranean flood prone Watersheds using high-resolution quality data

    Science.gov (United States)

    Eirini Vozinaki, Anthi; Alexakis, Dimitrios; Pappa, Polixeni; Tsanis, Ioannis

    2015-04-01

    Flooding is a significant threat causing lots of inconveniencies in several societies, worldwide. The fact that the climatic change is already happening, increases the flooding risk, which is no longer a substantial menace to several societies and their economies. The improvement of spatial-resolution and accuracy of the topography and land use data due to remote sensing techniques could provide integrated flood inundation simulations. In this work hydrological analysis of several historic flood events in Mediterranean flood prone watersheds (island of Crete/Greece) takes place. Satellite images of high resolution are elaborated. A very high resolution (VHR) digital elevation model (DEM) is produced from a GeoEye-1 0.5-m-resolution satellite stereo pair and is used for floodplain management and mapping applications such as watershed delineation and river cross-section extraction. Sophisticated classification algorithms are implemented for improving Land Use/ Land Cover maps accuracy. In addition, soil maps are updated with means of Radar satellite images. The above high-resolution data are innovatively used to simulate and validate several historical flood events in Mediterranean watersheds, which have experienced severe flooding in the past. The hydrologic/hydraulic models used for flood inundation simulation in this work are HEC-HMS and HEC-RAS. The Natural Resource Conservation Service (NRCS) curve number (CN) approach is implemented to account for the effect of LULC and soil on the hydrologic response of the catchment. The use of high resolution data provides detailed validation results and results of high precision, accordingly. Furthermore, the meteorological forecasting data, which are also combined to the simulation model results, manage the development of an integrated flood forecasting and early warning system tool, which is capable of confronting or even preventing this imminent risk. The research reported in this paper was fully supported by the

  12. Remote parallel rendering for high-resolution tiled display walls

    KAUST Repository

    Nachbaur, Daniel

    2014-11-01

    © 2014 IEEE. We present a complete, robust and simple to use hardware and software stack delivering remote parallel rendering of complex geometrical and volumetric models to high resolution tiled display walls in a production environment. We describe the setup and configuration, present preliminary benchmarks showing interactive framerates, and describe our contributions for a seamless integration of all the software components.

  13. Input variable selection for interpolating high-resolution climate ...

    African Journals Online (AJOL)

    2010-10-20

    Oct 20, 2010 ... Accurate climate surfaces are vital for applications relating to groundwater recharge modelling, evapotranspiration estima- ... with distance to oceans and elevation to generate 8 sets of high-resolution (i.e. 3 arc second) climate surfaces of the Western .... ANUSPLIN, developed by the Australian National.

  14. High resolution numerical weather prediction over the Indian ...

    Indian Academy of Sciences (India)

    In this study, the Florida State University Global Spectral Model (FSUGSM), in association with a high-resolution nested regional spectral model (FSUNRSM), is used for short-range weather forecasts over the Indian domain. Three-day forecasts for each day of August 1998 were performed using different versions of the ...

  15. Track prediction of very severe cyclone 'Nargis' using high resolution ...

    Indian Academy of Sciences (India)

    In the present study, a detailed diagnostic analysis of the system 'Nargis' is carried out initially to investigate the features associated with this unusual movement and subsequently the real time forecast of VSCS 'Nargis' using high resolution advanced version weather research forecasting (WRF) model is presented.

  16. HIGH RESOLUTION RESISTIVITY LEAK DETECTION DATA PROCESSING & EVALUATION MEHTODS & REQUIREMENTS

    Energy Technology Data Exchange (ETDEWEB)

    SCHOFIELD JS

    2007-10-04

    This document has two purposes: {sm_bullet} Describe how data generated by High Resolution REsistivity (HRR) leak detection (LD) systems deployed during single-shell tank (SST) waste retrieval operations are processed and evaluated. {sm_bullet} Provide the basic review requirements for HRR data when Hrr is deployed as a leak detection method during SST waste retrievals.

  17. Workshop on high-resolution, large-acceptance spectrometers

    Energy Technology Data Exchange (ETDEWEB)

    Zeidman, B. (ed.)

    1981-01-01

    The purpose of the Workshop on High-Resolution, Large-Acceptance Spectrometers was to provide a means for exchange of information among those actively engaged in the design and construction of these new spectrometers. Thirty-seven papers were prepared for the data base.

  18. High-resolution seismic imaging of the Sohagpur Gondwana basin ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Earth System Science; Volume 122; Issue 6 ... In this study, we illustrate how Gondwana tectonics affected the Sohagpur Gondwana basin that occurs at the junction of the Mahanadi and Son–Narmada rift systems in the central India, through a high-resolution seismic reflection study along six ...

  19. Bombs at High Resolution. I. Morphological Evidence for Photospheric Reconnection

    NARCIS (Netherlands)

    Watanabe, H.; Vissers, G.; Kitai, R.; Rouppe van der Voort, L.H.M.; Rutten, R.J.|info:eu-repo/dai/nl/074143662

    2011-01-01

    High-resolution imaging-spectroscopy movies of solar active region NOAA 10998 obtained with the Crisp Imaging Spectropolarimeter at the Swedish 1-m Solar Telescope show very bright, rapidly flickering, flame-like features that appear intermittently in the wings of the Balmer Hα line in a region with

  20. Calibration of a High Resolution Airborne 3-D SAR

    DEFF Research Database (Denmark)

    Dall, Jørgen; Grinder-Pedersen, Jan; Madsen, S.N.

    1997-01-01

    The potential of across-track interferometric (XTI) synthetic aperture radar (SAR) for producing high resolution 3D imagery has been demonstrated by several airborne systems including EMISAR, the dual frequency, polarimetric, and interferometric SAR developed at the Dept. of Electromagnetic Systems...

  1. Comparison of High Resolution Negative Electron Beam Resists

    DEFF Research Database (Denmark)

    Olsen, Brian Bilenberg; Schøler, Mikkel; Shi, Peixiong

    2006-01-01

    Four high resolution negative electron beam resists are compared: TEBN-1 from Tokuyama Corp. Japan, ma-N 2401XP and mr-L 6000AXP from microresist technology GmbH Germany, and SU-8 2000 series from MicroChem Corp., USA. Narrow linewidth high density patterns are defined by 100 kV electron beam...

  2. High-resolution spectroscopy of gases for industrial applications

    DEFF Research Database (Denmark)

    Fateev, Alexander; Clausen, Sønnik

    for analysis of complex experimental data and further development of the databases. High-temperature gas cell facilities available at DTU Chemical Engineering are presented and described. The gas cells and high-resolution spectrometers allow us to perform high-quality reference measurements of gases relevant...

  3. High Resolution Digital Imaging of Paintings: The Vasari Project.

    Science.gov (United States)

    Martinez, Kirk

    1991-01-01

    Describes VASARI (the Visual Art System for Archiving and Retrieval of Images), a project funded by the European Community to show the feasibility of high resolution colormetric imaging directly from paintings. The hardware and software used in the system are explained, storage on optical disks is described, and initial results are reported. (five…

  4. High resolution spectroscopy of the disk chromosphere. I - Observing procedures.

    Science.gov (United States)

    Beckers, J. M.; Mauter, H. A.; Mann, G. R.; Brown, D. R.

    1972-01-01

    Review of some of the main features of a high resolution spectroscopy program aimed at the precise photometric observation of chromospheric fine structures using the Sacramento Peak vacuum telescope. The observing procedures are described, and a sample of the first observational results is presented.

  5. High-Resolution Stamp Fabrication by Edge Lithography

    NARCIS (Netherlands)

    Zhao, Yiping

    2010-01-01

    The aim of the project was to create high resolution stamps for thermal nanoimprint applications. The creation of nanoridges with sub-100 nm resolutions was explored by means of edge lithography via top-down routes, i.e. in combination with micromachining technology. Edge lithography is an add-on

  6. High resolution STEM of quantum dots and quantum wires

    DEFF Research Database (Denmark)

    Kadkhodazadeh, Shima

    2013-01-01

    This article reviews the application of high resolution scanning transmission electron microscopy (STEM) to semiconductor quantum dots (QDs) and quantum wires (QWRs). Different imaging and analytical techniques in STEM are introduced and key examples of their application to QDs and QWRs...

  7. High resolution EPR applications to metalloenzymes and metals in medicine

    CERN Document Server

    Berliner, Lawrence

    2009-01-01

    EPR spectroscopy has an important role in the geometric structural characterization of the redox cofactors in metalloproteins and their electronic structure, as this is crucial for their reactivity. This title covers high-resolution EPR methods, iron proteins, nickel and copper enzymes, and metals in medicine.

  8. A high resolution powder diffractometer using focusing optics

    Indian Academy of Sciences (India)

    Research Centre, Mumbai 400 085, India. *Corresponding author. E-mail: siruguri@csr.ernet.in. Abstract. In this paper, we describe the design, construction and performance of a new high resolution neutron powder diffractometer that has been installed at the Dhruva reactor, Trombay, India. The instrument employs novel ...

  9. High resolution reflection seismic mapping of shallow coal seams

    CSIR Research Space (South Africa)

    Mngadi, SB

    2013-10-01

    Full Text Available Subsidence and collapse of unmapped shallow coal mine workings poses a risk to the public and hampers the development of valuable property. A high-resolution reflection seismic survey was conducted to determine whether it is possible to map...

  10. High resolution mid-infrared spectroscopy based on frequency upconversion

    DEFF Research Database (Denmark)

    Dam, Jeppe Seidelin; Hu, Qi; Tidemand-Lichtenberg, Peter

    2013-01-01

    We present high resolution upconversion of incoherent infrared radiation by means of sum-frequency mixing with a laser followed by simple CCD Si-camera detection. Noise associated with upconversion is, in strong contrast to room temperature direct mid-IR detection, extremely small, thus very faint...

  11. Interpretation of high resolution aeromagnetic data over southern ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Earth System Science; Volume 125; Issue 2 ... High resolution airborne magnetic data of parts of the southern Benue Trough were digitally processed and analyzed in order to estimate the depth of magnetic sources and to map the distribution and orientation of subsurface structural features.

  12. High resolution resist-free lithography in the SEM

    NARCIS (Netherlands)

    Hari, S.

    2017-01-01

    Focussed Electron Beam Induced Processing is a high resolution direct-write nanopatterning technique. Its ability to fabricate sub-10 nm structures together with its versatility and ease of use, in that it is resist-free and implementable inside a Scanning Electron Microscope, make it attractive for

  13. Systematic high-resolution assessment of global hydropower potential

    NARCIS (Netherlands)

    Hoes, Olivier A C; Meijer, Lourens J J; Van Der Ent, Ruud J.; Van De Giesen, Nick C.

    2017-01-01

    Population growth, increasing energy demand and the depletion of fossil fuel reserves necessitate a search for sustainable alternatives for electricity generation. Hydropower could replace a large part of the contribution of gas and oil to the present energy mix. However, previous high-resolution

  14. Dual camera system for acquisition of high resolution images

    Science.gov (United States)

    Papon, Jeremie A.; Broussard, Randy P.; Ives, Robert W.

    2007-02-01

    Video surveillance is ubiquitous in modern society, but surveillance cameras are severely limited in utility by their low resolution. With this in mind, we have developed a system that can autonomously take high resolution still frame images of moving objects. In order to do this, we combine a low resolution video camera and a high resolution still frame camera mounted on a pan/tilt mount. In order to determine what should be photographed (objects of interest), we employ a hierarchical method which first separates foreground from background using a temporal-based median filtering technique. We then use a feed-forward neural network classifier on the foreground regions to determine whether the regions contain the objects of interest. This is done over several frames, and a motion vector is deduced for the object. The pan/tilt mount then focuses the high resolution camera on the next predicted location of the object, and an image is acquired. All components are controlled through a single MATLAB graphical user interface (GUI). The final system we present will be able to detect multiple moving objects simultaneously, track them, and acquire high resolution images of them. Results will demonstrate performance tracking and imaging varying numbers of objects moving at different speeds.

  15. pattern of interstitial lung disease as seen by high resolution

    African Journals Online (AJOL)

    2012-09-01

    Sep 1, 2012 ... Background: Diffuse lung diseases constitute a major cause of morbidity and mortality worldwide. High Resolution Computed Tomography (HRCT) is the recommended imaging technique in the diagnosis, assessment and followup of these diseases. Objectives: To describe the pattern of HRCT findings in ...

  16. FMC cameras, high resolution films and very large scale mapping

    Science.gov (United States)

    Tachibana, Kikuo; Hasegawa, Hiroyuki

    1988-06-01

    Very large scale mapping (1/250) was experimented on the basis of FMC camera, high resolution film and total station surveying. The future attractive combination of precision photogrammetry and personal computer assisted terrestrial surveying was investigated from the point of view of accuracy, time effectiveness and total procedures control.

  17. Signal Processing for High Resolution FMCW SAR and Moving Target

    NARCIS (Netherlands)

    Meta, A.; Hoogeboom, P.

    2005-01-01

    The combination of Frequency Modulated ContinuousWave (FMCW) technology and Synthetic Aperture Radar (SAR) leads to lightweight, cost-effective imaging sensors of high resolution. In FMCW SAR applications the conventional stop-and-go approximation used in pulse radar algorithms cannot be considered

  18. Impact of high resolution land surface initialization in Indian summer ...

    Indian Academy of Sciences (India)

    The direct impact of high resolution land surface initialization on the forecast bias in a regional climatemodel in recent years over Indian summer monsoon region is investigated. Two sets of regional climatemodel simulations are performed, one with a coarse resolution land surface initial conditions and secondone used a ...

  19. Plant respirometer enables high resolution of oxygen consumption rates

    Science.gov (United States)

    Foster, D. L.

    1966-01-01

    Plant respirometer permits high resolution of relatively small changes in the rate of oxygen consumed by plant organisms undergoing oxidative metabolism in a nonphotosynthetic state. The two stage supply and monitoring system operates by a differential pressure transducer and provides a calibrated output by digital or analog signals.

  20. Towards high resolution data assimilation and ensemble forecasting

    NARCIS (Netherlands)

    Stappers, R.J.J.

    2013-01-01

    Due the increase in computational power of supercomputers the grid resolution of high resolution numerical weather prediction models is now reaching the 1 km scale. As a result, mesoscale processes related to high impact weather (such as deep convection) can now explicitly be resolved by the models.

  1. Reproducible high-resolution multispectral image acquisition in dermatology

    Science.gov (United States)

    Duliu, Alexandru; Gardiazabal, José; Lasser, Tobias; Navab, Nassir

    2015-07-01

    Multispectral image acquisitions are increasingly popular in dermatology, due to their improved spectral resolution which enables better tissue discrimination. Most applications however focus on restricted regions of interest, imaging only small lesions. In this work we present and discuss an imaging framework for high-resolution multispectral imaging on large regions of interest.

  2. High-resolution axial MR imaging of tibial stress injuries

    Science.gov (United States)

    2012-01-01

    Purpose To evaluate the relative involvement of tibial stress injuries using high-resolution axial MR imaging and the correlation with MR and radiographic images. Methods A total of 33 patients with exercise-induced tibial pain were evaluated. All patients underwent radiograph and high-resolution axial MR imaging. Radiographs were taken at initial presentation and 4 weeks later. High-resolution MR axial images were obtained using a microscopy surface coil with 60 × 60 mm field of view on a 1.5T MR unit. All images were evaluated for abnormal signals of the periosteum, cortex and bone marrow. Results Nineteen patients showed no periosteal reaction at initial and follow-up radiographs. MR imaging showed abnormal signals in the periosteal tissue and partially abnormal signals in the bone marrow. In 7 patients, periosteal reaction was not seen at initial radiograph, but was detected at follow-up radiograph. MR imaging showed abnormal signals in the periosteal tissue and entire bone marrow. Abnormal signals in the cortex were found in 6 patients. The remaining 7 showed periosteal reactions at initial radiograph. MR imaging showed abnormal signals in the periosteal tissue in 6 patients. Abnormal signals were seen in the partial and entire bone marrow in 4 and 3 patients, respectively. Conclusions Bone marrow abnormalities in high-resolution axial MR imaging were related to periosteal reactions at follow-up radiograph. Bone marrow abnormalities might predict later periosteal reactions, suggesting shin splints or stress fractures. High-resolution axial MR imaging is useful in early discrimination of tibial stress injuries. PMID:22574840

  3. High-resolution x-ray imaging using a structured scintillator

    Energy Technology Data Exchange (ETDEWEB)

    Hormozan, Yashar, E-mail: hormozan@kth.se; Sychugov, Ilya; Linnros, Jan [Materials and Nano Physics, School of Information and Communication Technology, KTH Royal Institute of Technology, Electrum 229, Kista, Stockholm SE-16440 (Sweden)

    2016-02-15

    Purpose: In this study, the authors introduce a new generation of finely structured scintillators with a very high spatial resolution (a few micrometers) compared to conventional scintillators, yet maintaining a thick absorbing layer for improved detectivity. Methods: Their concept is based on a 2D array of high aspect ratio pores which are fabricated by ICP etching, with spacings (pitches) of a few micrometers, on silicon and oxidation of the pore walls. The pores were subsequently filled by melting of powdered CsI(Tl), as the scintillating agent. In order to couple the secondary emitted photons of the back of the scintillator array to a CCD device, having a larger pixel size than the pore pitch, an open optical microscope with adjustable magnification was designed and implemented. By imaging a sharp edge, the authors were able to calculate the modulation transfer function (MTF) of this finely structured scintillator. Results: The x-ray images of individually resolved pores suggest that they have been almost uniformly filled, and the MTF measurements show the feasibility of a few microns spatial resolution imaging, as set by the scintillator pore size. Compared to existing techniques utilizing CsI needles as a structured scintillator, their results imply an almost sevenfold improvement in resolution. Finally, high resolution images, taken by their detector, are presented. Conclusions: The presented work successfully shows the functionality of their detector concept for high resolution imaging and further fabrication developments are most likely to result in higher quantum efficiencies.

  4. Constructing forward price curves in electricity markets

    DEFF Research Database (Denmark)

    Fleten, S.-E.; Lemming, Jørgen Kjærgaard

    2003-01-01

    We present and analyze a method for constructing approximated high-resolution forward price curves in electricity markets. Because a limited number of forward or futures contracts are traded in the market, only a limited picture of the theoretical continuous forward price curve is available...

  5. High resolution reservoir geological modelling using outcrop information

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Changmin; Lin Kexiang; Liu Huaibo [Jianghan Petroleum Institute, Hubei (China)] [and others

    1997-08-01

    This is China`s first case study of high resolution reservoir geological modelling using outcrop information. The key of the modelling process is to build a prototype model and using the model as a geological knowledge bank. Outcrop information used in geological modelling including seven aspects: (1) Determining the reservoir framework pattern by sedimentary depositional system and facies analysis; (2) Horizontal correlation based on the lower and higher stand duration of the paleo-lake level; (3) Determining the model`s direction based on the paleocurrent statistics; (4) Estimating the sandbody communication by photomosaic and profiles; (6) Estimating reservoir properties distribution within sandbody by lithofacies analysis; and (7) Building the reservoir model in sandbody scale by architectural element analysis and 3-D sampling. A high resolution reservoir geological model of Youshashan oil field has been built by using this method.

  6. Fabricating High-Resolution X-Ray Collimators

    Science.gov (United States)

    Appleby, Michael; Atkinson, James E.; Fraser, Iain; Klinger, Jill

    2008-01-01

    A process and method for fabricating multi-grid, high-resolution rotating modulation collimators for arcsecond and sub-arcsecond x-ray and gamma-ray imaging involves photochemical machining and precision stack lamination. The special fixturing and etching techniques that have been developed are used for the fabrication of multiple high-resolution grids on a single array substrate. This technology has application in solar and astrophysics and in a number of medical imaging applications including mammography, computed tomography (CT), single photon emission computed tomography (SPECT), and gamma cameras used in nuclear medicine. This collimator improvement can also be used in non-destructive testing, hydrodynamic weapons testing, and microbeam radiation therapy.

  7. The theory and practice of high resolution scanning electron microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Joy, D.C. (Tennessee Univ., Knoxville, TN (USA) Oak Ridge National Lab., TN (USA))

    1990-01-01

    Recent advances in instrumentation have produced the first commercial examples of what can justifiably be called High Resolution Scanning Electron Microscopes. The key components of such instruments are a cold field emission gun, a small-gap immersion probe-forming lens, and a clean dry-pumped vacuum. The performance of these microscopes is characterized by several major features including a spatial resolution, in secondary electron mode on solid specimens, which can exceed 1nm on a routine basis; an incident probe current density of the order of 10{sup 6} amps/cm{sup 2}; and the ability to maintain these levels of performance over an accelerating voltage range of from 1 to 30keV. This combination of high resolution, high probe current, low contamination and flexible electron-optical conditions provides many new opportunitites for the application of the SEM to materials science, physics, and the life sciences. 27 refs., 14 figs.

  8. Automatic Matching of High Resolution Satellite Images Based on RFM

    Directory of Open Access Journals (Sweden)

    JI Shunping

    2016-02-01

    Full Text Available A matching method for high resolution satellite images based on RFM is presented.Firstly,the RFM parameters are used to predict the initial parallax of corresponding points and the prediction accuracy is analyzed.Secondly,the approximate epipolar equation is constructed based on projection tracking and its accuracy is analyzed.Thirdly,approximate 1D image matching is executed on pyramid images and least square matching on base images.At last RANSAC is imbedded to eliminate mis-matching points and matching results are obtained.Test results verified the method more robust and with higher matching rate,compared to 2D gray correlation method and the popular SIFT matching method,and the method preferably solved the question of high resolution satellite image matching with different stereo model,different time and large rotation images.

  9. High-Resolution Fluorescence Microscope Imaging of Erythroblast Structure.

    Science.gov (United States)

    Smith, Alyson S; Nowak, Roberta B; Fowler, Velia M

    2018-01-01

    During erythropoiesis, erythroblasts undergo dramatic morphological changes to produce mature erythrocytes. Many unanswered questions regarding the molecular mechanisms behind these changes can be addressed with high-resolution fluorescence imaging. Immunofluoresence staining enables localization of specific molecules, organelles, and membrane components in intact cells at different phases of erythropoiesis. Confocal laser scanning microscopy can provide high-resolution, three-dimensional images of stained structures, which can be used to dissect the molecular mechanisms driving erythropoiesis. The sample preparation, staining procedure, imaging parameters, and image analysis methods used directly affect the quality of the confocal images and the amount and accuracy of information that they can provide. Here, we describe methods to dissect erythropoietic tissues from mice, to perform immunofluorescence staining and confocal imaging of various molecules, organelles and structures of interest in erythroblasts, and to present and quantitatively analyze the data obtained in these fluorescence images.

  10. Sparse Recovery Analysis of High-Resolution Climate Data

    Science.gov (United States)

    Archibald, R.

    2013-12-01

    The field of compressed sensing is vast and currently very active, with new results, methods, and algorithms appearing almost daily. The first notions of compressed sensing began with Prony's method, which was designed by the French mathematician Gaspard Riche de Prony to extract signal information from a limited number of measurements. Since then, sparsity has been used empirically in a variety of applications, including geology and geophysics, spectroscopy, signal processing, radio astronomy, and medical ultrasound. High-resolution climate studies performed on large scale high performance computing have been producing large amounts of data that can benefit from unique mathematical methods for analysis. This work demonstrates how sparse recovery and L1 regularization can be used effectively on large datasets from high-resolution climate studies.

  11. Design of UAV high resolution image transmission system

    Science.gov (United States)

    Gao, Qiang; Ji, Ming; Pang, Lan; Jiang, Wen-tao; Fan, Pengcheng; Zhang, Xingcheng

    2017-02-01

    In order to solve the problem of the bandwidth limitation of the image transmission system on UAV, a scheme with image compression technology for mini UAV is proposed, based on the requirements of High-definition image transmission system of UAV. The video codec standard H.264 coding module and key technology was analyzed and studied for UAV area video communication. Based on the research of high-resolution image encoding and decoding technique and wireless transmit method, The high-resolution image transmission system was designed on architecture of Android and video codec chip; the constructed system was confirmed by experimentation in laboratory, the bit-rate could be controlled easily, QoS is stable, the low latency could meets most applied requirement not only for military use but also for industrial applications.

  12. A Procedure for High Resolution Satellite Imagery Quality Assessment

    Directory of Open Access Journals (Sweden)

    Mattia Crespi

    2009-05-01

    Full Text Available Data products generated from High Resolution Satellite Imagery (HRSI are routinely evaluated during the so-called in-orbit test period, in order to verify if their quality fits the desired features and, if necessary, to obtain the image correction parameters to be used at the ground processing center. Nevertheless, it is often useful to have tools to evaluate image quality also at the final user level. Image quality is defined by some parameters, such as the radiometric resolution and its accuracy, represented by the noise level, and the geometric resolution and sharpness, described by the Modulation Transfer Function (MTF. This paper proposes a procedure to evaluate these image quality parameters; the procedure was implemented in a suitable software and tested on high resolution imagery acquired by the QuickBird, WorldView-1 and Cartosat-1 satellites.

  13. Scalable, flexible and high resolution patterning of CVD graphene.

    Science.gov (United States)

    Hofmann, Mario; Hsieh, Ya-Ping; Hsu, Allen L; Kong, Jing

    2014-01-07

    The unique properties of graphene make it a promising material for interconnects in flexible and transparent electronics. To increase the commercial impact of graphene in those applications, a scalable and economical method for producing graphene patterns is required. The direct synthesis of graphene from an area-selectively passivated catalyst substrate can generate patterned graphene of high quality. We here present a solution-based method for producing patterned passivation layers. Various deposition methods such as ink-jet deposition and microcontact printing were explored, that can satisfy application demands for low cost, high resolution and scalable production of patterned graphene. The demonstrated high quality and nanometer precision of grown graphene establishes the potential of this synthesis approach for future commercial applications of graphene. Finally, the ability to transfer high resolution graphene patterns onto complex three-dimensional surfaces affords the vision of graphene-based interconnects in novel electronics.

  14. Space to Think: Large, High-Resolution Displays for Sensemaking

    Energy Technology Data Exchange (ETDEWEB)

    Andrews, Christopher P.; Endert, Alexander; North, Chris

    2010-05-05

    Space supports human cognitive abilities in a myriad of ways. The note attached to the side of the monitor, the papers spread out on the desk, diagrams scrawled on a whiteboard, and even the keys left out on the counter are all examples of using space to recall, reveal relationships, and think. Technological advances have made it possible to construct large display environments in which space has real meaning. This paper examines how increased space affects the way displays are regarded and used within the context of the cognitively demanding task of sensemaking. A study was conducted observing analysts using a prototype large, high-resolution display to solve an analytic problem. This paper reports on the results of this study and suggests a number of potential design criteria for future sensemaking tools developed for large, high-resolution displays.

  15. Automated data processing of high-resolution mass spectra

    DEFF Research Database (Denmark)

    Hansen, Michael Adsetts Edberg; Smedsgaard, Jørn

    infusion of crude extracts into the source taking advantage of the high sensitivity, high mass resolution and accuracy and the limited fragmentation. Unfortunately, there has not been a comparable development in the data processing techniques to fully exploit gain in high resolution and accuracy...... of the massive amounts of data. We present an automated data processing method to quantitatively compare large numbers of spectra from the analysis of complex mixtures, exploiting the full quality of high-resolution mass spectra. By projecting all detected ions - within defined intervals on both the time...... infusion analyses of crude extract to find the relationship between species from several species terverticillate Penicillium, and also that the ions responsible for the segregation can be identified. Furthermore the process can automate the process of detecting unique species and unique metabolites....

  16. High-resolution dynamical modelling of the Antarctic stratospheric vortex

    Science.gov (United States)

    Haynes, P. H.

    1988-01-01

    Progress is reported on the high-resolution three-dimensional numerical simulation of flows characteristic of the Antarctic wintertime stratosphere. The numerical model is a modified version of the Reading University sigma-coordinate used previously for tropospheric studies. Physical parameterizations are kept to a minimum in order to concentrate as much computing power as possible on simulating details of the dynamical processes. The major question addressed is whether the features observed in recent high-resolution two-dimensional simulations - namely: (1) the formation of a sharp edge to the vortex (seen in the potential vorticity field), (2) the survival of the polar vortex in a material entity, and (3) the formation of small-scale eddies rough the break-up of tongues of high potential vorticity drawn out from the polar vortex - are realized in three-dimensional simulations.

  17. Quantitative High-Resolution Transmission Electron Microscopy of Single Atoms

    OpenAIRE

    Gamm, B.; Popescu, R.; Blank, H.; Schneider, R; Beyer, A.; Gölzhäuser, A.; Gerthsen, D.

    2010-01-01

    Single atoms can be considered as basic objects for electron microscopy to test the microscope performance and basic concepts for modeling of image contrast. In this work high-resolution transmission electron microscopy was applied to image single platinum atoms in an aberration-corrected transmission electron microscope. The atoms are deposited on a self-assembled monolayer substrate which induces only negligible contrast. Single-atom contrast simulations were performed on the basis of Weick...

  18. High-resolution Imaging Techniques for the Assessment of Osteoporosis

    OpenAIRE

    Krug, Roland; Burghardt, Andrew J.; Majumdar, Sharmila; Link, Thomas M.

    2010-01-01

    The importance of assessing the bone’s microarchitectural make-up in addition to its mineral density in the context of osteoporosis has been emphasized in a number of publications. The high spatial resolution required to resolve the bone’s microstructure in a clinically feasible scan time is challenging. Currently, the best suited modalities meeting these requirements in vivo are high-resolution peripheral quantitative imaging (HR-pQCT) and magnetic resonance imaging (MRI). Whereas HR-pQCT is...

  19. Concept for a new high resolution high intensity diffractometer

    Energy Technology Data Exchange (ETDEWEB)

    Stuhr, U. [Paul Scherrer Inst. (PSI), Villigen (Switzerland)

    1997-09-01

    A concept of a new time-of-flight powder-diffractometer for a thermal neutral beam tube at SINQ is presented. The design of the instrument optimises the contradictory conditions of high intensity and high resolution. The high intensity is achieved by using many neutron pulses simultaneously. By analysing the time-angle-pattern of the detected neutrons an assignment of the neutrons to a single pulse is possible. (author) 3 figs., tab., refs.

  20. A high resolution cavity BPM for the CLIC Test Facility

    CERN Document Server

    Chritin, N.; Soby, L.; Lunin, A.; Solyak, N.; Wendt, M.; Yakovlev, V.

    2012-01-01

    In frame of the development of a high resolution BPM system for the CLIC Main Linac we present the design of a cavity BPM prototype. It consists of a waveguide loaded dipole mode resonator and a monopole mode reference cavity, both operating at 15 GHz, to be compatible with the bunch frequencies at the CLIC Test Facility. Requirements, design concept, numerical analysis, and practical considerations are discussed.

  1. Automatic Matching of High Resolution Satellite Images Based on RFM

    OpenAIRE

    JI Shunping; YUAN Xiuxiao

    2016-01-01

    A matching method for high resolution satellite images based on RFM is presented.Firstly,the RFM parameters are used to predict the initial parallax of corresponding points and the prediction accuracy is analyzed.Secondly,the approximate epipolar equation is constructed based on projection tracking and its accuracy is analyzed.Thirdly,approximate 1D image matching is executed on pyramid images and least square matching on base images.At last RANSAC is imbedded to eliminate mis-matching points...

  2. Tuberculous otitis media: findings on high-resolution CT

    Energy Technology Data Exchange (ETDEWEB)

    Lungenschmid, D. [Dept. of Radiodiagnostics, University Hospital Innsbruck (Austria)]|[Dept. of Magnetic Resonance and Spectroscopy, University Hospital of Innsbruck (Austria); Buchberger, W. [Dept. of Radiodiagnostics, University Hospital Innsbruck (Austria)]|[Dept. of Magnetic Resonance and Spectroscopy, University Hospital of Innsbruck (Austria); Schoen, G. [Dept. of Radiodiagnostics, University Hospital Innsbruck (Austria); Schoepf, R. [Radiologic Inst., Landeck (Austria); Mihatsch, T. [Dept. of Oto-Rhino-Laryngology, University Hospital of Innsbruck (Austria); Birbamer, G. [Dept. of Magnetic Resonance and Spectroscopy, University Hospital of Innsbruck (Austria); Wicke, K. [Inst. of Computed Tomography, University Hospital of Innsbruck (Austria)

    1993-12-01

    We describe two cases of tuberculous otitis media studied with high-resolution computed tomography (CT). Findings included extensive soft tissue densities with fluid levels in the tympanic cavity, the antrum, the mastoid and petrous air cells. Multifocal bony erosions and reactive bone sclerosis were seen as well. CT proved valuable for planning therapy by accurately displaying the involvement of the various structures of the middle and inner ear. However, the specific nature of the disease could only be presumed. (orig.)

  3. High-Resolution Wind Measurements for Offshore Wind Energy Development

    Science.gov (United States)

    Nghiem, Son V.; Neumann, Gregory

    2011-01-01

    A mathematical transform, called the Rosette Transform, together with a new method, called the Dense Sampling Method, have been developed. The Rosette Transform is invented to apply to both the mean part and the fluctuating part of a targeted radar signature using the Dense Sampling Method to construct the data in a high-resolution grid at 1-km posting for wind measurements over water surfaces such as oceans or lakes.

  4. Acute pulmonary injury: high-resolution CT and histopathological spectrum

    Science.gov (United States)

    Obadina, E T; Torrealba, J M

    2013-01-01

    Acute lung injury usually causes hypoxaemic respiratory failure and acute respiratory distress syndrome (ARDS). Although diffuse alveolar damage is the hallmark of ARDS, other histopathological patterns of injury, such as acute and fibrinoid organising pneumonia, can be associated with acute respiratory failure. Acute eosinophilic pneumonia can also cause acute hypoxaemic respiratory failure and mimic ARDS. This pictorial essay reviews the high-resolution CT findings of acute lung injury and the correlative histopathological findings. PMID:23659926

  5. High Resolution Analysis of Copy Number Mutation in Breast Cancer

    Science.gov (United States)

    2005-05-01

    Pon , in Polysaccharides in Medic- copy number at high resolution throughout the other diseases, we must distinguish abnormal inal Applications, S...was determined to in- leles . In all experiments, there were a total of silico from the human genome sequence as- volve an interchromosomal duplication...well (3), although we do not explore that approach here. PON ) = e -pb o#regular( - )#deviated [1] The negative log likelihood function satisfies an

  6. High Resolution Wavenumber Standards for the Infrared. (IUPAC Recommendations 1995)

    Science.gov (United States)

    Guelachvili, G.; Birk, M.; Bord, C.; Brault, J.; Brown, L.; Carli, B.; Cole, A.; Evenson, D.; Fayt, A.; Hausamann, D.; hide

    1995-01-01

    The calibration of high resolution infrared spectra is generally more precise than accurate even when they are recorded with Fourier interferometers. In order to improve the consistency of the spectral measurements, an IUPAC project has been undertaken. Its aim was to recommend a selection of spectral lines as wavenumber standards for absolute calibration in the infrared. This paper will report the final recommendations in the spectral range extending from about 4 to about 7000 cm(be).

  7. Validation of AIRS high-resolution stratospheric temperature retrievals

    Science.gov (United States)

    Meyer, Catrin I.; Hoffmann, Lars

    2014-10-01

    This paper focuses on stratospheric temperature observations by the Atmospheric InfraRed Sounder (AIRS) aboard NASA's Aqua satellite. We validate a nine-year record (2003 - 2011) of data retrieved with a scientific retrieval processor independent from the operational processor operated by NASA. The retrieval discussed here provides stratospheric temperature profiles for each individual AIRS footprint and has nine times better horizontal sampling than the operational data provided by NASA. The high-resolution temperature data are considered optimal for for gravity wave studies. For validation the high-resolution retrieval data are compared with results from the AIRS operational Level-2 data and the ERA-Interim meteorological reanalysis. Due to the large amount of data we performed statistical comparisons of monthly zonal mean cross-sections and time series. The comparisons show that the high-resolution temperature data are in good agreement with the validation data sets. The bias in the zonal averages is mostly within ±2K. The bias reaches a maximum of 7K to ERA-Interim and 4K to the AIRS operational data at the stratopause, it is related to the different resolutions of the data sets. Variability is nearly the same in all three data sets, having maximum standard deviations around the polar vortex in the mid and upper stratosphere. The validation presented here indicates that the high-resolution temperature retrievals are well-suited for scientific studies. In particular, we expect that they will become a valuable asset for future studies of stratospheric gravity waves.

  8. High-resolution x-ray photoemission spectra of silver

    DEFF Research Database (Denmark)

    Barrie, A.; Christensen, N. E.

    1976-01-01

    An electron spectrometer fitted with an x-ray monochromator for Al Kα1,2 radiation (1486.6 eV) has been used to record high-resolution x-ray photoelectron spectra for the 4d valence band as well as the 3d spin doublet in silver. The core-level spectrum has a line shape that can be described...

  9. High Resolution Measurements and Electronic Structure Calculations of a Diazanaphthalene

    Science.gov (United States)

    Gruet, Sébastien; Goubet, Manuel; Pirali, Olivier

    2014-06-01

    Polycyclic Aromatic Hydrocarbons (PAHs) have long been suspected to be the carriers of so called Unidentified Infrared Bands (UIBs). Most of the results published in the literature report rotationally unresolved spectra of pure carbon as well as heteroatom-containing PAHs species. To date for this class of molecules, the principal source of rotational informations is ruled by microwave (MW) spectroscopy while high resolution measurements reporting rotational structure of the infrared (IR) vibrational bands are very scarce. Recently, some high resolution techniques provided interesting new results to rotationally resolve the IR and far-IR bands of these large carbonated molecules of astrophysical interest. One of them is to use the bright synchrotron radiation as IR continuum source of a high resolution Fourier transform (FTIR) spectrometer. We report the very complementary analysis of the [1,6] naphthyridine (a N-bearing PAH) for which we recorded the microwave spectrum at the PhLAM laboratory (Lille) and the high resolution far-infrared spectrum on the AILES beamline at synchrotron facility SOLEIL. MW spectroscopy provided highly accurate rotational constants in the ground state to perform Ground State Combinations Differences (GSCD) allowing the analysis of the two most intense FT-FIR bands in the 50-900 wn range. Moreover, during this presentation the negative value of the inertial defect in the GS of the molecule will be discussed. A. Leger, J. L. Puget, Astron. Astrophys. 137, L5-L8 (1984) L. J. Allamandola et al. Astrophys. J. 290, L25-L28 (1985). Z. Kisiel et al. J. Mol. Spectrosc. 217, 115 (2003) S. Thorwirth et al. Astrophys. J. 662, 1309 (2007) D. McNaughton et al. J. Chem. Phys. 124, 154305 (2011). S. Albert et al. Faraday Discuss. 150, 71-99 (2011) B. E. Brumfield et al. Phys. Chem. Lett. 3, 1985-1988 (2012) O. Pirali et al. Phys. Chem. Chem. Phys. 15, 10141 (2013).

  10. High-resolution neutron microtomography with noiseless neutron counting detector

    Science.gov (United States)

    Tremsin, A. S.; McPhate, J. B.; Vallerga, J. V.; Siegmund, O. H. W.; Feller, W. B.; Lehmann, E.; Butler, L. G.; Dawson, M.

    2011-10-01

    The improved collimation and intensity of thermal and cold neutron beamlines combined with recent advances in neutron imaging devices enable high-resolution neutron radiography and microtomography, which can provide information on the internal structure of objects not achievable with conventional X-ray imaging techniques. Neutron detection efficiency, spatial and temporal resolution (important for the studies of dynamic processes) and low background count rate are among the crucial parameters defining the quality of radiographic images and tomographic reconstructions. The unique capabilities of neutron counting detectors with neutron-sensitive microchannel plates (MCPs) and with Timepix CMOS readouts providing high neutron detection efficiency (˜70% for cold neutrons), spatial resolutions ranging from 15 to 55 μm and a temporal resolution of ˜1 μs—combined with the virtual absence of readout noise—make these devices very attractive for high-resolution microtomography. In this paper we demonstrate the capabilities of an MCP-Timepix detection system applied to microtomographic imaging, performed at the ICON cold neutron facility of the Paul Scherrer Institute. The high resolution and the absence of readout noise enable accurate reconstruction of texture in a relatively opaque wood sample, differentiation of internal tissues of a fly and imaging of individual ˜400 μm grains in an organic powder encapsulated in a ˜700 μm thick metal casing.

  11. High-resolution CT findings in Streptococcus milleri pulmonary infection.

    Science.gov (United States)

    Okada, F; Ono, A; Ando, Y; Nakayama, T; Ishii, H; Hiramatsu, K; Sato, H; Kira, A; Otabe, M; Mori, H

    2013-06-01

    To assess pulmonary high-resolution computed tomography (CT) findings in patients with acute Streptococcus milleri pulmonary infection. Sixty consecutive patients with acute S. milleri pneumonia who had undergone high-resolution CT chest examinations between January 2004 and March 2010 were retrospectively identified. Twenty-seven patients with concurrent infections were excluded. The final study group comprised 33 patients (25 men, 8 women; aged 20-88 years, mean 63.1 years) with S. milleri infection. The patients' clinical findings were assessed. Parenchymal abnormalities, enlarged lymph nodes, and pleural effusion were evaluated on high-resolution CT. Underlying conditions included malignancy (n = 15), a smoking habit (n = 11), and diabetes mellitus (n = 8). CT images of all patients showed abnormal findings, including ground-glass opacity (n = 24), bronchial wall thickening (n = 23), consolidation (n = 17), and cavities (n = 7). Pleural effusion was found in 18 patients, and complex pleural effusions were found in seven patients. Pulmonary infection caused by S. milleri was observed mostly in male patients with underlying conditions such as malignancy or a smoking habit. The CT findings in patients with S. milleri consisted mainly of ground-glass opacity, bronchial wall thickening, pleural effusions, and cavities. Copyright © 2013 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

  12. Progress in high-resolution x-ray holographic microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Jacobsen, C.; Kirz, J.; Howells, M.; McQuaid, K.; Rothman, S.; Feder, R.; Sayre, D.

    1987-07-01

    Among the various types of x-ray microscopes that have been demonstrated, the holographic microscope has had the largest gap between promise and performance. The difficulties of fabricating x-ray optical elements have led some to view holography as the most attractive method for obtaining the ultimate in high resolution x-ray micrographs; however, we know of no investigations prior to 1987 that clearly demonstrated submicron resolution in reconstructed images. Previous efforts suffered from problems such as limited resolution and dynamic range in the recording media, low coherent x-ray flux, and aberrations and diffraction limits in visible light reconstruction. We have addressed the recording limitations through the use of an undulator x-ray source and high-resolution photoresist recording media. For improved results in the readout and reconstruction steps, we have employed metal shadowing and transmission electron microscopy, along with numerical reconstruction techniques. We believe that this approach will allow holography to emerge as a practical method of high-resolution x-ray microscopy. 30 refs., 4 figs.

  13. Adaptive optics with pupil tracking for high resolution retinal imaging.

    Science.gov (United States)

    Sahin, Betul; Lamory, Barbara; Levecq, Xavier; Harms, Fabrice; Dainty, Chris

    2012-02-01

    Adaptive optics, when integrated into retinal imaging systems, compensates for rapidly changing ocular aberrations in real time and results in improved high resolution images that reveal the photoreceptor mosaic. Imaging the retina at high resolution has numerous potential medical applications, and yet for the development of commercial products that can be used in the clinic, the complexity and high cost of the present research systems have to be addressed. We present a new method to control the deformable mirror in real time based on pupil tracking measurements which uses the default camera for the alignment of the eye in the retinal imaging system and requires no extra cost or hardware. We also present the first experiments done with a compact adaptive optics flood illumination fundus camera where it was possible to compensate for the higher order aberrations of a moving model eye and in vivo in real time based on pupil tracking measurements, without the real time contribution of a wavefront sensor. As an outcome of this research, we showed that pupil tracking can be effectively used as a low cost and practical adaptive optics tool for high resolution retinal imaging because eye movements constitute an important part of the ocular wavefront dynamics.

  14. Sharpening high resolution information in single particle electron cryomicroscopy.

    Science.gov (United States)

    Fernández, J J; Luque, D; Castón, J R; Carrascosa, J L

    2008-10-01

    Advances in single particle electron cryomicroscopy have made possible to elucidate routinely the structure of biological specimens at subnanometer resolution. At this resolution, secondary structure elements are discernable by their signature. However, identification and interpretation of high resolution structural features are hindered by the contrast loss caused by experimental and computational factors. This contrast loss is traditionally modeled by a Gaussian decay of structure factors with a temperature factor, or B-factor. Standard restoration procedures usually sharpen the experimental maps either by applying a Gaussian function with an inverse ad hoc B-factor, or according to the amplitude decay of a reference structure. EM-BFACTOR is a program that has been designed to widely facilitate the use of the novel method for objective B-factor determination and contrast restoration introduced by Rosenthal and Henderson [Rosenthal, P.B., Henderson, R., 2003. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721-745]. The program has been developed to interact with the most common packages for single particle electron cryomicroscopy. This sharpening method has been further investigated via EM-BFACTOR, concluding that it helps to unravel the high resolution molecular features concealed in experimental density maps, thereby making them better suited for interpretation. Therefore, the method may facilitate the analysis of experimental data in high resolution single particle electron cryomicroscopy.

  15. Glacial lake mapping with very high resolution satellite SAR data

    Science.gov (United States)

    Strozzi, T.; Wiesmann, A.; Kääb, A.; Joshi, S.; Mool, P.

    2012-08-01

    Floods resulting from the outbursts of glacial lakes are among the most far-reaching disasters in high mountain regions. Glacial lakes are typically located in remote areas and space-borne remote sensing data are an important source of information about the occurrence and development of such lakes. Here we show that very high resolution satellite Synthetic Aperture Radar (SAR) data can be employed for reliably mapping glacial lakes. Results in the Alps, Pamir and Himalaya using TerraSAR-X and Radarsat-2 data are discussed in comparison to in-situ information, and high-resolution satellite optical and radar imagery. The performance of the satellite SAR data is best during the snow- and ice-free season. In the broader perspective of hazard management, the detection of glacial lakes and the monitoring of their changes from very high-resolution satellite SAR intensity images contributes to the initial assessment of hazards related to glacial lakes, but a more integrated, multi-level approach needs also to include other relevant information such as glacier outlines and outline changes or the identification of unstable slopes above the lake and the surrounding area, information types to which SAR analysis techniques can also contribute.

  16. High Resolution Observations of Escaping Ions in the Martian Magnetotail

    Science.gov (United States)

    Halekas, J. S.; Raman, C.; Brain, D.; DiBraccio, G. A.; Harada, Y.; McFadden, J. P.; Mitchell, D. L.; Connerney, J. E. P.; Jakosky, B. M.

    2016-12-01

    Ions escape from the Martian upper atmosphere via a number of channels, including the central plasmasheet of the magnetotail. Mars Express observations show that the heavy ions O+ and O2+ escaping through the central tail often have approximately the same energy, suggesting acceleration in a quasi-static electric field, which has been interpreted as a Hall electric field. The Solar Wind Ion Analyzer (SWIA) on MAVEN was designed to measure the upstream solar wind. However, during orbit segments with appropriate spacecraft attitude, SWIA can also make high resolution measurements of escaping ions in the tail. During the prime mission, these observations were only returned sporadically, during periods of intense escaping fluxes that fortuitously triggered a mode switch. Now, in the extended mission, we return high resolution observations from SWIA routinely. Some of these high resolution measurements reveal slight differences in both the direction and energy of escaping O+ and O2+ ions, which may help determine the acceleration process(es). We investigate the location and solar wind conditions for which the escaping ions separate in energy and angle and the systematics of their energies and flow vectors, and discuss the implications for ion acceleration and the overall picture of Martian atmospheric escape.

  17. High resolution, MRI-based, segmented, computerized head phantom

    Energy Technology Data Exchange (ETDEWEB)

    Zubal, I.G.; Harrell, C.R.; Smith, E.O.; Smith, A.L.; Krischlunas, P. [Yale Univ., New Haven, CT (United States). Dept. of Diagnostic Radiology

    1999-01-01

    The authors have created a high-resolution software phantom of the human brain which is applicable to voxel-based radiation transport calculations yielding nuclear medicine simulated images and/or internal dose estimates. A software head phantom was created from 124 transverse MRI images of a healthy normal individual. The transverse T2 slices, recorded in a 256x256 matrix from a GE Signa 2 scanner, have isotropic voxel dimensions of 1.5 mm and were manually segmented by the clinical staff. Each voxel of the phantom contains one of 62 index numbers designating anatomical, neurological, and taxonomical structures. The result is stored as a 256x256x128 byte array. Internal volumes compare favorably to those described in the ICRP Reference Man. The computerized array represents a high resolution model of a typical human brain and serves as a voxel-based anthropomorphic head phantom suitable for computer-based modeling and simulation calculations. It offers an improved realism over previous mathematically described software brain phantoms, and creates a reference standard for comparing results of newly emerging voxel-based computations. Such voxel-based computations lead the way to developing diagnostic and dosimetry calculations which can utilize patient-specific diagnostic images. However, such individualized approaches lack fast, automatic segmentation schemes for routine use; therefore, the high resolution, typical head geometry gives the most realistic patient model currently available.

  18. Adaptive optics high resolution spectroscopy: present status and future direction

    Energy Technology Data Exchange (ETDEWEB)

    Alcock, C; Angel, R; Ciarlo, D; Fugate, R O; Ge, J; Kuzmenko, P; Lloyd-Hart, M; Macintosh, B; Najita, J; Woolf, N

    1999-07-27

    High resolution spectroscopy experiments with visible adaptive optics (AO) telescopes at Starfire Optical Range and Mt. Wilson have demonstrated that spectral resolution can be routinely improved by a factor of - 10 over the seeing-limited case with no extra light losses at visible wavelengths. With large CCDs now available, a very wide wavelength range can be covered in a single exposure. In the near future, most large ground-based telescopes will be equipped with powerful A0 systems. Most of these systems are aimed primarily at diffraction-limited operation in the near IR. An exciting new opportunity will thus open up for high resolution IR spectroscopy. Immersion echelle gratings with much coarser grooves being developed by us at LLNL will play a critical role in achieving high spectral resolution with a compact and low cost IR cryogenically cooled spectrograph and simultaneous large wavelength coverage on relatively small IR detectors. We have constructed a new A0 optimized spectrograph at Steward Observatory to provide R = 200,000 in the optical, which is being commissioned at the Starfire Optical Range 3.5m telescope. We have completed the optical design of the LLNL IR Immersion Spectrograph (LISPEC) to take advantage of improved silicon etching technology. Key words: adaptive optics, spectroscopy, high resolution, immersion gratings

  19. Climatologies at high resolution for the earth's land surface areas

    Science.gov (United States)

    Karger, Dirk Nikolaus; Conrad, Olaf; Böhner, Jürgen; Kawohl, Tobias; Kreft, Holger; Soria-Auza, Rodrigo Wilber; Zimmermann, Niklaus E.; Linder, H. Peter; Kessler, Michael

    2017-09-01

    High-resolution information on climatic conditions is essential to many applications in environmental and ecological sciences. Here we present the CHELSA (Climatologies at high resolution for the earth's land surface areas) data of downscaled model output temperature and precipitation estimates of the ERA-Interim climatic reanalysis to a high resolution of 30 arc sec. The temperature algorithm is based on statistical downscaling of atmospheric temperatures. The precipitation algorithm incorporates orographic predictors including wind fields, valley exposition, and boundary layer height, with a subsequent bias correction. The resulting data consist of a monthly temperature and precipitation climatology for the years 1979-2013. We compare the data derived from the CHELSA algorithm with other standard gridded products and station data from the Global Historical Climate Network. We compare the performance of the new climatologies in species distribution modelling and show that we can increase the accuracy of species range predictions. We further show that CHELSA climatological data has a similar accuracy as other products for temperature, but that its predictions of precipitation patterns are better.

  20. Space Curves

    CERN Document Server

    Peskine, Christian; Sernesi, Edoardo

    1987-01-01

    The main topics of the conference on "Curves in Projective Space" were good and bad families of projective curves, postulation of projective space curves and classical problems in enumerative geometry.

  1. Identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by righ-resolution melting analysis.

    Directory of Open Access Journals (Sweden)

    Hllytchaikra Ferraz Fehlberg

    Full Text Available The objective of this study was to standardize the high-resolution melting method for identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by amplification of 18S ribosomal DNA (rDNA using a single primer pair. The analyses were performed on individual reactions (containing DNA from a single species of a protozoan, on duplex reactions (containing DNA from two species of protozoa in each reaction, and on a multiplex reaction (containing DNA of four parasites in a single reaction. The proposed method allowed us to identify and discriminate the four species by analyzing the derivative, normalized, and difference melting curves, with high reproducibility among and within the experiments, as demonstrated by low coefficients of variation (less than 2.2% and 2.0%, respectively. This is the first study where this method is used for discrimination of these four species of protozoa in a single reaction.

  2. Continuous online field deployable high precision and high resolution water isotope analysis from ice cores

    Science.gov (United States)

    Gkinis, Vasileios; Popp, Trevor; Johnsen, Sigfus; Blunier, Thomas; Stowasser, Christopher; Kettner, Ernesto

    2010-05-01

    One of the most important features of ice cores as climate and atmospheric paleoarchives is their potential for high temporal resolution. The measurement of chemical impurities in ice cores that fully exploits this high resolution has become standard with the advent of Continuous Flow Analysis (CFA) from continuously melted ice core segments, often performed in the field. Meanwhile, the measurement of water stable isotopic composition (deuterium and oxygen-18) continues, for the most part, to be performed offline via discrete sampling with traditional mass spectroscopy. These offline procedures are time consuming, expensive, and do not fully exploit the temporal resolution available in the ice core. Here we describe a new method and the first results for the determination of water isotopic content measured from a continuous melted rod with a commercially available near-infrared cavity ring down spectrometer (CRDS; http://www.picarro.com/) coupled to a CFA system both in the field on the Greenland ice sheet and in the laboratory. For the measurement of water isotopes a carefully controlled fraction of the CFA melt stream is evaporated directly into the laser cavity for simultaneous analysis of both deuterium and oxygen-18 content. The details of the system have been tested with a deployment to the North Greenland Eemian Ice Drilling (NEEM) coupled to the University of Bern CFA system and in the laboratory with NGRIP ice coupled to the University of Copenhagen CFA system. The results show that the system already produces the highest interpretable resolution records that are comparable or somewhat less precise than traditional mass spectroscopy discrete sample methods. The enormous potential of the system is being realized in the density and resolution of the produced records in connection with other highly resolved series (e.g. chemical impurities), and also in the efficiency by which the records can be produced. Among other things, time series from this type of

  3. High resolution solar observations from first principles to applications

    Science.gov (United States)

    Verdoni, Angelo P.

    2009-10-01

    The expression "high-resolution observations" in Solar Physics refers to the spatial, temporal and spectral domains in their entirety. High-resolution observations of solar fine structure are a necessity to answer many of the intriguing questions related to solar activity. However, a researcher building instruments for high-resolution observations has to cope with the fact that these three domains often have diametrically opposed boundary conditions. Many factors have to be considered in the design of a successful instrument. Modern post-focus instruments are more closely linked with the solar telescopes that they serve than in past. In principle, the quest for high-resolution observations already starts with the selection of the observatory site. The site survey of the Advanced Technology Solar Telescope (ATST) under the stewardship of the National Solar Observatory (NSO) has identified Big Bear Solar Observatory (BBSO) as one of the best sites for solar observations. In a first step, the seeing characteristics at BBSO based on the data collected for the ATST site survey are described. The analysis will aid in the scheduling of high-resolution observations at BBSO as well as provide useful information concerning the design and implementation of a thermal control system for the New Solar Telescope (NST). NST is an off-axis open-structure Gregorian-style telescope with a 1.6 m aperture. NST will be housed in a newly constructed 5/8-sphere ventilated dome. With optics exposed to the surrounding air, NST's open-structure design makes it particularly vulnerable to the effects of enclosure-related seeing. In an effort to mitigate these effects, the initial design of a thermal control system for the NST dome is presented. The goal is to remediate thermal related seeing effects present within the dome interior. The THermal Control System (THCS) is an essential component for the open-telescope design of NST to work. Following these tasks, a calibration routine for the

  4. Proceedings of the workshop on high resolution computed microtomography (CMT)

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-02-01

    The purpose of the workshop was to determine the status of the field, to define instrumental and computational requirements, and to establish minimum specifications required by possible users. The most important message sent by implementers was the remainder that CMT is a tool. It solves a wide spectrum of scientific problems and is complementary to other microscopy techniques, with certain important advantages that the other methods do not have. High-resolution CMT can be used non-invasively and non-destructively to study a variety of hierarchical three-dimensional microstructures, which in turn control body function. X-ray computed microtomography can also be used at the frontiers of physics, in the study of granular systems, for example. With high-resolution CMT, for example, three-dimensional pore geometries and topologies of soils and rocks can be obtained readily and implemented directly in transport models. In turn, these geometries can be used to calculate fundamental physical properties, such as permeability and electrical conductivity, from first principles. Clearly, use of the high-resolution CMT technique will contribute tremendously to the advancement of current R and D technologies in the production, transport, storage, and utilization of oil and natural gas. It can also be applied to problems related to environmental pollution, particularly to spilling and seepage of hazardous chemicals into the Earth's subsurface. Applications to energy and environmental problems will be far-ranging and may soon extend to disciplines such as materials science--where the method can be used in the manufacture of porous ceramics, filament-resin composites, and microelectronics components--and to biomedicine, where it could be used to design biocompatible materials such as artificial bones, contact lenses, or medication-releasing implants. Selected papers are indexed separately for inclusion in the Energy Science and Technology Database.

  5. Measuring Large-Scale Social Networks with High Resolution

    DEFF Research Database (Denmark)

    Stopczynski, Arkadiusz; Sekara, Vedran; Sapiezynski, Piotr

    2014-01-01

    , telecommunication, social networks, location, and background information (personality, demographics, health, politics) for a densely connected population of 1 000 individuals, using state-of-the-art smartphones as social sensors. Here we provide an overview of the related work and describe the motivation...... and research agenda driving the study. Additionally, the paper details the data-types measured, and the technical infrastructure in terms of both backend and phone software, as well as an outline of the deployment procedures. We document the participant privacy procedures and their underlying principles....... The paper is concluded with early results from data analysis, illustrating the importance of multi-channel high-resolution approach to data collection....

  6. High resolution wind turbine wake measurements with a scanning lidar

    DEFF Research Database (Denmark)

    Herges, T. G.; Maniaci, D. C.; Naughton, B. T.

    2017-01-01

    High-resolution lidar wake measurements are part of an ongoing field campaign being conducted at the Scaled Wind Farm Technology facility by Sandia National Laboratories and the National Renewable Energy Laboratory using a customized scanning lidar from the Technical University of Denmark. One...... of the primary objectives is to collect experimental data to improve the predictive capability of wind plant computational models to represent the response of the turbine wake to varying inflow conditions and turbine operating states. The present work summarizes the experimental setup and illustrates several...

  7. High-resolution AMLCD for the electronic library system

    Science.gov (United States)

    Martin, Russel A.; Middo, Kathy; Turner, William D.; Lewis, Alan; Thompson, Malcolm J.; Silverstein, Louis D.

    1994-06-01

    The Electronic Library System (ELS), is a proposed data resource for the cockpit which can provide the aircrew with a vast array of technical information on their aircraft and flight plan. This information includes, but is not limited to, approach plates, Jeppeson Charts, and aircraft technical manuals. Most of these data are appropriate for digitization at high resolution (300 spi). Xerox Corporation has developed a flat panel active matrix liquid crystal display, AMLCD, that is an excellent match to the ELS, due to its innovative and aggressive design.

  8. High resolution study of magnetic ordering at absolute zero.

    Science.gov (United States)

    Lee, M; Husmann, A; Rosenbaum, T F; Aeppli, G

    2004-05-07

    High resolution pressure measurements in the zero-temperature limit provide a unique opportunity to study the behavior of strongly interacting, itinerant electrons with coupled spin and charge degrees of freedom. Approaching the precision that has become the hallmark of experiments on classical critical phenomena, we characterize the quantum critical behavior of the model, elemental antiferromagnet chromium, lightly doped with vanadium. We resolve the sharp doubling of the Hall coefficient at the quantum critical point and trace the dominating effects of quantum fluctuations up to surprisingly high temperatures.

  9. High-Resolution Reciprocal Space Mapping for Characterizing Deformation Structures

    DEFF Research Database (Denmark)

    Pantleon, Wolfgang; Wejdemann, Christian; Jakobsen, Bo

    2014-01-01

    With high-angular resolution three-dimensional X-ray diffraction (3DXRD), quantitative information is gained about dislocation structures in individual grains in the bulk of a macroscopic specimen by acquiring reciprocal space maps. In high-resolution 3D reciprocal space maps of tensile...... dynamics is followed in situ during varying loading conditions by reciprocal space mapping: during uninterrupted tensile deformation, formation of subgrains is observed concurrently with broadening of Bragg reflections shortly after the onset of plastic deformation. When the traction is terminated, stress...

  10. High resolution X-ray spectroscopy in light antiprotonic atoms

    CERN Document Server

    Borchert, G L; Augsburger, M A; Castelli, C M; Chatellard, D; Egger, J P; El-Khoury, P; Elble, M; Gorke, H; Gotta, D; Hauser, P R; Indelicato, P J; Kirch, K; Lenz, S; Nelms, N; Rashid, K; Schult, O W B; Siems, T; Simons, L M

    2000-01-01

    At the LEAR facility, CERN, antiprotonic L alpha transitions in light elements have been investigated with a focussing crystal spectrometer. The high resolution of the experiment allowed for the first time to resolve in pH/pH the 2/sup 3/P/sub 0/ state from the close-lying states 2/sup 3/P/sub 2/, 2/sup 1/P/sub 1/, and 2/sup 3/P /sub 1/. In pD the corresponding transitions were found to be more than an order of magnitude broader. To a large extent the results for pH support the meson exchange model. (15 refs).

  11. Laser ablated hydantoin: A high resolution rotational study

    Science.gov (United States)

    Alonso, Elena R.; Kolesniková, Lucie; Alonso, José L.

    2017-09-01

    Laser ablation techniques coupled with broadband and narrowband Fourier transform microwave spectroscopies have allowed the high resolution rotational study of solid hydantoin, an important target in astrochemistry as a possible precursor of glycine. The complicated hyperfine structure arising from the presence of two 14N nuclei in non-equivalent positions has been resolved and interpreted in terms of the nuclear quadrupole coupling interactions. The results reported in this work provide a solid base for the interstellar searches of hydantoin in the astrophysical surveys. The values of the nuclear quadrupole coupling constants have been also discussed in terms of the electronic environment around the respective nitrogen atom.

  12. High Resolution-Resonance Ionization Spectroscopy on uranium

    Energy Technology Data Exchange (ETDEWEB)

    Hakimi, Amin, E-mail: hakimi@uni-mainz.de; Fischbach, Thomas [Johannes Gutenberg-Universitaet Mainz, Institut fuer Physik (Germany); Raeder, Sebastian [TRIUMF (Canada); Trautmann, Norbert [Johannes Gutenberg-Universitaet Mainz, Institut fuer Kernchemie (Germany); Wendt, Klaus [Johannes Gutenberg-Universitaet Mainz, Institut fuer Physik (Germany)

    2013-04-15

    High Resolution-Resonance Ionization Spectroscopy (HR-RIS) allows for sensitive probing of atomic structures and energy level schemes even for highly complex systems. This work explores the applicability of commercial diode lasers for isotope selective spectroscopy of uranium. Using narrow bandwidth continuous-wave (cw) diode lasers, multi step excitation processes were investigated involving levels which could be populated with the radiation of 405 nm BluRay{sup Copyright-Sign} laser diodes as a first step for ultra trace analysis of uranium.

  13. Invited article: High resolution digital camera for infrared reflectography.

    Science.gov (United States)

    Falco, Charles M

    2009-07-01

    This paper describes the characteristics of a high resolution infrared (IR) imaging system operating over the wavelength range of 830-1100 nm, based on a modified 8 Mpixels commercial digital camera, with which nonspecialists can obtain IR reflectograms of works of art in situ in a museum environment. The relevant imaging properties of sensitivity, resolution, noise, and contrast are characterized and the capabilities of this system are illustrated with an example that has revealed important new information about the working practices of a 16th century artist.

  14. Achieving High Resolution Timer Events in Virtualized Environment.

    Science.gov (United States)

    Adamczyk, Blazej; Chydzinski, Andrzej

    2015-01-01

    Virtual Machine Monitors (VMM) have become popular in different application areas. Some applications may require to generate the timer events with high resolution and precision. This however may be challenging due to the complexity of VMMs. In this paper we focus on the timer functionality provided by five different VMMs-Xen, KVM, Qemu, VirtualBox and VMWare. Firstly, we evaluate resolutions and precisions of their timer events. Apparently, provided resolutions and precisions are far too low for some applications (e.g. networking applications with the quality of service). Then, using Xen virtualization we demonstrate the improved timer design that greatly enhances both the resolution and precision of achieved timer events.

  15. High resolution projection micro stereolithography system and method

    Energy Technology Data Exchange (ETDEWEB)

    Spadaccini, Christopher M.; Farquar, George; Weisgraber, Todd; Gemberling, Steven; Fang, Nicholas; Xu, Jun; Alonso, Matthew; Lee, Howon

    2016-11-15

    A high-resolution P.mu.SL system and method incorporating one or more of the following features with a standard P.mu.SL system using a SLM projected digital image to form components in a stereolithographic bath: a far-field superlens for producing sub-diffraction-limited features, multiple spatial light modulators (SLM) to generate spatially-controlled three-dimensional interference holograms with nanoscale features, and the integration of microfluidic components into the resin bath of a P.mu.SL system to fabricate microstructures of different materials.

  16. Clickstream data yields high-resolution maps of science

    Energy Technology Data Exchange (ETDEWEB)

    Bollen, Johan [Los Alamos National Laboratory; Van De Sompel, Herbert [Los Alamos National Laboratory; Hagberg, Aric [Los Alamos National Laboratory; Bettencourt, Luis [Los Alamos National Laboratory; Chute, Ryan [Los Alamos National Laboratory; Rodriguez, Marko A [Los Alamos National Laboratory; Balakireva, Lyudmila [Los Alamos National Laboratory

    2009-01-01

    Intricate maps of science have been created from citation data to visualize the structure of scientific activity. However, most scientific publications are now accessed online. Scholarly web portals record detailed log data at a scale that exceeds the number of all existing citations combined. Such log data is recorded immediately upon publication and keeps track of the sequences of user requests (clickstreams) that are issued by a variety of users across many different domains. Given these advantagees of log datasets over citation data, we investigate whether they can produce high-resolution, more current maps of science.

  17. Tests of a High Resolution Beam Profile Monitor

    Energy Technology Data Exchange (ETDEWEB)

    Norem, J.

    2004-10-28

    High energy linear colliders require very small beams at the interaction point to produce high luminosities, and these beams must be measured and monitored. We have developed and tested a technique where the profile can be obtained from an extension of pinhole camera optics using thick, single sided collimators and slits. Very high resolutions (a few nm) should be possible. Gamma beams can be obtained from bremsstrahlung, Compton or beamstrahlung radiation. We describe tests of the technique using bremsstrahlung from an 800 MeV electron beam at Bates/MIT, Compton scattered photons from 47 GeV Final Focus Test Beam (FFTB) at SLAC, and other applications, such as linear colliders.

  18. High-resolution seismic wave propagation using local time stepping

    KAUST Repository

    Peter, Daniel

    2017-03-13

    High-resolution seismic wave simulations often require local refinements in numerical meshes to accurately capture e.g. steep topography or complex fault geometry. Together with explicit time schemes, this dramatically reduces the global time step size for ground-motion simulations due to numerical stability conditions. To alleviate this problem, local time stepping (LTS) algorithms allow an explicit time stepping scheme to adapt the time step to the element size, allowing nearoptimal time steps everywhere in the mesh. This can potentially lead to significantly faster simulation runtimes.

  19. High-resolution overtone spectra of molecular complexes

    Science.gov (United States)

    Didriche, K.; Földes, T.

    2013-02-01

    A high-resolution spectrum of the acetylene-water complex has been recorded in the overtone range. Two bands of C2H2-D2O were analysed, corresponding to the overtone excitations of either the acetylene or the water units. The vibrational shifts and the upper states rotational constants were retrieved, demonstrating that the geometry of the complex is only slightly modified by the excitation. A larger linewidth was observed for the 2CH band than for the 2OD + DOD band, probably due to the direct coupling of the 2CH excitation with the dissociation coordinate.

  20. High resolution skin colorimetry, strain mapping and mechanobiology.

    Science.gov (United States)

    Devillers, C; Piérard-Franchimont, C; Schreder, A; Docquier, V; Piérard, G E

    2010-08-01

    Skin colours are notoriously different between individuals. They are governed by ethnicities and phototypes, and further influenced by a variety of factors including photoexposures and sustained mechanical stress. Indeed, mechanobiology is a feature affecting the epidermal melanization. High-resolution epiluminescence colorimetry helps in deciphering the effects of forces generated by Langer's lines or relaxed skin tension lines on the melanocyte activity. The same procedure shows a prominent laddering pattern of melanization in striae distensae contrasting with the regular honeycomb pattern in the surrounding skin.

  1. On the optical stability of high-resolution transmission electron microscopes

    Energy Technology Data Exchange (ETDEWEB)

    Barthel, J., E-mail: ju.barthel@fz-juelich.de [Central Facility for Electron Microscopy (GFE), Aachen University (RWTH), Ahornstr. 55, 52074 Aachen (Germany); Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons (ER-C), Forschungszentrum Jülich GmbH, 52425 Jülich (Germany); Thust, A., E-mail: a.thust@fz-juelich.de [Peter Grünberg Institute (PGI), Forschungszentrum Jülich GmbH, 52425 Jülich (Germany); Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons (ER-C), Forschungszentrum Jülich GmbH, 52425 Jülich (Germany)

    2013-11-15

    In the recent two decades the technique of high-resolution transmission electron microscopy experienced an unprecedented progress through the introduction of hardware aberration correctors and by the improvement of the achievable resolution to the sub-Ångström level. The important aspect that aberration correction at a given resolution requires also a well defined amount of optical stability has received little attention so far. Therefore we investigate the qualification of a variety of high-resolution electron microscopes to maintain an aberration corrected optical state in terms of an optical lifetime. We develop a comprehensive statistical framework for the estimation of the optical lifetime and find remarkably low values between tens of seconds and a couple of minutes. Probability curves are introduced, which inform the operator about the chance to work still in the fully aberration corrected state. - Highlights: • We investigate the temporal stability of optical aberrations in HRTEM. • We develop a statistical framework for the estimation of optical lifetimes. • We introduce plots showing the success probability for aberration-free work. • Optical lifetimes in sub-Ångström electron microscopy are surprisingly low. • The success of aberration correction depends strongly on the optical stability.

  2. Innovations in contrast enhanced high resolution ultrasound improve sonographic imaging of the intestine.

    Science.gov (United States)

    Girlich, C; Schacherer, D; Lamby, P; Scherer, M N; Schreyer, A G; Jung, E M

    2010-01-01

    The aim was to describe the perfusion pattern of the inflamed bowel wall and the surrounding tissue in inflammatory bowel disease and diverticulitis of the sigmoid colon applying a high resolution matrix transducer and the new hybrid technique. We performed contrast enhanced ultrasound (CEUS) using an updated version of the 1-5 MHz (C1-5-D convex probe) and the 6-9 MHz probe (9L-D linear probe) as well as a matrix 6-15 MHz transducer (ML 6-15-D Matrix Array Linear Probe) and updated post-processing procedures to examine microvascularization of inflamed bowel wall in Crohn's disease (11 patients), ulcerative colitis (1 patient) and diverticulitis of the sigmoid colon (2 patients). Assessment of mural microvascularization was successful as well as identification of fistulas (2 patients) and covered perforation (1 patient). Moreover analysis of time intensity curves revealed increase of signal intensity up to 20 dB. Summarizing, application of high resolution linear probes and use of updated post-processing methods substantially improve detection of inflammation-caused increased microcirculation of the bowel wall and the surrounding tissue as well as identification of complications as fistulas or covered perforations.

  3. High-resolution Imaging Techniques for the Assessment of Osteoporosis

    Science.gov (United States)

    Krug, Roland; Burghardt, Andrew J.; Majumdar, Sharmila; Link, Thomas M.

    2010-01-01

    Synopsis The importance of assessing the bone’s microarchitectural make-up in addition to its mineral density in the context of osteoporosis has been emphasized in a number of publications. The high spatial resolution required to resolve the bone’s microstructure in a clinically feasible scan time is challenging. Currently, the best suited modalities meeting these requirements in vivo are high-resolution peripheral quantitative imaging (HR-pQCT) and magnetic resonance imaging (MRI). Whereas HR-pQCT is limited to peripheral skeleton regions like the wrist and ankle, MRI can also image other sites like the proximal femur but usually with lower spatial resolution. In addition Multidetector-CT has been used for high-resolution imaging of trabecular bone structure, however, the radiation dose is a limiting factor. This article provides an overview of the different modalities, technical requirements and recent developments in this emerging field. Details regarding imaging protocols as well as image post-processing methods for bone structure quantification are discussed. PMID:20609895

  4. Providing Internet Access to High-Resolution Mars Images

    Science.gov (United States)

    Plesea, Lucian

    2008-01-01

    The OnMars server is a computer program that provides Internet access to high-resolution Mars images, maps, and elevation data, all suitable for use in geographical information system (GIS) software for generating images, maps, and computational models of Mars. The OnMars server is an implementation of the Open Geospatial Consortium (OGC) Web Map Service (WMS) server. Unlike other Mars Internet map servers that provide Martian data using an Earth coordinate system, the OnMars WMS server supports encoding of data in Mars-specific coordinate systems. The OnMars server offers access to most of the available high-resolution Martian image and elevation data, including an 8-meter-per-pixel uncontrolled mosaic of most of the Mars Global Surveyor (MGS) Mars Observer Camera Narrow Angle (MOCNA) image collection, which is not available elsewhere. This server can generate image and map files in the tagged image file format (TIFF), Joint Photographic Experts Group (JPEG), 8- or 16-bit Portable Network Graphics (PNG), or Keyhole Markup Language (KML) format. Image control is provided by use of the OGC Style Layer Descriptor (SLD) protocol. The OnMars server also implements tiled WMS protocol and super-overlay KML for high-performance client application programs.

  5. Providing Internet Access to High-Resolution Lunar Images

    Science.gov (United States)

    Plesea, Lucian

    2008-01-01

    The OnMoon server is a computer program that provides Internet access to high-resolution Lunar images, maps, and elevation data, all suitable for use in geographical information system (GIS) software for generating images, maps, and computational models of the Moon. The OnMoon server implements the Open Geospatial Consortium (OGC) Web Map Service (WMS) server protocol and supports Moon-specific extensions. Unlike other Internet map servers that provide Lunar data using an Earth coordinate system, the OnMoon server supports encoding of data in Moon-specific coordinate systems. The OnMoon server offers access to most of the available high-resolution Lunar image and elevation data. This server can generate image and map files in the tagged image file format (TIFF) or the Joint Photographic Experts Group (JPEG), 8- or 16-bit Portable Network Graphics (PNG), or Keyhole Markup Language (KML) format. Image control is provided by use of the OGC Style Layer Descriptor (SLD) protocol. Full-precision spectral arithmetic processing is also available, by use of a custom SLD extension. This server can dynamically add shaded relief based on the Lunar elevation to any image layer. This server also implements tiled WMS protocol and super-overlay KML for high-performance client application programs.

  6. High-resolution diffraction grating interferometric transducer of linear displacements

    Science.gov (United States)

    Shang, Ping; Xia, Haojie; Fei, Yetai

    2016-01-01

    A high-resolution transducer of linear displacements is presented. The system is based on semiconductor laser illumination and a diffraction grating applied as a length master. The theory of the optical method is formulated using Doppler description. The relationship model among the interference strips, measurement errors, grating deflection around the X, Y and Z axes and translation along the Z axis is built. The grating interference strips' direction and space is not changed with movement along the X (direction of grating movement), Y (direction of grating line), Z axis, and the direction and space has a great effect when rotating around the X axis. Moreover the space is little affected by deflection around the Z axis however the direction is changed dramatically. In addition, the strips' position shifted rightward or downwards respectively for deflection around the X or Y axis. Because the emitted beams are separated on the grating plane, the tilt around the X axis error of the stage during motion will lead to the optical path difference of the two beams resulting in phase shift. This study investigates the influence of the tilt around the X axis error. Experiments show that after yaw error compensation, the high-resolution diffraction grating interferometric transducer readings can be significantly improved. The error can be reduced from +/-80 nm to +/-30 nm in maximum.

  7. High resolution multiplexed functional imaging in live embryos (Conference Presentation)

    Science.gov (United States)

    Xu, Dongli; Zhou, Weibin; Peng, Leilei

    2017-02-01

    Fourier multiplexed fluorescence lifetime imaging (FmFLIM) scanning laser optical tomography (FmFLIM-SLOT) combines FmFLIM and Scanning laser optical tomography (SLOT) to perform multiplexed 3D FLIM imaging of live embryos. The system had demonstrate multiplexed functional imaging of zebrafish embryos genetically express Foster Resonant Energy Transfer (FRET) sensors. However, previous system has a 20 micron resolution because the focused Gaussian beam diverges quickly from the focused plane, makes it difficult to achieve high resolution imaging over a long projection depth. Here, we present a high-resolution FmFLIM-SLOT system with achromatic Bessel beam, which achieves 3 micron resolution in 3D deep tissue imaging. In Bessel-FmFLIM-SLOT, multiple laser excitation lines are firstly intensity modulated by a Michelson interferometer with a spinning polygon mirror optical delay line, which enables Fourier multiplexed multi-channel lifetime measurements. Then, a spatial light modulator and a prism are used to transform the modulated Gaussian laser beam to an achromatic Bessel beam. The achromatic Bessel beam scans across the whole specimen with equal angular intervals as sample rotated. After tomography reconstruction and the frequency domain lifetime analysis method, both the 3D intensity and lifetime image of multiple excitation-emission can be obtained. Using Bessel-FmFLIM-SLOT system, we performed cellular-resolution FLIM tomography imaging of live zebrafish embryo. Genetically expressed FRET sensors in these embryo will allow non-invasive observation of multiple biochemical processes in vivo.

  8. Hyperresolution: an hyperspectral and high resolution imager for Earth observation

    Science.gov (United States)

    De Vidi, R.; Chiarantini, L.; Bini, A.

    2017-11-01

    Hyperspectral space imagery is an emerging technology that supports many scientific, civil, security and defence operational applications. The main advantage of this remote sensing technique is that it allows the so-called Feature Extraction: in fact the spectral signature allows the recognition of the materials composing the scene. Hyperspectral Products and their applications have been investigated in the past years by Galileo Avionica to direct the instrument characteristics design. Sample products have been identified in the civil / environment monitoring fields (such as coastal monitoring, vegetation, hot spot and urban classification) and in defense / security applications: their performances have been verified by means of airborne flight campaigns. The Hyperspectral and High Resolution Imager is a space-borne instrument that implement a pushbroom technique to get strip spectral images over the Hyperspectral VNIR and SWIR bands, with a ground sample distance at nadir of 20m in a 20 km wide ground swath, with 200 spectral channels, realizing an average spectral resolution of 10nm. The High Resolution Panchromatic Channel insists in the same swath to allow for multiresolution data fusion of hyperspectral imagery.

  9. Quantitative analysis of cholesteatoma using high resolution computed tomography

    Energy Technology Data Exchange (ETDEWEB)

    Kikuchi, Shigeru; Yamasoba, Tatsuya (Kameda General Hospital, Chiba (Japan)); Iinuma, Toshitaka

    1992-05-01

    Seventy-three cases of adult cholesteatoma, including 52 cases of pars flaccida type cholesteatoma and 21 of pars tensa type cholesteatoma, were examined using high resolution computed tomography, in both axial (lateral semicircular canal plane) and coronal sections (cochlear, vestibular and antral plane). These cases were classified into two subtypes according to the presence of extension of cholesteatoma into the antrum. Sixty cases of chronic otitis media with central perforation (COM) were also examined as controls. Various locations of the middle ear cavity were measured in terms of size in comparison with pars flaccida type cholesteatoma, pars tensa type cholesteatoma and COM. The width of the attic was significantly larger in both pars flaccida type and pars tensa type cholesteatoma than in COM. With pars flaccida type cholesteatoma there was a significantly larger distance between the malleus and lateral wall of the attic than with COM. In contrast, the distance between the malleus and medial wall of the attic was significantly larger with pars tensa type cholesteatoma than with COM. With cholesteatoma extending into the antrum, regardless of the type of cholesteatoma, there were significantly larger distances than with COM at the following sites: the width and height of the aditus ad antrum, and the width, height and anterior-posterior diameter of the antrum. However, these distances were not significantly different between cholesteatoma without extension into the antrum and COM. The hitherto demonstrated qualitative impressions of bone destruction in cholesteatoma were quantitatively verified in detail using high resolution computed tomography. (author).

  10. High-resolution electron microscopy of advanced materials

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, T.E.; Kung, H.H.; Sickafus, K.E.; Gray, G.T. III; Field, R.D.; Smith, J.F. [Los Alamos National Lab., NM (United States). Materials Science and Technology Div.

    1997-11-01

    This final report chronicles a three-year, Laboratory Directed Research and Development (LDRD) project at Los Alamos National Laboratory (LANL). The High-Resolution Electron Microscopy Facility has doubled in size and tripled in quality since the beginning of the three-year period. The facility now includes a field-emission scanning electron microscope, a 100 kV field-emission scanning transmission electron microscope (FE-STEM), a 300 kV field-emission high-resolution transmission electron microscope (FE-HRTEM), and a 300 kV analytical transmission electron microscope. A new orientation imaging microscope is being installed. X-ray energy dispersive spectrometers for chemical analysis are available on all four microscopes; parallel electron energy loss spectrometers are operational on the FE-STEM and FE-HRTEM. These systems enable evaluation of local atomic bonding, as well as chemical composition in nanometer-scale regions. The FE-HRTEM has a point-to-point resolution of 1.6 {angstrom}, but the resolution can be pushed to its information limit of 1 {angstrom} by computer reconstruction of a focal series of images. HRTEM has been used to image the atomic structure of defects such as dislocations, grain boundaries, and interfaces in a variety of materials from superconductors and ferroelectrics to structural ceramics and intermetallics.

  11. High-Resolution Mars Camera Test Image of Moon (Infrared)

    Science.gov (United States)

    2005-01-01

    This crescent view of Earth's Moon in infrared wavelengths comes from a camera test by NASA's Mars Reconnaissance Orbiter spacecraft on its way to Mars. The mission's High Resolution Imaging Science Experiment camera took the image on Sept. 8, 2005, while at a distance of about 10 million kilometers (6 million miles) from the Moon. The dark feature on the right is Mare Crisium. From that distance, the Moon would appear as a star-like point of light to the unaided eye. The test verified the camera's focusing capability and provided an opportunity for calibration. The spacecraft's Context Camera and Optical Navigation Camera also performed as expected during the test. The Mars Reconnaissance Orbiter, launched on Aug. 12, 2005, is on course to reach Mars on March 10, 2006. After gradually adjusting the shape of its orbit for half a year, it will begin its primary science phase in November 2006. From the mission's planned science orbit about 300 kilometers (186 miles) above the surface of Mars, the high resolution camera will be able to discern features as small as one meter or yard across.

  12. MR-Venography Using High Resolution True-FISP

    Energy Technology Data Exchange (ETDEWEB)

    Spuentrup, E. [Technische Hochschule Aachen (Germany). Klinik fuer Radiologische Diagnostik; Beth Israel Deaconess Medical Center, Boston, MA (United States). Dept. of Medicine; Harvard Medical School, Boston, MA (United States); Buecker, A.; Guenther, R.W. [Technische Hochschule Aachen (Germany). Klinik fuer Radiologische Diagnostik; Stuber, M. [Beth Israel Deaconess Medical Center, Boston, MA (United States). Dept. of Medicine; Harvard Medical School, Boston, MA (United States); Philips Med. Syst., Best (Netherlands)

    2001-08-01

    A new fast MR-venography approach using a high resolution True-FISP imaging sequence was investigated in 20 patients suffering from 23 deep vein thromboses. Diagnosis was proven by X-ray venography, CT or ultrasound examination. The presented technique allowed for clear thrombus visualization with a high contrast to the surrounding blood pool even in calf veins. Acquisition time was less than 10 minutes for imaging the pelvis and the legs. No contrast media was needed. The presented high resolution True-FISP MR-veography is a promising non-invasive, fast MR-venography approach for detection of deep venous thrombosis. (orig.) [German] Eine neue schnelle, oertlich hochaufgeloeste MR-Phlebographietechnik mit einer axialen True-FISP Bildgebungssequenz wurde an 20 Patienten mit 23 nach-gewiesenen tiefen Beinvenenthrombosen untersucht. Die Befunde wurden mit einer konventionellen Roentgenphlebographie, einer CT oder einer Sonographie gesichert. Die vorgestellte Technik erlaubte in allen Faellen eine Thrombusdarstellung mit hohem Kontrast zum umgebenden venoesen Blut, wobei aufgrund der hohen Ortsaufloesung auch die Unterschenkelvenen beurteilt werden konnten. Die Datenaufnahmezeit zur Untersuchung des Beckens und der Beine betrug weniger als 10 Minuten. Kontrastmittel wurde nicht benoetigt. Die vorgestellte MR-Phlebographietechnik unter Verwendung einer oertlich hochauf-geloesten True-FISP Sequenz ist eine neue, vielversprechende, nicht-invasive Technik zur Diagnostik der tiefen Bein- und Beckenvenenthrombose. (orig.)

  13. High-resolution digital imaging with storage phosphors.

    Science.gov (United States)

    Fuhrman, C R; Gur, D; Schaetzing, R

    1990-01-01

    This article describes the current status and potential applications of high-resolution storage phosphor for imaging of the chest. Digital imaging that uses storage phosphor technology is easily adaptable to existing x-ray--generating equipment and can also be used with mobile equipment. The wide latitude of the storage phosphor technique permits satisfactory imaging in situations in which exposure factors cannot be accurately estimated or easily controlled. Early experience with an experimental Kodak high-resolution (4K x 4K) storage phosphor system suggests that standard and portal chest images of excellent quality can be obtained. Many issues must be resolved, however, before digital radiology with a storage phosphor can be advocated as being preferable to conventional film-screen systems. These issues, which include display modalities (film or television monitor), resolution requirements, and the effects of image processing, can only be resolved by further large-scale accuracy studies. The change to a digital imaging system will involve major expenditures for equipment and computers. Cost will be related largely to the level of spatial resolution required for primary radiographic diagnosis.

  14. High resolution neutron diffractometer HRND at research reactor CMRR

    Science.gov (United States)

    Zhang, J.; Xia, Y.; Wang, Y.; Xie, C.; Sun, G.; Liu, L.; Pang, B.; Li, J.; Huang, C.; Liu, Y.; Gong, J.

    2018-01-01

    The high resolution neutron diffractometer HRND is located at the 20 MW China Mianyang Research Reactor (CMRR), which is a neutron powder diffractometer especially dedicated to crystal and magnetic structure studies for polycrystalline powder samples. A vertical focusing Ge (511) monochromator produce a monochromatic neutron beam with a wavelength of 1.885 Å at a fixed take-off angle of 120o. An array of 64 equidistant 3He filled proportional counters can acquire diffraction patterns with a large-scale diffraction angle range over 160o. As all the Soller slit collimators of HRND have a collimation angle of 10' and the monochromator has an average mosaicity of 0.359o, HRND obtains a best resolution of about 1.6\\textperthousand based on experiments, which makes the resolution of HRND can compete with the mainstream-level high resolution neutron powder diffractometers in the world. Equipped with a cryostat and a furnace, HRND allows structural characterization in an extremely broad temperature range. The details of the configuration and performance of the instrument are reported along with its specifications and performance assessments in the present paper.

  15. Characterization of ceramic archaeological by high resolution X ray microtomography

    Energy Technology Data Exchange (ETDEWEB)

    Machado, Alessandra C.; Freitas, Renato; Calza, Cristiane F.; Lopes, Ricardo T.; Lima, Inaya, E-mail: alecastro@lin.ufrj.br [Coordenacao dos Programas de Pos-Graduacao de Engenharia (COPPE/UFRJ), Rio de Janeiro, RJ (Brazil). Lab. de Instrumentacao Nuclear; Carvalho, Daniele D.; Gaspar, Maria D. [Museu Nacional (MN/UFRJ), RJ (Brazil). Centro de Tecnologia

    2013-07-01

    Characterization of ceramic fragments is a very important area of research in art and archeometry area because it enables a greater understanding of how ancient civilizations behave and what were their traditions and customs. Petrography and chemical analyses are commonly used, but these techniques are destructive, which is not interesting for this type of sample. Through the exchange of multidisciplinary scientific knowledge and new partnerships, high resolution X-ray microtomography has been introduced in archaeological area as a great possibility of 3D inspection in a non-destructive way. The goal of this work is to investigate the internal microstructures of four samples of archeological ceramic, from the Archaeological Site of Macacu - RJ. The X-ray microtomography were performed in a high resolution setup, and can be used to infer the nature of organic temper even with all plant remains completely burnt out during the firing process and also to ensure the homogeneity of samples envisaged for geochemical analyses, especially with respect to the distribution of chemically diverse fabric compounds. In this way this study intends to contribute to our understanding of the archaeological and historical formations of this region. (author)

  16. Segmentation of Striatal Brain Structures from High Resolution PET Images

    Directory of Open Access Journals (Sweden)

    Ricardo J. P. C. Farinha

    2009-01-01

    Full Text Available We propose and evaluate an automatic segmentation method for extracting striatal brain structures (caudate, putamen, and ventral striatum from parametric C11-raclopride positron emission tomography (PET brain images. We focus on the images acquired using a novel brain dedicated high-resolution (HRRT PET scanner. The segmentation method first extracts the striatum using a deformable surface model and then divides the striatum into its substructures based on a graph partitioning algorithm. The weighted kernel k-means algorithm is used to partition the graph describing the voxel affinities within the striatum into the desired number of clusters. The method was experimentally validated with synthetic and real image data. The experiments showed that our method was able to automatically extract caudate, ventral striatum, and putamen from the images. Moreover, the putamen could be subdivided into anterior and posterior parts. An automatic method for the extraction of striatal structures from high-resolution PET images allows for inexpensive and reproducible extraction of the quantitative information from these images necessary in brain research and drug development.

  17. A High-Resolution Aeromagnetic Survey to Identify Buried Faults at Dixie Valley, Nevada

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Richard Paul; Grauch, V. J. S.; Blackwell, David D.

    2002-09-01

    Preliminary results from a high-resolution aeromagnetic survey (200m line spacing) acquired in Dixie Valley early in 2002 provide confirmation of intra-basin faulting based on subtle surface indications. In addition the data allow identification of the locations and trends of many faults that have not been recognized at the surface, and provide a picture of intrabasin faulting patterns not possible using other techniques. The data reveal a suite of northeasterly-trending curving and branching faults that surround a relatively coherent block in the area of Humboldt Salt Marsh, the deepest part of the basin. The producing reservoir occurs at the north end of this coherent block, where rampart faults from the northwest side of the valley merge with anthithetic faults from the central and southeast parts of the valley.

  18. A high-resolution record of Greenland mass balance

    NARCIS (Netherlands)

    McMillan, Malcolm; Leeson, Amber; Shepherd, Andrew; Briggs, Kate; Armitage, Thomas; Hogg, Anna; Kuipers Munneke, P.|info:eu-repo/dai/nl/304831891; van den Broeke, M.R.|info:eu-repo/dai/nl/073765643; Noël, B.P.Y.|info:eu-repo/dai/nl/370612345; van de Berg, W.J.|info:eu-repo/dai/nl/304831611; Ligtenberg, S.R.M.|info:eu-repo/dai/nl/32821177X; Horwath, M.; Groh, Andreas; Muir, A.; Gilbert, Lin

    2016-01-01

    We map recent Greenland Ice Sheet elevation change at high spatial (5 km) and temporal (monthly) resolution using CryoSat-2 altimetry. After correcting for the impact of changing snowpack properties associated with unprecedented surface melting in 2012, we find good agreement (3 cm/yr bias) with

  19. Submarine melt rates under Greenland's ice tongues

    Science.gov (United States)

    Wilson, Nat; Straneo, Fiametta; Heimbach, Patrick; Cenedese, Claudia

    2017-04-01

    The few remaining ice tongues (ice-shelf like extensions) of Greenland's glaciers are undergoing rapid changes with potential implications for the stability of the ice sheet. Submarine melting is recognized as a major contributor to mass loss, yet the magnitude and spatial distribution of melt are poorly known or understood. Here, we use high resolution satellite imagery to infer the magnitude and spatial variability of melt rates under Greenland's largest remaining ice tongues: Ryder Glacier, Petermann Glacier and Nioghalvfjerdsbræ (79 North Glacier). We find that submarine plus aerial melt approximately balance the ice flux from the grounded ice sheet for the first two while at Nioghalvfjerdsbræ the total melt flux exceeds the inflow of ice indicating thinning of the ice tongue. We also show that melt rates under the ice tongues vary considerably, exceeding 60 m yr-1 near the grounding zone and decaying rapidly downstream. Channels, likely originating from upstream subglacial channels, give rise to large melt variations across the ice tongues. Using derived melt rates, we test simplified melt parameterizations appropriate for ice sheet models and find the best agreement with those that incorporate ice tongue geometry in the form of depth and slope.

  20. Evaluating Corneal Collagen Organization Using High Resolution Non Linear Optical (NLO) Macroscopy*

    Science.gov (United States)

    Jester, James V.; Winkler, Moritz; Jester, Bryan E.; Nien, Chyong; Chai, Dongyul; Brown, Donald J.

    2010-01-01

    Purpose Recent developments in non-linear optical (NLO) imaging using femtosecond lasers provides a non-invasive method for detecting collagen fibers by imaging second harmonic generated (SHG) signals. However, this technique is limited by the small field of view (FoV) necessary to generate SHG signals. The purpose of this report is to review our efforts to greatly extend the FoV in order to assess the entire collagen structure using high resolution macroscopic (HRMac) imaging. Methods Intact human eyes were fixed under pressure and the whole cornea (13 mm diameter) excised and embedded in low melting point agar for vibratome sectioning (200–300 μm). Sections were then optically scanned using a Zeiss LSM 510 Meta and Chameleon femtosecond laser to generate SHG images. For each vibratome section, an overlapping series of 3-D data sets (466 × 466 × 150 μm) were taken covering the entire tissue (15 mm × 6 mm area) using a motorized, mechanical stage. The 3-D data sets were then concatenated to generate an NLO based tomograph. Results HRMac of the cornea yielded large macroscopic (80 Meg Pixels per plane), 3-dimensional tomographs with high resolution (0.81 μm later, 2.0 μm axial) in which individual collagen fibers (stromal lamellae) could be traced, segmented and extracted. 3-D reconstructions suggested that the anterior cornea is comprised of highly intertwined lamellae that insert into the anterior limiting lamina (Bowman’s Layer). Conclusion We conclude that HRMac using NLO based tomography provides a powerful new tool to assess collagen structural organization within the cornea. PMID:20724856

  1. High resolution infrared acquisitions droning over the LUSI mud eruption.

    Science.gov (United States)

    Di Felice, Fabio; Romeo, Giovanni; Di Stefano, Giuseppe; Mazzini, Adriano

    2016-04-01

    The use of low-cost hand-held infrared (IR) thermal cameras based on uncooled micro-bolometer detector arrays became more widespread during the recent years. Thermal cameras have the ability to estimate temperature values without contact and therefore can be used in circumstances where objects are difficult or dangerous to reach such as volcanic eruptions. Since May 2006 the Indonesian LUSI mud eruption continues to spew boiling mud, water, aqueous vapor, CO2, CH4 and covers a surface of nearly 7 km2. At this locality we performed surveys over the unreachable erupting crater. In the framework of the LUSI Lab project (ERC grant n° 308126), in 2014 and 2015, we acquired high resolution infrared images using a specifically equipped remote-controlled drone flying at an altitude of m 100. The drone is equipped with GPS and an autopilot system that allows pre-programming the flying path or designing grids. The mounted thermal camera has peak spectral sensitivity in LW wavelength (μm 10) that is characterized by low water vapor and CO2 absorption. The low distance (high resolution) acquisitions have a temperature detail every cm 40, therefore it is possible to detect and observe physical phenomena such as thermodynamic behavior, hot mud and fluids emissions locations and their time shifts. Despite the harsh logistics and the continuously varying gas concentrations we managed to collect thermal images to estimate the crater zone spatial thermal variations. We applied atmosphere corrections to calculate infrared absorption by high concentration of water vapor. Thousands of images have been stitched together to obtain a mosaic of the crater zone. Regular monitoring with heat variation measurements collected, e.g. every six months, could give important information about the volcano activity estimating its evolution. A future data base of infrared high resolution and visible images stored in a web server could be a useful monitoring tool. An interesting development will be

  2. High resolution remote sensing of water surface patterns

    Science.gov (United States)

    Woodget, A.; Visser, F.; Maddock, I.; Carbonneau, P.

    2012-12-01

    The assessment of in-stream habitat availability within fluvial environments in the UK traditionally includes the mapping of patterns which appear on the surface of the water, known as 'surface flow types' (SFTs). The UK's River Habitat Survey identifies ten key SFTs, including categories such as rippled flow, upwelling, broken standing waves and smooth flow. SFTs result from the interaction between the underlying channel morphology, water depth and velocity and reflect the local flow hydraulics. It has been shown that SFTs can be both biologically and hydraulically distinct. SFT mapping is usually conducted from the river banks where estimates of spatial coverage are made by eye. This approach is affected by user subjectivity and inaccuracies in the spatial extent of mapped units. Remote sensing and specifically the recent developments in unmanned aerial systems (UAS) may now offer an alternative approach for SFT mapping, with the capability for rapid and repeatable collection of very high resolution imagery from low altitudes, under bespoke flight conditions. This PhD research is aimed at investigating the mapping of SFTs using high resolution optical imagery (less than 10cm) collected from a helicopter-based UAS flown at low altitudes (less than 100m). This paper presents the initial findings from a series of structured experiments on the River Arrow, a small lowland river in Warwickshire, UK. These experiments investigate the potential for mapping SFTs from still and video imagery of different spatial resolutions collected at different flying altitudes and from different viewing angles (i.e. vertical and oblique). Imagery is processed using 3D mosaicking software to create orthophotos and digital elevation models (DEM). The types of image analysis which are tested include a simple, manual visual assessment undertaken in a GIS environment, based on the high resolution optical imagery. In addition, an object-based image analysis approach which makes use of the

  3. A PICTORIAL PRESENTATION OF ESOPHAGEAL HIGH RESOLUTION MANOMETRY CURRENT PARAMETERS.

    Science.gov (United States)

    Lafraia, Fernanda M; Herbella, Fernando A M; Kalluf, Julia R; Patti, Marco G

    2017-01-01

    High resolution manometry is the current technology used to the study of esophageal motility and is replacing conventional manometry in important centers for esophageal motility with parameters used on esophageal motility, following the Chicago Classification. This classification unifies high resolution manometry interpretation and classifies esophageal disorders. This review shows, in a pictorial presentation, the new parameters established by the Chicago Classification, version 3.0, aimed to allow an easy comprehension and interpretation of high resolution manometry. Esophageal manometries performed by the authors were reviewed to select illustrative tracings representing Chicago Classification parameters. The parameters are: Esophagogastric Morphology, that classifies this junction according to its physiology and anatomy; Integrated Relaxation Pressure, that measures the lower esophageal sphincter relaxation; Distal Contractile Integral, that evaluates the contraction vigor of each wave; and, Distal Latency, that measures the peristalsis velocity from the beginning of the swallow to the epiphrenic ampulla. Clinical applications of these new concepts is still under evaluation. Mostrar, de forma pictórica, os novos parâmetros compilados na versão 3.0 da Classificação de Chicago, buscando facilitar a compreensão e interpretação da manometria de alta resolução. Foram revistas as manometrias da casuística dos autores e selecionados os traçados representativos dos parâmetros da Classificação de Chicago. Entre os parâmetros apresentados foram considerados a Morfologia da Transição Gastroesofágica, que classifica o segmento de acordo com sua fisiologia e anatomia; a Integral da Pressão de Relaxamento, que mede o relaxamento do esfíncter esofagiano inferior; a Integral Contrátil Distal, que avalia o vigor contrátil da onda peristáltica; e, a Latência Distal, que mede o tempo da peristalse, desde o início da deglutição até a ampola epifr

  4. Optical Histology: High-Resolution Visualization of Tissue Microvasculature

    Science.gov (United States)

    Moy, Austin Jing-Ming

    Mammalian tissue requires the delivery of nutrients, growth factors, and the exchange of oxygen and carbon dioxide gases to maintain normal function. These elements are delivered by the blood, which travels through the connected network of blood vessels, known as the vascular system. The vascular system consists of large feeder blood vessels (arteries and veins) that are connected to the small blood vessels (arterioles and venules), which in turn are connected to the capillaries that are directly connected to the tissue and facilitate gas exchange and nutrient delivery. These small blood vessels and capillaries make up an intricate but organized network of blood vessels that exist in all mammalian tissues known as the microvasculature and are very important in maintaining the health and proper function of mammalian tissue. Due to the importance of the microvasculature in tissue survival, disruption of the microvasculature typically leads to tissue dysfunction and tissue death. The most prevalent method to study the microvasculature is visualization. Immunohistochemistry (IHC) is the gold-standard method to visualize tissue microvasculature. IHC is very well-suited for highly detailed interrogation of the tissue microvasculature at the cellular level but is unwieldy and impractical for wide-field visualization of the tissue microvasculature. The objective my dissertation research was to develop a method to enable wide-field visualization of the microvasculature, while still retaining the high-resolution afforded by optical microscopy. My efforts led to the development of a technique dubbed "optical histology" that combines chemical and optical methods to enable high-resolution visualization of the microvasculature. The development of the technique first involved preliminary studies to quantify optical property changes in optically cleared tissues, followed by development and demonstration of the methodology. Using optical histology, I successfully obtained high

  5. High resolution modelling of extreme precipitation events in urban areas

    Science.gov (United States)

    Siemerink, Martijn; Volp, Nicolette; Schuurmans, Wytze; Deckers, Dave

    2015-04-01

    The present day society needs to adjust to the effects of climate change. More extreme weather conditions are expected, which can lead to longer periods of drought, but also to more extreme precipitation events. Urban water systems are not designed for such extreme events. Most sewer systems are not able to drain the excessive storm water, causing urban flooding. This leads to high economic damage. In order to take appropriate measures against extreme urban storms, detailed knowledge about the behaviour of the urban water system above and below the streets is required. To investigate the behaviour of urban water systems during extreme precipitation events new assessment tools are necessary. These tools should provide a detailed and integral description of the flow in the full domain of overland runoff, sewer flow, surface water flow and groundwater flow. We developed a new assessment tool, called 3Di, which provides detailed insight in the urban water system. This tool is based on a new numerical methodology that can accurately deal with the interaction between overland runoff, sewer flow and surface water flow. A one-dimensional model for the sewer system and open channel flow is fully coupled to a two-dimensional depth-averaged model that simulates the overland flow. The tool uses a subgrid-based approach in order to take high resolution information of the sewer system and of the terrain into account [1, 2]. The combination of using the high resolution information and the subgrid based approach results in an accurate and efficient modelling tool. It is now possible to simulate entire urban water systems using extreme high resolution (0.5m x 0.5m) terrain data in combination with a detailed sewer and surface water network representation. The new tool has been tested in several Dutch cities, such as Rotterdam, Amsterdam and The Hague. We will present the results of an extreme precipitation event in the city of Schiedam (The Netherlands). This city deals with

  6. Do high-resolution convection-permitting experiments on Europe need to be driven by high resolution global runs?

    Science.gov (United States)

    Berthou, Segolene; Chan, Steven; Kendon, Elizabeth; Roberts, Malcolm; Lee, Robert; Vanniere, Benoit

    2017-04-01

    Challenges of getting appropriate climate-change scenarios over Europe both come from having a good representation of the synoptic systems reaching Europe and having a good-enough representation of local and orographic processes in Europe. Therefore we perform both the evaluation of the driving global model and its dynamical downscaling with a 2.2km regional model on the present day period, in the perspective of using this configuration in a future climate scenario. 20-year long atmosphere-only simulations with the Unified Model of the Met Office were run at different global resolutions (130km, 60km and 25km) and the highest resolution was chosen to give the boundaries of a European-wide convection permitting simulation with a 2.2km resolution. The synoptic situation of the different global resolutions are comparable in terms of latitudinal distribution of the jets and weather regimes but there is consistent improvement in the frequency of storms reaching Europe at 25km resolution. High resolution global runs therefore mainly show added value in the high-frequency synoptic drivers. Compared to high resolution precipitation datasets, the 25km resolution is showing good representation of winter precipitation distribution, although with too many days of moderate precipitation in Western Europe. It shows a dry bias in summer, consistent with a mean jet too north.

  7. Quantitative high-resolution transmission electron microscopy of single atoms.

    Science.gov (United States)

    Gamm, Björn; Blank, Holger; Popescu, Radian; Schneider, Reinhard; Beyer, André; Gölzhäuser, Armin; Gerthsen, Dagmar

    2012-02-01

    Single atoms can be considered as the most basic objects for electron microscopy to test the microscope performance and basic concepts for modeling image contrast. In this work high-resolution transmission electron microscopy was applied to image single platinum, molybdenum, and titanium atoms in an aberration-corrected transmission electron microscope. The atoms are deposited on a self-assembled monolayer substrate that induces only negligible contrast. Single-atom contrast simulations were performed on the basis of Weickenmeier-Kohl and Doyle-Turner form factors. Experimental and simulated image intensities are in quantitative agreement on an absolute intensity scale, which is provided by the vacuum image intensity. This demonstrates that direct testing of basic properties such as form factors becomes feasible.

  8. High-Resolution Transmission Electron Microscopy - and Associated Techniques

    Science.gov (United States)

    Buseck, Peter; Cowley, John; Eyring, Leroy

    1989-02-01

    This book provides an introduction to the fundamental concepts, techniques, and methods used for electron microscopy at high resolution in space, energy, and even in time. It delineates the theory of elastic scattering, which is most useful for spectroscopic and chemical analyses. There are also discussions of the theory and practice of image calculations, and applications of HRTEM to the study of solid surfaces, highly disordered materials, solid state chemistry, mineralogy, semiconductors and metals. Contributors include J. Cowley, J. Spence, P. Buseck, P. Self, and M.A. O'Keefe. Compiled by experts in the fields of geology, physics and chemistry, this comprehensive text will be the standard reference for years to come.

  9. High-resolution imaging methods in array signal processing

    DEFF Research Database (Denmark)

    Xenaki, Angeliki

    The purpose of this study is to develop methods in array signal processing which achieve accurate signal reconstruction from limited observations resulting in high-resolution imaging. The focus is on underwater acoustic applications and sonar signal processing both in active (transmit and receive...... in active sonar signal processing for detection and imaging of submerged oil contamination in sea water from a deep-water oil leak. The submerged oil _eld is modeled as a uid medium exhibiting spatial perturbations in the acoustic parameters from their mean ambient values which cause weak scattering......-of-arrival (DOA) of the associated wavefronts from a limited number of observations. Usually, there are only a few sources generating the acoustic wavefield such that DOA estimation is essentially a sparse signal reconstruction problem. Conventional methods for DOA estimation (i.e., beamforming) suffer from...

  10. Sparse Representation Denoising for Radar High Resolution Range Profiling

    Directory of Open Access Journals (Sweden)

    Min Li

    2014-01-01

    Full Text Available Radar high resolution range profile has attracted considerable attention in radar automatic target recognition. In practice, radar return is usually contaminated by noise, which results in profile distortion and recognition performance degradation. To deal with this problem, in this paper, a novel denoising method based on sparse representation is proposed to remove the Gaussian white additive noise. The return is sparsely described in the Fourier redundant dictionary and the denoising problem is described as a sparse representation model. Noise level of the return, which is crucial to the denoising performance but often unknown, is estimated by performing subspace method on the sliding subsequence correlation matrix. Sliding window process enables noise level estimation using only one observation sequence, not only guaranteeing estimation efficiency but also avoiding the influence of profile time-shift sensitivity. Experimental results show that the proposed method can effectively improve the signal-to-noise ratio of the return, leading to a high-quality profile.

  11. Object-Based Building Extraction from High Resolution Satellite Imagery

    Directory of Open Access Journals (Sweden)

    R. Attarzadeh

    2012-07-01

    Full Text Available Automatic building extraction from high resolution satellite imagery is considered as an important field of research in remote sensing and machine vision. Many algorithms for extraction of buildings from satellite images have been presented so far. These algorithms mainly have considered radiometric, geometric, edge detection and shadow criteria approaches to perform the building extraction. In this paper, we propose a novel object based approach for automatic and robust detection and extraction of building in high spatial resolution images. To achieve this goal, we use stable and variable features together. Stable features are derived from inherent characteristics of building phenomenon and variable features are extracted using SEparability and THresholds analysis tool. The proposed method has been applied on a QuickBird imagery of an urban area in Isfahan city and visual validation demonstrates that the proposed method provides promising results.

  12. High-Resolution Data for a Low-Resolution World

    Energy Technology Data Exchange (ETDEWEB)

    Brady, Brendan Williams [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2016-05-10

    In the past 15 years, the upper section of Cañon de Valle has been severely altered by wildfires and subsequent runoff events. Loss of root structures on high-angle slopes results in debris flow and sediment accumulation in the narrow canyon bottom. The original intent of the study described here was to better understand the changes occurring in watershed soil elevations over the course of several post-fire years. An elevation dataset from 5 years post-Cerro Grande fire was compared to high-resolution LiDAR data from 14 years post-Cerro Grande fire (also 3 years post-Las Conchas fire). The following analysis was motivated by a problematic comparison of these datasets of unlike resolution, and therefore focuses on what the data reveals of itself. The objective of this study is to highlight the effects vegetation can have on remote sensing data that intends to read ground surface elevation.

  13. Localized corrosion information using high resolution measurement devices

    DEFF Research Database (Denmark)

    Ambat, Rajan

    2005-01-01

    High performance demand for several engineering alloys and components, and miniaturization of electronics and development of MEMS requires better understanding of local corrosion characteristics frequently down to µm scale. This is because in metallic materials corrosion is a sensitive function...... in conjunction with microstructural analysis, using advanced microscopic tools, becomes very important. Corrosion of microelectronics circuits and MEMs is also a recent problem, which demands measurement resolution down to few microns as the components are extremely small, and measurement needs to be carried out...... of the technique could be further enhanced by adding new features such as high resolution video visualization systems, fretting/tribo-corroson attachments, and also by integrating it with stress corrosion testing, corrosion investigation of concrete for a few to name with. The corrosion group in MPT, Technical...

  14. High-Resolution Characterization of UMo Alloy Microstructure

    Energy Technology Data Exchange (ETDEWEB)

    Devaraj, Arun [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Kovarik, Libor [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Joshi, Vineet V. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Jana, Saumyadeep [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Manandhar, Sandeep [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Arey, Bruce W. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States); Lavender, Curt A. [Pacific Northwest National Lab. (PNNL), Richland, WA (United States)

    2016-11-30

    This report highlights the capabilities and procedure for high-resolution characterization of UMo fuels in PNNL. Uranium-molybdenum (UMo) fuel processing steps, from casting to forming final fuel, directly affect the microstructure of the fuel, which in turn dictates the in-reactor performance of the fuel under irradiation. In order to understand the influence of processing on UMo microstructure, microstructure characterization techniques are necessary. Higher-resolution characterization techniques like transmission electron microscopy (TEM) and atom probe tomography (APT) are needed to interrogate the details of the microstructure. The findings from TEM and APT are also directly beneficial for developing predictive multiscale modeling tools that can predict the microstructure as a function of process parameters. This report provides background on focused-ion-beam–based TEM and APT sample preparation, TEM and APT analysis procedures, and the unique information achievable through such advanced characterization capabilities for UMo fuels, from a fuel fabrication capability viewpoint.

  15. High resolution capacitance detection circuit for rotor micro-gyroscope

    Directory of Open Access Journals (Sweden)

    Ming-Yuan Ren

    2014-03-01

    Full Text Available Conventional methods for rotor position detection of micro-gyroscopes include common exciting electrodes (single frequency and common sensing electrodes (frequency multiplex, but they have encountered some problems. So we present a high resolution and low noise pick-off circuit for micro-gyroscopes which utilizes the time multiplex method. The detecting circuit adopts a continuous-time current sensing circuit for capacitance measurement, and its noise analysis of the charge amplifier is introduced. The equivalent output noise power spectral density of phase-sensitive demodulation is 120 nV/Hz1/2. Tests revealed that the whole circuitry has a relative capacitance resolution of 1 × 10−8.

  16. High-resolution structure of viruses from random diffraction snapshots

    CERN Document Server

    Hosseinizadeh, A; Dashti, A; Fung, R; D'Souza, R M; Ourmazd, A

    2014-01-01

    The advent of the X-ray Free Electron Laser (XFEL) has made it possible to record diffraction snapshots of biological entities injected into the X-ray beam before the onset of radiation damage. Algorithmic means must then be used to determine the snapshot orientations and thence the three-dimensional structure of the object. Existing Bayesian approaches are limited in reconstruction resolution typically to 1/10 of the object diameter, with the computational expense increasing as the eighth power of the ratio of diameter to resolution. We present an approach capable of exploiting object symmetries to recover three-dimensional structure to high resolution, and thus reconstruct the structure of the satellite tobacco necrosis virus to atomic level. Our approach offers the highest reconstruction resolution for XFEL snapshots to date, and provides a potentially powerful alternative route for analysis of data from crystalline and nanocrystalline objects.

  17. Study of CME Properties Using High Resolution Data

    Science.gov (United States)

    Egorov, Ya. I.; Fainshtein, V. G.

    The joint use of high-resolution data from SDO and PROBA2 satellites and LASCO/SOHO coronographs enabled us to examine early stages of initiation and propagation of six limb CMEs registered in June 2010 - June 2011. For five events under consideration, the CME initiation is marked by filament (prominence) eruption or by a loop-like structure having another nature. Subsequently, several loop-like structures having higher brightness and following each other at different velocities appear in the region of the CME initiation. The CME frontal structure is formed by these loop-like structures. The CME kinematics and such CME characteristics as angular size and longitudinal to latitudinal size ratio was found for considered all events. We have drawn a conclusion about the possible existence of two CME types dependent on the velocity profile.

  18. High-Resolution Mammography Detector Employing Optical Switching Readout

    Science.gov (United States)

    Irisawa, Kaku; Kaneko, Yasuhisa; Yamane, Katsutoshi; Sendai, Tomonari; Hosoi, Yuichi

    Conceiving a new detector structure, FUJIFILM Corporation has successfully put its invention of an X-ray detector employing "Optical Switching" into practical use. Since Optical Switching Technology allows an electrode structure to be easily designed, both high resolution of pixel pitch and low electrical noise readout have been achieved, which have consequently realized the world's smallest pixel size of 50×50 μm2 from a Direct-conversion FPD system as well as high DQE. The digital mammography system equipped with this detector enables to acquire high definition images while maintaining granularity. Its outstanding feature is to be able to acquire high-precision images of microcalcifications which is an important index in breast examination.

  19. Optical diffraction tomography for high resolution live cell imaging

    Science.gov (United States)

    Sung, Yongjin; Choi, Wonshik; Fang-Yen, Christopher; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

    2009-01-01

    We report the experimental implementation of optical diffraction tomography for quantitative 3D mapping of refractive index in live biological cells. Using a heterodyne Mach-Zehnder interferometer, we record complex field images of light transmitted through a sample with varying directions of illumination. To quantitatively reconstruct the 3D map of complex refractive index in live cells, we apply optical diffraction tomography based on the Rytov approximation. In this way, the effect of diffraction is taken into account in the reconstruction process and diffraction-free high resolution 3D images are obtained throughout the entire sample volume. The quantitative refractive index map can potentially serve as an intrinsic assay to provide the molecular concentrations without the addition of exogenous agents and also to provide a method for studying the light scattering properties of single cells. PMID:19129896

  20. DMD based digital speckle illumination for high resolution imaging

    Science.gov (United States)

    Shinde, Anant; Mishra, Ayush; Perinchery, Sandeep M.; Murukeshan, V. M.

    2017-06-01

    Spatially non-uniform illumination patterns have shown significant potential to improve the imaging. Recent developments in the patterned illumination microscopy have demonstrated that the use of an optical speckle as an illumination pattern significantly improves the imaging resolution at the same time reducing the computational overheads. We present a DMD based method for generation of digital speckle pattern. The generated digital speckle and uniform white light illumination are used as two illuminations to acquire images. The image reconstruction algorithm for blind structured illumination microscopy is used to get the high resolution image. Our approach does not require any calibration step or stringent control of the illumination, and dramatically simplifies the experimental set-up.