Sample records for melting profile pcr

  1. Heterozygote PCR product melting curve prediction. (United States)

    Dwight, Zachary L; Palais, Robert; Kent, Jana; Wittwer, Carl T


    Melting curve prediction of PCR products is limited to perfectly complementary strands. Multiple domains are calculated by recursive nearest neighbor thermodynamics. However, the melting curve of an amplicon containing a heterozygous single-nucleotide variant (SNV) after PCR is the composite of four duplexes: two matched homoduplexes and two mismatched heteroduplexes. To better predict the shape of composite heterozygote melting curves, 52 experimental curves were compared with brute force in silico predictions varying two parameters simultaneously: the relative contribution of heteroduplex products and an ionic scaling factor for mismatched tetrads. Heteroduplex products contributed 25.7 ± 6.7% to the composite melting curve, varying from 23%-28% for different SNV classes. The effect of ions on mismatch tetrads scaled to 76%-96% of normal (depending on SNV class) and averaged 88 ± 16.4%. Based on uMelt ( with an expanded nearest neighbor thermodynamic set that includes mismatched base pairs, uMelt HETS calculates helicity as a function of temperature for homoduplex and heteroduplex products, as well as the composite curve expected from heterozygotes. It is an interactive Web tool for efficient genotyping design, heterozygote melting curve prediction, and quality control of melting curve experiments. The application was developed in Actionscript and can be found online at © 2013 WILEY PERIODICALS, INC.

  2. Real-time PCR gene expression profiling

    Czech Academy of Sciences Publication Activity Database

    Kubista, Mikael; Sjögreen, B.; Forootan, A.; Šindelka, Radek; Jonák, Jiří; Andrade, J.M.


    Roč. 1, - (2007), s. 56-60 ISSN 1360-8606 R&D Projects: GA AV ČR KJB500520601 Institutional research plan: CEZ:AV0Z50520514 Keywords : real - time PCR, * expression profiling * statistical analysis Subject RIV: EB - Genetics ; Molecular Biology

  3. Evaluation of PCR and high-resolution melt curve analysis for differentiation of Salmonella isolates. (United States)

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


    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.

  4. Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling (United States)

    Velez, Daniel Ortiz; Mack, Hannah; Jupe, Julietta; Hawker, Sinead; Kulkarni, Ninad; Hedayatnia, Behnam; Zhang, Yang; Lawrence, Shelley; Fraley, Stephanie I.


    In clinical diagnostics and pathogen detection, profiling of complex samples for low-level genotypes represents a significant challenge. Advances in speed, sensitivity, and extent of multiplexing of molecular pathogen detection assays are needed to improve patient care. We report the development of an integrated platform enabling the identification of bacterial pathogen DNA sequences in complex samples in less than four hours. The system incorporates a microfluidic chip and instrumentation to accomplish universal PCR amplification, High Resolution Melting (HRM), and machine learning within 20,000 picoliter scale reactions, simultaneously. Clinically relevant concentrations of bacterial DNA molecules are separated by digitization across 20,000 reactions and amplified with universal primers targeting the bacterial 16S gene. Amplification is followed by HRM sequence fingerprinting in all reactions, simultaneously. The resulting bacteria-specific melt curves are identified by Support Vector Machine learning, and individual pathogen loads are quantified. The platform reduces reaction volumes by 99.995% and achieves a greater than 200-fold increase in dynamic range of detection compared to traditional PCR HRM approaches. Type I and II error rates are reduced by 99% and 100% respectively, compared to intercalating dye-based digital PCR (dPCR) methods. This technology could impact a number of quantitative profiling applications, especially infectious disease diagnostics.

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

    Vietina, Michelangelo; Agrimonti, Caterina; Marmiroli, Nelson


    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.

  6. Probe-based real-time PCR method for multilocus melt typing of Xylella fastidiosa strains. (United States)

    Brady, Jeff A; Faske, Jennifer B; Ator, Rebecca A; Castañeda-Gill, Jessica M; Mitchell, Forrest L


    Epidemiological studies of Pierce's disease (PD) can be confounded by a lack of taxonomic detail on the bacterial causative agent, Xylella fastidiosa (Xf). PD in grape is caused by strains of Xylella fastidiosa subsp. fastidiosa, but is not caused by other subspecies of Xf that typically colonize plants other than grape. Detection assays using ELISA and qPCR are effective at detecting and quantifying Xf presence or absence, but offer no information on Xf subspecies or strain identity. Surveying insects or host plants for Xf by current ELISA or qPCR methods provides only presence/absence and quantity information for any and all Xf subspecies, potentially leading to false assessments of disease threat. This study uses a series of adjacent-hybridizing DNA melt analysis probes that are capable of efficiently discriminating Xf subspecies and strain relationships in rapid real-time PCR reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. Typing DNA profiles from previously enhanced fingerprints using direct PCR. (United States)

    Templeton, Jennifer E L; Taylor, Duncan; Handt, Oliva; Linacre, Adrian


    Fingermarks are a source of human identification both through the ridge patterns and DNA profiling. Typing nuclear STR DNA markers from previously enhanced fingermarks provides an alternative method of utilising the limited fingermark deposit that can be left behind during a criminal act. Dusting with fingerprint powders is a standard method used in classical fingermark enhancement and can affect DNA data. The ability to generate informative DNA profiles from powdered fingerprints using direct PCR swabs was investigated. Direct PCR was used as the opportunity to generate usable DNA profiles after performing any of the standard DNA extraction processes is minimal. Omitting the extraction step will, for many samples, be the key to success if there is limited sample DNA. DNA profiles were generated by direct PCR from 160 fingermarks after treatment with one of the following dactyloscopic fingerprint powders: white hadonite; silver aluminium; HiFi Volcano silk black; or black magnetic fingerprint powder. This was achieved by a combination of an optimised double-swabbing technique and swab media, omission of the extraction step to minimise loss of critical low-template DNA, and additional AmpliTaq Gold ® DNA polymerase to boost the PCR. Ninety eight out of 160 samples (61%) were considered 'up-loadable' to the Australian National Criminal Investigation DNA Database (NCIDD). The method described required a minimum of working steps, equipment and reagents, and was completed within 4h. Direct PCR allows the generation of DNA profiles from enhanced prints without the need to increase PCR cycle numbers beyond manufacturer's recommendations. Particular emphasis was placed on preventing contamination by applying strict protocols and avoiding the use of previously used fingerprint brushes. Based on this extensive survey, the data provided indicate minimal effects of any of these four powders on the chance of obtaining DNA profiles from enhanced fingermarks. Copyright © 2017

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

    Directory of Open Access Journals (Sweden)

    Lázcoz Paula


    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.

  9. [A rapid screening program on the resistance to streptomycin and ethambutol in Mycobacterium tuberculosis isolates by PCR melting curve analysis]. (United States)

    Wang, Feng; Hu, Si-yu; Gui, Jing; Cui, Yun-yong; Liu, Xiao-li; Li, Qing-ge


    To evaluate the effects of PCR melting curve analysis assay on a rapid screening program regarding the resistance of Mycobacterium tuberculosis (MTB) clinical isolates to streptomycin and ethambutol. A total of 331 clinical isolates of MTB had been collected since 2007-2009 in Shenzhen. Mutations at codon 306, 378-380, 406 and 497 of embB gene, codon 43, 88 of rpsL gene, and 513-517, 905-908 region of rrs gene were detected by PCR melting curve analysis. Results were compared with that of conventional drug susceptibility test. Compared to drug susceptibility test, sensitivity, specificity and accuracy for streptomycin resistance were 78.6%, 90.1% and 86.7%, respectively while 83.0%, 93.3% and 91.8%, respectively for ethambutol resistance detected by PCR melting curve analysis. PCR melting curve method was in good agreement with drug susceptibility test. PCR melting curve analysis on genetic regions associated with resistance to streptomycin and ethambutol seemed to be a rapid, specific and closed-tube method so it could be used for detection of streptomycin and ethambutol resistance in MTB.

  10. 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. (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


    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.

  11. 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


    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    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


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

    Araujo, Sergio; Goulart, Luiz Ricardo; Truman, Richard W; Goulart, Isabela Maria B; Vissa, Varalakshmi; Li, Wei; Matsuoka, Masanori; Suffys, Philip; Fontes, Amanda B; Rosa, Patricia S; Scollard, David M; Williams, Diana L


    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 to

  15. 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


    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

  16. Simplified strategy for rapid first-line screening of fragile X syndrome: closed-tube triplet-primed PCR and amplicon melt peak analysis. (United States)

    Rajan-Babu, Indhu-Shree; Law, Hai-Yang; Yoon, Chui-Sheun; Lee, Caroline G; Chong, Samuel S


    Premutation and full-mutation hyperexpansion of CGG-triplets in the X-linked Fragile X Mental Retardation 1 (FMR1) gene have been implicated in fragile X-associated tremor/ataxia syndrome, fragile X-associated primary ovarian insufficiency, and fragile X syndrome (FXS), respectively. The currently available molecular diagnostic tests are either costly or labour-intensive, which prohibits their application as a first-line FMR1 test in large-scale population-based screening programs. In this study, we demonstrate the utility of a simplified closed-tube strategy for rapid first-line screening of FXS based on melt peak temperature (Tm) analysis of direct triplet-primed polymerase chain reaction amplicons (dTP-PCR MCA). In addition, we also evaluated the correlation between Tm and CGG-repeat size based on capillary electrophoresis (CE) of dTP-PCR amplicons. The assays were initially tested on 29 FMR1 reference DNA samples, followed by a blinded validation on 107 previously characterised patient DNA samples. The dTP-PCR MCA produced distinct melt profiles of higher Tm for samples carrying an expanded allele. Among the samples tested, we also observed a good correlation between Tm and CGG-repeat size. In the blinded validation study, dTP-PCR MCA accurately classified all normal and expansion carriers, and the FMR1 genotypic classification of all samples was completely concordant with the previously determined genotypes as well as the dTP-PCR CE results. This simple and cost-effective MCA-based assay may be useful as a first-line FXS screening tool that could rapidly screen out the large majority of unaffected individuals, thus minimising the number of samples that need to be analysed by Southern blot analysis.

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


    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.

  18. Miltenberger blood group typing by real-time polymerase chain reaction (qPCR) melting curve analysis in Thai population. (United States)

    Vongsakulyanon, A; Kitpoka, P; Kunakorn, M; Srikhirin, T


    To develop reliable and convenient methods for Miltenberger (Mi(a) ) blood group typing. To apply real-time polymerase chain reaction (qPCR) melting curve analysis to Mi(a) blood group typing. The Mi(a) blood group is the collective set of glycophorin hybrids in the MNS blood group system. Mi(a+) blood is common among East Asians and is also found in the Thai population. Incompatible Mi(a) blood transfusions pose the risk of life-threatening haemolysis; therefore, Mi(a) blood group typing is necessary in ethnicities where the Mi(a) blood group is prevalent. One hundred and forty-three blood samples from Thai blood donors were used in the study. The samples included 50 Mi(a+) samples and 93 Mi(a-) samples, which were defined by serology. The samples were typed by Mi(a) typing qPCR, and 50 Mi(a+) samples were sequenced to identify the Mi(a) subtypes. Mi(a) subtyping qPCR was performed to define GP.Mur. Both Mi(a) typing and Mi(a) subtyping were tested on a conventional PCR platform. The results of Mi(a) typing qPCR were all concordant with serology. Sequencing of the 50 Mi(a+) samples revealed 47 GP.Mur samples and 3 GP.Hop or Bun samples. Mi(a) subtyping qPCR was the supplementary test used to further define GP.Mur from other Mi(a) subtypes. Both Mi(a) typing and Mi(a) subtyping performed well using a conventional PCR platform. Mi(a) typing qPCR correctly identified Mi(a) blood groups in a Thai population with the feasibility of Mi(a) subtype discrimination, and Mi(a) subtyping qPCR was able to further define GP.Mur from other Mi(a) subtypes. © 2015 British Blood Transfusion Society.

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

    Directory of Open Access Journals (Sweden)

    Hany Sady


    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.

  20. Application of PCR and High-Resolution Melting for Rapid Identification of Yeasts Routinely Isolated in a Clinical Microbiology Laboratory. (United States)

    Ninghui, Guo; Bing, Wang; Wei, Ren; Mengmeng, Liu; Meiling, Chu; Dongya, Meng; Liqiong, Yao; Wencheng, Xue


    This study aimed to develop a method for rapid and accurate identification of yeasts obtained in the clinic, especially from immunocompromised patients, in order to provide a timely and appropriate antifungal therapy. A total of 112 Candida isolates were analyzed in this study; 28 of them were used to validate the PCR-HRM method in species identification in a blinded manner. Strains were identified by conventional techniques that use VITEK 2 YST cards and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). These methods were compared to the newly developed technique based on real-time polymerase-chain reaction high-resolution melting (PCR-HRM). Discordant results were resolved with internal transcribed spacer (ITS) gene sequencing, the "golden standard" used to evaluate the reliability of all methods in identifying yeasts at the species level. PCR-HRM sensitivity was assessed with the isolated strains. VITEK 2, MALDI-TOF-MS, and PCR-HRM accurately identified 89.2% (74/83), 97.6% (81/83), 100% (83/83) of the isolates, respectively. PCR-HRM detection limit was 1fg/μl of yeasts. In validation assays, a 100% accuracy rate was achieved by the use of PCR-HRM. Therefore, the PCR-HRM method is a rapid, sensitive, and specific diagnostic approach, which provides a cost-effective and more suitable alternative for yeast identification in a clinical laboratory. Future research is needed for automation of data acquisition. © 2015 by the Association of Clinical Scientists, Inc.

  1. Melting profiles may affect detection of residual HPV L1 gene DNA fragments in Gardasil®. (United States)

    Lee, Sin Hang


    Gardasil® is a quadrivalent human papillomavirus (HPV) protein-based vaccine containing genotype-specific L1 capsid proteins of HPV-16, HPV-18, HPV-6 and HPV-11 in the form of virus-like-particles (VLPs) as the active ingredient. The VLPs are produced by a DNA recombinant technology. It is uncertain if the residual HPV L1 gene DNA fragments in the vaccine products are considered contaminants or excipients of the Gardasil® vaccine. Because naked viral DNA fragments, if present in the vaccine, may bind to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant which may help deliver the foreign DNA into macrophages, causing unintended pathophysiologic effects, experiments were undertaken to develop tests for HPV L1 gene DNA fragments in the final products of Gardasil® by polymerase chain reaction (PCR) and direct DNA sequencing. The results showed that while the HPV-11 and HPV-18 L1 gene DNA fragments in Gardasil® were readily amplified by the common GP6/MY11 degenerate consensus primers, the HPV-16 L1 gene DNA may need specially designed non-degenerate PCR primers for amplification at different regions of the L1 gene and different stringency conditions for detection. These variable melting profiles of HPV DNA in the insoluble fraction of the Gardasil® vaccine suggest that the HPV DNA fragments are firmly bound to the aluminum AAHS adjuvant. All methods developed for detecting residual HPV DNA in the vaccine Gardasil® for quality assurance must take into consideration the variable melting profiles of the DNA to avoid false negative results.

  2. Development of a real-time PCR melt curve assay for simultaneous detection of virulent and antibiotic resistant Salmonella. (United States)

    Singh, Prashant; Mustapha, Azlin


    Multiple drug resistance in Salmonella is an emerging problem in the area of food safety. Depending on the virulence and antibiotic resistance characteristics of the Salmonella strain, infections of varying severity could result. In this study, a multiplex melt curve real-time PCR assay for the detection of virulent and antibiotic resistance strains of Salmonella was developed with two primer sets. The first set targets the virulence gene, invasin (invA), and tetracycline (tetG), streptomycin (aadA2) and sulphonamide (sulI) antibiotic resistance genes, and the second set amplifies ampicillin (blaPSE,blaTEM) and chloramphenicol (floR) resistance genes. The multiplex assay was evaluated using 41 Salmonella strains and was further tested on eight different artificially inoculated food samples. The fluorescent DNA intercalating dye, SYTO9, generated high resolution melt curve peaks and, hence, was used for the development of the assay. This multiplex assay worked efficiently over a DNA concentration range of 20 ng-200 fg and showed a sensitivity of 290 CFU/mL with serially diluted broth cultures. The detection limit for un-enriched artificially inoculated food samples was 10(4) CFU/g, but an enrichment period of 6 h allowed for detection of 10 CFU/g of cells in the samples. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Image files of PCR data plotted on the FACS profiles - Plabrain DB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Plabrain DB Image files of PCR data plotted on the FACS profiles Data detail Data name Image files of PCR data plotte...s FACS-based single-cell PCR data was plotted on the FACS profile, in which cells are sorted by DNA content ...d License Update History of This Database Site Policy | Contact Us Image files of PCR data plotted on the FACS profiles - Plabrain DB | LSDB Archive ...

  4. Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array. (United States)

    Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina; Guldberg, Per; Dufva, Martin; Wang, Shan X; Hansen, Mikkel F


    Epigenetic modifications, in particular DNA methylation, are gaining increasing interest as complementary information to DNA mutations for cancer diagnostics and prognostics. We introduce a method to simultaneously profile DNA mutation and methylation events for an array of sites with single site specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection of DNA hybridization, which is insensitive to temperature variation. The melting curve approach further enhances the assay specificity and tolerance to variations in probe length. We demonstrate the utility of this method by simultaneously profiling five mutation and four methylation sites in human melanoma cell lines. The method correctly identified all mutation and methylation events and further provided quantitative assessment of methylation density validated by bisulphite pyrosequencing.

  5. PCR

    African Journals Online (AJOL)



    Jul 11, 2011 ... then in age group 31 to 40 years, 2.25% (2/89) CMV DNA were detected by PCR and 0% was recorded in age group of above 40 years. The overall prevalence of human cytomegalovirus (HCMV) infection in 16 ..... genome revisited: Comparison with the chimpanzee cytomegalovirus genome. J. Gen. Virol.

  6. A real-time ARMS PCR/high-resolution melt curve assay for the detection of the three primary mitochondrial mutations in Leber's hereditary optic neuropathy. (United States)

    Ryan, Siobhan Eustace; Ryan, Fergus; O'Dwyer, Veronica; Neylan, Derek


    Approximately 95% of patients who are diagnosed with Leber's hereditary optic neuropathy (LHON) have one of three mitochondrial point mutations responsible for the disease, G3460A, G11778A, and T14484C. The purpose of this study was to develop a novel multiplex real-time amplification-refractory mutation system (ARMS) PCR combined with high-resolution melt curves to identify the individual mutations involved. The study aimed to provide a more robust, cost- and time-effective mutation detection strategy than that offered with currently available methods. The assay reported in this study will allow diagnostic laboratories to avoid costly next-generation sequencing (NGS) assays for most patients with LHON and to focus resources on patients with unknown mutations that require further analysis. The test uses a combination of multiplex allele-specific PCR (ARMS PCR) in combination with a high-resolution melt curve analysis to detect the presence of the mutations in G3460A, G11778A, and T14484C. PCR primer sets were designed to produce a control PCR product and PCR products only in the presence of the mutations in 3460A, 11778A, and 14484C in a multiplex single tube format. Products produce discrete well-separated melt curves to clearly detect the mutations. This novel real-time ARMS PCR/high-resolution melt curve assay accurately detected 95% of the mutations that cause LHON. The test has proved to be robust, cost- and time-effective with the real-time closed tube system taking approximately 1 h to complete. A novel real-time ARMS PCR/high-resolution melt curve assay is described for the detection of the three primary mitochondrial mutations in LHON. This test provides a simple, robust, easy-to-read output that is cost- and time-effective, thus providing an alternative method to individual endpoint PCR-restriction fragment length polymorphism (RFLP), PCR followed by Sanger sequencing or pyrosequencing, and next-generation sequencing.

  7. Characterization of some Brucella species from Zimbabwe by biochemical profiling and AMOS-PCR

    Directory of Open Access Journals (Sweden)

    Skjerve Eystein


    Full Text Available Abstract Background Bovine brucellosis caused by Brucella abortus is endemic in most large commercial and smallholder cattle farms of Zimbabwe, while brucellosis in other domestic animals is rare. The diagnosis of brucellosis is mainly accomplished using serological tests. However, some Brucella spp. have been isolated from clinical cases in the field and kept in culture collection but their biochemical profiles were not documented. We report biochemical profiling and AMOS-PCR characterization of some of these field isolates of Brucella originating from both commercial and smallholder cattle farming sectors of Zimbabwe. Findings Fourteen isolates of Brucella from culture collection were typed using biochemical profiles, agglutination by monospecific antisera, susceptibility to Brucella-specific bacteriophages and by AMOS-PCR that amplifies species- specific IS711. The results of the biochemical profiles for B. abortus biovar 1 (11 isolates and biovar 2 (2 isolates were consistent with those of reference strains. A single isolate from a goat originating from a smallholder mixed animal farm was identified as B. melitensis biovar 1. The AMOS-PCR produced DNA products of sizes 498 bp and 731 bp for B. abortus (biovar 1 and 2 and B. melitensis biovar 1, respectively. Conclusion We concluded that the biochemical profiles and AMOS-PCR characterization were consistent with their respective species and biovars. B. abortus biovar 1 is likely to be the predominant cause of brucellosis in both commercial and smallholder cattle farms in Zimbabwe.

  8. Evaluation of RAPD-PCR and protein profile analysis to differentiate ...

    Indian Academy of Sciences (India)

    Sixty five isolates of Vibrio harveyi were subjected to random amplified polymorphic DNA (RAPD)-PCR analysis and protein profiling to investigate the genetic variability among V. harveyi prevalent along the coast and also assess the discriminating ability of these two molecular methods. A total of 10 RAPD primers were ...

  9. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature

    DEFF Research Database (Denmark)

    Guðnason, Haukur; Dufva, Hans Martin; Bang, Dang Duong


    The importance of real-time polymerase chain reaction (PCR) has increased steadily in clinical applications over the last decade. Many applications utilize SYBR Green I dye to follow the accumulation of amplicons in real time. SYBR Green I has, however, a number of limitations that include...... investigate 15 different intercalating DNA dyes for their inhibitory effects on PCR, effects on DNA melting temperature and possible preferential binding to GC-rich sequences. Our results demonstrated that in contrast to the results of SYBR Green I, two intercalating dyes SYTO-13 and SYTO-82 do not inhibit...

  10. High-resolution melt PCR analysis for rapid identification of Chlamydia abortus live vaccine strain 1B among C. abortus strains and field isolates. (United States)

    Vorimore, Fabien; Cavanna, Noémie; Vicari, Nadia; Magnino, Simone; Willems, Hermann; Rodolakis, Annie; Siarkou, Victoria I; Laroucau, Karine


    We describe a novel high-resolution melt assay that clearly differentiates Chlamydia abortus live vaccine strain 1B from field C. abortus strains and field wild-type isolates based on previously described single nucleotide polymorphisms. This modern genotyping technique is inexpensive, easy to use, and less time-consuming than PCR-RFLP. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. 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

  12. The workflow of single-cell expression profiling using quantitative real-time PCR

    Czech Academy of Sciences Publication Activity Database

    Stahlberg, A.; Kubista, Mikael


    Roč. 14, č. 3 (2014), s. 323-331 ISSN 1473-7159 R&D Projects: GA ČR GA13-02154S; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 Keywords : single-cell workflow * gene expression profiling * RT-qPCR Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.516, year: 2014

  13. Multiplex real-time PCR assay for the detection of extended-spectrum β-lactamase and carbapenemase genes using melting curve analysis. (United States)

    Singh, Prashant; Pfeifer, Yvonne; Mustapha, Azlin


    Real-time PCR melt curve assays for the detection of β-lactamase, extended-spectrum β-lactamase and carbapenemase genes in Gram-negative bacteria were developed. Two multiplex real-time PCR melt curve assays were developed for the detection of ten common β-lactamase genes: blaKPC-like, blaOXA-48-like, blaNDM-like, blaVIM-like, blaIMP-like, blaCTX-M-1+2-group, blaCMY-like, blaACC-like, blaSHV-like and blaTEM-like. The assays were evaluated using 25 bacterial strains and 31 DNA samples (total n=56) comprising different Enterobacteriaceae genera and Pseudomonas spp. These strains were previously characterized at five research institutes. Each resistance gene targeted in this study generated a non-overlapping and distinct melt curve peak. The assay worked effectively and detected the presence of additional resistance genes in 23 samples. The assays developed in this study offer a simple, low cost method for the detection of prevalent β-lactamase, ESBL and carbapenemase genes among Gram-negative pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. 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. (United States)

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


    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 .

  15. Ultrafast real-time PCR with integrated melting curve analysis and duplex capacities using a low-cost polymer lab-on-a-chip system (United States)

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


    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.

  16. Etiological profile of early neonatal bacterial sepsis by multiplex qPCR. (United States)

    Silva-Junior, Walter P; Martins, Almir S; Xavier, Paula C N; Appel, Kelly L A; Oliveira Junior, Silvio A; Palhares, Durval B


    Given the major impact in terms of morbidity and mortality that episodes of early neonatal sepsis (ENS) have on both newborns and health systems, this study aimed to identify the etiological profile of early neonatal bacterial sepsis by a multiplex quantitative real-time polymerase chain reaction (qPCR). Blood samples from newborns diagnosed with clinical ENS and hospitalized in neonatal intensive care units (NICUs) were collected and analyzed using the multiplex qPCR method to detect Streptococcus agalactiae, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter sp., Serratia sp., and Staphylococcus aureus. A universal primer was used in the analysis. A total of 150 neonates with clinical sepsis and 10 newborns as healthy controls were included in the study. The group with clinical sepsis was 100% positive for the presence of bacterial genomic DNA through the universal primer. The control group showed negativity by qPCR. The multiplex qPCR analysis showed that 76% of the samples were positive for Escherichia coli, 34% for Staphylococcus aureus, 13.3% for Streptococcus agalactiae, 7.3% for Pseudomonas aeruginosa, and 0.7% for Enterobacter sp. and Serratia sp. Multiplex qPCR of patients with clinical sepsis matched with 8.1% of the blood samples that tested positive by the microbiological method. Rapid and sensitive detection of the pathogens causing ENS by this new multi-target approach based on multiplex qPCR could potentially excel compared to microbiological methods, with the simple objective of facilitating the progression to a more rapid and specific antimicrobial therapy, avoiding the abuse of antibiotics in NICUs.

  17. Confirmed identification and toxin profiling of Campylobacter jejuni using a thermostabilized multiplex PCR formulation. (United States)

    Ramachandran, Nitya; Ramlal, Shylaja; Batra, Harsh Vardhan


    Cytolethal distending toxin (CDT) producing Campylobacter jejuni species are one of the leading causes of human gastroenteritis worldwide. The main intent of the study was to develop a multiplex PCR assay for the confirmed identification and toxin profiling of C. jejuni. The genes targeted were rpo B as genus specific, hip O for species; cdt A, cdt B, cdt C encoding respective subunit proteins of CDT with Internal Amplification Control (IAC). To enhance its application as a pre-mixed ready-to-use format, the master mix of developed mPCR was dried by lyophilization and stability was assessed. Thermostabilized reagents showed stability of 1.5 months at room-temperature and upto six months at 4 °C without any loss of functionality. The assay was evaluated on a number of presumptive Campylobacter isolates along with biochemical tests. Results obtained indicated the accurate identification of C. jejuni by developed mPCR format in contrast to misconception associated with biochemical assays. The assay was also tested on spiked samples for its real-time utility. Altogether, the room-temperature storable and ready-to- use mPCR format developed in this study could be preferred for rapid detection and confirmed identification of toxigenic strains of C. jejuni in place of conventional biochemical assays. © 2017 APMIS. Published by John Wiley & Sons Ltd.

  18. A real-time ARMS PCR/high-resolution melt curve assay for the detection of the three primary mitochondrial mutations in Leber?s hereditary optic neuropathy


    Ryan, Siobhan Eustace; Ryan, Fergus; O?Dwyer, Veronica; Neylan, Derek


    Purpose Approximately 95% of patients who are diagnosed with Leber?s hereditary optic neuropathy (LHON) have one of three mitochondrial point mutations responsible for the disease, G3460A, G11778A, and T14484C. The purpose of this study was to develop a novel multiplex real-time amplification-refractory mutation system (ARMS) PCR combined with high-resolution melt curves to identify the individual mutations involved. The study aimed to provide a more robust, cost- and time-effective mutation ...

  19. Automated Forensic Animal Family Identification by Nested PCR and Melt Curve Analysis on an Off-the-Shelf Thermocycler Augmented with a Centrifugal Microfluidic Disk Segment. (United States)

    Keller, Mark; Naue, Jana; Zengerle, Roland; von Stetten, Felix; Schmidt, Ulrike


    Nested PCR remains a labor-intensive and error-prone biomolecular analysis. Laboratory workflow automation by precise control of minute liquid volumes in centrifugal microfluidic Lab-on-a-Chip systems holds great potential for such applications. However, the majority of these systems require costly custom-made processing devices. Our idea is to augment a standard laboratory device, here a centrifugal real-time PCR thermocycler, with inbuilt liquid handling capabilities for automation. We have developed a microfluidic disk segment enabling an automated nested real-time PCR assay for identification of common European animal groups adapted to forensic standards. For the first time we utilize a novel combination of fluidic elements, including pre-storage of reagents, to automate the assay at constant rotational frequency of an off-the-shelf thermocycler. It provides a universal duplex pre-amplification of short fragments of the mitochondrial 12S rRNA and cytochrome b genes, animal-group-specific main-amplifications, and melting curve analysis for differentiation. The system was characterized with respect to assay sensitivity, specificity, risk of cross-contamination, and detection of minor components in mixtures. 92.2% of the performed tests were recognized as fluidically failure-free sample handling and used for evaluation. Altogether, augmentation of the standard real-time thermocycler with a self-contained centrifugal microfluidic disk segment resulted in an accelerated and automated analysis reducing hands-on time, and circumventing the risk of contamination associated with regular nested PCR protocols.

  20. 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.

  1. Meloidogyne javanica Chorismate Mutase Transcript Expression Profile Using Real-Time Quantitative RT-PCR


    Painter, Janet E.; Lambert, Kris N.


    A developmental expression profile of the Meloidodgyne javanica esophageal gland gene chorismate mutase-1 (Mj-cm-1) could suggest when in the lifecycle of the nematode the Mj-cm-1 product is functional. This study used real-time quantitative RT-PCR to examine the variation in Mj-cm-1 transcript levels over six timepoints in the nematode lifecycle: egg, infective second-stage juveniles (Inf-J2), 2-day post-inoculation (pi), 7-day pi, 14-day pi, and adult. The Mj-cm-1 mRNA levels peaked at 2-da...

  2. Simultaneous detection and quantitation of Chikungunya, dengue and West Nile viruses by multiplex RT-PCR assays and dengue virus typing using high resolution melting. (United States)

    Naze, F; Le Roux, K; Schuffenecker, I; Zeller, H; Staikowsky, F; Grivard, P; Michault, A; Laurent, P


    Chikungunya (CHIKV), Dengue (DENV) and West Nile (WNV) viruses are arthropod-borne viruses that are able to emerge or re-emerge in many regions due to climatic changes and increase in travel. Since these viruses produce similar clinical signs it is important for physicians and epidemiologists to differentiate them rapidly. A molecular method was developed for their detection and quantitation in plasma samples and a DENV typing technique were developed. The method consisted in performing two multiplex real-time one-step RT-PCR assays, to detect and quantify the three viruses. Both assays were conducted in a single run, from a single RNA extract containing a unique coextracted and coamplified composite internal control. The quantitation results were close to the best detection thresholds obtained with simplex RT-PCR techniques. The differentiation of DENV types was performed using a High Resolution Melting technique. The assays enable the early diagnosis of the three arboviruses during viremia, including cases of coinfection. The method is rapid, specific and highly sensitive with a potential for clinical diagnosis and epidemiological surveillance. A DENV positive sample can be typed conveniently using the High Resolution Melting technique using the same apparatus.

  3. Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection (United States)

    Kristensen, Lasse S.; Mikeska, Thomas; Krypuy, Michael; Dobrovic, Alexander


    DNA methylation changes that are recurrent in cancer have generated great interest as potential biomarkers for the early detection and monitoring of cancer. In such situations, essential information is missed if the methylation detection is purely qualitative. We describe a new probe-free quantitative methylation-specific PCR (MSP) assay that incorporates evaluation of the amplicon by high-resolution melting (HRM) analysis. Depending on amplicon design, different types of information can be obtained from the HRM analysis. Much of this information cannot be obtained by electrophoretic analysis. In particular, identification of false positives due to incomplete bisulphite conversion or false priming is possible. Heterogeneous methylation can also be distinguished from homogeneous methylation. As proof of principle, we have developed assays for the promoter regions of the CDH1, DAPK1, CDKN2A (p16INK4a) and RARB genes. We show that highly accurate quantification is possible in the range from 100% to 0.1% methylated template when 25 ng of bisulphite-modified DNA is used as a template for PCR. We have named this new approach to quantitative methylation detection, Sensitive Melting Analysis after Real Time (SMART)-MSP. PMID:18344521

  4. Effect of polymer melt wall slip on the flow balance of profile extrusion dies (United States)

    Carneiro, Olga S.; Ferrás, Luís L.; Pinho, Fernando T.; Nóbrega, João M.


    This work describes the implementation of the wall slip boundary condition in an in-house developed 3D numerical code based on the Finite Volume Method. For this purpose, several phenomenological models relating the velocity and the shear stress at the wall were implemented. This new feature is verified using a simple case study, by comparing the numerical results with those obtained through the corresponding analytical solution. Then, the potentialities of the new code are illustrated performing flow simulations of a polymer melt in a complex flow channel. The results obtained show that the slip at the wall influences the flow distribution at the die flow channel outlet. Therefore, and to assess the relevance of slippage in the optimal die geometry, the automatic optimization of a die flow channel, required for the production of a specific thermoplastic profile, is performed using both the no-slip and slip boundary conditions, together with two alternative optimization strategies. It is shown that slip favors the flow balance of the dies and also other issues of its performance.

  5. Complementary use of MALDI-TOF MS and real-time PCR-melt curve analysis for rapid identification of methicillin-resistant staphylococci and VRE. (United States)

    Chan, Wai-Sing; Chan, Tsz-Ming; Lai, Tsz-Wan; Chan, Jasper Fuk-Woo; Lai, Raymond Wai-Man; Lai, Christopher Koon-Chi; Tang, Bone Siu-Fai


    To develop a rapid method for routine screening of methicillin-resistant staphylococci and VRE for clinical isolates and positive blood cultures. Our method consisted of two parts: MALDI-TOF MS was used for identification of staphylococci and enterococci, followed by antibiotic resistance detection by real-time PCR-melt curve analysis without DNA extraction. The latter part included a triplex reaction for staphylococcal culture isolates (mecA, mecALGA251 and Panton-Valentine leucocidin genes), dual PCR of mecA/mecALGA251 and nuc genes for staphylococcal blood cultures, and a duplex reaction for enterococci (vanA and vanB genes). A total of 124 clinical isolates and 56 positive blood cultures were tested. MALDI-TOF MS was performed using Microflex LT (Bruker Daltonik, Bremen, Germany) and Rotor-Gene Q (Qiagen, Hilden, Germany) was used for real-time PCR-melt curve analysis. The total assay time was <2.5 h. The results revealed 100% concordance with antibiotic susceptibility testing or other reference methods for all culture isolates and enterococcal blood cultures. The percentage of concordance for staphylococcal blood cultures was 97.5%. The method described herein was fast, economical, reliable and capable of detecting mecALGA251, vanB1 and vanB2 genotypes, which are not included in most commercial assays. Large-scale screening is required to further test the performance of this protocol, especially for genotypes that are infrequently encountered. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail:

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

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


    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.

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

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


    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

  8. Detection of Campylobacter jejuni in naturally contaminated chicken skin by melting peak analysis of amplicons in real-time PCR. (United States)

    Oliveira, Tereza C R M; Barbut, Shai; Griffiths, Mansel W


    Contamination of poultry by Campylobacter spp. is a significant source of human diarrheal diseases. Traditional methods currently used to detect Campylobacter in foods are time-consuming and labor-intensive. In this study, primers designed for the Campylobacter jejuni cadF gene sequence were used in a SYBR Green I real-time PCR assay as an alternative to a conventional bacteriological method for the rapid detection of C. jejuni from poultry. Twelve portions of chicken purchased from two local grocery stores and 39 portions obtained from a commercial processing plant were examined. Samples of the skin were enriched in Bolton broth at 37 degrees C for 3 h and then at 42 degrees C for 9, 21, or 45 h under microaerobic conditions. DNA was extracted from 1-ml aliquots of the enrichment cultures using 1% Triton X-100. The DNA was used as the template in a real-time polymerase chain reaction (PCR) assay. After 24 h of enrichment, C. jejuni was isolated from 13 samples and all of the positive cultures were also detected by the real-time PCR procedure. C. jejuni was detected by both methods from samples artificially contaminated with 1 or 10 CFU of C. jejuni per 10 g, after 24 h of enrichment. The real-time PCR method was found to be sensitive and specific. It significantly reduced the time required for the detection of C. jejuni in poultry following enrichment of samples.

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

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


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

  10. Combined mutation and rearrangement screening by quantitative PCR high-resolution melting: is it relevant for hereditary recurrent Fever genes?

    Directory of Open Access Journals (Sweden)

    Nathalie Pallares-Ruiz


    Full Text Available The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase, NLRP3 (Nod like receptor family, pyrin domain containing 3 and TNFRSF1A (TNF receptor superfamily 1A, we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations and quantitative (rearrangement screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group and 88 selected (retrospective group patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.

  11. Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array

    DEFF Research Database (Denmark)

    Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina


    specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection...

  12. Molecular characterization of Salmonella enterica serotype Enteritidis isolates from food and human samples by serotyping, antimicrobial resistance, plasmid profiling, (GTG5-PCR and ERIC-PCR

    Directory of Open Access Journals (Sweden)

    F. Fardsanei


    Full Text Available In recent years, Salmonella enterica serovar Enteritidis has been a primary cause of human salmonellosis in many countries. The major objective of this study was to investigate genetic diversity among Salmonella Enteritidis strains from different origins (food and human by Enterobacterial Repetitive Intergenic Consensus (ERIC -PCR, as well as to assess their plasmid profiling and antimicrobial resistance. A total of 30 Salmonella Enteritidis isolates, 15 from food samples (chicken, lamb, beef and duck meats and 15 from clinical samples were collected in Tehran. Identification of isolates as Salmonella was confirmed by using conventional standard biochemical and serological tests. Multiplex-PCR was used for serotyping of isolates to identify Salmonella Enteritidis. Antimicrobial susceptibility testing to 16 agents founds drug resistance patterns among Salmonella Enteritidis isolates. No resistance was observed to cephalexin, ceftriaxone, ceftazidime and cefotaxime, ciprofloxacin, imipenem or meropenem, chloramphenicol and gentamicin. The highest resistance (96.7% was observed to nitrofurantoin. Seven plasmid profiles (P1–P7 were detected, and a 68-kb plasmid was found in all isolates. Two different primers; ERIC and (GTG5 were used for genotyping, which each produced four profiles. The majority of clinical and food isolates fell into two separate common types (CTs with a similar percentage of 95% by ERIC-PCR. Using primer (GTG5, 29 isolates incorporated in three CTs with 70% of isolates showing a single banding pattern. Limited genetic diversity among human and food isolates of Salmonella Enteritidis may indicate that contaminated foods were possibly the source of human salmonellosis. These results confirmed that ERIC-PCR genotyping has limited discriminatory power for Salmonella Enteritidis of different origin.

  13. Difference in melting profiles of gamma irradiated DNA from chicken erythrocytes and from Escherichia coli B/r

    International Nuclear Information System (INIS)

    Kopff, J.; Miller, G.; Leyko, W.


    Effects of gamma irradiation on melting curves of DNA from chicken erythrocytes and Escherichia coli B/r were compared. Considerable changes, following gamma irradiation in the case of chicken erythrocytes DNA and no changes in the case of DNA from Escherichia coli B/r were observed. To explain the lack of changes in gamma irradiated samples of DNA from Escherichia coli B/r it was assumed that the original effects of irradiation were obscured by the process of renaturation of DNA. To exclude the above mentioned effect, examination of gamma irradiated DNA from Escherichia coli B/r was carried out with the addition of formaldehyde immediately after irradiation of the sample. Using this procedure changes of melting profiles of DNA from Escherichia coli B/r were demonstrated. (author)

  14. The reproducibility of RAPD profiles: Effects of PCR components on RAPD analysis of four centaurium species

    Directory of Open Access Journals (Sweden)

    Skorić Marijana


    Full Text Available Random amplified polymorphic DNA (RAPD analysis is a simple and reliable method used to detect DNA polymorphism. Several factors can affect the amplification profiles, thereby causing false bands and non-reproducibility of the assay. In this study, we analyzed the effects of different concentrations of primer, magnesium chloride, template DNA and Taq DNA polymerase to develop and standardize a RAPD protocol for Centaurium species. The optimized PCR reaction mixture included: 50 ng of DNA extracted using a CTbased protocol, 2.5 mM MgCl2, 7.5 pmol primer and 2 U of Taq polymerase in a final volume of 25 μl. Each of the five primers used in experiments (OPB11, OPB15, OPB18, OPF05 and OPH02 generated reproducible and distinguishable fingerprinting patterns of four Centaurium species. The obtained optimized RAPD protocol and the selected primers are useful for our further work in the genetic diversity studies of Centaurium species.

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

    Directory of Open Access Journals (Sweden)

    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.

  16. Development of a SYBR Green real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of canine adenovirus type 1 and type 2. (United States)

    Balboni, Andrea; Dondi, Francesco; Prosperi, Santino; Battilani, Mara


    Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis (ICH) and infectious tracheobronchitis (ITB) in dogs, respectively. Cases of ICH have been documented in recent years and recent surveys have demonstrated a wide percentage of asymptomatic CAdV-1 infection in the canine population. Since both CAdV types are detectable in the same biological matrices, and viral coinfection with CAdV-1 and CAdV-2 are reported with high frequency, it is urgent to have available a rapid, highly sensitive and specific assay for the diagnosis of CAdV infection and distinction between CAdV-1 and CAdV-2. In order to detect canine adenovirus in biological samples and to rapidly distinguish the two viral types, a SYBR Green real-time PCR assay was optimized to discriminate CAdV-1 and CAdV-2 via a melting curve analysis. The developed assay showed high sensitivity and reproducibility and was highly efficient and specific in discriminating the two CAdV types. This reliable and rapid technique may represent a simple, useful and economic option for simultaneous CAdV types detection, which would be feasible and attractive for all diagnostic laboratories, both for clinical purposes and for epidemiological investigations. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Pathway-focused PCR array profiling of enriched populations of laser capture microdissected hippocampal cells after traumatic brain injury.

    Directory of Open Access Journals (Sweden)

    Deborah R Boone

    Full Text Available Cognitive deficits in survivors of traumatic brain injury (TBI are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive or surviving (Fluoro-Jade-negative pyramidal neurons obtained by laser capture microdissection (LCM. In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.

  18. Fluorescent duplex allele-specific PCR and amplicon melting for rapid homogeneous mtDNA haplogroup H screening and sensitive mixture detection.

    Directory of Open Access Journals (Sweden)

    Harald Niederstätter

    Full Text Available BACKGROUND: For large scale studies aiming at a better understanding of mitochondrial DNA (mtDNA, sequence variation in particular mt haplogroups (hgs and population structure, reliable low-cost high-throughput genotyping assays are needed. Furthermore, methods facilitating sensitive mixture detection and relative quantification of allele proportions are indispensable for the study of heteroplasmy, mitochondrial sequence evolution, and mitochondrial disorders. Here the properties of a homogeneous competitive duplex allele specific PCR (ARMS assay were scrutinized in the light of these requirements. METHODOLOGY/PRINCIPAL FINDINGS: A duplex ARMS assay amplifying either the ancestral mtDNA 2706G allele (non-hg H samples or the derived 7028C allele (hg H samples in the presence of SYBR Green fluorescent reporter dye was developed and characterized. Product detection, allele calling, and hg inference were based on the amplicon-characteristic melting-point temperatures obtained with on-line post-PCR fluorescent dissociation curve analysis (DCA. The analytical window of the assay covered at least 5 orders of magnitude of template DNA input with a detection limit in the low picogram range of genomic DNA. A set of forensically relevant test specimens was analyzed successfully. The presence of mtDNA mixtures was detected over a broad range of input DNA amounts and mixture ratios, and the estimation of allele proportions in samples with known total mtDNA content was feasible with limitations. A qualified DNA analyst successfully analyzed approximately 2,200 DNA extracts within three regular working days, without using robotic lab-equipment. By performing the amplification on-line, the assay also facilitated absolute mtDNA quantification. CONCLUSIONS: Although this assay was developed just for a particular purpose, the approach is general in that it is potentially suitable in a broad variety of assay-layouts for many other applications, including the

  19. Multiplex real-time PCR assays for detection of eight Shiga toxin-producing Escherichia coli in food samples by melting curve analysis. (United States)

    Singh, Prashant; Mustapha, Azlin


    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.

  20. PCR-based identification of methicillin–resistant Staphylococcus aureus strains and their antibiotic resistance profiles

    Directory of Open Access Journals (Sweden)

    Abazar Pournajaf


    Conclusions: This study is suggestive that the PCR for detection of mecA gene is a fast, accurate and valuable diagnostic tool, particularly in hospitals in areas where methicillin-resistant S. aureus is endemic.

  1. Accurate and objective copy number profiling using real-time quantitative PCR. (United States)

    D'haene, Barbara; Vandesompele, Jo; Hellemans, Jan


    Copy number changes are known to be involved in numerous human genetic disorders. In this context, qPCR-based copy number screening may serve as the method of choice for targeted screening of the relevant disease genes and their surrounding regulatory landscapes. qPCR has many advantages over alternative methods, such as its low consumable and instrumentation costs, fast turnaround and assay development time, high sensitivity and open format (independent of a single supplier). In this chapter we provide all relevant information for a successfully implement of qPCR-based copy number analysis. We emphasize the significance of thorough in silico and empirical validation of the primers, the need for a well thought-out experiment design, and the importance of quality controls along the entire workflow. Furthermore, we suggest an appropriate and practical way to calculate copy numbers and to objectively interpret the results. The provided guidelines will most certainly improve the quality and reliability of your qPCR-based copy number screening. Copyright 2010 Elsevier Inc. All rights reserved.

  2. Spatial expression profiles in the Xenopus laevis oocytes measured with qPCR tomography

    Czech Academy of Sciences Publication Activity Database

    Šindelka, R.; Šídová, Monika; Švec, David; Kubista, Mikael


    Roč. 51, - (2010), s. 87-91 ISSN 1046-2023 R&D Projects: GA AV ČR(CZ) IAA500970904; GA ČR GA301/09/1752 Institutional research plan: CEZ:AV0Z50520701 Keywords : Xenopus * Oocyte * qPCR tomography Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.527, year: 2010

  3. Molecular serotyping, virulence gene profiling and pathogenicity of Streptococcus agalactiae isolated from tilapia farms in Thailand by multiplex PCR. (United States)

    Kannika, K; Pisuttharachai, D; Srisapoome, P; Wongtavatchai, J; Kondo, H; Hirono, I; Unajak, S; Areechon, N


    This study aimed to biotype Streptococcus agalactiae isolated from tilapia farms in Thailand based on molecular biotyping methods and to determine the correlation between the serotype and virulence of bacteria. In addition to a biotyping (serotyping) technique based on multiplex PCR of cps genes, in this study, we developed multiplex PCR typing of Group B streptococcus (GBS) virulence genes to examine three clusters of virulence genes and their correlation with the pathogenicity of S. agalactiae. The epidemiology of S. agalactiae in Thailand was analysed to provide bacterial genetic information towards a future rational vaccine strategy for tilapia culture systems. Streptococcus agalactiae were isolated from diseased tilapia from different areas of Thailand. A total of 124 S. agalactiae isolates were identified by phenotypic analysis and confirmed by 16S rRNA PCR. Bacterial genotyping was conducted based on (i) molecular serotyping of the capsular polysaccharide (cps) gene cluster and (ii) virulence gene profiling using multiplex PCR analysis of 14 virulence genes (lmb, scpB, pavA, cspA, spb1, cyl, bca, rib, fbsA, fbsB, cfb, hylB, bac and pbp1A/ponA). Only serotypes Ia and III were found in this study; serotype Ia lacks the lmb, scpB and spb1 genes, whereas serotype III lacks only the bac gene. Virulence tests in juvenile Nile tilapia demonstrated a correlation between the pathogenicity of the bacteria and their virulence gene profile, with serotype III showing higher virulence than serotype Ia. Epidemiological analysis showed an almost equal distribution in all regions of Thailand, except serotype III was found predominantly in the southern areas. Only two serotypes of S. agalactiae were isolated from diseased tilapia in Thailand. Serotype Ia showed fewer virulence genes and lower virulence than serotype III. Both serotypes showed a similar distribution throughout Thailand. We identified two major serotypes of S. agalactiae isolates associated with the outbreak in


    Furlaneto-Maia, Luciana; Rocha, Kátia Real; Siqueira, Vera Lúcia Dias; Furlaneto, Márcia Cristina


    Enterococci are increasingly responsible for nosocomial infections worldwide. This study was undertaken to compare the identification and susceptibility profile using an automated MicrosScan system, PCR-based assay and disk diffusion assay of Enterococcus spp. We evaluated 30 clinical isolates of Enterococcus spp. Isolates were identified by MicrosScan system and PCR-based assay. The detection of antibiotic resistance genes (vancomycin, gentamicin, tetracycline and erythromycin) was also determined by PCR. Antimicrobial susceptibilities to vancomycin (30 µg), gentamicin (120 µg), tetracycline (30 µg) and erythromycin (15 µg) were tested by the automated system and disk diffusion method, and were interpreted according to the criteria recommended in CLSI guidelines. Concerning Enterococcus identification the general agreement between data obtained by the PCR method and by the automatic system was 90.0% (27/30). For all isolates of E. faecium and E. faecalis we observed 100% agreement. Resistance frequencies were higher in E. faecium than E. faecalis. The resistance rates obtained were higher for erythromycin (86.7%), vancomycin (80.0%), tetracycline (43.35) and gentamicin (33.3%). The correlation between disk diffusion and automation revealed an agreement for the majority of the antibiotics with category agreement rates of > 80%. The PCR-based assay, the van(A) gene was detected in 100% of vancomycin resistant enterococci. This assay is simple to conduct and reliable in the identification of clinically relevant enterococci. The data obtained reinforced the need for an improvement of the automated system to identify some enterococci. PMID:24626409

  5. Rhizosphere bacterial communities of potato cultivars evaluated through PCR-DGGE profiles Comunidades bacterianas associadas à rizosfera de cultivares de batata avaliadas por perfis de PCR-DGGE

    Directory of Open Access Journals (Sweden)

    Enderson Petrônio de Brito Ferreira


    Full Text Available The objective of this work was to determine the shifts on the PCR-DGGE profiles of bacterial communities associated to the rhizosphere of potato cultivars, in order to generate baseline information for further studies of environmental risk assessment of genetically modified potato plants. A greenhouse experiment was carried out with five potato cultivars (Achat, Bintje, Agata, Monalisa and Asterix, cultivated in pots containing soil from an integrated system for agroecological production. The experiment was conducted in a split plot randomized block design with five cultivars, three sampling periods and five replicates. Rhizosphere samples were collected in three sampling dates during plant development. DNA of rhizosphere microorganisms was extracted, amplified by PCR using bacterial universal primers, and analyzed through DGGE. Shifts on the rhizosphere bacterial communities associated to rhizosphere of different cultivars were related to both cultivar and plant age. Differences among rhizosphere bacterial communities were clearest at the earliest plant age, tending to decrease in later stages. This variation was detected among bacterial communities of the five tested cultivars. The characterization of soil microbial communities can be part of plant breeding programs to be used on studies of environmental risk assessment of genetically modified potatoes.O objetivo deste trabalho foi determinar as alterações nos perfis de PCR-DGGE das comunidades bacterianas associadas à rizosfera de cultivares de batata, para obter informações para futuros estudos de avaliação de risco ambiental de plantas de batatas geneticamente modificadas. Foi conduzido experimento em casa de vegetação com cinco cultivares de batata (Achat, Bintje, Ágata, Monalisa e Asterix, cultivadas em vasos com solo de um sistema integrado de produção agroecológica. O delineamento experimental foi o de blocos ao acaso, em parcelas subdivididas, com cinco cultivares, tr

  6. Use of a high resolution melt real-time polymerase chain reaction (PCR) assay for the environmental monitoring of Vibrio cholerae

    CSIR Research Space (South Africa)

    Le Rouw, Wouter J


    Full Text Available nucleotide sequencing (Inqaba Biotec, South Africa) and alignment of the obtained sequences to sequences in an existing database (GenBank at NCBI, BLASTn option). PCR sensitivity and proficiency evaluation For the evaluation of PCR sensitivity... culture based methods. Using the real-time PCR assay described here, problem areas and possible cholera outbreaks can be quickly identified reducing socio- economic impacts and the loss of lives. REFERENCES Albert MJ, Islam D, Nahar S, Qadri F...

  7. Comparison of lung cancer cell lines representing four histopathological subtypes with gene expression profiling using quantitative real-time PCR

    Directory of Open Access Journals (Sweden)

    Kawaguchi Makoto


    Full Text Available Abstract Background Lung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD, squamous cell carcinoma (SQ, large cell carcinoma (LC, and small cell carcinoma (SC. Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR. Results We selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA and a normal control lung cell line (MRC-9. From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, GAPDH, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L. Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA of gene expression profiling for 12 of the 19 genes (AMY2A, CDH1, FOXG1, IGSF3, ISL1, MALL, PLAU, RAB25, S100P, SLCO4A1, STMN1, and TGM2. The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. S100P in AD cells and CDH1 in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression. Conclusions These results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular S100P and CDH1, may be especially important for distinguishing the different subtypes. Our results

  8. A performance study on three qPCR quantification kits and their compatibilities with the 6-dye DNA profiling systems. (United States)

    Lin, Sze-Wah; Li, Christina; Ip, Stephen C Y


    DNA quantification plays an integral role in forensic DNA profiling. Not only does it estimate the total amount of amplifiable human autosomal and male DNA to ensure optimal amplification of target DNA for subsequent analysis, but also assesses the extraction efficiency and purity of the DNA extract. Latest DNA quantification systems even offer an estimate for the degree of DNA degradation in a sample. Here, we report the performance of three new generation qPCR kits, namely Investigator ® Quantiplex HYres Kit from QIAGEN, Quantifiler ® Trio DNA Quantification Kit from Applied Biosystems™, and PowerQuant ® System from Promega, and their compatibilities with three 6-dye DNA profiling systems. Our results have demonstrated that all three kits generate standard curves with satisfactory consistency and reproducibility, and are capable of screening out traces of male DNA in the presence of 30-fold excess of female DNA. They also exhibit a higher tolerance to PCR inhibition than Quantifiler ® Human DNA Quantification Kit from Applied Biosystems™ in autosomal DNA quantification. PowerQuant ® , as compared to Quantiplex HYres and Quantifiler ® Trio, shows a better precision for both autosomal and male DNA quantifications. Quantifiler ® Trio and PowerQuant ® in contrast to Quantiplex HYres offer better correlations with lower discrepancies between autosomal and male DNA quantification, and their additional degradation index features provide a detection platform for inhibited and/or degraded DNA template. Regarding the compatibility between these quantification and profiling systems: (1) both Quantifiler ® Trio and PowerQuant ® work well with GlobalFiler and Fusion 6C, allowing a fairly accurate prediction of their DNA typing results based on the quantification values; (2) Quantiplex HYres offers a fairly reliable IPC system for detecting any potential inhibitions on Investigator 24plex, whereas Quantifiler ® Trio and PowerQuant ® suit better for Global

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


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


    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 amplifie...

  10. A new general model for predicting melting thermodynamics of complementary and mismatched B-form duplexes containing locked nucleic acids: application to probe design for digital PCR detection of somatic mutations. (United States)

    Hughesman, Curtis; Fakhfakh, Kareem; Bidshahri, Roza; Lund, H Louise; Haynes, Charles


    Advances in real-time polymerase chain reaction (PCR), as well as the emergence of digital PCR (dPCR) and useful modified nucleotide chemistries, including locked nucleic acids (LNAs), have created the potential to improve and expand clinical applications of PCR through their ability to better quantify and differentiate amplification products, but fully realizing this potential will require robust methods for designing dual-labeled hydrolysis probes and predicting their hybridization thermodynamics as a function of their sequence, chemistry, and template complementarity. We present here a nearest-neighbor thermodynamic model that accurately predicts the melting thermodynamics of a short oligonucleotide duplexed either to its perfect complement or to a template containing mismatched base pairs. The model may be applied to pure-DNA duplexes or to duplexes for which one strand contains any number and pattern of LNA substitutions. Perturbations to duplex stability arising from mismatched DNA:DNA or LNA:DNA base pairs are treated at the Gibbs energy level to maintain statistical significance in the regressed model parameters. This approach, when combined with the model's accounting of the temperature dependencies of the melting enthalpy and entropy, permits accurate prediction of T(m) values for pure-DNA homoduplexes or LNA-substituted heteroduplexes containing one or two independent mismatched base pairs. Terms accounting for changes in solution conditions and terminal addition of fluorescent dyes and quenchers are then introduced so that the model may be used to accurately predict and thereby tailor the T(m) of a pure-DNA or LNA-substituted hydrolysis probe when duplexed either to its perfect-match template or to a template harboring a noncomplementary base. The model, which builds on classic nearest-neighbor thermodynamics, should therefore be of use to clinicians and biologists who require probes that distinguish and quantify two closely related alleles in either a

  11. Determination of volatiles in volcanic rocks and minerals with a Directly Coupled Evolved Gas Analyzing System (DEGAS -Part I: Interpretation of degassing profiles (DEGAS-profiles of minerals and rocks on the basis of melting experiments

    Directory of Open Access Journals (Sweden)

    C. M. Schmidt


    Full Text Available Volatile components in magma strongly influence many physical properties of melts and minerals. The temperature resolved degassing analysis of volcanic crystalline and vitreous rocks gives detailed information about volatile compounds in the melt. Aspecial high-temperature mass-spectrometry device in combination with a thermo-balance allows a quantitative determination of different volatile species. It enables a differentiation between the primary gas content in the magma and the gas released from decomposition of secondary alteration products. The gas release profiles give the following indications: i during the littoral explosions of Pahoehoe lava the content of volatiles is not changed by interaction with air or sea water; ii the degassing profiles of vitreous black sand verify the primary content of volatiles in the erupted melt, only CO2 was detected; iii the oxygen release profile gives significant indications for oxygen undersaturation of the erupted magma; iv remelting of black sand in air at 1450°C for 0.45 h causes an oxygen saturation of the basaltic melt; v remelting of black sand in argon atmosphere confirms the oxygen undersaturation of the melt; vi remelting of black sand-black shale mixtures affects a significant change in the degassing profiles, especially in CO2-release. With the first investigations we can demonstrate that gas release curves of volcanic rocks are qualified for a detection of the primary gas content of erupted magma; b detection of alteration processes of the igneous glass; c detection of contamination of the magma with adjacent rocks.

  12. Quantitative detection and differentiation of free-living amoeba species using SYBR green-based real-time PCR melting curve analysis. (United States)

    Behets, Jonas; Declerck, Priscilla; Delaedt, Yasmine; Verelst, Lieve; Ollevier, Frans


    Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.

  13. Multiway real-time PCR gene expression profiling in yeast. Saccharomyces cerevisiae reveals altered transcriptional response of ADH-genes to glucose stimuli

    Czech Academy of Sciences Publication Activity Database

    Stahlberg, A.; Elbing, K.; Andrade-Garda, J.M.; Sjögreen, B.; Forootan, A.; Kubista, Mikael


    Roč. 9, č. 170 (2008), s. 1-41 ISSN 1471-2164 Institutional research plan: CEZ:AV0Z50520701 Keywords : Expression Profiling * Real-time PCR * Yeast Subject RIV: EI - Biotechnology ; Bionics Impact factor: 3.926, year: 2008

  14. Conversion of a molecular classifier obtained by gene expression profiling into a classifier based on real-time PCR: a prognosis predictor for gliomas. (United States)

    Kawarazaki, Satoru; Taniguchi, Kazuya; Shirahata, Mitsuaki; Kukita, Yoji; Kanemoto, Manabu; Mikuni, Nobuhiro; Hashimoto, Nobuo; Miyamoto, Susumu; Takahashi, Jun A; Kato, Kikuya


    The advent of gene expression profiling was expected to dramatically improve cancer diagnosis. However, despite intensive efforts and several successful examples, the development of profile-based diagnostic systems remains a difficult task. In the present work, we established a method to convert molecular classifiers based on adaptor-tagged competitive PCR (ATAC-PCR) (with a data format that is similar to that of microarrays) into classifiers based on real-time PCR. Previously, we constructed a prognosis predictor for glioma using gene expression data obtained by ATAC-PCR, a high-throughput reverse-transcription PCR technique. The analysis of gene expression data obtained by ATAC-PCR is similar to the analysis of data from two-colour microarrays. The prognosis predictor was a linear classifier based on the first principal component (PC1) score, a weighted summation of the expression values of 58 genes. In the present study, we employed the delta-delta Ct method for measurement by real-time PCR. The predictor was converted to a Ct value-based predictor using linear regression. We selected UBL5 as the reference gene from the group of genes with expression patterns that were most similar to the median expression level from the previous profiling study. The number of diagnostic genes was reduced to 27 without affecting the performance of the prognosis predictor. PC1 scores calculated from the data obtained by real-time PCR showed a high linear correlation (r=0.94) with those obtained by ATAC-PCR. The correlation for individual gene expression patterns (r=0.43 to 0.91) was smaller than for PC1 scores, suggesting that errors of measurement were likely cancelled out during the weighted summation of the expression values. The classification of a test set (n=36) by the new predictor was more accurate than histopathological diagnosis (log rank p-values, 0.023 and 0.137, respectively) for predicting prognosis. We successfully converted a molecular classifier obtained by ATAC-PCR

  15. Evaluation of numerical analysis of PFGE-DNA profiles for differentiating Campylobacter fetus subspecies by comparison with phenotypic, PCR and 16S rDNA sequencing methods

    DEFF Research Database (Denmark)

    On, Stephen L.W.; Harrington, C.S.


    Aims: To assess the efficacy of numerical analysis of PFGE-DNA profiles for identification and differentiation of Campylobacter fetus subspecies. Methods and Results: 31 Camp. fetus strains were examined by phenotypic, PCR- and PFGE-based methods, and the 16S rDNA sequences of 18 strains compared....... Conclusions: Numerical analysis of PFGE-DNA profiles is an effective method for differentiating Camp. fetus subspecies. Significance and Impact of the Study: Critical comparison of PFGE, PCR, 16S rDNA sequencing and phenotypic methods for differentiation of Camp. fetus subspecies was attained. Novel....... Numerical analysis of PFGE-DNA profiles divided strains into two clusters at the 86% similarity level. One cluster contained 19 strains clearly identified as Camp. fetus subsp. venerealis. The other cluster comprised 12 strains, of which 10 were unambiguously identified as Camp. fetus subsp. fetus...

  16. RE-PCR variability and toxigenic profile of food poisoning, foodborne and soil-associated Bacillus cereus isolates from Brazil. (United States)

    Santos, C A; Almeida, F S; Guimarães, A G; Abrahão, W M; Arantes, O M N; Vilas-Bôas, G T


    Twenty-three Bacillus cereus isolates from food poisoning outbreaks associated with a diarrheal-type syndrome, fourteen foodborne isolates not associated with food poisoning and fifteen isolates from Brazilian soil samples were analyzed for the presence and genetic diversity (by RE-PCR) of the virulence genes ces (emetic toxin, cereulide), plcR-papR (pleiotropic regulator PlcR and peptide PapR), nheA (a component of the NHE complex), bceT (diarrheal enterotoxin bc-D-ENT), gyrB (B subunit of DNA gyrase), cytK-2 (necrotic enterotoxin cytotoxin K-2), and plcA (phosphatidylcholine-specific phospholipase C). Additionally, these isolates were phenotypically characterized for motility, hemolytic and lecithinase activities, as well as HBL enterotoxin production. The group of isolates associated with food poisoning had the highest occurrence of the phenotypically analyzed factors and the most frequent occurrence and highest genetic diversity of the plcR-papR, nheA, bceT, cytK-2, plcA, and gyrB genes. An analysis of molecular variance (AMOVA), in which all loci were analyzed, demonstrated that the genetic variation intragroup of isolates (92%) was significantly higher than that intergroup (8%) (Pfood poisoning outbreaks do not have a specific genetic profile characterized by the presence of a particular gene or allele among the genes assessed. On the contrary, different combinations of genes encoding virulence factors may be present in different isolates of B. cereus that potentially cause food poisoning outbreaks. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Genomic GC-content affects the accuracy of 16S rRNA gene sequencing bsed microbial profiling due to PCR bias

    DEFF Research Database (Denmark)

    Laursen, Martin F.; Dalgaard, Marlene Danner; Bahl, Martin Iain


    Profiling of microbial community composition is frequently performed by partial 16S rRNA gene sequencing on benchtop platforms following PCR amplification of specific hypervariable regions within this gene. Accuracy and reproducibility of this strategy are two key parameters to consider, which may...... be influenced during all processes from sample collection and storage, through DNA extraction and PCR based library preparation to the final sequencing. In order to evaluate both the reproducibility and accuracy of 16S rRNA gene based microbial profiling using the Ion Torrent PGM platform, we prepared libraries...... and performed sequencing of a well-defined and validated 20-member bacterial DNA mock community on five separate occasions and compared results with the expected even distribution. In general the applied method had a median coefficient of variance of 11.8% (range 5.5-73.7%) for all 20 included strains...

  18. A singleplex real-time fluorescence resonance energy transfer PCR with melting curve analysis for the differential detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs in faeces. (United States)

    Tantrawatpan, Chairat; Saijuntha, Weerachai; Manochantr, Sirikul; Kheolamai, Pakpoom; Thanchomnang, Tongjit; Sadaow, Lakkhana; Intapan, Pewpan M; Maleewong, Wanchai


    Because the eggs of Paragonimus, Echinostoma and Fasciola are very similar in size and shape, it is difficult to distinguish and accurately identify species by the morphology of their eggs, which is a standard diagnostic method. In this study, a novel assay combining a real-time fluorescence resonance energy transfer PCR and melting curve analysis using one set of primers and fluorophore-labelled hybridization probes specific for the 28S rDNA region was developed for the molecular detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs. This assay could detect and distinguish P. heterotremus, E. malayanum and F. gigantica DNA with the distinct melting temperature (Tm) values of 57.99±0.08, 62.12±0.15 and 74.10±0.18, respectively. The assay can also be used to detect and distinguish DNA from P. bangkokensis, P. harinasutai, P. machorchis, E. revolutum, Hypodereum conoideum and F. hepatica, which have different Tm values. The sensitivity of this assay enabled the detection of one egg of P. heterotremus, E. malayanum or F. gigantica per 100 mg of faeces. In addition, the specificity testing showed no fluorescence signal for other parasites. Due to the sensitivity and specificity of our assay in detecting P. heterotremus, E. malayanum and F. gigantica, our method could be used to accurately diagnose these three medically important parasitic groups and has potential implications for molecular epidemiological investigations of human and/or animal infections. © The Author 2015. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail:

  19. Preparation of sustained release co-extrudates by hot-melt extrusion and mathematical modelling of in vitro/in vivo drug release profiles. (United States)

    Quintavalle, U; Voinovich, D; Perissutti, B; Serdoz, F; Grassi, G; Dal Col, A; Grassi, M


    Aim of this work was to develop a cylindrical co-extrudate characterised by an in vivo sustained release profile by means of a hot-melt extrusion process. Co-extrudate was made up of two concentric extruded matrices: an inner one having a hydrophilic character, based on polyethylene glycol, and an outer one with lipophilic character, based on microcrystalline wax. Both segments contained theophylline as a model drug. A screening between several devices differing for dimensions (diameter and length) and relative proportions of the inner and outer part was carried out on the basis of their in vitro drug release and the release mechanism was studied by means of a mathematical model. The co-extrudate exhibiting the desired sustained release was selected for in vivo bioavailability studies. In vivo studies confirmed the achievement of the purpose of the research, demonstrating the desired release of theophylline on four healthy volunteers. Accordingly, hot-melt extrusion process is a viable method to produce in a single step co-extrudates showing a sustained release. In addition, the developed mathematical model proved to be a reliable descriptor of the both in vitro and in vivo experimental data.

  20. Close relatedness of Acanthamoeba pustulosa with Acanthamoeba palestinensis based on isoenzyme profiles and rDNA PCR-RFLP patterns. (United States)

    Kim, Y H; Ock, M S; Yun, H C; Hwang, M Y; Yu, H S; Kong, H H; Chung, D I


    The taxonomic validity of morphological group III Acanthamoeba spp. is uncertain. In the present study, six type strains of group III Acanthamoeba spp., A. culbertsoni, A. healyi, A. pustulosa, A. palestinensis, A. royreba and A. lenticulata were subjected for the evaluation of their taxonomic validity by comparison of the isoenzyme patterns by isoelectic focusing on polyacrylamide gels, mitochondrial DNA (Mt DNA) restriction fragment length polymorphism (RFLP), and small subunit ribosomal DNA (ssu rDNA) PCR-RFLP patterns. The Mt DNA RFLP patterns were heterogeneous between the species. The type strains of A. palestinensis and A. pustulosa showed almost identical patterns of isoenzymes and rDNA PCR RFLP with an estimated sequence divergence of 2.6%. The other species showed heterogeneous patterns of isoenzymes and rDNA PCR-RFLP. It is likely that A. pustulosa is closely related with A. palestinensis and that the former may be regarded as a junior synonym of the latter.

  1. Usefulness of Multiplex Real-Time PCR for Simultaneous Pathogen Detection and Resistance Profiling of Staphylococcal Bacteremia

    Directory of Open Access Journals (Sweden)

    Yousun Chung


    Full Text Available Staphylococci are the leading cause of nosocomial blood stream infections. Fast and accurate identification of staphylococci and confirmation of their methicillin resistance are crucial for immediate treatment with effective antibiotics. A multiplex real-time PCR assay that targets mecA, femA specific for S. aureus, femA specific for S. epidermidis, 16S rRNA for universal bacteria, and 16S rRNA specific for staphylococci was developed and evaluated with 290 clinical blood culture samples containing Gram-positive cocci in clusters (GPCC. For the 262 blood cultures identified to the species level with the MicroScan WalkAway system (Siemens Healthcare Diagnostics, USA, the direct real-time PCR assay of positive blood cultures showed very good agreement for the categorization of staphylococci into methicillin-resistant S. aureus (MRSA, methicillin-susceptible S. aureus (MSSA, methicillin-resistant S. epidermidis (MRSE, methicillin-susceptible S. epidermidis (MSSE, methicillin-resistant non-S. epidermidis CoNS (MRCoNS, and methicillin-susceptible non-S. epidermidis CoNS (MSCoNS (κ=0.9313. The direct multiplex real-time PCR assay of positive blood cultures containing GPCC can provide essential information at the critical point of infection with a turnaround time of no more than 4 h. Further studies should evaluate the clinical outcome of using this rapid real-time PCR assay in glycopeptide antibiotic therapy in clinical settings.

  2. 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. (United States)

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


    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.

  3. Differential gene expression profiling in aggressive bladder transitional cell carcinoma compared to the adjacent microscopically normal urothelium by microdissection-SMART cDNA PCR-SSH. (United States)

    Wang, H T; Ma, F L; Ma, X B; Han, R F; Zhang, Y B; Chang, J W


    Identifying novel and known genes that are differentially expressed in aggressive bladder transitional cell carcinoma (BTCC) has important implications in understanding the biology of bladder tumorigenesis and developing new diagnostic and therapeutic agents. In this study we identified the differential gene expression profiles comparing tumor to the adjacent microscopically normal mucosa by manual microdissection on frozen sections. The RNAs extracted from microdissected tissues were amplified by SMART cDNA PCR technology to generate forward subtractive cDNA library by suppressive subtractive hybridization (SSH). We obtained 376 positive clones, one hundred clones of aggressive BTCC subtracted cDNA library were selected at random and inserts were reamplified by PCR. After differential screening by reverse dot blotting, 73 positive clones, that contend inserts putatively upregulated in aggressive BTCC, were further analysed by DNA sequencing, GenBank and EST database searching. Sequencing results showed that 66 clones stand for 23 known genes and 7 clones for three new EST (Genbank number: DN236875, DN236874 and DN236873). In conclusion, microdissection-SMART cDNA PCR-SSH allowed for an efficient way to identify aggressive BTCC-specific differential expressed genes that may potentially be involved in the carcinogenesis and/or progression of aggressive BTCC. These differentially expressed genes may be of potential utility as therapeutic and diagnostic targets for aggressive BTCC.

  4. PCR-fingerprint profiles of mitochondrial and genomic DNA extracted from Fetus cervi using different extraction methods. (United States)

    Ai, Jinxia; Wang, Xuesong; Gao, Lijun; Xia, Wei; Li, Mingcheng; Yuan, Guangxin; Niu, Jiamu; Zhang, Lihua


    The use of Fetus cervi, which is derived from the embryo and placenta of Cervus Nippon Temminck or Cervs elaphus Linnaeus, has been documented for a long time in China. There are abundant species of deer worldwide. Those recorded by China Pharmacopeia (2010 edition) from all the species were either authentic or adulterants/counterfeits. Identification of their origins or authenticity became a key in the preparation of the authentic products. The traditional SDS alkaline lysis and salt-outing methods were modified to extract mt DNA and genomic DNA from fresh and dry Fetus cervi in addition to Fetus from false animals, respectively. A set of primers were designed by bioinformatics to target the intra-and inter-variation. The mt DNA and genomic DNA extracted from Fetus cervi using the two methods meet the requirement for authenticity. Extraction of mt DNA by SDS alkaline lysis is more practical and accurate than extraction of genomic DNA by salt-outing method. There were differences in length and number of segments amplified by PCR between mt DNA from authentic Fetus cervi and false animals Fetus. The distinctive PCR-fingerprint patterns can distinguish the Fetus cervi from adulterants and counterfeit animal Fetus.

  5. Molecular analysis of the genera eremopyrum (ledeb). jaub. and spach and agropyron gaertner (poaceae) by pcr methods

    International Nuclear Information System (INIS)

    Yilmaz, R.; Cabi, E.; Dogan, M.


    RAPD-PCR (Random Amplified Polymorphic DNA Polymerase Chain Reaction) and Post PCR (Polymerase Chain Reaction) Melting Curve Analysis (MCA) have been used to investigate the pattern of genetic variation among some species in the genera Eremopyrum (Ledeb.) Jaub. and Spach and Agropyron Gaertner (Poaceae). Thirteen primers have been used in the study based on the RAPD-PCR and MCA analyses. Each species produced a distinct pattern of DNA fragments which have been used as a measure of the degree of relationship between species by means of using the RAPD-PCR results with three primers selected for identifying the genetic similarities. Polymorphic melting profiles have been obtained with Post PCR MCA method using three primers. Genetic similarities are calculated for all the species studied with RAPD-PCR and MCA methods, the dendrograms are obtained with the MVSP (Multi Variate Statistical Package) software using UPGMA (Unweighted Pair Group Method with Arithmetic Averages) and Jaccard's Coefficient. Polymorphism between 18 populations of Eremopyrum and 6 Agropyron populations and within the species are determined by using RAPD-PCR and Post PCR melting curve analysis (MCA) respectively. (author)

  6. Changes in Growth, Genomic DNA, Protein Profiles in Wheat Plant Using Physiological and RAPD-PCR Techniques

    International Nuclear Information System (INIS)

    El-Tarras, A.


    Wheat is the major winter cereal crop in the world. The total cultivated area of this crop in Egypt is about two million feddans. Soil salinity represent a serious problem to agriculture in arid and semi-arid in the world. Mexico wheat (Triticum vulgar var. Ycora rojo) was imported in 1999 for cultivation. Mexico wheat was exposed to gamma rays (cobalt 60) from 10 to 80 Krad The unirradiated and irradiated wheat were cultivated in the presence of 0, 5000,10000 and 20000 mg/L of salt solution and 16 hour light /25 degree C. The previous treatment was repeated in combination with 5, 10 mg/l ABA and 10, 20 mg/l GA3 separately. Different accessed parameters were used for evaluation, these parameters were: germination percentage, length of shoots and roots, pigment contents (chl. a,b and a/b carotenoids and total pigments), total protein patterns and RAPD, PCR techniques. The results showed that both of radiation and salinity reduced the percentage of germination. Soaking grains in GA3 considerably increased the shoot and root lengths. Highest value of carotenoids obtained act as a defense mechanism against harmful salinity action. Also, the seedling exposed to 80 Krad and treated with ABA (5 or 10 mg/l) can survive during the experimental period, while plants treated with 10 and 20 mg/l GA3 and exposed to 80 Krad can not survive. At low radiation doses (10 and 20 Krad) there was no difference in the number and density of bands of the total protein patterns, while in the RAPD, PCR technique in presence and/or absence of DNA band in unirradiated and irradiated wheat seeds were observed

  7. RAPD PCR Profile, Antibiotic Resistance, Prevalence of armA Gene, and Detection of KPC Enzyme in Klebsiella pneumoniae Isolates

    Directory of Open Access Journals (Sweden)

    Arezoo Saadatian Farivar


    Full Text Available The increasing prevalence of multidrug-resistant Klebsiella pneumoniae strains isolated from hospitals shows the limitation of recent antibiotics used for bacterial eradication. In this study, 81 K. pneumoniae isolates were collected from three hospitals in Tehran. Antibiotic susceptibility test showed the highest rates of resistance to cefotaxim (85.5% and ceftazidime (78.3%, and the lowest rates of resistance were detected for colistin (16.9%, streptomycin (16.8%, and chloroamphenicol (21.7%. Eleven different resistance patterns were observed. Sixty-six out of 81 isolates (81.5% were found to be multidrug resistant (MDR, and 35.8% of them belonged to A3 resistance pattern. 7.4% and 66.7% were KPC enzyme and armA gene positive, respectively. RAPD PCR assay of these bacteria showed 5 clusters, 16 single types, and 14 common types, and there was not any correlation between genetic patterns of the isolates and presence of resistance agents. Simultaneous detection of resistance-creating agents could be an important challenge for combination therapy of MDR K. pneumoniae-caused infections.

  8. Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86. (United States)

    Nijhof, Ard M; Balk, Jesper A; Postigo, Milagros; Jongejan, Frans


    For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually beta-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in Rhipicephalus appendiculatus and Rhipicephalus (Boophilus) microplus ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against R. microplus were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against R. appendiculatus. In fact, Bm86-based vaccines give better protection against some tick species with lower Bm86 sequence homology. One possible explanation is the variation in Bm86 expression levels between R. microplus and R. appendiculatus. The most stable reference genes were therefore used for normalization of the Bm86 expression profile in all life stages of both species to examine whether antigen abundance plays a role in Bm86 vaccine susceptibility. The transcription levels of nine potential reference genes: beta-actin (ACTB), beta-tubulin (BTUB), elongation factor 1alpha (ELF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutathione S-transferase (GST), H3 histone family 3A (H3F3A), cyclophilin (PPIA), ribosomal protein L4 (RPL4) and TATA box binding protein (TBP) were measured in all life stages of R. microplus and R. appendiculatus. ELF1A was found to be the most stable expressed gene in both species following analysis by both geNorm and Normfinder software applications, GST showed the least stability. The expression profile of Bm86 in R. appendiculatus and R. microplus revealed a more continuous Bm86 antigen abundance in R. microplus throughout its one

  9. Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86

    Directory of Open Access Journals (Sweden)

    Jongejan Frans


    Full Text Available Abstract Background For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually β-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in Rhipicephalus appendiculatus and Rhipicephalus (Boophilus microplus ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against R. microplus were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against R. appendiculatus. In fact, Bm86-based vaccines give better protection against some tick species with lower Bm86 sequence homology. One possible explanation is the variation in Bm86 expression levels between R. microplus and R. appendiculatus. The most stable reference genes were therefore used for normalization of the Bm86 expression profile in all life stages of both species to examine whether antigen abundance plays a role in Bm86 vaccine susceptibility. Results The transcription levels of nine potential reference genes: β-actin (ACTB, β-tubulin (BTUB, elongation factor 1α (ELF1A, glyceraldehyde 3-phosphate dehydrogenase (GAPDH, glutathione S-transferase (GST, H3 histone family 3A (H3F3A, cyclophilin (PPIA, ribosomal protein L4 (RPL4 and TATA box binding protein (TBP were measured in all life stages of R. microplus and R. appendiculatus. ELF1A was found to be the most stable expressed gene in both species following analysis by both geNorm and Normfinder software applications, GST showed the least stability. The expression profile of Bm86 in R. appendiculatus and R. microplus revealed a more continuous Bm86 antigen abundance in R

  10. High Throughput qPCR Expression Profiling of Circulating MicroRNAs Reveals Minimal Sex- and Sample Timing-Related Variation in Plasma of Healthy Volunteers.

    Directory of Open Access Journals (Sweden)

    Catherine Mooney

    Full Text Available MicroRNAs are a class of small non-coding RNA that regulate gene expression at a post-transcriptional level. MicroRNAs have been identified in various body fluids under normal conditions and their stability as well as their dysregulation in disease opens up a new field for biomarker study. However, diurnal and day-to-day variation in plasma microRNA levels, and differential regulation between males and females, may affect biomarker stability. A QuantStudio 12K Flex Real-Time PCR System was used to profile plasma microRNA levels using OpenArray in male and female healthy volunteers, in the morning and afternoon, and at four time points over a one month period. Using this system we were able to run four OpenArray plates in a single run, the equivalent of 32 traditional 384-well qPCR plates or 12,000 data points. Up to 754 microRNAs can be identified in a single plasma sample in under two hours. 108 individual microRNAs were identified in at least 80% of all our samples which compares favourably with other reports of microRNA profiles in serum or plasma in healthy adults. Many of these microRNAs, including miR-16-5p, miR-17-5p, miR-19a-3p, miR-24-3p, miR-30c-5p, miR-191-5p, miR-223-3p and miR-451a are highly expressed and consistent with previous studies using other platforms. Overall, microRNA levels were very consistent between individuals, males and females, and time points and we did not detect significant differences in levels of microRNAs. These results suggest the suitability of this platform for microRNA profiling and biomarker discovery and suggest minimal confounding influence of sex or sample timing. However, the platform has not been subjected to rigorous validation which must be demonstrated in future biomarker studies where large differences may exist between disease and control samples.

  11. Gene expression profiling of acute myeloid leukemia samples from adult patients with AML-M1 and -M2 through boutique microarrays, real-time PCR and droplet digital PCR. (United States)

    Handschuh, Luiza; Kaźmierczak, Maciej; Milewski, Marek C; Góralski, Michał; Łuczak, Magdalena; Wojtaszewska, Marzena; Uszczyńska-Ratajczak, Barbara; Lewandowski, Krzysztof; Komarnicki, Mieczysław; Figlerowicz, Marek


    Acute myeloid leukemia (AML) is the most common and severe form of acute leukemia diagnosed in adults. Owing to its heterogeneity, AML is divided into classes associated with different treatment outcomes and specific gene expression profiles. Based on previous studies on AML, in this study, we designed and generated an AML-array containing 900 oligonucleotide probes complementary to human genes implicated in hematopoietic cell differentiation and maturation, proliferation, apoptosis and leukemic transformation. The AML-array was used to hybridize 118 samples from 33 patients with AML of the M1 and M2 subtypes of the French-American‑British (FAB) classification and 15 healthy volunteers (HV). Rigorous analysis of the microarray data revealed that 83 genes were differentially expressed between the patients with AML and the HV, including genes not yet discussed in the context of AML pathogenesis. The most overexpressed genes in AML were STMN1, KITLG, CDK6, MCM5, KRAS, CEBPA, MYC, ANGPT1, SRGN, RPLP0, ENO1 and SET, whereas the most underexpressed genes were IFITM1, LTB, FCN1, BIRC3, LYZ, ADD3, S100A9, FCER1G, PTRPE, CD74 and TMSB4X. The overexpression of the CPA3 gene was specific for AML with mutated NPM1 and FLT3. Although the microarray-based method was insufficient to differentiate between any other AML subgroups, quantitative PCR approaches enabled us to identify 3 genes (ANXA3, S100A9 and WT1) whose expression can be used to discriminate between the 2 studied AML FAB subtypes. The expression levels of the ANXA3 and S100A9 genes were increased, whereas those of WT1 were decreased in the AML-M2 compared to the AML-M1 group. We also examined the association between the STMN1, CAT and ABL1 genes, and the FLT3 and NPM1 mutation status. FLT3+/NPM1- AML was associated with the highest expression of STMN1, and ABL1 was upregulated in FLT3+ AML and CAT in FLT3- AML, irrespectively of the NPM1 mutation status. Moreover, our results indicated that CAT and WT1

  12. Quantitative Real Time PCR approach to study gene expression profile during prenatal growth of skeletal muscle in pig of Duroc and Pietrain breeds

    Directory of Open Access Journals (Sweden)

    M. Cagnazzo


    Full Text Available The quantitative real time-PCR (QRT-PCR is a very sensitive method used to quantify mRNA level in gene expression analysis. Combining amplification, detection and quantification in a single step, allows a more accurate measurement compared to the traditional PCR end point analysis (Pfaffl, 2001; Bustin, 2002.

  13. Effects of melt percolation on highly siderophile elements and Os isotopes in subcontinental lithospheric mantle: A study of the upper mantle profile beneath Central Europe

    Czech Academy of Sciences Publication Activity Database

    Ackerman, Lukáš; Walker, R. J.; Puchtel, I. S.; Pitcher, L.; Jelínek, E.; Strnad, L.


    Roč. 73, č. 8 (2009), s. 2400-2414 ISSN 0016-7037 R&D Projects: GA AV ČR IAA3013403; GA AV ČR KJB300130612 Institutional research plan: CEZ:AV0Z30130516 Keywords : peridotite * dunite-wehrlite * melt-rock reaction Subject RIV: DD - Geochemistry Impact factor: 4.385, year: 2009

  14. Salmonella isolated from chicken carcasses from a slaughterhouse in the state of Mato Grosso, Brazil: antibiotic resistance profile, serotyping, and characterization by repetitive sequence-based PCR system. (United States)

    Cunha-Neto, Adelino da; Carvalho, Larrayane Albuês; Carvalho, Ricardo César Tavares; Dos Prazeres Rodrigues, Dália; Mano, Sergio Borges; Figueiredo, Eduardo Eustáquio de Souza; Conte-Junior, Carlos Adam


    Salmonella is one of the major causative agents of foodborne infections. Salmonellosis becomes more dangerous when strains resistant to several antibiotics are found in food, especially in chicken, one of the primary transmission vehicles of this pathogen for humans. The present study aimed to estimate the occurrence of Salmonella in chicken carcasses from the state of Mato Grosso, Brazil, as well as determine the antibiotic resistance profile and genotypic characteristic of multi-drug resistant (MDR) isolates. During a 15-month period, from 01/2014 to 05/2015, 850 samples of chilled fresh chicken carcasses were sampled from a slaughterhouse and submitted to Salmonella determinations according to the ISO-6579/2002 method, serotyping and multiplex PCR. The disc diffusion test was applied for 17 antibiotics, according to CLSI (2014). Five isolates were genotyped by repetitive sequence-based PCR using the semi-automated DiversiLab (bioMérieux®) system. The occurrence of Salmonella in chicken carcasses was of 3.7% (31/850), with only 4 strains (12.9%) presenting as MDR, and 6 strains (19.35%) displaying ESBL. The predominant serovars were Salmonella Infantis (35.4%, 11/31), and S. Abony (25.8%, 8/31), followed by serovars S. Agona (12.9%, 4/31), S. Schwarzengrund (9.7%, 3/31), S. Anatum and Salmonella enterica O:4,5 (6.5%, 2/31), and only one Salmonella enterica O:6,7 strain (3.2%, 1/31). All isolates were resistant to one to 5 classes of antibiotics in decreasing order: folate pathway inhibitors, β-lactams (cephalosporins, penicillin, monobactams), tetracyclines, chloramphenicol, and gentamicin. However, strains sensitive to florfenicol, streptomycin, nalidixic acid, ciprofloxacin, enrofloxacin, and nitrofurantoin were also found in this study. Genotyping revealed 98 to 99% homology between 3 Salmonella strains, which displayed high phenotypic resistance similarity to β-lactams and folate pathway inhibitors. Detection of MDR non-typhoid Salmonella in chicken

  15. 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


    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.

  16. The Effect of Pressure on Iron Speciation in Silicate Melts at a Fixed Oxygen Fugacity: The Possibility of a Redox Profile Through a Terrestrial Magma Ocean (United States)

    Armstrong, K.; Frost, D. J.; McCammon, C. A.; Rubie, D. C.; Boffa Ballaran, T.


    As terrestrial planets accreted, mantle silicates equilibrated with core-forming metallic iron, which would have imposed a mantle oxygen fugacity below the iron-wüstite oxygen buffer. Throughout Earth's history, however, the oxygen fugacity of at least the accessible portions of the upper mantle has been 4-5 orders of magnitude higher. The process that caused the rapid increase in the redox state of the mantle soon after core formation is unclear. Here we test the possibility that pressure stabilises ferric iron in silicate melts, as has been observed in silicate minerals. A deep magma ocean, which would have likely existed towards the end of accretion, could then develop a gradient in oxygen fugacity for a fixed ferric-ferrous ratio as a result of pressure. We have equilibrated an andesitic melt with a Ru-RuO2 buffer in a multianvil press between 5 and 24 GPa. Further experiments were performed on the same melt in equilibrium with iron metal. The recovered melts were then analysed using Mössbauer spectroscopy to determine the ferric/ferrous ratio. The results show that for the Ru-RuO2 buffer at lower pressures, the ferric iron content decreases with pressure, due to a positive volume change of the reaction FeO + 1/4O2 = FeO1.5. Ferric iron content also appears to be sensitive to water content at lower pressures. However, above 15 GPa this trend apparently reverses and the ferric iron content increases with pressure. This reversal in pressure dependence would drive the oxygen fugacity of a deep magma ocean with a fixed ferric/ferrous ratio down with increasing depth. This would create a redox gradient, where the magma ocean could potentially be in equilibrium with metallic iron at its base but more oxidised in its shallower regions. Crystallisation of this magma ocean could render an upper mantle oxygen fugacity similar to that in the Earth's accessible mantle today.

  17. 16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice. (United States)

    Walker, Alan W; Martin, Jennifer C; Scott, Paul; Parkhill, Julian; Flint, Harry J; Scott, Karen P


    Characterisation of the bacterial composition of the gut microbiota is increasingly carried out with a view to establish the role of different bacterial species in causation or prevention of disease. It is thus essential that the methods used to determine the microbial composition are robust. Here, several widely used molecular techniques were compared to establish the optimal methods to assess the bacterial composition in faecal samples from babies, before weaning. The bacterial community profile detected in the faeces of infants is highly dependent on the methodology used. Bifidobacteria were the most abundant bacteria detected at 6 weeks in faeces from two initially breast-fed babies using fluorescent in situ hybridisation (FISH), in agreement with data from previous culture-based studies. Using the 16S rRNA gene sequencing approach, however, we found that the detection of bifidobacteria in particular crucially depended on the optimisation of the DNA extraction method, and the choice of primers used to amplify the V1-V3 regions of 16S rRNA genes prior to subsequent sequence analysis. Bifidobacteria were only well represented among amplified 16S rRNA gene sequences when mechanical disruption (bead-beating) procedures for DNA extraction were employed together with optimised "universal" PCR primers. These primers incorporate degenerate bases at positions where mismatches to bifidobacteria and other bacterial taxa occur. The use of a DNA extraction kit with no bead-beating step resulted in a complete absence of bifidobacteria in the sequence data, even when using the optimised primers. This work emphasises the importance of sample processing methodology to downstream sequencing results and illustrates the value of employing multiple approaches for determining microbiota composition.

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




    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...

  19. Real-Time PCR (qPCR) Primer Design Using Free Online Software (United States)

    Thornton, Brenda; Basu, Chhandak


    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  20. Profiles

    International Nuclear Information System (INIS)


    Profiles is a synthetic overview of more than 100 national energy markets in the world, providing insightful facts and key energy statistics. A Profile is structured around 6 main items and completed by key statistics: Ministries, public agencies, energy policy are concerned; main companies in the oil, gas, electricity and coal sectors, status, shareholders; reserve, production, imports and exports, electricity and refining capacities; deregulation of prices, subsidies, taxes; consumption trends by sector, energy market shares; main energy projects, production and consumption prospects. Statistical Profiles are present in about 3 pages the main data and indicators on oil, gas, coal and electricity. (A.L.B.)

  1. MELTING, a flexible platform to predict the melting temperatures of nucleic acids

    Directory of Open Access Journals (Sweden)

    Dumousseau Marine


    Full Text Available Abstract Background Computing accurate nucleic acid melting temperatures has become a crucial step for the efficiency and the optimisation of numerous molecular biology techniques such as in situ hybridization, PCR, antigene targeting, and microarrays. MELTING is a free open source software which computes the enthalpy, entropy and melting temperature of nucleic acids. MELTING 4.2 was able to handle several types of hybridization such as DNA/DNA, RNA/RNA, DNA/RNA and provided corrections to melting temperatures due to the presence of sodium. The program can use either an approximative approach or a more accurate Nearest-Neighbor approach. Results Two new versions of the MELTING software have been released. MELTING 4.3 is a direct update of version 4.2, integrating newly available thermodynamic parameters for inosine, a modified adenine base with an universal base capacity, and incorporates a correction for magnesium. MELTING 5 is a complete reimplementation which allows much greater flexibility and extensibility. It incorporates all the thermodynamic parameters and corrections provided in MELTING 4.x and introduces a large set of thermodynamic formulae and parameters, to facilitate the calculation of melting temperatures for perfectly matching sequences, mismatches, bulge loops, CNG repeats, dangling ends, inosines, locked nucleic acids, 2-hydroxyadenines and azobenzenes. It also includes temperature corrections for monovalent ions (sodium, potassium, Tris, magnesium ions and commonly used denaturing agents such as formamide and DMSO. Conclusions MELTING is a useful and very flexible tool for predicting melting temperatures using approximative formulae or Nearest-Neighbor approaches, where one can select different sets of Nearest-Neighbor parameters, corrections and formulae. Both versions are freely available at at the terms of the GPL license.

  2. Quantifying variant differences in DNA melting curves: Effects of length, melting rate, and curve overlay. (United States)

    Li, M; Palais, R A; Zhou, L; Wittwer, C T


    High resolution DNA melting of PCR products is a simple technique for sequence variant detection and analysis. However, sensitivity and specificity vary and depend on many factors that continue to be defined. We introduce the area between normalized melting curves as a metric to quantify genotype discrimination. The effects of amplicon size (51-547 bp), melting rate (0.01-0.64 °C/s) and analysis method (curve shape by overlay vs absolute temperature differences) were qualitatively and quantitatively analyzed. To limit experimental variance, we studied a single nucleotide variant with identical predicted wild type and homozygous variant stabilities by nearest neighbor thermodynamic theory. Heterozygotes were easier to detect in smaller amplicons, at faster melting rates, and after curve overlay (superimposition), with some p-values overlay, PCR product size, and analysis method is complicated for homozygote genotype discrimination and is difficult to predict. Similar to temperature cycling in PCR, if the temperature control and temperature homogeneity of the solution are adequate, faster rates improve melting analysis, just like faster rates improve PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Multiway real-time PCR gene expression profiling in yeast Saccharomyces cerevisiae reveals altered transcriptional response of ADH-genes to glucose stimuli

    Czech Academy of Sciences Publication Activity Database

    Stahlberg, A.; Elbing, K.; Andrade-Garda, J.M.; Sjögreen, B.; Forootan, A.; Kubista, Mikael


    Roč. 9, č. 170 (2008), s. 1-15 ISSN 1471-2164 Institutional research plan: CEZ:AV0Z50520701 Keywords : qPCR * Sacharomyces cerevisiae * gene expression Subject RIV: EB - Genetic s ; Molecular Biology Impact factor: 3.926, year: 2008

  4. Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. (United States)

    Czechowski, Tomasz; Bari, Rajendra P; Stitt, Mark; Scheible, Wolf-Rüdiger; Udvardi, Michael K


    Summary To overcome the detection limits inherent to DNA array-based methods of transcriptome analysis, we developed a real-time reverse transcription (RT)-PCR-based resource for quantitative measurement of transcripts for 1465 Arabidopsis transcription factors (TFs). Using closely spaced gene-specific primer pairs and SYBR Green to monitor amplification of double-stranded DNA (dsDNA), transcript levels of 83% of all target genes could be measured in roots or shoots of young Arabidopsis wild-type plants. Only 4% of reactions produced non-specific PCR products. The amplification efficiency of each PCR was determined from the log slope of SYBR Green fluorescence versus cycle number in the exponential phase, and was used to correct the readout for each primer pair and run. Measurements of transcript abundance were quantitative over six orders of magnitude, with a detection limit equivalent to one transcript molecule in 1000 cells. Transcript levels for different TF genes ranged between 0.001 and 100 copies per cell. Only 13% of TF transcripts were undetectable in these organs. For comparison, 22K Arabidopsis Affymetrix chips detected less than 55% of TF transcripts in the same samples, the range of transcript levels was compressed by a factor more than 100, and the data were less accurate especially in the lower part of the response range. Real-time RT-PCR revealed 35 root-specific and 52 shoot-specific TF genes, most of which have not been identified as organ-specific previously. Finally, many of the TF transcripts detected by RT-PCR are not represented in Arabidopsis EST (expressed sequence tag) or Massively Parallel Signature Sequencing (MPSS) databases. These genes can now be annotated as expressed.

  5. Melting under shock compression

    International Nuclear Information System (INIS)

    Bennett, B.I.


    A simple model, using experimentally measured shock and particle velocities, is applied to the Lindemann melting formula to predict the density, temperature, and pressure at which a material will melt when shocked from room temperature and zero pressure initial conditions

  6. Improvement of the sensitivity and resolution of PCR-SSCP analysis ...

    Indian Academy of Sciences (India)

    25-dihydroxyvitamin D3) re- ceptor (VDR) to ... C for 5 min. After amplifi- cation, PCR product was purified with low-melting agarose. Keywords. single-strand conformation polymorphism; mutation screening; primer; PCR. Journal of Genetics ...

  7. High similarity of Trypanosoma cruzi kDNA genetic profiles detected by LSSP-PCR within family groups in an endemic area of Chagas disease in Brazil

    Directory of Open Access Journals (Sweden)

    Sandra Maria Alkmim-Oliveira


    Full Text Available Introduction Determining the genetic similarities among Trypanosoma cruzi populations isolated from different hosts and vectors is very important to clarify the epidemiology of Chagas disease. Methods An epidemiological study was conducted in a Brazilian endemic area for Chagas disease, including 76 chronic chagasic individuals (96.1% with an indeterminate form; 46.1% with positive hemoculture. Results T. cruzi I (TcI was isolated from one child and TcII was found in the remaining (97.1% subjects. Low-stringency single-specific-primer-polymerase chain reaction (LSSP-PCR showed high heterogeneity among TcII populations (46% of shared bands; however, high similarities (80-100% among pairs of mothers/children, siblings, or cousins were detected. Conclusions LSSP-PCR showed potential for identifying similar parasite populations among individuals with close kinship in epidemiological studies of Chagas disease.

  8. [Solder melting torches]. (United States)

    Cubero Postigo, G


    In this study about melting and torchs employed in solder in fixed prosthodontics, it's analysed the accurate melting, adequate quantity, as well as protection of adjacent tissues with an accurate anti-melting. The torch chosen is the oxyacetylene burner, because its greater calorific power.

  9. Effect of the distribution of saturated fatty acids in the melting and crystallization profiles of high-oleic high-stearic oils

    Directory of Open Access Journals (Sweden)

    Bootello, M. A.


    Full Text Available The composition and distribution of fatty acids in triacylglycerol (TAG molecules are commonly considered as factors that determine the physical properties of a given oil or fat. The distribution of any fatty acid in fats and oils can be described through the α coefficient of asymmetry, which can be calculated from the TAG composition and fatty acid composition of the sn-2 position of the TAGs determined through lipase hydrolysis. High-oleic high-stearic oils and fats are considered stable and healthy, and they are good substitutes for hydrogenated vegetable oils and palm fractions in many food products, such as spreads and confectionery. Here, different high-oleic high-stearic acid oils were formulated which contained different distributions of saturated fatty acids in their TAGs, while maintaining a similar fatty acid composition. The aim of this work was to discuss the possibility of using the α coefficient to predict the physical properties of fats in function of their chemical composition and their melting and crystallization behavior as examined by differential scanning calorimetry.La composición y distribución de los ácidos grasos en las moléculas de triglicéridos se consideran factores determinantes en las propiedades físicas de los aceites y grasas. La distribución de ácidos grasos en un determinado aceite o grasa puede caracterizarse mediante un coeficiente de asimetría α, calculado a partir de las composiciones de triglicéridos y de ácidos grasos en la posición sn-2 de la molécula de triglicérido mediante hidrólisis con lipasa. El aceite de girasol alto oleico-alto esteárico es una grasa estable y saludable, adecuada para reemplazar a los aceites vegetales hidrogenados y fracciones de palma en muchos productos alimentarios, como grasas plásticas y grasas de confitería. En el presente trabajo, se formularon diferentes aceites alto oleico-alto esteárico con diferente distribución de los ácidos grasos saturados en

  10. Classification of Fowl Adenovirus Serotypes by Use of High-Resolution Melting-Curve Analysis of the Hexon Gene Region▿ (United States)

    Steer, Penelope A.; Kirkpatrick, Naomi C.; O'Rourke, Denise; Noormohammadi, Amir H.


    Identification of fowl adenovirus (FAdV) serotypes is of importance in epidemiological studies of disease outbreaks and the adoption of vaccination strategies. In this study, real-time PCR and subsequent high-resolution melting (HRM)-curve analysis of three regions of the hexon gene were developed and assessed for their potential in differentiating 12 FAdV reference serotypes. The results were compared to previously described PCR and restriction enzyme analyses of the hexon gene. Both HRM-curve analysis of a 191-bp region of the hexon gene and restriction enzyme analysis failed to distinguish a number of serotypes used in this study. In addition, PCR of the region spanning nucleotides (nt) 144 to 1040 failed to amplify FAdV-5 in sufficient quantities for further analysis. However, HRM-curve analysis of the region spanning nt 301 to 890 proved a sensitive and specific method of differentiating all 12 serotypes. All melt curves were highly reproducible, and replicates of each serotype were correctly genotyped with a mean confidence value of more than 99% using normalized HRM curves. Sequencing analysis revealed that each profile was related to a unique sequence, with some sequences sharing greater than 94% identity. Melting-curve profiles were found to be related mainly to GC composition and distribution throughout the amplicons, regardless of sequence identity. The results presented in this study show that the closed-tube method of PCR and HRM-curve analysis provides an accurate, rapid, and robust genotyping technique for the identification of FAdV serotypes and can be used as a model for developing genotyping techniques for other pathogens. PMID:19036935

  11. Application of SSH and quantitative real time PCR to construction of gene expression profiles from scallop Chlamys farreri in response to exposure to tetrabromobisphenol A. (United States)

    Gong, Xiaoli; Pan, Luqing; Miao, Jingjing; Liu, Na


    TBBPA-induced genes were identified using suppression subtractive hybridization (SSH) from Chlamys farreri. A total of 203 and 44 clones from SSH forward and reverse library were respectively obtained including cellular process, immune system process, response to stimulus, metabolic process and signaling etc. Differential gene expressions were compared between scallops from control and TBBPA treatment groups (400 μg/L, 15 days) using quantitative real time RT-PCR. For further research, eight significant genes expression from scallops exposed to TBBPA (0; 100; 200; 400 μg/L) sampling at 0, 1, 3, 6 and 15 days, were utilized for Q-RT-PCR. The results revealed that the expression level of most selected cDNAs was dominantly up-regulated or down-regulated in the TBBPA-induced scallops. These findings provide basic genomic information of the bivalve and the selected genes may be the potential molecular biomarkers for TBBPA pollution in aquatic environment. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  12. High resolution melting for mutation scanning of TP53 exons 5–8

    International Nuclear Information System (INIS)

    Krypuy, Michael; Dobrovic, Alexander; Ahmed, Ahmed Ashour; Etemadmoghadam, Dariush; Hyland, Sarah J; Australian Ovarian Cancer Study Group; Fazio, Anna de; Fox, Stephen B; Brenton, James D; Bowtell, David D


    p53 is commonly inactivated by mutations in the DNA-binding domain in a wide range of cancers. As mutant p53 often influences response to therapy, effective and rapid methods to scan for mutations in TP53 are likely to be of clinical value. We therefore evaluated the use of high resolution melting (HRM) as a rapid mutation scanning tool for TP53 in tumour samples. We designed PCR amplicons for HRM mutation scanning of TP53 exons 5 to 8 and tested them with DNA from cell lines hemizygous or homozygous for known mutations. We assessed the sensitivity of each PCR amplicon using dilutions of cell line DNA in normal wild-type DNA. We then performed a blinded assessment on ovarian tumour DNA samples that had been previously sequenced for mutations in TP53 to assess the sensitivity and positive predictive value of the HRM technique. We also performed HRM analysis on breast tumour DNA samples with unknown TP53 mutation status. One cell line mutation was not readily observed when exon 5 was amplified. As exon 5 contained multiple melting domains, we divided the exon into two amplicons for further screening. Sequence changes were also introduced into some of the primers to improve the melting characteristics of the amplicon. Aberrant HRM curves indicative of TP53 mutations were observed for each of the samples in the ovarian tumour DNA panel. Comparison of the HRM results with the sequencing results revealed that each mutation was detected by HRM in the correct exon. For the breast tumour panel, we detected seven aberrant melt profiles by HRM and subsequent sequencing confirmed the presence of these and no other mutations in the predicted exons. HRM is an effective technique for simple and rapid scanning of TP53 mutations that can markedly reduce the amount of sequencing required in mutational studies of TP53

  13. Role of Melt Curve Analysis in Interpretation of Nutrigenomics' MicroRNA Expression Data. (United States)

    Ahmed, Farid E; Gouda, Mostafa M; Hussein, Laila A; Ahmed, Nancy C; Vos, Paul W; Mohammad, Mahmoud A


    This article illustrates the importance of melt curve analysis (MCA) in interpretation of mild nutrogenomic micro(mi)RNA expression data, by measuring the magnitude of the expression of key miRNA molecules in stool of healthy human adults as molecular markers, following the intake of Pomegranate juice (PGJ), functional fermented sobya (FS), rich in potential probiotic lactobacilli, or their combination. Total small RNA was isolated from stool of 25 volunteers before and following a three-week dietary intervention trial. Expression of 88 miRNA genes was evaluated using Qiagen's 96 well plate RT 2 miRNA qPCR arrays. Employing parallel coordinates plots, there was no observed significant separation for the gene expression (Cq) values, using Roche 480® PCR LightCycler instrument used in this study, and none of the miRNAs showed significant statistical expression after controlling for the false discovery rate. On the other hand, melting temperature profiles produced during PCR amplification run, found seven significant genes (miR-184, miR-203, miR-373, miR-124, miR-96, miR-373 and miR-301a), which separated candidate miRNAs that could function as novel molecular markers of relevance to oxidative stress and immunoglobulin function, for the intake of polyphenol (PP)-rich, functional fermented foods rich in lactobacilli (FS), or their combination. We elaborate on these data, and present a detailed review on use of melt curves for analyzing nutigenomic miRNA expression data, which initially appear to show no significant expressions, but are actually more subtle than this simplistic view, necessitating the understanding of the role of MCA for a comprehensive understanding of what the collective expression and MCA data collectively imply. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  14. Expression Profile of IL-35 mRNA in Gingiva of Chronic Periodontitis and Aggressive Periodontitis Patients: A Semiquantitative RT-PCR Study

    Directory of Open Access Journals (Sweden)

    Nagaraj B. Kalburgi


    Full Text Available Background. Proinflammatory and anti-inflammatory cytokines play a key role in the pathogenesis of periodontal diseases. Secretion of bioactive IL-35 has been described by T regulatory cells ( and is required for their maximal suppressive activity. are involved in the modulation of local immune response in chronic periodontitis patients. Objective. Hence, the present study was aimed to investigate the expression of IL-35 mRNA in chronic periodontitis and aggressive periodontitis patients. Materials and Methods. The present study was carried out in 60 subjects, which included 20 chronic periodontitis patients, 20 aggressive periodontitis patients, and 20 periodontally healthy controls. IL-35 mRNA expression in gingival tissue samples of all subjects was semiquantitatively analyzed using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR. Results. The present study demonstrated the expression of IL-35 mRNA in gingival tissues of all the three groups. IL-35 mRNA expression was highest in chronic periodontitis subjects ( as compared to the aggressive periodontitis group ( and least seen in healthy patients (. Conclusion. The increased expression of IL-35 in chronic and aggressive periodontitis suggests its possible role in pathogenesis of periodontitis. Future studies done on large samples with intervention will strengthen our result.

  15. Determination of haemolytic and non haemolytic genes profiles of Bacillus cereus strains isolated from food samples by polymerase chain reaction (pcr) technique (United States)

    Jawad, Nisreen; Ahemd, Asmat; Abdullah, Aminah


    The aim of this study was to investigate the presence of Bacillus cereus and detection of enterotoxigenic genes in food samples by utilizing a Polymerase Chain Reaction technique (PCR). In this study the providence of B. cereus was carried out to food samples. The B. cereus isolates were investigated for enterotoxigenic gene. The cooked seafood, and raw milk samples were purchased from several restaurants and market in the area of (Bangi, Kajang, Serdang and UKM) Selangor, Malaysia. A total of 60 samples have been analyzed. B. cereus contamination has been formed between 1.4×105 - 3×105 cfu/mL of cooked seafood and raw milk samples. Five colonies have been detected as B. cereus using biochemical test. All B. cereus isolates named BC1 to BC27, were characterized for haemolytic enterotoxin (HBL) complex encoding genes (hblA), non-haemolytic enterotoxin encoding gene (NheA). 10 isolates have been reported to be positive towards hblA and 12 isolates were positive towards NheA. The presence of B. cereus and their enterotoxigenic genes in cooked seafood and raw milk from to food samples obtained may pose a potential risk for public health.

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

    African Journals Online (AJOL)

    PKD1 and PKD2 are the two genes responsible for the development of autosomal dominant polycystic kidney disease (ADPKD). PKD1 gene mutations accounts for ≈85% of all ADPKD cases, while the remaining ≈15% of cases is caused by mutations in the PKD2 gene. Genotyping for PKD1 and PKD2 mutations was ...

  17. 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


    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....

  18. Clinical profile of uveitis in Hansen’s disease after completion of treatment – A study of 50 cases using Polymerase Chain Reaction (PCR on aqueous humour

    Directory of Open Access Journals (Sweden)

    Radha Annamalai


    Full Text Available Chronic low grade anterior uveitis is the commonest cause of blindness in leprosy. It is usually asymptomatic until the late stages and patients seek help only after irreversible visual loss. We analysed patients who had a recurrence of uveitis after completion of treatment with anti-leprosy drugs and had been proven as histopathologically negative. The presence of chronic uveitis, complications and the extent of ocular damage it may cause, can continue even after treatment, emphasising the importance of follow-up, early detection and treatment. This is a prospective cohort study. Ophthalmic evaluation was performed using slit lamp examination, biomicroscopy, indirect ophthalmoscopy, applanation tonometry, corneal sensation and Schirmer’s test. Split skin microscopy was done to confirm the activity of leprosy. In patients with recalcitrant iridocyclitis, anterior chamber paracentesis was performed. The sample was analysed both by smear and polymerase chain reaction. The sequences that were targeted using PCR included genes encoding the DNA of 36-kDa antigen, 18-kDa antigen, 65-kDa antigen and the repetitive sequences among other M. leprae genes. Aqueous aspirate showed copies of mycobacterium leprae DNA in five out of twelve patients with recalcitrant anterior uveitis. Direct smear and staining with Ziehl- Neelson staining for mycobacteria was positive showing both live and dead bacilli. Live bacilli can persist in the aqueous humour even after completion of treatment. In our study this was more frequently observed in tuberculoid leprosy. This is possibly due to an immune mediated response combined with inadequate treatment dose in these patients.

  19. Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression

    Directory of Open Access Journals (Sweden)

    Řičicová Markéta


    Full Text Available Abstract Background Candida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein and of genes belonging to the ALS (agglutinin-like sequence, SAP (secreted aspartyl protease, PLB (phospholipase B and LIP (lipase gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP or in the Centres for Disease Control (CDC reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR model, and on mucosal surfaces in the reconstituted human epithelium (RHE model. Results HWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model. Conclusions Our findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression

  20. Melt Cast High Explosives

    Directory of Open Access Journals (Sweden)

    Stanisław Cudziło


    Full Text Available [b]Abstract[/b]. This paper reviews the current state and future developments of melt-cast high explosives. First the compositions, properties and methods of preparation of trinitrotoluene based (TNT conventional mixtures with aluminum, hexogen (RDX or octogen (HMX are described. In the newer, less sensitive explosive formulations, TNT is replaced with dinitroanisole (DNANDNANDNAN and nitrotriazolone (NTONTONTO, nitroguanidine (NG or ammonium perchlorate (AP are the replacement for RDRDX and HMX. Plasticized wax or polymer-based binder systems for melt castable explosives are also included. Hydroxyl terminated polybutadiene (HPTB is the binder of choice, but polyethylene glycol, and polycaprolactone with energetic plasticizers are also used. The most advanced melt-cast explosives are compositions containing energetic thermoplastic elastomers and novel highly energetic compounds (including nitrogen rich molecules in whose particles are nanosized and practically defect-less.[b]Keywords[/b]: melt-cast explosives, detonation parameters

  1. Melt fracture revisited

    Energy Technology Data Exchange (ETDEWEB)

    Greenberg, J. M.


    In a previous paper the author and Demay advanced a model to explain the melt fracture instability observed when molten linear polymer melts are extruded in a capillary rheometer operating under the controlled condition that the inlet flow rate was held constant. The model postulated that the melts were a slightly compressible viscous fluid and allowed for slipping of the melt at the wall. The novel feature of that model was the use of an empirical switch law which governed the amount of wall slip. The model successfully accounted for the oscillatory behavior of the exit flow rate, typically referred to as the melt fracture instability, but did not simultaneously yield the fine scale spatial oscillations in the melt typically referred to as shark skin. In this note a new model is advanced which simultaneously explains the melt fracture instability and shark skin phenomena. The model postulates that the polymer is a slightly compressible linearly viscous fluid but assumes no slip boundary conditions at the capillary wall. In simple shear the shear stress {tau}and strain rate d are assumed to be related by d = F{tau} where F ranges between F{sub 2} and F{sub 1} > F{sub 2}. A strain rate dependent yield function is introduced and this function governs whether F evolves towards F{sub 2} or F{sub 1}. This model accounts for the empirical observation that at high shears polymers align and slide more easily than at low shears and explains both the melt fracture and shark skin phenomena.

  2. Radioactive waste melting furnace

    International Nuclear Information System (INIS)

    Nakayama, Junpei.


    The present invention provides a radioactive waste melting furnace excellent in heat insulating property, capable of exchanging only refractory materials with lesser amount of contamination. Namely, an heat insulation layer is disposed on the outer wall of the melting furnace. A refractory layer is disposed on the inner wall being in contact with molten materials in the melting furnace. A metal vessel covering the refractory layer is interposed between the heat insulation layer and the refractory layer. In addition, a metal outer shell covering the heat insulation layer is disposed on the heat insulation layer on the outer wall of the melting furnace. Bricks comprising, for example, alumina, carbon, zircon, magnesia or chromia having a low heat conductivity are used for the outer wall heat insulation layer irrespective of the melting performance. The refractory layer on the inner wall is made of bricks comprising chromia, alumina and zircon as molten materials of low basicity and chromia and magnesia as molten materials of high basicity. The materials of the metal vessel may be ordinary carbon steels, cast irons, or stainless steels. The refractory layer is taken out from the melting furnace together with the metal vessel, and only the refractory layer can be removed. Radiation contamination is eliminated. The metal vessel can be used again. (I.S.)

  3. Multicomponent Diffusion in Experimentally Cooled Melt Inclusions (United States)

    Saper, L.; Stolper, E.


    Glassy olivine-hosted melt inclusions are compositionally zoned, characterized by a boundary layer depleted in olivine-compatible components that extends into the melt inclusion from its wall. The boundary layer forms in response to crystallization of olivine and relaxes with time due to diffusive exchange with the interior of the inclusion. At magmatic temperatures, the time scale for homogenization of inclusions is minutes to hours. Preservation of compositional gradients in natural inclusions results from rapid cooling upon eruption. A model of MgO concentration profiles that couples crystal growth and diffusive relaxation of a boundary layer can be used to solve for eruptive cooling rates [1]. Controlled cooling-rate experiments were conducted to test the accuracy of the model. Mauna Loa olivine containing >80 µm melt inclusions were equilibrated at 1225°C in a 1-atm furnace for 24 hours, followed by linear cooling at rates of 102 - 105 °C/hr. High-resolution concentration profiles of 40 inclusions were obtained using an electron microprobe. The model of [1] fits the experimental data with low residuals and the best-fit cooling rates are within 30% of experimental values. The initial temperature of 1225 °C is underestimated by 65°C. The model was modified using (i) MELTS to calculate the interface melt composition as a function of temperature, and (ii) a concentration-dependent MgO diffusion coefficient using the functional form of [2]. With this calibration the best-fit starting temperatures are within 5°C of the experimental values and the best-fit cooling rates are within 20% of experimental rates. The evolution of the CaO profile during cooling is evidence for strong diffusive coupling between melt components. Because CaO is incompatible in olivine, CaO concentrations are expected to be elevated in the boundary layer adjacent to the growing olivine. Although this is observed at short time scales, as the profile evolves the CaO concentration near the


    Directory of Open Access Journals (Sweden)

    L. V. Golubeva


    Full Text Available Summary. Melted butter is made from dairy butter by rendering the fat phase. It has specific taste and aroma, high-calorie content and good assimilability. Defects of butter which appeared during the storage causes by the development of microbiological processes or by the chemical oxidation. On the development of these processes influence quality and composition of fresh butter, its physical structure, content of the increased amount of gas phase and content of heavy metals, storage conditions. Microbiological spoilage of butter occurs generally due to damage of plasma which is good environment for the development of microorganisms. Defects of microbiological origin include: unclean, sour, moldy, yeasty, cheesy, bitter taste. Defects of test and smell chemical origin are formed due to hydrolytic digestion of lipids. It's prevailed at long storage of butter in the conditions of freezing temperatures. It's picked out the following main processes of spoiling: souring, acidifying and sallowness. Often these processes take place simultaneously.It has been investigated melted butter with lactated additive. The latter improves the microbiological and toxicological safety, prolongs the storage condition of the products. Technological efficiency of the additives is achieved by a multilayer products formation from the inactive bound water, preventing microorganisms growth and by the barrier layer with lactate inhibiting hydrolytic reactions. Oil samples were obtained with the batch-type butter maker application, then they were melted and after that lactated additive were supplemented. It has been studied organoleptic and physico-chemical indices of the melted butter samples. The fatty-acid composition of melted butter were studied. Comparative analysis of fatty-acid composition of cow's milk fat and produced melted butter has shown their similarity. Also in the last sample there is increased weight fraction of linoleic and linolenic acids. The obtained

  5. Melting temperature of graphite

    International Nuclear Information System (INIS)

    Korobenko, V.N.; Savvatimskiy, A.I.


    Full Text: Pulse of electrical current is used for fast heating (∼ 1 μs) of metal and graphite specimens placed in dielectric solid media. Specimen consists of two strips (90 μm in thick) placed together with small gap so they form a black body model. Quasy-monocrystal graphite specimens were used for uniform heating of graphite. Temperature measurements were fulfilled with fast pyrometer and with composite 2-strip black body model up to melting temperature. There were fulfilled experiments with zirconium and tungsten of the same black body construction. Additional temperature measurements of liquid zirconium and liquid tungsten are made. Specific heat capacity (c P ) of liquid zirconium and of liquid tungsten has a common feature in c P diminishing just after melting. It reveals c P diminishing after melting in both cases over the narrow temperature range up to usual values known from steady state measurements. Over the next wide temperature range heat capacity for W (up to 5000 K) and Zr (up to 4100 K) show different dependencies of heat capacity on temperature in liquid state. The experiments confirmed a high quality of 2-strip black body model used for graphite temperature measurements. Melting temperature plateau of tungsten (3690 K) was used for pyrometer calibration area for graphite temperature measurement. As a result, a preliminary value of graphite melting temperature of 4800 K was obtained. (author)

  6. Development of -Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Species

    Directory of Open Access Journals (Sweden)

    Jing Zhang


    Full Text Available Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan- Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan- Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis , and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.


    Directory of Open Access Journals (Sweden)

    Němec L.


    Full Text Available Four aspects of effective glass melting have been defined – namely the fast kinetics of partial melting phenomena, a consideration of the melting phenomena ordering, high utilisation of the melting space, and effective utilisation of the supplied energy. The relations were defined for the specific melting performance and specific energy consumption of the glass melting process which involve the four mentioned aspects of the process and indicate the potentials of effective melting. The quantity “space utilisation” has been treated in more detail as an aspect not considered in practice till this time. The space utilisation was quantitatively defined and its values have been determined for the industrial melting facility by mathematical modelling. The definitions of the specific melting performance and specific energy consumption have been used for assessment of the potential impact of a controlled melt flow and high space utilisation on the melting process efficiency on the industrial scale. The results have shown that even the partial control of the melt flow, leading to the partial increase of the space utilisation, may considerably increase the melting performance, whereas a decrease of the specific energy consumption was determined to be between 10 - 15 %.

  8. Viscosity Measurement for Tellurium Melt (United States)

    Lin, Bochuan; Li, Chao; Ban, Heng; Scripa, Rosalia N.; Su, Ching-Hua; Lehoczky, Sandor L.


    The viscosity of high temperature Te melt was measured using a new technique in which a rotating magnetic field was applied to the melt sealed in a suspended ampoule, and the torque exerted by rotating melt flow on the ampoule wall was measured. Governing equations for the coupled melt flow and ampoule torsional oscillation were solved, and the viscosity was extracted from the experimental data by numerical fitting. The computational result showed good agreement with experimental data. The melt velocity transient initiated by the rotating magnetic field reached a stable condition quickly, allowing the viscosity and electrical conductivity of the melt to be determined in a short period.

  9. MELT-IIIB: an updated version of the melt code

    International Nuclear Information System (INIS)

    Tabb, K.K.; Lewis, C.H.; O'Dell, L.D.; Padilla, A. Jr.; Smith, D.E.; Wilburn, N.P.


    The MELT series is a reactor modeling code designed to investigate a wide variety of hypothetical accident conditions, particularly the transient overpower sequence. MELT-IIIB is the latest in the series

  10. PCR-RFLP

    African Journals Online (AJOL)


    Mar 30, 2012 ... The. cpDNA PCR-RFLP based genetic distance (GD) among 30 tea accessions ranged from 0 to 0.071, with the mean of 0.049. This study suggests that the optimization system was suitable for PCR-RFLP analysis of. cpDNA in tea. Key words: Camellia sinensis, PCR-RFLP, chloroplast DNA, establishment.

  11. CODEHOP PCR and CODEHOP PCR primer design. (United States)

    Staheli, Jeannette P; Boyce, Richard; Kovarik, Dina; Rose, Timothy M


    While PCR primer design for the amplification of known sequences is usually quite straightforward, the design, and successful application of primers aimed at the detection of as yet unknown genes is often not. The search for genes that are presumed to be distantly related to a known gene sequence, such as homologous genes in different species, paralogs in the same genome, or novel pathogens in diverse hosts, often turns into the proverbial search for the needle in the haystack. PCR-based methods commonly used to address this issue involve the use of either consensus primers or degenerate primers, both of which have significant shortcomings regarding sensitivity and specificity. We have developed a novel primer design approach that diminishes these shortcomings and instead takes advantage of the strengths of both consensus and degenerate primer designs, by combining the two concepts into a Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) approach. CODEHOP PCR primers contain a relatively short degenerate 3' core and a 5' nondegenerate clamp. The 3' degenerate core consists of a pool of primers containing all possible codons for a 3-4 aminoacid motif that is highly conserved in multiply aligned sequences from known members of a protein family. Each primer in the pool also contains a single 5' nondegenerate nucleotide sequence derived from a codon consensus across the aligned aminoacid sequences flanking the conserved motif. During the initial PCR amplification cycles, the degenerate core is responsible for specific binding to sequences encoding the conserved aminoacid motif. The longer consensus clamp region serves to stabilize the primer and allows the participation of all primers in the pool in the efficient amplification of products during later PCR cycles. We have developed an interactive web site and algorithm (iCODEHOP) for designing CODEHOP PCR primers from multiply aligned protein sequences, which is freely available online. Here, we describe the

  12. Melting graft wound syndrome

    Directory of Open Access Journals (Sweden)

    Shiou-Mei Chen


    Full Text Available Melting graft wound syndrome is characterized by progressive epidermal loss from a previously well-taken skin graft, healed burn, or donor site. It may result in considerable morbidity and require prolonged treatment. We report a 23-year-old flame-burned patient with second- to third-degree burns involving more than 70% of the total body surface area, whose condition was complicated with septic shock. The patient presented with erosions and ulcers occurring on previously well-taken skin graft recipient sites over both legs and progressive epidermal loss on donor sites over the back. The patient's presentation was compatible with the diagnosis of melting graft wound syndrome, and we successfully treated the patient with debridement and supportive treatment.

  13. Influence of PCR reagents on DNA polymerase extension rates measured on real-time PCR instruments. (United States)

    Montgomery, Jesse L; Wittwer, Carl T


    Radioactive DNA polymerase activity methods are cumbersome and do not provide initial extension rates. A simple extension rate assay would enable study of basic assumptions about PCR and define the limits of rapid PCR. A continuous assay that monitors DNA polymerase extension using noncovalent DNA dyes on common real-time PCR instruments was developed. Extension rates were measured in nucleotides per second per molecule of polymerase. To initiate the reaction, a nucleotide analog was heat activated at 95 °C for 5 min, the temperature decreased to 75 °C, and fluorescence monitored until substrate exhaustion in 30-90 min. The assay was linear with time for over 40% of the reaction and for polymerase concentrations over a 100-fold range (1-100 pmol/L). Extension rates decreased continuously with increasing monovalent cation concentrations (lithium, sodium, potassium, cesium, and ammonium). Melting-temperature depressors had variable effects. DMSO increased rates up to 33%, whereas glycerol had little effect. Betaine, formamide, and 1,2-propanediol decreased rates with increasing concentrations. Four common noncovalent DNA dyes inhibited polymerase extension. Heat-activated nucleotide analogs were 92% activated after 5 min, and hot start DNA polymerases were 73%-90% activated after 20 min. Simple DNA extension rate assays can be performed on real-time PCR instruments. Activity is decreased by monovalent cations, DNA dyes, and most melting temperature depressors. Rational inclusion of PCR components on the basis of their effects on polymerase extension is likely to be useful in PCR, particularly rapid-cycle or fast PCR.

  14. Emerging melt quality control solution technologies for aluminium melt

    Directory of Open Access Journals (Sweden)

    Arturo Pascual, Jr


    Full Text Available The newly developed “MTS 1500” Melt Treatment System is performing the specifi cally required melt treatment operations like degassing, cleaning, modification and/or grain refinement by an automated process in one step and at the same location. This linked process is saving time, energy and metal losses allowing - by automated dosage of the melt treatment agents - the production of a consistent melt quality batch after batch. By linking the MTS Metal Treatment System with sensors operating on-line in the melt, i.e., with a hydrogen sensor “Alspek H”, a fully automated control of parts of the process chain like degassing is possible. This technology does guarantee a pre-specifi ed and documented melt quality in each melt treatment batch. Furthermore, to ensure that castings are consistent and predictable there is a growing realization that critical parameters such as metal cleanliness must be measured prior to casting. There exists accepted methods for measuring the cleanliness of an aluminum melt but these can be both slow and costly. A simple, rapid and meaningful method of measuring and bench marking the cleanliness of an aluminum melt has been developed to offer the foundry a practical method of measuring melt cleanliness. This paper shows the structure and performance of the integrated MTS melt treatment process and documents achieved melt quality standards after degassing, cleaning, modifi cation and grain refi nement operations under real foundry conditions. It also provides an insight on a melt cleanliness measuring device “Alspek MQ” to provide foundry men better tools in meeting the increasing quality and tighter specifi cation demand from the industry.

  15. External PCR, ASN's decision

    International Nuclear Information System (INIS)



    The French law imposes in some situations the presence of a person skilled in radiation protection (PCR). This article describes the cases when this person must belong to the staff of the enterprise or when this person may be sub-contracted. For instance in most nuclear facilities the PCR must be on the payroll, for enterprises dedicated to nuclear transport the PCR's job can be sub-contracted. A decision given by the ASN (French Nuclear Safety Authority) sets the minimal requests (in terms of training, job contract, activities) of the sub-contracted PCR. (A.C.)

  16. High-resolution melting analysis for detection of MYH9 mutations. (United States)

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


    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.

  17. Real-time PCR detection of Campylobacter spp. In free-ranging mountain gorillas (Gorilla beringei beringei). (United States)

    Whittier, Christopher A; Cranfield, Michael R; Stoskopf, Michael K


    Health monitoring of wildlife populations can greatly benefit from rapid, local, noninvasive molecular assays for pathogen detection. Fecal samples collected from free-living Virunga mountain gorillas (Gorilla beringei beringei) between August 2002 and February 2003 were tested for Campylobacter spp. DNA using a portable, real-time polymerase chain reaction (PCR) instrument. A high prevalence of Campylobacter spp. was detected in both individually identified (22/26=85%) and nest-collected samples (68/114=59.6%), with no statistically significant differences among different gorilla sexes or age classes or between tourist-visited versus research gorilla groups. The PCR instrument was able to discriminate two distinct groups of Campylobacter spp. in positive gorilla samples based on the PCR product fluorescent-probe melting profiles. The rare type (6/90 positives, 7%, including three mixed cases) matched DNA sequences of Campylobacter jejuni and was significantly associated with abnormally soft stools. The more common type of positive gorilla samples (87/90 positives, 97%) were normally formed and contained a Campylobacter sp. with DNA matching no published sequences. We speculate that the high prevalence of Campylobacter spp. detected in gorilla fecal samples in this survey mostly reflects previously uncharacterized and nonpathogenic intestinal flora. The real-time PCR assay was more sensitive than bacterial culture with Campylobacter-specific media and commercially available, enzyme immunoassay tests for detecting Campylobacter spp. in human samples.

  18. Temperature profile detector (United States)

    Tokarz, Richard D.


    Temperature profiles at elevated temperature conditions are monitored by use of an elongated device having two conductors spaced by the minimum distance required to normally maintain an open circuit between them. The melting point of one conductor is selected at the elevated temperature being detected, while the melting point of the other is higher. As the preselected temperature is reached, liquid metal will flow between the conductors, creating short circuits which are detectable as to location.

  19. Temperature-profile detector (United States)

    Not Available


    Temperature profiles at elevated temperature conditions are monitored by use of an elongated device having two conductors spaced by the minimum distance required to normally maintain an open circuit between them. The melting point of one conductor is selected at the elevated temperature being detected, while the melting point of the other is higher. As the preselected temperature is reached, liquid metal will flow between the conductors creating short circuits which are detectable as to location.

  20. 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.

  1. Elastic properties of silicate melts

    DEFF Research Database (Denmark)

    Clark, Alisha N.; Lesher, Charles E.


    Low seismic velocity regions in the mantle and crust are commonly attributed to the presence of silicate melts. Determining melt volume and geometric distribution is fundamental to understanding planetary dynamics. We present a new model for seismic velocity reductions that accounts for the anoma......Low seismic velocity regions in the mantle and crust are commonly attributed to the presence of silicate melts. Determining melt volume and geometric distribution is fundamental to understanding planetary dynamics. We present a new model for seismic velocity reductions that accounts...... for the anomalous compressibility of silicate melt, rendering compressional wave velocities more sensitive to melt fraction and distribution than previous estimates. Forward modeling predicts comparable velocity reductions for compressional and shear waves for partially molten mantle, and for low velocity regions...

  2. Thermodynamics of freezing and melting


    Pedersen, Ulf Rørbæk; Costigliola, Lorenzo; Bailey, Nicholas; Schrøder, Thomas; Dyre, Jeppe C.


    Although the freezing of liquids and melting of crystals are fundamental for many areas of the sciences, even simple properties like the temperature?pressure relation along the melting line cannot be predicted today. Here we present a theory in which properties of the coexisting crystal and liquid phases at a single thermodynamic state point provide the basis for calculating the pressure, density and entropy of fusion as functions of temperature along the melting line, as well as the variatio...

  3. Logistics Reduction: Heat Melt Compactor (United States)

    National Aeronautics and Space Administration — The Advanced Exploration Systems (AES) Logistics Reduction (LR) project Heat Melt Compactor (HMC) technology is a waste management technology. Currently, there are...

  4. Melting of contaminated metallic waste

    International Nuclear Information System (INIS)

    Lee, Y.-S.; Cheng, S.-Y.; Kung, H.-T.; Lin, L.-F.


    Approximately 100 tons of contaminated metallic wastes were produced each year due to maintenance for each TPC's nuclear power reactor and it was roughly estimated that there will be 10,000 tons of metallic scraps resulted from decommissioning of each reactor in the future. One means of handling the contaminated metal is to melt it. Melting process owns not only volume reduction which saves the high cost of final disposal but also resource conservation and recycling benefits. Melting contaminated copper and aluminum scraps in the laboratory scale have been conducted at INER. A total of 546 kg copper condenser tubes with a specific activity of about 2.7 Bq/g was melted in a vacuum induction melting facility. Three types of products, ingot, slag and dust were derived from the melting process, with average activities of 0.10 Bq/g, 2.33 Bq/g and 84.3 Bq/g respectively. After the laboratory melting stage, a pilot plant with a 500 kg induction furnace is being designed to melt the increasingly produced contaminated metallic scraps from nuclear facilities and to investigate the behavior of different radionuclides during melting. (author)

  5. ‘‘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


    High resolution melting curve analysis (HRM) measures dissociation of double stranded DNA of a PCR product amplified in the presence of a saturating fluorescence dye. Recently, HRM proved successful to genotype DNA sequence polymorphisms such as SSRs and SNPs based on the shape of the melting...

  6. Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species. (United States)

    Zhang, Jing; Hung, Guo-Chiuan; Nagamine, Kenjiro; Li, Bingjie; Tsai, Shien; Lo, Shyh-Ching


    Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan-Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan-Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

  7. Detection of Salmonella in Shellfish Using SYBR Green™ I-Based Real-Time Multiplexed PCR Assay Targeting invA and spvB

    KAUST Repository

    Gangwar, Maulshree


    A SYBR Green™ I-based real-time multiplexed PCR assay was developed targeting invA and spvB for the detection of Salmonella strains in shellfish after both hns and invA genes were identified in all Salmonella strains. Simultaneously, the 16S rRNA gene was used as a PCR internal amplification control (IAC). All 89 Salmonella strains tested in this study exhibited amplification of invA, whereas only 21 (23. 6 %) were PCR positive for spvB. The sensitivity of detection of all three targeted genes was 1 ng, which is equivalent to approximately 105 colony-forming unit (CFU) of Salmonella enterica. The analysis showed specific PCR products that were identified by reproducible melt temperature profiles (invA, 84. 27 ± 1. 7 °C; spvB, 88. 76 ± 1. 0 °C; and 16S rRNA gene, 87. 16 ± 0. 8 °C). The sensitivity of detection was 10 pg purified DNA (invA) or 105 CFU in 1 mL pure culture of S. enterica ATCC 14028. The above molecular detection method for Salmonella strains was successfully applied to the oyster homogenates (food matrix). An initial inoculum of 106 and 102 CFU Salmonella in 1 ml seeded oyster tissue homogenate was detected by multiplexed PCR for all three genes after 5 and 24 h of enrichment, respectively. Natural oysters isolated from Gulf of Mexico during the winter months exhibited negative PCR amplification results suggesting the absence of Salmonella. In contrast to conventional PCR, real-time multiplex PCR assay developed in this study is rapid and sensitive and will help Interstate Shellfish Sanitation Conference undertake appropriate measures to monitor Salmonella in oysters, thereby preventing disease outbreaks and consequently protecting consumer health. © 2012 Springer Science+Business Media, LLC.

  8. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    Directory of Open Access Journals (Sweden)

    Susan D'Costa


    Full Text Available Clinical trials using recombinant adeno-associated virus (rAAV vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR is now the most common method to titer vector genomes (vg; however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

  9. Molecular diagnostic PCR handbook

    International Nuclear Information System (INIS)

    Viljoen, G.J.; Crowther, J.R.; Nel, L.H.


    The uses of nucleic acid-directed methods have increased significantly in the past five years and have made important contributions to disease control country programmes for improving national and international trade. These developments include the more routine use of PCR as a diagnostic tool in veterinary diagnostic laboratories. However, there are many problems associated with the transfer and particularly, the application of this technology. These include lack of consideration of: the establishment of quality-assured procedures, the required set-up of the laboratory and the proper training of staff. This can lead to a situation where results are not assured. This book gives a comprehensive account of the practical aspects of PCR and strong consideration is given to ensure its optimal use in a laboratory environment. This includes the setting-up of a PCR laboratory; Good Laboratory Practice and standardised PCR protocols to detect animal disease pathogens. Examples of Standard Operating Procedures as used in individual specialist laboratories and an outline of training materials necessary for PCR technology transfer are presented. The difficulties, advantages and disadvantages in PCR applications are explained and placed in context with other test systems. Emphasis is placed on the use of PCR for detection of pathogens, with a particular focus on diagnosticians and scientists from the developing world. It is hoped that this book will enable readers from various disciplines and levels of expertise to better judge the merits of PCR and to increase their skills and knowledge in order to assist in a more logical, efficient and assured use of this technology

  10. The investigation of the truncated mbtA gene within the mycobactin cluster of Mycobacterium avium subspecies paratuberculosis as a novel diagnostic marker for real-time PCR. (United States)

    de Kruijf, Marcel; Coffey, Aidan; O'Mahony, Jim


    The inability of Mycobacterium avium subspecies paratuberculosis (MAP) to produce endogenous mycobactin in-vitro is most likely due to the presence of a truncated mbtA gene within the mycobactin cluster of MAP. The main goal of this study was to investigate this unique mbtA truncation as a potential novel PCR diagnostic marker for MAP. Novel primers were designed that were located within the truncated region and the contiguous MAP2179 gene. Primers were evaluated against non-MAP isolates and no amplicons were generated. The detection limit of this mbtA-MAP2179 target was evaluated using a range of MAP DNA concentrations, MAP inoculated faecal material and 20 MAP isolates. The performance of mbtA-MAP2179 was compared to the established f57 target. The detection limits recorded for MAP K-10 DNA and from MAP K-10 inoculated faecal samples were 0.34pg and 10 4 CFU/g respectively for both f57 and mbtA-MAP2179. A detection limit of 10 3 CFU/g was recorded for both targets, but not achieved consistently. The detection limit of MAP from inoculated faecal material was successful at 10 3 CFU/g for mbtA-MAP2179 when FAM probe real-time PCR was used. A MAP cell concentration of 10 2 CFU/g was detected successfully, but again not consistently achieved. All 20 mycobacterial isolates were successfully identified as MAP by f57 and mbtA-MAP2179. Interestingly, the mbtA-MAP2179 real-time PCR assay resulted in the formation of a unique melting curve profile that contained two melting curve peaks rather than one single peak. This melting curve phenomenon was attributed towards the asymmetrical GC% distribution within the mbtA-MAP2179 amplicon. This study investigated the implementation of the mbtA-MAP2179 target as a novel diagnostic marker and the detection limits obtained with mbtA-MAP2179 were comparable to the established f57 target, making the mbtA-MAP2179 an adequate confirmatory target. Moreover, the mbtA-MAP2179 target could be implemented in multiplex real-time PCR assays and

  11. Nanorheology of Entangled Polymer Melts (United States)

    Ge, Ting; Grest, Gary S.; Rubinstein, Michael


    We use molecular simulations to probe the local viscoelasticity of an entangled polymer melt by tracking the motion of embedded nonsticky nanoparticles (NPs). As in conventional microrheology, the generalized Stokes-Einstein relation is employed to extract an effective stress relaxation function GGSE(t ) from the mean square displacement of NPs. GGSE(t ) for different NP diameters d are compared with the stress relaxation function G (t ) of a pure polymer melt. The deviation of GGSE(t ) from G (t ) reflects the incomplete coupling between NPs and the dynamic modes of the melt. For linear polymers, a plateau in GGSE(t ) emerges as d exceeds the entanglement mesh size a and approaches the entanglement plateau in G (t ) for a pure melt with increasing d . For ring polymers, as d increases towards the spanning size R of ring polymers, GGSE(t ) approaches G (t ) of the ring melt with no entanglement plateau.

  12. Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification. (United States)

    Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang


    Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

  13. Three dimensional numerical simulation for air flow field in melt blowing (United States)

    Xie, Sheng; Han, Wanli; Jiang, Guojun


    Melt blowing is one-step approach for producing microfibrous nonwovens. In this study, the characteristics of air flow field in different die melt blowing were studied. CFD simulation analysis was adopted to complete the air flow field simulation. The characteristics of the air flow fields in different die melt blowing were revealed and compared. Meanwhile, the fiber paths in these two melt-blowing processes are recorded with a high-speed camera. The fiber path profiles, especially the whipping, are obtained and presented.

  14. Development of duplex SYBR Green I-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses (United States)

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

  15. Nitrogen Control in VIM Melts (United States)

    Jablonski, P. D.; Hawk, J. A.

    NETL has developed a design and control philosophy for the addition of nitrogen to austenitic and ferritic steels. The design approach uses CALPHAD as the centerpiece to predict the level to which nitrogen is soluble in both the melt and the solid. Applications of this technique have revealed regions of "exclusion" in which the alloy, while within specification limits of prescribed, cannot be made by conventional melt processing. Furthermore, other investigations have found that substantial retrograde solubility of nitrogen exists, which can become problematic during subsequent melt processing and/or other finishing operations such as welding. Additionally, the CALPHAD method has been used to adjust primary melt conditions. To that end, nitrogen additions have been made using chrome nitride, silicon nitride, high-nitrogen ferrochrome as well as nitrogen gas. The advantages and disadvantages of each approach will be discussed and NETL experience in this area will be summarized with respect to steel structure.

  16. [Pelletization of melts and liquids]. (United States)

    Rabisková, Miloslava


    During the second half of the last century, pelletization methods based on wetting were developed, e.g. agglomeration in coating pans, pelletization plates or fluid-bed equipment, layering of the drug in solution or suspension on inactive spherical cores, extrusion/spheronization and later on also rotoagglomeration in rotogranulators or rotoprocessors. These technologies have become a requisite part of industrial production of solid dosage forms. At present, numerous experimental papers deal with pellet preparation from melts and liquids. These new pelletization methods are the topic of the present article. Pellet preparation from melts is represented by three methods, i.e. fluid hot melt agglomeration, hot melt extrusion, and freeze pelletization. Jet cutting and cryopelletization are the techniques dealing with pellet preparation from liquids.

  17. Identification of feline polycystic kidney disease mutation using fret probes and melting curve analysis. (United States)

    Criado-Fornelio, A; Buling, A; Barba-Carretero, J C


    We developed and validated a real-time polymerase chain reaction (PCR) assay using fluorescent hybridization probes and melting curve analysis to identify the PKD1 exon 29 (C-->A) mutation, which is implicated in polycystic kidney disease of cats. DNA was isolated from peripheral blood of 20 Persian cats. The employ of the new real-time PCR and melting curve analysis in these samples indicated that 13 cats (65%) were wild type homozygotes and seven cats (35%) were heterozygotes. Both PCR-RFLP and sequencing procedures were in full agreement with real-time PCR test results. Sequence analysis showed that the mutant gene had the expected base change compared to the wild type gene. The new procedure is not only very reliable but also faster than the techniques currently applied for diagnosis of the mutation.

  18. Real-time PCR (qPCR) primer design using free online software. (United States)

    Thornton, Brenda; Basu, Chhandak


    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

  19. Scaleable Clean Aluminum Melting Systems

    Energy Technology Data Exchange (ETDEWEB)

    Han, Q.; Das, S.K. (Secat, Inc.)


    The project entitled 'Scaleable Clean Aluminum Melting Systems' was a Cooperative Research and Development Agreements (CRADAs) between Oak Ridge National Laboratory (ORNL) and Secat Inc. The three-year project was initially funded for the first year and was then canceled due to funding cuts at the DOE headquarters. The limited funds allowed the research team to visit industrial sites and investigate the status of using immersion heaters for aluminum melting applications. Primary concepts were proposed on the design of furnaces using immersion heaters for melting. The proposed project can continue if the funding agency resumes the funds to this research. The objective of this project was to develop and demonstrate integrated, retrofitable technologies for clean melting systems for aluminum in both the Metal Casting and integrated aluminum processing industries. The scope focused on immersion heating coupled with metal circulation systems that provide significant opportunity for energy savings as well as reduction of melt loss in the form of dross. The project aimed at the development and integration of technologies that would enable significant reduction in the energy consumption and environmental impacts of melting aluminum through substitution of immersion heating for the conventional radiant burner methods used in reverberatory furnaces. Specifically, the program would couple heater improvements with furnace modeling that would enable cost-effective retrofits to a range of existing furnace sizes, reducing the economic barrier to application.

  20. PCR and real-time PCR assays to detect fungi of Alternaria alternata species. (United States)

    Kordalewska, Milena; Brillowska-Dąbrowska, Anna; Jagielski, Tomasz; Dworecka-Kaszak, Bożena


    Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of β-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata pathogens.

  1. Numerical Model based Reliability Estimation of Selective Laser Melting Process

    DEFF Research Database (Denmark)

    Mohanty, Sankhya; Hattel, Jesper Henri


    Selective laser melting is developing into a standard manufacturing technology with applications in various sectors. However, the process is still far from being at par with conventional processes such as welding and casting, the primary reason of which is the unreliability of the process. While...... of the selective laser melting process. A validated 3D finite-volume alternating-direction-implicit numerical technique is used to model the selective laser melting process, and is calibrated against results from single track formation experiments. Correlation coefficients are determined for process input...... parameters such as laser power, speed, beam profile, etc. Subsequently, uncertainties in the processing parameters are utilized to predict a range for the various outputs, using a Monte Carlo method based uncertainty analysis methodology, and the reliability of the process is established....

  2. Melt analysis of mismatch amplification mutation assays (Melt-MAMA): a functional study of a cost-effective SNP genotyping assay in bacterial models. (United States)

    Birdsell, Dawn N; Pearson, Talima; Price, Erin P; Hornstra, Heidie M; Nera, Roxanne D; Stone, Nathan; Gruendike, Jeffrey; Kaufman, Emily L; Pettus, Amanda H; Hurbon, Audriana N; Buchhagen, Jordan L; Harms, N Jane; Chanturia, Gvantsa; Gyuranecz, Miklos; Wagner, David M; Keim, Paul S


    Single nucleotide polymorphisms (SNPs) are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA), is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg). Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs) and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of Melt-MAMA, which

  3. Microbial diversity in a thermophilic aerobic biofilm process: analysis by length heterogeneity PCR (LH-PCR). (United States)

    Tiirola, Marja A; Suvilampi, Juhani E; Kulomaa, Markku S; Rintala, Jukka A


    A two-stage pilot-scale thermophilic aerobic suspended carrier biofilm process (SCBP) was set up for the on-site treatment of pulp and paper mill whitewater lining. The microbial diversity in this process was analyzed by length heterogeneity analysis of PCR-amplified 16S ribosomal DNA. The primer pair selected for PCR amplification was first evaluated by a computational analysis of fragment lengths in ten main phylogenetical eubacterial groups. The fragment contained the first third of the 16S rRNA gene, which was shown to vary naturally between 465 and 563 bp in length. The length heterogeneity analysis of polymerase chain reaction (LH-PCR) profile of the biomass attached to carrier elements was found to be diverse in both stages of the SCBP. During normal operating conditions, sequences belonging to beta-Proteobacteria, Cytophaga/Flexibacter/Bacteroides group and gamma-Proteobacteria were assigned to the most prominent LH-PCR peak. Samples from the suspended biomass consisted of completely different bacterial populations, which were, however, similar in the serial reactors. The pilot process experienced alkaline shocks, after which Bacillus-like sequences were detected in both the biofilm and suspended biomass. However, when the conditions were reversed, the normal microbial population in the biofilm recovered rapidly without further biomass inoculations. This study shows that LH-PCR is a valuable method for profiling microbial diversity and dynamics in industrial wastewater processes.

  4. PCR in forensic genetics

    DEFF Research Database (Denmark)

    Morling, Niels


    Since the introduction in the mid-1980s of analyses of minisatellites for DNA analyses, a revolution has taken place in forensic genetics. The subsequent invention of the PCR made it possible to develop forensic genetics tools that allow both very informative routine investigations and still more...... and more advanced, special investigations in cases concerning crime, paternity, relationship, disaster victim identification etc. The present review gives an update on the use of DNA investigations in forensic genetics....

  5. A real-time PCR assay for the differentiation of Candida species. (United States)

    Fricke, S; Fricke, C; Schimmelpfennig, C; Oelkrug, C; Schönfelder, U; Blatz, R; Zilch, C; Faber, S; Hilger, N; Ruhnke, M; Rodloff, A C


    We established a real-time PCR assay for the detection and strain identification of Candida species and demonstrated the ability to differentiate between Candida albicans the most common species, and also Candida parapsilosis, Candida glabrata, Candida tropicalis and Candida dubliniensis by LightCycler PCR and melting curve analysis. The DNA isolation from cultures and serum was established using the QIAmp Tissue Kit. The sensitivity of the assay was ≥ 2 genome equivalents/assay. It was possible to differentiate all investigated Candida species by melting curve analysis, and no cross-reaction to human DNA or Aspergillus species could be observed. The established real-time PCR assay is a useful tool for the rapid identification of Candida species and a base technology for more complex PCR assays. We carried out initial steps in validation of a PCR assay for the detection and differentiation of medically relevant Candida species. The PCR was improved by generating PCR standards, additional generation of melting curves for species identification and the possibility to investigate different specimens simultaneously. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

  6. Molecular thermodynamics of polymer melts at interfaces

    Energy Technology Data Exchange (ETDEWEB)

    Theodorou, D.N.


    A lattice model is developed for the prediction of structure and thermodynamic properties at free polymer melt surfaces and polymer melt/solid interfaces. Density variations in the interfacial region are taken into account by introducing voids in the lattice, in the spirit of the equation of state theory of Sanchez and Lacombe. Intramolecular energy (chain stiffness) effects are explicitly incorporated. The model is derived through a rigorous statistical mechanical and thermodynamic analysis, which is based on the concept of availability. Two cases are considered: ''full equilibrium,'' whereby the interfacial polymer is taken as free to exchange heat, work and mass with a bulk polymer phase at given temperature and pressure; and ''restricted equilibrium,'' whereby a thin polymer film is allowed to equilibrate locally in response to ambient temperature and pressure, but in which chains do not necessarily have the same chemical potential as in the unconstrained bulk. Techniques are developed for calculating surface tension, adhesion tension, density profiles, chain shape, bond orientation, as well as the distribution of segments of various orders in the interfacial region. 28 refs., 6 figs.

  7. One-stop polymerase chain reaction (PCR): An improved PCR ...

    African Journals Online (AJOL)

    -cycling steps to visualize amplicons, decelerating PCR sample processing and result calling. “One-stop PCR” was developed by including both the loading buffer and nontoxic staining dye within a single PCR tube, allowing direct loading and ...

  8. One-stop polymerase chain reaction (PCR): An improved PCR ...

    African Journals Online (AJOL)



    Dec 21, 2011 ... improved PCR method with speedy operation and ... novel PCR method is desired to compatibilize Taq DNA .... as template. A 20 ul traditional PCR mixture included 10×PCR reaction buffer, 2 µl, 40 mM dNTP (10mM each), 0.5 µl; Taq DNA polymerase, 0.2 µl (1 unit), 0.5 µl forward and reverse primer mix ...

  9. Lithium diffusion in silicate melts (United States)

    Cunningham, G. J.; Henderson, P.; Lowry, R. K.; Nolan, J.; Reed, S. J. B.; Long, J. V. P.


    The diffusion properties of Li in an andesitic and pitchstone melt have been determined over the temperature range 1300-1400°C. The diffusion data have been fitted to an Arrhenius relationship between log D0 and 1/ T, and give relatively small activation energies of diffusion: 21.4±5.8 kcal mol -1 in the andesite and 20.1±2.8 kcal mol -1 in the pitchstone. Li +, unlike several other cations, shows similar diffusivities in these melt compositions to that in a basaltic melt. Despite the similar ionic radius of Li + to that of Co 2+, the diffusion properties of the two ions are very different from each other.

  10. Enhancement of PCR Detection Limit by Single-Tube Restriction Endonuclease-PCR (RE-PCR). (United States)

    Datta, Sibnarayan; Budhauliya, Raghvendra; Chatterjee, Soumya; Vanlalhmuaka; Veer, Vijay; Chakravarty, Runu


    Polymerase chain reaction (PCR) is widely used in biological research and diagnostics because of its high sensitivity and specificity. However, the sensitivity of PCR is strongly influenced by topological characteristics of the template. Supercoiled templates are known to inhibit PCR, whereas linearized forms of the same supercoiled templates facilitate PCR. This study was conducted to compare the PCR efficiency of circular supercoiled DNA templates to their restriction endonuclease (RE)-mediated linearized forms. Additionally, we also evaluated the possibility of RE digestion of the circular supercoiled templates within the complete PCR buffer. Following a systematic approach, we demonstrated that circular supercoiled templates could be efficiently linearized by RE in the complete PCR buffer itself. This allowed linearization of circular supercoiled templates and their subsequent amplification in the PCR buffer in a single-tube format. Using this extremely simple RE-PCR approach, we documented up to tenfold increases in detection efficiency of PCR with two different circular supercoiled templates of clinical origin, including an international calibration standard. This inexpensive and easy approach to increasing PCR sensitivity can be easily adapted to any standard PCR protocol aimed at amplifying circular supercoiled genomes. Apart from its application in the development of sensitive clinical diagnostic PCR assays for a large number of organisms, this method could also prove to be very useful in simplifying the existing protocols for other applications where pre-PCR restriction digestion is required, such as mutation detection, genotyping, and selective template amplification.

  11. Identification and Quantification of Celery Allergens Using Fiber Optic Surface Plasmon Resonance PCR (United States)

    Daems, Devin; Peeters, Bernd; Delport, Filip; Remans, Tony; Lammertyn, Jeroen; Spasic, Dragana


    Accurate identification and quantification of allergens is key in healthcare, biotechnology and food quality and safety. Celery (Apium graveolens) is one of the most important elicitors of food allergic reactions in Europe. Currently, the golden standards to identify, quantify and discriminate celery in a biological sample are immunoassays and two-step molecular detection assays in which quantitative PCR (qPCR) is followed by a high-resolution melting analysis (HRM). In order to provide a DNA-based, rapid and simple detection method suitable for one-step quantification, a fiber optic PCR melting assay (FO-PCR-MA) was developed to determine different concentrations of celery DNA (1 pM–0.1 fM). The presented method is based on the hybridization and melting of DNA-coated gold nanoparticles to the FO sensor surface in the presence of the target gene (mannitol dehydrogenase, Mtd). The concept was not only able to reveal the presence of celery DNA, but also allowed for the cycle-to-cycle quantification of the target sequence through melting analysis. Furthermore, the developed bioassay was benchmarked against qPCR followed by HRM, showing excellent agreement (R2 = 0.96). In conclusion, this innovative and sensitive diagnostic test could further improve food quality control and thus have a large impact on allergen induced healthcare problems. PMID:28758965

  12. Identification and Quantification of Celery Allergens Using Fiber Optic Surface Plasmon Resonance PCR

    Directory of Open Access Journals (Sweden)

    Devin Daems


    Full Text Available Abstract: Accurate identification and quantification of allergens is key in healthcare, biotechnology and food quality and safety. Celery (Apium graveolens is one of the most important elicitors of food allergic reactions in Europe. Currently, the golden standards to identify, quantify and discriminate celery in a biological sample are immunoassays and two-step molecular detection assays in which quantitative PCR (qPCR is followed by a high-resolution melting analysis (HRM. In order to provide a DNA-based, rapid and simple detection method suitable for one-step quantification, a fiber optic PCR melting assay (FO-PCR-MA was developed to determine different concentrations of celery DNA (1 pM–0.1 fM. The presented method is based on the hybridization and melting of DNA-coated gold nanoparticles to the FO sensor surface in the presence of the target gene (mannitol dehydrogenase, Mtd. The concept was not only able to reveal the presence of celery DNA, but also allowed for the cycle-to-cycle quantification of the target sequence through melting analysis. Furthermore, the developed bioassay was benchmarked against qPCR followed by HRM, showing excellent agreement (R2 = 0.96. In conclusion, this innovative and sensitive diagnostic test could further improve food quality control and thus have a large impact on allergen induced healthcare problems.

  13. Eutectic melting temperature of the lowermost Earth's mantle (United States)

    Andrault, D.; Lo Nigro, G.; Bolfan-Casanova, N.; Bouhifd, M.; Garbarino, G.; Mezouar, M.


    Partial melting of the Earth's deep mantle probably occurred at different stages of its formation as a consequence of meteoritic impacts and seismology suggests that it even continues today at the core-mantle boundary. Melts are important because they dominate the chemical evolution of the different Earth's reservoirs and more generally the dynamics of the whole planet. Unfortunately, the most critical parameter, that is the temperature profile inside the deep Earth, remains poorly constrained accross the planet history. Experimental investigations of the melting properties of materials representative of the deep Earth at relevant P-T conditions can provide anchor points to refine past and present temperature profiles and consequently determine the degree of melting at the different geological periods. Previous works report melting relations in the uppermost lower mantle region, using the multi-anvil press [1,2]. On the other hand, the pyrolite solidus was determined up to 65 GPa using optical observations in the laser-heated diamond anvil cell (LH-DAC) [3]. Finally, the melting temperature of (Mg,Fe)2SiO4 olivine is documented at core-mantle boundary (CMB) conditions by shock wave experiments [4]. Solely based on these reports, experimental data remain too sparse to draw a definite melting curve for the lower mantle in the relevant 25-135 GPa pressure range. We reinvestigated melting properties of lower mantle materials by means of in-situ angle dispersive X-ray diffraction measurements in the LH-DAC at the ESRF [5]. Experiments were performed in an extended P-T range for two starting materials: forsterite and a glass with chondrite composition. In both cases, the aim was to determine the onset of melting, and thus the eutectic melting temperatures as a function of pressure. Melting was evidenced from drastic changes of diffraction peak shape on the image plate, major changes in diffraction intensities in the integrated pattern, disappearance of diffraction rings

  14. Challenges in Melt Furnace Tests (United States)

    Belt, Cynthia


    Measurement is a critical part of running a cast house. Key performance indicators such as energy intensity, production (or melt rate), downtime (or OEE), and melt loss must all be understood and monitored on a weekly or monthly basis. Continuous process variables such as bath temperature, flue temperature, and furnace pressure should be used to control the furnace systems along with storing the values in databases for later analysis. While using measurement to track furnace performance over time is important, there is also a time and place for short-term tests.

  15. Development of simple multiplex real-time PCR assays for foodborne pathogens detection and identification on LightCycler

    Directory of Open Access Journals (Sweden)

    Avo Karus1


    Full Text Available Most acute intestinal diseases are caused by food-borne pathogens. A fast and simple real-time PCR-based procedure for simultaneous detection of food contamination by any of the five food-borne pathogens: Campylobacter jejuni, Mycobacterium bovis, Enterobacter sakazaki, Shigella boydii, Clostridium perfrigens using multiplex EvaGreen real-time PCR for LightCycler was developed and evaluated. Real-time qPCR showed excellent sensitivity. Tm calling and Melting Curve Genotyping (MCG were used for analysis of PCR product melting curves. The Melting Curve Genotyping option showed good performance for discrimination of positive samples containing DNA of single pathogen or pathogen mixtures from negative samples.

  16. Strain-level characterization of nonstarter lactic acid bacteria in Norvegia cheese by high-resolution melt analysis. (United States)

    Porcellato, D; Østlie, H M; Liland, K H; Rudi, K; Isaksson, T; Skeie, S B


    The nonstarter lactic acid bacteria (NSLAB) constitute an important microbial group found during cheese ripening and they are thought to be fundamental to the quality of cheese. Rapid and accurate diagnostic tests for NSLAB are important for cheese quality control and in understanding the cheese ripening process. Here, we present a novel rapid approach for strain-level characterization through combined 16S rRNA gene and repetitive sequence-based high-resolution melt analysis (HRM). The approach was demonstrated through the characterization of 94 isolates from Norvegia, a Gouda-type cheese. The HRM profiles of the V1 and V3 variable regions of the 16S rRNA gene of the isolates were compared with the HRM profiles of 13 reference strains. The HRM profile comparison of the V1 and V3 regions of the 16S rRNA gene allowed discrimination of isolates and reference strains. Among the cheese isolates, Lactobacillus casei/paracasei (62 isolates) and Lactobacillus plantarum/Lactobacillus pentosus (27 isolates) were the dominant species, whereas Lactobacillus curvatus/Lactobacillus sakei were found occasionally (5 isolates). The HRM profiling of repetitive sequence-based PCR using the (GTG)(5) primer was developed for strain-level characterization. The clustering analysis of the HRM profiles showed high discriminatory power, similar to that of cluster analysis based on the gel method. In conclusion, the HRM approach in this study may be applied as a fast, accurate, and reproducible method for characterization of the NSLAB microflora in cheese and may be applicable to other microbial environments following selective plate culturing. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  17. Detection of Treponema pallidum in the vitreous by PCR (United States)

    Müller, M; Ewert, I; Hansmann, F; Tiemann, C; Hagedorn, H J; Solbach, W; Roider, J; Nölle, B; Laqua, H; Hoerauf, H


    Background Ocular involvement of syphilis still poses a clinical challenge due to the chameleonic behaviour of the disease. As the serodiagnosis has significant limitations, the direct detection of Treponema pallidum (TP) in the vitreous represents a desirable diagnostic tool. Methods Real‐time polymerase chain reaction (PCR) for the detection of TP was applied in diagnostic vitrectomies of two patients with acute chorioretinitis. Qualitative verification of TP by real‐time PCR and melting point analysis according to a modified protocol was ruled out. Patients underwent complete ophthalmological examination with fundus photographs, fluorescein angiography, serological examination, antibiotic treatment and follow‐up. Results In two cases of acute chorioretinitis of unknown origin, real‐time PCR of vitreous specimens of both patients provided evidence of TP and was 100% specific. Initial diagnosis of presumed viral retinitis was ruled out by PCR of vitreous specimen. Patients were treated with systemic antibiotics and showed prompt improvement in visual function and resolution of fundus lesions. Conclusions With real‐time PCR, detection of TP in the vitreous was possible and delivered a sensitive, quick and inexpensive answer to a disease rather difficult to assess. In cases of acute chorioretinitis, the use of PCR‐based assays of vitreous specimens in the diagnostic evaluation of patients is advisable. Although syphilitic chorioretinitis is a rare disease, PCR should include search for TP, as diagnostic dilemmas prolong definitive treatment in a sight‐threatening disease. PMID:17108014

  18. PCR, exit stage left ...

    CERN Multimedia


    The Prevessin Control Room during LEP's start up in 1989. The Prévessin Control Room (PCR) was recently engulfed in a wave of nostalgia. The PCR, scene of some of the greatest moments in CERN's history, is being dismantled to prepare for a complete overhaul. In February 2006, a new combined control centre for all the accelerators will open its doors on the same site, together with a new building currently under construction (see Bulletin issue 27/2004 of 28 June 2004). This marks the end of an important chapter in CERN's history. The Prévessin Control Room saw its first momentous event 28 years ago when the 400 GeV beam for the SPS was commissioned in the presence of Project Leader John Adams. It was also here that the first proton-antiproton collisions were observed, in 1981. Eight years later, in 1989, operators and directors alike jumped for joy at the announcement of the first electron-positron collisions at the start up of LEP, the biggest accelerator in the world. Today the 80 terminals and PCs have b...

  19. Thermodynamics of freezing and melting

    DEFF Research Database (Denmark)

    Pedersen, Ulf Rørbæk; Costigliola, Lorenzo; Bailey, Nicholas


    phases at a single thermodynamic state point provide the basis for calculating the pressure, density and entropy of fusion as functions of temperature along the melting line, as well as the variation along this line of the reduced crystalline vibrational mean-square displacement (the Lindemann ratio...

  20. The infidelity of melt inclusions? (United States)

    Kent, A. J.


    Melt inclusions provide important information about magmatic systems and represent unique records of magma composition and evolution. However, it is also clear that melt inclusions do not necessarily constitute a petrological 'magic bullet', and potential exists for trapped melt compositions to be modified by a range of inclusion-specific processes. These include trapping of diffusional boundary layers, crystallization of the host mineral after trapping and dissolution of co-trapped minerals during homogenization, diffusional exchange between trapped liquid and the host mineral and external melt, and cryptic alteration of trapped material during weathering or hydrothermal alteration. It clearly important to identify when melt inclusions are unmodified, and which compositional indices represent the most robust sources of petrogenetic information. In this presentation I review and discuss various approaches for evaluating compositions and compositional variations in inclusion suites. An overriding principle is that the variations evident in melt inclusions should be able to be understood in terms of petrological processes that are known, or can be reasonably inferred to also effect bulk magma compositions. One common approach is to base petrological conclusions on species that should be more robust, and many workers use variations in incompatible trace elements for this purpose. However important information may also be obtained from a comparison of variations in melt inclusions and the lavas that host them, and in most cases this comparison is the key to identifying inclusions and suites that are potentially suspect. Comparisons can be made between individual inclusions and lavas, although comparison of average inclusion composition and the host lava, after correction for differences in crystal fractionation, may also be valuable. An important extension of this is the comparison of the variability of different species in inclusions and host lavas. This also provides

  1. Diluted melt proton exchange slab waveguides in LiNbO3: A new fabrication and characterization method

    DEFF Research Database (Denmark)

    Veng, Torben; Skettrup, Torben


    A method of dilute-melt proton exchange employing a mixture of glycerol and KHSO4 with lithium benzoate added is used to fabricate planar waveguides in c-cut LiNbO3. With this exchange melt system the waveguide refractive index profiles can be fabricated with a high degree of reproducibility. In ...

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


    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. 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


    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.

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


    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).

  5. 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


    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

  6. Petrological Geodynamics of Mantle Melting I. AlphaMELTS + Multiphase Flow: Dynamic Equilibrium Melting, Method and Results

    Directory of Open Access Journals (Sweden)

    Massimiliano Tirone


    Full Text Available The complex process of melting in the Earth's interior is studied by combining a multiphase numerical flow model with the program AlphaMELTS which provides a petrological description based on thermodynamic principles. The objective is to address the fundamental question of the effect of the mantle and melt dynamics on the composition and abundance of the melt and the residual solid. The conceptual idea is based on a 1-D description of the melting process that develops along an ideal vertical column where local chemical equilibrium is assumed to apply at some level in space and time. By coupling together the transport model and the chemical thermodynamic model, the evolution of the melting process can be described in terms of melt distribution, temperature, pressure and solid and melt velocities but also variation of melt and residual solid composition and mineralogical abundance at any depth over time. In this first installment of a series of three contributions, a two-phase flow model (melt and solid assemblage is developed under the assumption of complete local equilibrium between melt and a peridotitic mantle (dynamic equilibrium melting, DEM. The solid mantle is also assumed to be completely dry. The present study addresses some but not all the potential factors affecting the melting process. The influence of permeability and viscosity of the solid matrix are considered in some detail. The essential features of the dynamic model and how it is interfaced with AlphaMELTS are clearly outlined. A detailed and explicit description of the numerical procedure should make this type of numerical models less obscure. The general observation that can be made from the outcome of several simulations carried out for this work is that the melt composition varies with depth, however the melt abundance not necessarily always increases moving upwards. When a quasi-steady state condition is achieved, that is when melt abundance does not varies significantly

  7. Filament stretching rheometry of polymer melts

    DEFF Research Database (Denmark)

    Hassager, Ole; Nielsen, Jens Kromann; Rasmussen, Henrik Koblitz


    The Filament Stretching Rheometry (FSR) method developed by Sridhar, McKinley and coworkers for polymer solutions has been extended to be used also for polymer melts. The design of a melt-FSR will be described and differences to conventional melt elongational rheometers will be pointed out. Results...

  8. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

    DEFF Research Database (Denmark)

    Hummelshoj, Lone; Ryder, Lars P; Madsen, Hans O


    and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking activity. More...

  9. Excised radicle tips as a source of genomic DNA for PCR-based ...

    Indian Academy of Sciences (India)


    Dec 13, 2012 ... and endogenous locus are proprietory information of Nuzi- veedu Seeds and Monsanto India Ltd. 2.4 Melting curve analysis for establishing hybridity. 50 ng genomic DNA each of the parental lines (NC105 and. NC1108Bt) and their hybrid (NCHB-990Bt) were used as templates for PCR amplification in a ...

  10. Expression profiles of the immune genes CD4, CD8β, IFNγ, IL-4, IL-6 and IL-10 in mitogen-stimulated koala lymphocytes (Phascolarctos cinereus by qRT-PCR

    Directory of Open Access Journals (Sweden)

    Iona E. Maher


    Full Text Available Investigation of the immune response of the koala (Phascolarctos cinereus is needed urgently, but has been limited by scarcity of species-specific reagents and methods for this unique and divergent marsupial. Infectious disease is an important threat to wild populations of koalas; the most widespread and important of these is Chlamydial disease, caused by Chlamydia pecorum and Chlamydia pneumoniae. In addition, koala retrovirus (KoRV, which is of 100% prevalence in northern Australia, has been proposed as an important agent of immune suppression that could explain the koala’s susceptibility to disease. The correct balance of T regulatory, T helper 1 (Th1 and Th2 lymphocyte responses are important to an individual’s susceptibility or resistance to chlamydial infection. The ability to study chlamydial or KoRV pathogenesis, effects of environmental stressors on immunity, and the response of koalas to vaccines under development, by examining the koala’s adaptive response to natural infection or in-vitro stimulation, has been limited to date by a paucity of species- specific reagents. In this study we have used cytokine sequences from four marsupial genomes to identify mRNA sequences for key T regulatory, Th1 and Th2 cytokines interleukin 4 (IL-4, interleukin 6 (IL-6, interleukin 10 (IL-10 and interferon gamma (IFNγ along with CD4 and CD8β. The koala sequences used for primer design showed >58% homology with grey short-tailed opossum, >71% with tammar wallaby and 78% with Tasmanian devil amino acid sequences. We report the development of real-time RT-PCR assays to measure the expression of these genes in unstimulated cells and after three common mitogen stimulation protocols (phorbol myristate acetate/ionomycin, phorbol myristate acetate/phytohemagglutinin and concanavalin A. Phorbol myristate acetate/ionomycin was found to be the most effective mitogen to up-regulate the production of IL-4, IL-10 and IFNγ. IL-6 production was not

  11. Grain boundary melting in ice


    Thomson, E. S.; Hansen-Goos, Hendrik; Wilen, L. A.; Wettlaufer, J. S.


    We describe an optical scattering study of grain boundary premelting in water ice. Ubiquitous long ranged attractive polarization forces act to suppress grain boundary melting whereas repulsive forces originating in screened Coulomb interactions and classical colligative effects enhance it. The liquid enhancing effects can be manipulated by adding dopant ions to the system. For all measured grain boundaries this leads to increasing premelted film thickness with increasing electrolyte concentr...

  12. Quantitative (real-time) PCR

    International Nuclear Information System (INIS)

    Denman, S.E.; McSweeney, C.S.


    Many nucleic acid-based probe and PCR assays have been developed for the detection tracking of specific microbes within the rumen ecosystem. Conventional PCR assays detect PCR products at the end stage of each PCR reaction, where exponential amplification is no longer being achieved. This approach can result in different end product (amplicon) quantities being generated. In contrast, using quantitative, or real-time PCR, quantification of the amplicon is performed not at the end of the reaction, but rather during exponential amplification, where theoretically each cycle will result in a doubling of product being created. For real-time PCR, the cycle at which fluorescence is deemed to be detectable above the background during the exponential phase is termed the cycle threshold (Ct). The Ct values obtained are then used for quantitation, which will be discussed later

  13. A cost-effective melting temperature assay for the detection of single-nucleotide polymorphism in the MBL2 gene of HIV-1-infected children

    Directory of Open Access Journals (Sweden)

    Arraes L.C.


    Full Text Available We report a fast (less than 3 h and cost-effective melting temperature assay method for the detection of single-nucleotide polymorphisms in the MBL2 gene. The protocol, which is based on the Corbett Rotor Gene real time PCR platform and SYBR Green I chemistry, yielded, in the cohorts studied, sensitive (100% and specific (100% PCR amplification without the use of costly fluorophore-labeled probes or post-PCR manipulation. At the end of the PCR, the dissociation protocol included a slow heating from 60º to 95ºC in 0.2ºC steps, with an 8-s interval between steps. Melting curve profiles were obtained using the dissociation software of the Rotor Gene-3000 apparatus. Samples were analyzed in duplicate and in different PCR runs to test the reproducibility of this technique. No supplementary data handling is required to determine the MBL2 genotype. MBL2 genotyping performed on a cohort of 164 HIV-1-positive Brazilian children and 150 healthy controls, matched for age and sex and ethnic origin, yielded reproducible results confirmed by direct sequencing of the amplicon performed in blind. The three MBL2 variants (Arg52Cys, Gly54Asp, Gly57Glu were grouped together and called allele 0, while the combination of three wild-type alleles was called allele A. The frequency of the A/A homozygotes was significantly higher among healthy controls (0.68 than in HIV-infected children (0.55; P = 0.0234 and the frequency of MBL2 0/0 homozygotes was higher among HIV-1-infected children than healthy controls (P = 0.0296. The 0 allele was significantly more frequent among the 164 HIV-1-infected children (0.29 than among the 150 healthy controls (0.18; P = 0.0032. Our data confirm the association between the presence of the mutated MBL2 allele (allele 0 and HIV-1 infection in perinatally exposed children. Our results are in agreement with the literature data which indicate that the presence of the allele 0 confers a relative risk of 1.37 for HIV-1 infection through

  14. Setup of a PCR laboratory. (United States)

    Khan, Zaheer


    PCR represents an extremely powerful and central molecular biology method. At the heart of its power is the exquisite sensitivity offered: single molecule detection in certain contexts. However, with great power comes great responsibility. Contamination of reagents or test samples with amplifiable material, such as previous reaction products, can be crippling to scientists applying PCR protocols. Prevention of PCR contamination is far and away preferred over eradication. This chapter sets out to offer guidance as to how to use PCR while minimising contamination problems.

  15. Bubble Formation in Basalt-like Melts

    DEFF Research Database (Denmark)

    Jensen, Martin; Keding, Ralf; Yue, Yuanzheng


    The effect of the melting temperature on bubble size and bubble formation in an iron bearing calcium aluminosilicate melt is studied by means of in-depth images acquired by optical microscopy. The bubble size distribution and the total bubble volume are determined by counting the number of bubbles...... and their diameter. The variation in melting temperature has little influence on the overall bubble volume. However, the size distribution of the bubbles varies with the melting temperature. When the melt is slowly cooled, the bubble volume increases, implying decreased solubility of the gaseous species. Mass...

  16. Triacylglycerol and melting profiles of milk fat from several species

    NARCIS (Netherlands)

    Smiddy, M.A.; Huppertz, T.; Ruth, van S.M.


    Gas chromatography and differential scanning calorimetry (DSC) were used to differentiate the fats of cow, goat, sheep, water buffalo, donkey, horse and camel milk (n = 20 for each species). Principal component analysis of triacylglycerol (TAG) composition allowed classification into groups

  17. Industrial opportunities of controlled melt flow during glass melting, part 1: Melt flow evaluation

    Czech Academy of Sciences Publication Activity Database

    Dyrčíková, Petra; Hrbek, Lukáš; Němec, Lubomír


    Roč. 58, č. 2 (2014), s. 111-117 ISSN 0862-5468 R&D Projects: GA TA ČR TA01010844 Institutional support: RVO:67985891 Keywords : glass melting * controlled flow * space utilization Subject RIV: JH - Ceramics, Fire-Resistant Materials and Glass Impact factor: 0.435, year: 2014

  18. [Development of a rapid molecular typing method for Vibrio cholerae using melting curve-based multilocus melt typing]. (United States)

    Huang, Shijie; Liu, Zanzan; Wen, Huixin; Li, Li; Li, Qingge; Huang, Jianwei


    To develop a high-throughput rapid method for Vibrio (V.) cholerae molecular typing based on Melting Curve-based Multilocus Melt Typing (McMLMT). Seven housekeeping genes of V.cholerae were screened out, and for each gene, the specific primers were designed for correspondent genes as well as 4 probes covering polymorphism loci of sequences. After optimizing all parameters, a method of melting-curve analysis following asymmetric PCR was established with dual-fluorescent-reporter in two reaction tubes for each gene. A set of 28 Tm-values was obtained for each strain and then translated into a set of code of allelic genes, standing for the strain's McMLMT type (MT). Meanwhile, sequences of the 7-locus polymorphism were typed according to the method of MLST. To evaluate the efficiency and reliability of McMLMT, the data were compared with that of sequence-typing and PFGE using BioNumerics software. McMLMT method was established and refined for rapid typing of V. cholerae that a dozen of strains can be finished testing in a 3-hours PCR running using 96-well plates. 108 strains were analyzed and 28-Tm-values could be grouped and encoded according to 7 housekeeping gene to obtain the code set of allelic genes, and classified into 18 types (D = 0.723 3). Sequences of the 7 genes' polymorphism areas were directly clustered into the same 18 types with reference to MLST method. 46 of the strains, each represented a different PFGE type, could be classified into 13 types (D = 0.614 5) with McMLMT method and A- K groups at 85% similarity (D = 0.858 9) with PFGE method. McMLMT method is a rapid high-throughput molecular typing method for batches of strains with a resolution equal to MLST method and comparable to PFGE group.

  19. Rapid Simultaneous Amplification and Detection of the MBR/JH Chromosomal Translocation by Fluorescence Melting Curve Analysis (United States)

    Bohling, Sandra D.; King, Thomas C.; Wittwer, Carl T.; Elenitoba-Johnson, Kojo S. J.


    Polymerase chain reaction (PCR) amplification and product analysis for the detection of chromosomal translocations, such as the t(14;18), has traditionally been a two-step process. PCR product detection has generally entailed gel electrophoresis and/or hybridization or sequencing for confirmation of assay specificity. Using a microvolume fluorimeter integrated with a thermal cycler and a PCR-compatible double-stranded DNA (dsDNA) binding fluorescent dye (SYBR Green I), we investigated the feasibility of simultaneous thermal amplification and detection of MBR/JH translocation products by fluorescence melting curve analysis. We analyzed DNA from 30 cases of lymphoproliferative disorders comprising 19 cases of previously documented MBR/JH-positive follicle center lymphoma and 11 reactive lymphadenopathies. The samples were coded and analyzed blindly for the presence of MBR/JH translocations by fluorescence melting curve analysis. We also performed dilutional assays using the MBR/JH-positive cell line SUDHL-6. Multiplex PCR for MBR/JH and β-globin was used to simultaneously assess sample adequacy. All (100%) of the 19 cases previously determined to be MBR/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at ∼90°C by melting curve analysis after amplification. Fluorescence melting peaks obtained by plotting the negative derivative of fluorescence over temperature (−dF/dT) versus temperature (T) showed melting temperatures (Tm) at 88.85 ± 1.15°C. In addition, multiplex assays using both MBR/JH and β-globin primers yielded easily distinguishable fluorescence melting peaks at ∼90°C and 81.2°C, respectively. Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 100-fold. Simultaneous amplification and fluorescence melting curve analysis is a simple, reliable, and sensitive method

  20. Ice shelf melt rates and 3D imaging (United States)

    Lewis, Cameron Scott

    Ice shelves are sensitive indicators of climate change and play a critical role in the stability of ice sheets and oceanic currents. Basal melting of ice shelves plays an important role in both the mass balance of the ice sheet and the global climate system. Airborne- and satellite based remote sensing systems can perform thickness measurements of ice shelves. Time separated repeat flight tracks over ice shelves of interest generate data sets that can be used to derive basal melt rates using traditional glaciological techniques. Many previous melt rate studies have relied on surface elevation data gathered by airborne- and satellite based altimeters. These systems infer melt rates by assuming hydrostatic equilibrium, an assumption that may not be accurate, especially near an ice shelf's grounding line. Moderate bandwidth, VHF, ice penetrating radar has been used to measure ice shelf profiles with relatively coarse resolution. This study presents the application of an ultra wide bandwidth (UWB), UHF, ice penetrating radar to obtain finer resolution data on the ice shelves. These data reveal significant details about the basal interface, including the locations and depth of bottom crevasses and deviations from hydrostatic equilibrium. While our single channel radar provides new insight into ice shelf structure, it only images a small swatch of the shelf, which is assumed to be an average of the total shelf behavior. This study takes an additional step by investigating the application of a 3D imaging technique to a data set collected using a ground based multi channel version of the UWB radar. The intent is to show that the UWB radar could be capable of providing a wider swath 3D image of an ice shelf. The 3D images can then be used to obtain a more complete estimate of the bottom melt rates of ice shelves.

  1. Differential identification of Sporothrix spp. and Leishmania spp. by conventional PCR and qPCR in multiplex format. (United States)

    Rodríguez-Brito, Sabrina; Camacho, Emma; Mendoza, Mireya; Niño-Vega, Gustavo A


    Sporotrichosis and cutaneous leishmaniasis are skin infections with similar clinical manifestations but different treatment methods. The present study aimed to evaluate qPCR and conventional PCR for differential detection of the etiological agents of both infections in multiplex format. Assays were designed using two sets of reported primers: SS1/SS2, designed on the 18S ribosomal RNA gene from Sporothrix spp., and JW11/JW12, designed on the kinetoplast DNA (kDNA) minicircles of Leishmania spp. qPCR detected 200 fg of DNA per reaction for both Sporothrix and Leishmania. Melting curve analysis revealed two distinctive Tm peaks for Sporothrix spp. (85.5°C), and Leishmania spp. (82.6°C). A detection limit of 20 pg was determined for the diagnosis of both with conventional PCR. No other clinically important organisms were detected by either PCR or qPCR. However, a Blast analysis on GenBank databases, using as query the sequence of the PCR fragment obtained with primers SS1/SS2, showed 100% identity to environmental fungi of the Ophiostomales order. Lower percentages of identity (≤80%), with mismatches at primers' sequence regions were obtained for other environmental or clinically important fungi. Proper handling of clinical samples is required to avoid false negatives due to contamination with environmental fungi of the Ophiostomales order. © The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail:

  2. Measuring melting capacity with calorimetry


    Betten, Linda


    Road salting is an important aspect of winter maintenance. There has been an increase in the usage of salt in later years to keep the road safe and accessible. It is a desire to reduce the amount of salt due to environmental aspects. To achieve better practices for winter maintenance it is necessary to obtain more knowledge about the different properties of salt. The motivation for this thesis is to develop a better method for determining the melting capacity for salt, which is an important p...

  3. Retrieving original melt compositions in migmatites (United States)

    Kriegsman, L. M.; Nyström, A. I.


    The final textures and mineral modes of anatectic migmatites are affected by four successive processes: (i) prograde partial melting and small-scale segregation into melt-rich domains and restitic domains; (ii) partial melt extraction; (iii) partial retrograde reactions (back reaction) between in situ crystallizing melt and the restite; (iv) crystallization of remaining melt at the water-saturated solidus, releasing volatiles (Kriegsman, 2001). These processes are investigated using mass balance calculations in the KFMASH chemical system. Starting from a fixed bulk composition, fluid-absent melting reactions are considered along an isobaric heating path, followed by mineral-melt reactions during isobaric cooling (path 1), and uplift (path 2). Variables in the model are the restite fraction X and the melt fraction Y involved in back reaction, the melt fraction Z extracted from the system, and the melt fraction 1-Y-Z crystallized in situ at the water-saturated solidus. Incongruent phases are considered to be part of the restite. To facilitate calculations, mineral and melt compositions are taken to be constant. It is shown that melanosome, leucosome, and mesosome compositions generally do not show linear compositional trends in a closed system. Instead, mesosome, neosome, protolith and melt compositions lie on a hyperplane and form linear trends in any compositional diagram. Several methods are proposed to retrieve the melt composition from neosome and mesosome compositions. Applications to natural examples (dataset of Ashworth, 1976; and our new data from SW Finland) strengthen the migmatite-to-granite connection which may notably be obscured by the back reaction process. References: Kriegsman, L.M., 2001, Lithos 56, 75-96 Ashworth, J.R., 1976. Mineralogical Magazine 40, 661-682

  4. Shape based kinetic outlier detection in real-time PCR

    Directory of Open Access Journals (Sweden)

    D'Atri Mario


    Full Text Available Abstract Background Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD. Results Richards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molecules was found, due to the presence of biological inhibitors such as tannic acid, IgG or quercitin, SOD efficiently marked these amplification profiles as outliers. SOD was subsequently compared with KOD, the current approach based on PCR efficiency estimation. The data obtained showed that SOD was more sensitive than KOD, whereas SOD and KOD were equally specific. Conclusion Our results demonstrated, for the first time, that outlier detection can be based on amplification shape instead of PCR efficiency. SOD represents an improvement in real-time PCR analysis because it decreases the variance of data thus increasing the reliability of quantification.

  5. Melt migration in a silicate liquid-olivine system - An experimental test of compaction theory (United States)

    Riley, G. N., Jr.; Kohlstedt, D. L.; Richter, F. M.


    An experimentally derived melt migration profile is compared with those obtained from compaction theory. A couple is prepared in which a source-for-melt (a disk composed of a silicate glass and olivine) was placed in contact with a sink-for-melt (a disk of polycrystalline olivine) in order to induce melt migration under laboratory conditions. It is noted that melt infiltrates into the sink disk along triple junctions driven by capillary forces. In order to analyze the melt migration profile in terms of compaction theory, the equations developed by McKenzie (1984) to describe porous flow of a liquid in a deformable matrix were modified. The bouyancy force term is replaced with a capillary force term and governing equations are then solved numerically with the initial boundary conditions specified by the experimental design. Also, it is determined that compaction theory provides a good description of the experimental results provided that the permeability of these solid-liquid materials increases linearly with increasing liquid fraction.

  6. Monitoring of polymer melt processing

    International Nuclear Information System (INIS)

    Alig, Ingo; Steinhoff, Bernd; Lellinger, Dirk


    The paper reviews the state-of-the-art of in-line and on-line monitoring during polymer melt processing by compounding, extrusion and injection moulding. Different spectroscopic and scattering techniques as well as conductivity and viscosity measurements are reviewed and compared concerning their potential for different process applications. In addition to information on chemical composition and state of the process, the in situ detection of morphology, which is of specific interest for multiphase polymer systems such as polymer composites and polymer blends, is described in detail. For these systems, the product properties strongly depend on the phase or filler morphology created during processing. Examples for optical (UV/vis, NIR) and ultrasonic attenuation spectra recorded during extrusion are given, which were found to be sensitive to the chemical composition as well as to size and degree of dispersion of micro or nanofillers in the polymer matrix. By small-angle light scattering experiments, process-induced structures were detected in blends of incompatible polymers during compounding. Using conductivity measurements during extrusion, the influence of processing conditions on the electrical conductivity of polymer melts with conductive fillers (carbon black or carbon nanotubes) was monitored. (topical review)

  7. Chemical decontamination and melt densification

    International Nuclear Information System (INIS)

    Dillon, R.L.; Griggs, B.; Kemper, R.S.; Nelson, R.G.


    Preliminary studies on the chemical decontamination and densification of Zircaloy, stainless steel, and Inconel undissolved residues remaining after dissolution of the UO 2 --PuO 2 spent fuel material from sheared fuel bundles are reported. The studies were made on cold or very small samples to demonstrate the feasibility of the processes developed before proceeding to hot cell demonstrations with kg level of the sources. A promising aqueous decontamination method for Zr alloy cladding was developed in which oxidized surfaces are conditioned with HF prior to leaching with ammonium oxalate, ammonium citrate, ammonium fluoride, and hydrogen peroxide. Feasibility of molten salt decontamination of oxidized Zircaloy was demonstrated. A low melting alloy of Zircaloy, stainless steel, and Inconel was obtained in induction heated graphite crucibles. Segregated Zircaloy cladding sections were directly melted by the inductoslag process to yield a metal ingot suitable for storage. Both Zircaloy and Zircaloy--stainless steel--Inconel alloys proved to be highly satisfactory getters and sinks for recovered tritium

  8. Viscosity model for aluminosilicate melt

    Directory of Open Access Journals (Sweden)

    Zhang G.H.


    Full Text Available The structurally based viscosity model proposed in our previous study is extended to include more components, e.g. SiO2, Al2O3, FeO, MnO, MgO, CaO, Na2O and K2O. A simple method is proposed to calculate the numbers of different types of oxygen ions classified by the different cations they bonded with, which is used to characterize the influence of composition on viscosity. When dealing with the aluminosilicate melts containing several basic oxides, the priority order is established for different cations for charge compensating Al3+ ions, according to the coulombic force between cation and oxygen anion. It is indicated that basic oxides have two paradox influences on viscosity: basic oxide with a higher basicity decreases viscosity more greatly by forming weaker non-bridging oxygen bond; while it increases viscosity more greatly by forming stronger bridging oxygen bond in tetrahedron after charge compensating Al3+ ion. The present model can extrapolate its application range to the system without SiO2. Furthermore, it could also give a satisfy interpretation to the abnormal phenomenon that viscosity increases when adding K2O to CaO-Al2O3-SiO2 melt within a certain composition range.

  9. Digital PCR: A brief history


    Morley, Alexander A.


    Digital PCR for quantification of a target of interest has been independently developed several times, being described in 1990 and 1991 using the term “limiting dilution PCR” and in 1999 using the term “digital PCR”. It came into use in the decade following its first development but its use was cut short by the description of real-time PCR in 1996. However digital PCR has now had a renaissance due to the recent development of new instruments and chemistry which have made it a much simpler and...

  10. Lead extraction from cathode ray tube funnel glass melted under different oxidizing conditions. (United States)

    Okada, Takashi


    Lead was extracted into hydrochloric acid from cathode ray tube funnel glass melted under reducing atmosphere, oxidizing atmosphere, or a sequential combination of both to mechanistically investigate effects of the melting atmosphere on lead extraction. Melting funnel glass in a reductive atmosphere led to the generation of metallic lead particles that were readily soluble in the acid, increasing the quantity of lead extracted into the acid. Meanwhile, the glass product obtained after melting funnel glass in an oxidative atmosphere exhibited higher corrosion resistance in the acid, and the quantity of lead extracted from the treated glass decreased. However, Na2CO3 addition to the glass during melting hindered the enhancement of corrosion resistance and the immobilization of lead in the acid. X-ray photoelectron spectroscopic analysis of the treated glass samples showed that the positions of the peak or the profiles of the spectra attributed to Pb 4f, Si 2p, and O 1s signals were modified by oxidative melting, an indication that oxidative melting results in structural changes in the SiO2 framework of the glass. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Sensory and analytical characterization of the "cool-melting" perception of commercial spreads. (United States)

    Galindo-Cuspinera, Verónica; Valenҁa de Sousa, Joana; Knoop, Marcia


    Butters, margarines and table spreads are water-in-oil emulsions. Melting characteristics of these products are important for flavor release and consumer acceptance. One characteristic that is believed to discriminate butters from margarines is a cooling sensation perceived in-mouth while consuming these products. Here, we investigated different methods to characterize sensorically and analytically the "cool-melting" properties of commercial butter and margarines. Our results show that butter indeed can be distinguished from margarines based on their "cool-melting" properties. Furthermore, changes in enthalpy as measured through DSC and solid fat content are good predictors of the "cool-melting" effect of spreads. By understanding the mechanisms of the "cool-melting" perception of spreads, and linking them to analytical measurements, we can create an in-vitro quantification method of "cool-melting." This method can eventually help directing product development to achieve the desire product profile and increase consumer acceptance and liking of margarines and low-fat spread products. In this study we did not assess the impact of "cool-melting" on consumer perception, which would be the next step in understanding the drivers of liking of spread products. © 2017 The Authors. Journal of Texture Studies published by Wiley Periodicals, Inc.

  12. Multiscale Models of Melting Arctic Sea Ice (United States)


    Sea ice reflectance or albedo , a key parameter in climate modeling, is primarily determined by melt pond and ice floe configurations. Ice - albedo ...determine their albedo - a key parameter in climate modeling. Here we explore the possibility of a conceptual sea ice climate model passing through a...bifurcation points. Ising model for melt ponds on Arctic sea ice Y. Ma, I. Sudakov, and K. M. Golden Abstract: The albedo of melting

  13. Sound propagation in selenium and tellurium melts

    International Nuclear Information System (INIS)

    Glazov, V.M.; Kim, S.G.; Sulejmenov, T.


    Methods, that under similar frequences of ten MHz and using one sample permit to study temperature dependences and propagation velocities, the absorption coefficient of the sound in melts, are described. As a result studying selenium and tellurium melts intricate polytherms of sound propagation velocity and absorption coefficient, that are interpreted usiung representations on breaking chains in associated liquid(selenium) and dissolution of short chains in the melt(tellurium) atomic matrix, are constructed

  14. Direct writing by way of melt electrospinning. (United States)

    Brown, Toby D; Dalton, Paul D; Hutmacher, Dietmar W


    Melt electrospun fibers of poly(ϵ-caprolactone) are accurately deposited using an automated stage as the collector. Matching the translation speed of the collector to the speed of the melt electrospinning jet establishes control over the location of fiber deposition. In this sense, melt electrospinning writing can be seen to bridge the gap between solution electrospinning and direct writing additive manufacturing processes. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Surface melting of deuterium hydride thick films


    Zeppenfeld, P.; Bienfait, M.; Feng Chuan Liu,; Vilches, O.E.; Coddens, G.


    Quasi-elastic neutron scattering has been used to measure, below the bulk melting temperature, the thickness and the diffusion coefficient of the mobile surface layer of 8 and 10 layer thick films of deuterium hydride (HD) condensed on MgO(100). The measurements show that the close-packed surface of solid HD surface melts gradually, with the thickness of the melted layer increasing from 0.5 to 6 molecular layers as the temperature rises from 4 K to 0.05 K below the bulk melting temperature. T...

  16. Melt analysis of mismatch amplification mutation assays (Melt-MAMA: a functional study of a cost-effective SNP genotyping assay in bacterial models.

    Directory of Open Access Journals (Sweden)

    Dawn N Birdsell

    Full Text Available Single nucleotide polymorphisms (SNPs are abundant in genomes of all species and biologically informative markers extensively used across broad scientific disciplines. Newly identified SNP markers are publicly available at an ever-increasing rate due to advancements in sequencing technologies. Efficient, cost-effective SNP genotyping methods to screen sample populations are in great demand in well-equipped laboratories, but also in developing world situations. Dual Probe TaqMan assays are robust but can be cost-prohibitive and require specialized equipment. The Mismatch Amplification Mutation Assay, coupled with melt analysis (Melt-MAMA, is flexible, efficient and cost-effective. However, Melt-MAMA traditionally suffers from high rates of assay design failures and knowledge gaps on assay robustness and sensitivity. In this study, we identified strategies that improved the success of Melt-MAMA. We examined the performance of 185 Melt-MAMAs across eight different pathogens using various optimization parameters. We evaluated the effects of genome size and %GC content on assay development. When used collectively, specific strategies markedly improved the rate of successful assays at the first design attempt from ~50% to ~80%. We observed that Melt-MAMA accurately genotypes across a broad DNA range (~100 ng to ~0.1 pg. Genomic size and %GC content influence the rate of successful assay design in an independent manner. Finally, we demonstrated the versatility of these assays by the creation of a duplex Melt-MAMA real-time PCR (two SNPs and conversion to a size-based genotyping system, which uses agarose gel electrophoresis. Melt-MAMA is comparable to Dual Probe TaqMan assays in terms of design success rate and accuracy. Although sensitivity is less robust than Dual Probe TaqMan assays, Melt-MAMA is superior in terms of cost-effectiveness, speed of development and versatility. We detail the parameters most important for the successful application of

  17. Rapid discrimination of Isaria javanica and Isaria poprawskii from Isaria spp. using high resolution DNA melting assays (United States)

    The current study evaluates the potential of using high resolution DNA melting assays to discriminate species in the genus, Isaria. The study utilizes a previously identified 103 base pair PCR amplicon, which was reported to be selective for Isaria fumosorosea. Our study finds the amplicon selective...

  18. Quantification and Optimization of Candida albicans DNA in Blood Samples Using Real- Time PCR

    Directory of Open Access Journals (Sweden)

    Mojtaba Nabili


    Full Text Available Background: Candida albicans (C. albicans is a major cause of candidaemia in people with impaired immunity. Blood culture is a “gold standard” for candidaemia detection but is time-consuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis. Methods: Five milliliter blood samples from healthy volunteers were spiked with 100-106 C. albicans cells to determine the detection limit of our method. DNA was extracted from whole blood using glass beads and the QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany. DNA from C. albicans isolates were amplified with primers and inserted into Escherichia coli (E. coli DH5α.1 cells with the TA cloning vector (Invitrogen. The plasmid was used for standardization and optimization. A quantitative PCR assay with the LightCycler amplification and detection system based on fluorescence resonance energy transfer (FRET with two different specific probes was established. To assess the precision and reproducibility of real-time PCR the intra-assay precision was determined in six consecutive assays. Results: No cross-reactivity of the hybridization probes with the DNA of non-C. albicans species or human genomic DNA was observed, which confirmed its 100% specificity. The minimum limit detected was one C. albicans cell or 100 CFU/ml (10 fg per PCR reaction. The real-time PCR efficiency rate for Candida was high (E = 1.95. Melting curve analysis of C. albicans showed a specific melting peak temperature of 65.76 °C. Conclusion: The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis.

  19. Quantification and optimization of Candida albicans DNA in blood samples using Real- Time PCR. (United States)

    Nabili, Mojtaba; Ashrafi, Mohsen; Janbabaie, Ghasem; Hedayati, Mohamad Taghi; Ali-Moghaddam, Kamran; Shokohi, Tahereh


    Candida albicans (C. albicans) is a major cause of candidaemia in people with impaired immunity. Blood culture is a "gold standard" for candidaemia detection but is time-consuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis. Five milliliter blood samples from healthy volunteers were spiked with 10(0)-10(6) C. albicans cells to determine the detection limit of our method. DNA was extracted from whole blood using glass beads and the QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany). DNA from C. albicans isolates were amplified with primers and inserted into Escherichia coli (E. coli) DH5α.1 cells with the TA cloning vector (Invitrogen). The plasmid was used for standardization and optimization. A quantitative PCR assay with the LightCycler amplification and detection system based on fluorescence resonance energy transfer (FRET) with two different specific probes was established. To assess the precision and reproducibility of real-time PCR the intra-assay precision was determined in six consecutive assays. No cross-reactivity of the hybridization probes with the DNA of non-C. albicans species or human genomic DNA was observed, which confirmed its 100% specificity. The minimum limit detected was one C. albicans cell or 10(0) CFU/ml (10 fg) per PCR reaction. The real-time PCR efficiency rate for Candida was high (E = 1.95). Melting curve analysis of C. albicans showed a specific melting peak temperature of 65.76 °C. The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis.

  20. Grain boundary melting in ice (United States)

    Thomson, E. S.; Hansen-Goos, Hendrik; Wettlaufer, J. S.; Wilen, L. A.


    We describe an optical scattering study of grain boundary premelting in water ice. Ubiquitous long ranged attractive polarization forces act to suppress grain boundary melting whereas repulsive forces originating in screened Coulomb interactions and classical colligative effects enhance it. The liquid enhancing effects can be manipulated by adding dopant ions to the system. For all measured grain boundaries this leads to increasing premelted film thickness with increasing electrolyte concentration. Although we understand that the interfacial surface charge densities qs and solute concentrations can potentially dominate the film thickness, we cannot directly measure them within a given grain boundary. Therefore, as a framework for interpreting the data we consider two appropriate qs dependent limits; one is dominated by the colligative effect and other is dominated by electrostatic interactions.

  1. Electrolysis of simulated lunar melts (United States)

    Lewis, R. H.; Lindstrom, D. J.; Haskin, L. A.


    Electrolysis of molten lunar soil or rock is examined as an attractive means of wresting useful raw materials from lunar rocks. It requires only hat to melt the soil or rock and electricity to electrolyze it, and both can be developed from solar power. The conductivities of the simple silicate diopside, Mg CaSi2O6 were measured. Iron oxide was added to determine the effect on conductivity. The iron brought about substantial electronic conduction. The conductivities of simulated lunar lavas were measured. The simulated basalt had an AC conductivity nearly a fctor of two higher than that of diopside, reflecting the basalt's slightly higher total concentration of the 2+ ions Ca, Mg, and Fe that are the dominant charge carriers. Electrolysis was shown to be about 30% efficient for the basalt composition.

  2. O-5S quantitative real-time PCR: a new diagnostic tool for laboratory confirmation of human onchocerciasis


    Mekonnen, Solomon A.; Beissner, Marcus; Saar, Malkin; Ali, Solomon; Zeynudin, Ahmed; Tesfaye, Kassahun; Adbaru, Mulatu G.; Battke, Florian; Poppert, Sven; Hoelscher, Michael; Löscher, Thomas; Bretzel, Gisela; Herbinger, Karl-Heinz


    Background Onchocerciasis is a parasitic disease caused by the filarial nematode Onchocerca volvulus. In endemic areas, the diagnosis is commonly confirmed by microscopic examination of skin snip samples, though this technique is considered to have low sensitivity. The available melting-curve based quantitative real-time PCR (qPCR) using degenerated primers targeting the O-150 repeat of O. volvulus was considered insufficient for confirming the individual diagnosis, especially in elimination ...

  3. Reaction of soda-lime-silica glass melt with water vapour at melting temperatures

    Czech Academy of Sciences Publication Activity Database

    Vernerová, Miroslava; Kloužek, Jaroslav; Němec, Lubomír


    Roč. 416, MAY 15 (2015), s. 21-30 ISSN 0022-3093 R&D Projects: GA TA ČR TA01010844 Institutional support: RVO:67985891 Keywords : glass melt * sulfate * water vapour * bubble nucleation * melt foaming * glass melting Subject RIV: JH - Ceramics, Fire-Resistant Materials and Glass Impact factor: 1.825, year: 2015

  4. Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots (United States)

    Sang, Fuming; Yang, Yang; Yuan, Lin; Ren, Jicun; Zhang, Zhizhou


    Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour preincubation at 50 °C before real time PCR. Moreover, the results obtained by QD-based HS PCR were comparable to a commercial Taq antibody DNA polymerase. However, no obvious HS effect of QDs was found in real time PCR using Taq DNA polymerase. The findings of this study demonstrated that a cost-effective high-throughput real time PCR based on QD triggered HS PCR could be established with high consistency, sensitivity and accuracy.Hot start (HS) PCR is an excellent alternative for high-throughput real time PCR due to its ability to prevent nonspecific amplification at low temperature. Development of a cost-effective and simple HS PCR technique to guarantee high-throughput PCR specificity and consistency still remains a great challenge. In this study, we systematically investigated the HS characteristics of QDs triggered in real time PCR with EvaGreen and SYBR Green I dyes by the analysis of amplification curves, standard curves and melting curves. Two different kinds of DNA polymerases, Pfu and Taq, were employed. Here we showed that high specificity and efficiency of real time PCR were obtained in a plasmid DNA and an error-prone two-round PCR assay using QD-based HS PCR, even after an hour

  5. Permeability and 3-D melt geometry in shear-induced high melt fraction conduits (United States)

    Zhu, W.; Cordonnier, B.; Qi, C.; Kohlstedt, D. L.


    Observations of dunite channels in ophiolites and uranium-series disequilibria in mid-ocean ridge basalt suggest that melt transport in the upper mantle beneath mid-ocean ridges is strongly channelized. Formation of high melt fraction conduits could result from mechanical shear, pyroxene dissolution, and lithological partitioning. Deformation experiments (e.g. Holtzman et al., 2003) demonstrate that shear stress causes initially homogeneously distributed melt to segregate into an array of melt-rich bands, flanked by melt-depleted regions. At the same average melt fraction, the permeability of high melt fraction conduits could be orders of magnitude higher than that of their homogenous counterparts. However, it is difficult to determine the permeability of melt-rich bands. Using X-ray synchrotron microtomography, we obtained high-resolution images of 3-dimensional (3-D) melt distribution in a partially molten rock containing shear-induced high melt fraction conduits. Sample CQ0705, an olivine-alkali basalt aggregate with a nominal melt fraction of 4%, was deformed in torsion at a temperature of 1473 K and a confining pressure of 300 MPa to a shear strain of 13.3. A sub-volume of CQ0705 encompassing 3-4 melt-rich bands was imaged. Microtomography data were reduced to binary form so that solid olivine is distinguishable from basalt glass. At a spatial resolution of 160 nm, the 3-D images reveal the shape and connectedness of melt pockets in the melt-rich bands. Thin melt channels formed at grain edges are connected at large melt nodes at grain corners. Initial data analysis shows a clear preferred orientation of melt pockets alignment subparallel to the melt-rich band. We use the experimentally determined geometrical parameters of melt topology to create a digital rock with identical 3-D microstructures. Stokes flow simulations are conducted on the digital rock to obtain the permeability tensor. Using this digital rock physics approach, we determine how deformation

  6. Development of melting system for Measurement of trace elements and ions in ice core

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Sang Bum; Lee, Khang Hyun; Hur, Soon Do; Soyol-Erene, Tseren-Ochir; Kim, Sun Mee; Chung, Ji Woong; Jun, Seong Joon [Korea Polar Research Institute, KIOST, Incheon (Korea, Republic of); Hong, Sung Min [Dept. of Ocean Sciences, Inha University, Incheon (Korea, Republic of); Kang, Chang Hee [Dept. of Chemistry and Research Institute for Basic Sciences, Jeju National University, Jeju (Korea, Republic of)


    We present a titanium (Ti) melting head divided into three zones as an improved melting system for decontaminating ice-core samples. This system was subjected to performance tests using short ice-core samples (4 × 4 cm{sup 2}, ⁓5 cm long). The procedural blanks (PBs) and detection limits of ionic species, with the exception of math formula, were comparable with published values, but for elements the experimental procedures should be refined to obtain valid Zn concentrations due to the PB of ⁓90.0 ± 16.2 ng/L. The improved melting system efficiently decontaminated the samples, as verified by the concentration profiles of elements and ions in the melted samples from the three melting-head zones. The recovery of trace elements in ice-core samples was ⁓70–120% at ⁓100 ng/L in artificial ice cores. Because of the memory effects between ice-core samples melted in series, the melting system should be rinsed at least 5–6 times (in a total volume of ⁓2.5 mL deionized water) after each melting procedure. Finally, as an application of this technique, trace elements were measured in ice-core samples recovered from the East Rongbuk Glacier, Mount Everest, (28°03′N, 86°96′E, 6518 m a.s.l.), and the concentrations of trace elements following mechanical chiseling and the melting method were compared.

  7. Stick and Slip Behaviour of Confined Oligomer Melts under Shear. A Molecular-Dynamics Study.

    NARCIS (Netherlands)

    Manias, E.; Hadziioannou, G.; Bitsanis, I.; Brinke, G. ten


    The flow behaviour of melts of short chains, confined in molecularly thin Couette flow geometries, is studied with molecular-dynamics simulations. The effect of wall attraction and confinement on the density and velocity profiles is analysed. In these highly inhomogeneous films, a strong correlation


    NARCIS (Netherlands)



    The flow behaviour of melts of short chains, confined in molecularly thin Couette flow geometries, is studied with molecular-dynamics simulations. The effect of wall attraction and confinement on the density and velocity profiles is analysed. In these highly inhomogeneous films, a strong correlation

  9. Disordering and Melting of Aluminum Surfaces

    DEFF Research Database (Denmark)

    Stoltze, Per; Nørskov, Jens Kehlet; Landman, U.


    We report on a molecular-dynamics simulation of an Al(110) surface using the effective-medium theory to describe the interatomic interactions. The surface region is found to start melting ≅200 K below the bulk melting temperature with a gradual increase in the thickness of the disordered layer as...

  10. Recent Changes in the Arctic Melt Season (United States)

    Stroeve, Julienne; Markus, Thorsten; Meier, Walter N.; Miller, Jeff


    Melt-season duration, melt-onset and freeze-up dates are derived from satellite passive microwave data and analyzed from 1979 to 2005 over Arctic sea ice. Results indicate a shift towards a longer melt season, particularly north of Alaska and Siberia, corresponding to large retreats of sea ice observed in these regions. Although there is large interannual and regional variability in the length of the melt season, the Arctic is experiencing an overall lengthening of the melt season at a rate of about 2 weeks decade(sup -1). In fact, all regions in the Arctic (except for the central Arctic) have statistically significant (at the 99% level or higher) longer melt seasons by greater than 1 week decade(sup -1). The central Arctic shows a statistically significant trend (at the 98% level) of 5.4 days decade(sup -1). In 2005 the Arctic experienced its longest melt season, corresponding with the least amount of sea ice since 1979 and the warmest temperatures since the 1880s. Overall, the length of the melt season is inversely correlated with the lack of sea ice seen in September north of Alaska and Siberia, with a mean correlation of -0.8.

  11. Recharging "Hot-Melt" Adhesive Film (United States)

    Progar, D. J.


    Technique for recharging surface with "hot-melt" film makes use of one sided, high-temperature, pressure-sensitive adhesive tape. Purpose of the one-sided tape is to hold hot-melt charge in place until fused to surface. After adhesive has fused to surface and cooled, tape is removed, leaving adhesive on surface.

  12. Summer Melts Immigrant Students' College Plans (United States)

    Naranjo, Melissa M.; Pang, Valerie Ooka; Alvarado, Jose Luis


    Many college-intending students find themselves dealing with the undermatch and summer melt phenomena. Undermatch refers to the situation where academically-successful high-school graduates choose not to go to any college or to go to a local community college not commensurate with their academic achievements. Summer melt describes how students may…

  13. 1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer

    Directory of Open Access Journals (Sweden)

    Dráberová Lubica


    Full Text Available Abstract Background Quantitative real-time PCR (qPCR is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI. In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify. Results We found that several DNA dyes (SGI, SYTO-9, SYTO-13, SYTO-82, EvaGreen, LCGreen or ResoLight exhibited optimum qPCR performance in buffers of different salt composition. Fidelity assays demonstrated that the observed differences were not caused by changes in Taq DNA polymerase induced mutation frequencies in PCR mixes of different salt composition or containing different DNA dyes. In search for a PCR mix compatible with all the DNA dyes, and suitable for efficient amplification of difficult-to-amplify DNA templates, such as those in whole blood, of medium size and/or GC-rich, we found excellent performance of a PCR mix supplemented with 1 M 1,2-propanediol and 0.2 M trehalose (PT enhancer. These two additives together decreased DNA melting temperature and efficiently neutralized PCR inhibitors present in blood samples. They also made possible more efficient amplification of GC-rich templates than betaine and other previously described additives. Furthermore, amplification in the presence of PT enhancer increased the robustness and performance of routinely used qPCRs with short amplicons. Conclusions The combined data indicate that PCR mixes supplemented with PT enhancer are suitable for DNA amplification in the presence of various DNA dyes and for a variety of templates which otherwise can be amplified with difficulty.

  14. Modeling the summertime evolution of sea-ice melt ponds

    DEFF Research Database (Denmark)

    Lüthje, Mikael; Feltham, D.L.; Taylor, P.D.


    We present a mathematical model describing the summer melting of sea ice. We simulate the evolution of melt ponds and determine area coverage and total surface ablation. The model predictions are tested for sensitivity to the melt rate of unponded ice, enhanced melt rate beneath the melt ponds...

  15. Nanotexturing of surfaces to reduce melting point.

    Energy Technology Data Exchange (ETDEWEB)

    Garcia, Ernest J.; Zubia, David (University of Texas at El Paso El Paso, TX); Mireles, Jose (Universidad Aut%C3%94onoma de Ciudad Ju%C3%94arez Ciudad Ju%C3%94arez, Mexico); Marquez, Noel (University of Texas at El Paso El Paso, TX); Quinones, Stella (University of Texas at El Paso El Paso, TX)


    This investigation examined the use of nano-patterned structures on Silicon-on-Insulator (SOI) material to reduce the bulk material melting point (1414 C). It has been found that sharp-tipped and other similar structures have a propensity to move to the lower energy states of spherical structures and as a result exhibit lower melting points than the bulk material. Such a reduction of the melting point would offer a number of interesting opportunities for bonding in microsystems packaging applications. Nano patterning process capabilities were developed to create the required structures for the investigation. One of the technical challenges of the project was understanding and creating the specialized conditions required to observe the melting and reshaping phenomena. Through systematic experimentation and review of the literature these conditions were determined and used to conduct phase change experiments. Melting temperatures as low as 1030 C were observed.

  16. Shape evolution of a melting nonspherical particle. (United States)

    Kintea, Daniel M; Hauk, Tobias; Roisman, Ilia V; Tropea, Cameron


    In this study melting of irregular ice crystals was observed in an acoustic levitator. The evolution of the particle shape is captured using a high-speed video system. Several typical phenomena have been discovered: change of the particle shape, appearance of a capillary flow of the melted liquid on the particle surface leading to liquid collection at the particle midsection (where the interface curvature is smallest), and appearance of sharp cusps at the particle tips. No such phenomena can be observed during melting of spherical particles. An approximate theoretical model is developed which accounts for the main physical phenomena associated with melting of an irregular particle. The agreement between the theoretical predictions for the melting time, for the evolution of the particle shape, and the corresponding experimental data is rather good.

  17. Melting Can Hinder Impact-Induced Adhesion (United States)

    Hassani-Gangaraj, Mostafa; Veysset, David; Nelson, Keith A.; Schuh, Christopher A.


    Melting has long been used to join metallic materials, from welding to selective laser melting in additive manufacturing. In the same school of thought, localized melting has been generally perceived as an advantage, if not the main mechanism, for the adhesion of metallic microparticles to substrates during a supersonic impact. Here, we conduct the first in situ supersonic impact observations of individual metallic microparticles aimed at the explicit study of melting effects. Counterintuitively, we find that under at least some conditions melting is disadvantageous and hinders impact-induced adhesion. In the parameter space explored, i.e., ˜10 μ m particle size and ˜1 km /s particle velocity, we argue that the solidification time is much longer than the residence time of the particle on the substrate, so that resolidification cannot be a significant factor in adhesion.

  18. Volatile diffusion in silicate melts and its effects on melt inclusions

    Directory of Open Access Journals (Sweden)

    P. Scarlato


    Full Text Available A compendium of diffusion measurements and their Arrhenius equations for water, carbon dioxide, sulfur, fluorine, and chlorine in silicate melts similar in composition to natural igneous rocks is presented. Water diffusion in silicic melts is well studied and understood, however little data exists for melts of intermediate to basic compositions. The data demonstrate that both the water concentration and the anhydrous melt composition affect the diffusion coefficient of water. Carbon dioxide diffusion appears only weakly dependent, at most, on the volatilefree melt composition and no effect of carbon dioxide concentration has been observed, although few experiments have been performed. Based upon one study, the addition of water to rhyolitic melts increases carbon dioxide diffusion by orders of magnitude to values similar to that of 6 wt% water. Sulfur diffusion in intermediate to silicic melts depends upon the anhydrous melt composition and the water concentration. In water-bearing silicic melts sulfur diffuses 2 to 3 orders of magnitude slower than water. Chlorine diffusion is affected by both water concentration and anhydrous melt composition; its values are typically between those of water and sulfur. Information on fluorine diffusion is rare, but the volatile-free melt composition exerts a strong control on its diffusion. At the present time the diffusion of water, carbon dioxide, sulfur and chlorine can be estimated in silicic melts at magmatic temperatures. The diffusion of water and carbon dioxide in basic to intermediate melts is only known at a limited set of temperatures and compositions. The diffusion data for rhyolitic melts at 800°C together with a standard model for the enrichment of incompatible elements in front of growing crystals demonstrate that rapid crystal growth, greater than 10-10 ms-1, can significantly increase the volatile concentrations at the crystal-melt interface and that any of that melt trapped

  19. Melt Rate Improvement for DWPF MB3: Melt Rate Furnace Testing

    Energy Technology Data Exchange (ETDEWEB)

    Stone, M.E.


    The Defense Waste Processing Facility (DWPF) would like to increase its canister production rate. The goal of this study is to improve the melt rate in DWPF specifically for Macrobatch 3. However, the knowledge gained may result in improved melting efficiencies translating to future DWPF macrobatches and in higher throughput for other Department of Energy's (DOE) melters. Increased melting efficiencies decrease overall operational costs by reducing the immobilization campaign time for a particular waste stream. For melt rate limited systems, a small increase in melting efficiency translates into significant hard dollar savings by reducing life cycle operational costs.

  20. Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay. (United States)

    Wu, Qingqing; Xiang, Shengnan; Wang, Wenjun; Zhao, Jinyan; Xia, Jinhua; Zhen, Yueran; Liu, Bang


    Various detection methods have been developed to date for identification of animal species. New techniques based on PCR approach have raised the hope of developing better identification methods, which can overcome the limitations of the existing methods. PCR-based methods used the mitochondrial DNA (mtDNA) as well as nuclear DNA sequences. In this study, by targeting nuclear DNA, multiplex PCR and real-time PCR methods were developed to assist with qualitative and quantitative analysis. The multiplex PCR was found to simultaneously and effectively distinguish four species (fox, dog, mink, and rabbit) ingredients by the different sizes of electrophoretic bands: 480, 317, 220, and 209 bp. Real-time fluorescent PCR's amplification profiles and standard curves showed good quantitative measurement responses and linearity, as indicated by good repeatability and coefficient of determination R 2  > 0.99. The quantitative results of quaternary DNA mixtures including mink, fox, dog, and rabbit DNA are in line with our expectations: R.D. (relative deviation) varied between 1.98 and 12.23% and R.S.D. (relative standard deviation) varied between 3.06 and 11.51%, both of which are well within the acceptance criterion of ≤ 25%. Combining the two methods is suitable for the rapid identification and accurate quantification of fox-, dog-, mink-, and rabbit-derived ingredients in the animal products.

  1. Terrestrial impact melt rocks and glasses (United States)

    Dressler, B. O.; Reimold, W. U.


    The effects of meteorite and comet impact on Earth are rock brecciation, the formation of shock metamorphic features, rock melting, and the formation of impact structures, i.e. simple craters, complex craters, and multi-ring basins. Large events, such as the 65-Ma Chicxulub impact, are believed to have had catastrophic environmental effects that profoundly influenced the development of life on Earth. In this review, an attempt is made to summarize some of the voluminous literature on impact melting, one important aspect of planetary impact, provide some comments on this process, and to make suggestions for future research. The products of impact melting are glasses, impact melt rocks, and pseudotachylites. Our treatise deals mainly with the geological setting, petrography, and major-element chemistry of melt rocks and glasses. Impact glasses, in several petrographic aspects, are similar to volcanic glasses, but they are associated with shock metamorphosed mineral and rock fragments and, in places, with siderophile element anomalies suggestive of meteoritic contamination. They are found in allogenic breccia deposits within (fall-back 'suevite') and outside (fall-out 'suevite') impact craters and, as spherules, in distal ejecta. Large events, such as the K/T boundary Chicxulub impact, are responsible for the formation of worldwide ejecta horizons which are associated with siderophile element anomalies and shock metamorphosed mineral and rock debris. Impact glasses have a bulk chemical composition that is homogeneous but exemptions to this rule are common. On a microscopic scale, however, impact glasses are commonly strikingly heterogeneous. Tektites are glasses ejected from craters over large distances. They are characterized by very low water and volatile contents and element abundances and ratios that are evidence that tektites formed by melting of upper crustal, sedimentary rocks. Four tektite strewn-fields are known, three of which can be tied to specific impact

  2. Viscosity of ring polymer melts

    KAUST Repository

    Pasquino, Rossana


    We have measured the linear rheology of critically purified ring polyisoprenes, polystyrenes, and polyethyleneoxides of different molar masses. The ratio of the zero-shear viscosities of linear polymer melts η0,linear to their ring counterparts η0,ring at isofrictional conditions is discussed as a function of the number of entanglements Z. In the unentangled regime η0,linear/η 0,ring is virtually constant, consistent with the earlier data, atomistic simulations, and the theoretical expectation η0,linear/ η0,ring = 2. In the entanglement regime, the Z-dependence of ring viscosity is much weaker than that of linear polymers, in qualitative agreement with predictions from scaling theory and simulations. The power-law extracted from the available experimental data in the rather limited range 1 < Z < 20, η0,linear/η0,ring ∼ Z 1.2±0.3, is weaker than the scaling prediction (η0,linear/η0,ring ∼ Z 1.6±0.3) and the simulations (η0,linear/ η0,ring ∼ Z2.0±0.3). Nevertheless, the present collection of state-of-the-art experimental data unambiguously demonstrates that rings exhibit a universal trend clearly departing from that of their linear counterparts, and hence it represents a major step toward resolving a 30-year-old problem. © 2013 American Chemical Society.

  3. Dynamics of Melting and Melt Migration as Inferred from Incompatible Trace Element Abundance in Abyssal Peridotites (United States)

    Peng, Q.; Liang, Y.


    To better understand the melting processes beneath the mid-ocean ridge, we developed a simple model for trace element fractionation during concurrent melting and melt migration in an upwelling steady-state mantle column. Based on petrologic considerations, we divided the upwelling mantle into two regions: a double- lithology upper region where high permeability dunite channels are embedded in a lherzolite/harzburgite matrix, and a single-lithology lower region that consists of partially molten lherzolite. Melt generated in the single lithology region migrates upward through grain-scale diffuse porous flow, whereas melt in the lherzolite/harzburgite matrix in the double-lithology region is allowed to flow both vertically through the overlying matrix and horizontally into its neighboring dunite channels. There are three key dynamic parameters in our model: degree of melting experienced by the single lithology column (Fd), degree of melting experienced by the double lithology column (F), and a dimensionless melt suction rate (R) that measures the accumulated rate of melt extraction from the matrix to the channel relative to the accumulated rate of matrix melting. In terms of trace element fractionation, upwelling and melting in the single lithology column is equivalent to non-modal batch melting (R = 0), whereas melting and melt migration in the double lithology region is equivalent to a nonlinear combination of non-modal batch and fractional melting (0 abyssal peridotite, we showed, with the help of Monte Carlo simulations, that it is difficult to invert for all three dynamic parameters from a set of incompatible trace element data with confidence. However, given Fd, it is quite possible to constrain F and R from incompatible trace element abundances in residual peridotite. As an illustrative example, we used the simple melting model developed in this study and selected REE and Y abundance in diopside from abyssal peridotites to infer their melting and melt migration

  4. Methylation-Specific PCR Unraveled

    Directory of Open Access Journals (Sweden)

    Sarah Derks


    Full Text Available Methylation‐specific PCR (MSP is a simple, quick and cost‐effective method to analyze the DNA methylation status of virtually any group of CpG sites within a CpG island. The technique comprises two parts: (1 sodium bisulfite conversion of unmethylated cytosine's to uracil under conditions whereby methylated cytosines remains unchanged and (2 detection of the bisulfite induced sequence differences by PCR using specific primer sets for both unmethylated and methylated DNA. This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical applications.

  5. Molecular typing for blood group antigens within 40 minutes by direct PCR from plasma or serum (United States)

    Wagner, Franz Friedrich; Flegel, Willy Albert; Bittner, Rita; Döscher, Andrea


    Determining blood group antigens by serological methods may be unreliable in certain situations, such as in patients after chronic or massive transfusion. Red cell genotyping offers a complementary approach, but current methods may take much longer than conventional serological typing, limiting their utility in urgent situations. To narrow this gap, we devised a rapid method using direct polymerase chain reaction (PCR) amplification while avoiding the DNA extraction step. DNA was amplified by PCR directly from plasma or serum of blood donors followed by a melting curve analysis in a capillary rapid-cycle PCR assay. We evaluated the single nucleotide polymorphisms underlying the clinically relevant Fya, Fyb, Jka and Jkb antigens, with our analysis being completed within 40 min of receiving a plasma or serum sample. The positive predictive value was 100% and the negative predictive value at least 84%. Direct PCR with melting point analysis allowed faster red cell genotyping to predict blood group antigens than any previous molecular method. Our assay may be used as a screening tool with subsequent confirmatory testing, within the limitations of the false-negative rate. With fast turnaround times, the rapid-cycle PCR assay may eventually be developed and applied to red cell genotyping in the hospital setting. PMID:27991657

  6. Locked nucleic acid inhibits amplification of contaminating DNA in real-time PCR

    DEFF Research Database (Denmark)

    Hummelshoj, Lone; Ryder, Lars P; Madsen, Hans O


    in intronic DNA, the aim was to inhibit the amplification of genomic DNA without affecting the amplification of reverse-transcribed spliced mRNA. LNA was designed to bind within intron 5 in the x-box binding protein 1 (XBP1) gene. An irrelevant LNA oligonucleotide served as a negative control. In both PCR...... and real-time PCR, the addition of LNA showed blocking of the amplification of genomic XBP1 but not cDNA XBP1. To test the effect of melting temperature (Tm) on the LNA, we investigated the number of LNA nucleotides that could be replaced with DNA nucleotides and still retain the blocking activity. More...

  7. Melt processed high-temperature superconductors

    CERN Document Server


    The achievement of large critical currents is critical to the applications of high-temperature superconductors. Recent developments have shown that melt processing is suitable for producing high J c oxide superconductors. Using magnetic forces between such high J c oxide superconductors and magnets, a person could be levitated.This book has grown largely out of research works on melt processing of high-temperature superconductors conducted at ISTEC Superconductivity Research Laboratory. The chapters build on melt processing, microstructural characterization, fundamentals of flux pinning, criti

  8. Bayesian estimation of core-melt probability

    International Nuclear Information System (INIS)

    Lewis, H.W.


    A very simple application of the canonical Bayesian algorithm is made to the problem of estimation of the probability of core melt in a commercial power reactor. An approximation to the results of the Rasmussen study on reactor safety is used as the prior distribution, and the observation that there has been no core melt yet is used as the single experiment. The result is a substantial decrease in the mean probability of core melt--factors of 2 to 4 for reasonable choices of parameters. The purpose is to illustrate the procedure, not to argue for the decrease

  9. Corium melt researches at VESTA test facility


    Hwan Yeol Kim; Sang Mo An; Jaehoon Jung; Kwang Soon Ha; Jin Ho Song


    VESTA (Verification of Ex-vessel corium STAbilization) and VESTA-S (-small) test facilities were constructed at the Korea Atomic Energy Research Institute in 2010 to perform various corium melt experiments. Since then, several tests have been performed for the verification of an ex-vessel core catcher design for the EU-APR1400. Ablation tests of an impinging ZrO2 melt jet on a sacrificial material were performed to investigate the ablation characteristics. ZrO2 melt in an amount of 65–70 kg w...

  10. Corium melt researches at VESTA test facility

    Directory of Open Access Journals (Sweden)

    Hwan Yeol Kim


    Full Text Available VESTA (Verification of Ex-vessel corium STAbilization and VESTA-S (-small test facilities were constructed at the Korea Atomic Energy Research Institute in 2010 to perform various corium melt experiments. Since then, several tests have been performed for the verification of an ex-vessel core catcher design for the EU-APR1400. Ablation tests of an impinging ZrO2 melt jet on a sacrificial material were performed to investigate the ablation characteristics. ZrO2 melt in an amount of 65–70 kg was discharged onto a sacrificial material through a well-designed nozzle, after which the ablation depths were measured. Interaction tests between the metallic melt and sacrificial material were performed to investigate the interaction kinetics of the sacrificial material. Two types of melt were used: one is a metallic corium melt with Fe 46%, U 31%, Zr 16%, and Cr 7% (maximum possible content of U and Zr for C-40, and the other is a stainless steel (SUS304 melt. Metallic melt in an amount of 1.5–2.0 kg was delivered onto the sacrificial material, and the ablation depths were measured. Penetration tube failure tests were performed for an APR1400 equipped with 61 in-core instrumentation penetration nozzles and extended tubes at the reactor lower vessel. ZrO2 melt was generated in a melting crucible and delivered down into an interaction crucible where the test specimen is installed. To evaluate the tube ejection mechanism, temperature distributions of the reactor bottom head and in-core instrumentation penetration were measured by a series of thermocouples embedded along the specimen. In addition, lower vessel failure tests for the Fukushima Daiichi nuclear power plant are being performed. As a first step, the configuration of the molten core in the plant was investigated by a melting and solidification experiment. Approximately 5 kg of a mixture, whose composition in terms of weight is UO2 60%, Zr 10%, ZrO2 15%, SUS304 14%, and B4C 1%, was melted in a

  11. Real-Time PCR Identification of Six Malassezia Species. (United States)

    Ilahi, Amin; Hadrich, Inès; Neji, Sourour; Trabelsi, Houaida; Makni, Fattouma; Ayadi, Ali


    Lipophilic yeast Malassezia species is widely found on the skin surface of humans and other animals. This fungus can cause pityriasis versicolor, Malassezia folliculitis, and seborrheic dermatitis. Still now, there is a problem with species identification of Malassezia with conventional methods. We developed a real-time polymerase chain reaction (PCR) assay with multiple hybridization probes for detecting M. globosa, M. furfur, M. restricta, M. sympodialis, M. slooffiae, and M. pachydermatis. The amplification curves and specific melting peaks of the probes hybridized with real-time PCR product were used for species identifications. The assay was further evaluated on 120 samples which were performed by swabbing from 60 domestic animals (23 goats, 10 dogs, 15 cows, 3 cats, 8 rabbits, and 1 donkey) and in 70 human samples (28 patients with pityriasis versicolor, 17 breeders, and 25 control group). Fifteen M. pachydermatis were identified from animals. From human, 61 isolates were identified as M. globosa (28), M. furfur (15), M. restricta (6), M. sympodialis (8), M. slooffiae (2), and M. pachydermatis (2). Eight cases of co-detection from 6 patients and 2 breeders were revealed. Our findings show that the assay was highly effective in identifying Malassezia species. The application of multiplex real-time PCR provides a sensitive and rapid identification system for Malassezia species, which may be applied in further epidemiological surveys from clinical samples.

  12. Decompression Melting beneath the Indonesian Volcanic Front (United States)

    Kelley, K. A.; Colabella, A.; Sisson, T. W.; Hauri, E. H.; Sigurdsson, H.


    Subduction zone magmas are typically characterized by high concentrations of dissolved H2O (up to 6-7 wt%), presumably derived from the subducted plate and ultimately responsible for melt generation in this tectonic setting. Pressure-release melting from upward mantle flow, however, is increasingly cited as a secondary driver of mantle wedge melting. Here we report new SIMS volatile and LA-ICP-MS trace element data for olivine-hosted melt inclusions from Galunggung (GG) and Tambora (TB) volcanoes in the Indonesian subduction zone to evaluate the relative importance of decompression vs. H2O-flux melting beneath arc volcanoes. Prior studies of melt inclusions from Galunggung showed unusually low primary H2O concentrations (~0.5 wt%), implicating decompression as a significant mechanism of mantle melting beneath this volcano (Sisson &Bronto, 1998). Our new data from a larger suite of Galunggung melt inclusions show a bimodal distribution of H2O concentrations: a dominant population with ~0.5 wt% H2O, and a small group with 1.5-2.5 wt% H2O, indicating that a small amount of H2O addition from the slab may also contribute to mantle melting here. New volatile data from Tambora melt inclusions also indicate low primary H2O contents (1-2 wt%), suggesting that decompression melting may be a large-scale characteristic of the Indonesian volcanic front. Our new trace element data show both volcanoes are LREE enriched relative to MORB, but Tambora melts show greater LREE enrichment (La/Sm=1.7-2.7[GG]; 6.0- 9.5[TB]). Galunggung melts have Nb/Y in the range of NMORB (0.1-0.2), whereas Tambora Nb/Y is similar to EMORB (0.3-0.5). Most Tambora melt inclusions also have H2O/Y (Y (200-1000) and H2O/Ce (100-1400) relative to NMORB, suggesting a larger influence from slab-derived H2O despite having lower average H2O concentrations than Tambora. The range of H2O/Y and H2O/Ce at Galunggung, however, is largely within the range of back-arc basin basalts and does not preclude a major

  13. Production, pathways and budgets of melts in mid-ocean ridges: An enthalpy based thermo-mechanical model (United States)

    Mandal, Nibir; Sarkar, Shamik; Baruah, Amiya; Dutta, Urmi


    Using an enthalpy based thermo-mechanical model we provide a theoretical evaluation of melt production beneath mid-ocean ridges (MORs), and demonstrate how the melts subsequently develop their pathways to sustain the major ridge processes. Our model employs a Darcy idealization of the two-phase (solid-melt) system, accounting enthalpy (ΔH) as a function of temperature dependent liquid fraction (ϕ). Random thermal perturbations imposed in this model set in local convection that drive melts to flow through porosity controlled pathways with a typical mushroom-like 3D structure. We present across- and along-MOR axis model profiles to show the mode of occurrence of melt-rich zones within mushy regions, connected to deeper sources by single or multiple feeders. The upwelling of melts experiences two synchronous processes: 1) solidification-accretion, and 2) eruption, retaining a large melt fraction in the framework of mantle dynamics. Using a bifurcation analysis we determine the threshold condition for melt eruption, and estimate the potential volumes of eruptible melts (∼3.7 × 106 m3/yr) and sub-crustal solidified masses (∼1-8.8 × 106 m3/yr) on an axis length of 500 km. The solidification process far dominates over the eruption process in the initial phase, but declines rapidly on a time scale (t) of 1 Myr. Consequently, the eruption rate takes over the solidification rate, but attains nearly a steady value as t > 1.5 Myr. We finally present a melt budget, where a maximum of ∼5% of the total upwelling melt volume is available for eruption, whereas ∼19% for deeper level solidification; the rest continue to participate in the sub-crustal processes.

  14. Effect of the packing structure of silicon chunks on the melting process and carbon reduction in Czochralski silicon crystal growth (United States)

    Liu, Xin; Nakano, Satoshi; Kakimoto, Koichi


    Carbon (C) contamination in Czochralski silicon (CZ-Si) crystal growth mainly originates from carbon monoxide (CO) generation on the graphite components, which reaches a maximum during the melting stage. Loading a crucible with poly-Si feedstock includes many technical details for optimization of the melting and growth processes. To investigate the effect of the packing structure of Si chunks on C accumulation in CZ-Si crystal growth, transient global simulations of heat and mass transport were performed for the melting process with different packing structures of poly-Si. The heat transport modeling took into account the effective thermal conductivity (ETC) of the Si feedstock, which is affected by the packing structure. The effect of the chunk size on the melting process and C accumulation were investigated by parametric studies of different packing structures. The heat transport and melting process in the crucible were affected by the ETC and the emissivity of the Si feedstock. It was found that smaller Si chunks packed in the upper part could speed up the melting process and smooth the power profile. Decreasing the duration of the melting process is favorable for reduction of C contamination in the Si feedstock. Parametric studies indicated that optimization of the melting process by the packing structure is possible and essential for C reduction in CZ-Si crystal growth.

  15. PCR

    African Journals Online (AJOL)



    Oct 2, 2006 ... RP-N (15941482) ..... Cloning of the nucleocapsid protein gene of peste des petits ruminants virus: relationship to other Morbillivuruses. J. General Virol. 75:233-237. Diallo A, Barrett T, Barbron M, Shaila MS, Taylor WP ...

  16. pcr

    African Journals Online (AJOL)


    These include DNA cloning for sequencing, DNA based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic finger prints (used in ... Strand separation (denaturing) of the double stranded sample .... study with 30 clinical specimen 18AH and 12VF from. 20 eyes with the ...

  17. Interaction between sulphide and H 2O in silicate melts (United States)

    Stelling, Jan; Behrens, Harald; Wilke, Max; Göttlicher, Jörg; Chalmin-Aljanabi, Emilie


    Reaction between dissolved water and sulphide was experimentally investigated in soda-lime-silicate (NCS) and sodium trisilicate (NS3) melts at temperatures from 1000 to 1200 °C and pressures of 100 or 200 MPa in internally heated gas pressure vessels. Diffusion couple experiments were conducted at water-undersaturated conditions with one half of the couple being doped with sulphide (added as FeS or Na 2S; 1500-2000 ppm S by weight) and the other with H 2O (˜3.0 wt.%). Additionally, two experiments were performed using a dry NCS glass cylinder and a free H 2O fluid. Here, the melt was water-saturated at least at the melt/fluid interface. Profiling by electron microprobe (sulphur) and infrared microscopy (H 2O) demonstrate that H 2O diffusion in the melts is faster by 1.5-2.3 orders of magnitude than sulphur diffusion and, hence, H 2O can be considered as a rapidly diffusing oxidant while sulphur is quasi immobile in these experiments. In Raman spectra a band at 2576 cm -1 appears in the sulphide - H 2O transition zone which is attributed to fundamental S-H stretching vibrations. Formation of new IR absorption bands at 5025 cm -1 (on expense of the combination band of molecular H 2O at 5225 cm -1) and at 3400 cm -1 was observed at the front of the in-diffusing water in the sulphide bearing melt. The appearance and intensity of these two IR bands is correlated with systematic changes in S K-edge XANES spectra. A pre-edge excitation at 2466.5 eV grows with increasing H 2O concentration while the sulphide peak at 2474.0 eV decreases in intensity relative to the peak at 2477.0 eV and the feature at 2472.3 eV becomes more pronounced (all energies are relative to the sulphate excitation, calibrated to 2482.5 eV). The observations by Raman, IR and XANES spectroscopy indicate a well coordinated S 2- - H 2O complex which was probably formed in the glasses during cooling at the glass transition. No oxidation of sulphide was observed in any of the diffusion couple

  18. Molecular profile of 5-fluorouracil pathway genes in colorectal carcinoma

    International Nuclear Information System (INIS)

    Kunicka, T.; Prochazka, P.; Krus, I.; Bendova, P.; Protivova, M.; Susova, S.; Hlavac, V.; Liska, V.; Novak, P.; Schneiderova, M.; Pitule, P.; Bruha, J.; Vycital, O.; Vodicka, P.; Soucek, P.


    This study addresses involvement of major 5-fluorouracil (5-FU) pathway genes in the prognosis of colorectal carcinoma patients. Testing set and two validation sets comprising paired tumor and adjacent mucosa tissue samples from 151 patients were used for transcript profiling of 15 5-FU pathway genes by quantitative real-time PCR and DNA methylation profiling by high resolution melting analysis. Intratumoral molecular profiles were correlated with clinical data of patients. Protein levels of two most relevant candidate markers were assessed by immunoblotting. Downregulation of DPYD and upregulation of PPAT, UMPS, RRM2, and SLC29A1 transcripts were found in tumors compared to adjacent mucosa in testing and validation sets of patients. Low RRM2 transcript level significantly associated with poor response to the first-line palliative 5-FU-based chemotherapy in the testing set and with poor disease-free interval of patients in the validation set irrespective of 5-FU treatment. UPP2 was strongly methylated while its transcript absent in both tumors and adjacent mucosa. DPYS methylation level was significantly higher in tumor tissues compared to adjacent mucosa samples. Low intratumoral level of UPB1 methylation was prognostic for poor disease-free interval of the patients (P = 0.0002). The rest of the studied 5-FU genes were not methylated in tumors or adjacent mucosa. The observed overexpression of several 5-FU activating genes and DPYD downregulation deduce that chemotherapy naïve colorectal tumors share favorable gene expression profile for 5-FU therapy. Low RRM2 transcript and UPB1 methylation levels present separate poor prognosis factors for colorectal carcinoma patients and should be further investigated. The online version of this article (doi:10.1186/s12885-016-2826-8) contains supplementary material, which is available to authorized users

  19. Humboldt Glacier, Greenland sub-marine melt rates derived from CTD/current casts (United States)

    Lauchman, E.; Box, J. E.; Howat, I. M.; Hubbard, A.; Bates, R.


    A larger de-stabilization of ice sheets is threatened from oceanic than atmospheric climate change. Yet, little is known about the magnitude of sub-marine melt rates. A chain of oceanographic profiles were cast in front of the Humboldt Glacier terminus, northwest Greenland summer 2009 with the Greenpeace ship Arctic Sunrise. A heat and mass budget closure scheme is applied to derive effective sub-marine ice melt rates. The results are discussed in context of seasonal climate and recent oceanic and atmospheric climate change.

  20. Investigation of Melting Dynamics of Hafnium Clusters. (United States)

    Ng, Wei Chun; Lim, Thong Leng; Yoon, Tiem Leong


    Melting dynamics of hafnium clusters are investigated using a novel approach based on the idea of the chemical similarity index. Ground state configurations of small hafnium clusters are first derived using Basin-Hopping and Genetic Algorithm in the parallel tempering mode, employing the COMB potential in the energy calculator. These assumed ground state structures are verified by using the Low Lying Structures (LLS) method. The melting process is carried out either by using the direct heating method or prolonged simulated annealing. The melting point is identified by a caloric curve. However, it is found that the global similarity index is much more superior in locating premelting and total melting points of hafnium clusters.

  1. Electron beam melting of bearing materials

    Energy Technology Data Exchange (ETDEWEB)

    Goldschmied, G.; Schuler, A. (Technische Univ., Vienna (Austria). Inst. fuer Allgemeine Elektrotechnik); Elsinger, G.; Koroschetz, F. (MIBA Gleitlager AG, Laakirchen (Austria)); Tschegg, E.K. (Technische Univ., Vienna (Austria). Inst. fuer Angewandte und Technische Physik)


    This paper reports on a surface treatment method for the bearing materials AlSn6 which permits the use of this material without the overlay usually required. Microstructural refinement is achieved by means of a surface melting technique using an electron beam with successive rapid solidification. Extremely fine tin precipitates are formed in the melted surface layer which lead to significantly better tribological properties of the bearing material. Tests compared the tribological properties for AlSn6 bearings treated by the surface melting technique with those of untreated bearings. Whereas all untreated bearings failed by seizure after only 2 h of testing, 30% of the tested bearings which had been surface melted survived the entire testing program without damage.

  2. ESR melting under constant voltage conditions

    Energy Technology Data Exchange (ETDEWEB)

    Schlienger, M.E.


    Typical industrial ESR melting practice includes operation at a constant current. This constant current operation is achieved through the use of a power supply whose output provides this constant current characteristic. Analysis of this melting mode indicates that the ESR process under conditions of constant current is inherently unstable. Analysis also indicates that ESR melting under the condition of a constant applied voltage yields a process which is inherently stable. This paper reviews the process stability arguments for both constant current and constant voltage operation. Explanations are given as to why there is a difference between the two modes of operation. Finally, constant voltage process considerations such as melt rate control, response to electrode anomalies and impact on solidification will be discussed.

  3. Selective Laser Ablation and Melting, Phase I (United States)

    National Aeronautics and Space Administration — In this project Advratech will develop a new additive manufacturing (AM) process called Selective Laser Ablation and Melting (SLAM). The key innovation in this...

  4. Are Entangled Polymer Melts Different From Solutions?

    DEFF Research Database (Denmark)

    Huang, Qian; Mednova, Olga; Rasmussen, Henrik K.

    to further investigate the extensional steady state viscosity of polymer melts, we carefully synthesized two monodisperse polystyrenes with molar masses of 248 and 484 kg/mole. The start-up and steady uniaxial elongational viscosity have been measured for the two melts using a filament stretching rheometer....... We then compared the measurements with the bi-disperse polystyrene melts made from the above two polymers. The influence and sensitivity of impurities were studied by adding different percentages of 484k into 248k polystyrene melt. Furthermore a polydisperse polystyrene with weight average molecular...... weight 230 kg/mole was also measured for comparison. Possible reasons for the differences shown in the previously mentioned experiments are discussed....

  5. Melt Stirring by Horizontal Crucible Vibration (United States)

    Wolf, M. F.; Elwell, D.; Feigelson, R. S.


    Horizontal vibration suggested as technique for more effective stirring of melts in crystal-growth apparatus. Vibrational technique may replace accelerated crucible rotation. Potential superiority of vibrational technique shown by preliminary experiments in which ink stirred into water.

  6. Energy Saving Melting and Revert Reduction Technology (E-SMARRT): Melting Efficiency Improvement

    Energy Technology Data Exchange (ETDEWEB)

    Principal Investigator Kent Peaslee; Co-PI’s: Von Richards, Jeffrey Smith


    Steel foundries melt recycled scrap in electric furnaces and typically consume 35-100% excess energy from the theoretical energy requirement required to pour metal castings. This excess melting energy is multiplied by yield losses during casting and finishing operations resulting in the embodied energy in a cast product typically being three to six times the theoretical energy requirement. The purpose of this research project was to study steel foundry melting operations to understand energy use and requirements for casting operations, define variations in energy consumption, determine technologies and practices that are successful in reducing melting energy and develop new melting techniques and tools to improve the energy efficiency of melting in steel foundry operations.

  7. DWPF Macrobatch 2 Melt Rate Tests

    Energy Technology Data Exchange (ETDEWEB)

    Stone, M.E.


    The Defense Waste Processing Facility (DWPF) canister production rate must be increased to meet canister production goals. Although a number of factors exist that could potentially increase melt rate, this study focused on two: (1) changes in frit composition and (2) changes to the feed preparation process to alter the redox of the melter feed. These two factors were investigated for Macrobatch 2 (sludge batch 1B) utilizing crucible studies and a specially designed ''melt rate'' furnace. Other potential factors that could increase melt rate include: mechanical mixing via stirring or the use of bubblers, changing the power skewing to redistribute the power input to the melter, and elimination of heat loss (e.g. air in leakage). The melt rate testing in FY00 demonstrated that melt rate can be improved by adding a different frit or producing a much more reducing glass by the addition of sugar as a reductant. The frit that melted the fastest in the melt rate testing was Frit 165. A paper stud y was performed using the Product Composition Control System (PCCS) to determine the impact on predicted glass viscosity, liquidus, durability, and operating window if the frit was changed from Frit 200 to Frit 165. PCCS indicated that the window was very similar for both frits. In addition, the predicted viscosity of the frit 165 glass was 46 poise versus 84 poise for the Frit 200 glass. As a result, a change from Frit 200 to Frit 165 is expected to increase the melt rate in DWPF without decreasing waste loading.

  8. Grain boundary disordering just before partial melting (United States)

    Takei, Y.


    Recent experimental studies by using a rock analogue (organic polycrystals) have shown that significant enhancement of anelastic relaxation and steady-state creep in the partially molten aggregates starts from considerably below the solidus temperature in the absence of melt (Takei et al, 2014; Yamauchi & Takei, 2016, JGR). These results suggest that melt is not necessary to explain the seismic low velocity, high attenuation, and weak viscosity regions in the upper mantle. Indeed, Priestley & McKenzie (2006, 2013, EPSL) captured a steep reduction of seismic Vs just below the dry peridotite solidus, which was explained well by the empirical model of Yamauchi & Takei (2016). In spite of many geophysical implications (Takei, 2017, Ann. Rev. EPS, in press), however, underlying physics for the mechanical weakening just before partial melting remains unclear. The purpose of this study is to develop a physical model. Anelasticity and viscosity measured by Yamauchi & Takei (2016) are both rate-controlled by grain-boundary diffusion. Therefore, their observations suggest that the dynamic properties of grain boundary change just before partial melting. Significant disordering of grain boundary just before partial melting has been predicted theoretically in the area of material sciences (sometimes called `pre-melting'). I will summarize the thermodynamic models of grain boundary developed in these studies, and compare the predictions of these models to the experimental observations by Yamauchi & Takei (2016). Using these models, I will also clarify a relationship between grain-boundary disordering and grain-boundary wetting, and a different behavior between pure and binary systems in pre-melting. Acknowledgement: I thank R. Cooper for letting me know about the theoretical studies of pre-melting in binary eutectic system.

  9. Uniaxial Elongational viscosity of bidisperse polystyrene melts

    DEFF Research Database (Denmark)

    Nielsen, Jens Kromann; Rasmussen, Henrik K.; Hassager, Ole


    The startup and steady uniaxial elongational viscosity have been measured for three bidisperse polystyrene (PS) melts, consisting of blends of monodisperse PS with molecular weights of 52 kg/mole or 103 kg/mole and 390 kg/mole. The bidisperse melts have a maximum in the steady elongational...... viscosity, of up to a factor of 7 times the Trouton limit of 3 times the zero-shear viscosity....

  10. Electrodepositions on Tantalum in Alkali Halide Melts

    DEFF Research Database (Denmark)

    Barner, Jens H. Von; Jensen, Annemette Hindhede; Christensen, Erik


    Surface layers of tantalum metal were electrodeposited on steel from K2TaF7-LiF-NaF-KF melts. With careful control of the oxide contents dense and adherent deposits could be obtained by pulse plating. In NaCl-KCl-NaF-Na2CO3 and NaCl-KCl-Na2CO3 melts carbonate ions seems to be reduced to carbon in...

  11. Electrodepositions on Tantalum in alkali halide melts

    DEFF Research Database (Denmark)

    Barner, Jens H. Von; Jensen, Annemette Hindhede; Christensen, Erik


    Surface layers of tantalum metal were electrodeposited on steel from K 2TaF7-LiF-NaF-KF melts. With careful control of the oxide contents dense and adherent deposits could be obtained by pulse plating. In NaCl-KCl-NaF-Na2CO3 and NaCl-KCl-Na2CO 3 melts carbonate ions seems to be reduced to carbon ...

  12. Design and optimization of reverse-transcription quantitative PCR experiments. (United States)

    Tichopad, Ales; Kitchen, Rob; Riedmaier, Irmgard; Becker, Christiane; Ståhlberg, Anders; Kubista, Mikael


    Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

  13. Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis (United States)

    Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco


    Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains. PMID:10508059

  14. Behavior of metals in ash melting and gasification-melting of municipal solid waste (MSW). (United States)

    Jung, C H; Matsuto, T; Tanaka, N


    In this study, metal behavior in ash-melting and municipal solid waste (MSW) gasification-melting facilities were investigated. Eight ash-melting and three MSW gasification-melting facilities with a variety of melting processes and feedstocks were selected. From each facility, melting furnace fly ash (MFA) and molten slag were sampled, and feedstock of the ash-melting processes was also taken. For the ash melting process, the generation rate of MFA was well correlated with the ratio of incineration fly ash (IFA) in feedstock, and this was because MFA was formed mostly by mass transfer from IFA and a limited amount from bottom ash (BA). Distribution ratios of metal elements to MFA were generally determined by volatility of the metal element, but chlorine content in feedstock had a significant effect on Cu and a marginal effect on Pb. Distribution ratio of Zn to MFA was influenced by the oxidizing atmosphere in the furnace. High MFA generation and distribution ratio of non-volatile metals to MFA in gasification-melting facilities was probably caused by carry-over of fine particles to the air pollution control system due to large gas volume. Finally, dilution effect was shown to have a significant effect on metal concentration in MFA.

  15. Rapakivi texture formation via disequilibrium melting in a contact partial melt zone, Antarctica (United States)

    Currier, R. M.


    In the McMurdo Dry Valleys of Antarctica, a Jurassic aged dolerite sill induced partial melting of granite in the shallow crust. The melt zone can be traced in full, from high degrees of melting (>60%) along the dolerite contact, to no apparent signs of melting, 10s of meters above the contact. Within this melt zone, the well-known rapakivi texture is found, arrested in various stages of development. High above the contact, and at low degrees of melting, K-feldspar crystals are slightly rounded and unmantled. In the lower half of the melt zone, mantles of cellular textured plagioclase appear on K-feldspar, and thicken towards the contact heat source. At the highest degrees of melting, cellular-textured plagioclase completely replaces restitic K-feldspar. Because of the complete exposure and intact context, the leading models of rapakivi texture formation can be tested against this system. The previously proposed mechanisms of subisothermal decompression, magma-mixing, and hydrothermal exsolution all fail to adequately describe rapakivi generation in this melt zone. Preferred here is a closed system model that invokes the production of a heterogeneous, disequilibrium melt through rapid heating, followed by calcium and sodium rich melt reacting in a peritectic fashion with restitic K-feldspar crystals. This peritectic reaction results in the production of plagioclase of andesine-oligoclase composition—which is consistent with not just mantles in the melt zone, but globally as well. The thickness of the mantle is diffusion limited, and thus a measure of the diffusive length scale of sodium and calcium over the time scale of melting. Thermal modeling provides a time scale of melting that is consistent with the thickness of observed mantles. Lastly, the distribution of mantled feldspars is highly ordered in this melt zone, but if it were mobilized and homogenized—mixing together cellular plagioclase, mantled feldspars, and unmantled feldspars—the result would be

  16. Development and validation of two SYBR green PCR assays and a multiplex real-time PCR for the detection of Shiga toxin-producing Escherichia coli in meat. (United States)

    Brusa, Victoria; Galli, Lucía; Linares, Luciano H; Ortega, Emanuel E; Lirón, Juan P; Leotta, Gerardo A


    Shiga toxin-producing Escherichia coli (STEC) are recognized as food-borne pathogens. We developed and validated two SYBR green PCR (SYBR-PCR) and a real-time multiplex PCR (RT-PCR) to detect stx1 and stx2 genes in meat samples, and compared these techniques in ground beef samples from retail stores. One set of primers and one hydrolysis probe were designed for each stx gene. For RT-PCR, an internal amplification control (IAC) was used. All PCR intra-laboratory validations were performed using pure strains and artificially contaminated ground beef samples. A total of 50 STEC and 30 non-STEC strains were used. Naturally contaminated ground beef samples (n=103) were obtained from retail stores and screened with SYBR-PCR and RT-PCR, and stx-positive samples were processed for STEC isolation. In the intra-laboratory validation, each PCR obtained a 1×10(2) CFU mL(-1) limit of detection and 100% inclusivity and exclusivity. The same results were obtained when different laboratory analysts in alternate days performed the assay. The level of agreement obtained with SYBR-PCR and RT-PCR was kappa=0.758 and 0.801 (P<0.001) for stx1 and stx2 gene detection, respectively. Two PCR strategies were developed and validated, and excellent performance with artificially contaminated ground beef samples was obtained. However, the efforts made to isolate STEC from retail store samples were not enough. Only 11 STEC strains were isolated from 35 stx-positive ground beef samples identically detected by all PCRs. The combination of molecular approaches based on the identification of a virulence genotypic profile of STEC must be considered to improve isolation. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Assessment of high resolution melting analysis as a potential SNP genotyping technique in forensic casework. (United States)

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


    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.

  18. Simultaneous detection and differentiation of Entamoeba histolytica, E. dispar, E. moshkovskii, Giardia lamblia and Cryptosporidium spp. in human fecal samples using multiplex PCR and qPCR-MCA. (United States)

    Zebardast, Nozhat; Yeganeh, Farshid; Gharavi, Mohammad Javad; Abadi, Alireza; Seyyed Tabaei, Seyyed Javad; Haghighi, Ali


    Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. are common causes of diarrheal and intestinal diseases all over the world. Microscopic methods are useful in the diagnosis of intestinal parasites (IPs), but their sensitivity was assessed approximately 60 percent. Recently, molecular techniques have been used increasingly for the identification and characterization of the parasites. Among those, in this study we have used multiplex PCR and Real-time PCR with melting curve analysis (qPCR-MCA) for simultaneous detection and differentiation of E. histolytica, E. dispar, E. moshkovskii, G. lamblia and Cryptosporidium spp. in human fecal samples. Twenty DNA samples from 12 E. histolytica and 8 E. dispar samples and twenty stool samples confirmed positive for G. lamblia and Cryptosporidium spp. were analyzed. After DNA extraction from the samples, multiplex PCR was done for detection and differentiation of above mentioned parasites. QPCR-MCA was also performed for the detection and differentiation of 11 isolates of above mentioned parasite in a cycle with a time and temperature. Multiplex PCR was able to simultaneous detect and differentiate of above mentioned parasite in a single reaction. QPCR-MCA was able to differentiate genus and species those five protozoa using melting temperature simultaneously at the same time and temperature programs. In total, qPCR-MCA diagnosed 7/11 isolation of E. histolytica, 6/8 isolation of E. dispar, 1/1 E. moshkovskii Laredo, 10/11 G. Lamblia and 6/11 Cryptosporidium spp. Application of multiplex PCR for detection of more than one species in a test in developing countries, at least in reference laboratories has accurate diagnosis and plays a critical role in differentiation of protozoan species. Multiplex PCR assay with a template and multi template had different results and it seems that using a set of primers with one template has higher diagnostic capability in compare with multi template. The results of this study

  19. Introduction on Using the FastPCR Software and the Related Java Web Tools for PCR and Oligonucleotide Assembly and Analysis. (United States)

    Kalendar, Ruslan; Tselykh, Timofey V; Khassenov, Bekbolat; Ramanculov, Erlan M


    This chapter introduces the FastPCR software as an integrated tool environment for PCR primer and probe design, which predicts properties of oligonucleotides based on experimental studies of the PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations. These include the standard PCR as well as the multiplex, long-distance, inverse, real-time, group-specific, unique, overlap extension PCR for multi-fragments assembling cloning and loop-mediated isothermal amplification (LAMP). It also contains a built-in program to design oligonucleotide sets both for long sequence assembly by ligase chain reaction and for design of amplicons that tile across a region(s) of interest. The software calculates the melting temperature for the standard and degenerate oligonucleotides including locked nucleic acid (LNA) and other modifications. It also provides analyses for a set of primers with the prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, linguistic complexity as well as a primer dilution and resuspension calculator. The program consists of various bioinformatical tools for analysis of sequences with the GC or AT skew, CG% and GA% content, and the purine-pyrimidine skew. It also analyzes the linguistic sequence complexity and performs generation of random DNA sequence as well as restriction endonucleases analysis. The program allows to find or create restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It performs efficient and complete detection of various repeat types with visual display. The FastPCR software allows the sequence file batch processing that is essential for automation. The program is available for download at , and its online version is located at .

  20. Internal stress-induced melting below melting temperature at high-rate laser heating

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Yong Seok, E-mail: [Department of Aerospace Engineering, Iowa State University, Ames, Iowa 50011 (United States); Levitas, Valery I., E-mail: [Departments of Aerospace Engineering, Mechanical Engineering, and Material Science and Engineering, Iowa State University, Ames, Iowa 50011 (United States)


    In this Letter, continuum thermodynamic and phase field approaches (PFAs) predicted internal stress-induced reduction in melting temperature for laser-irradiated heating of a nanolayer. Internal stresses appear due to thermal strain under constrained conditions and completely relax during melting, producing an additional thermodynamic driving force for melting. Thermodynamic melting temperature for Al reduces from 933.67 K for a stress-free condition down to 898.1 K for uniaxial strain and to 920.8 K for plane strain. Our PFA simulations demonstrated barrierless surface-induced melt nucleation below these temperatures and propagation of two solid-melt interfaces toward each other at the temperatures very close to the corresponding predicted thermodynamic equilibrium temperatures for the heating rate Q≤1.51×10{sup 10}K/s. At higher heating rates, kinetic superheating competes with a reduction in melting temperature and melting under uniaxial strain occurs at 902.1 K for Q = 1.51 × 10{sup 11 }K/s and 936.9 K for Q = 1.46 × 10{sup 12 }K/s.

  1. Energy Saving Melting and Revert Reduction Technology: Melting Efficiency in Die Casting Operations

    Energy Technology Data Exchange (ETDEWEB)

    David Schwam


    This project addressed multiple aspects of the aluminum melting and handling in die casting operations, with the objective of increasing the energy efficiency while improving the quality of the molten metal. The efficiency of melting has always played an important role in the profitability of aluminum die casting operations. Consequently, die casters need to make careful choices in selecting and operating melting equipment and procedures. The capital cost of new melting equipment with higher efficiency can sometimes be recovered relatively fast when it replaces old melting equipment with lower efficiency. Upgrades designed to improve energy efficiency of existing equipment may be well justified. Energy efficiency is however not the only factor in optimizing melting operations. Melt losses and metal quality are also very important. Selection of melting equipment has to take into consideration the specific conditions at the die casting shop such as availability of floor space, average quantity of metal used as well as the ability to supply more metal during peaks in demand. In all these cases, it is essential to make informed decisions based on the best available data.

  2. Effect of melting conditions on striae in iron-bearing silicate melts

    DEFF Research Database (Denmark)

    Jensen, Martin; Yue, Yuanzheng


    of melt temperature and/or a decrease of viscosity play a more important role in decreasing the stria content. We also demonstrate that the extent of striation is influenced by the crucible materials that causes a change of redox state of the melt, and hence its viscosity. We discuss the effect of other...

  3. Olivine/melt transition metal partitioning, melt composition, and melt structure—Melt polymerization and Qn-speciation in alkaline earth silicate systems (United States)

    Mysen, Bjorn O.


    The two most abundant network-modifying cations in magmatic liquids are Ca 2+ and Mg 2+. To evaluate the influence of melt structure on exchange of Ca 2+ and Mg 2+ with other geochemically important divalent cations ( m-cations) between coexisting minerals and melts, high-temperature (1470-1650 °C), ambient-pressure (0.1 MPa) forsterite/melt partitioning experiments were carried out in the system Mg 2SiO 4-CaMgSi 2O 6-SiO 2 with ⩽1 wt% m-cations (Mn 2+, Co 2+, and Ni 2+) substituting for Ca 2+ and Mg 2+. The bulk melt NBO/Si-range ( NBO/Si: nonbridging oxygen per silicon) of melt in equilibrium with forsterite was between 1.89 and 2.74. In this NBO/Si-range, the NBO/Si(Ca) (fraction of nonbridging oxygens, NBO, that form bonds with Ca 2+, Ca 2+- NBO) is linearly related to NBO/Si, whereas fraction of Mg 2+- NBO bonds is essentially independent of NBO/Si. For individual m-cations, rate of change of KD( m-Mg) with NBO/Si(Ca) for the exchange equilibrium, mmelt + Mg olivine ⇌ molivine + Mg melt, is linear. KD( m-Mg) decreases as an exponential function of increasing ionic potential, Z/ r2 ( Z: formal electrical charge, r: ionic radius—here calculated with oxygen in sixfold coordination around the divalent cations) of the m-cation. The enthalpy change of the exchange equilibrium, Δ H, decreases linearly with increasing Z/ r2 [Δ H = 261(9)-81(3)· Z/ r2 (Å -2)]. From existing information on (Ca,Mg)O-SiO 2 melt structure at ambient pressure, these relationships are understood by considering the exchange of divalent cations that form bonds with nonbridging oxygen in individual Qn-species in the melts. The negative ∂ KD( m-Mg) /∂( Z/ r2) and ∂(Δ H)/∂( Z/ r2) is because increasing Z/ r2 is because the cations forming bonds with nonbridging oxygen in increasingly depolymerized Qn-species where steric hindrance is decreasingly important. In other words, principles of ionic size/site mismatch commonly observed for trace and minor elements in crystals, also

  4. Bloomington (LL6) chondrite and its shock melt glasses

    International Nuclear Information System (INIS)

    Dodd, R.T.; Olsen, E.J.; Clarke, R.S. Jr.; National Museum of Natural History, Washington, DC; Field Museum of Natural History, Chicago, IL)


    The shock melt glasses of the Bloomington LL-group chondrite were examined using electron-beam microscopy and compared with data from studies of other shock melt glasses. Petrologic and mineralogic characterizations were also performed of the samples. The metal contents of the meteorite were almost wholly Ni-rich martensite. The glasses resembled shock melt glasses in L-group chondrites, and were indicative of isochemical melting during one melt phase, i.e., a very simple history. 12 references

  5. Melting of the Earth's inner core. (United States)

    Gubbins, David; Sreenivasan, Binod; Mound, Jon; Rost, Sebastian


    The Earth's magnetic field is generated by a dynamo in the liquid iron core, which convects in response to cooling of the overlying rocky mantle. The core freezes from the innermost surface outward, growing the solid inner core and releasing light elements that drive compositional convection. Mantle convection extracts heat from the core at a rate that has enormous lateral variations. Here we use geodynamo simulations to show that these variations are transferred to the inner-core boundary and can be large enough to cause heat to flow into the inner core. If this were to occur in the Earth, it would cause localized melting. Melting releases heavy liquid that could form the variable-composition layer suggested by an anomaly in seismic velocity in the 150 kilometres immediately above the inner-core boundary. This provides a very simple explanation of the existence of this layer, which otherwise requires additional assumptions such as locking of the inner core to the mantle, translation from its geopotential centre or convection with temperature equal to the solidus but with composition varying from the outer to the inner core. The predominantly narrow downwellings associated with freezing and broad upwellings associated with melting mean that the area of melting could be quite large despite the average dominance of freezing necessary to keep the dynamo going. Localized melting and freezing also provides a strong mechanism for creating seismic anomalies in the inner core itself, much stronger than the effects of variations in heat flow so far considered.

  6. Melt electrospinning of biodegradable polyurethane scaffolds. (United States)

    Karchin, Ari; Simonovsky, Felix I; Ratner, Buddy D; Sanders, Joan E


    Electrospinning from a melt, in contrast to from a solution, is an attractive tissue engineering scaffold manufacturing process as it allows for the formation of small diameter fibers while eliminating potentially cytotoxic solvents. Despite this, there is a dearth of literature on scaffold formation via melt electrospinning. This is likely due to the technical challenges related to the need for a well-controlled high-temperature setup and the difficulty in developing an appropriate polymer. In this paper, a biodegradable and thermally stable polyurethane (PU) is described specifically for use in melt electrospinning. Polymer formulations of aliphatic PUs based on (CH(2))(4)-content diisocyanates, polycaprolactone (PCL), 1,4-butanediamine and 1,4-butanediol (BD) were evaluated for utility in the melt electrospinning process. The final polymer formulation, a catalyst-purified PU based on 1,4-butane diisocyanate, PCL and BD in a 4/1/3M ratio with a weight-average molecular weight of about 40kDa, yielded a nontoxic polymer that could be readily electrospun from the melt. Scaffolds electrospun from this polymer contained point bonds between fibers and mechanical properties analogous to many in vivo soft tissues. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  7. Improved quantification accuracy for duplex real-time PCR detection of genetically modified soybean and maize in heat processed foods

    Directory of Open Access Journals (Sweden)

    CHENG Fang


    Full Text Available Real-time PCR technique has been widely used in quantitative GMO detection in recent years.The accuracy of GMOs quantification based on the real-time PCR methods is still a difficult problem,especially for the quantification of high processed samples.To develop the suitable and accurate real-time PCR system for high processed GM samples,we made ameliorations to several real-time PCR parameters,including re-designed shorter target DNA fragment,similar lengths of amplified endogenous and exogenous gene targets,similar GC contents and melting temperatures of PCR primers and TaqMan probes.Also,one Heat-Treatment Processing Model (HTPM was established using soybean flour samples containing GM soybean GTS 40-3-2 to validate the effectiveness of the improved real-time PCR system.Tested results showed that the quantitative bias of GM content in heat processed samples were lowered using the new PCR system.The improved duplex real-time PCR was further validated using processed foods derived from GM soybean,and more accurate GM content values in these foods was also achieved.These results demonstrated that the improved duplex real-time PCR would be quite suitable in quantitative detection of high processed food products.

  8. Two-temperature PCR for Microfluidics

    KAUST Repository

    Kodzius, Rimantas


    Since its invention in 1983, polymerase chain reaction (PCR) has been the method of choice for DNA amplification. Successful PCR depends on the optimization of several parameters, which is a cumbersome task due to the many variables (conditions and compon

  9. 7-Deaza-2'-deoxyguanosine allows PCR and sequencing reactions from CpG islands. (United States)

    Jung, A; Ruckert, S; Frank, P; Brabletz, T; Kirchner, T


    CpG islands are GC rich sequences that are found in the promoters of many genes in higher eukaryotes. They contain a high frequency of CG dinucleotides, which are substrates for DNA methylases. Methylation leads to transcriptional silencing of promoters. Owing to their high GC content CpG islands exhibit strong base-base interactions, which lead to superstructures and consequently to regions with higher melting temperatures. Therefore, Taq polymerases (especially sequenases) fall off their templates, causing premature termination of the polymerase chain reaction (PCR) or sequencing reactions. The results from such reactions are thus insufficient for further analysis. Therefore, we have evaluated the use of 7-deaza-2'-deoxyguanosine for PCR amplification of the human p16(INK4A) promoter and sequencing of HUMARA exon 1 PCR products. Our results show that the addition of 7-deaza-2'-deoxyguanosine significantly improves results, particularly when small amounts of poor quality DNA are available as starting material.

  10. 7-Deaza-2`-deoxyguanosine allows PCR and sequencing reactions from CpG islands (United States)

    Jung, A; Ruckert, S; Frank, P; Brabletz, T; Kirchner, T


    CpG islands are GC rich sequences that are found in the promoters of many genes in higher eukaryotes. They contain a high frequency of CG dinucleotides, which are substrates for DNA methylases. Methylation leads to transcriptional silencing of promoters. Owing to their high GC content CpG islands exhibit strong base–base interactions, which lead to superstructures and consequently to regions with higher melting temperatures. Therefore, Taq polymerases (especially sequenases) fall off their templates, causing premature termination of the polymerase chain reaction (PCR) or sequencing reactions. The results from such reactions are thus insufficient for further analysis. Therefore, we have evaluated the use of 7-deaza-2`-deoxyguanosine for PCR amplification of the human p16INK4A promoter and sequencing of HUMARA exon 1 PCR products. Our results show that the addition of 7-deaza-2`-deoxyguanosine significantly improves results, particularly when small amounts of poor quality DNA are available as starting material. PMID:11836448

  11. Determination of enthalpy–temperature–composition relations in incongruent-melting phase change materials

    International Nuclear Information System (INIS)

    Desgrosseilliers, Louis; Allred, Paul; Groulx, Dominic; White, Mary Anne


    This paper demonstrates that liquidus line (T-x) data can be obtained from calorimetric determinations of phase transition enthalpy profiles (H-T) for incongruent-melting phase change materials (PCMs) more efficiently than using traditional cooling curves. An accurate and reliable equilibrium mixture enthalpy model bridges the H-T and T-x gap to provide a full suite of high density H-T-x data to assist latent heat energy storage researchers to evaluate composition-dependent two-phase equilibrium processes. The proposed method is validated for T-history method H-T determinations of 1:1 diluted sodium acetate trihydrate in water, and can also be used with other laboratory calorimetric techniques used to determine the phase transition enthalpy profiles of incongruent-melting compounds. -- Highlights: • H-T data can also be used to obtain valuable liquidus region T-x data. • Applies to all incongruent-melting compounds with known thermodynamic properties. • Reduces the effort and cost of assessing full suite H-T-x data for PCMs. • Uses existing T-x or H-T data of incongruent-melting PCMs to determine the other

  12. Pathogen Causing Disease of Diagnosis PCR Tecnology


    SEVİNDİK, Emre; KIR, A. Çağrı; BAŞKEMER, Kadir; UZUN, Veysel


    Polimerase chain reaction (PCR) with which, the development of recombinant DNA tecnology, a technique commonly used in field of moleculer biology and genetic. Duplication of the target DNA is provided with this technique without the need for cloning. Some fungus species, bacteria, viruses constitutent an important group of pathogenicity in human, animals and plants. There are routinely applied types of PCR in the detection of pathogens infections diseases. These Nested- PCR, Real- Time PCR, M...

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

    DEFF Research Database (Denmark)

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


    -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......In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High...... 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...

  14. Entangled Polymer Melts in Extensional Flow

    DEFF Research Database (Denmark)

    Hengeller, Ludovica

    . On the other hand, addition of low-volatility solvents to polymers is also a common industrial practice that others a means for lowering the Tg of the polymers. Moreover industrial polymers present a wide distribution of chain lengths and/or branched architectures that strongly influence their response....... Understanding the behaviour of polymer melts and solutions in complex non-linearflows is crucial for the design of polymeric materials and polymer processes. Through rheological characterization, in shear and extensional flow, of model polymer systems,i.e. narrow molar mass distribution polymer melts......Many commercial materials derived from synthetic polymers exhibit a complex response under different processing operations such as fiber formation, injection moulding,film blowing, film casting or coatings. They can be processed both in the solid or in the melted state. Often they may contain two...

  15. Melting of peridotite to 140 gigapascals. (United States)

    Fiquet, G; Auzende, A L; Siebert, J; Corgne, A; Bureau, H; Ozawa, H; Garbarino, G


    Interrogating physical processes that occur within the lowermost mantle is a key to understanding Earth's evolution and present-day inner composition. Among such processes, partial melting has been proposed to explain mantle regions with ultralow seismic velocities near the core-mantle boundary, but experimental validation at the appropriate temperature and pressure regimes remains challenging. Using laser-heated diamond anvil cells, we constructed the solidus curve of a natural fertile peridotite between 36 and 140 gigapascals. Melting at core-mantle boundary pressures occurs at 4180 ± 150 kelvin, which is a value that matches estimated mantle geotherms. Molten regions may therefore exist at the base of the present-day mantle. Melting phase relations and element partitioning data also show that these liquids could host many incompatible elements at the base of the mantle.

  16. Analysis of Picosecond Pulsed Laser Melted Graphite (United States)

    Steinbeck, J.; Braunstein, G.; Speck, J.; Dresselhaus, M. S.; Huang, C. Y.; Malvezzi, A. M.; Bloembergen, N.


    A Raman microprobe and high resolution TEM have been used to analyze the resolidified region of liquid carbon generated by picosecond pulse laser radiation. From the relative intensities of the zone center Raman-allowed mode for graphite at 1582 cm{sup -1} and the disorder-induced mode at 1360 cm{sup -1}, the average graphite crystallite size in the resolidified region is determined as a function of position. By comparison with Rutherford backscattering spectra and Raman spectra from nanosecond pulsed laser melting experiments, the disorder depth for picosecond pulsed laser melted graphite is determined as a function of irradiating energy density. Comparisons of TEM micrographs for nanosecond and picosecond pulsed laser melting experiments show that the structure of the laser disordered regions in graphite are similar and exhibit similar behavior with increasing laser pulse fluence.

  17. Analysis of picosecond pulsed laser melted graphite

    Energy Technology Data Exchange (ETDEWEB)

    Steinbeck, J.; Braunstein, G.; Speck, J.; Dresselhaus, M.S.; Huang, C.Y.; Malvezzi, A.M.; Bloembergen, N.


    A Raman microprobe and high resolution TEM have been used to analyze the resolidified region of liquid carbon generated by picosecond pulse laser radiation. From the relative intensities of the zone center Raman-allowed mode for graphite at 1582 cm/sup -1/ and the disorder-induced mode at 1360 cm/sup -1/, the average graphite crystallite size in the resolidified region is determined as a function of position. By comparison with Rutherford backscattering spectra and Raman spectra from nonosecond pulsed laser melting experiments, the disorder depth for picosecond pulsed laser melted graphite is determined as a function of irradiating energy density. Comparisons of TEM micrographs for nanosecond and picosecond pulsed laser melting experiments show that the structure of the laser disordered regions in graphite are similar and exhibit similar behavior with increasing laser pulse fluence.

  18. Scrap uranium recycling via electron beam melting

    International Nuclear Information System (INIS)

    McKoon, R.


    A program is underway at the Lawrence Livermore National Laboratory (LLNL) to recycle scrap uranium metal. Currently, much of the material from forging and machining processes is considered radioactive waste and is disposed of by oxidation and encapsulation at significant cost. In the recycling process, uranium and uranium alloys in various forms will be processed by electron beam melting and continuously cast into ingots meeting applicable specifications for virgin material. Existing vacuum processing facilities at LLNL are in compliance with all current federal and state environmental, safety and health regulations for the electron beam melting and vaporization of uranium metal. One of these facilities has been retrofitted with an auxiliary electron beam gun system, water-cooled hearth, crucible and ingot puller to create an electron beam melt furnace. In this furnace, basic process R ampersand D on uranium recycling will be performed with the goal of eventual transfer of this technology to a production facility

  19. Scleral melt following Retisert intravitreal fluocinolone implant

    Directory of Open Access Journals (Sweden)

    Georgalas I


    Full Text Available Ilias Georgalas,1 Chrysanthi Koutsandrea,1 Dimitrios Papaconstantinou,1 Dimitrios Mpouritis,1 Petros Petrou1,2 1Ophthalmology Department, University of Athens, Athens, Greece; 2Moorfields Eye Hospital, London, UKAbstract: Intravitreal fluocinolone acetonide implant (Retisert has a high potency, a low solubility, and a very short duration of action in the systemic circulation, enabling the steroid pellet to be small and reducing the risk of systemic side effects. Scleral melt has not been reported as a possible complication of Retisert implant. The authors describe the occurrence of scleral melt 18 months after the implantation of fluocinolone acetonide implant in a 42-year-old Caucasian woman. To the authors’ knowledge, this is the first report of this possible complication.Keywords: Retisert, scleral melt, complication, surgical management

  20. Analysis of picosecond pulsed laser melted graphite

    International Nuclear Information System (INIS)

    Steinbeck, J.; Braunstein, G.; Speck, J.; Dresselhaus, M.S.; Huang, C.Y.; Malvezzi, A.M.; Bloembergen, N.


    A Raman microprobe and high resolution TEM have been used to analyze the resolidified region of liquid carbon generated by picosecond pulse laser radiation. From the relative intensities of the zone center Raman-allowed mode for graphite at 1582 cm -1 and the disorder-induced mode at 1360 cm -1 , the average graphite crystallite size in the resolidified region is determined as a function of position. By comparison with Rutherford backscattering spectra and Raman spectra from nonosecond pulsed laser melting experiments, the disorder depth for picosecond pulsed laser melted graphite is determined as a function of irradiating energy density. Comparisons of TEM micrographs for nanosecond and picosecond pulsed laser melting experiments show that the structure of the laser disordered regions in graphite are similar and exhibit similar behavior with increasing laser pulse fluence

  1. Selective Laser Melting of Pure Copper (United States)

    Ikeshoji, Toshi-Taka; Nakamura, Kazuya; Yonehara, Makiko; Imai, Ken; Kyogoku, Hideki


    Appropriate building parameters for selective laser melting of 99.9% pure copper powder were investigated at relatively high laser power of 800 W for hatch pitch in the range from 0.025 mm to 0.12 mm. The highest relative density of the built material was 99.6%, obtained at hatch pitch of 0.10 mm. Building conditions were also studied using transient heat analysis in finite element modeling of the liquidation and solidification of the powder layer. The estimated melt pool length and width were comparable to values obtained by observations using a thermoviewer. The trend for the melt pool width versus the hatch pitch agreed with experimental values.

  2. Melt spun aluminium alloys for moulding optics (United States)

    Gubbels, G.; Tegelaers, L.; Senden, R.


    Melt spinning is a rapid quenching process that makes it possible to create materials with a very fine microstructure. Due to this very fine microstructure the melt spinning process is an enabler for diamond turning optics and moulds without the need of post-polishing. Using diamond turning of melt spun aluminium one can achieve coating concept RSA-905 can be upgraded to a competitive alternative to steel in terms of price, performance and logistics. This paper presents some recent developments for improved mould performance of such concept. Hardness, wear resistance and adhesion are topics of interest and they can be applied by special coatings such as diamond-like carbon (DLC) and chromium nitride (CrN). These coatings make the aluminium alloy suitable for moulding mass production of small as well as larger optics, such as spectacle lenses.

  3. Rapid Screening of Topoisomerase Gene Mutations by a Novel Melting Curve Analysis Method for Early Warning of Fluoroquinolone-Resistant Streptococcus pneumoniae Emergence▿


    Fukushima, Kazuko Y.; Hirakata, Yoichi; Sugahara, Kazuyuki; Yanagihara, Katsunori; Kondo, Akira; Kohno, Shigeru; Kamihira, Shimeru


    We developed a real-time PCR assay combined with melting curve analysis for rapidly genotyping quinolone resistance-determining regions (QRDR) of topoisomerase genes in Streptococcus pneumoniae. This assay was not only accurate for the screening of fluoroquinolone (FQ) resistance but also relevant as an early warning system for detecting preexisting single QRDR mutations.

  4. Principles and technical aspects of PCR amplification

    National Research Council Canada - National Science Library

    Pelt-Verkuil, Elizabeth van; Belkum, Alex van; Hays, John P


    ... to illustrate any particularly important concepts or comments. Indeed, all commercial PCR biotechnology companies offer information about their products on internet sites and in online technical manuals. These online resources will be invaluable for any readers requiring more detailed PCR protocols. The authors have provided references for many PCR co...

  5. The PCR revolution: basic technologies and applications

    National Research Council Canada - National Science Library

    Bustin, Stephen A


    ... by leading authorities on the many applications of PCR and how this technology has revolutionized their respective areas of interest. This book conveys the ways in which PCR has overcome many obstacles in life science and clinical research and also charts the PCR's development from time-consuming, low throughput, nonquantitative proced...

  6. Identification of the GST-T1 and GST-M1 null genotypes using high resolution melting analysis. (United States)

    Drobná, Zuzana; Del Razo, Luz Maria; Garcia-Vargas, Gonzalo; Sánchez-Ramírez, Blanca; González-Horta, Carmen; Ballinas-Casarrubias, Lourdes; Loomis, Dana; Stýblo, Miroslav


    Glutathione S-transferases, including GST-T1 and GST-M1, are known to be involved in the phase II detoxification pathways for xenobiotics as well as in the metabolism of endogenous compounds. Polymorphisms in these genes have been linked to an increased susceptibility to carcinogenesis and associated with risk factors that predispose to certain inflammatory diseases. In addition, GST-T1 and GST-M1 null genotypes have been shown to be responsible for interindividual variations in the metabolism of arsenic, a known human carcinogen. To assess the specific GST genotypes in the Mexican population chronically exposed to arsenic, we have developed a multiplex High Resolution Melting PCR (HRM-PCR) analysis using a LightCycler480 instrument. This method is based on analysis of the PCR product melting curve that discriminates PCR products according to their lengths and base sequences. Three pairs of primers that specifically recognize GST-T1, GST-M1, and β-globin, an internal control, to produce amplicons of different length were designed and combined with LightCycler480 High Resolution Melting Master Mix containing ResoLight, a completely saturating DNA dye. Data collected from melting curve analysis were evaluated using LightCycler480 software to determine specific melting temperatures of individual melting curves representing target genes. Using this newly developed multiplex HRM-PCR analysis, we evaluated GST-T1 and GST-M1 genotypes in 504 DNA samples isolated from the blood of individuals residing in Zimapan, Lagunera, and Chihuahua regions in Mexico. We found that the Zimapan and Lagunera populations have similar GST-T1 and GST-M1 genotype frequencies which differ from those of the Chihuahua population. In addition, 14 individuals have been identified as carriers of the double null genotype, i.e., null genotypes in both GST-T1 and GST-M1 genes. Although this procedure does not distinguish between biallelic (+/+) and monoallelic (+/-) genotypes, it can be used in an

  7. The rock melting approach to drilling

    Energy Technology Data Exchange (ETDEWEB)

    Cort, G.E.; Goff, S.J.; Rowley, J.C.; Neudecker, J.W. Jr.; Dreesen, D.S.; Winchester, W.


    During the early and mid-1970`s the Los Alamos National Laboratory demonstrated practical applications of drilling and coring using an electrically-heated graphite, tungsten, or molybdenum penetrator that melts a hole as it is slowly pushed through the rock or soil. The molten material consolidates into a rugged glass lining that prevents hole collapse; minimizes the potential for cross-flow, lost circulation, or the release of hazardous materials without casing operations; and produces no cuttings in porous or low density (<1.7 g/cc) formations. Because there are no drilling fluids required, the rock melting approach reduces waste handling, treatment and disposal. Drilling by rock melting has been demonstrated to depths up to 30 m in caliche, clay, alluvium, cobbles, sand, basalt, granite, and other materials. Penetrating large cobbles without debris removal was achieved by thermal stress fracturing and lateral extrusion of portions of the rock melt into the resulting cracks. Both horizontal and vertical holes in a variety of diameters were drilled in these materials using modular, self-contained field units that operate in remote areas. Because the penetrator does not need to rotate, steering by several simple approaches is considered quite feasible. Melting is ideal for obtaining core samples in alluvium and other poorly consolidated soils since the formed-in-place glass liner stabilizes the hole, encapsulates volatile or hazardous material, and recovers an undisturbed core. Because of the relatively low thermal conductivity of rock and soil materials, the heat-affected zone beyond the melt layer is very small, <1 inch thick. Los Alamos has begun to update the technology and this paper will report on the current status of applications and designs for improved drills.

  8. Structure of polyvalent metal halide melts

    International Nuclear Information System (INIS)

    Tosi, M.P.


    A short review is given of recent progress in determining and understanding the structure of molten halide salts involving polyvalent metal ions. It covers the following three main topics: (i) melting mechanisms and types of liquid structure for pure polyvalent-metal chlorides; (ii) geometry and stability of local coordination for polyvalent metal ions in molten mixtures of their halides with alkali halides; and (iii) structure breaking and electron localization on addition of metal to the melt. (author). 28 refs, 3 figs, 1 tab

  9. 3He melting pressure temperature scale

    DEFF Research Database (Denmark)

    Halperin, W.P.; Archie, C.N.; Richardson, R.C.


    The latent heat for solidification of **3He has been measured along the **3He melting curve between 23 and 1 mK. A temperature scale is established which depends only on measurements of heat, pressure and volume, and on the condition that the entropy of solid **3He approaches R ln 2 at high...... temperatures. The A feature of the melting curve which suggests itself as a thermometric fixed point is found to be T//A equals 2. 75 plus or minus 0. 11 mK. The agreement between this value and independent measurements of T//A, based on nuclear or electronic paramagnetism, Johnson noise thermometry...

  10. Chemical reactions in solvents and melts

    CERN Document Server

    Charlot, G


    Chemical Reactions in Solvents and Melts discusses the use of organic and inorganic compounds as well as of melts as solvents. This book examines the applications in organic and inorganic chemistry as well as in electrochemistry. Organized into two parts encompassing 15 chapters, this book begins with an overview of the general properties and the different types of reactions, including acid-base reactions, complex formation reactions, and oxidation-reduction reactions. This text then describes the properties of inert and active solvents. Other chapters consider the proton transfer reactions in

  11. A one-step real-time multiplex PCR for screening Y-chromosomal microdeletions without downstream amplicon size analysis.

    Directory of Open Access Journals (Sweden)

    Viviana Kozina

    Full Text Available BACKGROUND: Y-chromosomal microdeletions (YCMD are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. PRINCIPAL FINDINGS: The widely established "EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004" were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. SIGNIFICANCE: With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection.

  12. Real-time LightCycler polymerase chain reaction and melting temperature analysis for identification of clinically important Candida spp. (United States)

    Khan, Ziauddin; Mustafa, Abu Salim; Alam, Fasahat Fakhar


    Invasive candidiasis is a major fungal infection occurring in patients who have prolonged hospital admissions. The rapid detection and confirmation of Candida spp. in clinical specimens is essential for efficient management and improved prognosis of these patients. The purpose of this study was to develop a real-time LightCycler polymerase chain reaction (PCR) assay for the identification of Candida spp. commonly associated with invasive infections. Using the LightCycler PCR System, the targets of genomic DNA isolated from the reference strains of 6 Candida spp. were amplified using genus- and species-specific primers, and detected in real-time employing SYBR Green fluorescent dye. The identity of Candida spp. was established by melting curve analysis. A similar analysis was performed with clinical isolates (n = 72) previously identified by conventional methods. The melting curve analysis of amplified DNA from the reference strains could differentiate between Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Candida krusei, and Candida dubliniensis. The specificity of the real-time PCR assay was validated by testing 72 clinical isolates of Candida spp. with 100% concordance, as compared with conventional identification methods. The notable findings of the study were differentiation of C. krusei from all other Candida spp. tested and of C. dubliniensis from C. albicans by melting temperature analysis; the latter 2 species share common phenotypic characteristics of germ-tube formation and chlamydospore production, so are often misidentified. Real-time PCR using LightCycler and melting curve analysis are reliable methods for rapid identification of 6 Candida spp. frequently associated with candidemia and invasive candidiasis.

  13. Real-time PCR in Food Science: PCR Diagnostics. (United States)

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta


    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

  14. The Microwave Properties of Simulated Melting Precipitation Particles: Sensitivity to Initial Melting (United States)

    Johnson, B. T.; Olson, W. S.; Skofronick-Jackson, G.


    A simplified approach is presented for assessing the microwave response to the initial melting of realistically shaped ice particles. This paper is divided into two parts: (1) a description of the Single Particle Melting Model (SPMM), a heuristic melting simulation for ice-phase precipitation particles of any shape or size (SPMM is applied to two simulated aggregate snow particles, simulating melting up to 0.15 melt fraction by mass), and (2) the computation of the single-particle microwave scattering and extinction properties of these hydrometeors, using the discrete dipole approximation (via DDSCAT), at the following selected frequencies: 13.4, 35.6, and 94.0GHz for radar applications and 89, 165.0, and 183.31GHz for radiometer applications. These selected frequencies are consistent with current microwave remote-sensing platforms, such as CloudSat and the Global Precipitation Measurement (GPM) mission. Comparisons with calculations using variable-density spheres indicate significant deviations in scattering and extinction properties throughout the initial range of melting (liquid volume fractions less than 0.15). Integration of the single-particle properties over an exponential particle size distribution provides additional insight into idealized radar reflectivity and passive microwave brightness temperature sensitivity to variations in size/mass, shape, melt fraction, and particle orientation.

  15. [Progress in digital PCR technology and application]. (United States)

    Lin, Jiaqi; Su, Guocheng; Su, Wenjin; Zhou, Changyi


    Digital PCR is an emerging analysis technology for absolute quantification after realtime-PCR. Through digital PCR, single DNA molecules are distributed into isolated reactions, and the product with fluorescence signal can be detected and analyzed after amplification. With the advantages of higher sensitivity and accuracy, digital PCR, independent of a standard curve, is developing rapidly and applied widely to the next generation sequencing and detection fields, such as gene mutation, copy number variation, microorganism, and genetically modified food. In this article, we reviewed the quantitative method and research progress of digital PCR technology in the main application fields.

  16. Quasi-equilibrium melting of quartzite upon extreme friction (United States)

    Lee, Sung Keun; Han, Raehee; Kim, Eun Jeong; Jeong, Gi Young; Khim, Hoon; Hirose, Takehiro


    The friction on fault planes that controls how rocks slide during earthquakes decreases significantly as a result of complex fault-lubrication processes involving frictional melting. Fault friction has been characterized in terms of the preferential melting of minerals with low melting points--so-called disequilibrium melting. Quartz, which has a high melting temperature of about 1,726 °C and is a major component of crustal rocks, is not expected to melt often during seismic slip. Here we use high-velocity friction experiments on quartzite to show that quartz can melt at temperatures of 1,350 to 1,500 °C. This implies that quartz within a fault plane undergoing rapid friction sliding could melt at substantially lower temperatures than expected. We suggest that depression of the melting temperature is caused by the preferential melting of ultra-fine particles and metastable melting of β-quartz at about 1,400 °C during extreme frictional slip. The results for quartzite are applicable to complex rocks because of the observed prevalence of dynamic grain fragmentation, the preferential melting of smaller grains and the kinetic preference of β-quartz formation during frictional sliding. We postulate that frictional melting of quartz on a fault plane at temperatures substantially below the melting temperature could facilitate slip-weakening and lead to large earthquakes.

  17. Recent Changes in Arctic Sea Ice Melt Onset, Freeze-Up, and Melt Season Length (United States)

    Markus, Thorsten; Stroeve, Julienne C.; Miller, Jeffrey


    In order to explore changes and trends in the timing of Arctic sea ice melt onset and freeze-up and therefore melt season length, we developed a method that obtains this information directly from satellite passive microwave data, creating a consistent data set from 1979 through present. We furthermore distinguish between early melt (the first day of the year when melt is detected) and the first day of continuous melt. A similar distinction is made for the freeze-up. Using this method we analyze trends in melt onset and freeze-up for 10 different Arctic regions. In all regions except for the Sea of Okhotsk, which shows a very slight and statistically insignificant positive trend (O.4 days/decade), trends in melt onset are negative, i.e. towards earlier melt. The trends range from -1.0day/decade for the Bering Sea to -7.3 days/decade for the East Greenland Sea. Except for the Sea of Okhotsk all areas also show a trend towards later autumn freeze onset. The Chukchi/Beaufort Seas and Laptev/East Siberian Seas observe the strongest trends with 7 days/decade. For the entire Arctic, the melt season length has increased by about 20 days over the last 30 years. Largest trends of over 1O days/decade are seen for Hudson Bay, the East Greenland Sea the Laptev/East Siberian Seas, and the Chukchi/Beaufort Seas. Those trends are statistically significant a1 the 99% level.

  18. A melt evolution model for Kerimasi volcano, Tanzania: Evidence from carbonate melt inclusions in jacupirangite (United States)

    Káldos, Réka; Guzmics, Tibor; Mitchell, Roger H.; Dawson, John Barry; Milke, Ralf; Szabó, Csaba


    This study presents compositional data for a statistically significant number (n = 180) of heated and quenched (recreated) carbonate melt inclusions trapped in magnetite and clinopyroxene in jacupirangite from Kerimasi volcano (Tanzania). On the basis of homogenization experiments for clinopyroxene-hosted melt inclusions and forsterite-monticellite-calcite phase relations, a range of 1000 to 900 °C is estimated for their crystallization temperatures. Petrographic observations and geochemical data show that during jacupirangite crystallization, a CaO-rich and alkali-"poor" carbonate melt (relative to Oldoinyo Lengai natrocarbonatite) existed and was entrapped in the precipitating magnetite, forming primary melt inclusions, and was also enclosed in previously crystallized clinopyroxene as secondary melt inclusions. The composition of the trapped carbonate melts in magnetite and clinopyroxene is very similar to the parental melt of Kerimasi calciocarbonatite; i.e., enriched in Na2O, K2O, F, Cl and S, but depleted in SiO2 and P2O5 relative to carbonate melts entrapped at an earlier stage and higher temperature (1050-1100 °C) during the formation of Kerimasi afrikandite. Significant compositional variation is shown by the major minerals of Kerimasi plutonic rocks (afrikandite, jacupirangite and calciocarbonatite). Magnetite and clinopyroxene in the jacupirangite are typically transitional in composition between those of afrikandite and calciocarbonatite. These data suggest that the jacupirangite represents an intermediate stage between the formation of afrikandite and calciocarbonatite. Jacupirangite most probably formed when immiscible silicate and carbonate melts separated from the afrikandite body, although the carbonate melt was not separated completely from the silicate melt fraction. In general, during the evolution of the carbonate melt at Kerimasi, concentrations of P2O5 and SiO2 decreased, whereas volatile content (alkalis, S, F, Cl and H2O) increased

  19. Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar enteritidis

    DEFF Research Database (Denmark)

    Garaizar, J.; Lopez-Molina, N.; Laconcha, I.


    Strains of Salmonella enterica (n = 212) of different serovars and phage types were used to establish a library typing computerized system for serovar Enteritidis on the basis of PCR fingerprinting, infrequent-restriction-site PCR (IRS-PCR), or pulsed-field gel electrophoresis (PFGE). The rate...... showed an intercenter reproducibility value of 93.3%. The high reproducibility of PFGE combined with the previously determined high discrimination directed its use for library typing. The use of PFGE with enzymes XbaI, BlnI, and SpeI for library typing of serovar Enteritidis was assessed with GelCompar 4.......0 software, Three computer libraries of PFGE DNA profiles were constructed, and their ability to recognize new DNA profiles was analyzed. The results obtained pointed out that the combination of PFGE with computerized analysis could be suitable in long-term epidemiological comparison and surveillance...

  20. Pressure Melting and Ice Skating / Bunsen Burner

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 1; Issue 5. Pressure Melting and Ice Skating / Bunsen Burner - Revisited. Classroom Volume 1 Issue 5 May 1996 pp 71-78. Fulltext. Click here to view fulltext PDF. Permanent link: Resonance ...

  1. Record Summer Melt in Greenland in 2010

    NARCIS (Netherlands)

    Tedesco, M.; Fettweis, X.; van den Broeke, M.R.; van de Wal, R.S.W.; Smeets, C.J.P.P.; van de Berg, W.J.; Serreze, M.C.; Box, J.E.


    As Arctic temperatures increase, there is growing concern about the melting of the Greenland ice sheet, which reached a new record during the summer of 2010. Understanding the changing surface mass balance of the Greenland ice sheet requires appreciation of the close links among changes in surface

  2. Hot-Melt Adhesive Attachment System (United States)

    Fox, R. L.; Frizzell, A. W.; Little, B. D.; Progar, D. J.; Coultrip, R. H.; Couch, R. H.; Stein, B. A.; Buckley, J. D.; St. Clair, T. L.; Gleason, J. R.


    Adhesive system is as effective on Earth as in space. Fiberglass cloth mounted in head assembly. When adhesive reaches melt temperature head is attached to metals composites, ceramics, and other materials. Once attached, head cooled rapidly for quick stick. Used to tether tools or attach temporary scaffolding to walls, buildings, or beams.

  3. Stress Relaxation in Entangled Polymer Melts

    DEFF Research Database (Denmark)

    Hou, Ji-Xuan; Svaneborg, Carsten; Everaers, Ralf


    We present an extensive set of simulation results for the stress relaxation in equilibrium and step-strained bead-spring polymer melts. The data allow us to explore the chain dynamics and the shear relaxation modulus, G(t), into the plateau regime for chains with Z=40 entanglements and into the t...

  4. Can Text Messages Mitigate Summer Melt? (United States)

    Castleman, Benjamin L.; Page, Lindsay C.


    Higher education officials have long been familiar with the concept of "summer melt," where students who have paid a deposit to attend one college or university instead matriculate at a different institution, usually presumed to be of comparable quality. In previous research, drawing on longitudinal data from various urban school…

  5. Radiation polymerized hot melt pressure sensitive adhesives

    International Nuclear Information System (INIS)

    Pastor, S.D.; Skoultchi, M.M.


    Hot melt pressure sensitive adhesive compositions formed by copolymerizing at least one 3-(chlorinated aryloxy)-2-hydroxypropyl ester of an alpha, beta unsaturated carboxylic acid with acrylate based copolymerizable monomers, are described. The resultant ethylenically saturated prepolymer is heated to a temperature sufficient to render it fluid and flowable. This composition is coated onto a substrate and exposed to ultraviolet radiation

  6. Catastrophic failure of polymer melts during extension

    DEFF Research Database (Denmark)

    Rasmussen, Henrik K.


    Numerical flow modeling has been applied to study the break of monodisperse polymer melts during extension. These continuum mechanical based computations are within the ideas of the microstructural ’interchain pressure’ theory. Calculated breaks, a result of small initial sample imperfections, ag...

  7. Pressure-Induced Melting of Confined Ice

    NARCIS (Netherlands)

    Sotthewes, Kai; Bampoulis, Pantelis; Zandvliet, Henricus J.W.; Lohse, Detlef; Poelsema, B.


    The classic regelation experiment of Thomson in the 1850s deals with cutting an ice cube, followed by refreezing. The cutting was attributed to pressure-induced melting but has been challenged continuously, and only lately consensus emerged by understanding that compression shortens the O:H nonbond

  8. Melting Metal on a Playing Card (United States)

    Greenslade, Thomas B., Jr.


    Many of us are familiar with the demonstration of boiling water in a paper cup held over a candle or a Bunsen burner; the ignition temperature of paper is above the temperature of 100°C at which water boils under standard conditions. A more dramatic demonstration is melting tin held in a playing card. This illustration is from Tissandier's book on…

  9. Needleless Melt-Electrospinning of Polypropylene Nanofibres

    Directory of Open Access Journals (Sweden)

    Jian Fang


    Full Text Available Polypropylene (PP nanofibres have been electrospun from molten PP using a needleless melt-electrospinning setup containing a rotary metal disc spinneret. The influence of the disc spinneret (e.g., disc material and diameter, operating parameters (e.g., applied voltage, spinning distance, and a cationic surfactant on the fibre formation and average fibre diameter were examined. It was shown that the metal material used for making the disc spinneret had a significant effect on the fibre formation. Although the applied voltage had little effect on the fibre diameter, the spinning distance affected the fibre diameter considerably, with shorter spinning distance resulting in finer fibres. When a small amount of cationic surfactant (dodecyl trimethyl ammonium bromide was added to the PP melt for melt-electrospinning, the fibre diameter was reduced considerably. The finest fibres produced from this system were 400±290 nm. This novel melt-electrospinning setup may provide a continuous and efficient method to produce PP nanofibres.

  10. Differential transcript abundance and genotypic variation of four putative allergen-encoding gene families in melting peach

    NARCIS (Netherlands)

    Yang, Zhaowei; Ma, Yingtao; Chen, Lin; Xie, Rangjin; Zhang, Xianqi; Zhang, Bo; Lu, Meidan; Wu, Shandong; Gilissen, Luud J. W. J.; van Ree, Ronald; Gao, Zhongshan


    We analysed the temporal and spatial transcript expression of the panel of 18 putative isoallergens from four gene families (Pru p 1-4) in the peach fruit, anther and leaf of two melting cultivars, to gain insight into their expression profiles and to identify the key family members. Genotypic

  11. Differential transcript abundance and genotypic variation of four putative allergen-encoding gene families in melting peach

    NARCIS (Netherlands)

    Yang, Z.; Ma, Y.; Chen, L.; Xie, R.; Zhang, X.; Zhang, B.; Lu, M.; Wu, S.; Gilissen, L.J.W.J.; Ree, van R.; Gao, Z.


    We analysed the temporal and spatial transcript expression of the panel of 18 putative isoallergens from four gene families (Pru p 1–4) in the peach fruit, anther and leaf of two melting cultivars, to gain insight into their expression profiles and to identify the key family members. Genotypic

  12. Shallow mantle melt stagnation under Gakkel Ridge (United States)

    von der Handt, A.; Snow, J. E.; Hellebrand, E.; Dick, H. J. B.; Michael, P.


    Few studies have been devoted to abyssal plagioclase peridotites, despite their relatively high abundance (30% of AP). Their origin is still unresolved, probably because intense alteration sets limits to spatially controlled geochemical analysis and obliterates textural relationships. Impregnation by a melt is the most widely accepted theory whereas other studies propose an origin by retrogression from spinel to plagioclase facies conditions. During the AMORE cruise along Gakkel Ridge in summer 2001, a dredge haul recovered spinel and plagioclase lherzolites in the axial valley of the amagmatic area. Their exceptional freshness has allowed to analyse all mineral phases. Plagioclase-bearing and -free samples are coarse-grained cpx-rich lherzolites. The plagioclase lherzolites show a wide range of modal plagioclase-contents and often showes textures related to impregnation. Noticeable are the common symplectite textures in the plagioclase peridotites, mostly opx-plag around cpx grains but also one ol-plag around cpx, suggesting a breakdown origin. The spinel lherzolites are characterised by low spinel-Cr# (˜16) and homogeneous flat cpx REE-patterns (~6 x CI). The plagioclase peridotites display strong compositional heterogeneities with pronounced core-rim variations in major and trace elements. Trace element variations in cpx show consistent correlations with textures as contact with plagioclase or symplectite formation. The An-contents of plagioclase range from 76 to 94, spinel Cr# from 10 to 48. Plagioclase trace element data reveal low concentrations for the LREE and no positive Sr-anomaly. Therefore it suggests an impregnation origin for most of the plagioclase by an already fractionated and depleted melt. Yet a minor breakdown component can be observed which was probably triggered by the impregnation. The inferred composition of this melt cannot be correlated with the nearest basalts in this region nor with a melt produced by melting of the spinel lherzolites.

  13. Local and bulk melting of Cu at grain boundaries

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Shengnian [Los Alamos National Laboratory; Han, Li - Bo [USTC; An, Qi [USTC/CALTECH; Fu, Rong - Shan [USTC; Zheng, Lianqing [FSU


    We investigate gain boundary (GB) melting using molecular dynamics simulations on face-centered-cubic Cu bicrystals with symmetric {l_angle}110{r_angle} tilt grain boundaries. Two representative types of GBs are explored: {Sigma} = 11/(113)/50.48{sup o} (low GB energy) and {Sigma} = 27/(552)/148.41{sup o} (high GB energy). The temperature and temporal evolutions of the Cu bicrystals under stepped heating are characterized in terms of order parameters and diffusion coefficients, as ell as the nucleation and growth of melt. Within the GB region, continuous local melting precedes discontinuous bulk melting, while continuous solid state disordering may precede local melting. Premelting may occur for local melting but not for bulk melting. For {Sigma} = 11/(113)/50.48{sup o}, premelting of the GB region is negligible, and local melting occurs near the thermodynamic melting temperature. The GB region as a whole is superheated by about 13% before its bulk melting. In the case of {Sigma} = 27/(552)/148.41, considerable premelting is observed for local melting, while the bulk melting occurs with negligible superheating. The exact melting behavior of a general GB depends on the GB energy, but is likely bracketed within these two cases.

  14. The influence of partial melting and melt migration on the rheology of the continental crust (United States)

    Cavalcante, Geane Carolina G.; Viegas, Gustavo; Archanjo, Carlos José; da Silva, Marcos Egydio


    The presence of melt during deformation produces a drastic change in the rheological behavior of the continental crust; rock strength is decreased even for melt fractions as low as ∼7%. At pressure/temperature conditions typical of the middle to lower crust, melt-bearing systems may play a critical role in the process of strain localization and in the overall strength of the continental lithosphere. In this contribution we focus on the role and dynamics of melt flow in two different mid-crustal settings formed during the Brasiliano orogeny: (i) a large-scale anatectic layer in an orthogonal collision belt, represented by the Carlos Chagas anatexite in southeastern Brazil, and (ii) a strike-slip setting, in which the Espinho Branco anatexite in the Patos shear zone (northeast Brazil) serves as an analogue. Both settings, located in eastern Brazil, are part of the Neoproterozoic tectonics that resulted in widespread partial melting, shear zone development and the exhumation of middle to lower crustal layers. These layers consist of compositionally heterogeneous anatexites, with variable former melt fractions and leucosome structures. The leucosomes usually form thick interconnected networks of magma that reflect a high melt content (>30%) during deformation. From a comparison of previous work based on detailed petrostructural and AMS studies of the anatexites exposed in these areas, we discuss the rheological implications caused by the accumulation of a large volume of melt ;trapped; in mid-crustal levels, and by the efficient melt extraction along steep shear zones. Our analyses suggest that rocks undergoing partial melting along shear settings exhibit layers with contrasting competence, implying successive periods of weakening and strengthening. In contrast, regions where a large amount of magma accumulates lack clear evidence of competence contrast between layers, indicating that they experienced only one major stage of dramatic strength drop. This comparative

  15. Lipid Profile (United States)

    ... Known As Coronary Risk Panel Formal Name Lipid Profile This article was last reviewed on June 29, ... phospholipid molecules. The particles measured with a lipid profile are classified by their density into high-density ...

  16. Data Profiling


    Hladíková, Radka


    Title: Data Profiling Author: Radka Hladíková Department: Department of Software Engineering Supervisor: Ing. Vladimír Kyjonka Supervisor's e-mail address: Abstract: This thesis puts mind on problems with data quality and data profiling. This Work analyses and summarizes problems of data quality, data defects, process of data quality, data quality assessment and data profiling. The main topic is data profiling as a process of researching data available in existing...

  17. Can Nano-Particle Melt below the Melting Temperature of Its Free Surface Partner?

    International Nuclear Information System (INIS)

    Sui Xiao-Hong; Qin Shao-Jing; Wang Zong-Guo; Kang Kai; Wang Chui-Lin


    The phonon thermal contribution to the melting temperature of nano-particles is inspected. The discrete summation of phonon states and its corresponding integration form as an approximation for a nano-particle or for a bulk system have been analyzed. The discrete phonon energy levels of pure size effect and the wave-vector shifts of boundary conditions are investigated in detail. Unlike in macroscopic thermodynamics, the integration volume of zero-mode of phonon for a nano-particle is not zero, and it plays an important role in pure size effect and boundary condition effect. We find that a nano-particle will have a rising melting temperature due to purely finite size effect; a lower melting temperature bound exists for a nano-particle in various environments, and the melting temperature of a nano-particle with free boundary condition reaches this lower bound. We suggest an easy procedure to estimation the melting temperature, in which the zero-mode contribution will be excluded, and only several bulk quantities will be used as input. We would like to emphasize that the quantum effect of discrete energy levels in nano-particles, which is not present in early thermodynamic studies on finite size corrections to melting temperature in small systems, should be included in future researches. (condensed matter: structural, mechanical, and thermal properties)

  18. Sea Ice Melt Pond Data from the Canadian Arctic (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set contains observations of albedo, depth, and physical characteristics of melt ponds on sea ice, taken during the summer of 1994. The melt ponds studied...

  19. Improved procedures for separating crystals from the melt

    NARCIS (Netherlands)

    Verdoes, D.; Arkenbout, G.J.; Bruinsma, O.S.L.; Koutsoukos, P.G.; Ulrich, J.


    Innovative separation techniques like melt crystallization have the potential to fulfil two important demands, namely: a significant reduction of energy consumption by the chemical industry, and the production of high quality products required by industry. Several industrial applications of melt

  20. Causes of Glacier Melt Extremes in the Alps Since 1949 (United States)

    Thibert, E.; Dkengne Sielenou, P.; Vionnet, V.; Eckert, N.; Vincent, C.


    Recent record-breaking glacier melt values are attributable to peculiar extreme events and long-term warming trends that shift averages upward. Analyzing one of the world's longest mass balance series with extreme value statistics, we show that detrending melt anomalies makes it possible to disentangle these effects, leading to a fairer evaluation of the return period of melt extreme values such as 2003, and to characterize them by a more realistic bounded behavior. Using surface energy balance simulations, we show that three independent drivers control melt: global radiation, latent heat, and the amount of snow at the beginning of the melting season. Extremes are governed by large deviations in global radiation combined with sensible heat. Long-term trends are driven by the lengthening of melt duration due to earlier and longer-lasting melting of ice along with melt intensification caused by trends in long-wave irradiance and latent heat due to higher air moisture.

  1. A 2D double-porosity model for melting and melt migration beneath mid-oceanic ridges (United States)

    Liu, B.; Liang, Y.; Parmentier, E.


    Several lines of evidence suggest that the melting and melt extraction region of the MORB mantle is heterogeneous consisting of an interconnected network of high permeability dunite channels in a low porosity harzburgite or lherzolite matrix. In principle, one can include channel formation into the tectonic-scale geodynamic models by solving conservation equations for a chemically reactive and viscously deformable porous medium. Such an approach eventually runs into computational limitations such as resolving fractal-like channels that have a spectrum of width. To better understand first order features of melting and melt-rock interaction beneath MOR, we have formulated a 2D double porosity model in which we treat the triangular melting region as two overlapping continua occupied by the low-porosity matrix and interconnected high-porosity channels. We use melt productivity derived from a thermodynamic model and melt suction rate to close our problem. We use a high-order accurate numerical method to solve the conservation equations in 2D for porosity, solid and melt velocities and concentrations of chemical tracers in the melting region. We carry out numerical simulations to systematically study effects of matrix-to-channel melt suction and spatially distributed channels on the distributions of porosity and trace element and isotopic ratios in the melting region. For near fractional melting with 10 vol% channel in the melting region, the flow field of the matrix melt follows closely to that of the solid because the small porosity (exchange between the melt and the solid. The smearing effect can be approximated by dispersion coefficient. For slowly diffusing trace elements (e.g., LREE and HFSE), the melt migration induced dispersion can be as effective as thermal diffusion. Therefore, sub-kilometer scale heterogeneities of Nd and Hf isotopes are significantly damped or homogenized in the melting region.

  2. Application of reverse transcription-PCR and real-time PCR in nanotoxicity research. (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei


    Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq(®) DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix.

  3. Rapid detection of carbapenemase genes by multiplex real-time PCR. (United States)

    Monteiro, Jussimara; Widen, Raymond H; Pignatari, Antonio C C; Kubasek, Carly; Silbert, Suzane


    To develop a single multiplex real-time PCR assay to detect six different genetic types of carbapenemases already identified in Enterobacteriaceae (KPC, GES, NDM, IMP, VIM and OXA-48). A total of 58 bacterial isolates were tested. Thirty were previously characterized as resistant to carbapenems and documented by PCR and sequencing analysis to carry the following genes: bla(KPC) type, bla(GES) type, bla(IMP) type, bla(VIM) type, bla(OXA-48) and bla(NDM-1). These positive strains included 21 Enterobacteriaceae, 1 Acinetobacter baumannii and 8 Pseudomonas aeruginosa isolates. The remaining 28 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial DNA was extracted using the easyMag extractor (bioMérieux, France). The real-time PCR was performed using the Rotor-Gene 6000 instrument (Corbett Life Science, Australia) and specific primers for each carbapenemase target were designed using the DNAStar software (Madison, WI, USA). Each one of the six carbapenemase genes tested presented a different melting curve after PCR amplification. The melting temperature (T(m)) analysis of the amplicons identified was as follows: bla(IMP) type (T(m) 80.1°C), bla(OXA-48) (T(m) 81.6°C), bla(NDM-1) (T(m) 84°C), bla(GES) type (T(m) 88.6°C), bla(VIM) type (T(m) 90.3°C) and bla(KPC) type (T(m) 91.6°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. The new assay was able to detect the presence of six different carbapenemase gene types in a single 3 h PCR.

  4. Enzootic bovine leukosis real time PCR


    Dias, Natanael Lamas; Fonseca Júnior, Antônio Augusto; Rodrigues, Daniel Sobreira; Camargos, Marcelo Fernandes


    O objetivo deste trabalho foi realizar a validação de uma reação em cadeia da polimerase em tempo real com o sistema Plexor® (qPCR) para o diagnóstico da Leucose Enzoótica Bovina (LEB), por meio da comparação com testes de diagnóstico recomendados pela Organização Mundial de Saúde Animal (OIE). A qPCR foi comparada com duas outras técnicas: a PCR nested (nPCR) e a imunodifusão em gel de ágar (IDGA). Das 82 amostras analisadas pela qPCR e nPCR, 79 apresentaram resultados concordantes, sendo a ...

  5. Contaminations Occurring in Fungal PCR Assays (United States)

    Loeffler, Juergen; Hebart, Holger; Bialek, Ralf; Hagmeyer, Lars; Schmidt, Diethard; Serey, Francois-Prâseth; Hartmann, Matthias; Eucker, Jan; Einsele, Hermann


    Successful in vitro amplification of fungal DNA in clinical specimens has been reported recently. In a collaboration among five European centers, the frequency and risk of contamination due to airborne spore inoculation or carryover contamination in fungal PCR were analyzed. The identities of all contaminants were specified by cycle sequencing and GenBank analysis. Twelve of 150 PCR assays that together included over 2,800 samples were found to be contaminated (3.3% of the negative controls were contaminated during the DNA extraction, and 4.7% of the PCR mixtures were contaminated during the amplification process). Contaminants were specified as Aspergillus fumigatus, Saccharomyces cerevisiae, and Acremonium spp. Further analysis showed that commercially available products like zymolyase powder or 10× PCR buffer may contain fungal DNA. In conclusion, the risk of contamination is not higher in fungal PCR assays than in other diagnostic PCR-based assays if general precautions are taken. PMID:10074553

  6. Electrochemistry of silicon in chloro-fluoride and carbonate melts

    Directory of Open Access Journals (Sweden)

    Devyatkin S.V.


    Full Text Available The electrochemical behavior of K2SiF6 in chloro-fluoride melts and that of SiO2 in carbonate melts has been studied. Silicon, titanium silicides, boron silicide and ternary compounds Ti-Si-B have been deposited from chloro-fluoride melts. Only SiC was deposited from carbonate-silica melts under carbon dioxide atmosphere (that is, excessive pressure of CO2.

  7. Nanoparticle-induced unusual melting and solidification behaviours of metals


    Ma, Chao; Chen, Lianyi; Cao, Chezheng; Li, Xiaochun


    Effective control of melting and solidification behaviours of materials is significant for numerous applications. It has been a long-standing challenge to increase the melted zone (MZ) depth while shrinking the heat-affected zone (HAZ) size during local melting and solidification of materials. In this paper, nanoparticle-induced unusual melting and solidification behaviours of metals are reported that effectively solve this long-time dilemma. By introduction of Al2O3 nanoparticles, the MZ dep...

  8. Investigating the vortex melting phenomenon in BSCCO crystals ...

    Indian Academy of Sciences (India)

    To study the fluctuations in the local melting temperature/field, we have constructed maps of the melting landscape m(, ), viz., the melting temperature (m) at ... Department of Condensed Matter Physics, The Weizmann Institute of Science, Rehovot 76100, Israel; Department of Applied Physics, The University of Tokyo, ...

  9. Melt flow characteristics in gas-assisted laser cutting

    Indian Academy of Sciences (India)

    R. Narasimhan (Krishtel eMaging) 1461 1996 Oct 15 13:05:22

    Abstract. We present a study on laser cutting of mild steel with oxygen as an assist gas. We correlate the cut surface quality with the melt film thickness. We estimate the optimum pressure required for melt ejection under laminar flow regime. The thickness of melt film inside the kerf is estimated using mass balance and the ...

  10. Investigation of the stability of melt flow in gating systems

    DEFF Research Database (Denmark)

    Tiedje, Niels Skat; Larsen, Per


    Melt flow in four different gating systems designed for production of brake discs was analysed experimentally and by numerical modelling. In the experiments moulds were fitted with glass fronts and melt flow was recorded on video. The video recordings were compared with modelling of melt flow in ...

  11. Elongational viscosity of monodisperse and bidisperse polystyrene melts

    DEFF Research Database (Denmark)

    Nielsen, Jens Kromann; Rasmussen, Henrik K.; Hassager, Ole


    The start-up and steady uniaxial elongational viscosity have been measured for two monodisperse polystyrene melts with molecular weights of 52 and 103 kg/mole, and for three bidisperse polystyrene melts. The monodisperse melts show a maximum in the steady elongational viscosity vs. the elongational...

  12. Melt pool vorticity in deep penetration laser material welding

    Indian Academy of Sciences (India)

    this value, convective heat transfer indicates melting and evaporation occurring in the weld pool during laser ... while surface tension and hydrostatic pressure help to retain the melt in the keyhole cavity in this high .... so does the strength of the shock wave and the Mach angle becomes increasingly narrow. As the melt flow ...

  13. Pitfalls in PCR troubleshooting: Expect the unexpected?

    Directory of Open Access Journals (Sweden)

    Livia Schrick


    Full Text Available PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings.

  14. Absolute quantification by droplet digital PCR versus analog real-time PCR (United States)

    Hindson, Christopher M; Chevillet, John R; Briggs, Hilary A; Gallichotte, Emily N; Ruf, Ingrid K; Hindson, Benjamin J; Vessella, Robert L; Tewari, Muneesh


    Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer. PMID:23995387

  15. Karolinske psychodynamic profile (KAPP)

    DEFF Research Database (Denmark)

    Mathiesen, Birgit Bork; Søgaard, Ulf


    psykologiske testmetoder, assesment, Karolinska psychodynamic profile (KAPP), psykodynamisk profil......psykologiske testmetoder, assesment, Karolinska psychodynamic profile (KAPP), psykodynamisk profil...

  16. Free dendritic growth in viscous melts - Cyclohexanol (United States)

    Singh, N. B.; Glicksman, M. E.


    Experiments were carried out to measure the growth speed, V, and dendritic tip radius, R, of highly purified cyclohexanol. The data show that VR-squared = constant over the entire experimentally observed supercooling range, Delta T is between 0.1 and 1 K. The stability parameter estimated from this result indicates that sigma(asterisk) = 0.027, a value in good agreement with the values of sigma(asterisk) found for the cubic plastic crystals succinonitrile pivalic acid. Cyclohexanol differs from other carefully measured plastic crystals in that the viscosity of its melt at the melting point is about 20 times higher, so gravity-induced convection remains weak even at small supercoolings.

  17. Melting of metallic intermediate level waste

    International Nuclear Information System (INIS)

    Huutoniemi, Tommi; Larsson, Arne; Blank, Eva


    This report presents a feasibility study of a melting facility for core components and reactor internals. An overview is given of how such a facility for treatment of intermediate level waste might be designed, constructed and operated and highlights both the possibilities and challenges. A cost estimate and a risk analysis are presented in order to make a conclusion of the technical feasibility of such a facility. Based on the authors' experience in operating a low level waste melting facility, their conclusion is that without technical improvements such a facility is not feasible today. This is based on the cost of constructing and operating such a facility, in conjunction with the radiological risks associated with operation and the uncertain benefits to disposal and long term safety

  18. Detection of structural heterogeneity of glass melts

    DEFF Research Database (Denmark)

    Yue, Yuanzheng


    The structural heterogeneity of both supercooled liquid and molten states of silicate has been studied using calorimetric method. The objects of this study are basaltic glasses and liquids. Two experimental approaches are taken to detect the structural heterogeneity of the liquids. One...... is discussed. The ordered structure of glass melts above the liquidus temperature is indirectly characterized by use of X-ray diffraction method. The new approaches are of importance for monitoring the glass melting and forming process and for improving the physical properties of glasses and glass fibers....... is the hyperquench-anneal-calorimetric scan approach, by which the structural information of a basaltic supercooled liquid and three binary silicate liquids is acquired. Another is the calorimetrically repeated up- and downscanning approach, by which the structural heterogeneity, the intermediate range order...

  19. Melting of metallic intermediate level waste

    Energy Technology Data Exchange (ETDEWEB)

    Huutoniemi, Tommi; Larsson, Arne; Blank, Eva [Studsvik Nuclear AB, Nykoeping (Sweden)


    This report presents a feasibility study of a melting facility for core components and reactor internals. An overview is given of how such a facility for treatment of intermediate level waste might be designed, constructed and operated and highlights both the possibilities and challenges. A cost estimate and a risk analysis are presented in order to make a conclusion of the technical feasibility of such a facility. Based on the authors' experience in operating a low level waste melting facility, their conclusion is that without technical improvements such a facility is not feasible today. This is based on the cost of constructing and operating such a facility, in conjunction with the radiological risks associated with operation and the uncertain benefits to disposal and long term safety.

  20. Stress Relaxation in Entangled Polymer Melts

    DEFF Research Database (Denmark)

    Hou, Ji-Xuan; Svaneborg, Carsten; Everaers, Ralf


    We present an extensive set of simulation results for the stress relaxation in equilibrium and step-strained bead-spring polymer melts. The data allow us to explore the chain dynamics and the shear relaxation modulus, G(t), into the plateau regime for chains with Z=40 entanglements and into the t......We present an extensive set of simulation results for the stress relaxation in equilibrium and step-strained bead-spring polymer melts. The data allow us to explore the chain dynamics and the shear relaxation modulus, G(t), into the plateau regime for chains with Z=40 entanglements...... excellent agreement for the Likhtman-McLeish theory using the double reptation approximation for constraint release, if we remove the contribution of high-frequency modes to contour length fluctuations of the primitive chain....

  1. Quality improvement of melt extruded laminar systems using mixture design. (United States)

    Hasa, D; Perissutti, B; Campisi, B; Grassi, M; Grabnar, I; Golob, S; Mian, M; Voinovich, D


    This study investigates the application of melt extrusion for the development of an oral retard formulation with a precise drug release over time. Since adjusting the formulation appears to be of the utmost importance in achieving the desired drug release patterns, different formulations of laminar extrudates were prepared according to the principles of Experimental Design, using a design for mixtures to assess the influence of formulation composition on the in vitro drug release from the extrudates after 1h and after 8h. The effect of each component on the two response variables was also studied. Ternary mixtures of theophylline (model drug), monohydrate lactose and microcrystalline wax (as thermoplastic binder) were extruded in a lab scale vertical ram extruder in absence of solvents at a temperature below the melting point of the binder (so that the crystalline state of the drug could be maintained), through a rectangular die to obtain suitable laminar systems. Thanks to the desirability approach and a reliability study for ensuring the quality of the formulation, a very restricted optimal zone was defined within the experimental domain. Among the mixture components, the variation of microcrystalline wax content played the most significant role in overall influence on the in vitro drug release. The formulation theophylline:lactose:wax, 57:14:29 (by weight), selected based on the desirability zone, was subsequently used for in vivo studies. The plasma profile, obtained after oral administration of the laminar extruded system in hard gelatine capsules, revealed the typical trend of an oral retard formulation. The application of the mixture experimental design associated to a desirability function permitted to optimize the extruded system and to determine the composition space that ensures final product quality. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. BWR core melt progression phenomena: Experimental analyses

    International Nuclear Information System (INIS)

    Ott, L.J.


    In the BWR Core Melt in Progression Phenomena Program, experimental results concerning severe fuel damage and core melt progression in BWR core geometry are used to evaluate existing models of the governing phenomena. These include control blade eutectic liquefaction and the subsequent relocation and attack on the channel box structure; oxidation heating and hydrogen generation; Zircaloy melting and relocation; and the continuing oxidation of zirconium with metallic blockage formation. Integral data have been obtained from the BWR DF-4 experiment in the ACRR and from BWR tests in the German CORA exreactor fuel-damage test facility. Additional integral data will be obtained from new CORA BWR test, the full-length FLHT-6 BWR test in the NRU test reactor, and the new program of exreactor experiments at Sandia National Laboratories (SNL) on metallic melt relocation and blockage formation. an essential part of this activity is interpretation and use of the results of the BWR tests. The Oak Ridge National Laboratory (ORNL) has developed experiment-specific models for analysis of the BWR experiments; to date, these models have permitted far more precise analyses of the conditions in these experiments than has previously been available. These analyses have provided a basis for more accurate interpretation of the phenomena that the experiments are intended to investigate. The results of posttest analyses of BWR experiments are discussed and significant findings from these analyses are explained. The ORNL control blade/canister models with materials interaction, relocation and blockage models are currently being implemented in SCDAP/RELAP5 as an optional structural component

  3. In vessel core melt progression phenomena

    International Nuclear Information System (INIS)

    Courtaud, M.


    For all light water reactor (LWR) accidents, including the so called severe accidents where core melt down can occur, it is necessary to determine the amount and characteristics of fission products released to the environment. For existing reactors this knowledge is used to evaluate the consequences and eventual emergency plans. But for future reactors safety authorities demand decrease risks and reactors designed in such a way that fission products are retained inside the containment, the last protective barrier. This requires improved understanding and knowledge of all accident sequences. In particular it is necessary to be able to describe the very complex phenomena occurring during in vessel core melt progression because they will determine the thermal and mechanical loads on the primary circuit and the timing of its rupture as well as the fission product source term. On the other hand, in case of vessel failure, knowledge of the physical and chemical state of the core melt will provide the initial conditions for analysis of ex-vessel core melt progression and phenomena threatening the containment. Finally a good understanding of in vessel phenomena will help to improve accident management procedures like Emergency Core Cooling System water injection, blowdown and flooding of the vessel well, with their possible adverse effects. Research and Development work on this subject was initiated a long time ago and is still in progress but now it must be intensified in order to meet the safety requirements of the next generation of reactors. Experiments, limited in scale, analysis of the TMI 2 accident which is a unique source of global information and engineering judgment are used to establish and assess physical models that can be implemented in computer codes for reactor accident analysis

  4. Low-melting point heat transfer fluid (United States)

    Cordaro, Joseph Gabriel; Bradshaw, Robert W.


    A low-melting point, heat transfer fluid made of a mixture of five inorganic salts including about 29.1-33.5 mol % LiNO.sub.3, 0-3.9 mol % NaNO.sub.3, 2.4-8.2 mol % KNO.sub.3, 18.6-19.9 mol % NaNO.sub.2, and 40-45.6 mol % KNO.sub.2. These compositions can have liquidus temperatures below C. for some compositions.

  5. Conditioning of nuclear cladding wastes by melting

    International Nuclear Information System (INIS)

    Puyou, M.; Jouan, A.; Jacquet-Francillon, N.


    This paper discusses a cold-crucible induction melting process to condition cladding waste from irradiated fast breeder reactor fuel. The process has been developed by the CEA at Marcoule (France) as part of a major R and D program. It has been qualified at industrial scale on nonradioactive waste, and at laboratory scale on radioactive waste: several radioactive ingots have been produced from actual stainless steel or zircaloy hulls. The results confirm the numerous advantages of this containment method

  6. Holographic picture of heavy vector meson melting

    Energy Technology Data Exchange (ETDEWEB)

    Braga, Nelson R.F.; Diles, Saulo [Universidade Federal do Rio de Janeiro, Instituto de Fisica, Rio de Janeiro, RJ (Brazil); Martin Contreras, Miguel Angel [Universidad de los Andes, High Energy Group, Department of Physics, Bogota (Colombia)


    The fraction of heavy vector mesons produced in a heavy ion collision, as compared to a proton-proton collision, serves as an important indication of the formation of a thermal medium, the quark-gluon plasma. This sort of analysis strongly depends on understanding the thermal effects of a medium like the plasma on the states of heavy mesons. In particular, it is crucial to know the temperature ranges where they undergo a thermal dissociation, or melting. AdS/QCD models are know to provide an important tool for the calculation of hadronic masses, but in general are not consistent with the observation that decay constants of heavy vector mesons decrease with excitation level. It has recently been shown that this problem can be overcome using a soft wall background and introducing an extra energy parameter, through the calculation of correlation functions at a finite position of anti-de Sitter space. This approach leads to the evaluation of masses and decay constants of S wave quarkonium states with just one flavor dependent and one flavor independent parameter. Here we extend this more realistic model to finite temperatures and analyze the thermal behavior of the states 1S, 2S and 3S of bottomonium and charmonium. The corresponding spectral function exhibits a consistent picture for the melting of the states where, for each flavor, the higher excitations melt at lower temperatures. We estimate for these six states the energy ranges in which the heavy vector mesons undergo a transition from a well-defined peak in the spectral function to complete melting in the thermal medium. A very clear distinction between the heavy flavors emerges, with the bottomonium state Υ(1S) surviving a deconfinement transition at temperatures much larger than the critical deconfinement temperature of the medium. (orig.)

  7. The electrical conductivity of sodium polysulfide melts

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Meihui [Univ. of California, Berkeley, CA (United States)


    The sodium polysulfide melt has been described by a macroscopic model. This model considers the melt to be composed of sodium cations, monosulfide anions, and neutral sulfur solvent. The transport equations of concentrated-solution theory are used to derived the governing equations for this binaryelectrolyte melt model. These equations relate measurable transport properties to fundamental transport parameters. The focus of this research is to measure the electrical conductivity of sodium polysulfide melts and calculate one of fundamental transport parameters from the experimental data. The conductance cells used in the conductivity measurements are axisymmetric cylindrical cells with a microelectrode. The electrode effects, including double-layer capacity, charge transfer resistance, and concentration overpotential, were minimized by the use of the alternating current at an adequately high frequency. The high cell constants of the conductance cells not only enhanced the experimental accuracy but also made the electrode effects negligible. The electrical conductivities of sodium polysulfide Na2S4 and Na2S5 were measured as a function of temperature (range: 300 to 360°C). Variations between experiments were only up to 2%. The values of the Arrhenius activation energy derived from the experimental data are about 33 kJ/mol. The fundamental transport parameter which quantifies the interaction within sodium cations and monosulfide anions are of interest and expected to be positive. Values of it were calculated from the experimental conductivity data and most of them are positive. Some negative values were obtained probably due to the experimental errors of transference number, diffusion coefficient, density or conductivity data.

  8. The electrical conductivity of sodium polysulfide melts

    Energy Technology Data Exchange (ETDEWEB)

    Meihui Wang.


    The sodium polysulfide melt has been described by a macroscopic model. This model considers the melt to be composed of sodium cations, monosulfide anions, and neutral sulfur solvent. The transport equations of concentrated-solution theory are used to derived the governing equations for this binaryelectrolyte melt model. These equations relate measurable transport properties to fundamental transport parameters. The focus of this research is to measure the electrical conductivity of sodium polysulfide melts and calculate one of fundamental transport parameters from the experimental data. The conductance cells used in the conductivity measurements are axisymmetric cylindrical cells with a microelectrode. The electrode effects, including double-layer capacity, charge transfer resistance, and concentration overpotential, were minimized by the use of the alternating current at an adequately high frequency. The high cell constants of the conductance cells not only enhanced the experimental accuracy but also made the electrode effects negligible. The electrical conductivities of sodium polysulfide Na{sub 2}S{sub 4} and Na{sub 2}S{sub 5} were measured as a function of temperature (range: 300 to 360{degree}C). Variations between experiments were only up to 2%. The values of the Arrhenius activation energy derived from the experimental data are about 33 kJ/mol. The fundamental transport parameter which quantifies the interaction within sodium cations and monosulfide anions are of interest and expected to be positive. Values of it were calculated from the experimental conductivity data and most of them are positive. Some negative values were obtained probably due to the experimental errors of transference number, diffusion coefficient, density or conductivity data.

  9. "Chemical contraction" in rubidium-bismuth melts (United States)

    Khairulin, R. A.; Abdullaev, R. N.; Stankus, S. V.


    The density and thermal expansion of liquid rubidium and rubidium-bismuth alloy containing 25.0 at % Bi were measured by the gamma-ray attenuation technique at temperatures from liquidus to 1000 K. The results of this study were compared with the data obtained by other authors. The molar volume of the Rb75Bi25 melt strongly deviates from the additivity rule for ideal solutions.

  10. Vacuum induction melting of uranium ingots

    International Nuclear Information System (INIS)

    Hussain, M.M.; Bagchi, S.N.; Singh, S.P.


    Massive uranium ingot is produced from green salt (UF 4 ) using calciothermic reduction (CTR) or magnesiothermic reduction (MTR) process. CTR process has been replaced by MTR process at Trombay due to economic considerations. This paper highlights problems associated with the vacuum induction melting of MTR ingots and the remedial measures taken to produce good quality billets. Details of metallographic examination of inclusions in ingots and billets have been incorporated. (author). 3 figs

  11. Properties of aspirin modified enteric polymer prepared by hot-melt mixing. (United States)

    Chomcharn, Nonjaros; Xanthos, Marino


    Melt mixing in batch equipment or continuous extruders is a technique that recently gained the attention of the pharmaceutical industry. The present work has employed hot-melt mixing to prepare a modified enteric matrix, as a delayed-release dosage form. Different concentrations of aspirin (ASP) ranging from 10 to -30% (w/w) were melt-mixed with a plasticized methacrylic acid copolymer, Eudragit(®) L100-55 in a batch mixer for 5 min at 100 °C which is above the plasticized polymer's glass transition temperature and below the ASP's melting point. The samples were cooled down to room temperature and compressed to thin discs. Processing ASP with Eudragit(®) L100-55 did not promote hydrolysis of ASP. X-ray diffraction spectra obtained at room temperature revealed that aspirin was present in a crystalline state. However, at elevated temperatures the dissolved aspirin displayed a plasticizing effect by reducing the glass transition temperature (Tg) and lowering the viscosity of the polymer in proportion to its increasing concentration. The addition of ASP altered to some extent the rheological behavior of the polymer from rubbery to viscous. However, the amount of ASP loading had no significant impact on the dissolution profiles. The samples met USP delayed-release requirements and the API release mechanism was shown to be diffusion dominant. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Influence of snowpack internal structure on snow metamorphism and melting intensity on Hansbreen, Svalbard

    Directory of Open Access Journals (Sweden)

    Laska Michał


    Full Text Available This paper presents a detailed study of melting processes conducted on Hansbreen – a tidewater glacier terminating in the Hornsund fjord, Spitsbergen. The fieldwork was carried out from April to July 2010. The study included observations of meltwater distribution within snow profiles in different locations and determination of its penetration time to the glacier ice surface. In addition, the variability of the snow temperature and heat transfer within the snow cover were measured. The main objective concerns the impact of meltwater on the diversity of physical characteristics of the snow cover and its melting dynamics. The obtained results indicate a time delay between the beginning of the melting processes and meltwater reaching the ice surface. The time necessary for meltwater to percolate through the entire snowpack in both, the ablation zone and the equilibrium line zone amounted to c. 12 days, despite a much greater snow depth at the upper site. An elongated retention of meltwater in the lower part of the glacier was caused by a higher amount of icy layers (ice formations and melt-freeze crusts, resulting from winter thaws, which delayed water penetration. For this reason, a reconstruction of rain-on-snow events was carried out. Such results give new insight into the processes of the reactivation of the glacier drainage system and the release of freshwater into the sea after the winter period.

  13. Semi-continuous casting of magnesium alloy AZ91 using a filtered melt delivery system

    Directory of Open Access Journals (Sweden)

    Mainul Hasan


    Full Text Available A 3-D numerical simulation of an industrial-sized slab caster for magnesium alloy AZ91 has been carried out for the steady state operational phase of the caster. The simulated model consists of an open-top melt delivery system fitted with a porous filter near the hot-top. The melt flow through the porous filter was modeled on the basis of Brinkmann-Forchimier-Extended non-Darcy model for turbulent flow. An in-house 3-D CFD code was modified to account for the melt flow through the porous filter. Results are obtained for four casting speeds namely, 40, 60, 80, and 100 mm/min. The metal-mold contact region as well as the convective heat transfer coefficient at the mold wall were also varied. In addition to the above, the Darcy number for the porous media was also changed. All parametric studies were performed for a fixed inlet melt superheat of 64 °C. The results are presented pictorially in the form of temperature and velocity fields. The sump depth, mushy region thickness, solid shell thickness at the exit of the mold and axial temperature profiles are also presented and correlated with the casting speed through regression analysis.

  14. Laser melting of groove defect repair on high thermal conductivity steel (HTCS-150) (United States)

    Norhafzan, B.; Aqida, S. N.; Fazliana, F.; Reza, M. S.; Ismail, I.; Khairil, C. M.


    This paper presents laser melting repair of groove defect on HTCS-150 surface using Nd:YAG laser system. Laser melting process was conducted using JK300HPS Nd:YAG twin lamp laser source with 1064 nm wavelength and pulsed mode. The parameters are pulse repetition frequency (PRF) that is set from 70 to 100 Hz, average power ( P A) of 50-70 W, and laser spot size of 0.7 mm. HTCS-150 samples were prepared with groove dimension of 0.3 mm width and depths of 0.5 mm using EDM wire cut. Groove defect repaired using laser melting process on groove surface area with various parameters' process. The melted surface within the groove was characterized for subsurface hardness profile, roughness, phase identification, chemical composition, and metallographic study. The roughness analysis indicates high PRF at large spot size caused high surface roughness and low surface hardness. Grain refinement of repaired layer was analyzed within the groove as a result of rapid heating and cooling. The hardness properties of modified HTCS inside the groove and the bulk surface increased two times from as received HTCS due to grain refinement which is in agreement with Hall-Petch equation. These findings are significant to parameter design of die repair for optimum surface integrity and potential for repairing crack depth and width of less than 0.5 and 0.3 mm, respectively.

  15. Strong morphological and crystallographic texture and resulting yield strength anisotropy in selective laser melted tantalum

    International Nuclear Information System (INIS)

    Thijs, Lore; Montero Sistiaga, Maria Luz; Wauthle, Ruben; Xie, Qingge; Kruth, Jean-Pierre; Van Humbeeck, Jan


    Selective laser melting (SLM) makes use of a high energy density laser beam to melt successive layers of metallic powders in order to create functional parts. The energy density of the laser is high enough to melt refractory metals like Ta and produce mechanically sound parts. Furthermore, the localized heat input causes a strong directional cooling and solidification. Epitaxial growth due to partial remelting of the previous layer, competitive growth mechanism and a specific global direction of heat flow during SLM of Ta result in the formation of long columnar grains with a 〈1 1 1〉 preferential crystal orientation along the building direction. The microstructure was visualized using both optical and scanning electron microscopy equipped with electron backscattered diffraction and the global crystallographic texture was measured using X-ray diffraction. The thermal profile around the melt pool was modeled using a pragmatic model for SLM. Furthermore, rotation of the scanning direction between different layers was seen to promote the competitive growth. As a result, the texture strength increased to as large as 4.7 for rotating the scanning direction 90° every layer. By comparison of the yield strength measured by compression tests in different orientations and the averaged Taylor factor calculated using the viscoplastic self-consistent model, it was found that both the morphological and crystallographic texture observed in SLM Ta contribute to yield strength anisotropy

  16. Melts of garnet lherzolite: experiments, models and comparison to melts of pyroxenite and carbonated lherzolite (United States)

    Grove, Timothy L.; Holbig, Eva S.; Barr, Jay A.; Till, Christy B.; Krawczynski, Michael J.


    Phase equilibrium experiments on a compositionally modified olivine leucitite from the Tibetan plateau have been carried out from 2.2 to 2.8 GPa and 1,380–1,480 °C. The experiments-produced liquids multiply saturated with spinel and garnet lherzolite phase assemblages (olivine, orthopyroxene, clinopyroxene and spinel ± garnet) under nominally anhydrous conditions. These SiO2-undersaturated liquids and published experimental data are utilized to develop a predictive model for garnet lherzolite melting of compositionally variable mantle under anhydrous conditions over the pressure range of 1.9–6 GPa. The model estimates the major element compositions of garnet-saturated melts for a range of mantle lherzolite compositions and predicts the conditions of the spinel to garnet lherzolite phase transition for natural peridotite compositions at above-solidus temperatures and pressures. We compare our predicted garnet lherzolite melts to those of pyroxenite and carbonated lherzolite and develop criteria for distinguishing among melts of these different source types. We also use the model in conjunction with a published predictive model for plagioclase and spinel lherzolite to characterize the differences in major element composition for melts in the plagioclase, spinel and garnet facies and develop tests to distinguish between melts of these three lherzolite facies based on major elements. The model is applied to understand the source materials and conditions of melting for high-K lavas erupted in the Tibetan plateau, basanite–nephelinite lavas erupted early in the evolution of Kilauea volcano, Hawaii, as well as younger tholeiitic to alkali lavas from Kilauea.

  17. Electric melting furnace for waste solidification

    International Nuclear Information System (INIS)

    Masaki, Toshio.


    To avoid electric troubles or reduction of waste processing performance even when platinum group elements are contained in wastes to be applied with glass solidification. For this purpose, a side electrode is disposed to the side wall of a melting vessel and a central electrode serving as a counter electrode is disposed about at the center inside the melting vessel. With such a constitution, if conductive materials are deposited at the bottom of the furnace or the bottom of the melting vessel, heating currents flow selectively between the side electrode and the central electrode. Accordingly, no electric currents flow through the conductive deposits thereby enabling to prevent abnormal heating in the bottom of the furnace. Further, heat generated by electric supply between the side electrode and the central electrode is supplied efficiently to raw material on the surface of the molten glass liquid to improve the processing performance. Further, disposition of the bottom electrode at the bottom of the furnace enables current supply between the central electrode and the bottom electrode to facilitate the temperature control for the molten glass in the furnace than in the conventional structure. (I.S.)

  18. The kinetic fragility of natural silicate melts

    International Nuclear Information System (INIS)

    Giordano, Daniele; Dingwell, Donald B


    Newtonian viscosities of 19 multicomponent natural and synthetic silicate liquids, with variable contents of SiO 2 (41-79 wt%), Al 2 O 3 (10-19 wt%), TiO 2 (0-3 wt%), FeO tot (0-11 wt%); alkali oxides (5-17 wt%), alkaline-earth oxides (0-35 wt%), and minor oxides, obtained at ambient pressure using the high-temperature concentric cylinder, the low-temperature micropenetration, and the parallel plates techniques, have been analysed. For each silicate liquid, regression of the experimentally determined viscosities using the well known Vogel-Fulcher-Tammann (VFT) equation allowed the viscosity of all these silicates to be accurately described. The results of these fits, which provide the basis for the subsequent analysis here, permit qualitative and quantitative correlations to be made between the VFT adjustable parameters (A VFT , B VFT , and T 0 ). The values of B VFT and T 0 , calibrated via the VFT equation, are highly correlated. Kinetic fragility appears to be correlated with the number of non-bridging oxygens per tetrahedrally coordinated cation (NBO/T). This is taken to infer that melt polymerization controls melt fragility in liquid silicates. Thus NBO/T might form an useful ingredient of a structure-based model of non-Arrhenian viscosity in multicomponent silicate melts

  19. Melt processing of Yb-123 tapes

    International Nuclear Information System (INIS)

    Athur, S. P.; Balachandran, U.; Salama, K.


    The innovation of a simple, scalable process for manufacturing long-length conductors of HTS is essential to potential commercial applications such as power cables, magnets, and transformers. In this paper the authors demonstrate that melt processing of Yb-123 tapes made by the PIT route is an alternative to the coated conductor and Bi-2223 PIT tape fabrication techniques. Ag-clad Yb-123 tapes were fabricated by groove rolling and subsequently, melt processed in different oxygen partial pressures in a zone-melting furnace with a gradient of 140 C/cm. The transition temperatures measured were found to be around 81 K undermost processing conditions. EPMA of the tapes processed under different conditions show the 123 phase to be Ba deficient and Cu and Yb rich. Critical current was measured at various temperatures from 77 K to 4.2 K. The J c increased with decrease in pO 2 . The highest I c obtained was 52 A at 4.2 K

  20. Physical properties of Al-R melts

    International Nuclear Information System (INIS)

    Sidorov, V.; Gornov, O.; Bykov, V.; Son, L.; Ryltsev, R.; Uporov, S.; Shevchenko, V.; Kononenko, V.; Shunyaev, K.; Ilynykh, N.; Moiseev, G.; Kulikova, T.; Sordelet, D.


    In this work, we present experimental data of physical properties (viscosity, surface tension, magnetic susceptibility and electroresistivity) studies for Al-R (R = Ce, Pr, Sm, Gd, Dy, Ho, Yb and Y) alloys and first intermetallic compounds from aluminum side, Al 11 R 3 (Al 3 R). All properties were measured during heating up to 2000 K and the following cooling down under helium atmosphere. The main results are: (1) the electronic characteristics of the objects are in good correlation with R positions in the periodic table, but rather lower than for pure elements. The conclusion is that through all investigated temperature range, the rare-earth elements have partly covalent but not only metallic states; (2) all the melts remain strongly microheterogeneous even at high overheatings above liquidus. The existence of associations with Al 2 R type is highly probable here. Some destruction of these associations takes place with increasing temperature above melting point at the composition of Al 2 R compound. However, the transformation into true solution state is somewhere above 1900 K. To check the idea, the thermodynamic modeling of the melts was performed. It was shown that associates with Al 2 R type are stable up to 2000 K

  1. Vortex lattice melting, pinning and kinetics

    International Nuclear Information System (INIS)

    Doniach, S.; Ryu, S.; Kapitulnik, A.


    The phenomenology of the high T c superconductors is discussed both at the level of the thermodynamics of melting of the Abrikosov flux lattice and in terms of the melting and kinetics of the flux lattice for a pinned system. The authors review results on 3D melting obtained by a Monte Carlo simulation approach in which the 2D open-quotes pancakeclose quotes vortices are treated as statistical variables. The authors discuss pinning in the context of the strong pinning regime in which the vortex density given in terms of the applied field B is small compared to that represented by an effective field B pin measuring the pinning center density. The authors introduce a new criterion for the unfreezing of a vortex glass on increase of magnetic field or temperature, in the strong pinning, small field unit. The authors model this limit in terms of a single flux line interacting with a columnar pin. This model is studied both analytically and by computer simulation. By applying a tilt potential, the authors study the kinetics of the vortex motion in an external current and show that the resulting current-voltage characteristic follows a basic vortex glass-like scaling relation in the vicinity of the depinning transition

  2. Rapid-melt Dynamic Nuclear Polarization (United States)

    Sharma, M.; Janssen, G.; Leggett, J.; Kentgens, A. P. M.; van Bentum, P. J. M.


    In recent years, Dynamic Nuclear Polarization (DNP) has re-emerged as a means to ameliorate the inherent problem of low sensitivity in nuclear magnetic resonance (NMR). Here, we present a novel approach to DNP enhanced liquid-state NMR based on rapid melting of a solid hyperpolarized sample followed by 'in situ' NMR detection. This method is applicable to small (10 nl to 1 μl) sized samples in a microfluidic setup. The method combines generic DNP enhancement in the solid state with the high sensitivity of stripline 1 H NMR detection in the liquid state. Fast cycling facilitates options for signal averaging or 2D structural analysis. Preliminary tests show solid-state 1 H enhancement factors of up to 500 for H2O/D2O/d6-glycerol samples doped with TEMPOL radicals. Fast paramagnetic relaxation with nitroxide radicals, In nonpolar solvents such as toluene, we find proton enhancement factors up to 400 with negligible relaxation losses in the liquid state, using commercially available BDPA radicals. A total recycling delay (including sample freezing, DNP polarization and melting) of about 5 s can be used. The present setup allows for a fast determination of the hyper-polarization as function of the microwave frequency and power. Even at the relatively low field of 3.4 T, the method of rapid melting DNP can facilitate the detection of small quantities of molecules in the picomole regime.

  3. Glass forming ability of calcium aluminosilicate melts

    DEFF Research Database (Denmark)

    Moesgaard, Mette; Yue, Yuanzheng


    The glass forming ability (GFA) of two series of calcium aluminosilicate melts is studied by measuring their viscous behavior and crystallization tendency. The first series consists of five compositions on the joining line between the eutectic point of anorthite-wollastonite-tridymite and that of......The glass forming ability (GFA) of two series of calcium aluminosilicate melts is studied by measuring their viscous behavior and crystallization tendency. The first series consists of five compositions on the joining line between the eutectic point of anorthite......-wollastonite-tridymite and that of anorthite-wollastonite-gehlenite. The series includes the eutectic compositions as end members. The second series consists of five compositions on a line parallel to the joining line on the alumina rich side. In the present work, GFA is described in terms of glass stability, i.e., the ability of a glass...... to resist crystallization during reheating. In addition, the fragility index (m) is derived by fitting the viscosity data with the Avramov-Milchev equation. The results show that m is inversely proportional to the glass stability for the two series of melts, implying that m is an indirect measure of GFA...

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

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


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

  5. High resolution melting analysis for identification of commercially-important Mytilus species. (United States)

    Jilberto, Felipe; Araneda, Cristián; Larraín, María Angélica


    Mytilus are edible mussels, including commercially-significant species such as M. chilensis, M. galloprovincialis and M. edulis. The scientific name of the species must be indicated on commercial products to satisfy labelling and traceability requirements. Species identification using morphological criteria is difficult due the plasticity of these characteristics and the absence of shells in processed products, and conventional PCR-based methods are laborious and time-intensive. As alternative, we propose high resolution melting (HRM) analysis as a simple tool to detect and identify SNP (single nucleotide polymorphisms) and length polymorphisms in Mytilus spp. We designed HRM-specific primers for the Mytilus genus to identify M. chilensis, M. galloprovincialis, M. edulis and their hybrids through clearly-distinguishable melting curves. HRM analysis showed high sensitivity (0.9639), specificity (1.0000) and precision (1.0000) compared to a conventional PCR-RFLP test. HRM is a fast and low cost method, being a reliable tool for species identification within the Mytilus genus. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Qiuying Huang


    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.

  7. Development of multiplex real-time PCR assays for identification of members of the Anopheles funestus species group

    Directory of Open Access Journals (Sweden)

    Field Linda M


    Full Text Available Abstract Background The malaria vector and non-vector species of the Anopheles funestus group are morphologically very similar and accurate identification is required as part of effective control strategies. In the past, this has relied on morphological and cytogenetic methods but these have been largely superseded by a robust allele-specific PCR (AS-PCR. One disadvantage of AS-PCR is the requirement for post-PCR processing by gel electrophoresis of PCR products. In this study, three new high-throughput 'closed-tube' assays were developed and compared with the previously described AS-PCR technique. Methods Protocols for three fluorescence-based assays based on Melt Curve Analysis (MCA, High Resolution Melt (HRM and TaqMan SNP genotyping were developed to detect and discriminate Anopheles parensis, Anopheles leesoni, Anopheles vaneedeni, Anopheles rivulorum and An. funestus s.s. The sensitivity and specificity of these assays were compared with the widely used AS-PCR in a blind trial using DNA extracted from wild-caught mosquitoes. Results The TaqMan assay proved to be the most sensitive and specific of the three new assays. The MCA and HRM assays initially gave promising results, but were more sensitive to both DNA quality and quantity and consequently showed a higher rate of incorrect identifications. Conclusion The TaqMan assay proved to be the most robust of the three protocols tested in this study. This assay very effectively identified all five members of the An. funestus group using fluorescently-labeled probes with distinct emission and excitation spectra allowing their independent detection in a single reaction. This method is at least as sensitive and specific as the gold standard AS-PCR approach and because it has no requirement for post-PCR processing is simpler and more rapid to run. The one disadvantage of the TaqMan assay is the cost of this assay, both in terms of initial capital outlay and running cost per sample, which is higher

  8. Mutation spectrum of 122 hemophilia A families from Taiwanese population by LD-PCR, DHPLC, multiplex PCR and evaluating the clinical application of HRM

    Directory of Open Access Journals (Sweden)

    Chang Chieh-Ting


    Full Text Available Abstract Background Hemophilia A represents the most common and severe inherited hemorrhagic disorder. It is caused by mutations in the F8 gene, which leads to a deficiency or dysfunctional factor VIII protein, an essential cofactor in the factor X activation complex. Methods We used long-distance polymerase chain reaction and denaturing high performance liquid chromatography for mutation scanning of the F8 gene. We designed the competitive multiplex PCR to identify the carrier with exonal deletions. In order to facilitate throughput and minimize the cost of mutation scanning, we also evaluated a new mutation scanning technique, high resolution melting analysis (HRM, as an alternative screening method. Results We presented the results of detailed screening of 122 Taiwanese families with hemophilia A and reported twenty-nine novel mutations. There was one family identified with whole exons deletion, and the carriers were successfully recognized by multiplex PCR. By HRM, the different melting curve patterns were easily identified in 25 out of 28 cases (89% and 15 out of 15 (100% carriers. The sensitivity was 93 % (40/43. The overall mutation detection rate of hemophilia A was 100% in this study. Conclusion We proposed a diagnostic strategy for hemophilia A genetic diagnosis. We consider HRM as a powerful screening tool that would provide us with a more cost-effective protocol for hemophilia A mutation identification.

  9. Fabrication and subband gap optical properties of silicon supersaturated with chalcogens by ion implantation and pulsed laser melting

    International Nuclear Information System (INIS)

    Bob, Brion P.; Aziz, Michael J.; Kohno, Atsushi; Charnvanichborikarn, Supakit; Williams, James S.; Warrender, Jeffrey M.; Umezu, Ikurou; Tabbal, Malek


    Topographically flat, single crystal silicon supersaturated with the chalcogens S, Se, and Te was prepared by ion implantation followed by pulsed laser melting and rapid solidification. The influences of the number of laser shots on the atomic and carrier concentration-depth profiles were measured with secondary ion mass spectrometry and spreading resistance profiling, respectively. We found good agreement between the atomic concentration-depth profiles obtained from experiments and a one-dimensional model for plane-front melting, solidification, liquid-phase diffusion, with kinetic solute trapping, and surface evaporation. Broadband subband gap absorption is exhibited by all dopants over a wavelength range from 1 to 2.5 microns. The absorption did not change appreciably with increasing number of laser shots, despite a measurable loss of chalcogen and of electronic carriers after each shot.

  10. A sensitive, reproducible, and economic real-time reverse transcription PCR detecting avian metapneumovirus subtypes A and B. (United States)

    Franzo, G; Drigo, M; Lupini, C; Catelli, E; Laconi, A; Listorti, V; Bonci, M; Naylor, C J; Martini, M; Cecchinato, M


    Use of real-time PCR is increasing in the diagnosis of infectious disease due to its sensitivity, specificity, and speed of detection. These characteristics make it particularly suited for the diagnosis of viral infections, like avian metapneumovirus (AMPV), for which effective control benefits from continuously updated knowledge of the epidemiological situation. Other real-time reverse transcription (RT)-PCRs have been published based on highly specific fluorescent dye-labeled probes, but they have high initial cost, complex validation, and a marked susceptibility to the genetic variability of their target sequence. With this in mind, we developed and validated a SYBR Green I-based quantitative RT-PCR for the detection of the two most prevalent AMPV subtypes (i.e., subtypes A and B). The assay demonstrated an analytical sensitivity comparable with that of a previously published real-time RT-PCR and the ability to detect RNA equivalent to approximately 0.5 infectious doses for both A and B subtypes. The high efficiency and linearity between viral titer and crossing point displayed for both subtypes make it suited for viral quantification. Optimization of reaction conditions and the implementation of melting curve analysis guaranteed the high specificity of the assay. The stable melting temperature difference between the two subtypes indicated the possibility of subtyping through melting temperature analysis. These characteristics make our assay a sensitive, specific, and rapid tool, enabling contemporaneous detection, quantification, and discrimination of AMPV subtype A and B.

  11. Contrasting melt equilibration conditions across Anatolia (United States)

    Reid, Mary; Delph, Jonathan; Schleiffarth, W. Kirk; Cosca, Michael


    The widespread mafic volcanism, elevated crustal temperatures, and plateau-type topography in Central Anatolia, Turkey, could collectively be the result of lithospheric delamination, mantle upwelling, and tectonic escape in response to Arabian-Anatolian plate collision. We used the results from basalt geochemistry and a passive-source broadband seismic experiment obtained as part of an international collaborative effort (Continental Dynamics - Central Anatolia Tectonics) to investigate the crust-mantle structure and melting conditions associated with the Quaternary Hasandag Monogenic Cluster (HMC) south and west of Hasandag volcano. The HMC is unusually mafic, not only for Central Anatolia but globally, enabling meaningful comparisons between geochemical and seismic interpretations of mantle conditions. HMC basalts are characterized by orogenic signatures that could have originated (1) in mantle wedge that, after stagnating because of collision, was remobilized south and upward as a result of rollback of the African slab or, alternatively (2) by piecemeal foundering of residual mantle lithosphere into convecting upper mantle, producing small-scale convection and associated decompression melting. Melt equilibration conditions for the HMC are hot (TP ˜1335-1250˚ C, assuming 1-4 wt.% H2O) and shallow (P = 1.1 to 1.6 GPa), approaching those for MORB. Shear wave velocities are relatively constant at ˜4.1 km/s between the Moho and a depth of ˜45-50 km (˜1.4 GPa; Fig. 6), below which Vs increases with increasing depth. We infer that a melt-perfused mantle lid could be locally present between 40 and 55 km. In contrast to Central Anatolia, estimated equilibration conditions for Western Anatolia and Eastern Anatolia (east of the Inner Tauride Suture) mantle melts are hotter (by ≥60˚ C) and deeper (mostly by 0.6-1.0 GPa). They also have chemical signatures that, unlike Central Anatolia, are similar to those of intraplate basalts. These differences are likely related

  12. A FRET-based real-time PCR assay to identify the main causal agents of New World tegumentary leishmaniasis.

    Directory of Open Access Journals (Sweden)

    Pablo Tsukayama

    Full Text Available In South America, various species of Leishmania are endemic and cause New World tegumentary leishmaniasis (NWTL. The correct identification of these species is critical for adequate clinical management and surveillance activities. We developed a real-time polymerase chain reaction (PCR assay and evaluated its diagnostic performance using 64 archived parasite isolates and 192 prospectively identified samples collected from individuals with suspected leishmaniasis enrolled at two reference clinics in Lima, Peru. The real-time PCR assay was able to detect a single parasite and provided unambiguous melting peaks for five Leishmania species of the Viannia subgenus that are highly prevalent in South America: L. (V. braziliensis, L. (V. panamensis, L. (V. guyanensis, L. (V. peruviana and L. (V. lainsoni. Using kinetoplastid DNA-based PCR as a gold standard, the real-time PCR had sensitivity and specificity values of 92% and 77%, respectively, which were significantly higher than those of conventional tests such as microscopy, culture and the leishmanin skin test (LST. In addition, the real-time PCR identified 147 different clinical samples at the species level, providing an overall agreement of 100% when compared to multilocus sequence typing (MLST data performed on a subset of these samples. Furthermore, the real-time PCR was three times faster and five times less expensive when compared to PCR - MLST for species identification from clinical specimens. In summary, this new assay represents a cost-effective and reliable alternative for the identification of the main species causing NWTL in South America.

  13. Effect of the distribution of saturated fatty acids in the melting and crystallization profiles of high-oleic high-stearic oils; Efecto de la distribución de los ácidos grasos saturados en los perfiles de fusión y cristalización de los aceites alto esteárico alto oleico

    Energy Technology Data Exchange (ETDEWEB)

    Bootello, M.A.; Garces, R.; Martinez-Force, E.; Salas, J.J.


    The composition and distribution of fatty acids in triacylglycerol (TAG) molecules are commonly considered as factors that determine the physical properties of a given oil or fat. The distribution of any fatty acid in fats and oils can be described through the α coefficient of asymmetry, which can be calculated from the TAG composition and fatty acid composition of the Sn-2 position of the TAGs determined through lipase hydrolysis. High-oleic high-stearic oils and fats are considered stable and healthy, and they are good substitutes for hydrogenated vegetable oils and palm fractions in many food products, such as spreads and confectionery. Here, different high-oleic high-stearic acid oils were formulated which contained different distributions of saturated fatty acids in their TAGs, while maintaining a similar fatty acid composition. The aim of this work was to discuss the possibility of using the α coefficient to predict the physical properties of fats in function of their chemical composition and their melting and crystallization behavior as examined by differential scanning calorimetry.

  14. Y-STR genetic screening by high-resolution melting analysis. (United States)

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


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

  15. Laser post-processing of Inconel 625 made by selective laser melting (United States)

    Witkin, David; Helvajian, Henry; Steffeney, Lee; Hansen, William


    The effect of laser remelting of surfaces of as-built Selective Laser Melted (SLM) Inconel 625 was evaluated for its potential to improve the surface roughness of SLM parts. Many alloys made by SLM have properties similar to their wrought counterparts, but surface roughness of SLM-made parts is much higher than found in standard machine shop operations. This has implications for mechanical properties of SLM materials, such as a large debit in fatigue properties, and in applications of SLM, where surface roughness can alter fluid flow characteristics. Because complexity and netshape fabrication are fundamental advantages of Additive Manufacturing (AM), post-processing by mechanical means to reduce surface roughness detracts from the potential utility of AM. Use of a laser to improve surface roughness by targeted remelting or annealing offers the possibility of in-situ surface polishing of AM surfaces- the same laser used to melt the powder could be amplitude modulated to smooth the part during the build. The effects of remelting the surfaces of SLM Inconel 625 were demonstrated using a CW fiber laser (IPG: 1064 nm, 2-50 W) that is amplitude modulated with a pulse profile to induce remelting without spallation or ablation. The process achieved uniform depth of melting and improved surface roughness. The results show that with an appropriate pulse profile that meters the heat-load, surface features such as partially sintered powder particles and surface connected porosity can be mitigated via a secondary remelting/annealing event.

  16. Crystallization, recrystallization, and melting lines in syndiotactic polypropylene crystallized from quiescent melt and semicrystalline state due to stress-induced localized melting and recrystallization. (United States)

    Lu, Ying; Wang, Yaotao; Fu, Lianlian; Jiang, Zhiyong; Men, Yongfeng


    Crystalline lamellar thickness in syndiotactic polypropylene (sPP) during crystallization from either isothermal molten or stretching induced localized melt states and during subsequent heating was investigated by means of temperature dependent small-angle X-ray scattering techniques. Well-defined crystallization lines where the reciprocal lamellar thickness is linearly dependent on crystallization temperature were observed. Unlike in the case of polybutene-1 where stretching crystallization line was shifted to direction of much smaller lamellar thickness (Macromolecules 2013, 46, 7874), the stretching induced crystallization line for sPP deviates from its corresponding isothermal crystallization line only slightly. Such phenomenon could be attributed to the fact that both crystallization processes from quiescent melt and stress induced localized melt are mediated in a mesomorphic phase in sPP. Subsequent heating of sPP after crystallization revealed the same melting behavior in both systems for the two kinds of crystallites obtained from either quiescent melt or stretching induced localized melt. Both of them underwent melting and recrystallization when the lamellar thickness was smaller than a critical value and melting directly without changing in thickness when the lamellar thickness was larger than the critical value. The melting behavior in sPP systems can be understood by considering the chain relaxation ability within crystalline phase and also can be used as evidence that the crystallization from molten state and stress-induced crystallization passed through the intermediate phase before forming crystallites.

  17. PCR protocols: current methods and applications

    National Research Council Canada - National Science Library

    White, Bruce Alan


    ..." between "small" and "big" labs, since its use makes certain projects, especially those related to molecular cloning, now far more feasible for the small lab with a modest budget. This new volume on PCR Protocols does not attempt the impossible task of representing all PCR-based protocols. Rather, it presents a range of protocols, both analytical ...

  18. [E-MTAB-587] PCR_artifacts

    NARCIS (Netherlands)

    Muino Acuna, J.M.


    WARNING: This library was yield low amount of material and it was over-amplified by PCR. This libraries are used study the robustness of several statitical methods against PCR artifacts. ChIP experiments were performed on Arabidopsis wildtype inflorescences using an antibody raised against a

  19. PCR specific for Actinobacillus pleuropneumoniae serotype 3

    DEFF Research Database (Denmark)

    Zhou, L.; Jones, S.C.P.; Angen, Øystein


    , but the method has liminations, for example, cross-reactions between serotypes 3, 6, and 8. This study describes the development of a serotype 3-specific PCR, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The PCR test was evaluated on 266 strains...

  20. Testing for Genetically Modified Foods Using PCR (United States)

    Taylor, Ann; Sajan, Samin


    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  1. (PCR) and loop-mediated isothermal amplification

    African Journals Online (AJOL)



    Mar 26, 2014 ... better alternative for PCR, even in low technology laboratories. In addition, these findings revealed that the possibility of fatal fusariosis due to F. solani is not so rare in HIV positive patients. Key words: Fusarium solani, loop-mediated isothermal amplification (LAMP), HIV, polymerase chain reaction. (PCR).

  2. Validation of RNAi by real time PCR

    DEFF Research Database (Denmark)

    Josefsen, Knud; Lee, Ying Chiu


    Real time PCR is the analytic tool of choice for quantification of gene expression, while RNAi is concerned with downregulation of gene expression. Together, they constitute a powerful approach in any loss of function studies of selective genes. We illustrate here the use of real time PCR to verify...

  3. Bioinformatic tools for PCR Primer design

    African Journals Online (AJOL)


    reaction (PCR), oligo hybridization and DNA sequencing. Proper primer design is actually one of the most important factors/steps in successful DNA sequencing. Various bioinformatics programs are available for selection of primer pairs from a template sequence. The plethora programs for PCR primer design reflects the.

  4. Comparative analysis of conventional PCR and real-time PCR to diagnose shrimp WSD

    Directory of Open Access Journals (Sweden)

    C.A.G. Leal


    Full Text Available The aims of this study were to standard and optimize a qPCR protocol with FAM-BHQ1 probe, and to compare its sensitivity against TaqMan qPCR and PCR methods to diagnose shrimp WSD. The FAM-BHQ1 qPCR presented higher clinical sensitivity and showed to be a robust alternative to detect WSSV in clinical samples.

  5. Lessons learnt from FARO/TERMOS corium melt quenching experiments

    Energy Technology Data Exchange (ETDEWEB)

    Magallon, D.; Huhtiniemi, I.; Hohmann, H. [Commission of the European Communities, Ispra (Italy). Joint Research Center


    The influence of melt quantity, melt composition, water depth and initial pressure on quenching is assessed on the basis of seven tests performed in various conditions in the TERMOS vessel of the FARO facility at JRC-Ispra. Tests involved UO{sub 2}-based melt quantities in the range 18-176 kg at a temperature of approximately 3000 K poured into saturated water. The results suggest that erosion of the melt jet column is an efficient contributor to the amount of break-up, and thus quenching, for large pours of corium melt. The presence of Zr metal in the melt induced a much more efficient quenching than in a similar test with no Zr metal, attributed to the oxidation of the Zr. Significant amounts of H{sub 2} were produced also in tests with pure oxidic melts (e.g. about 300 g for 157 kg melt). In the tests at 5.0 and 2.0 MPa good mixing with significant melt break-up and quenching was obtained during the penetration in the water. At 0.5 MPa, good penetration of the melt into the water could still be achieved, but a jump in the vessel pressurisation occurred when the melt contacted the bottom and part (5 kg) of the debris was re-ejected from the water. (author)

  6. Development of a multiplex real-time PCR assay for phylogenetic analysis of Uropathogenic Escherichia coli. (United States)

    Hasanpour, Mojtaba; Najafi, Akram


    Uropathogenic Escherichia coli (UPEC) is among major pathogens causing 80-90% of all episodes of urinary tract infections (UTIs). Recently, E. coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F, and clade I. This study was aimed to develop a rapid, sensitive, and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. This study was carried out on E. coli strains (isolated from the patient with UTI) in which the presence of all seven target genes had been confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons with melting temperatures (T m ) ranging from 82°C to 93°C and temperature difference of more than 1.5°C between each peak.The multiplex real-time PCR assays that have been developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated, with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for arpAgpE genes, and 91±0.5 for the internal control. To our knowledge, this study is the first melting curve-based real-time PCR assay developed for simultaneous and discrete detection of these seven target genes. Our findings showed that this assay has the potential to be a rapid, reliable and cost-effective alternative for routine phylotyping of E. coli strains. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Molecular dynamics simulations on the melting of gold nanoparticles (United States)

    Qiao, Zhiwei; Feng, Haijun; Zhou, Jian


    Molecular dynamics is employed to study the melting of bulk gold and gold nanoparticles. PCFF, Sutton-Chen and COMPASS force fields are adopted to study the melting point of bulk gold and we find out that the Sutton-Chen force field is the most accurate model in predicting the melting point of bulk gold. Consequently, the Sutton-Chen force field is applied to study the melting points of spherical gold nanoparticles with different diameters. Variations of diffusion coefficient, potential energy and translational order parameter with temperature are analyzed. The simulated melting points of gold nanoparticles are between 615∼1115 K, which are much lower than that of bulk gold (1336 K). As the diameter of gold nanoparticle drops, the melting point also descends. The melting mechanism is also analyzed for gold nanoparticles.

  8. Nanoparticle-induced unusual melting and solidification behaviours of metals (United States)

    Ma, Chao; Chen, Lianyi; Cao, Chezheng; Li, Xiaochun


    Effective control of melting and solidification behaviours of materials is significant for numerous applications. It has been a long-standing challenge to increase the melted zone (MZ) depth while shrinking the heat-affected zone (HAZ) size during local melting and solidification of materials. In this paper, nanoparticle-induced unusual melting and solidification behaviours of metals are reported that effectively solve this long-time dilemma. By introduction of Al2O3 nanoparticles, the MZ depth of Ni is increased by 68%, while the corresponding HAZ size is decreased by 67% in laser melting at a pulse energy of 0.18 mJ. The addition of SiC nanoparticles shows similar results. The discovery of the unusual melting and solidification of materials that contain nanoparticles will not only have impacts on existing melting and solidification manufacturing processes, such as laser welding and additive manufacturing, but also on other applications such as pharmaceutical processing and energy storage.

  9. Polymerase chain reaction methods (PCR in agrobiotechnology

    Directory of Open Access Journals (Sweden)

    Taški-Ajduković Ksenija


    Full Text Available The agricultural biotechnology applies polymerase chain reaction (PCR technology at numerous steps throughout product development. The major uses of PCR technology during product development include gene discovery and cloning, vector construction, transformant identification, screening and characterization as well as seed quality control. Commodity and food companies as well as testing laboratories rely on PCR technology to verify the presence or absence of genetically modification (GM in a product or to quantify the amount of GM material present in the product. This article describes the fundamental elements of PCR analysis and its application to the testing of grains and highlights some of areas to which attention must be paid in order to produce reliable test results. The article also discuses issues related to the analysis of different matrixes and the effect they may have on the accuracy of the PCR analytical results.

  10. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman, Johan Frederik; Pierik, A.


    Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the

  11. Effect of apple pectin on gut microbiota - qPCR in applied microbiology

    DEFF Research Database (Denmark)

    Bergström, Anders; Wilcks, Andrea; Poulsen, Morten

    community in rats, as well as on a number of cecal parameters, which could be influenced by a changed microbiota. Principal Component Analysis (PCA) of cecal microbiota profiles obtained by PCR-DGGE targeting bacterial 16S rRNA genes showed an effect of whole apples in a long-term feeding study (14 weeks...

  12. A Novel Teaching-Learning-Based Optimization for Improved Mutagenic Primer Design in Mismatch PCR-RFLP SNP Genotyping. (United States)

    Cheng, Yu-Huei


    Many single nucleotide polymorphisms (SNPs) for complex genetic diseases are genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in small-scale basic research studies. It is an essential work to design feasible PCR-RFLP primer pair and find out available restriction enzymes to recognize the target SNP for PCR experiments. However, many SNPs are incapable of performing PCR-RFLP makes SNP genotyping become unpractical. A genetic algorithm (GA) had been proposed for designing mutagenic primer and get available restriction enzymes, but it gives an unrefined solution in mutagenic primers. In order to improve the mutagenic primer design, we propose TLBOMPD (TLBO-based Mutagenic Primer Design) a novel computational intelligence-based method that uses the notion of "teaching and learning" to search for more feasible mutagenic primers and provide the latest available restriction enzymes. The original Wallace's formula for the calculation of melting temperature is maintained, and more accurate calculation formulas of GC-based melting temperature and thermodynamic melting temperature are introduced into the proposed method. Mutagenic matrix is also reserved to increase the efficiency of judging a hypothetical mutagenic primer if involve available restriction enzymes for recognizing the target SNP. Furthermore, the core of SNP-RFLPing version 2 is used to enhance the mining work for restriction enzymes based on the latest REBASE. Twenty-five SNPs with mismatch PCR-RFLP screened from 288 SNPs in human SLC6A4 gene are used to appraise the TLBOMPD. Also, the computational results are compared with those of the GAMPD. In the future, the usage of the mutagenic primers in the wet lab needs to been validated carefully to increase the reliability of the method. The TLBOMPD is implemented in JAVA and it is freely available at

  13. Water isotopic ratios from a continuously melted ice core sample

    Directory of Open Access Journals (Sweden)

    V. Gkinis


    Full Text Available A new technique for on-line high resolution isotopic analysis of liquid water, tailored for ice core studies is presented. We built an interface between a Wavelength Scanned Cavity Ring Down Spectrometer (WS-CRDS purchased from Picarro Inc. and a Continuous Flow Analysis (CFA system. The system offers the possibility to perform simultaneuous water isotopic analysis of δ18O and δD on a continuous stream of liquid water as generated from a continuously melted ice rod. Injection of sub μl amounts of liquid water is achieved by pumping sample through a fused silica capillary and instantaneously vaporizing it with 100% efficiency in a~home made oven at a temperature of 170 °C. A calibration procedure allows for proper reporting of the data on the VSMOW–SLAP scale. We apply the necessary corrections based on the assessed performance of the system regarding instrumental drifts and dependance on the water concentration in the optical cavity. The melt rates are monitored in order to assign a depth scale to the measured isotopic profiles. Application of spectral methods yields the combined uncertainty of the system at below 0.1‰ and 0.5‰ for δ18O and δD, respectively. This performance is comparable to that achieved with mass spectrometry. Dispersion of the sample in the transfer lines limits the temporal resolution of the technique. In this work we investigate and assess these dispersion effects. By using an optimal filtering method we show how the measured profiles can be corrected for the smoothing effects resulting from the sample dispersion. Considering the significant advantages the technique offers, i.e. simultaneuous measurement of δ18O and δD, potentially in combination with chemical components that are traditionally measured on CFA systems, notable reduction on analysis time and power consumption, we consider it as an alternative to traditional isotope ratio mass spectrometry with the possibility to

  14. Simulation of melt spreading in consideration of phase transitions

    International Nuclear Information System (INIS)

    Spengler, C.


    The analysis of melt spreading and relocation phenomena in the containment of LWR power plants in case of hypothetical severe accidents leading to core melting is an important issue for reactor safety investigations. For the simulation of melt spreading the code LAVA has been developed on the basis of a method from the related subject of volcanology by adding more detailed models for heat transfer phenomena and flow rheology. The development is supported by basic analysis of the spreading of gravity currents as well as experimental investigations of the rheology of solidifying melts. These exhibit strong non-Newtonian effects in case of a high content of solids in the freezing melt. The basic model assumption in LAVA is the ideal Bingham plastic approach to the non-Newtonian, shear-thinning characteristic of solidifying melts. For the recalculation of melt spreading experiments, the temperature-dependent material properties for solidifying melt mixtures have been calculated using correlations from the literature. With the parameters and correlations for the rheological material properties approached by results from literature, it was possible to recalculate successfully recent spreading experiments with simulant materials and prototypic reactor core materials. An application to the behaviour of core melt in the reactor cavity assumed a borderline case for the issue of spreading. This limit is represented by melt conditions (large solid fraction, low volume flux), under which the melt is hardly spreadable. Due to the persistent volume flux the reactor cavity is completely, but inhomogeneously filled with melt. The degree of inhomogeneity is rather small, so it is concluded, that for the long-term coolability of a melt pool in narrow cavities the spreading of melt will probably have only negligible influence. (orig.)

  15. Quantitative real-time RT-PCR and chromogenic in situ hybridization

    DEFF Research Database (Denmark)

    Rosa, Fabíola E; Silveira, Sara M; Silveira, Cássia G T


    HER-2 expression and morphological features. However, FISH signals are labile and fade over time, making post-revision of the tumor difficult. CISH (chromogenic in situ hybridization) is an alternative procedure, with certain advantages, although still limited as a diagnostic tool in breast carcinomas....... METHODS: To elucidate the molecular profile of HER-2 status, mRNA and protein expression in 75 invasive breast carcinomas were analyzed by real time quantitative RT-PCR (qRT-PCR) and IHC, respectively. Amplifications were evaluated in 43 of these cases by CISH and in 11 by FISH. RESULTS: The concordance...... rate between IHC and qRT-PCR results was 78.9%, and 94.6% for qRT-PCR and CISH. Intratumoral heterogeneity of HER-2 status was identified in three cases by CISH. The results of the three procedures were compared and showed a concordance rate of 83.8%; higher discordances were observed in 0 or 1...

  16. Evaluation of PCR and nested PCR for laboratory diagnosis of hepatitis C virus infection. (United States)

    Aslanzadeh, J; Padilla, B B; Shanley, J D


    The detection of hepatitis C virus (HCV) RNA by nested polymerase chain reaction (PCR) is believed to be the most reliable method to diagnose HCV infections. A pitfall of nested PCR is that it is prone to contamination. Single step reverse transcription-PCR (RT-PCR) was performed, prospectively, on 80 sera from 59 patients with a set of primers that amplified a 273 bp sequence unique to the 5' noncoding (NC) region of the HCV genome. Nested PCR, was performed on all PCR negative specimens with a set of primers that amplified a 255 bp internal to the original primers. Single step RT-PCR was positive on 45 sera from 35 patients following gel electrophoresis and on two additional sera from two patients following Southern blot hybridization. Nested PCR was positive on two more sera following gel electrophoresis of the nested PCR products. These two patients were seropositive and subsequent serum from one patient was positive by single step PCR. Three additional sera were positive following Southern blot analysis of the nested PCR products. Two patients were seropositive and had elevated serum alanine aminotransferase (ALT) levels. The third patient was seronegative with normal ALT level and was considered a false positive. The remaining seronegative control specimens were PCR negative by both methods. The majority of PCR positive patients (82%) had elevated ALT levels, while the majority of PCR negative seropositive patients had normal ALT levels. We conclude that single step PCR is a sensitive test for the laboratory diagnoses of the majority of the HCV infections.

  17. Assessment of the real-time PCR and different digital PCR platforms for DNA quantification. (United States)

    Pavšič, Jernej; Žel, Jana; Milavec, Mojca


    Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method.

  18. Melting Behavior of ashes from the co-combustion of coal and straw

    DEFF Research Database (Denmark)

    Arvelakis, Stelios; Frandsen, Flemming


    the temperature profile in the different parts of the boiler to reduce the deposition of the ash material. The results show that the straw in the co-combustion mixture changes the viscosity characteristics of the produced ash fractions. The viscosity of the different ash fractions is lowered, as the percentage...... and costly repairs, increasing the operational costs and the cost of the produced power. In this paper, the melting characteristics of several ash fractions sampled from different parts of a pilot-scale pulverized fuel (PF) boiler operating with different coal/straw mixtures is determined by measuring...... the ash viscosity using a high-temperature rotational viscometer. The produced data provide information on the melting of the ash material, its flow characteristics, and the rates of crystallization and recrystallization, as a function of the temperature. This information may be used to modify...

  19. A high-throughput assay for quantitative measurement of PCR errors. (United States)

    Shagin, Dmitriy A; Shagina, Irina A; Zaretsky, Andrew R; Barsova, Ekaterina V; Kelmanson, Ilya V; Lukyanov, Sergey; Chudakov, Dmitriy M; Shugay, Mikhail


    The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.

  20. Recent results in characterization of melt-grown and quench-melt- grown YBCO superconductors

    International Nuclear Information System (INIS)

    Balachandran, U.; Poeppel, R.B.; Gangopadhyay, A.K.


    From the standpoint of applications, melt-grown (MG) and quench-melt-grown (QMG) bulk YBCO superconductors are of considerable interest. In this paper, we studied the intragranular critical current density (J c ), the apparent pinning potential (U o ), and the irreversibility temperature (T irr ) of MG and QMG samples and compared the results to those for conventionally sintered YBCO. A systematic increase in U o and a slower drop in J c with temperature indicate a systematic improvement in flux-pinning properties in progressing from the sintered YBCO to QMG and MG samples. Weaker pinning is observed in the QMG YBCO than in the MG samples

  1. A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp

    Energy Technology Data Exchange (ETDEWEB)

    Jothikumar, N., E-mail:; Hill, Vincent R.


    Highlights: •Uses a single-labeled fluorescent primer for real-time PCR. •The detection sensitivity of PET PCR was comparable to TaqMan PCR. •Melt curve analysis can be performed to confirm target amplicon production. •Conventional PCR primers can be converted to PET PCR primers. -- Abstract: We report the development of a fluorescently labeled oligonucleotide primer that can be used to monitor real-time PCR. The primer has two parts, the 3′-end of the primer is complimentary to the target and a universal 17-mer stem loop at the 5′-end forms a hairpin structure. A fluorescent dye is attached to 5′-end of either the forward or reverse primer. The presence of guanosine residues at the first and second position of the 3′ dangling end effectively quenches the fluorescence due to the photo electron transfer (PET) mechanism. During the synthesis of nucleic acid, the hairpin structure is linearized and the fluorescence of the incorporated primer increases several-fold due to release of the fluorescently labeled tail and the absence of guanosine quenching. As amplicons are synthesized during nucleic acid amplification, the fluorescence increase in the reaction mixture can be measured with commercially available real-time PCR instruments. In addition, a melting procedure can be performed to denature the double-stranded amplicons, thereby generating fluorescence peaks that can differentiate primer dimers and other non-specific amplicons if formed during the reaction. We demonstrated the application of PET-PCR for the rapid detection and quantification of Cryptosporidium parvum DNA. Comparison with a previously published TaqMan® assay demonstrated that the two real-time PCR assays exhibited similar sensitivity for a dynamic range of detection of 6000–0.6 oocysts per reaction. PET PCR primers are simple to design and less-expensive than dual-labeled probe PCR methods, and should be of interest for use by laboratories operating in resource

  2. 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.

  3. Simulation of core melt spreading with lava: theoretical background and status of validation

    International Nuclear Information System (INIS)

    Allelein, H.-J.; Breest, A.; Spengler, C.


    The goal of this paper is to present the GRS R and D achievements and perspectives of its approach to simulate ex-vessel core melt spreading. The basic idea followed by GRS is the analogy of core melt spreading to volcanic lava flows. A fact first proposed by Robson (1967) and now widely accepted is that lava rheologically behaves as a Bingham fluid, which is characterized by yield stress and plastic viscosity. Recent experimental investigations by Epstein (1996) reveal that corium-concrete mixtures may be described as Bingham fluids. The GRS code LAVA is based on a successful lava flow model, but is adapted to prototypic corium and corium-simulation spreading. Furthermore some detailed physical models such as a thermal crust model on the free melt surface and a model for heat conduction into the substratum are added. Heat losses of the bulk, which is represented by one mean temperature, are now determined by radiation and by temperature profiles in the upper crust and in the substratum. In order to reduce the weak mesh dependence of the original algorithm, a random space method of cellular automata is integrated, which removes the mesh bias without increasing calculation time. LAVA is successfully validated against a lot of experiments using different materials spread. The validation process has shown that LAVA is a robust and fast running code to simulate corium-type spreading. LAVA provides all integral information of practical interest (spreading length, height of the melt after stabilization) and seems to be an appropriate tool for handling large core melt masses within a plant application. (orig.)

  4. High-resolution melting analysis of MED12 mutations in uterine leiomyomas in Chinese patients. (United States)

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


    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.

  5. Material Biocompatibility for PCR Microfluidic Chips

    KAUST Repository

    Kodzius, Rimantas


    As part of the current miniaturization trend, biological reactions and processes are being adapted to microfluidics devices. PCR is the primary method employed in DNA amplification, its miniaturization is central to efforts to develop portable devices for diagnostics and testing purposes. A problem is the PCR-inhibitory effect due to interaction between PCR reagents and the surrounding environment, which effect is increased in high-surface-are-to-volume ration microfluidics. In this study, we evaluated the biocompatibility of various common materials employed in the fabrication of microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most of the cases, addition of bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, whereas they did show noticeable interaction with the DNA polymerase. Our test, instead of using microfluidic devices, can be easily conducted in common PCR tubes using a standard bench thermocycler. Our data supports an overview of the means by which the materials most bio-friendly to microfluidics can be selected.

  6. Reversed Extension Flow of Polymer melts

    DEFF Research Database (Denmark)

    Rasmussen, Henrik K.; Nielsen, Jens Kromann


    The measurement of the startup of uni axial elongational flow (potentially until steady state) followed by reversed bi axial flow, both with a constant elongational rate was made possible using a Filament Stretching Rheometer (FSR). The filament stretching rheometer rheometer is surrounded by a t...... by a thermostated environment and allows measurements on polymeric melts and liquids from room temperatures until 200 °C. In the experiments the Hencky strain at which the stress becomes zero (the recovery strain) of the reversed flow can be identified....

  7. Pressure-induced melting of micellar crystal

    DEFF Research Database (Denmark)

    Mortensen, K.; Schwahn, D.; Janssen, S.


    that pressure improves the solvent quality of water, thus resulting in decomposition of the micelles and consequent melting of the micellar crystal. The combined pressure and temperature dependence reveals that in spite of the apparent increase of order on the 100 angstrom length scale upon increasing......Aqueous solutions of triblock copolymers of poly(ethylene oxide) and poly(propylene oxide) aggregate at elevated temperatures into micelles which for polymer concentrations greater-than-or-equal-to 20% make a hard sphere crystallization to a cubic micellar crystal. Structural studies show...... temperature (decreasing pressure) the overall entropy increases through the inverted micellar crystallization characteristic....

  8. Manufacturing of implants by selective laser melting

    Directory of Open Access Journals (Sweden)

    Cosma Sorin Cosmin


    Full Text Available In recent years, digitizing and automation have gained an important place in fabrication of medical parts. Rapid Prototyping could be very suitable for medical applications due to their complex geometry, low volume and strong individualization. The presented study investigates the possibility to produce medical or dental parts by Selective Laser Melting (SLM. The SLM process is optimized and fully characterized for different biocompatible metal alloys, such as: TiAl6V4 and CoCrMo. The potential of SLM as medical manufacturing technique is proved by a developed procedure to fabricate frameworks for complex dental prostheses.

  9. Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in milk. (United States)

    Wang, Meng; Yang, Junjie; Gai, Zhongtao; Huo, Shengnan; Zhu, Jianhua; Li, Jun; Wang, Ranran; Xing, Sheng; Shi, Guosheng; Shi, Feng; Zhang, Lei


    As a kind of zero-tolerance foodborne pathogens, Salmonella typhimurium poses a great threat to quality of food products and public health. Hence, rapid and efficient approaches to identify Salmonella typhimurium are urgently needed. Combined with PCR and fluorescence technique, real-time PCR (qPCR) and digital PCR (ddPCR) are regarded as suitable tools for detecting foodborne pathogens. To compare the effect between qPCR and ddPCR in detecting Salmonella typhimurium, a series of nucleic acid, pure strain culture and spiking milk samples were applied and the resistance to inhibitors referred in this article as well. Compared with qPCR, ddPCR exhibited more sensitive (10 -4 ng/μl or 10 2 cfu/ml) and less pre-culturing time (saving 2h). Moreover, ddPCR had stronger resistance to inhibitors than qPCR, yet absolute quantification hardly performed when target's concentration over 1ng/μl or 10 6 cfu/ml. This study provides an alternative strategy in detecting foodborne Salmonella typhimurium. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Cross-platform comparison of SYBR® Green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray Quality Control (MAQC study

    Directory of Open Access Journals (Sweden)

    Dial Stacey L


    Full Text Available Abstract Background The MicroArray Quality Control (MAQC project evaluated the inter- and intra-platform reproducibility of seven microarray platforms and three quantitative gene expression assays in profiling the expression of two commercially available Reference RNA samples (Nat Biotechnol 24:1115-22, 2006. The tested microarrays were the platforms from Affymetrix, Agilent Technologies, Applied Biosystems, GE Healthcare, Illumina, Eppendorf and the National Cancer Institute, and quantitative gene expression assays included TaqMan® Gene Expression PCR Assay, Standardized (Sta RT-PCR™ and QuantiGene®. The data showed great consistency in gene expression measurements across different microarray platforms, different technologies and test sites. However, SYBR® Green real-time PCR, another common technique utilized by half of all real-time PCR users for gene expression measurement, was not addressed in the MAQC study. In the present study, we compared the performance of SYBR Green PCR with TaqMan PCR, microarrays and other quantitative technologies using the same two Reference RNA samples as the MAQC project. We assessed SYBR Green real-time PCR using commercially available RT2 Profiler™ PCR Arrays from SuperArray, containing primer pairs that have been experimentally validated to ensure gene-specificity and high amplification efficiency. Results The SYBR Green PCR Arrays exhibit good reproducibility among different users, PCR instruments and test sites. In addition, the SYBR Green PCR Arrays have the highest concordance with TaqMan PCR, and a high level of concordance with other quantitative methods and microarrays that were evaluated in this study in terms of fold-change correlation and overlap of lists of differentially expressed genes. Conclusion These data demonstrate that SYBR Green real-time PCR delivers highly comparable results in gene expression measurement with TaqMan PCR and other high-density microarrays.

  11. Identification and quantification of gabbro cumulate partial melting in mafic igneous complexes (United States)

    Leuthold, J.


    In mafic magma chambers, olivine, plagioclase and clinopyroxene fractionate along the basaltic liquid line of descent. Ol-rich, troctolite and gabbro cumulates crystallize. Hot primitive magma sills are regularly injected into igneous complexes where they heat and partially melt surrounding hot rocks, percolate, hybridize and crystallize new and secondary phases (see Figure [1]). Here, I quantify the effect of gabbro cumulate partial melting and hybridization with invading primitive basalt using field observations, Cpx microtexture and core-rim geochemical profiles from the Rum sill complex (Scotland). I have run gabbro-basalt hybrid equilibrium and kinetic experiments to test the effect of gabbro assimilation on the basalt liquid and solid lines of descent. Rum poikilitic gabbro resorbed Cpx cores are overgrown by Cr-, Al-, Zr- and REE-depleted interstitial rim with high Mg#, Eu* and Sr* (see [2]). Plg is reversely zoned. Fractional crystallization fails to explain the combined dissolution texture and incoherent compatible and incompatible elements zoning. Gabbro cumulate partial melting produces a Cpx-depleted residue and a melt that is saturated in Cpx, depleted in Cr, Al, Zr and REE and with high SiO2 and Mg# contents [1]. REE-poor Cpx rim crystallized from a hybrid basalt-gabbro magma, despite a lower DREE. In picrite-gabbro kinetic experiments reacted at conditions where Ol, Plg and Cpx are stable in gabbro but only Ol is stable in primitive basalt (1210°C, NNO-2), gabbro Ol and Cpx are anhedral and reversely zoned and Plg is euhedral. In the reaction rim, Cpx is absent, Ol anhedral grains are small and Cr-Spl abundance strongly increased (to 1.8 vol%). Troctolite small-scale partial melting and hybridization with primitive basalt produces a hybrid Al-rich melt that is Spl over-saturated. Gabbro partial melting products can be very difficult to distinguish from cumulates crystallized along the basalt liquid line of descent, because of the limited

  12. Additive Manufacturing Processes: Selective Laser Melting, Electron Beam Melting and Binder Jetting-Selection Guidelines. (United States)

    Gokuldoss, Prashanth Konda; Kolla, Sri; Eckert, Jürgen


    Additive manufacturing (AM), also known as 3D printing or rapid prototyping, is gaining increasing attention due to its ability to produce parts with added functionality and increased complexities in geometrical design, on top of the fact that it is theoretically possible to produce any shape without limitations. However, most of the research on additive manufacturing techniques are focused on the development of materials/process parameters/products design with different additive manufacturing processes such as selective laser melting, electron beam melting, or binder jetting. However, we do not have any guidelines that discuss the selection of the most suitable additive manufacturing process, depending on the material to be processed, the complexity of the parts to be produced, or the design considerations. Considering the very fact that no reports deal with this process selection, the present manuscript aims to discuss the different selection criteria that are to be considered, in order to select the best AM process (binder jetting/selective laser melting/electron beam melting) for fabricating a specific component with a defined set of material properties.

  13. Additive Manufacturing Processes: Selective Laser Melting, Electron Beam Melting and Binder Jetting—Selection Guidelines (United States)

    Konda Gokuldoss, Prashanth; Kolla, Sri; Eckert, Jürgen


    Additive manufacturing (AM), also known as 3D printing or rapid prototyping, is gaining increasing attention due to its ability to produce parts with added functionality and increased complexities in geometrical design, on top of the fact that it is theoretically possible to produce any shape without limitations. However, most of the research on additive manufacturing techniques are focused on the development of materials/process parameters/products design with different additive manufacturing processes such as selective laser melting, electron beam melting, or binder jetting. However, we do not have any guidelines that discuss the selection of the most suitable additive manufacturing process, depending on the material to be processed, the complexity of the parts to be produced, or the design considerations. Considering the very fact that no reports deal with this process selection, the present manuscript aims to discuss the different selection criteria that are to be considered, in order to select the best AM process (binder jetting/selective laser melting/electron beam melting) for fabricating a specific component with a defined set of material properties. PMID:28773031

  14. Melt Electrospinning Writing of Highly Ordered Large Volume Scaffold Architectures. (United States)

    Wunner, Felix M; Wille, Marie-Luise; Noonan, Thomas G; Bas, Onur; Dalton, Paul D; De-Juan-Pardo, Elena M; Hutmacher, Dietmar W


    The additive manufacturing of highly ordered, micrometer-scale scaffolds is at the forefront of tissue engineering and regenerative medicine research. The fabrication of scaffolds for the regeneration of larger tissue volumes, in particular, remains a major challenge. A technology at the convergence of additive manufacturing and electrospinning-melt electrospinning writing (MEW)-is also limited in thickness/volume due to the accumulation of excess charge from the deposited material repelling and hence, distorting scaffold architectures. The underlying physical principles are studied that constrain MEW of thick, large volume scaffolds. Through computational modeling, numerical values variable working distances are established respectively, which maintain the electrostatic force at a constant level during the printing process. Based on the computational simulations, three voltage profiles are applied to determine the maximum height (exceeding 7 mm) of a highly ordered large volume scaffold. These thick MEW scaffolds have fully interconnected pores and allow cells to migrate and proliferate. To the best of the authors knowledge, this is the first study to report that z-axis adjustment and increasing the voltage during the MEW process allows for the fabrication of high-volume scaffolds with uniform morphologies and fiber diameters. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Proton NMR relaxation in hydrous melts

    International Nuclear Information System (INIS)

    Braunstein, J.; Bacarella, A.L.; Benjamin, B.M.; Brown, L.L.; Girard, C.


    Pulse and continuous wave NMR measurements are reported for protons in hydrous melts of calcium nitrate at temperatures between -4 and 120 0 C. Although measured in different temperature ranges, spin-lattice (T 1 ) and spin-spin (T 2 ) relaxation times appear to be nearly equal to each other and proportional to the self-diffusion coefficients of solute metal cations such as Cd 2+ . At temperatures near 50 0 C, mean Arrhenius coefficients Δ H/sub T 1 / (kcal/mol) are 7.9, 7.3, and 4.8, respectively, for melts containing 2.8, 4.0, and 8.0 moles of water per mole of calcium nitrate, compared to 4.6 kcal/mol for pure water. Temperature dependence of T 1 and T 2 in Ca(NO 3 ) 2 -2.8 H 2 O between -4 and 120 0 C are non-Arrhenius and can be represented by a Fulcher-type equation with a ''zero mobility temperature'' (T 0 ) of 225 0 K, close to the value of T 0 for solute diffusion, electrical conductance and viscosity. Resolution of the relaxation rates into correlation times for intramolecular (rotational) and intermolecular (translational) diffusional motion is discussed in terms of the Bloembergen-Purcell-Pound and more recent models for dipolar relaxation

  16. Biomass preservation in impact melt ejecta (United States)

    Howard, Kieren Torres; Bailey, Melanie J.; Berhanu, Deborah; Bland, Phil A.; Cressey, Gordon; Howard, Lauren E.; Jeynes, Chris; Matthewman, Richard; Martins, Zita; Sephton, Mark A.; Stolojan, Vlad; Verchovsky, Sasha


    Meteorites can have played a role in the delivery of the building blocks of life to Earth only if organic compounds are able to survive the high pressures and temperatures of an impact event. Although experimental impact studies have reported the survival of organic compounds, there are uncertainties in scaling experimental conditions to those of a meteorite impact on Earth and organic matter has not been found in highly shocked impact materials in a natural setting. Impact glass linked to the 1.2-km-diameter Darwin crater in western Tasmania is strewn over an area exceeding 400km2 and is thought to have been ejected by a meteorite impact about 800kyr ago into terrain consisting of rainforest and swamp. Here we use pyrolysis-gas chromatography-mass spectrometry to show that biomarkers representative of plant species in the local ecosystem--including cellulose, lignin, aliphatic biopolymer and protein remnants--survived the Darwin impact. We find that inside the impact glass the organic components are trapped in porous carbon spheres. We propose that the organic material was captured within impact melt and preserved when the melt quenched to glass, preventing organic decomposition since the impact. We suggest that organic material can survive capture and transport in products of extreme impact processing, at least for a Darwin-sized impact event.

  17. Do cracks melt their way through solids?

    International Nuclear Information System (INIS)

    Okamoto, P. R.


    Real-time, in situ fracture studies in the high-voltage electron microscope (HVEM) show that microscopically thin regions of amorphous NiTi form ahead of moving crack tips in the B2-NiTi intermetallic compound during tensile straining at temperatures equal to or below 600K. The upper cutoff temperature of 600K for this stress-induced melting (or amorphization) is identical to the upper cutoff temperatures reported in the literature for both heavy-ion-induced amorphization of the intermetallic NiTi and ion-beam-mixing-induced amorphization of Ni and Ti multilayer. These results, together with the fact that the higher crystallization temperatures (∼800K)of unrelaxed amorphous NiTi alloys obtained by rapid quenching can also be reduced to, but not lower than 600K, by heavy-ion irradiation, strongly suggest that structural relaxation processes enhanced or induced by dynamic atomic disordering allow the formation of a unique, fully-relaxed glassy state which is characterized by a unique isothermal crystallization temperature. We believe that this unique temperature is the Kauzmann glass-transition temperature, corresponding to the ideal glass having the same entropy as the crystalline state. As the glassy state with the lowest global free energy, the preferential formation of this ideal glass by disorder-induced amorphization processes can be understood as the most energetically-favored, kinetically-constrained melting response of crystalline materials driven far from equilibrium at low temperatures

  18. Synthesis of ammonia using sodium melt. (United States)

    Kawamura, Fumio; Taniguchi, Takashi


    Research into inexpensive ammonia synthesis has increased recently because ammonia can be used as a hydrogen carrier or as a next generation fuel which does not emit CO 2 . Furthermore, improving the efficiency of ammonia synthesis is necessary, because current synthesis methods emit significant amounts of CO 2 . To achieve these goals, catalysts that can effectively reduce the synthesis temperature and pressure, relative to those required in the Haber-Bosch process, are required. Although several catalysts and novel ammonia synthesis methods have been developed previously, expensive materials or low conversion efficiency have prevented the displacement of the Haber-Bosch process. Herein, we present novel ammonia synthesis route using a Na-melt as a catalyst. Using this route, ammonia can be synthesized using a simple process in which H 2 -N 2 mixed gas passes through the Na-melt at 500-590 °C under atmospheric pressure. Nitrogen molecules dissociated by reaction with sodium then react with hydrogen, resulting in the formation of ammonia. Because of the high catalytic efficiency and low-cost of this molten-Na catalyst, it provides new opportunities for the inexpensive synthesis of ammonia and the utilization of ammonia as an energy carrier and next generation fuel.

  19. Detection of Salmonella spp. Using a Generic and Differential FRET-PCR (United States)

    Zhang, Jilei; Wei, Lanjing; Kelly, Patrick; Freeman, Mark; Jaegerson, Kirsten; Gong, Jiansen; Xu, Bu; Pan, Zhiming; Xu, Chuanling; Wang, Chengming


    To facilitate the detection of Salmonella and to be able to rapidly and conveniently determine the species/subspecies present, we developed and tested a generic and differential FRET-PCR targeting their tetrathionate reductase response regulator gene. The differential pan-Salmonella FRET-PCR we developed successfully detected seven plasmids that contained partial sequences of S. bongori and the six S. enterica subspecies. The detection limit varied from ∼5 copies of target gene/per PCR reaction for S. enterica enterica to ∼200 for S. bongori. Melting curve analysis demonstrated a Tm of ∼68°C for S. enterica enterica, ∼62.5°C for S. enterica houtenae and S. enterica diarizonae, ∼57°C for S. enterica indica, and ∼54°C for S. bongori, S. enterica salamae and S. enterica arizonae. The differential pan-Salmonella FRET-PCR also detected and determined the subspecies of 4 reference strains and 47 Salmonella isolated from clinically ill birds or pigs. Finally, we found it could directly detect and differentiate Salmonella in feline (5/50 positive; 10%; one S. enterica salamae and 4 S. enterica enterica) and canine feces (15/114 positive; 13.2%; all S. enterica enterica). The differential pan-Salmonella FRET-PCR failed to react with 96 non-Salmonella bacterial strains. Our experiments show the differential pan-Salmonella FRET-PCR we developed is a rapid, sensitive and specific method to detect and differentiate Salmonella. PMID:24146814


    Directory of Open Access Journals (Sweden)

    Nicole SELLESKI


    Full Text Available Background Celiac disease is an autoimmune enteropathy triggered by the ingestion of gluten in genetically susceptible individuals. Genetic susceptibility is associated with two sets of alleles, DQA1*05 - DQB1*02 and DQA1*03 - DQB1*03:02, which code for class II MHC DQ2 and DQ8 molecules, respectively. Approximately 90%-95% of celiac patients are HLA-DQ2 positive, and half of the remaining patients are HLA-DQ8 positive. In fact, during a celiac disease diagnostic workup, the absence of these specific DQA and DQB alleles has a near perfect negative predictive value. Objective Improve the detection of celiac disease predisposing alleles by combining the simplicity and sensitivity of real-time PCR (qPCR and melting curve analysis with the specificity of sequence-specific primers (SSP. Methods Amplifications of sequence-specific primers for DQA1*05 (DQ2, DQB1*02 (DQ2, and DQA1*03 (DQ8 were performed by the real time PCR method to determine the presence of each allele in independent reactions. Primers for Human Growth Hormone were used as an internal control. A parallel PCR-SSP protocol was used as a reference method to validate our results. Results Both techniques yielded equal results. From a total of 329 samples the presence of HLA predisposing alleles was determined in 187 (56.8%. One hundred fourteen samples (61% were positive for a single allele, 68 (36.3% for two alleles, and only 5 (2.7% for three alleles. Conclusion Results obtained by qPCR technique were highly reliable with no discordant results when compared with those obtained using PCR-SSP.

  1. Ferric iron partitioning between pyroxene and melt during partial melting of the Earth's upper mantle (United States)

    Rudra, A.; Hirschmann, M. M.


    The oxidation state of the Earth's mantle influences melt production, volatile behavior, partitioning of key trace elements and possible saturation of alloy at depth. Average Fe3+/FeT ratios in MORBs indicate oxygen fugacitiy of the source regions is close to QFM, in contrast to a 3 log unit variation of fO2 recorded by abyssal peridotites. Quantification of the relationship between basalt and source Fe3+/FeT, oxygen fugacity, and melting requires constraints on Fe3+ partitioning between melt and mantle minerals and in particular the principal Fe3+ host, pyroxene. McCanta et al. (2004) investigated valence dependent partitioning of Fe between Martian ferroan pigeonites and melt, but behavior in terrestrial pyroxene compositions relevant to MORB petrogenesis has not been investigated. We are conducting 1 atm controlled fO2 experiments over 4 log unit variation of fO2 between ΔQFM = 2.5 to -1.5 to grow pyroxenes of variable tetrahedral and octahedral cationic population from andesitic melts of varying Mg#, alumina and alkali content. Dynamic crystallization technique facilitates growth of pyroxene crystals (100-200 um) that EPMA analyses show to be compositionally homogeneous and in equilibrium with the melt. Fe3+/FeT ratio of the synthetic pyroxenes have been analyzed by XAFS spectroscopy at the APS (GSECARS) synchrotron. To quantify the x-ray anisotropy in pyroxenes, we collected Fe K-edge XAFS spectra of oriented natural single crystals for a wide range compositions whose Fe3+/FeT ratios we determined by Mossbauer spectroscopy. We have collected both XANES and EXAFS spectral regions spanning from 7020-7220 eV to explore predictive capabilities of different spectral regions about ferric iron concentration and site occupancy. Our results will document the Fe3+ compatibility in pyroxenes of different compositions under a variety of fO2 conditions, which in turn will better constrain the interrelationship between mantle redox and melting.

  2. High-resolution melting analysis for prenatal diagnosis of beta-thalassemia in northern Thailand. (United States)

    Charoenkwan, Pimlak; Sirichotiyakul, Supatra; Phusua, Arunee; Suanta, Sudjai; Fanhchaksai, Kanda; Sae-Tung, Rattika; Sanguansermsri, Torpong


    High-resolution melting (HRM) analysis is a rapid mutation analysis which assesses the pattern of reduction of fluorescence signal after subjecting the amplified PCR product with saturated fluorescence dye to an increasing temperature. We used HRM analysis for prenatal diagnosis of beta-thalassemia disease in northern Thailand. Five PCR-HRM protocols were used to detect point mutations in five different segments of the beta-globin gene, and one protocol to detect the 3.4 kb beta-globin deletion. We sought to characterize the mutations in carriers and to enable prenatal diagnosis in 126 couples at risk of having a fetus with beta-thalassemia disease. The protocols identified 18 common mutations causing beta-thalassemia, including the rare codon 132 (A-T) mutation. Each mutation showed a specific HRM pattern and all results were in concordance with those from direct DNA sequencing or gap-PCR methods. In cases of beta-thalassemia disease resulting from homozygosity for a mutation or compound heterozygosity for two mutations on the same amplified segment, the HRM patterns were different to those of a single mutation and were specific for each combination. HRM analysis is a simple and useful method for mutation identification in beta-thalassemia carriers and prenatal diagnosis of beta-thalassemia in northern Thailand.

  3. A high-resolution melting analysis for genotyping urogenital Chlamydia trachomatis. (United States)

    Li, Jian-Hong; Yin, Yue-Ping; Zheng, He-Ping; Zhong, Ming-Ying; Peng, Rui-Rui; Wang, Baoxi; Chen, Xiang-Sheng


    We have developed a high-resolution melting analysis (HRMA) for the genotyping of Chlamydia trachomatis and applied it specifically to the 11 sexually transmitted infection-related genotypes: D through K and L1 through L3. The variable segment 2 (VS2) was selected as the target for HRMA genotype identification. Eleven C. trachomatis genotypes were amplified by a nested real-time polymerase chain reaction (PCR) in the presence of the LCGreen saturating dye and showed no cross-reaction with 10 pathogenic bacteria or commensals from urogenital tract. The detection limit of HRMA method was 100 elementary bodies (EB)/mL. All of the 11 genotypes can be distinguished from each other by following an HRMA workflow. Genotype F, G, H, I, J, K, L2, and L3 could be directly identified from each other, whereas D, E, and L1 could be distinguished from each other by a second analysis with fewer curves or by heteroduplex formation with a known reference strain. In the validation panel of 36 C. trachomatis-positive urogenital samples genotyped by VS1-VS2 sequencing, nested real-time VS2 PCR followed by HRMA was able to discriminate between all samples correctly. This assay requires no fluorescence-labeled probes or separate post-PCR analysis and provides a simple and rapid approach for genotyping the C. trachomatis strains that are the most commonly sexually transmitted. Copyright © 2010 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Azar D. Khosravi


    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.

  5. The potential advantages of digital PCR for clinical virology diagnostics. (United States)

    Hall Sedlak, Ruth; Jerome, Keith R


    Digital PCR (dPCR), a new nucleic acid amplification technology, offers several potential advantages over real-time or quantitative PCR (qPCR), the current workhorse of clinical molecular virology diagnostics. Several studies have demonstrated dPCR assays for human cytomegalovirus or HIV, which give more precise and reproducible results than qPCR assays without sacrificing sensitivity. Here we review the literature comparing dPCR and qPCR performance in viral molecular diagnostic assays and offer perspective on the future of dPCR in clinical virology diagnostics.

  6. Premixing and steam explosion phenomena in the tests with stratified melt-coolant configuration and binary oxidic melt simulant materials

    Energy Technology Data Exchange (ETDEWEB)

    Kudinov, Pavel, E-mail:; Grishchenko, Dmitry, E-mail:; Konovalenko, Alexander, E-mail:; Karbojian, Aram, E-mail:


    Highlights: • Steam explosion in stratified melt-coolant configuration is studied experimentally. • Different binary oxidic melt simulant materials were used. • Five spontaneous steam explosions were observed. • Instability of melt-coolant interface and formation of premixing layer was observed. • Explosion strength is influenced by melt superheat and water subcooling. - Abstract: Steam explosion phenomena in stratified melt-coolant configuration are considered in this paper. Liquid corium layer covered by water on top can be formed in severe accident scenarios with (i) vessel failure and release of corium melt into a relatively shallow water pool; (ii) with top flooding of corium melt layer. In previous assessments of potential energetics in stratified melt-coolant configuration, it was assumed that melt and coolant are separated by a stable vapor film and there is no premixing prior to the shock wave propagation. This assumption was instrumental for concluding that the amount of energy that can be released in such configuration is not of safety importance. However, several recent experiments carried out in Pouring and Under-water Liquid Melt Spreading (PULiMS) facility with up to 78 kg of binary oxidic corium simulants mixtures have resulted in spontaneous explosions with relatively high conversion ratios (order of one percent). The instability of the melt-coolant interface, melt splashes and formation of premixing layer were observed in the tests. In this work, we present results of experiments carried out more recently in steam explosion in stratified melt-coolant configuration (SES) facility in order to shed some light on the premixing phenomena and assess the influence of the test conditions on the steam explosion energetics.

  7. Introduction of Electrostatically Charged Particles into Metal Melts (United States)

    Kudryashova, Olga; Vorozhtsov, Sergey; Stepkina, Maria; Khrustalev, Anton


    One of the possible methods to produce composite alloys with improved mechanical characteristics is the modification of metal melts using submicron- or nanosized particles. Different methods, like ultrasonic or vibration processing, have been used to introduce these particles into the metal melt. The introduction of particles into a metal melt is prevented by the poor wettability of the liquid metal. The present study explores the use of electrostatic charge for increasing the wettability of the particles and preventing their agglomeration. The wettability of electrostatically charged particles by the metal melt under the impact of ultrasound has been studied. The relationships between the impact time and the physical and chemical properties of the particles and the melt along with the characteristics of the acoustic radiation have been studied. It was experimentally demonstrated that the introduction of electrostatically charged particles into the metal melt reduces the porosity and the crystal grain size.

  8. Reaction between YBCO/Ag superconductor and melted silver

    International Nuclear Information System (INIS)

    Maeda, Junya; Izumi, Teruo; Shiohara, Yuh


    In order to study the feasibility of applying liquid phase processing using melted silver to fabricate YBCO/silver contacts, the chemical reaction at the boundaries between the Y-Ba-Cu-O (YBCO) superconducting matrix and melted silver was observed. YBCO superconducting current leads prepared by the unidirectional solidification method were dipped into liquid silver melted by an electric furnace. Copper-poor layers were formed at the boundary between the YBCO matrix and melted silver, although the temperatures were lower than the YBCO/Ag melting temperature (∼970 deg. C). It was considered that melted silver took up copper from the YBa 2 Cu 3 O x (Y123) crystal, and it is considered that the phase equilibrium at the boundary between the YBCO matrix and molten silver was changed from Y 2 Ba 1 Cu 1 O 5 (Y211)-Y123 to another phase. (author)

  9. Influence of gas-generation on melt/concrete interaction

    International Nuclear Information System (INIS)

    Powers, D.A.


    Gases formed during the interaction of a high-temperature melt with concrete are shown to stem from the thermal dehydration and decarboxylation of the concrete. The kinetics of these decomposition reactions are described. Gases within the melt cause an apparent swelling of the melt. The observed swelling is not easily correlated to the rate of gas evolution. Metallic melts cause CO 2 /CO and H 2 O liberated from the melt to be reduced to CO and hydrogen. When these gases escape from the melt they assist in aerosol formation. As the gases cool they react along a pathway whose oxygen fugacity is apparently buffered by the iron-Wuestite equilibrium. Methane is a product of the gas-phase reaction. (orig./HP) [de

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

    International Nuclear Information System (INIS)

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


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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  12. Calibrated user-friendly reverse transcriptase-PCR assay

    DEFF Research Database (Denmark)

    Bor, M V; Sørensen, B S; Rammer, P


    We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CURT...

  13. Increase of Unsaturated Fatty Acids (Low Melting Point) of Broiler Fatty Waste Obtained Through Staphylococcus xylosus Fermentation. (United States)

    Marques, Roger V; Duval, Eduarda H; Corrêa, Luciara B; Corrêa, Érico K


    The increasing rise in the production of meat around the world causes a significant generation of agro-industrial waste--most of it with a low value added. Fatty wastes have the potential of being converted into biodiesel, given the overcome of technological and economical barriers, as well as its presentation in solid form. Therefore, the aim of this work was to investigate the capacity of Staphylococcus xylosus strains to modify the chemical structure of chicken fatty wastes intending to reduce the melting points of the wastes to mild temperatures, thereby breaking new ground in the production of biodiesel from these sources in an economically attractive and sustainable manner. The effects in time of fermentation and concentration of the fat in the medium were investigated, assessing the melting point and profile of fatty acids. The melting temperature showed a decrease of approximately 22 °C in the best operational conditions, due to reduction in the content of saturated fatty acids (high melting point) and increase of unsaturated fatty acids (low melting point).

  14. Composition profile determination in isomorphous binary alloys

    International Nuclear Information System (INIS)

    An, C.Y.; Bandeira, I.N.


    The inhomogeneity along the growth axis of the pseudo-binary alloys is due to the segregation of the solute which will be mixed in the melt due to convective and diffusive flows. A process for determination of the exact composition profile by measurements of the crystal density, for alloys of the type A sub(1-x) B sub(x), is shown. (Author) [pt

  15. Differential melt scaling for oblique impacts on terrestrial planets (United States)

    Abramov, Oleg; Wong, Stephanie M. Wong; Kring, David A. Kring


    Analytical estimates of melt volumes produced by a given projectile and contained in a given impact crater are derived as a function of impact velocity, impact angle, planetary gravity, target and projectile densities, and specific internal energy of melting. Applications to impact events and impact craters on the Earth, Moon, and Mars are demonstrated and discussed. The most probable oblique impact (45°) produces ∼1.6 times less melt volume than a vertical impact, and ∼1.6 and 3.7 times more melt volume than impacts with 30° and 15° trajectories, respectively. The melt volume for a particular crater diameter increases with planetary gravity, so a crater on Earth should have more melt than similar-size craters on Mars and the Moon. The melt volume for a particular projectile diameter does not depend on gravity, but has a strong dependence on impact velocity, so the melt generated by a given projectile on the Moon is significantly larger than on Mars. Higher surface temperatures and geothermal gradients increase melt production, as do lower energies of melting. Collectively, the results imply thinner central melt sheets and a smaller proportion of melt particles in impact breccias on the Moon and Mars than on Earth. These effects are illustrated in a comparison of the Chicxulub crater on Earth, linked to the Cretaceous–Tertiary mass extinction, Gusev crater on Mars, where the Mars Exploration Rover Spirit landed, and Tsiolkovsky crater on the Moon. The results are comparable to those obtained from field and spacecraft observations, other analytical expressions, and hydrocode simulations.

  16. Melting tests for recycling of radioactive metal wastes

    International Nuclear Information System (INIS)

    Nakamura, Hisashi; Kanazawa, Katsuo; Fujiki, Kazuo


    To allow the future recycling of decommissioning wastes to promote smoothly, melting tests were conducted using metal wastes and simulated wastes with radioisotopes. The test results indicate that the transfer behavior of radionuclides during melting is basically understood by considering the volatility and oxidizable tendency of each radionuclide. The partitioning of some radionuclides into products was influenced by the melting process of wastes. The radioactivity distribution in ingots was uniform regardless of the kinds of radionuclide. (author)

  17. Observation of a prewetting transition during surface melting of caprolactam (United States)

    Chandavarkar, Sumant; Geertman, Rob M.; de Jeu, Wim H.


    The surface-induced melting of the closed-packed (100) face of the anisotropic molecular crystal caprolactam has been studied using x-ray reflectivity. A thin-to-thick film prewetting transition is observed at about 13 K below the bulk melting point. Only above this transition does the thickness of the quasiliquid layer increase continuously with temperature. We speculate that initially the surface melting proceeds via layering transitions.

  18. Relationships between melting point and boiling point of organic compounds

    Energy Technology Data Exchange (ETDEWEB)

    Yalkowsky, S.H.; Krzyzaniak, J.F.; Myrdal, P.B. (Univ. of Arizona, Tucson, AZ (United States). College of Pharmacy)


    Relationships between melting point and boiling point are shown to be dependent upon the molecular symmetry number and a modified count of the total number of atoms in the molecule. Using the above relationships, the boiling and melting points of nearly 1,000 non-hydrogen-bonding organic compounds have been correlated. The correlations for boiling point and melting point have root mean square errors of 28 and 36 C, respectively.

  19. Retrograde Melting and Internal Liquid Gettering in Silicon

    Energy Technology Data Exchange (ETDEWEB)

    Hudelson, Steve; Newman, Bonna K.; Bernardis, Sarah; Fenning, David P.; Bertoni, Mariana I.; Marcus, Matthew A.; Fakra, Sirine C.; Lai, Barry; Buonassisi, Tonio


    Retrograde melting (melting upon cooling) is observed in silicon doped with 3d transition metals, via synchrotron-based temperature-dependent X-ray microprobe measurements. Liquid metal-silicon droplets formed via retrograde melting act as efficient sinks for metal impurities dissolved within the silicon matrix. Cooling results in decomposition of the homogeneous liquid phase into solid multiple-metal alloy precipitates. These phenomena represent a novel pathway for engineering impurities in semiconductor-based systems.

  20. Nitrogen distribution between aqueous fluids and silicate melts (United States)

    Li, Yuan; Huang, Ruifang; Wiedenbeck, Michael; Keppler, Hans


    The partitioning of nitrogen between hydrous fluids and haplogranitic, basaltic, or albitic melts was studied at 1-15 kbar, 800-1200 °C, and oxygen fugacities (fO2) ranging from the Fe-FeO buffer to 3log units above the Ni-NiO buffer. The nitrogen contents in quenched glasses were analyzed either by electron microprobe or by secondary ion mass spectrometry (SIMS), whereas the nitrogen contents in fluids were determined by mass balance. The results show that the nitrogen content in silicate melt increases with increasing nitrogen content in the coexisting fluid at given temperature, pressure, and fO2. Raman spectra of the silicate glasses suggest that nitrogen species change from molecular N2 in oxidized silicate melt to molecular ammonia (NH3) or the ammonium ion (NH4+) in reduced silicate melt, and the normalized Raman band intensities of the nitrogen species linearly correlate with the measured nitrogen content in silicate melt. Elevated nitrogen contents in silicate melts are observed at reduced conditions and are attributed to the dissolution of NH3/NH4+. Measured fluid/melt partition coefficients for nitrogen (DNfluid/ melt) range from 60 for reduced haplogranitic melts to about 10 000 for oxidized basaltic melts, with fO2 and to a lesser extent melt composition being the most important parameters controlling the partitioning of nitrogen. Pressure appears to have only a minor effect on DNfluid/ melt in the range of conditions studied. Our data imply that degassing of nitrogen from both mid-ocean ridge basalts and arc magmas is very efficient, and predicted nitrogen abundances in volcanic gases match well with observations. Our data also confirm that nitrogen degassing at present magma production rates is insufficient to accumulate the atmosphere. Most of the nitrogen in the atmosphere must have degassed very early in Earth's history and degassing was probably enhanced by the oxidation of the mantle.

  1. Melt electrospinning vs. solution electrospinning: A comparative study of drug-loaded poly (ε-caprolactone) fibres. (United States)

    Lian, He; Meng, Zhaoxu


    Curcumin-loaded poly (ε-caprolactone) (PCL) fibres prepared by melt and solution electrospinning methods were both fabricated to investigate their difference in characterization and drug release behaviour. The increasing curcumin content did not influence the morphologies of melt electrospun fibre, but enhanced the range of diameter distribution of solution electrospun fibre owing to the curcumin aggregates in the spinning solution which disturbed the stability of jet. Moreover, a large amount of curcumin with amorphous state could be loaded in the melt electrospun fibre. Whereas the limited solubility of curcumin in the solvent led to the drug aggregates dispersing within the solution electrospun fibre. In addition, the melt electrospun fibres had low drug release rate without burst release on the profiles due to the high crystallinity in the fibre, but high drug release rate and burst release occurred on the release profiles of the solution electrospun fibres because of their low crystallinity, porous structure and roughness surface. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. A naked-eye colorimetric "PCR developer" (United States)

    Valentini, Paola; Pompa, Pier Paolo


    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated market of several billion dollars/year. Nevertheless, PCR still relies on the laborious, time-consuming, and multi-step gel electrophoresis-based detection, which includes gel casting, electrophoretic run, gel staining, and gel visualization. In this work, we propose a "PCR developer", namely a universal one-step, one-tube method, based on controlled aggregation of gold nanoparticles (AuNPs), to detect PCR products by naked eye in few minutes, with no need for any instrumentation. We demonstrated the specificity and sensitivity of the PCR developer on different model targets, suitable for a qualitative detection in real-world diagnostics (i.e., gene rearrangements, genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  3. Real-time PCR using SYBR Green for the detection of Shigella spp. in food and stool samples. (United States)

    Mokhtari, W; Nsaibia, S; Gharbi, A; Aouni, M


    Shigella spp are exquisitely fastidious Gram negative organisms that frequently get missed in the detection by traditional culture methods. For this reason, this work has adapted a classical PCR for detection of Shigella in food and stool specimens to real-time PCR using the SYBR Green format. This method follows a melting curve analysis to be more rapid and provide both qualitative and quantitative data about the targeted pathogen. A total of 117 stool samples with diarrhea and 102 food samples were analyzed in Public Health Regional Laboratory of Nabeul by traditional culture methods and real-time PCR. To validate the real-time PCR assay, an experiment was conducted with both spiked and naturally contaminated stool samples. All Shigella strains tested were ipaH positive and all non-Shigella strains yielded no amplification products. The melting temperature (T(m) = 81.5 ± 0.5 °C) was consistently specific for the amplicon. Correlation coefficients of standard curves constructed using the quantification cycle (C(q)) versus copy numbers of Shigella showed good linearity (R² = 0.995; slope = 2.952) and the minimum level of detection was 1.5 × 10³ CFU/g feces. All food samples analyzed were negative for Shigella by standard culture methods, whereas ipaH was detected in 8.8% culture negative food products. Moreover, the ipaH specific PCR system increased the detection rate over that by culture alone from 1.7% to 11.1% among patients with diarrhea. The data presented here shows that the SYBR Green I was suitable for use in the real-time PCR assay, which provided a specific, sensitive and efficient method for the detection and quantification of Shigella spp in food and stool samples. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Biophysical Profile (United States)

    ... and pregnancy High-risk pregnancy Biophysical profile About Advertisement Mayo Clinic does not endorse companies or products. ... a Job Site Map About This Site Twitter Facebook Google YouTube Pinterest Mayo Clinic is a not- ...

  5. Erosion of melt layers developed during a plasma disruption

    International Nuclear Information System (INIS)

    Hassanein, A.; Konkashbaev, A.; Konkashbaev, I.


    Material erosion of plasma-facing components during a tokamak disruption is a serious problem that limits reactor operation and economical reactor lifetime. In particular, metallic low-Z components such as Be will be subjected to severe melting during disruptions and edge localized modes (ELMs). Loss of the developed melt layer will critically shorten the lifetime of these components, severely contaminate the plasma, and seriously inhibit successful and reliable operation of the reactor. In this study mechanisms responsible for melt-layer loss during a disruption are modeled and evaluated. Implications of melt-layer loss on the performance of metallic facing components in the reactor environment are discussed. (orig.)

  6. Constraints on melt migration in the Earth's upper mantle (United States)

    Garapic, Gordana

    Melting and melt segregation are key processes in the geochemical evolution of the Earth. However, mechanism and time scale of melt transport from the source to the surface are still not well understood and are dependent on the grain-scale distribution of melt. A related question is the retention of melt in partially molten regions of the Earth's upper mantle. Seismic observations from mid-ocean ridges (MOR) and subduction zones are interpreted to show in-situ melt contents up to 3%, while geochemical observations from MOR basalts are inferred to indicate very efficient extraction of melt (porosities of order of 0.1%). Earlier theoretical models of the melt distribution were based on the balance of surface tension between melt and uniform crystalline grains, predicting a simple network of melt along three-grain edges. Analyses of experimentally produced samples of olivine and basaltic melt show that the melt geometry is much more complex, and includes wetted two-grain boundaries. I reconstructed the 3-D model of melt geometry of two experimentally produced samples by serial sectioning and rendering of the pore space which demonstrates for the first time that melt exists in thin layers on two-grain boundaries. This confirms the inferences from previous 2-D observations and has significant implications for physical properties of partially molten regions, for example seismic velocities and attenuation. The wetted two-grain boundaries are inferred to be a consequence of continuous grain growth. Due to the complexity of the 3-D melt geometry the permeability of partially molten rocks can not be predicted from simple models. I therefore investigated the permeability as a function of porosity for both synthetic and experimentally determined pore geometries using a lattice-Boltzmann method. The calculated permeability is not a simple function of porosity, but increases rapidly at a critical fraction of wetted two-grain boundaries. To extrapolate the experimentally based

  7. Isothermal Gas assisted displacement of a polystyrene melt

    DEFF Research Database (Denmark)

    Eriksson, Torbjörn Gerhard; Rasmussen, Henrik K.


    Isothermal gas displacements of a polystyrene melt (shaped as circular cylinder with a radius of 2.5mm) placed inside a circular steel annulus were performed. A flow valve ensures a constant flow rate and rotational symmetric flow during the displacement. The experiments show an increase...... in the steady fractional coverage above a Newtonian level at very low Deborah numbers. At higher Deborah numbers, where the melt behaves as an entangled melt system, the steady fractional coverage decreases. The fractional coverage is the fraction of melt left in the cylinder during steady displacement...

  8. Investigation of the core melt accident in light water reactors

    International Nuclear Information System (INIS)

    Koerber, H.


    In the thesis the core melt accident, heating up and collapsing of the reactor core were investigated. The most important parameters of influence were found and their effect on the development of the accident were shown. A causal diagram was developed representing the great number of events occurring in the course of the core melt accident as well as their mutual dependences. Models were developed and applied for a detailed description of the collapse process, melting of materials, heat and material transport at flow-off of the melted mass and for taking into account steam blocking in the destroyed core sections. (orig.) [de

  9. Does buoyancy matter in the melting dynamics of ice? (United States)

    Guo, Jicheng; Ordu, Mustafa; Basu, Soumendra; Bird, James


    Ice in a horizontal cylindrical container will melt when placed in a sufficient warm environment. Because of the density difference between the ice and the continuously forming water, the ice can rise close to the boundary, separated by a thin gap of water. The melting dynamics of the ice appear qualitatively similar to the evaporation of a drop under Leidenfrost conditions; however, the extent of the analogy is unclear. Here we investigate the melting dynamics of ice in thin-walled cylindrical containers. Through a combination of experiments and physical modeling, we identify a characteristic melting time and gap thickness, which we compare to evaporating droplets.

  10. Experimental study of thermocapillary convection in a germanium melt (United States)

    Gorbunov, Leonid A.


    The present paper is dedicated to the experimental investigation of thermocapillary convection (TCC) in semiconductor melts. The investigation showed that in the process of single crystal growth under terrestrial conditions TCC could be compared to thermogravity convection (TGC) for a number of semiconductor melts such as Ge, Si, GaAs. But in comparatively thin layers with H container radius) it can dominate over TGC. The experiments were conducted with a Ge melt. Oxide particle tracers were used to measure the melt motion rate. The results obtained emphasize the significance of TCC in the process of single crystal growth under terrestrial conditions.

  11. Surface reconstruction precursor to melting in Au309 clusters

    Directory of Open Access Journals (Sweden)

    Fuyi Chen


    Full Text Available The melting of gold cluster is one of essential properties of nanoparticles and revisited to clarify the role played by the surface facets in the melting transition by molecular dynamics simulations. The occurrence of elaborate surface reconstruction is observed using many-body Gupta potential as energetic model for 309-atom (2.6 nm decahedral, cuboctahedral and icosahedral gold clusters. Our results reveal for the first time a surface reconstruction as precursor to the melting transitions. The surface reconstruction lead to an enhanced melting temperature for (100 faceted decahedral and cuboctahedral cluster than (111 faceted icosahedral gold cluster, which form a liquid patch due to surface vacancy.

  12. Electrochemistry of the Oxofluoro Complexes of Boron in Fluoride Melts

    DEFF Research Database (Denmark)

    Polyakova, L.P.; Bukatova, G.A.; Polyakov, E.G.


    Electrochemical behavior of oxofluoro complexes of boron, synthesized both in situ in FLINAK melt and added into the melt as Na3B3O3F6 compound, was by linear voltammetry within the range of 570-750 oC. It was shown that in lower part of this range the electrochemical reduction of BOF2- complexes...... energy of 61.6 kJ/mol. Study of the thermal stability of boron containing oxofluoro melts showed that O/B ratio changes in time due to evaporation of BF3. As a result borate complexes emerge in the melt alongside with oxofluoro ones....

  13. Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing Escherichia coli (United States)

    Reischl, Udo; Youssef, Mohammad T.; Kilwinski, Jochen; Lehn, Norbert; Zhang, Wen Lan; Karch, Helge; Strockbine, Nancy A.


    PCR assays have proved useful for detecting and characterizing Shiga toxin-producing Escherichia coli (STEC). Recent advances in PCR technology have facilitated the development of real-time fluorescence PCR assays with greatly reduced amplification times and improved methods for the detection of amplified target sequences. We developed and evaluated two such assays for the LightCycler instrument: one that simultaneously detects the genes for Shiga toxins 1 and 2 (stx1 and stx2) and another that simultaneously detects the genes for intimin (eae) and enterohemolysin (E-hly). Amplification and sequence-specific detection of the two target genes were completed within 60 min. Findings from the testing of 431 STEC isolates of human and animal origin, 73 isolates of E. coli negative for stx genes, and 118 isolates of other bacterial species with the LightCycler PCR (LC-PCR) assays were compared with those obtained by conventional block cycler PCR analysis. The sensitivities and specificities of the LC-PCR assays were each 100% for the stx1, eae, and E-hly genes and 96 and 100%, respectively, for the stx2 gene. No stx2 genes were detected from 10 stx2f-positive isolates because of significant nucleotide differences in their primer annealing regions. Melting curve analyses of the amplified Shiga toxin genes revealed sequence variation within each of the tested genes that correlated with described and novel gene variants. The performance characteristics of the LC-PCR assays, such as their speed, detection method, and the potential subtyping information available from melting curve analyses, make them attractive alternatives to block cycler PCR assays for detecting and characterizing STEC strains. PMID:12089277

  14. Optimization of the Divergent method for genotyping single nucleotide variations using SYBR Green-based single-tube real-time PCR. (United States)

    Gentilini, Fabio; Turba, Maria E


    A novel technique, called Divergent, for single-tube real-time PCR genotyping of point mutations without the use of fluorescently labeled probes has recently been reported. This novel PCR technique utilizes a set of four primers and a particular denaturation temperature for simultaneously amplifying two different amplicons which extend in opposite directions from the point mutation. The two amplicons can readily be detected using the melt curve analysis downstream to a closed-tube real-time PCR. In the present study, some critical aspects of the original method were specifically addressed to further implement the technique for genotyping the DNM1 c.G767T mutation responsible for exercise-induced collapse in Labrador retriever dogs. The improved Divergent assay was easily set up using a standard two-step real-time PCR protocol. The melting temperature difference between the mutated and the wild-type amplicons was approximately 5°C which could be promptly detected by all the thermal cyclers. The upgraded assay yielded accurate results with 157pg of genomic DNA per reaction. This optimized technique represents a flexible and inexpensive alternative to the minor grove binder fluorescently labeled method and to high resolution melt analysis for high-throughput, robust and cheap genotyping of single nucleotide variations. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Identification of genes showing differential expression profile ...

    Indian Academy of Sciences (India)

    in pig with genetic propensity for higher growth rate were identified by sequence analysis of 12 differentially expressed clones selected by differential screening following the generation of the subtracted cDNA population. Real-time PCR analysis con- firmed difference in expression profiles of the identified genes in ...

  16. Molecular characterization of Salmonella isolates by REP-PCR and RAPD analysis. (United States)

    Albufera, U; Bhugaloo-Vial, P; Issack, M I; Jaufeerally-Fakim, Y


    Eighteen Salmonella isolates from both human and food (non-human) sources (fish, meat, and poultry) were characterized using conventional culture methods, biochemical, serological, and molecular analyses. REP-PCR and RAPD produced DNA profiles for differentiation purposes. Enterobacterial repetitive intergenic consensus (ERIC), repetitive extragenic palindronic (REP) and BOXAIR primers were selected for REP-PCR and two arbitrary primers, namely OPP-16 and OPS-11 were used for RAPD to generate DNA fingerprints from the Salmonella isolates. REP-PCR method showed greater discriminatory power in differentiating closely related strains of the related strains of Salmonella and produced more complex banding patterns as compared with RAPD. A dendogram was constructed with both sets of profiles using SPSS Version 13.0 computer software and showed that most human isolates were separately clustered from the non-human isolates. Two of the human isolates were closely related to some of the non-human isolates. A good correlation was also observed between the serogrouping of the O antigen and the molecular profiles obtained from REP-PCR and RAPD data of the Salmonella isolates. The results of a principal coordinate analysis (PCA) corresponded to the clustering in the dendrogram.

  17. Profiling cancer

    DEFF Research Database (Denmark)

    Ciro, Marco; Bracken, Adrian P; Helin, Kristian


    In the past couple of years, several very exciting studies have demonstrated the enormous power of gene-expression profiling for cancer classification and prediction of patient survival. In addition to promising a more accurate classification of cancer and therefore better treatment of patients......, gene-expression profiling can result in the identification of novel potential targets for cancer therapy and a better understanding of the molecular mechanisms leading to cancer....

  18. Direct genotyping of single nucleotide polymorphisms in methyl metabolism genes using probe-free high-resolution melting analysis. (United States)

    Kristensen, Lasse S; Dobrovic, Alexander


    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.

  19. Use of the melting curve assay as a means for high-throughput quantification of Illumina sequencing libraries

    Directory of Open Access Journals (Sweden)

    Hiroshi Shinozuka


    Full Text Available Background. Multiplexed sequencing is commonly performed on massively parallel short-read sequencing platforms such as Illumina, and the efficiency of library normalisation can affect the quality of the output dataset. Although several library normalisation approaches have been established, none are ideal for highly multiplexed sequencing due to issues of cost and/or processing time. Methods. An inexpensive and high-throughput library quantification method has been developed, based on an adaptation of the melting curve assay. Sequencing libraries were subjected to the assay using the Bio-Rad Laboratories CFX ConnectTM Real-Time PCR Detection System. The library quantity was calculated through summation of reduction of relative fluorescence units between 86 and 95 °C. Results.PCR-enriched sequencing libraries are suitable for this quantification without pre-purification of DNA. Short DNA molecules, which ideally should be eliminated from the library for subsequent processing, were differentiated from the target DNA in a mixture on the basis of differences in melting temperature. Quantification results for long sequences targeted using the melting curve assay were correlated with those from existing methods (R2 > 0.77, and that observed from MiSeq sequencing (R2 = 0.82. Discussion.The results of multiplexed sequencing suggested that the normalisation performance of the described method is equivalent to that of another recently reported high-throughput bead-based method, BeNUS. However, costs for the melting curve assay are considerably lower and processing times shorter than those of other existing methods, suggesting greater suitability for highly multiplexed sequencing applications.

  20. Evaluation of the MeltPro TB/STR assay for rapid detection of streptomycin resistance in Mycobacterium tuberculosis. (United States)

    Zhang, Ting; Hu, Siyu; Li, Guoli; Li, Hui; Liu, Xiaoli; Niu, Jianjun; Wang, Feng; Wen, Huixin; Xu, Ye; Li, Qingge


    Rapid and comprehensive detection of drug-resistance is essential for the control of tuberculosis, which has facilitated the development of molecular assays for the detection of drug-resistant mutations in Mycobacterium tuberculosis. We hereby assessed the analytical and clinical performance of an assay for streptomycin-resistant mutations. MeltPro TB/STR is a closed-tube, dual-color, melting curve analysis-based, real-time PCR test designed to detect 15 streptomycin-resistant mutations in rpsL 43, rpsL 88, rrs 513, rrs 514, rrs 517, and rrs 905-908 of M. tuberculosis. Analytical studies showed that the accuracy was 100%, the limit of detection was 50-500 bacilli per reaction, the reproducibility in the form of Tm variation was within 1.0 °C, and we could detect 20% STR resistance in mixed bacterial samples. The cross-platform study demonstrated that the assay could be performed on six models of real-time PCR instruments. A multicenter clinical study was conducted using 1056 clinical isolates, which were collected from three geographically different healthcare units, including 709 STR-susceptible and 347 STR-resistant isolates characterized on Löwenstein-Jensen solid medium by traditional drug susceptibility testing. The results showed that the clinical sensitivity and specificity of the MeltPro TB/STR was 88.8% and 95.8%, respectively. Sequencing analysis confirmed the accuracy of the mutation types. Among all the 8 mutation types detected, rpsL K43R (AAG → AGG), rpsL K88R (AAG → AGG) and rrs 514 A → C accounted for more than 90%. We concluded that MeltPro TB/STR represents a rapid and reliable assay for the detection of STR resistance in clinical isolates. Copyright © 2014. Published by Elsevier Ltd.

  1. A One-Step Real-Time Multiplex PCR for Screening Y-Chromosomal Microdeletions without Downstream Amplicon Size Analysis (United States)

    Kozina, Viviana; Cappallo-Obermann, Heike; Gromoll, Jörg; Spiess, Andrej-Nikolai


    Backgound Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. Principal Findings The widely established “EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)” were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. Significance With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection. PMID:21887237

  2. Investigations of slip in capillary flow by laser-Doppler velocimetry and their relations to melt fracture (United States)

    Münstedt, Helmut


    Flow profiles within a slit capillary are measured by laser-Doppler velocimetry. They allow the direct determination of the slip velocity at the wall. It is demonstrated that the flow profile of the melt of a high density polyethylene (HDPE) already shows slip components at small shear rates. At high shear rates the slip is dominant and a plug flow is found. Furthermore, it is shown that the surface irregularity called "shark skin" is generated at the slit exit by the stretching of surface layers at pronounced elongational rates. These elongational rates are due to the differences between the flow velocities at the wall of the slit and those of the extruded strand. It is shown how "shark skin" may be avoided when the elongational rate is reduced by introducing slip of the melt in the slit using special additives.

  3. Process for forming retrograde profiles in silicon (United States)

    Weiner, Kurt H.; Sigmon, Thomas W.


    A process for forming retrograde and oscillatory profiles in crystalline and polycrystalline silicon. The process consisting of introducing an n- or p-type dopant into the silicon, or using prior doped silicon, then exposing the silicon to multiple pulses of a high-intensity laser or other appropriate energy source that melts the silicon for short time duration. Depending on the number of laser pulses directed at the silicon, retrograde profiles with peak/surface dopant concentrations which vary from 1-1e4 are produced. The laser treatment can be performed in air or in vacuum, with the silicon at room temperature or heated to a selected temperature.

  4. Spontaneous rotation of a melting ice disk (United States)

    Dorbolo, Stephane; Vandewalle, Nicolas; Darbois-Texier, Baptiste; Grasp Team

    Ice disks were released at the surface of a thermalised aluminium plate. The fusion of the ice creates a lubrication film between the ice disk and the plate. The situation is similar to the Leidenfrost effect reported for liquid droplet evaporating at the surface of a plate which temperature is above the boiling temperature of the liquid. An analogy is depicted between the Leidenfrost phenomenon and the rapid fusion of a solid at the contact of a hot plate. Similarly to Leidenfrost droplet, we observe that, while the ice disks were melting, the disks were very mobile: translation and rotation. SD acknowledges support from FNRS as Senior Research Associate. This research has been funded by the Interuniversity Attraction Pole Programme (IAP 7/38 MicroMAST) initiated by the Belgian Science Policy Office.

  5. Reduced energy consumption for melting in foundries

    Energy Technology Data Exchange (ETDEWEB)

    Skov-Hansen, S.


    By improving the gating technology in traditional gating systems it is possible to reduce the amount of metal to be re-melted, and hence reduce the energy consumption for melting in foundries. Traditional gating systems are known for a straight tapered down runner a well base and 90 deg. bends in the runner system. In the streamlined gating systems there are no sharp changes in direction and a large effort is done to confine and control the flow of the molten metal during mould filling. Experiments in real production lines have proven that using streamlined gating systems improves yield by decreasing the poured weight compared to traditional layouts. In a layout for casting of valve housings in a vertically parted mould the weight of the gating system was reduced by 1,1kg which is a 20% weight reduction for the gating system. In a layout for horizontally parted moulds the weight of the gating system has been reduced by 3,7kg which is a weight reduction of 60% for the gating system. The experiments casting valve housings in ductile iron also proved that it is possible to lower the pouring temperature from 1400 deg. C to 1300 deg. C without the risk of cold runs. Glass plate fronted moulds have been used to study the flow of melt during mould filling. These experiments have also been used for studying the flow pattern when ceramic filters are used. The thorough study of the use of filters revealed that the metal passing through the filter is divided into a number of small jets. This proves that filters do not have the claimed positive effect on the flow of metal. The volumes necessary on either side of the filter is not filled till a backpressure is build up and results in formation of pressure shocks when backfilled. These pressure shocks result in more turbulence inside the casting than the same gating system with no filter. Not using filters can mean a reduction in poured weight of 0,6kg. To examine if the experiments using glass plate fronted moulds give

  6. Antibacterial Titanium Produced Using Selective Laser Melting (United States)

    Macpherson, Andrew; Li, Xiaopeng; McCormick, Paul; Ren, Ling; Yang, Ke; Sercombe, Timothy B.


    Titanium and titanium alloys used in current medical and dental applications do not possess antibacterial properties, and therefore, postoperative infection remains a significant risk. Recently, the addition of silver and copper to conventional biomaterials has been shown to produce a material with good antibacterial properties. In this article, we investigate selective laser melting as a method of producing antibacterial Ti-6Al-4V containing elemental additions of Cu or Ag. The addition of Ag had no effect on the microstructure or strength, but it did result in a 300% increase in the ductility of the alloy. In contrast, the addition of Cu resulted in an increase in strength but in a decrease in ductility, along with a change in the structure of the material. The Cu-containing alloy also showed moderate antibacterial properties and was superior to the Ag-containing alloy.

  7. SASqPCR: robust and rapid analysis of RT-qPCR data in SAS.

    Directory of Open Access Journals (Sweden)

    Daijun Ling

    Full Text Available Reverse transcription quantitative real-time PCR (RT-qPCR is a key method for measurement of relative gene expression. Analysis of RT-qPCR data requires many iterative computations for data normalization and analytical optimization. Currently no computer program for RT-qPCR data analysis is suitable for analytical optimization and user-controllable customization based on data quality, experimental design as well as specific research aims. Here I introduce an all-in-one computer program, SASqPCR, for robust and rapid analysis of RT-qPCR data in SAS. This program has multiple macros for assessment of PCR efficiencies, validation of reference genes, optimization of data normalizers, normalization of confounding variations across samples, and statistical comparison of target gene expression in parallel samples. Users can simply change the macro variables to test various analytical strategies, optimize results and customize the analytical processes. In addition, it is highly automatic and functionally extendable. Thus users are the actual decision-makers controlling RT-qPCR data analyses. SASqPCR and its tutorial are freely available at

  8. Signal and noise in bridging PCR

    Directory of Open Access Journals (Sweden)

    Thaler David S


    Full Text Available Abstract Background In a variant of the standard PCR reaction termed bridging, or jumping, PCR the primer-bound sequences are originally on separate template molecules. Bridging can occur if, and only if, the templates contain a region of sequence similarity. A 3' end of synthesis in one round of synthesis that terminates in this region of similarity can prime on the other. In principle, Bridging PCR (BPCR can detect a subpopulation of one template that terminates synthesis in the region of sequence shared by the other template. This study considers the sensitivity and noise of BPCR as a quantitative assay for backbone interruptions. Bridging synthesis is also important to some methods for computing with DNA. Results In this study, BPCR was tested over a 328 base pair segment of the E. coli lac operon and a signal to noise ratio (S/N of approximately 10 was obtained under normal PCR conditions with Taq polymerase. With special precautions in the case of Taq or by using the Stoffel fragment the S/N was improved to 100, i.e. 1 part of cut input DNA yielded the same output as 100 parts of intact input DNA. Conclusions In the E. coli lac operator region studied here, depending on details of protocol, between 3 and 30% per kilobase of final PCR product resulted from bridging. Other systems are expected to differ in the proportion of product that is bridged consequent to PCR protocol and the sequence analyzed. In many cases physical bridging during PCR will have no informational consequence because the bridged templates are of identical sequence, but in a number of special cases bridging creates, or, destroys, information.

  9. Vapor segregation and loss in basaltic melts (United States)

    Edmonds, M.; Gerlach, T.M.


    Measurements of volcanic gases at Pu'u'O??'o??, Kilauea Volcano, Hawai'i, reveal distinct degassing regimes with respect to vapor segregation and loss during effusive activity in 2004-2005. Three styles of vapor loss are distinguished by the chemical character of the emitted volcanic gases, measured by open path Fourier transform infrared spectroscopy: 1 persistent continuous gas emission, 2 gas piston events, and 3 lava spattering. Persistent continuous gas emission is associated with magma ascent and degassing beneath the crater vents, then eruption of the degassed magma from flank vents. Gas piston events are the result of static gas accumulation at depths of 400-900 m beneath Pu'u'O??'o??. A CO2-rich gas slug travels up the conduit at a few meters per second, displacing magma as it expands. Lava spattering occurs due to dynamic bubble coalescence in a column of relatively stagnant magma. The Large gas bubbles are H2O rich and are generated by open-system degassing at depths of gas accumulation and dynamic bubble coalescence are both manifestations of vapor segregation in basaltic melts, but their implications differ. Accumulation and segregation of CO2-rich vapor at depth does not deplete the melt of H2O (required to drive lava fountains near to the surface) and therefore gas piston events can occur interspersed with lava fountaining activity. Lava spattering, however, efficiently strips H2O-rich vapor from magma beneath the crater vents; the magma must then erupt effusively from vents on the flank of the cone. ?? 2007 The Geological Society of America.

  10. Rapid identification of bovine mastitis pathogens by high-resolution melt analysis of 16S rDNA sequences. (United States)

    Ajitkumar, Praseeda; Barkema, Herman W; De Buck, Jeroen


    Accurate identification of mastitis pathogens is often compromised when using conventional culture-based methods. Here, we report a novel, rapid assay tested for speciation of bacterial mastitis pathogens using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time PCR amplification of 16S rRNA gene fragment, spanning the variable region V5 and V6 was performed with a resulting amplicon of 290bp. First, a library was generated of melt curves of 9 common pathogens that are implicated in bovine mastitis. Six of the isolates, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus uberis, Staphylococcus aureus and Mycoplasma bovis, were type strains while the other 3, Arcanobacterium pyogenes, Corynebacterium bovis and Streptococcus dysgalactiae, were bovine mastitis field isolates. Four of the type strains, E. coli, S. agalactiae, K. pneumoniae and S. aureus, were found to be of human origin, while the other 3 type strains were isolated from bovine infections. Secondly, the melt curves and corresponding amplicon sequences of A. pyogenes, E. coli, S. agalactiae, S. dysgalactiae, K. pneumoniae, S. uberis and S. aureus were compared with 10 bovine mastitis field isolates of each pathogen. Based on the distinct differences in melt curves and sequences between human and bovine isolates of E. coli and K. pneumoniae, it was deemed necessary to select a set of bovine strains for these pathogens to be used as reference strains in the HRMA. Next, the HRMA was validated by three interpreters analyzing the differential clustering pattern of melt curves of 60 bacterial cultures obtained from mastitis milk samples. The three test interpreters were blinded to the culture and sequencing results of the isolates. Overall accuracy of the validation assay was 95% as there was difficulty in identifying the streptococci due to heterogeneity observed in the PCR amplicons of S. uberis. The present study revealed that broad-range real-time PCR with

  11. γ-ray compton profiles of liquid and crystalline aluminum

    International Nuclear Information System (INIS)

    Honda, Toshihisa; Itoh, Fumitake; Suzuki, Kenji


    Compton profiles of liquid and crystalline aluminum metal have been measured using 59.54 keV γ-rays emitted from 241 Am and Ge(Li) solid state detectors. The difference of the Compton profile between the liquid and crystalline aluminum has been found and can be qualitatively understood in terms of the change in the electronic density upon melting. The experimental profile of crystalline aluminum is in good agreement with the result of the LCAO band calculation, while the profile of liquid aluminum is in poor agreement with the free electron model and the Green function method. (author)

  12. Optimization of hot melt extrusion parameters for sphericity and hardness of polymeric face-cut pellets (United States)

    Alshetaili, Abdullah S.; Almutairy, Bjad K.; Alshahrani, Saad M.; Ashour, Eman A.; Tiwari, Roshan V.; Alshehri, Sultan M.; Feng, Xin; Alsulays, Bader B.; Majumdar, Soumyajit; Langley, Nigel; Kolter, Karl; Gryczke, Andreas; Martin, Scott T.; Repka, Michael A.


    The aim of this study was to formulate face-cut, melt extruded pellets and to optimize hot melt process parameters to obtain maximized sphericity and hardness by utilizing Soluplus® as a polymeric carrier and carbamazepine (CBZ) as a model drug. Thermal gravimetric analysis (TGA) was used to detect thermal stability of CBZ. The Box-Behnken design for response surface methodology was developed using three factors, processing temperature (°C), feeding rate (%), and screw speed (rpm), which resulted in 17 experimental runs. The influence of these factors on pellet sphericity and mechanical characteristics was assessed and evaluated for each experimental run. Pellets with optimal sphericity and mechanical properties were chosen for further characterization. This included differential scanning calorimetry, drug release, hardness friability index (HFI), flowability, bulk density, tapped density, Carr’s index, and fourier transform infrared radiation (FTIR) spectroscopy. TGA data showed no drug degradation upon heating to 190°C. Hot melt extrusion (HME) processing conditions were found to have a significant effect on the pellet shape and hardness profile. Pellets with maximum sphericity and hardness exhibited no crystalline peak after extrusion. The rate of drug release was affected mainly by pellet size, where smaller pellets released the drug faster. All optimized formulations were found to be of superior hardness and not friable. The flow properties of optimized pellets were excellent with high bulk and tapped density. PMID:27080252

  13. The principle and application of new PCR Technologies (United States)

    Yu, Miao; Cao, Yue; Ji, Yubin


    Polymerase chain reaction (PCR) is essentially a selective DNA amplification technique commonlyapplied for genetic testing and molecular diagnosis because of its high specificity and sensitivity.PCR technologies as the key of molecular biology, has realized that the qualitative detection of absolute quantitative has been changed. It has produced a variety of new PCR technologies, such as extreme PCR, photonic PCR, o-amplification at lower denaturation temperature PCR, nanoparticle PCR and so on. In this paper, the principle and application of PCR technologies are reviewed, and its development is prospected too.

  14. Uranium and neodymium partitioning in alkali chloride melts using low-melting gallium-based alloys

    Directory of Open Access Journals (Sweden)

    Melchakov Stanislav Yu.


    Full Text Available Partitioning of uranium and neodymium was studied in a ‘molten chloride salt - liquid Ga-X (X = In or Sn alloy’ system. Chloride melts were based on the low-melting ternary LiCl-KCl-CsCl eutectic. Nd/U separation factors were calculated from the thermodynamic data as well as determined experimentally. Separation of uranium and neodymium was studied using reductive extraction with neodymium acting as a reducing agent. Efficient partitioning of lanthanides (Nd and actinides (U, simulating fission products and fissile materials in irradiated nuclear fuels, was achieved in a single stage process. The experimentally observed Nd/U separation factor valued up to 106, depending on the conditions.

  15. Melting process by means of the hollow electrode melting system. Einschmelzverfahren mit dem Hohlelektroden-Schmelzsystem

    Energy Technology Data Exchange (ETDEWEB)

    Saleh, F. (Mannesmann Anlagenbau AG, Duesseldorf (Germany)); Schubert, J. (Mannesmann Anlagenbau AG, Duesseldorf (Germany)); Staeubner, H. (Mannesmann Demag AG, Duisburg (Germany)); Lembgen, E. (Mannesmann Demag AG, Duisburg (Germany))


    It is shown that the proven hollow electrode melting system permits conversion of flue ash from waste incineration plants to reusable materials in full complience with the legal regulations, thus providing its recirculation as a raw or final product in the materials flow. This not only reduces the disposal of toxic substances, i.e., counteracts the practice of putting off problems, but also permits the recovery of a large fraction of the reusable materials. Moreover, it helps conserve resources. The glass-like product of this process is similar in composition to natural products and environmentally neutral. The process also provides a sink for toxic substances such as dioxins, which otherwise persist beyond disposal. The specific costs of a complete plant with all the necessary components for melting and cleaning systems are low. (orig./EF)

  16. On melting dynamics and the glass transition. II. Glassy dynamics as a melting process. (United States)

    Krzakala, Florent; Zdeborová, Lenka


    There are deep analogies between the melting dynamics in systems with a first-order phase transition and the dynamics from equilibrium in super-cooled liquids. For a class of Ising spin models undergoing a first-order transition--namely p-spin models on the so-called Nishimori line--it can be shown that the melting dynamics can be exactly mapped to the equilibrium dynamics. In this mapping the dynamical--or mode-coupling--glass transition corresponds to the spinodal point, while the Kauzmann transition corresponds to the first-order phase transition itself. Both in mean field and finite dimensional models this mapping provides an exact realization of the random first-order theory scenario for the glass transition. The corresponding glassy phenomenology can then be understood in the framework of a standard first-order phase transition.

  17. PCR+ In Diesel Fuels and Emissions Research

    Energy Technology Data Exchange (ETDEWEB)

    McAdams, H.T.


    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  18. Industrial opportunities of controlled melt flow during glass melting, part 2: Potential applications

    Czech Academy of Sciences Publication Activity Database

    Hrbek, Lukáš; Dyrčíková, Petra; Němec, Lubomír; Jebavá, Marcela


    Roč. 58, č. 3 (2014), s. 202-209 ISSN 0862-5468 R&D Projects: GA TA ČR TA01010844 Institutional support: RVO:67985891 Keywords : glass melting * controlled flow * space utilization * homogenization processess Subject RIV: JH - Ceramics, Fire-Resistant Materials and Glass Impact factor: 0.435, year: 2014

  19. Optimization of ultrahigh-speed multiplex PCR for forensic analysis. (United States)

    Gibson-Daw, Georgiana; Crenshaw, Karin; McCord, Bruce


    In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl 2 , and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.

  20. Observation of melting conditions in selective laser melting of metals (SLM) (United States)

    Thombansen, U.; Abels, Peter


    Process observation in 3D printing of metals currently is one of the central challenges. Many companies strive to employ this additive manufacturing process in their production chains in order to gain competitive advantages through added flexibility in product design and embedded features. The new degrees of freedom are accompanied with the challenge to manufacture every detail of the product to the predefined specifications. Products with filigree internal structures for example require a perfect build to deliver the performance that was designed into these structures. Melting conditions determine properties such as grain structure and density of the finished part before it is sent to post processing steps. Monitoring of such melting conditions is still a challenge where the use of photodiodes, pyrometry and camera systems contribute to an overall picture that might identify errors or deviations during the build process. Additional considerations must be made to decide if these sensors are applied coaxially or from a lateral perspective. Furthermore, setting parameters of focal plane array (FPA) sensors are discussed and events that are seen in the machine vision image are compared against the pyrometry data. The resume of the experiments suggests the application of multiple sensors to the selective laser melting process (SLM) as they jointly contribute to an identification of events. These events need to be understood in order to establish cause effect relationships in the future.