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.

2014-01-01

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)

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

DEFF Research Database (Denmark)

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

2009-01-01

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...... curves. In this study, HRM was used for simultaneous screening and genotyping of genic DNA sequence polymorphisms identified in the Lolium perenne F2 mapping population VrnA. Melting profiles of PCR products amplified from previously published gene loci and from a novel gene putatively involved...

Isotopic fractionation and profile evolution of a melting snowcover

Institute of Scientific and Technical Information of China (English)

周石硚; 中尾正义; 桥本重将; 坂井亚规子; 成田英器; 石川信敬

2001-01-01

Successive snow pits were dug intensively in a melting snowcover. Water was successfully separated from snow grains in the field for the first time. By measuring δ18O values of water and snow grain samples as well as comparing isotopic profiles, it is found that meltwater percolating down in snow develops quick and clear isotopic fractionation with snow grains, but exerts no clear impact on the δ18O profile of the snowcover through which the meltwater percolates.

Probe-based real-time PCR method for multilocus melt typing of Xylella fastidiosa strains.

Science.gov (United States)

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

2012-04-01

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.

Direct-to-PCR tissue preservation for DNA profiling.

Science.gov (United States)

Sorensen, Amy; Berry, Clare; Bruce, David; Gahan, Michelle Elizabeth; Hughes-Stamm, Sheree; McNevin, Dennis

2016-05-01

Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition.

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

Science.gov (United States)

Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

2016-10-19

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

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

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Hany Sady

2015-07-01

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

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

Science.gov (United States)

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

2008-05-01

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

[Rapid detection of hot spot mutations of FGFR3 gene with PCR-high resolution melting assay].

Science.gov (United States)

Li, Shan; Wang, Han; Su, Hua; Gao, Jinsong; Zhao, Xiuli

2017-08-10

To identify the causative mutations in five individuals affected with dyschondroplasia and develop an efficient procedure for detecting hot spot mutations of the FGFR3 gene. Genomic DNA was extracted from peripheral blood samples with a standard phenol/chloroform method. PCR-Sanger sequencing was used to analyze the causative mutations in the five probands. PCR-high resolution melting (HRM) was developed to detect the identified mutations. A c.1138G>A mutation in exon 8 was found in 4 probands, while a c.1620C>G mutation was found in exon 11 of proband 5 whom had a mild phenotype. All patients were successfully distinguished from healthy controls with the PCR-HRM method. The results of HRM analysis were highly consistent with that of Sanger sequencing. The Gly380Arg and Asn540Lys are hot spot mutations of the FGFR3 gene among patients with ACH/HCH. PCR-HRM analysis is more efficient for detecting hot spot mutations of the FGFR3 gene.

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

2007-01-01

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......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...... the inhibition of PCR, preferential binding to GC-rich sequences and effects on melting curve analysis. Although a few alternative dyes without some of these limitations have been recently proposed, no large-scale investigation into the properties of intercalating dyes has been performed. In this study, we...

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

Science.gov (United States)

Singh, Prashant; Pfeifer, Yvonne; Mustapha, Azlin

2016-05-01

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.

DNA profiling of spermatozoa by laser capture microdissection and low volume-PCR.

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Cai-xia Li

Full Text Available Genetic profiling of sperm from complex biological mixtures such as sexual assault casework samples requires isolation of a pure sperm population and the ability to analyze low abundant samples. Current standard procedure for sperm isolation includes preferential lysis of epithelial contaminants followed by collection of intact sperm by centrifugation. While effective for samples where sperm are abundant, this method is less effective when samples contain few spermatozoa. Laser capture microdissection (LCM is a proven method for the isolation of cells biological mixtures, even when found in low abundance. Here, we demonstrate the efficacy of LCM coupled with on-chip low volume PCR (LV-PCR for the isolation and genotyping of low abundance sperm samples. Our results indicate that this method can obtain complete profiles (13-16 loci from as few as 15 sperm cells with 80% reproducibility, whereas at least 40 sperm cells are required to profile 13-16 loci by standard 'in-tube' PCR. Further, LCM and LV-PCR of a sexual assault casework sample generated a DNA genotype that was consistent with that of the suspect. This method was unable, however, to analyze a casework sample from a gang rape case in which two or more sperm contributors were in a mixed population. The results indicate that LCM and LV-PCR is sensitive and effective for genotyping sperm from sperm/epithelial cell mixtures when epithelial lysis may be insufficient due to low abundance of sperm; LCM and LV-PCR, however, failed in a casework sample when spermatozoa from multiple donors was present, indicating that further study is necessitated.

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

Science.gov (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

2017-06-01

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

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

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Skjerve Eystein

2009-12-01

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.

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

DEFF Research Database (Denmark)

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

2010-01-01

A new test for pathogenic Leptospira isolates, based on RAPD-PCR and high-resolution melt (HRM) analysis (which measures the melting temperature of amplicons in real time, using a fluorescent DNA-binding dye), has recently been developed. A characteristic profile of the amplicons can be used...... typed against 13 previously published RAPD primers, using a real-time cycler (the Corbett Life Science RotorGene 6000) and the optimised reagents from a commercial kit (Quantace SensiMix). RAPD-HRM at specific temperatures generated defining amplicon melt profiles for each of the tested serovars....... These profiles were evaluated as difference-curve graphs generated using the RotorGene software package, with a cut-off of at least 8 'U' (plus or minus). The results demonstrated that RAPD-HRM can be used to measure serovar diversity and establish identity, with a high degree of stability. The characterisation...

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.

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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

Thermodynamics of Oligonucleotide Duplex Melting

Science.gov (United States)

Schreiber-Gosche, Sherrie; Edwards, Robert A.

2009-01-01

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

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

Directory of Open Access Journals (Sweden)

Erin K. Hanson

2013-12-01

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

Identification of circulating miRNA biomarkers based on global quantitative real-time PCR profiling

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Kang Kang

2012-02-01

Full Text Available Abstract MicroRNAs (miRNAs are small noncoding RNAs (18-25 nucleotides that regulate gene expression at the post-transcriptional level. Recent studies have demonstrated the presence of miRNAs in the blood circulation. Deregulation of miRNAs in serum or plasma has been associated with many diseases including cancers and cardiovascular diseases, suggesting the possible use of miRNAs as diagnostic biomarkers. However, the detection of the small amount of miRNAs found in serum or plasma requires a method with high sensitivity and accuracy. Therefore, the current study describes polymerase chain reaction (PCR-based methods for measuring circulating miRNAs. Briefly, the procedure involves four major steps: (1 sample collection and preparation; (2 global miRNAs profiling using quantitative real-time PCR (qRT-PCR; (3 data normalization and analysis; and (4 selection and validation of miRNA biomarkers. In conclusion, qRT-PCR is a promising method for profiling of circulating miRNAs as biomarkers.

Product differentiation during continuous-flow thermal gradient PCR.

Science.gov (United States)

Crews, Niel; Wittwer, Carl; Palais, Robert; Gale, Bruce

2008-06-01

A continuous-flow PCR microfluidic device was developed in which the target DNA product can be detected and identified during its amplification. This in situ characterization potentially eliminates the requirement for further post-PCR analysis. Multiple small targets have been amplified from human genomic DNA, having sizes of 108, 122, and 134 bp. With a DNA dye in the PCR mixture, the amplification and unique melting behavior of each sample is observed from a single fluorescent image. The melting behavior of the amplifying DNA, which depends on its molecular composition, occurs spatially in the thermal gradient PCR device, and can be observed with an optical resolution of 0.1 degrees C pixel(-1). Since many PCR cycles are within the field of view of the CCD camera, melting analysis can be performed at any cycle that contains a significant quantity of amplicon, thereby eliminating the cycle-selection challenges typically associated with continuous-flow PCR microfluidics.

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini using real-time PCR and high resolution melting analysis.

Science.gov (United States)

Cai, Xian-Quan; Yu, Hai-Qiong; Li, Rong; Yue, Qiao-Yun; Liu, Guo-Hua; Bai, Jian-Shan; Deng, Yan; Qiu, De-Yi; Zhu, Xing-Quan

2014-01-01

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.

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

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Xian-Quan Cai

2014-01-01

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

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

Science.gov (United States)

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

2015-12-01

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.

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

OpenAIRE

Cai, Xian-Quan; Yu, Hai-Qiong; Li, Rong; Yue, Qiao-Yun; Liu, Guo-Hua; Bai, Jian-Shan; Deng, Yan; Qiu, De-Yi; Zhu, Xing-Quan

2014-01-01

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...

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C).

Science.gov (United States)

Fong, Wai-Ying; Ho, Chi-Chun; Poon, Wing-Tat

2017-05-12

Thiopurine intolerance and treatment-related toxicity, such as fatal myelosuppression, is related to non-function genetic variants encoding thiopurine S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15). Genetic testing of the common variants NUDT15:NM_018283.2:c.415C>T (Arg139Cys, dbSNP rs116855232 T allele) and TPMT: NM_000367.4:c.719A>G (TPMT*3C, dbSNP rs1142345 G allele) in East Asians including Chinese can potentially prevent treatment-related complications. Two complementary genotyping approaches, real-time PCR-high resolution melt (PCR-HRM) and PCR-restriction fragment length morphism (PCR-RFLP) analysis were evaluated using conventional PCR and Sanger sequencing genotyping as the gold standard. Sixty patient samples were tested, revealing seven patients (11.7%) heterozygous for NUDT15 c.415C>T, one patient homozygous for the variant and one patient heterozygous for the TPMT*3C non-function allele. No patient was found to harbor both variants. In total, nine out of 60 (15%) patients tested had genotypic evidence of thiopurine intolerance, which may require dosage adjustment or alternative medication should they be started on azathioprine, mercaptopurine or thioguanine. The two newly developed assays were more efficient and showed complete concordance (60/60, 100%) compared to the Sanger sequencing results. Accurate and cost-effective genotyping assays by real-time PCR-HRM and PCR-RFLP for NUDT15 c.415C>T and TPMT*3C were successfully developed. Further studies may establish their roles in genotype-informed clinical decision-making in the prevention of morbidity and mortality due to thiopurine intolerance.

High-resolution melting-curve (HRM) analysis for C. meleagridis identification in stool samples.

Science.gov (United States)

Chelbi, Hanen; Essid, Rym; Jelassi, Refka; Bouzekri, Nesrine; Zidi, Ines; Ben Salah, Hamza; Mrad, Ilhem; Ben Sghaier, Ines; Abdelmalek, Rym; Aissa, Sameh; Bouratbine, Aida; Aoun, Karim

2018-02-01

Cryptosporidiosis represents a major public health problem. This infection, caused by a protozoan parasite of the genus Cryptosporidium, has been reported worldwide as a frequent cause of diarrhoea. In the immunocompetent host, the typical watery diarrhea can be self-limiting. However, it is severe and chronic, in the immunocompromised host and may cause death. Cryptosporidium spp. are coccidians, which complete their life cycle in both humans and animals. The two species C. hominis and C. parvum are the major cause of human infection. Compared to studies on C. hominis and C. parvum, only a few studies have developed methods to identify C. meleagridis. To develop a new real time PCR-coupled High resolution melting assay allowing the detection for C. meleagridis, in addition of the other dominant species (C. hominis and C. parvum). The polymorphic sequence on the dihydrofolate reductase gene (DHFR) of three species was sequenced to design primers pair and establish a sensitive real-time PCR coupled to a high-resolution melting-curve (HRM) analysis method, allowing the detection of Cryptosporidium sp. and discrimination between three prevalent species in Tunisia. We analyzed a collection of 42 archived human isolates of the three studied species. Real-time PCR coupled to HRM assay allowed detection of Cryptosporidium, using the new designed primers, and basing on melting profile, we can distinguish C. meleagridis species in addition to C. parvum and C. hominis. We developed a qPCR-HRM assay that allows Cryptosporidium genotyping. This method is sensitive and able to distinguish three Cryptosporidium species. Copyright © 2017. Published by Elsevier Ltd.

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

Science.gov (United States)

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

2016-01-01

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

Rapid detection of Opisthorchis viverrini and Strongyloides stercoralis in human fecal samples using a duplex real-time PCR and melting curve analysis.

Science.gov (United States)

Janwan, Penchom; Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Anamnart, Witthaya; Maleewong, Wanchai

2011-12-01

Human opisthorchiasis caused by the liver fluke Opisthorchis viverrini is an endemic disease in Southeast Asian countries including the Lao People's Democratic Republic, Cambodia, Vietnam, and Thailand. Infection with the soil-transmitted roundworm Strongyloides stercoralis is an important problem worldwide. In some areas, both parasitic infections are reported as co-infections. A duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis was developed for the rapid detection of O. viverrini and S. stercoralis in human fecal samples. Duplex real-time FRET PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two genera of DNA elements: the 162 bp pOV-A6 DNA sequence specific to O. viverrini and the 244 bp 18S rRNA sequence specific to S. stercoralis, and two pairs of specific fluorophore-labeled probes. Both O. viverrini and S. stercoralis can be differentially detected in infected human fecal samples by this process through their different fluorescence channels and melting temperatures. Detection limit of the method was as little as two O. viverrini eggs and four S. stercoralis larvae in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasite materials, as well as from the DNA of human leukocytes and other control parasites. The technique showed 100% sensitivity and specificity. The introduced duplex real-time FRET PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The method is important for simultaneous detection especially in areas where both parasites overlap incidence and is useful as the screening tool in the returning travelers and immigrants to industrialized countries where number of samples in the diagnostic units will become increasing.

Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

Science.gov (United States)

Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

2009-06-01

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

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

OpenAIRE

sprotocols

2015-01-01

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

A Real-Time PCR with Melting Curve Analysis for Molecular Typing of Vibrio parahaemolyticus.

Science.gov (United States)

He, Peiyan; Wang, Henghui; Luo, Jianyong; Yan, Yong; Chen, Zhongwen

2018-05-23

Foodborne disease caused by Vibrio parahaemolyticus is a serious public health problem in many countries. Molecular typing has a great scientific significance and application value for epidemiological research of V. parahaemolyticus. In this study, a real-time PCR with melting curve analysis was established for molecular typing of V. parahaemolyticus. Eighteen large variably presented gene clusters (LVPCs) of V. parahaemolyticus which have different distributions in the genome of different strains were selected as targets. Primer pairs of 18 LVPCs were distributed into three tubes. To validate this newly developed assay, we tested 53 Vibrio parahaemolyticus strains, which were classified in 13 different types. Furthermore, cluster analysis using NTSYS PC 2.02 software could divide 53 V. parahaemolyticus strains into six clusters at a relative similarity coefficient of 0.85. This method is fast, simple, and conveniently for molecular typing of V. parahaemolyticus.

Molecular differentiation of Opisthorchis viverrini and Clonorchis sinensis eggs by multiplex real-time PCR with high resolution melting analysis.

Science.gov (United States)

Kaewkong, Worasak; Intapan, Pewpan M; Sanpool, Oranuch; Janwan, Penchom; Thanchomnang, Tongjit; Laummaunwai, Porntip; Lulitanond, Viraphong; Doanh, Pham Ngoc; Maleewong, Wanchai

2013-12-01

Opisthorchis viverrini and Clonorchis sinensis are parasites known to be carcinogenic and causative agents of cholangiocarcinoma in Asia. The standard method for diagnosis for those parasite infections is stool examination to detect parasite eggs. However, the method has low sensitivity, and eggs of O. viverrini and C. sinensis are difficult to distinguish from each other and from those of some other trematodes. Here, we report a multiplex real-time PCR coupled with high resolution melting (HRM) analysis for the differentiation of O. viverrini and C. sinensis eggs in fecal samples. Using 2 pairs of species-specific primers, DNA sequences from a portion of the mitochondrial NADH dehydrogenase subunit 2 (nad 2) gene, were amplified to generate 209 and 165 bp products for O. viverrini and C. sinensis, respectively. The distinct characteristics of HRM patterns were analyzed, and the melting temperatures peaked at 82.4±0.09℃ and 85.9±0.08℃ for O. viverrini and C. sinensis, respectively. This technique was able to detect as few as 1 egg of O. viverrini and 2 eggs of C. sinensis in a 150 mg fecal sample, which is equivalent to 7 and 14 eggs per gram of feces, respectively. The method is species-specific, rapid, simple, and does not require fluorescent probes or post-PCR processing for discrimination of eggs of the 2 species. It offers a new tool for differentiation and detection of Asian liver fluke infections in stool specimens.

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

Science.gov (United States)

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

2012-09-01

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.

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

Czech Academy of Sciences Publication Activity Database

Stahlberg, A.; Kubista, Mikael

2014-01-01

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

Real-Time PCR (qPCR) Primer Design Using Free Online Software

Science.gov (United States)

Thornton, Brenda; Basu, Chhandak

2011-01-01

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…

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

2016-11-01

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.

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

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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.

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

Science.gov (United States)

Druml, Barbara; Cichna-Markl, Margit

2014-09-01

DNA based methods play an increasing role in food safety control and food adulteration detection. Recent papers show that high resolution melting (HRM) analysis is an interesting approach. It involves amplification of the target of interest in the presence of a saturation dye by the polymerase chain reaction (PCR) and subsequent melting of the amplicons by gradually increasing the temperature. Since the melting profile depends on the GC content, length, sequence and strand complementarity of the product, HRM analysis is highly suitable for the detection of single-base variants and small insertions or deletions. The review gives an introduction into HRM analysis, covers important aspects in the development of an HRM analysis method and describes how HRM data are analysed and interpreted. Then we discuss the potential of HRM analysis based methods in food analysis, i.e. for the identification of closely related species and cultivars and the identification of pathogenic microorganisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

2017-01-01

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...... be explained partly by premature read truncation, but to larger degree their genomic GC-content, which correlated negatively with the observed relative abundances, suggesting a PCR bias against GC-rich species during library preparation. Increasing the initial denaturation time during the PCR amplification...

Rapid Simultaneous Amplification and Detection of the MBR/JH Chromosomal Translocation by Fluorescence Melting Curve Analysis

Science.gov (United States)

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

1999-01-01

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

Role of Melt Curve Analysis in Interpretation of Nutrigenomics' MicroRNA Expression Data.

Science.gov (United States)

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

2017-01-01

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.

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

Science.gov (United States)

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

2017-07-31

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 ( R ² = 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.

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.

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

Directory of Open Access Journals (Sweden)

Devin Daems

2017-07-01

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.

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.

Development of a real-time PCR melt curve assay for simultaneous detection of virulent and antibiotic resistant Salmonella.

Science.gov (United States)

Singh, Prashant; Mustapha, Azlin

2014-12-01

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.

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.

Science.gov (United States)

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

2015-02-17

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

Rapid detection of Echinococcus species by a high-resolution melting (HRM) approach.

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Santos, Guilherme Brzoskowski; Espínola, Sergio Martín; Ferreira, Henrique Bunselmeyer; Margis, Rogerio; Zaha, Arnaldo

2013-11-14

High-resolution melting (HRM) provides a low-cost, fast and sensitive scanning method that allows the detection of DNA sequence variations in a single step, which makes it appropriate for application in parasite identification and genotyping. The aim of this work was to implement an HRM-PCR assay targeting part of the mitochondrial cox1 gene to achieve an accurate and fast method for Echinococcus spp. differentiation. For melting analysis, a total of 107 samples from seven species were used in this study. The species analyzed included Echinococcus granulosus (n = 41) and Echinococcus ortleppi (n = 50) from bovine, Echinococcus vogeli (n = 2) from paca, Echinococcus oligarthra (n = 3) from agouti, Echinococcus multilocularis (n = 6) from monkey and Echinococcus canadensis (n = 2) and Taenia hydatigena (n = 3) from pig. DNA extraction was performed, and a 444-bp fragment of the cox1 gene was amplified. Two approaches were used, one based on HRM analysis, and a second using SYBR Green Tm-based. In the HRM analysis, a specific profile for each species was observed. Although some species exhibited almost the same melting temperature (Tm) value, the HRM profiles could be clearly discriminated. The SYBR Green Tm-based analysis showed differences between E. granulosus and E. ortleppi and between E. vogeli and E. oligarthra. In this work, we report the implementation of HRM analysis to differentiate species of the genus Echinococcus using part of the mitochondrial gene cox1. This method may be also potentially applied to identify other species belonging to the Taeniidae family.

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

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

2017-10-01

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

Detection and characterization of Newcastle disease virus in clinical samples using real time RT-PCR and melting curve analysis based on matrix and fusion genes amplification

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Saad Sharawi

2013-10-01

Full Text Available Aim: Newcastle disease is still one of the major threats for poultry industry allover the world. Therefore, attempt was made in this study to use the SYBR Green I real-time PCR with melting curves analysis as for detection and differentiation of NDV strains in suspected infected birds. Materials and Methods: Two sets of primers were used to amplify matrix and fusion genes in samples collected from suspectly infected birds (chickens and pigeons. Melting curve analysis in conjunction with real time PCR was conducted for identifying different pathotypes of the isolated NDVs. Clinical samples were propagated on specific pathogen free ECE and tested for MDT and ICIP. Results: The velogenic NDVs isolated from chickens and pigeons were distinguished with mean T 85.03±0.341 and m 83.78±0.237 respectively for M-gene amplification and for F-gene amplification the mean T were 84.04±0.037 and m 84.53±0.223. On the other hand the lentogenic NDV isolates including the vaccinal strains (HB1 and LaSota have a higher mean T (86.99±0.021 for M-gene amplification and 86.50±0.063 for F-gene amplification. The test showed no reaction with m unrelated RNA samples. In addition, the results were in good agreement with both virus isolation and biological pathotyping (MDT and ICIP. The assay offers an attractive alternative method for the diagnosis of NDV that can be easily applied in laboratory diagnosis as a screening test for the detection and differentiation of NDV infections. Conclusion: As was shown by the successful rapid detection and pathotyping of 15 NDV strains in clinical samples representing velogenic and lentogenic NDV strains, and the agreement with the results of virus isolation , biological pathotyping and pathogenicity indices. The results of this report suggests that the described SybrGreen I real-time RT-PCR assay in conjunction with Melting curve analysis used as a rapid, specific and simple diagnostic tools for detection and pathotyping of

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.

1977-01-01

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)

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

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

2014-01-01

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

High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms.

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Steven Y C Tong

Full Text Available We have developed a single nucleotide polymorphism (SNP nucleated high-resolution melting (HRM technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE and an allele specific real-time PCR (AS kinetic PCR SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs and provides a Simpson's Index of Diversity (D of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.

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

2007-01-01

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

Multicomponent Diffusion in Experimentally Cooled Melt Inclusions

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Saper, L.; Stolper, E.

2017-12-01

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

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.

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

2017-05-01

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

Rapid detection of the H275Y oseltamivir resistance mutation in influenza A/H1N1 2009 by single base pair RT-PCR and high-resolution melting.

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Steven Y C Tong

Full Text Available We aimed to design a real-time reverse-transcriptase-PCR (rRT-PCR, high-resolution melting (HRM assay to detect the H275Y mutation that confers oseltamivir resistance in influenza A/H1N1 2009 viruses.A novel strategy of amplifying a single base pair, the relevant SNP at position 823 of the neuraminidase gene, was chosen to maintain specificity of the assay. Wildtype and mutant virus were differentiated when using known reference samples of cell-cultured virus. However, when dilutions of these reference samples were assayed, amplification of non-specific primer-dimer was evident and affected the overall melting temperature (T(m of the amplified products. Due to primer-dimer appearance at >30 cycles we found that if the cycle threshold (C(T for a dilution was >30, the HRM assay did not consistently discriminate mutant from wildtype. Where the C(T was 32.98 would have an H275Y assay C(T>30. Analysis of the TaqMan C(T values for 609 consecutive clinical samples predicted that 207 (34% of the samples would result in an HRM assay C(T>30 and therefore not be amenable to the HRM assay.The use of single base pair PCR and HRM can be useful for specifically interrogating SNPs. When applied to H1N1 09, the constraints this placed on primer design resulted in amplification of primer-dimer products. The impact primer-dimer had on HRM curves was adjusted for by plotting T(m against C(T. Although less sensitive than TaqMan assays, the HRM assay can rapidly, and at low cost, screen samples with moderate viral concentrations.

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

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Thornton, Brenda; Basu, Chhandak

2011-01-01

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.

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

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

2018-03-01

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.

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

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

2017-06-01

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

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

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

2016-01-01

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.

High resolution melting analysis (HRM) for the assessment of clonality in feline B-cell lymphomas.

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Henrich, Manfred; Scheffold, Svenja; Hecht, Werner; Reinacher, Manfred

2018-06-01

Analysis of clonality is gaining importance in diagnosing lymphomas in veterinary medicine. Usually, PCR for the analysis of antigen receptor rearrangement (PARR) is followed by electrophoretic separation of the PCR products. Aim of this study was to test the feasibility of HRM for the assessment of clonality in B-cell lymphomas of cats. High resolution melting analysis differentiates PCR products by their different melting point using the decrease in fluorescence of an intercalating dye during melting of the PCR product. Additionally, the method is easy to use with no post-PCR manipulation of the samples. Forty-seven feline B-cell lymphomas and 31 reactive lymphatic proliferations of cats were investigated by PARR followed either by capillary electrophoresis or an HRM assay. To objectify the interpretation of the HRM results a recently published mathematical approach was applied to the melting curve. To overcome discrepancies between the visual interpretation and the mathematical approach, the latter was modified to include testing of reproducibility and recognition of pseudoclonality. In 11 of 47 lymphoma cases clonal populations were detectable by HRM assay compared to 14 of 47 lymphomas in which clonal populations were detected by capillary electrophoresis assay. Neither of the methods showed a clonal pattern in any of the reactive samples. However, the HRM assay showed a unique pattern in cases of follicular lymphatic hyperplasia that had no corresponding pattern in capillary electrophoresis. The capillary electrophoresis assay could identify 3 lymphomas that were not detected by the HRM assay and is therefore regarded superior to the HRM assay. The comparison however, was hampered by the overall bad performance of the PARR, that might be the consequence of insufficient primer binding due to somatic hypermutation of the binding sites during antigen stimulated proliferation of the B lymphocytes. Copyright © 2018 Elsevier B.V. All rights reserved.

High-throughput STR analysis for DNA database using direct PCR.

Science.gov (United States)

Sim, Jeong Eun; Park, Su Jeong; Lee, Han Chul; Kim, Se-Yong; Kim, Jong Yeol; Lee, Seung Hwan

2013-07-01

Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high-throughput and cost-effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter-loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to <3%. In conclusion, the newly developed direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time- and cost-saving manner. © 2013 American Academy of Forensic Sciences Published 2013. This article is a U.S. Government work and is in the public domain in the U.S.A.

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

2008-01-01

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

Rapid detection of most frequent Slovenian germ-line mutations in BRCA1 gene using real-time PCR and melting curve analysis

International Nuclear Information System (INIS)

Novakovic, S.; Stegel, V.

2005-01-01

Background. Detection of inherited mutations in cancer susceptibility genes is of great importance in some types of cancers including the colorectal cancer (mutations of APC gene in familial adenomatous polyposis - FAP, mutations in mismatch repair genes in hereditary nonpolyposis colorectal cancer - HNPCC), malignant melanoma (mutations in CDKN2A and CDK4 genes) and breast cancer (mutations in BRCA1 and BRCA2 genes). Methods. This article presents the technical data for the detection of five mutations in BRCA1 gene in breast cancer patients and their relatives. The mutations - 1806C>T, 300T>G, 300T>A, 310G>A, 5382insC - were determined by the real-time PCR and the melting curve analysis. Results and conclusion. In comparison to direct sequencing, this method proved to be sensitive and rapid enough for the routine daily determination of mutations in DNA isolated from the peripheral blood. (author)

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

Science.gov (United States)

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

2017-06-01

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

Shock induced melting of lead (experimental study)

International Nuclear Information System (INIS)

Mabire, Catherine; Hereil, Pierre L.

2002-01-01

To investigate melting on release of lead, two shock compression measurements have been carried out at 51 GPa. In the first one, a pyrometric measurement has been performed at the Pb/LiF interface. In the second one, the Pb/LiF interface velocity has been recorded using VISAR measurement technique. VISAR and radiance profile are in good agreement and seem to show melting on release of lead

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

2016-01-01

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.

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

Science.gov (United States)

D'Costa, Susan; Blouin, Veronique; Broucque, Frederic; Penaud-Budloo, Magalie; François, Achille; Perez, Irene C; Le Bec, Christine; Moullier, Philippe; Snyder, Richard O; Ayuso, Eduard

2016-01-01

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.

Melting in super-earths.

Science.gov (United States)

Stixrude, Lars

2014-04-28

We examine the possible extent of melting in rock-iron super-earths, focusing on those in the habitable zone. We consider the energetics of accretion and core formation, the timescale of cooling and its dependence on viscosity and partial melting, thermal regulation via the temperature dependence of viscosity, and the melting curves of rock and iron components at the ultra-high pressures characteristic of super-earths. We find that the efficiency of kinetic energy deposition during accretion increases with planetary mass; considering the likely role of giant impacts and core formation, we find that super-earths probably complete their accretionary phase in an entirely molten state. Considerations of thermal regulation lead us to propose model temperature profiles of super-earths that are controlled by silicate melting. We estimate melting curves of iron and rock components up to the extreme pressures characteristic of super-earth interiors based on existing experimental and ab initio results and scaling laws. We construct super-earth thermal models by solving the equations of mass conservation and hydrostatic equilibrium, together with equations of state of rock and iron components. We set the potential temperature at the core-mantle boundary and at the surface to the local silicate melting temperature. We find that ancient (∼4 Gyr) super-earths may be partially molten at the top and bottom of their mantles, and that mantle convection is sufficiently vigorous to sustain dynamo action over the whole range of super-earth masses.

Identification of the GST-T1 and GST-M1 null genotypes using high resolution melting analysis.

Science.gov (United States)

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

2012-01-13

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

Shock Compression and Melting of an Fe-Ni-Si Alloy: Implications for the Temperature Profile of the Earth's Core and the Heat Flux Across the Core-Mantle Boundary

Science.gov (United States)

Zhang, Youjun; Sekine, Toshimori; Lin, Jung-Fu; He, Hongliang; Liu, Fusheng; Zhang, Mingjian; Sato, Tomoko; Zhu, Wenjun; Yu, Yin

2018-02-01

Understanding the melting behavior and the thermal equation of state of Fe-Ni alloyed with candidate light elements at conditions of the Earth's core is critical for our knowledge of the region's thermal structure and chemical composition and the heat flow across the liquid outer core into the lowermost mantle. Here we studied the shock equation of state and melting curve of an Fe-8 wt% Ni-10 wt% Si alloy up to 250 GPa by hypervelocity impacts with direct velocity and reliable temperature measurements. Our results show that the addition of 10 wt% Si to Fe-8 wt% Ni alloy slightly depresses the melting temperature of iron by 200-300 (±200) K at the core-mantle boundary ( 136 GPa) and by 600-800 (±500) K at the inner core-outer core boundary ( 330 GPa), respectively. Our results indicate that Si has a relatively mild effect on the melting temperature of iron compared with S and O. Our thermodynamic modeling shows that Fe-5 wt% Ni alloyed with 6 wt% Si and 2 wt% S (which has a density-velocity profile that matches the outer core's seismic profile well) exhibits an adiabatic profile with temperatures of 3900 K and 5300 K at the top and bottom of the outer core, respectively. If Si is a major light element in the core, a geotherm modeled for the outer core indicates a thermal gradient of 5.8-6.8 (±1.6) K/km in the D″ region and a high heat flow of 13-19 TW across the core-mantle boundary.

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

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Dafni-Maria Kagkli

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

Technical aspects and recommendations for single-cell qPCR.

Science.gov (United States)

Ståhlberg, Anders; Kubista, Mikael

2018-02-01

Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

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

2017-04-01

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

Incorporation of Certain Hydrophobic Excipients in the Core of Melt ...

African Journals Online (AJOL)

Patrick Erah

incorporation of hydrophobic materials (talc or magnesium stearate) in the core of such granules may further retard .... (500mg) was filled into a capsule shell and ... of the drug particles. The effect of melt granulation on the release profiles of paracetamol is shown in Fig 1. The melt granulations displayed a retarded release.

Candidate DNA Barcode Tags Combined With High Resolution Melting (Bar-HRM) Curve Analysis for Authentication of Senna alexandrina Mill. With Validation in Crude Drugs.

Science.gov (United States)

Mishra, Priyanka; Shukla, Ashutosh K; Sundaresan, Velusamy

2018-01-01

Senna alexandrina (Fabaceae) is a globally recognized medicinal plant for its laxative properties as well as the only source of sennosides, and is highly exported bulk herb from India. Its major procurement is exclusively from limited cultivation, which leads to risks of deliberate or unintended adulteration. The market raw materials are in powdered or finished product form, which lead to difficulties in authentication. Here, DNA barcode tags based on chloroplast genes ( rbcL and matK ) and intergenic spacers ( psbA-trnH and ITS ) were developed for S. alexandrina along with the allied species. The ability and performance of the ITS1 region to discriminate among the Senna species resulted in the present proposal of the ITS1 tags as successful barcode. Further, these tags were coupled with high-resolution melting (HRM) curve analysis in a real-time PCR genotyping method to derive Bar-HRM (Barcoding-HRM) assays. Suitable HRM primer sets were designed through SNP detection and mutation scanning in genomic signatures of Senna species. The melting profiles of S. alexandrina and S . italica subsp. micrantha were almost identical and the remaining five species were clearly separated so that they can be differentiated by HRM method. The sensitivity of the method was utilized to authenticate market samples [Herbal Sample Assays (HSAs)]. HSA01 ( S. alexandrina crude drug sample from Bangalore) and HSA06 ( S. alexandrina crude drug sample from Tuticorin, Tamil Nadu, India) were found to be highly contaminated with S . italica subsp. micrantha . Species admixture samples mixed in varying percentage was identified sensitively with detection of contamination as low as 1%. The melting profiles of PCR amplicons are clearly distinct, which enables the authentic differentiation of species by the HRM method. This study reveals that DNA barcoding coupled with HRM is an efficient molecular tool to authenticate Senna herbal products in the market for quality control in the drug supply

Candidate DNA Barcode Tags Combined With High Resolution Melting (Bar-HRM Curve Analysis for Authentication of Senna alexandrina Mill. With Validation in Crude Drugs

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Priyanka Mishra

2018-03-01

Full Text Available Senna alexandrina (Fabaceae is a globally recognized medicinal plant for its laxative properties as well as the only source of sennosides, and is highly exported bulk herb from India. Its major procurement is exclusively from limited cultivation, which leads to risks of deliberate or unintended adulteration. The market raw materials are in powdered or finished product form, which lead to difficulties in authentication. Here, DNA barcode tags based on chloroplast genes (rbcL and matK and intergenic spacers (psbA-trnH and ITS were developed for S. alexandrina along with the allied species. The ability and performance of the ITS1 region to discriminate among the Senna species resulted in the present proposal of the ITS1 tags as successful barcode. Further, these tags were coupled with high-resolution melting (HRM curve analysis in a real-time PCR genotyping method to derive Bar-HRM (Barcoding-HRM assays. Suitable HRM primer sets were designed through SNP detection and mutation scanning in genomic signatures of Senna species. The melting profiles of S. alexandrina and S. italica subsp. micrantha were almost identical and the remaining five species were clearly separated so that they can be differentiated by HRM method. The sensitivity of the method was utilized to authenticate market samples [Herbal Sample Assays (HSAs]. HSA01 (S. alexandrina crude drug sample from Bangalore and HSA06 (S. alexandrina crude drug sample from Tuticorin, Tamil Nadu, India were found to be highly contaminated with S. italica subsp. micrantha. Species admixture samples mixed in varying percentage was identified sensitively with detection of contamination as low as 1%. The melting profiles of PCR amplicons are clearly distinct, which enables the authentic differentiation of species by the HRM method. This study reveals that DNA barcoding coupled with HRM is an efficient molecular tool to authenticate Senna herbal products in the market for quality control in the drug

Quantitative PCR Profiling of Escherichia coli in Livestock Feces Reveals Increased Population Resilience Relative to Culturable Counts under Temperature Extremes.

Science.gov (United States)

Oliver, David M; Bird, Clare; Burd, Emmy; Wyman, Michael

2016-09-06

The relationship between culturable counts (CFU) and quantitative PCR (qPCR) cell equivalent counts of Escherichia coli in dairy feces exposed to different environmental conditions and temperature extremes was investigated. Fecal samples were collected in summer and winter from dairy cowpats held under two treatments: field-exposed versus polytunnel-protected. A significant correlation in quantified E. coli was recorded between the qPCR and culture-based methods (r = 0.82). Evaluation of the persistence profiles of E. coli over time revealed no significant difference in the E. coli numbers determined as either CFU or gene copies during the summer for the field-exposed cowpats, whereas significantly higher counts were observed by qPCR for the polytunnel-protected cowpats, which were exposed to higher ambient temperatures. In winter, the qPCR returned significantly higher counts of E. coli for the field-exposed cowpats, thus representing a reversal of the findings from the summer sampling campaign. Results from this study suggest that with increasing time post-defecation and with the onset of challenging environmental conditions, such as extremes in temperature, culture-based counts begin to underestimate the true resilience of viable E. coli populations in livestock feces. This is important not only in the long term as the Earth changes in response to climate-change drivers but also in the short term during spells of extremely cold or hot weather.

Detection of polyoma virus in brain tissue of patients with progressive multifocal leukoencephalopathy by real-time PCR and pyrosequencing.

Science.gov (United States)

Beck, Rose C; Kohn, Debra J; Tuohy, Marion J; Prayson, Richard A; Yen-Lieberman, Belinda; Procop, Gary W

2004-03-01

We evaluated 2 methods, a LightCycler PCR assay and pyrosequencing for the detection of the JC polyoma virus (JCV) in fixed brain tissue of 10 patients with and 3 control patients without progressive multifocal leukoencephalopathy (PML). Nucleic acid extraction was performed after deparaffinization and proteinase K digestion. The LightCycler assay differentiates the BK virus (BKV), JCV, and SV40 using melt curve analysis. Conventional PCR was used with the same primers to generate products for pyrosequencing. Two sequencing primers were used that differentiate the polyoma viruses. Seven of 11 biopsies (1 patient had 2 biopsies) with PML were positive for JCV by real-time PCR and/or PCR/pyrosequencing. Three of 4 remaining biopsies were positive by real-time PCR but had melting points between JCV and SV40. The 4 specimens that were negative or atypical by LightCycler PCR were positive by traditional PCR, but 1 had an amplicon of lower molecular weight by gel electrophoresis. These were shown to represent JCV by at least 1 of the 2 pyrosequencing primers. The biopsies from patients without PML were PCR negative. Both the LightCycler and pyrosequencing assays are useful for confirming JCV in brain biopsies from patients with PML, but variant JCVs may require supplementary methods to confirm JCV infection.

Amplification of nonspecific products in quantitative polymerase chain reactions (qPCR

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Adrián Ruiz-Villalba

2017-12-01

Full Text Available Quantitative PCR allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, a survey with 93 validated assays for genes in the Wnt-pathway showed that the amplification of nonspecific products occurs frequently and is unrelated to Cq or PCR efficiency values. Titration experiments showed that the occurrence of low and high melting temperature artifacts was shown to be determined by annealing temperature, primer concentration and cDNA input. To explore the range of input variations that occur in the normal use of the Cre assay these conditions were mimicked in a complete two-way design of template −plasmid DNA- and non-template −mouse cDNA- concentrations. These experiments showed that the frequency of the amplification of the correct product and the artifact, as well as the valid quantification of the correct product, depended on the concentration of the non-template cDNA. This finding questions the interpretation of dilution series in which template as well as non-template concentrations are simultaneously decreasing. Repetition of this cDNA concentration experiment with other templates revealed that exact reproduction qPCR experiments was affected by the time it takes to complete the pipetting of a qPCR plate. Long bench times were observed to lead to significantly more artifacts. However, the measurement of artifact-associated fluorescence can be avoided by inclusion of a small heating step after the elongation phase in the amplification protocol. Taken together, this trouble-shooting journey showed that reliability and reproducibility of qPCR experiments not only depends on standardization and reporting of the biochemistry and technical aspects but also on hitherto neglected factors as sample dilution and waiting times in the laboratory work flow. Keywords: RT-qPCR, Melting curve analysis, Reaction parameters, Artifacts

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

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Kawaguchi Makoto

2010-01-01

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

Molecular typing for blood group antigens within 40 minutes by direct PCR from plasma or serum

Science.gov (United States)

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

2016-01-01

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

Hydroxyl and molecular H2O diffusivity in a haploandesitic melt

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Ni, Huaiwei; Xu, Zhengjiu; Zhang, Youxue

2013-02-01

H2O diffusion in a haploandesitic melt (a high-silica and Fe-free andesitic melt, NBO/T = 0.173) has been investigated at 1 GPa in a piston-cylinder apparatus. We adopted a double diffusion couple technique, in which one couple was composed of a nominally anhydrous glass with 0.01 wt.% H2O and a hydrous glass with 5.7 wt.% H2O, and the other contained the same nominally anhydrous glass and a hydrous glass with 3.3 wt.% H2O. Both couples were annealed in a single experimental run and hence experienced exactly the same P-T history, which is crucial for constraining the dependence of H2O diffusivity on water content. H2O concentration profiles were measured by both Fourier transform infrared (FTIR) microspectroscopy and confocal Raman microspectroscopy. Nearly identical profiles were obtained from Raman and FTIR methods for profile length >1 mm (produced at 1619-1842 K). By contrast, for profile lengths <100 μm (produced at 668-768 K), FTIR profiles show marked convolution effects compared to Raman profiles. A comparison between the short FTIR and Raman profiles indicates that the real spatial resolution (FWHM) of FTIR analyses is about 28 μm for a 7 μm wide aperture on ˜200 μm thick glasses. While the short profiles are not reliable for quantitative modeling, the long diffusion profiles at superliquidus temperatures can be fit reasonably well by a diffusivity model previously developed for felsic melts, in which molecular H2O (H2Om) is the only diffusive species and its diffusivity (D) increases exponentially with the content of total water (H2Ot). However, there is noticeable misfit of the data at low H2Ot concentrations, suggesting that OH diffusivity (DOH) cannot be neglected in this andesitic melt at high temperatures and low water contents. We hence develop a new fitting procedure that simultaneously fits both diffusion profiles from a single experimental run and accounts for the roles of both OH and H2Om diffusion. With this procedure, DOH/D is constrained

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

Science.gov (United States)

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

2016-06-01

We report the development of an automated genetic analyzer for human sample testing based on microfluidic rapid polymerase chain reaction (PCR) with high-resolution melting analysis (HRMA). The integrated DNA microfluidic cartridge was used on a platform designed with a robotic pipettor system that works by sequentially picking up different test solutions from a 384-well plate, mixing them in the tips, and delivering mixed fluids to the DNA cartridge. A novel image feedback flow control system based on a Canon 5D Mark II digital camera was developed for controlling fluid movement through a complex microfluidic branching network without the use of valves. The same camera was used for measuring the high-resolution melt curve of DNA amplicons that were generated in the microfluidic chip. Owing to fast heating and cooling as well as sensitive temperature measurement in the microfluidic channels, the time frame for PCR and HRMA was dramatically reduced from hours to minutes. Preliminary testing results demonstrated that rapid serial PCR and HRMA are possible while still achieving high data quality that is suitable for human sample testing. © 2015 Society for Laboratory Automation and Screening.

Simplifying the detection of MUTYH mutations by high resolution melting analysis

International Nuclear Information System (INIS)

López-Villar, Isabel; Martínez-López, Joaquín; Ayala, Rosa; Wesselink, Jan; Morillas, Juan Diego; López, Elena; Marín, José Carlos; Díaz-Tasende, José; González, Sara; Robles, Luis

2010-01-01

MUTYH-associated polyposis (MAP) is a disorder caused by bi-allelic germline MUTYH mutation, characterized by multiple colorectal adenomas. In order to identify mutations in MUTYH gene we applied High Resolution Melting (HRM) genotyping. HRM analysis is extensively employed as a scanning method for the detection of heterozygous mutations. Therefore, we applied HRM to show effectiveness in detecting homozygous mutations for these clinically important and frequent patients. In this study, we analyzed phenotype and genotype data from 82 patients, with multiple (>= 10) synchronous (19/82) or metachronous (63/82) adenomas and negative APC study (except one case). Analysis was performed by HRM-PCR and direct sequencing, in order to identify mutations in MUTYH exons 7, 12 and 13, where the most prevalent mutations are located. In monoallelic mutation carriers, we evaluated entire MUTYH gene in search of another possible alteration. HRM-PCR was performed with strict conditions in several rounds: the first one to discriminate the heteroduplex patterns and homoduplex patterns and the next ones, in order to refine and confirm parameters. The genotypes obtained were correlated to phenotypic features (number of adenomas (synchronous or metachronous), colorectal cancer (CRC) and family history). MUTYH germline mutations were found in 15.8% (13/82) of patients. The hot spots, Y179C (exon 7) and G396D (exon 13), were readily identified and other mutations were also detected. Each mutation had a reproducible melting profile by HRM, both heterozygous mutations and homozygous mutations. In our study of 82 patients, biallelic mutation is associated with being a carrier of ≥10 synchronous polyps (p = 0.05) and there is no association between biallelic mutation and CRC (p = 0.39) nor family history (p = 0.63). G338H non-pathogenic polymorphism (exon 12) was found in 23.1% (19/82) of patients. In all cases there was concordance between HRM (first and subsequent rounds) and sequencing

Development of a multiplex real-time PCR assay for phylogenetic analysis of Uropathogenic Escherichia coli.

Science.gov (United States)

Hasanpour, Mojtaba; Najafi, Akram

2017-06-01

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.

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

Variability of Basal Melt Beneath the Pine Island Glacier Ice Shelf, West Antarctica

Science.gov (United States)

Bindschadler, Robert; Vaughan, David G.; Vornberger, Patricia

2011-01-01

Observations from satellite and airborne platforms are combined with model calculations to infer the nature and efficiency of basal melting of the Pine Island Glacier ice shelf, West Antarctica, by ocean waters. Satellite imagery shows surface features that suggest ice-shelf-wide changes to the ocean s influence on the ice shelf as the grounding line retreated. Longitudinal profiles of ice surface and bottom elevations are analyzed to reveal a spatially dependent pattern of basal melt with an annual melt flux of 40.5 Gt/a. One profile captures a persistent set of surface waves that correlates with quasi-annual variations of atmospheric forcing of Amundsen Sea circulation patterns, establishing a direct connection between atmospheric variability and sub-ice-shelf melting. Ice surface troughs are hydrostatically compensated by ice-bottom voids up to 150m deep. Voids form dynamically at the grounding line, triggered by enhanced melting when warmer-than-average water arrives. Subsequent enlargement of the voids is thermally inefficient (4% or less) compared with an overall melting efficiency beneath the ice shelf of 22%. Residual warm water is believed to cause three persistent polynyas at the ice-shelf front seen in Landsat imagery. Landsat thermal imagery confirms the occurrence of warm water at the same locations.

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

2013-01-01

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

Effect of polymer melt wall slip on the flow balance of profile extrusion dies

Science.gov (United States)

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

2013-04-01

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.

Eutectic melting temperature of the lowermost Earth's mantle

Science.gov (United States)

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

2009-12-01

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

Diffusion of hydrous species in model basaltic melt

Science.gov (United States)

Zhang, Li; Guo, Xuan; Wang, Qinxia; Ding, Jiale; Ni, Huaiwei

2017-10-01

Water diffusion in Fe-free model basaltic melt with up to 2 wt% H2O was investigated at 1658-1846 K and 1 GPa in piston-cylinder apparatus using both hydration and diffusion couple techniques. Diffusion profiles measured by FTIR are consistent with a model in which both molecular H2O (H2Om) and hydroxyl (OH) contribute to water diffusion. OH diffusivity is roughly 13% of H2Om diffusivity, showing little dependence on temperature or water concentration. Water diffusion is dominated by the motion of OH until total H2O (H2Ot) concentration reaches 1 wt%. The dependence of apparent H2Ot diffusivity on H2Ot concentration appears to be overestimated by a previous study on MORB melt, but H2Ot diffusivity at 1 wt% H2Ot in basaltic melt is still greater than those in rhyolitic to andesitic melts. The appreciable contribution of OH to water diffusion in basaltic melt can be explained by enhanced mobility of OH, probably associated with the development of free hydroxyl bonded with network-modifying cations, as well as higher OH concentration. Calculation based on the Nernst-Einstein equation demonstrates that OH may serve as an effective charge carrier in hydrous basaltic melt, which could partly account for the previously observed strong influence of water on electrical conductivity of basaltic melt.

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

KAUST Repository

Gangwar, Maulshree

2012-09-23

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.

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

KAUST Repository

Gangwar, Maulshree; Waters, Alicia M.; Bej, Gautam A.; Bej, Asim K.; Mojib, Nazia

2012-01-01

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.

DNA microarray-based PCR ribotyping of Clostridium difficile.

Science.gov (United States)

Schneeberg, Alexander; Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

2015-02-01

This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Petrological Geodynamics of Mantle Melting II. AlphaMELTS + Multiphase Flow: Dynamic Fractional Melting

Science.gov (United States)

Tirone, Massimiliano

2018-03-01

In this second installment of a series that aims to investigate the dynamic interaction between the composition and abundance of the solid mantle and its melt products, the classic interpretation of fractional melting is extended to account for the dynamic nature of the process. A multiphase numerical flow model is coupled with the program AlphaMELTS, which provides at the moment possibly the most accurate petrological description of melting based on thermodynamic principles. The conceptual idea of this study is based on a description of the melting process taking place along a 1-D vertical ideal column where chemical equilibrium is assumed to apply in two local sub-systems separately on some spatial and temporal scale. The solid mantle belongs to a local sub-system (ss1) that does not interact chemically with the melt reservoir which forms a second sub-system (ss2). The local melt products are transferred in the melt sub-system ss2 where the melt phase eventually can also crystallize into a different solid assemblage and will evolve dynamically. The main difference with the usual interpretation of fractional melting is that melt is not arbitrarily and instantaneously extracted from the mantle, but instead remains a dynamic component of the model, hence the process is named dynamic fractional melting (DFM). Some of the conditions that may affect the DFM model are investigated in this study, in particular the effect of temperature, mantle velocity at the boundary of the mantle column. A comparison is made with the dynamic equilibrium melting (DEM) model discussed in the first installment. The implications of assuming passive flow or active flow are also considered to some extent. Complete data files of most of the DFM simulations, four animations and two new DEM simulations (passive/active flow) are available following the instructions in the supplementary material.

Introduction on Using the FastPCR Software and the Related Java Web Tools for PCR and Oligonucleotide Assembly and Analysis.

Science.gov (United States)

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

2017-01-01

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 http://primerdigital.com/fastpcr.html , and its online version is located at http://primerdigital.com/tools/pcr.html .

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

DEFF Research Database (Denmark)

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

2005-01-01

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...

GLASS MELTING PHENOMENA, THEIR ORDERING AND MELTING SPACE UTILISATION

Directory of Open Access Journals (Sweden)

Němec L.

2013-12-01

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 %.

Molecular characterization of Salmonella isolates by REP-PCR and RAPD analysis.

Science.gov (United States)

Albufera, U; Bhugaloo-Vial, P; Issack, M I; Jaufeerally-Fakim, Y

2009-05-01

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.

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.

2006-01-01

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

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

Directory of Open Access Journals (Sweden)

Massimiliano Tirone

2017-10-01

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

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: jin2@cdc.gov; Hill, Vincent R.

2013-06-28

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

A novel photoinduced electron transfer (PET) primer technique for rapid real-time PCR detection of Cryptosporidium spp

International Nuclear Information System (INIS)

Jothikumar, N.; Hill, Vincent R.

2013-01-01

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

Microstructure and properties of high chrome steel roller after laser surface melting

International Nuclear Information System (INIS)

Li Meiyan; Wang Yong; Han Bin; Zhao Weimin; Han Tao

2009-01-01

Laser surface melting of high chrome steels was achieved by a 5 kW continuous wave CO 2 laser. The microstructure of the laser surface-melted steels was investigated by scanning electron microscopy, transmission electron microscopy and X-ray diffractometry, and the hardness profiles were determined by a Vickers hardness tester. The corrosion behavior in 3.5% NaCl solution was studied by electrochemical corrosion equipment. The large carbides of high chrome steels are completely dissolved and ultrafine dendrites of austenite with submicroscopic M 23 C 6 carbides precipitation are formed in the melted zone. The austenite in the melted zone has a high tempering stability. The corrosion resistance of the laser surface-melted steels is significantly improved due to the dissolution of carbides and the increase of the alloying elements in the solid solution as well as the large amount of austenite.

Quantitative real-time RT-PCR and chromogenic in situ hybridization

DEFF Research Database (Denmark)

Rosa, Fabíola E; Silveira, Sara M; Silveira, Cássia G T

2009-01-01

. 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...

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

Science.gov (United States)

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

2014-01-01

A high resolution melting (HRM) curve method was developed to identify canine parvovirus type 2 (CPV-2) strains by nested PCR. Two sets of primers, CPV-426F/426R and CPV-87R/87F, were designed that amplified a 52 bp and 53 bp product from the viral VP2 capsid gene. The region amplified by CPV-426F/426R included the A4062G and T4064A mutations in CPV-2a, CPV-2b and CPV-2c. The region amplified by CPV-87F/87R included the A3045T mutation in the vaccine strains of CPV-2 and CPV-2a, CPV-2b and CPV-2c. Faecal samples were obtained from 30 dogs that were CPV antigen-positive. The DNA was isolated from the faecal samples and PCR-amplified using the two sets of primers, and genotyped by HRM curve analysis. The PCR-HRM assay was able to distinguish single nucleotide polymorphisms between CPV-2a, CPV-2b and CPV-2c using CPV-426F/426R. CPV-2a was distinguished from CPV-2b and CPV-2c by differences in the melting temperature. CPV-2b and CPV-2c could be distinguished based on the shape of the melting curve after generating heteroduplexes using a CPV-2b reference sample. The vaccine strains of CPV-2 were identified using CPV-87F/87R. Conventional methods for genotyping CPV strains are labor intensive, expensive or time consuming; the present PCR-based HRM assay might be an attractive alternative. Copyright © 2014 Elsevier Ltd. All rights reserved.

A sensitive, reproducible, and economic real-time reverse transcription PCR detecting avian metapneumovirus subtypes A and B.

Science.gov (United States)

Franzo, G; Drigo, M; Lupini, C; Catelli, E; Laconi, A; Listorti, V; Bonci, M; Naylor, C J; Martini, M; Cecchinato, M

2014-06-01

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.

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

Science.gov (United States)

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

2018-03-01

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

Mutation spectrum of 122 hemophilia A families from Taiwanese population by LD-PCR, DHPLC, multiplex PCR and evaluating the clinical application of HRM

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Chang Chieh-Ting

2008-06-01

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.

Emerging melt quality control solution technologies for aluminium melt

Directory of Open Access Journals (Sweden)

Arturo Pascual, Jr

2009-11-01

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.

Development of real-time PCR for detection of Mycoplasma hominis

DEFF Research Database (Denmark)

Baczynska, A.; Svenstrup, Helle Friis; Fedder, J.

2004-01-01

BACKGROUND: Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginosis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. Association with development of female infertility has also been suggested, but different....... Information on bacterial load in genital swabs can be obtained. The assay allowed detection of M. hominis in a closed system reducing the risk of contamination by amplicon carry-over......., glyceraldehyde-3-phosphate dehydrogenase (gap), as a target. RESULTS: Real-time PCR was optimized to detect 10 copies of M. hominis PG21 genomic DNA. A fluorescence signal was measured for all 20 other M. hominis isolates, and melting curves analysis showed variations in the melting temperature in agreement...

Microstructure and properties of high chrome steel roller after laser surface melting

Energy Technology Data Exchange (ETDEWEB)

Li Meiyan, E-mail: lmy_102411@163.com [College of Electromechanical Engineering, China University of Petroleum, 271 Bei' er Road, Dongying 257061 (China); Wang Yong; Han Bin; Zhao Weimin; Han Tao [College of Electromechanical Engineering, China University of Petroleum, 271 Bei' er Road, Dongying 257061 (China)

2009-06-15

Laser surface melting of high chrome steels was achieved by a 5 kW continuous wave CO{sub 2} laser. The microstructure of the laser surface-melted steels was investigated by scanning electron microscopy, transmission electron microscopy and X-ray diffractometry, and the hardness profiles were determined by a Vickers hardness tester. The corrosion behavior in 3.5% NaCl solution was studied by electrochemical corrosion equipment. The large carbides of high chrome steels are completely dissolved and ultrafine dendrites of austenite with submicroscopic M{sub 23}C{sub 6} carbides precipitation are formed in the melted zone. The austenite in the melted zone has a high tempering stability. The corrosion resistance of the laser surface-melted steels is significantly improved due to the dissolution of carbides and the increase of the alloying elements in the solid solution as well as the large amount of austenite.

Monitoring Metabolite Profiles of Cannabis sativa L. Trichomes during Flowering Period Using 1H NMR-Based Metabolomics and Real-Time PCR.

Science.gov (United States)

Happyana, Nizar; Kayser, Oliver

2016-08-01

Cannabis sativa trichomes are glandular structures predominantly responsible for the biosynthesis of cannabinoids, the biologically active compounds unique to this plant. To the best of our knowledge, most metabolomic works on C. sativa that have been reported previously focused their investigations on the flowers and leaves of this plant. In this study, (1)H NMR-based metabolomics and real-time PCR analysis were applied for monitoring the metabolite profiles of C. sativa trichomes, variety Bediol, during the last 4 weeks of the flowering period. Partial least squares discriminant analysis models successfully classified metabolites of the trichomes based on the harvest time. Δ (9)-Tetrahydrocannabinolic acid (1) and cannabidiolic acid (2) constituted the vital differential components of the organic preparations, while asparagine, glutamine, fructose, and glucose proved to be their water-extracted counterparts. According to RT-PCR analysis, gene expression levels of olivetol synthase and olivetolic acid cyclase influenced the accumulation of cannabinoids in the Cannabis trichomes during the monitoring time. Moreover, quantitative (1)H NMR and RT-PCR analysis of the Cannabis trichomes suggested that the gene regulation of cannabinoid biosynthesis in the C. sativa variety Bediol is unique when compared with other C. sativa varieties. Georg Thieme Verlag KG Stuttgart · New York.

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.

Science.gov (United States)

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

2016-07-01

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

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.

2000-01-01

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...

Detection and differentiation of Mycobacterium tuberculosis and nontuberculous mycobacterial isolates by real-time PCR.

Science.gov (United States)

Shrestha, Nabin K; Tuohy, Marion J; Hall, Gerri S; Reischl, Udo; Gordon, Steven M; Procop, Gary W

2003-11-01

Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35 degrees C (63.27 to 65.42 degrees C); M. kansasii, 59.20 degrees C (58.07 to 60.33 degrees C); M. avium, 57.82 degrees C (57.05 to 58.60 degrees C); M. intracellulare, 54.46 degrees C (53.69 to 55.23 degrees C); M. marinum, 58.91 degrees C (58.28 to 59.55 degrees C); rapidly growing mycobacteria, 53.09 degrees C (50.97 to 55.20 degrees C) or 43.19 degrees C (42.19 to 44.49 degrees C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.

Volatile diffusion in silicate melts and its effects on melt inclusions

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P. Scarlato

2005-06-01

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

Numerical Model based Reliability Estimation of Selective Laser Melting Process

DEFF Research Database (Denmark)

Mohanty, Sankhya; Hattel, Jesper Henri

2014-01-01

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....

Controlled localised melting in silicon by high dose germanium implantation and flash lamp annealing

International Nuclear Information System (INIS)

Voelskow, Matthias; Skorupa, Wolfgang; Pezoldt, Joerg; Kups, Thomas

2009-01-01

High intensity light pulse irradiation of monocrystalline silicon wafers is usually accompanied by inhomogeneous surface melting. The aim of the present work is to induce homogeneous buried melting in silicon by germanium implantation and subsequent flash lamp annealing. For this purpose high dose, high energy germanium implantation has been employed to lower the melting temperature of silicon in a predetermined depth region. Subsequent flash lamp irradiation at high energy densities leads to local melting of the germanium rich buried layer, whereby the thickness of the molten layer depends on the irradiation energy density. During the cooling down epitaxial crystallization takes place resulting in a largely defect-free layer. The combination of buried melting and dopant segregation has the potential to produce unusually buried doping profiles or to create strained silicon structures.

Trypanosoma rangeli: RAPD-PCR and LSSP-PCR analyses of isolates from southeast Brazil and Colombia and their relation with KPI minicircles.

Science.gov (United States)

Marquez, D S; Ramírez, L E; Moreno, J; Pedrosa, A L; Lages-Silva, E

2007-09-01

This study presents the first genetic characterization of five Trypanosoma rangeli isolates from Minas Gerais, in the southeast of Brazil and their comparison with Colombian populations by minicircle classification, RAPD-PCR and LSSP-PCR analyses. Our results demonstrated a homogenous T. rangeli population circulating among Didelphis albiventris as reservoir host in Brazil while heterogeneous populations were found in different regions of Colombia. KP1(+) minicircles were found in 100% isolates from Brazil and in 36.4% of the Colombian samples, whereas the KP2 and KP3 minicircles were detected in both groups. RAPD-PCR and LSSP-PCR profiles revealed a polymorphism within KP1(+) and KP1(-) T. rangeli populations and allowed the division of T. rangeli in two branches. The Brazilian KP1(+) isolates were more homogenous than the KP1(+) isolates from Colombia. The RAPD-PCR were entirely consistent with the distribution of KP1 minicircles while those obtained by LSSP-PCR were associated in 88.9% and 71.4% with KP1(+) and KP1(-) populations, respectively.

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

Science.gov (United States)

Ståhlberg, Anders; Elbing, Karin; Andrade-Garda, José Manuel; Sjögreen, Björn; Forootan, Amin; Kubista, Mikael

2008-04-16

The large sensitivity, high reproducibility and essentially unlimited dynamic range of real-time PCR to measure gene expression in complex samples provides the opportunity for powerful multivariate and multiway studies of biological phenomena. In multiway studies samples are characterized by their expression profiles to monitor changes over time, effect of treatment, drug dosage etc. Here we perform a multiway study of the temporal response of four yeast Saccharomyces cerevisiae strains with different glucose uptake rates upon altered metabolic conditions. We measured the expression of 18 genes as function of time after addition of glucose to four strains of yeast grown in ethanol. The data are analyzed by matrix-augmented PCA, which is a generalization of PCA for 3-way data, and the results are confirmed by hierarchical clustering and clustering by Kohonen self-organizing map. Our approach identifies gene groups that respond similarly to the change of nutrient, and genes that behave differently in mutant strains. Of particular interest is our finding that ADH4 and ADH6 show a behavior typical of glucose-induced genes, while ADH3 and ADH5 are repressed after glucose addition. Multiway real-time PCR gene expression profiling is a powerful technique which can be utilized to characterize functions of new genes by, for example, comparing their temporal response after perturbation in different genetic variants of the studied subject. The technique also identifies genes that show perturbed expression in specific strains.

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

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Andrade-Garda José

2008-04-01

Full Text Available Abstract Background The large sensitivity, high reproducibility and essentially unlimited dynamic range of real-time PCR to measure gene expression in complex samples provides the opportunity for powerful multivariate and multiway studies of biological phenomena. In multiway studies samples are characterized by their expression profiles to monitor changes over time, effect of treatment, drug dosage etc. Here we perform a multiway study of the temporal response of four yeast Saccharomyces cerevisiae strains with different glucose uptake rates upon altered metabolic conditions. Results We measured the expression of 18 genes as function of time after addition of glucose to four strains of yeast grown in ethanol. The data are analyzed by matrix-augmented PCA, which is a generalization of PCA for 3-way data, and the results are confirmed by hierarchical clustering and clustering by Kohonen self-organizing map. Our approach identifies gene groups that respond similarly to the change of nutrient, and genes that behave differently in mutant strains. Of particular interest is our finding that ADH4 and ADH6 show a behavior typical of glucose-induced genes, while ADH3 and ADH5 are repressed after glucose addition. Conclusion Multiway real-time PCR gene expression profiling is a powerful technique which can be utilized to characterize functions of new genes by, for example, comparing their temporal response after perturbation in different genetic variants of the studied subject. The technique also identifies genes that show perturbed expression in specific strains.

The PCR-GLOBWB global hydrological reanalysis product

Science.gov (United States)

Wanders, Niko; Bierkens, Marc; Sutanudjaja, Edwin; van Beek, Rens

2014-05-01

Accurate and long time series of hydrological data are important for understanding land surface water and energy budgets in many parts of the world, as well as for improving real-time hydrological monitoring and climate change anticipation. The ultimate goal of the present work is to produce a multi-decadal "land surface hydrological reanalysis" dataset with retrospective and updated hydrological states and fluxes that are constrained to available in-situ river discharge measurements. Here we use PCR-GLOBWB (van Beek et al., 2011), which is a large-scale hydrological model intended for global to regional studies. PCR-GLOBWB provides a grid-based representation of terrestrial hydrology with a typical spatial resolution of approximately 50×50 km (currently 0.5° globally) on a daily basis. For each grid cell, PCR-GLOBWB simulates moisture storage in two vertically stacked soil layers as well as the water exchange between the soil and the atmosphere and the underlying groundwater reservoir. Exchange to the atmosphere comprises precipitation, evaporation and transpiration, as well as snow accumulation and melt, which are all simulated by considering vegetation phenology and sub-grid variations of elevation, land cover and soil saturation distribution. The model includes improved schemes for runoff-infiltration partitioning, interflow, groundwater recharge and baseflow, as well as river routing of discharge. It also dynamically simulates water storage in reservoirs, water demand and the withdrawal, allocation and consumptive use of surface water and groundwater resources. By embedding the PCR-GLOBWB model in an Ensemble Kalman Filter framework, we calibrate the model parameters based on the discharge observations from the Global Runoff Data Centre. The parameters calibrated are related to snow accumulation and melt, runoff-infiltration partitioning, groundwater recharge, channel discharge and baseflow processes, as well as pre-factors to correct forcing precipitation

Double melting in polytetrafluoroethylene γ-irradiated above its melting point

International Nuclear Information System (INIS)

Serov, S.A.; Khatipov, S.A.; Sadovskaya, N.V.; Tereshenkov, A.V.; Chukov, N.A.

2012-01-01

Highlights: ► PTFE irradiation leads to formation of double melting peaks in DSC curves. ► This is connected to dual crystalline morphology typical for PTFE. ► Two crystalline types exist in the PTFE irradiated in the melt. - Abstract: PTFE irradiation above its melting point leads to formation of double melting and crystallization peaks in DSC curves. Splitting of melting peaks is connected to dual crystalline morphology typical for PTFE irradiated in the melt. According to electron microscopy, two crystalline types with different size and packing density exist in the irradiated PTFE.

Transgene detection by digital droplet PCR.

Directory of Open Access Journals (Sweden)

Dirk A Moser

Full Text Available Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR protocol for Insulin-Like Growth Factor 1 (IGF1 detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1 and Erythropoietin (EPO transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.

Rapid discrimination of Isaria javanica and Isaria poprawskii from Isaria spp. using high resolution DNA melting assays

Science.gov (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...

Melting method for miscellaneous radioactive solid waste and melting furnace

International Nuclear Information System (INIS)

Osaki, Toru; Furukawa, Hirofumi; Uda, Nobuyoshi; Katsurai, Kiyomichi

1998-01-01

A vessel containing miscellaneous solid wastes is inserted in a crucible having a releasable material on the inner surface, they are induction-heated from the outside of the crucible by way of low temperature heating coils to melt low melting point materials in the miscellaneous wastes within a temperature range at which the vessel does not melt. Then, they are induction-heated by way of high temperature heating coils to melt the vessel and not yet melted materials, those molten materials are cooled, solidified molten material and the releasable material are taken out, and then the crucible is used again. Then, the crucible can be used again, so that it can be applied to a large scaled melting furnace which treats wastes by a unit of drum. In addition, since the cleaning of the used crucible and the application of the releasable material can be conducted without interrupting the operation of the melting furnace, the operation cycle of the melting furnace can be shortened. (N.H.)

Development of a high-throughput real time PCR based on a hot-start alternative for Pfu mediated by quantum dots

Science.gov (United States)

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

2015-09-01

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

Melting heat transfer in boundary layer stagnation-point flow towards a stretching/shrinking sheet

International Nuclear Information System (INIS)

Bachok, Norfifah; Ishak, Anuar; Pop, Ioan

2010-01-01

An analysis is carried out to study the steady two-dimensional stagnation-point flow and heat transfer from a warm, laminar liquid flow to a melting stretching/shrinking sheet. The governing partial differential equations are converted into ordinary differential equations by similarity transformation, before being solved numerically using the Runge-Kutta-Fehlberg method. Results for the skin friction coefficient, local Nusselt number, velocity profiles as well as temperature profiles are presented for different values of the governing parameters. Effects of the melting parameter, stretching/shrinking parameter and Prandtl number on the flow and heat transfer characteristics are thoroughly examined. Different from a stretching sheet, it is found that the solutions for a shrinking sheet are non-unique.

Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay.

Science.gov (United States)

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

2018-05-01

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.

Tin in granitic melts: The role of melting temperature and protolith composition

Science.gov (United States)

Wolf, Mathias; Romer, Rolf L.; Franz, Leander; López-Moro, Francisco Javier

2018-06-01

Granite bound tin mineralization typically is seen as the result of extreme magmatic fractionation and late exsolution of magmatic fluids. Mineralization, however, also could be obtained at considerably less fractionation if initial melts already had enhanced Sn contents. We present chemical data and results from phase diagram modeling that illustrate the dominant roles of protolith composition, melting conditions, and melt extraction/evolution for the distribution of Sn between melt and restite and, thus, the Sn content of melts. We compare the element partitioning between leucosome and restite of low-temperature and high-temperature migmatites. During low-temperature melting, trace elements partition preferentially into the restite with the possible exception of Sr, Cd, Bi, and Pb, that may be enriched in the melt. In high-temperature melts, Ga, Y, Cd, Sn, REE, Pb, Bi, and U partition preferentially into the melt whereas Sc, V, Cr, Co, Ni, Mo, and Ba stay in the restite. This contrasting behavior is attributed to the stability of trace element sequestering minerals during melt generation. In particular muscovite, biotite, titanite, and rutile act as host phases for Sn and, therefore prevent Sn enrichment in the melt as long as they are stable phases in the restite. As protolith composition controls both the mineral assemblage and modal contents of the various minerals, protolith composition eventually also controls the fertility of a rock during anatexis, restite mineralogy, and partitioning behavior of trace metals. If a particular trace element is sequestered in a phase that is stable during partial melting, the resulting melt is depleted in this element whereas the restite becomes enriched. Melt generation at high temperature may release Sn when Sn-hosts become unstable. If melt has not been lost before the breakdown of Sn-hosts, Sn contents in the melt will increase but never will be high. In contrast, if melt has been lost before the decomposition of Sn

Comparative Study on Two Melting Simulation Methods: Melting Curve of Gold

International Nuclear Information System (INIS)

Liu Zhong-Li; Li Rui; Sun Jun-Sheng; Zhang Xiu-Lu; Cai Ling-Cang

2016-01-01

Melting simulation methods are of crucial importance to determining melting temperature of materials efficiently. A high-efficiency melting simulation method saves much simulation time and computational resources. To compare the efficiency of our newly developed shock melting (SM) method with that of the well-established two-phase (TP) method, we calculate the high-pressure melting curve of Au using the two methods based on the optimally selected interatomic potentials. Although we only use 640 atoms to determine the melting temperature of Au in the SM method, the resulting melting curve accords very well with the results from the TP method using much more atoms. Thus, this shows that a much smaller system size in SM method can still achieve a fully converged melting curve compared with the TP method, implying the robustness and efficiency of the SM method. (paper)

Thermal evolutions of two kinds of melt pond with different salinity

Science.gov (United States)

Kim, Joo-Hong; Wilkinson, Jeremy; Moon, Woosok; Hwang, Byongjun; Granskog, Mats

2016-04-01

Melt ponds are water pools on sea ice. Their formation reduces ice surface albedo and alter surface energy balance, by which the ice melting and freezing processes are regulated. Thus, better understanding of their radiative characteristics has been vital to improve the simulation of melting/freezing of sea ice in numerical models. A melt pond would preserve nearly fresh water if it formed on multi-year ice and no flooding of sea water occurred, whereas a melt pond would contain more salty water if it formed on thinner and porous first-year ice, if there were an inflow of sea water by streams or cracks. One would expect that the fluid dynamic/thermodynamic properties (e.g., turbulence, stability, etc.) of pond water are influenced by the salinity, so that the response of pond water to any heat input (e.g., shortwave radiation) would be different. Therefore, better understanding of the salinity-dependent thermal evolution also has significant potential to improve the numerical simulation of the sea ice melting/freezing response to radiative thermal forcing. To observe and understand the salinity-dependent thermal evolution, two ice mass balance buoys (IMBs) were deployed in two kinds (fresh and salty) of melt pond on a same ice floe on 13 August 2015 during Araon Arctic cruise. The thermistor chain, extending from the air through the pond and ice into the sea water, was deployed through a drilled borehole inside the pond. Besides, the IMBs were also accompanied with three broadband solar radiation sensors (two (up and down) in the air over melt pond and one upward-looking under sea ice) to measure the net shortwave radiation at the pond surface and the penetrating solar radiation through ice. Also, the web camera was installed to observe any updates in the conditions of equipment and surrounding environment (e.g., weather, surface state, etc.). On the date of deployment, the fresh pond had salinity of 2.3 psu, light blue color, lots of slush ice particles which

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

Directory of Open Access Journals (Sweden)

Skorić Marijana

2012-01-01

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.

Rapid Determination of Lymphogranuloma Venereum Serovars of Chlamydia trachomatis by Quantitative High-Resolution Melt Analysis (HRMA)

Science.gov (United States)

Stevens, Matthew P.; Garland, Suzanne M.; Zaia, Angelo M.; Tabrizi, Sepehr N.

2012-01-01

A quantitative high-resolution melt analysis assay was developed to differentiate lymphogranuloma venereum-causing serovars of Chlamydia trachomatis (L1 to L3) from other C. trachomatis serovars (D to K). The detection limit of this assay is approximately 10 copies per reaction, comparable to the limits of other quantitative-PCR-based methods. PMID:22933594

Amphibious Magnetotelluric Investigation of the Aleutian Arc: Mantle Melt Generation and Migration beneath Okmok Caldera

Science.gov (United States)

Zelenak, G.; Key, K.; Bennington, N. L.; Bedrosian, P.

2015-12-01

Understanding the factors controlling the release of volatiles from the downgoing slab, the subsequent generation of melt in the overlying mantle wedge, the migration of melt to the crust, and its evolution and emplacement within the crust are important for advancing our understanding of arc magmatism and crustal genesis. Because melt and aqueous fluids are a few orders of magnitude more electrically conductive than unmelted peridotite, the conductivity-mapping magnetotelluric (MT) method is well-suited to imaging fluids and melt beneath arc volcanoes. Here we present conductivity results from an amphibious MT profile crossing Okmok volcano in the central Aleutian arc. The Aleutian arc is one of the most volcanically active regions in North America, making it an ideal location for studying arc magnetism. Okmok volcano, located on the northeastern portion of Umnak Island, is among the most active volcanoes in the Aleutian chain. In addition to two caldera-forming events in the Holocene, numerous eruptions in the past century indicate a robust magmatic supply. Previous coarse resolution seismic studies have inferred a crustal magma reservoir. In order to investigate the role fluids play in melting the mantle wedge, how melts ascend through the corner flow regime of the mantle wedge, how melt migrates and is stored within the upper mantle and crust, and how this impacts explosive caldera forming eruptions, we carried out an amphibious geophysical survey across the arc in June-July 2015. Twenty-nine onshore MT stations and 10 offshore stations were collected in a 3D array covering Okmok, and 43 additional offshore MT stations completed a 300 km amphibious profile starting at the trench, crossing the forearc, arc and backarc. Thirteen onshore passive seismic stations were also installed and will remain in place for one year to supplement the twelve permanent stations on the island. Data collected by this project will be used to map seismic velocity and electrical

Olivine/melt transition metal partitioning, melt composition, and melt structure—Melt polymerization and Qn-speciation in alkaline earth silicate systems

Science.gov (United States)

Mysen, Bjorn O.

2008-10-01

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

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

DEFF Research Database (Denmark)

Veng, Torben; Skettrup, Torben

1997-01-01

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...... the waveguide refractive index profile from the measured mode indices is introduced. The main advantage of this characterization method compared with other methods is that it also applies to single-mode waveguides. Using the new characterization method we investigate in detail the relation between waveguide...

Permeability and 3-D melt geometry in shear-induced high melt fraction conduits

Science.gov (United States)

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

2017-12-01

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

Three-dimensional model of heat transport during In Situ Vitrification with melting and cool down

International Nuclear Information System (INIS)

Hawkes, G.L.

1993-01-01

A potential technology for permanent remediation of buried wastes is the In Situ Vitrification (ISV) process. This process uses electrical resistance heating to melt waste and contaminated soil in place to produce a durable, glasslike material that encapsulates and immobilizes buried wastes. The magnitude of the resulting electrical resistance heating is sufficient to cause soil melting. As the molten region grows, surface heat losses cause the soil near the surface to re solidify. This paper presents numerical results obtained by considering heat transport and melting when solving the conservation of mass and energy equations using finite element methods. A local heat source is calculated by solving the electric field equation and calculating a Joule Heat source term. The model considered is a three-dimensional model of the electrodes and surrounding soil. Also included in the model is subsidence; where the surface of the melted soil subsides due to the change in density when the soil melts. A power vs. time profile is implemented for typical ISV experiments. The model agrees well with experimental data for melt volume and melt shape

Detection of adenoviruses in shellfish by means of conventional-PCR, nested-PCR, and integrated cell culture PCR (ICC/PCR).

Science.gov (United States)

Rigotto, C; Sincero, T C M; Simões, C M O; Barardi, C R M

2005-01-01

We tested three PCR based methodologies to detect adenoviruses associated with cultivated oysters. Conventional-PCR, nested-PCR, and integrated cell culture-PCR (ICC/PCR) were first optimized using oysters seeded with know amounts of Adenovirus serotype 5 (Ad5). The maximum sensitivity for Ad5 detection was determined for each method, and then used to detect natural adenovirus contamination in oysters from three aquiculture farms in Florianopolis, Santa Catarina State, Brazil, over a period of 6 months. The results showed that the nested-PCR was more sensitive (limit of detection: 1.2 PFU/g of tissue) than conventional-PCR and ICC-PCR (limit of detection for both: 1.2 x 10(2)PFU/g of tissue) for detection of Ad5 in oyster extracts. Nested-PCR was able to detect 90% of Ad5 contamination in harvested oyster samples, while conventional-PCR was unable to detect Ad5 in any of the samples. The present work suggests that detection of human adenoviruses can be used as a tool to monitor the presence of human viruses in marine environments where shellfish grow, and that nested-PCR is the method of choice.

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

DEFF Research Database (Denmark)

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

2014-01-01

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

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

2013-04-01

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.

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

Science.gov (United States)

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

2016-06-01

Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ∼0.5 °C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi

Directory of Open Access Journals (Sweden)

Danielle C. Leal

2011-07-01

Full Text Available Conventional PCR (PCRTeq for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128, but did not differ significantly from the M/PCRTeq-Bc (1:64. In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780 and moderate agreement with N/PCR-Teq (k = 0.562 and M/PCRTeq-Bc (k = 0.488. PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05, and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05. PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

Selection and Validation of Reference Genes for qRT-PCR Expression Analysis of Candidate Genes Involved in Olfactory Communication in the Butterfly Bicyclus anynana

OpenAIRE

Arun, Alok; Bauml?, V?ronique; Amelot, Ga?l; Nieberding, Caroline M.

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at ident...

Methylation-Sensitive High Resolution Melting (MS-HRM).

Science.gov (United States)

Hussmann, Dianna; Hansen, Lise Lotte

2018-01-01

Methylation-Sensitive High Resolution Melting (MS-HRM) is an in-tube, PCR-based method to detect methylation levels at specific loci of interest. A unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1-1% methylated alleles in an unmethylated background.Primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays. Bisulfite treatment of the DNA prior to performing MS-HRM ensures a different base composition between methylated and unmethylated DNA, which is used to separate the resulting amplicons by high resolution melting.The high sensitivity of MS-HRM has proven useful for detecting cancer biomarkers in a noninvasive manner in urine from bladder cancer patients, in stool from colorectal cancer patients, and in buccal mucosa from breast cancer patients. MS-HRM is a fast method to diagnose imprinted diseases and to clinically validate results from whole-epigenome studies. The ability to detect few copies of methylated DNA makes MS-HRM a key player in the quest for establishing links between environmental exposure, epigenetic changes, and disease.

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.

Science.gov (United States)

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

2016-01-01

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: journals.permissions@oup.com.

Evaluation of Melt Behavior with initial Melt Velocity under SFR Severe Accidents

Energy Technology Data Exchange (ETDEWEB)

Heo, Hyo; Bang, In Cheol [UNIST, Ulsan (Korea, Republic of); Jerng, Dong Wook [Chung-Ang Univ, Seoul (Korea, Republic of)

2015-10-15

In the current Korean sodium-cooled fast reactor (SFR) program, early dispersion of the molten metallic fuel within a subchannel is suggested as one of the inherent safety strategies for the initiating phase of hypothetical core disruptive accident (HCDA). The safety strategy provides negative reactivity driven by the melt dispersal, so it could reduce the possibility of the recriticality event under a severe triple or more fault scenario for SFR. Since the behavior of the melt dispersion is unpredictable, it depends on the accident condition, particularly core region. While the voided coolant channel region is usually developed in the inner core, the unvoided coolant channel region is formed in the outer core. It is important to confirm the fuel dispersion with the core region, but there are not sufficient existing studies for them. From the existing studies, the coolant vapor pressure is considered as one of driving force to move the melt towards outside of the core. There is a complexity of the phenomena during intermixing of the melt with the coolant after the horizontal melt injections. It is too difficult to understand the several combined mechanisms related to the melt dispersion and the fragmentation. Thus, it could be worthwhile to study the horizontal melt injections at lower temperature as a preliminary study in order to identify the melt dispersion phenomena. For this reason, it is required to clarify whether the coolant vapor pressure is the driving force of the melt dispersion with the core region. The specific conditions to be well dispersed for the molten metallic fuel were discussed in the experiments with the simulant materials. The each melt behavior was compared to evaluate the melt dispersion under the coolant void condition and the boiling condition. As the results, the following results are remarked: 1. The upward melt dispersion did not occur for a given melt and coolant temperature in the nonboiling range. Over current range of conditions

Evaluation of Melt Behavior with initial Melt Velocity under SFR Severe Accidents

International Nuclear Information System (INIS)

Heo, Hyo; Bang, In Cheol; Jerng, Dong Wook

2015-01-01

In the current Korean sodium-cooled fast reactor (SFR) program, early dispersion of the molten metallic fuel within a subchannel is suggested as one of the inherent safety strategies for the initiating phase of hypothetical core disruptive accident (HCDA). The safety strategy provides negative reactivity driven by the melt dispersal, so it could reduce the possibility of the recriticality event under a severe triple or more fault scenario for SFR. Since the behavior of the melt dispersion is unpredictable, it depends on the accident condition, particularly core region. While the voided coolant channel region is usually developed in the inner core, the unvoided coolant channel region is formed in the outer core. It is important to confirm the fuel dispersion with the core region, but there are not sufficient existing studies for them. From the existing studies, the coolant vapor pressure is considered as one of driving force to move the melt towards outside of the core. There is a complexity of the phenomena during intermixing of the melt with the coolant after the horizontal melt injections. It is too difficult to understand the several combined mechanisms related to the melt dispersion and the fragmentation. Thus, it could be worthwhile to study the horizontal melt injections at lower temperature as a preliminary study in order to identify the melt dispersion phenomena. For this reason, it is required to clarify whether the coolant vapor pressure is the driving force of the melt dispersion with the core region. The specific conditions to be well dispersed for the molten metallic fuel were discussed in the experiments with the simulant materials. The each melt behavior was compared to evaluate the melt dispersion under the coolant void condition and the boiling condition. As the results, the following results are remarked: 1. The upward melt dispersion did not occur for a given melt and coolant temperature in the nonboiling range. Over current range of conditions

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

Directory of Open Access Journals (Sweden)

Qiuying Huang

2011-04-01

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

Complementary techniques: validation of gene expression data by quantitative real time PCR.

Science.gov (United States)

Provenzano, Maurizio; Mocellin, Simone

2007-01-01

Microarray technology can be considered the most powerful tool for screening gene expression profiles of biological samples. After data mining, results need to be validated with highly reliable biotechniques allowing for precise quantitation of transcriptional abundance of identified genes. Quantitative real time PCR (qrt-PCR) technology has recently reached a level of sensitivity, accuracy and practical ease that support its use as a routine bioinstrumentation for gene level measurement. Currently, qrt-PCR is considered by most experts the most appropriate method to confirm or confute microarray-generated data. The knowledge of the biochemical principles underlying qrt-PCR as well as some related technical issues must be beard in mind when using this biotechnology.

Rapakivi texture formation via disequilibrium melting in a contact partial melt zone, Antarctica

Science.gov (United States)

Currier, R. M.

2017-12-01

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

Experimental results for TiO2 melting and release using cold crucible melting

International Nuclear Information System (INIS)

Hong, S. W.; Min, B. T.; Park, I. G.; Kim, H. D.

2000-01-01

To simulate the severe accident phenomena using the real reactor material which melting point is about 2,800K, the melting and release method for materials with high melting point should be developed. This paper discusses the test results for TiO 2 materials using the cold crucible melting method to study the melting and release method of actual corium. To melt and release of few kg of TiO2, the experimental facility is manufactured through proper selection of design parameters such as frequency and capacity of R.F generator, crucible size and capacity of coolant. The melting and release of TiO 2 has been successfully performed in the cold crucible of 15cm in inner diameter and 30cm in height with 30kW RF power generator of 370 KHz. In the melt delivery experiment, about 2.6kg of molten TiO2, 60% of initial charged mass, is released. Rest of it is remained in the watercage in form of the rubble crust formed at the top of crucible and melt crust formed at the interface between the water-cage and melt. Especially, in the melt release test, the location of the working coil is important to make the thin crust at the bottom of the crucible

High-resolution melting analysis of the single nucleotide polymorphism hot-spot region in the rpoB gene as an indicator of reduced susceptibility to rifaximin in Clostridium difficile.

Science.gov (United States)

Pecavar, Verena; Blaschitz, Marion; Hufnagl, Peter; Zeinzinger, Josef; Fiedler, Anita; Allerberger, Franz; Maass, Matthias; Indra, Alexander

2012-06-01

Clostridium difficile, a Gram-positive, spore-forming, anaerobic bacterium, is the main causative agent of hospital-acquired diarrhoea worldwide. In addition to metronidazole and vancomycin, rifaximin, a rifamycin derivative, is a promising antibiotic for the treatment of recurring C. difficile infections (CDI). However, exposure of C. difficile to this antibiotic has led to the development of rifaximin-resistance due to point mutations in the β-subunit of the RNA polymerase (rpoB) gene. In the present study, 348 C. difficile strains with known PCR-ribotypes were investigated for respective single nucleotide polymorphisms (SNPs) within the proposed rpoB hot-spot region by using high-resolution melting (HRM) analysis. This method allows the detection of SNPs by comparing the altered melting behaviour of dsDNA with that of wild-type DNA. Discrimination between wild-type and mutant strains was enhanced by creating heteroduplexes by mixing sample DNA with wild-type DNA, leading to characteristic melting curve shapes from samples containing SNPs in the respective rpoB section. In the present study, we were able to identify 16 different rpoB sequence-types (ST) by sequencing analysis of a 325 bp fragment. The 16 PCR STs displayed a total of 24 different SNPs. Fifteen of these 24 SNPs were located within the proposed 151 bp SNP hot-spot region, resulting in 11 different HRM curve profiles (CP). Eleven SNPs (seven of which were within the proposed hot-spot region) led to amino acid substitutions associated with reduced susceptibility to rifaximin and 13 SNPs (eight of which were within the hot-spot region) were synonymous. This investigation clearly demonstrates that HRM analysis of the proposed SNP hot-spot region in the rpoB gene of C. difficile is a fast and cost-effective method for the identification of C. difficile samples with reduced susceptibility to rifaximin and even allows simultaneous SNP subtyping of the respective C. difficile isolates.

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

2012-07-31

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.

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.

1979-04-01

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

Soft fruit traceability in food matrices using real-time PCR.

Science.gov (United States)

Palmieri, Luisa; Bozza, Elisa; Giongo, Lara

2009-02-01

Food product authentication provides a means of monitoring and identifying products for consumer protection and regulatory compliance. There is a scarcity of analytical methods for confirming the identity of fruit pulp in products containing Soft Fruit. In the present work we have developed a very sensible qualitative and quantitative method to determine the presence of berry DNAs in different food matrices. To our knowledge, this is the first study that shows the applicability, to Soft Fruit traceability, of melting curve analysis and multiplexed fluorescent probes, in a Real-Time PCR platform. This methodology aims to protect the consumer from label misrepresentation.

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

2017-01-01

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...

The WAIS Melt Monitor: An automated ice core melting system for meltwater sample handling and the collection of high resolution microparticle size distribution data

Science.gov (United States)

Breton, D. J.; Koffman, B. G.; Kreutz, K. J.; Hamilton, G. S.

2010-12-01

profiles to guide the melting process. The combination of these data allow us to analyze melt head performance, minimize outer-to-inner fraction contamination and avoid melt head flooding. The WAIS Melt Monitor system allows the collection of real-time, sub-annual microparticle and electrical conductivity data while producing and storing enough sample for traditional Coulter-Counter particle measurements as well long term acid leaching of bioactive metals (e.g., Fe, Co, Cd, Cu, Zn) prior to chemical analysis.

Cloud screening and melt water detection over melting sea ice using AATSR/SLSTR

Science.gov (United States)

Istomina, Larysa; Heygster, Georg

2014-05-01

With the onset of melt in the Arctic Ocean, the fraction of melt water on sea ice, the melt pond fraction, increases. The consequences are: the reduced albedo of sea ice, increased transmittance of sea ice and affected heat balance of the system with more heat passing through the ice into the ocean, which facilitates further melting. The onset of melt, duration of melt season and melt pond fraction are good indicators of the climate state of the Arctic and its change. In the absence of reliable sea ice thickness retrievals in summer, melt pond fraction retrieval from satellite is in demand as input for GCM as an indicator of melt state of the sea ice. The retrieval of melt pond fraction with a moderate resolution radiometer as AATSR is, however, a non-trivial task due to a variety of subpixel surface types with very different optical properties, which give non-unique combinations if mixed. In this work this has been solved by employing additional information on the surface and air temperature of the pixel. In the current work, a concept of melt pond detection on sea ice is presented. The basis of the retrieval is the sensitivity of AATSR reflectance channels 550nm and 860nm to the amount of melt water on sea ice. The retrieval features extensive usage of a database of in situ surface albedo spectra. A tree of decisions is employed to select the feasible family of in situ spectra for the retrieval, depending on the melt stage of the surface. Reanalysis air temperature at the surface and brightness temperature measured by the satellite sensor are analyzed in order to evaluate the melting status of the surface. Case studies for FYI and MYI show plausible retrieved melt pond fractions, characteristic for both of the ice types. The developed retrieval can be used to process the historical AATSR (2002-2012) dataset, as well as for the SLSTR sensor onboard the future Sentinel-3 mission (scheduled for launch in 2015), to keep the continuity and obtain longer time sequence

Viscosity and diffusivity in melts: from unary to multicomponent systems

Science.gov (United States)

Chen, Weimin; Zhang, Lijun; Du, Yong; Huang, Baiyun

2014-05-01

Viscosity and diffusivity, two important transport coefficients, are systematically investigated from unary melt to binary to multicomponent melts in the present work. By coupling with Kaptay's viscosity equation of pure liquid metals and effective radii of diffusion species, the Sutherland equation is modified by taking the size effect into account, and further derived into an Arrhenius formula for the convenient usage. Its reliability for predicting self-diffusivity and impurity diffusivity in unary liquids is then validated by comparing the calculated self-diffusivities and impurity diffusivities in liquid Al- and Fe-based alloys with the experimental and the assessed data. Moreover, the Kozlov model was chosen among various viscosity models as the most reliable one to reproduce the experimental viscosities in binary and multicomponent melts. Based on the reliable viscosities calculated from the Kozlov model, the modified Sutherland equation is utilized to predict the tracer diffusivities in binary and multicomponent melts, and validated in Al-Cu, Al-Ni and Al-Ce-Ni melts. Comprehensive comparisons between the calculated results and the literature data indicate that the experimental tracer diffusivities and the theoretical ones can be well reproduced by the present calculations. In addition, the vacancy-wind factor in binary liquid Al-Ni alloys with the increasing temperature is also discussed. What's more, the calculated inter-diffusivities in liquid Al-Cu, Al-Ni and Al-Ag-Cu alloys are also in excellent agreement with the measured and theoretical data. Comparisons between the simulated concentration profiles and the measured ones in Al-Cu, Al-Ce-Ni and Al-Ag-Cu melts are further used to validate the present calculation method.

Laser post-processing of Inconel 625 made by selective laser melting

Science.gov (United States)

Witkin, David; Helvajian, Henry; Steffeney, Lee; Hansen, William

2016-04-01

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.

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.

Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases.

Science.gov (United States)

Rossetti, Lia; Giraffa, Giorgio

2005-11-01

About a thousand lactic acid bacteria (LAB) isolated from dairy products, especially cheeses, were identified and typed by species-specific PCR and RAPD-PCR, respectively. RAPD-PCR profiles, which were obtained by using the M13 sequence as a primer, allowed us to implement a large database of different fingerprints, which were analysed by BioNumerics software. Cluster analysis of the combined RAPD-PCR fingerprinting profiles enabled us to implement a library, which is a collection of library units, which in turn is a selection of representative database entries. A library unit, in this case, can be considered to be a definable taxon. The strains belonged to 11 main RAPD-PCR fingerprinting library units identified as Lactobacillus casei/paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus and Lactococcus lactis. The possibility to routinely identify newly typed, bacterial isolates by consulting the library of the software was valued. The proposed method could be suggested to refine previous strain identifications, eliminate redundancy and dispose of a technologically useful LAB strain collection. The same approach could also be applied to identify LAB strains isolated from other food ecosystems.

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

Directory of Open Access Journals (Sweden)

Ricardo Andrade Zampieri

2016-02-01

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

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

Science.gov (United States)

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

2016-02-01

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

Melting of Dense Sodium

International Nuclear Information System (INIS)

Gregoryanz, Eugene; Degtyareva, Olga; Hemley, Russell J.; Mao, Ho-kwang; Somayazulu, Maddury

2005-01-01

High-pressure high-temperature synchrotron diffraction measurements reveal a maximum on the melting curve of Na in the bcc phase at ∼31 GPa and 1000 K and a steep decrease in melting temperature in its fcc phase. The results extend the melting curve by an order of magnitude up to 130 GPa. Above 103 GPa, Na crystallizes in a sequence of phases with complex structures with unusually low melting temperatures, reaching 300 K at 118 GPa, and an increased melting temperature is observed with further increases in pressure

A 2D double-porosity model for melting and melt migration beneath mid-oceanic ridges

Science.gov (United States)

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

2017-12-01

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.

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

Energy Technology Data Exchange (ETDEWEB)

David Schwam

2012-12-15

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.

Differentiation of minute virus of mice and mouse parvovirus by high resolution melting curve analysis.

Science.gov (United States)

Rao, Dan; Wu, Miaoli; Wang, Jing; Yuan, Wen; Zhu, Yujun; Cong, Feng; Xu, Fengjiao; Lian, Yuexiao; Huang, Bihong; Wu, Qiwen; Chen, Meili; Zhang, Yu; Huang, Ren; Guo, Pengju

2017-12-01

Murine parvovirus is one of the most prevalent infectious pathogens in mouse colonies. A specific primer pair targeting the VP2 gene of minute virus of mice (MVM) and mouse parvovirus (MPV) was utilized for high resolution melting (HRM) analysis. The resulting melting curves could distinguish these two virus strains and there was no detectable amplification of the other mouse pathogens which included rat parvovirus (KRV), ectromelia virus (ECT), mouse adenovirus (MAD), mouse cytomegalovirus (MCMV), polyoma virus (Poly), Helicobactor hepaticus (H. hepaticus) and Salmonella typhimurium (S. typhimurium). The detection limit of the standard was 10 copies/μL. This study showed that the PCR-HRM assay could be an alternative useful method with high specificity and sensitivity for differentiating murine parvovirus strains MVM and MPV. Copyright © 2017 Elsevier B.V. All rights reserved.

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

Science.gov (United States)

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

2017-01-01

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

Fuel Rod Melt Progression Simulation Using Low-Temperature Melting Metal Alloy

International Nuclear Information System (INIS)

Seung Dong Lee; Suh, Kune Y.; GoonCherl Park; Un Chul Lee

2002-01-01

The TMI-2 accident and various severe fuel damage experiments have shown that core damage is likely to proceed through various states before the core slumps into the lower head. Numerous experiments were conducted to address when and how the core can lose its original geometry, what geometries are formed, and in what processes the core materials are transported to the lower plenum of the reactor pressure vessel. Core degradation progresses along the line of clad ballooning, clad oxidation, material interaction, metallic blockage, molten pool formation, melt progression, and relocation to the lower head. Relocation into the lower plenum may occur from the lateral periphery or from the bottom of the core depending upon the thermal and physical states of the pool. Determining the quantities and rate of molten material transfer to the lower head is important since significant amounts of molten material relocated to the lower head can threaten the vessel integrity by steam explosion and thermal and mechanical attack of the melt. In this paper the focus is placed on the melt flow regime on a cylindrical fuel rod utilizing the LAMDA (Lumped Analysis of Melting in Degrading Assemblies) facility at the Seoul National University. The downward relocation of the molten material is a combination of the external film flow and the internal pipe flow. The heater rods are 0.8 m long and are coated by a low-temperature melting metal alloy. The electrical internal heating method is employed during the test. External heating is adopted to simulate the exothermic Zircaloy-steam reaction. Tests are conducted in several quasi-steady-state conditions. Given the variable boundary conditions including the heat flux and the water level, observation is made for the melting location, progression, and the mass of molten material. Finally, the core melt progression model is developed from the visual inspection and quantitative analysis of the experimental data. As the core material relocates

Detection of Lymnaea columella infection by Fasciola hepatica through Multiplex-PCR

Directory of Open Access Journals (Sweden)

Kelly Grace Magalhães

2004-06-01

Full Text Available From complete mitochondrial DNA sequence of Fasciola hepatica available in Genbank, specific primers were designed for a conserved and repetitive region of this trematode. A pair of primers was used for diagnosis of infected Lymnaea columella by F. hepatica during the pre-patent period simultaneously with another pair of primers which amplified the internal transcribed spacer (ITS region of rDNA from L. columella in a single Multiplex-PCR. The amplification generated a ladder band profile specific for F. hepatica. This profile was observed in positive molluscs at different times of infection, including adult worms from the trematode. The Multiplex-PCR technique showed to be a fast and safe tool for fascioliasis diagnosis, enabling the detection of F. hepatica miracidia in L. columella during the pre-patent period and identification of transmission areas.

Sequence polymorphism can produce serious artefacts in real-time PCR assays: hard lessons from Pacific oysters

Directory of Open Access Journals (Sweden)

Camara Mark D

2008-05-01

Full Text Available Abstract Background Since it was first described in the mid-1990s, quantitative real time PCR (Q-PCR has been widely used in many fields of biomedical research and molecular diagnostics. This method is routinely used to validate whole transcriptome analyses such as DNA microarrays, suppressive subtractive hybridization (SSH or differential display techniques such as cDNA-AFLP (Amplification Fragment Length Polymorphism. Despite efforts to optimize the methodology, misleading results are still possible, even when standard optimization approaches are followed. Results As part of a larger project aimed at elucidating transcriptome-level responses of Pacific oysters (Crassostrea gigas to various environmental stressors, we used microarrays and cDNA-AFLP to identify Expressed Sequence Tag (EST fragments that are differentially expressed in response to bacterial challenge in two heat shock tolerant and two heat shock sensitive full-sib oyster families. We then designed primers for these differentially expressed ESTs in order to validate the results using Q-PCR. For two of these ESTs we tested fourteen primer pairs each and using standard optimization methods (i.e. melt-curve analysis to ensure amplification of a single product, determined that of the fourteen primer pairs tested, six and nine pairs respectively amplified a single product and were thus acceptable for further testing. However, when we used these primers, we obtained different statistical outcomes among primer pairs, raising unexpected but serious questions about their reliability. We hypothesize that as a consequence of high levels of sequence polymorphism in Pacific oysters, Q-PCR amplification is sub-optimal in some individuals because sequence variants in priming sites results in poor primer binding and amplification in some individuals. This issue is similar to the high frequency of null alleles observed for microsatellite markers in Pacific oysters. Conclusion This study highlights

Formation of D- and I-shaped geochemical profiles in saucer-shaped sills due to post- emplacement magma flow induced by thermal stresses

Science.gov (United States)

Aarnes, I.; Podladchikov, Y. Y.; Neumann, E.

2007-12-01

There are still unresolved problems in the processes of emplacement and crystallization of saucer shaped sill intrusions. We use geochemistry and numerical modelling in order to constrain identify processes in mafic sill intrusions. Profiles sampled through through a saucer-shaped sill complex in the Karoo igneous province, South Africa show a variety of geochemical variations. Some variations are observed repeatedly, i.e. the D- and I-shaped profiles. D-shaped profiles are recognized by having the least evolved composition in the center (high Mg#) with more evolved composition at the upper and lower margins (low Mg#), resulting in a D-shaped Mg# profile. I- shaped profiles are recognized by having no variation in the Mg# through the profile. The formation mechanism of D-shaped profiles is enigmatic, as classical fractional crystallization theory predicts C-shapes to occur. The least evolved composition will be at the margins where crystallization initiates, and with continued cooling and crystallization the center will be progressively more evolved. Hence, we need another formation mechanism. The most common explanation for D-shaped profiles is a movement of early formed phenocrysts towards the center due to flow segregation. However, petrographical evidences from a D-shaped profile in this study show no phenocryst assemblage in the center, and the modal composition is homogeneous through the profile. We propose that differentiation is caused by a melt flow from the central parts of the sill towards the margins driven by underpressure anomalies at the margins. The underpressures develop because of strong cooling gradients at the margins, assuming no volume change due to a rigid crystal network. The less compatible elements associated with the melt phase will be transported into the margins by advection, resulting in a more evolved total system composition from a higher total melt percentage. The central parts will progressively be depleted in the less compatible

HybProbes-based real-time PCR assay for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei, the potato common scab pathogens.

Science.gov (United States)

Xu, R; Falardeau, J; Avis, T J; Tambong, J T

2016-02-01

The aim of this study was to develop and validate a HybProbes-based real-time PCR assay targeting the trpB gene for specific identification of Streptomyces scabies and Streptomyces europaeiscabiei. Four primer pairs and a fluorescent probe were designed and evaluated for specificity in identifying S. scabies and Streptomyces europaeiscabiei, the potato common scab pathogens. The specificity of the HybProbes-based real-time PCR assay was evaluated using 46 bacterial strains, 23 Streptomyces strains and 23 non-Streptomyces bacterial species. Specific and strong fluorescence signals were detected from all nine strains of S. scabies and Streptomyces europaeiscabiei. No fluorescence signal was detected from 14 strains of other Streptomyces species and all non-Streptomyces strains. The identification was corroborated by the melting curve analysis that was performed immediately after the amplification step. Eight of the nine S. scabies and S. europaeiscabiei strains exhibited a unique melting peak, at Tm of 69·1°C while one strain, Warba-6, had a melt peak at Tm of 65·4°C. This difference in Tm peaks could be attributed to a guanine to cytosine mutation in strain Warba-6 at the region spanning the donor HybProbe. The reported HybProbes assay provides a more specific tool for accurate identification of S. scabies and S. europaeiscabiei strains. This study reports a novel assay based on HybProbes chemistry for rapid and accurate identification of the potato common scab pathogens. Since the HybProbes chemistry requires two probes for positive identification, the assay is considered to be more specific than conventional PCR or TaqMan real-time PCR. The developed assay would be a useful tool with great potential in early diagnosis and detection of common scab pathogens of potatoes in infected plants or for surveillance of potatoes grown in soil environment. © 2015 Her Majesty the Queen in Right of Canada © 2015 The Society for Applied Microbiology.

Molecular thermodynamics of polymer melts at interfaces

International Nuclear Information System (INIS)

Theodorou, D.N.

1988-09-01

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

Water jet intrusion into hot melt concomitant with direct-contact boiling of water

Energy Technology Data Exchange (ETDEWEB)

Sibamoto, Yasuteru [Japan Atomic Energy Research Inst., Tokai Research Establishment, Tokai, Ibaraki (Japan)

2005-08-01

Boiling of water poured on surface of high-temperature melt (molten metal or metal oxide) provides an efficient means for heat exchange or cooling of melt. The heat transfer surface area can be extended by forcing water into melt. Objectives of the present study are to elucidate key factors of the thermal and hydrodynamic interactions for the water jet injection into melt (Coolant Injection mode). Proposed applications include in in-vessel heat exchangers for liquid metal reactor and emergency measures for cooling of molten core debris in severe accidents of light water reactor. Water penetration into melt may occurs also as a result of fuel-coolant interaction (FCI) in modes other than CI, it is anticipated that the present study contributes to understand the fundamental mechanism of the FCI process. The previous works have been limited on understanding the melt-water interaction phenomena in the water-injection mode because of difficulty in experimental measurement where boiling occurs in opaque invisible hot melt unlike the melt-injection mode. We conducted visualization and measurement of melt-water-vapor multiphase flow phenomena by using a high-frame-rate neutron radiography technique and newly-developed probes. Although limited knowledge, however, has been gained even such an approach, the experimental data were analyzed deeply by comparing with the knowledge obtained from relevant matters. As a result, we succeeded in revealing several key phenomena and validity in the conditions under which stable heat transfer is established. Moreover, a non-intrusive technique for measurement of the velocity and pressure fields adjacent to a moving free surface is developed. The technique is based on the measurement of fluid surface profile, which is useful for elucidation of flow mechanism accompanied by a free surface like the present phenomena. (author)

STICK AND SLIP BEHAVIOR OF CONFINED OLIGOMER MELTS UNDER SHEAR - A MOLECULAR-DYNAMICS STUDY

NARCIS (Netherlands)

MANIAS, E; HADZIIOANNOU, G; BITSANIS, [No Value; TENBRINKE, G

1993-01-01

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

Shock melting method to determine melting curve by molecular dynamics: Cu, Pd, and Al.

Science.gov (United States)

Liu, Zhong-Li; Zhang, Xiu-Lu; Cai, Ling-Cang

2015-09-21

A melting simulation method, the shock melting (SM) method, is proposed and proved to be able to determine the melting curves of materials accurately and efficiently. The SM method, which is based on the multi-scale shock technique, determines melting curves by preheating and/or prepressurizing materials before shock. This strategy was extensively verified using both classical and ab initio molecular dynamics (MD). First, the SM method yielded the same satisfactory melting curve of Cu with only 360 atoms using classical MD, compared to the results from the Z-method and the two-phase coexistence method. Then, it also produced a satisfactory melting curve of Pd with only 756 atoms. Finally, the SM method combined with ab initio MD cheaply achieved a good melting curve of Al with only 180 atoms, which agrees well with the experimental data and the calculated results from other methods. It turned out that the SM method is an alternative efficient method for calculating the melting curves of materials.

Shock melting method to determine melting curve by molecular dynamics: Cu, Pd, and Al

International Nuclear Information System (INIS)

Liu, Zhong-Li; Zhang, Xiu-Lu; Cai, Ling-Cang

2015-01-01

A melting simulation method, the shock melting (SM) method, is proposed and proved to be able to determine the melting curves of materials accurately and efficiently. The SM method, which is based on the multi-scale shock technique, determines melting curves by preheating and/or prepressurizing materials before shock. This strategy was extensively verified using both classical and ab initio molecular dynamics (MD). First, the SM method yielded the same satisfactory melting curve of Cu with only 360 atoms using classical MD, compared to the results from the Z-method and the two-phase coexistence method. Then, it also produced a satisfactory melting curve of Pd with only 756 atoms. Finally, the SM method combined with ab initio MD cheaply achieved a good melting curve of Al with only 180 atoms, which agrees well with the experimental data and the calculated results from other methods. It turned out that the SM method is an alternative efficient method for calculating the melting curves of materials

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

Science.gov (United States)

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

2016-01-01

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

Design and optimization of reverse-transcription quantitative PCR experiments.

Science.gov (United States)

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

2009-10-01

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. .

An Evaluation of Quantitative PCR Assays (TaqMan® and SYBR Green for the Detection of Babesia bigemina and Babesia bovis, and a Novel Fluorescent-ITS1-PCR Capillary Electrophoresis Method for Genotyping B. bovis Isolates

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Bing Zhang

2016-09-01

Full Text Available Babesia spp. are tick-transmitted haemoparasites causing tick fever in cattle. In Australia, economic losses to the cattle industry from tick fever are estimated at AUD$26 Million per annum. If animals recover from these infections, they become immune carriers. Here we describe a novel multiplex TaqMan qPCR targeting cytochrome b genes for the identification of Babesia spp. The assay shows high sensitivity, specificity and reproducibility, and allows quantification of parasite DNA from Babesia bovis and B. bigemina compared to standard PCR assays. A previously published cytochrome b SYBR Green qPCR was also tested in this study, showing slightly higher sensitivity than the Taqman qPCRs but requires melting curve analysis post-PCR to confirm specificity. The SYBR Green assays were further evaluated using both diagnostic submissions and vaccinated cattle (at 7, 9, 11 and 14 days post-inoculation showed that B. bigemina can be detected more frequently than B. bovis. Due to fewer circulating parasites, B. bovis detection in carrier animals requires higher DNA input. Preliminary data for a novel fluorescent PCR genotyping based on the Internal Transcribed Spacer 1 region to detect vaccine and field alleles of B. bovis are described. This assay is capable of detecting vaccine and novel field isolate alleles in a single sample.

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

Science.gov (United States)

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

2012-01-01

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

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

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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.

Development of Quantitative Competitive PCR and Absolute Based Real-Time PCR Assays for Quantification of The Butyrate Producing Bacterium: Butyrivibrio fibrisolvens

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Mojtaba Tahmoorespur

2016-04-01

fibrisolvens were successfully used for amplifying the specific fragments from this bacteria. The main and important factors for increasing the accuracy of Q-C PCR is the degree of similarity between competitor and target fragment. In this study the competitor fragment was highest homology to target sequences. In this regards it seems obtained results have considerable accuracy. The intensity of bands was evaluated and analyzed using Image J software. The results of band intensity analysis showed linear trend between competitor and target in different serial dilution of competitors. The specific fragment from 16S rDNA region of Butyrivibrio fibriolvents Bacteria was amplified using specific primers and cloned in pTZ57R/T plasmid. After amplifying the competitor fragment and target sequence simultaneously, the two bands were detectable in gel electrophoresis. The range of 10-1 to 10-6 serial dilution from competitor was selected for QC-PCR reaction. The results of this section showed that the considerable linear correlation was exist between competitor and target fragment in QC-PCR reaction (R2=0.985. In this study, Real-time PCR was also used for quantification of Butyrivibrio fibriolvents Bacteria strain. Melting analysis was showed that the reaction in Real-time PCR had appropriate condition for amplifying the target sequence. We used a standard in this study. This standard was designed as a vector contain of competitor fragment. This kind of standard was successfully used for QC-PCR and Real-time PCR analyses. Standard competitor could be used for absolute quantification for this bacterial strain. Overall, our results showed that the designed standard and optimized QC-PCR have considerable potential for Butyrivibrio fibriolvents Bacteria strain. Conclusion In this study, the two methods of quantification of nucleotide acids in biological samples were optimized. These two methods were performed in order to optimize Butyrivibrio fibriolvents Bacteria strain quantification

The Microwave Properties of Simulated Melting Precipitation Particles: Sensitivity to Initial Melting

Science.gov (United States)

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

2016-01-01

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.

DEPENDENCY OF SULFATE SOLUBILITY ON MELT COMPOSITION AND MELT POLYMERIZATION

International Nuclear Information System (INIS)

JANTZEN, CAROL M.

2004-01-01

Sulfate and sulfate salts are not very soluble in borosilicate waste glass. When sulfate is present in excess it can form water soluble secondary phases and/or a molten salt layer (gall) on the melt pool surface which is purported to cause steam explosions in slurry fed melters. Therefore, sulfate can impact glass durability while formation of a molten salt layer on the melt pool can impact processing. Sulfate solubility has been shown to be compositionally dependent in various studies, (e.g. , B2O3, Li2O, CaO, MgO, Na2O, and Fe2O3 were shown to increase sulfate solubility while Al2O3 and SiO2 decreased sulfate solubility). This compositional dependency is shown to be related to the calculated melt viscosity at various temperatures and hence the melt polymerization

Shape based kinetic outlier detection in real-time PCR

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D'Atri Mario

2010-04-01

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.

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

Science.gov (United States)

Singh, Prashant; Mustapha, Azlin

2015-12-23

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

Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR.

Science.gov (United States)

Vitali, Beatrice; Pugliese, Ciro; Biagi, Elena; Candela, Marco; Turroni, Silvia; Bellen, Gert; Donders, Gilbert G G; Brigidi, Patrizia

2007-09-01

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.

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

Science.gov (United States)

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

2017-05-01

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

Modelling the geometry of a moving laser melt pool and deposition track via energy and mass balances

Energy Technology Data Exchange (ETDEWEB)

Pinkerton, Andrew J; Li Lin [Laser Processing Research Centre, Department of Mechanical, Aerospace and Manufacturing Engineering, University of Manchester Institute of Science and Technology, PO Box 88, Sackville Street, Manchester M60 1QD (United Kingdom)

2004-07-21

The additive manufacturing technique of laser direct metal deposition allows multiple tracks of full density metallic material to be built to form complex parts for rapid tooling and manufacture. Practical results and theoretical models have shown that the geometries of the tracks are governed by multiple factors. Original work with single layer cladding identified three basic clad profiles but, so far, models of multiple layer, powder-feed deposition have been based on only two of them. At higher powder mass flow rates, experimental results have shown that a layer's width can become greater than the melt pool width at the substrate surface, but previous analytical models have not been able to accommodate this. In this paper, a model based on this third profile is established and experimentally verified. The model concentrates on mathematical analysis of the melt pool and establishes mass and energy balances based on one-dimensional heat conduction to the substrate. Deposition track limits are considered as arcs of circles rather than of ellipses, as used in most established models, reflecting the dominance of surface tension forces in the melt pool, and expressions for elongation of the melt pool with increasing traverse speed are incorporated. Trends in layer width and height with major process parameters are captured and predicted layer dimensions correspond well to the experimental values.

Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene

DEFF Research Database (Denmark)

Candiloro, Ida Lm; Mikeska, Thomas; Hokland, Peter

2008-01-01

ABSTRACT: BACKGROUND: Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) t......ABSTRACT: BACKGROUND: Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (d......MS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation. We used this approach to study methylation of the CDKN2B (p15) cell cycle progression inhibitor gene which is inactivated by DNA methylation in haematological malignancies...... the methylated alleles and assess the degree of methylation. Direct sequencing of selected dMS-HRM products was used to determine the exact DNA methylation pattern and confirmed the degree of methylation estimated by dMS-HRM. CONCLUSION: dMS-HRM is a powerful technique for the analysis of methylation in CDKN2B...

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

Science.gov (United States)

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

2017-10-02

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 studies. This study aimed to improve detection of O. volvulus DNA in clinical samples through the development of a highly sensitive qPCR assay. A novel hydrolysis probe based qPCR assay was designed targeting the specific sequence of the O. volvulus O-5S rRNA gene. A total of 200 clinically suspected onchocerciasis cases were included from Goma district in South-west Ethiopia, from October 2012 through May 2013. Skin snip samples were collected and subjected to microscopy, O-150 qPCR, and the novel O-5S qPCR. Among the 200 individuals, 133 patients tested positive (positivity rate of 66.5%) and 67 negative by O-5S qPCR, 74 tested positive by microscopy (37.0%) and 78 tested positive by O-150 qPCR (39.0%). Among the 133 O-5S qPCR positive individuals, microscopy and O-150 qPCR detected 55.6 and 59.4% patients, respectively, implying a higher sensitivity of O-5S qPCR than microscopy and O-150 qPCR. None of the 67 individuals who tested negative by O-5S qPCR tested positive by microscopy or O-150 qPCR, implying 100% specificity of the newly designed O-5S qPCR assay. The novel O-5S qPCR assay is more sensitive than both microscopic examination and the existing O-150 qPCR for the detection of O. volvulus from skin snip samples. The newly designed assay is an important step towards appropriate individual diagnosis and control of onchocerciasis.

Production, pathways and budgets of melts in mid-ocean ridges: An enthalpy based thermo-mechanical model

Science.gov (United States)

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

2018-04-01

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.

Ribotypes and AP-PCR fingerprints of thermophilic campylobacters from marine recreational waters.

Science.gov (United States)

Hernández, J; Fayos, A; Alonso, J L; Owen, R J

1996-02-01

Thirty-two strains of thermophilic campylobacters isolated from marine recreational water and seven reference strains were biotyped and analysed by chromosomal DNA HaeIII ribopatterns and AP-PCR profiles based on a random 10-mer primer (5'-CAA TCG CCG T-3'). The majority of seawater isolates (90%) were Campylobacter coli, and three strains were Camp. jejuni. Southern blot hybridization analysis showed differences between the strains, and in a numerical analysis three main clusters were formed at the 45% similarity level, that corresponded to Camp. jejuni subsp. jejuni, Camp. coli, and a combination of Camp. coli and Camp. jejuni subsp. doylei. AP-PCR profiles also differentiated between the species but were less discriminatory than ribotyping because six strains (17%) could not be typed by this method. Numerical analysis gave four main clusters at the 45% similarity level, corresponding to Camp. jejuni subsp. jejuni, Camp. coli (two clusters) and Camp. lari. The study shows that strains within each species are diverse genomically. Both molecular methods were highly discriminatory, although some strains with identical ribotypes could be distinguished by AP-PCR, and they are valuable new alternatives to traditional typing in epidemiological studies of environmental campylobacters.

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

Science.gov (United States)

Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

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

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

Directory of Open Access Journals (Sweden)

Zhiyuan Wu

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

Shock Induced Melting in Aluminum: Wave Profile Measurements

Energy Technology Data Exchange (ETDEWEB)

Chhabildas, Lalit C.; Furnish, Michael D.; Reinhart, William D.

1999-06-23

We have developed launch capabilities that can propel macroscopic plates to hypervelocities (8 to 16 km/s). This capability has been used to determine the first time-resolved wave profile measurements using velocity interferometry techniques at impact velocities of 10 km/s. These measurements show that alu- minum continues to exhibit normal release behavior to 161 GPa with complete loss of strength in the shocked state. Results of these experiments are discussed and compared with the results of lower pressure experi- ments conducted at lower impact velocities.

Melting of contaminated metallic waste

International Nuclear Information System (INIS)

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

2004-01-01

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)

Dynamics of Melting and Melt Migration as Inferred from Incompatible Trace Element Abundance in Abyssal Peridotites

Science.gov (United States)

Peng, Q.; Liang, Y.

2008-12-01

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

The use of high resolution melting analysis to detect Fabry mutations in heterozygous females via dry bloodspots.

Science.gov (United States)

Tai, Chang-Long; Liu, Mei-Ying; Yu, Hsiao-Chi; Chiang, Chiang-Chuan; Chiang, Hung; Suen, Jeng-Hung; Kao, Shu-Min; Huang, Yu-Hsiu; Wu, Tina Jui-Ting; Yang, Chia-Feng; Tsai, Fang-Chih; Lin, Ching-Yuang; Chang, Jan-Gowth; Chen, Hong-Duo; Niu, Dau-Ming

2012-02-18

As an X-linked genetic disorder, Fabry disease was first thought to affect males only, and females were generally considered to be asymptomatic carriers. However, recent research suggests that female carriers of Fabry disease may still develop vital organ damage causing severe morbidity and mortality. In the previous newborn screening, from 299,007 newborns, we identified a total of 20 different Fabry mutations and 121 newborns with Fabry mutations. However, we found that most female carriers are not detected by enzyme assays. A streamlined method for high resolution melting (HRM) analysis was designed to screen for GLA gene mutations using a same PCR and melting program. Primer sets were designed to cover the 7 exons and the Chinese common intronic mutation, IVS4+919G>A of GLA gene. The HRM analysis was successful in identifying heterozygous and hemizygous patients with the 20 surveyed mutations. We were also successful in using this method to test dry blood spots of newborns afflicted with Fabry mutations without having to determine DNA concentration before PCR amplification. The results of this study show that HRM could be a reliable and sensitive method for use in the rapid screening of females for GLA mutations. Copyright © 2011 Elsevier B.V. All rights reserved.

Development of a single nucleotide polymorphism (SNP) marker for ...

African Journals Online (AJOL)

The nature of the single nucleotide polymorphism (SNP) marker was validated by DNA sequencing of the parental PCR products. Using high resolution melt (HRM) profiles and normalised difference plots, we successfully differentiated the homozygous dominant (wild type), homozygous recessive (LPA) and heterozygous ...

STR melting curve analysis as a genetic screening tool for crime scene samples.

Science.gov (United States)

Nguyen, Quang; McKinney, Jason; Johnson, Donald J; Roberts, Katherine A; Hardy, Winters R

2012-07-01

In this proof-of-concept study, high-resolution melt curve (HRMC) analysis was investigated as a postquantification screening tool to discriminate human CSF1PO and THO1 genotypes amplified with mini-STR primers in the presence of SYBR Green or LCGreen Plus dyes. A total of 12 CSF1PO and 11 HUMTHO1 genotypes were analyzed on the LightScanner HR96 and LS-32 systems and were correctly differentiated based upon their respective melt profiles. Short STR amplicon melt curves were affected by repeat number, and single-source and mixed DNA samples were additionally differentiated by the formation of heteroduplexes. Melting curves were shown to be unique and reproducible from DNA quantities ranging from 20 to 0.4 ng and distinguished identical from nonidentical genotypes from DNA derived from different biological fluids and compromised samples. Thus, a method is described which can assess both the quantity and the possible probative value of samples without full genotyping. 2012 American Academy of Forensic Sciences. Published 2012. This article is a U.S. Government work and is in the public domain in the U.S.A.

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

2010-11-01

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.

Modelling of the controlled melt flow in a glass melting space – Its melting performance and heat losses

Czech Academy of Sciences Publication Activity Database

Jebavá, Marcela; Dyrčíková, Petra; Němec, Lubomír

2015-01-01

Roč. 430, DEC 15 (2015), s. 52-63 ISSN 0022-3093 Institutional support: RVO:67985891 Keywords : glass melt flow * mathematical modelling * energy distribution * space utilizatios * melting performance Subject RIV: JH - Ceramics, Fire-Resistant Materials and Glass Impact factor: 1.825, year: 2015

Recent Changes in Arctic Sea Ice Melt Onset, Freeze-Up, and Melt Season Length

Science.gov (United States)

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

2010-01-01

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.

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

2015-01-01

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

Transition metal ions in silicate melts. I. Manganese in sodium silicate melts

Energy Technology Data Exchange (ETDEWEB)

Nelson, C; White, W B

1980-01-01

Optical absorption spectra obtained on glasses quenched from sodium silicate melts show Mn/sup 3 +/ to be the dominant species for melts heated in air and Mn/sup 2 +/ to be the dominant species for melts heated at P/sub O/sub 2// = 10/sup -17/ bar. The absorption spectrum of Mn/sup 3 +/ consists of an intense band at 20,000 cm/sup -1/ with a 15,000 cm/sup -1/ satellite possibly arising from the Jahn-Teller effect. The independence of the spectrum from melt composition and the high band intensity is offered as evidence for a distinct Mn/sup 3 +/ complex in the melt. The spectrum of Mn/sup 2 +/ is weak and many expected bands are not observed. A two-band luminescence spectrum from Mn/sup 2 +/ has been tentatively interpreted as due to Mn/sup 2 +/ in interstitial sites in the network and Mn/sup 2 +/ coordiated by non-bridging oxygens.

On high-pressure melting of tantalum

Science.gov (United States)

Luo, Sheng-Nian; Swift, Damian C.

2007-01-01

The issues related to high-pressure melting of Ta are discussed within the context of diamond-anvil cell (DAC) and shock wave experiments, theoretical calculations and common melting models. The discrepancies between the extrapolations of the DAC melting curve and the melting point inferred from shock wave experiments, cannot be reconciled either by superheating or solid-solid phase transition. The failure to reproduce low-pressure DAC melting curve by melting models such as dislocation-mediated melting and the Lindemann law, and molecular dynamics and quantum mechanics-based calculations, undermines their predictions at moderate and high pressures. Despite claims to the contrary, the melting curve of Ta (as well as Mo and W) remains inconclusive at high pressures.

PCR

African Journals Online (AJOL)

Elham

2013-07-03

Jul 3, 2013 ... was constructed with competitive strategy by PCR-cloning technique and the limitation range was determined. The PCR products of MTB and IAC were 245 and 660 bp, respectively on .... products' differentiation was easy.

Outbreak of hepatitis E virus infection in Darfur, Sudan: effectiveness of real-time reverse transcription-PCR analysis of dried blood spots.

Science.gov (United States)

Mérens, Audrey; Guérin, Philippe Jean; Guthmann, Jean-Paul; Nicand, Elisabeth

2009-06-01

Biological samples collected in refugee camps during an outbreak of hepatitis E were used to compare the accuracy of hepatitis E virus RNA amplification by real-time reverse transcription-PCR (RT-PCR) for sera and dried blood spots (concordance of 90.6%). Biological profiles (RT-PCR and serology) of asymptomatic individuals were also analyzed.

Melt inclusions: Chapter 6

Science.gov (United States)

,; Lowenstern, J. B.

2014-01-01

Melt inclusions are small droplets of silicate melt that are trapped in minerals during their growth in a magma. Once formed, they commonly retain much of their initial composition (with some exceptions) unless they are re-opened at some later stage. Melt inclusions thus offer several key advantages over whole rock samples: (i) they record pristine concentrations of volatiles and metals that are usually lost during magma solidification and degassing, (ii) they are snapshots in time whereas whole rocks are the time-integrated end products, thus allowing a more detailed, time-resolved view into magmatic processes (iii) they are largely unaffected by subsolidus alteration. Due to these characteristics, melt inclusions are an ideal tool to study the evolution of mineralized magma systems. This chapter first discusses general aspects of melt inclusions formation and methods for their investigation, before reviewing studies performed on mineralized magma systems.

Identification of feline immunodeficiency virus subtype-B on St. Kitts, West Indies by quantitative PCR.

Science.gov (United States)

Kelly, Patrick J; Stocking, Ruey; Gao, Dongya; Phillips, Nikol; Xu, Chuanling; Kaltenboeck, Bernhard; Wang, Chengming

2011-07-04

Although antibodies to the feline immunodeficiency virus (FIV) have been detected by SNAP assay in cats from St. Kitts, there have been no molecular studies to further confirm the infection and determine the FIV subtypes present. Total nucleic acids were extracted from EDTA whole blood specimens from 35 cats, followed by quantitative fluorescence resonance energy transfer (FRET) PCR under a six-channel LightCycler 2.0 Instrument with Software version 4.1. Four of 11 stray cats (36 %) but none of 24 owned cats were FIV positive by real-time PCR.  High-resolution melting curve analysis indicated that all four positive cats were infected with FIV subtype-B. This is the first molecular characterization of FIV subtypes on St. Kitts and the results confirm the high prevalence of FIV infection in stray cats on the island.

High-pressure melting curve of KCl: Evidence against lattice-instability theories of melting

International Nuclear Information System (INIS)

Ross, M.; Wolf, G.

1986-01-01

We show that the large curvature in the T-P melting curve of KCl is the result of a reordering of the liquid to a more densely packed arrangement. As a result theories of melting, such as the instability model, which do not take into account the structure of the liquid fail to predict the correct pressure dependence of the melting curve

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

Science.gov (United States)

Torriani, S; Zapparoli, G; Dellaglio, F

1999-10-01

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 6412(T), 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.

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

Science.gov (United States)

Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

1999-01-01

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

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

2008-07-01

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.

Hot melt extrusion versus spray drying: hot melt extrusion degrades albendazole.

Science.gov (United States)

Hengsawas Surasarang, Soraya; Keen, Justin M; Huang, Siyuan; Zhang, Feng; McGinity, James W; Williams, Robert O

2017-05-01

The purpose of this study was to enhance the dissolution properties of albendazole (ABZ) by the use of amorphous solid dispersions. Phase diagrams of ABZ-polymer binary mixtures generated from Flory-Huggins theory were used to assess miscibility and processability. Forced degradation studies showed that ABZ degraded upon exposure to hydrogen peroxide and 1 N NaOH at 80 °C for 5 min, and the degradants were albendazole sulfoxide (ABZSX), and ABZ impurity A, respectively. ABZ was chemically stable following exposure to 1 N HCl at 80 °C for one hour. Thermal degradation profiles show that ABZ, with and without Kollidon ® VA 64, degraded at 180 °C and 140 °C, respectively, which indicated that ABZ could likely be processed by thermal processing. Following hot melt extrusion, ABZ degraded up to 97.4%, while the amorphous ABZ solid dispersion was successfully prepared by spray drying. Spray-dried ABZ formulations using various types of acids (methanesulfonic acid, sulfuric acid and hydrochloric acid) and polymers (Kollidon ® VA 64, Soluplus ® and Eudragit ® E PO) were studied. The spray-dried ABZ with methanesulfonic acid and Kollidon ® VA 64 substantially improved non-sink dissolution in acidic media as compared to bulk ABZ (8-fold), physical mixture of ABZ:Kollidon ® VA 64 (5.6-fold) and ABZ mesylate salt (1.6-fold). No degradation was observed in the spray-dried product for up to six months and less than 5% after one-year storage. In conclusion, amorphous ABZ solid dispersions in combination with an acid and polymer can be prepared by spray drying to enhance dissolution and shelf-stability, whereas those made by melt extrusion are degraded.

Absolute quantification by droplet digital PCR versus analog real-time PCR

Science.gov (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

2014-01-01

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

Modulating laser intensity profile ellipticity for microstructural control during metal additive manufacturing

International Nuclear Information System (INIS)

Roehling, Tien T.; Wu, Sheldon S.Q.; Khairallah, Saad A.; Roehling, John D.; Soezeri, S. Stefan; Crumb, Michael F.; Matthews, Manyalibo J.

2017-01-01

Additively manufactured (AM) metals are often highly textured, containing large columnar grains that initiate epitaxially under steep temperature gradients and rapid solidification conditions. These unique microstructures partially account for the massive property disparity existing between AM and conventionally processed alloys. Although equiaxed grains are desirable for isotropic mechanical behavior, the columnar-to-equiaxed transition remains difficult to predict for conventional solidification processes, and much more so for AM. In this study, the effects of laser intensity profile ellipticity on melt track macrostructures and microstructures were studied in 316L stainless steel. Experimental results were supported by temperature gradients and melt velocities simulated using the ALE3D multi-physics code. As a general trend, columnar grains preferentially formed with increasing laser power and scan speed for all beam profiles. However, when conduction mode laser heating occurs, scan parameters that result in coarse columnar microstructures using Gaussian profiles produce equiaxed or mixed equiaxed-columnar microstructures using elliptical profiles. By modulating spatial laser intensity profiles on the fly, site-specific microstructures and properties can be directly engineered into additively manufactured parts.

Electron beam melting of sponge titanium

International Nuclear Information System (INIS)

Kanayama, Hiroshi; Kusamichi, Tatsuhiko; Muraoka, Tetsuhiro; Onouye, Toshio; Nishimura, Takashi

1991-01-01

Fundamental investigations were done on electron beam (EB) melting of sponge titanium by using 80 kW EB melting furnace. Results obtained are as follows: (1) To increase the melting yield of titanium in EB melting of sponge titanium, it is important to recover splashed metal by installation of water-cooled copper wall around the hearth and to decrease evaporation loss of titanium by keeping the surface temperature of molten metal just above the melting temperature of titanium without local heating. (2) Specific power consumption of drip melting of pressed sponge titanium bar and hearth melting of sponge titanium are approximately 0.9 kWh/kg-Ti and 0.5-0.7 kWh/kg-Ti, respectively. (3) Ratios of the heat conducted to water-cooled mould in the drip melting and to water-cooled hearth in the hearth melting to the electron beam input power are 50-65% and 60-65%, respectively. (4) Surface defects of EB-melted ingots include rap which occurs when the EB output is excessively great, and transverse cracks when the EB output is excessively small. To prevent surface defects, the up-down withdrawal method is effective. (author)

High-resolution melting analysis for prenatal diagnosis of beta-thalassemia in northern Thailand.

Science.gov (United States)

Charoenkwan, Pimlak; Sirichotiyakul, Supatra; Phusua, Arunee; Suanta, Sudjai; Fanhchaksai, Kanda; Sae-Tung, Rattika; Sanguansermsri, Torpong

2017-12-01

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.

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

Science.gov (United States)

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

2015-11-01

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

Melting point of yttria

International Nuclear Information System (INIS)

Skaggs, S.R.

1977-06-01

Fourteen samples of 99.999 percent Y 2 O 3 were melted near the focus of a 250-W CO 2 laser. The average value of the observed melting point along the solid-liquid interface was 2462 +- 19 0 C. Several of these same samples were then melted in ultrahigh-purity oxygen, nitrogen, helium, or argon and in water vapor. No change in the observed temperature was detected, with the exception of a 20 0 C increase in temperature from air to helium gas. Post test examination of the sample characteristics, clarity, sphericity, and density is presented, along with composition. It is suggested that yttria is superior to alumina as a secondary melting-point standard

Real-time PCR for Leishmania species identification: Evaluation and comparison with classical techniques.

Science.gov (United States)

de Morais, Rayana Carla Silva; da Costa Oliveira, Cintia Nascimento; de Albuquerque, Suênia da Cunha Gonçalves; Mendonça Trajano Silva, Lays Adrianne; Pessoa-E-Silva, Rômulo; Alves da Cruz, Heidi Lacerda; de Brito, Maria Edileuza Felinto; de Paiva Cavalcanti, Milena

2016-06-01

Cutaneous leishmaniasis (CL) is a parasitic disease caused by various Leishmania species. Several studies have shown that real time quantitative PCR (qPCR) can be used for Leishmania spp. identification by analyzing the melting temperature (Tm). Thus, the aim of this study was to evaluate the viability of qPCR for differentiating eight closely related Leishmania species that cause the same clinical form of the disease and to compare the results with classical techniques. qPCR assays for standardizing the Tm using reference strains were performed. After the CL diagnosis on blood samples of domestic animals, positive samples were analyzed by their Tm and qPCR products were purified and sequenced. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by Tm. A Restriction Fragment Length Polymorphism (RFLP) assay, a reference test, was also standardized, by using the reference strains. Through standardization of Tm for Leishmania spp., two Tm ranges were created for analysis: 1 (Tm = 78-79.99 °C) included Leishmania (V.) braziliensis, Leishmania (V.) panamensis, Leishmania (V.) lainsoni, Leishmania (V.) guyanensis and Leishmania (V.) shawi; and 2 (Tm = 80-82.2 °C) included Leishmania (V.) naiffi, Leishmania (L.) amazonensis and Leishmania (L.) mexicana. A total of 223 positive blood samples were analyzed, with 58 included in range 1 and 165 in range 2. L. (V.) braziliensis, L. (V.) panamensis and L. (V.) guyanensis were identified by sequencing, while L. (V.) braziliensis, L. (L.) mexicana and L. (V.) panamensis were identified by RFLP analysis. Ten human samples previously characterized by Multilocus Enzyme Electrophoresis (MLEE) were also analyzed by qPCR Tm analysis; five were classified in range 1 and five in range 2. A concordance of 80% was calculated between qPCR and the gold-standard (MLEE) with no significant difference between the methods (p = 0.6499); a similar result was observed for sequencing

Detection of Dirofilaria immitis and other arthropod-borne filarioids by an HRM real-time qPCR, blood-concentrating techniques and a serological assay in dogs from Costa Rica.

Science.gov (United States)

Rojas, Alicia; Rojas, Diana; Montenegro, Víctor M; Baneth, Gad

2015-03-23

Canine filarioids are important nematodes transmitted to dogs by arthropods. Diagnosis of canine filariosis is accomplished by the microscopic identification of microfilariae, serology or PCR for filarial-DNA. The aim of this study was to evaluate a molecular assay for the detection of canine filariae in dog blood, to compare its performance to other diagnostic techniques, and to determine the relationship between microfilarial concentration and infection with other vector-borne pathogens. Blood samples from 146 dogs from Costa Rica were subjected to the detection of canine filarioids by four different methods: the microhematocrit tube test (MCT), Knott's modified test, serology and a high resolution melt and quantitative real-time PCR (HRM-qPCR). Co-infection with other vector-borne pathogens was also evaluated. Fifteen percent of the dogs were positive to Dirofilaria immitis by at least one of the methods. The HRM-qPCR produced distinctive melting plots for the different filarial worms and revealed that 11.6% of dogs were infected with Acanthocheilonema reconditum. The latter assay had a limit of detection of 2.4x10⁻⁴ mf/μl and detected infections with lower microfilarial concentrations in comparison to the microscopic techniques and the serological assay. The MCT and Knott's test only detected dogs with D. immitis microfilaremias above 0.7 mf/μl. Nevertheless, there was a strong correlation between the microfilarial concentration obtained by the Knott's modified test and the HRM-qPCR (r = 0.906, p HRM-qPCR showed the most sensitive and reliable performance in the detection of blood filaroids in comparison to the Knott's modified test, the MCT test and a serological assay.

Two-temperature hydrodynamic expansion and coupling of strong elastic shock with supersonic melting front produced by ultrashort laser pulse

International Nuclear Information System (INIS)

Inogamov, Nail A; Khokhlov, Viktor A; Zhakhovsky, Vasily V; Khishchenko, Konstantin V; Demaske, Brian J; Oleynik, Ivan I

2014-01-01

Ultrafast processes, including nonmonotonic expansion of material into vacuum, supersonic melting and generation of super-elastic shock wave, in a surface layer of metal irradiated by an ultrashort laser pulse are discussed. In addition to the well-established two-temperature (2T) evolution of heated layer a new effect of electron pressure gradient on early stage of material expansion is studied. It is shown that the expanding material experiences an unexpected jump in flow velocity in a place where stress exceeds the effective tensile strength provided by used EoS of material. Another 2T effect is that supersonic propagation of homogeneous melting front results in distortion of spatial profile of ion temperature, which later imprints on ion pressure profile transforming in a super-elastic shock wave with time.

Method of melting solid waste

International Nuclear Information System (INIS)

Ootsuka, Katsuyuki; Mizuno, Ryokichi; Kuwana, Katsumi; Sawada, Yoshihisa; Komatsu, Fumiaki.

1982-01-01

Purpose: To enable the volume reduction treatment of a HEPA filter containing various solid wastes, particularly acid digestion residue, or an asbestos separator at a relatively low temperature range. Method: Solid waste to be heated and molten is high melting point material treated by ''acid digestion treatment'' for treating solid waste, e.g. a HEPA filter or polyvinyl chloride, etc. of an atomic power facility treated with nitric acid or the like. When this material is heated and molten by an electric furnace, microwave melting furnace, etc., boron oxide, sodium boride, sodium carbonate, etc. is added as a melting point lowering agent. When it is molten in this state, its melting point is lowered, and it becomes remarkably fluid, and the melting treatment is facilitated. Solidified material thus obtained through the melting step has excellent denseness and further large volume reduction rate of the solidified material. (Yoshihara, H.)

Effect of calcium and phosphorus, residual lactose, and salt-to-moisture ratio on the melting characteristics and hardness of cheddar cheese during ripening.

Science.gov (United States)

Chevanan, N; Muthukumarappan, K

2007-05-01

Meltability, melt profile parameters, and hardness of cheddar cheese prepared with varying levels of calcium (Ca) and phosphorus (P) content, residual lactose content, and salt-to-moisture ratio were studied at 0, 1, 2, 4, 6, and 8 mo of ripening. Meltability, melt profile parameters, and hardness of cheddar cheeses measured at 0, 1, 2, 4, 6, and 8 mo of ripening showed significant interaction between the levels of Ca and P, residual lactose, salt-to-moisture ratio, and ripening time for most of the properties studied. cheddar cheese prepared with high Ca and P (0.67% Ca and 0.53% P) resulted in up to 6.2%, 4.5%, 9.6%, 5.0%, and 22.8% increase in softening time, softening temperature, melting time, melting temperature, and hardness, respectively, and 23.5%, 9.6%, and 3.2% decrease in meltability, flow rate, and extent of flow, respectively, compared to the cheddar cheese prepared with low Ca and P (0.53% Ca and 0.39% P). cheddar cheese prepared with high lactose (1.4%) content resulted in up to 7.7%, 7.0%, 4.9%, 4.2%, and 24.6% increase in softening time, softening temperature, melting time, melting temperature, and hardness, respectively, and 14.7%, 12.7%, and 2.8% decrease in meltability, flow rate, and extent of flow respectively compared to the cheddar cheese prepared with low lactose (0.78%) content. cheddar cheese prepared with high salt-to-moisture ratio (6.4%) resulted in up to 21.8%, 11.3%, 12.9%, 4.1%, and 29.4% increase in softening time, softening temperature, melting time, melting temperature, and hardness, respectively, and 13.2%, 28.6%, and 2.6% decrease in meltability, flow rate, and extent of flow, respectively, compared to the cheddar cheese prepared with low salt-to-moisture ratio (4.8%) during ripening.

Automatic Control of Silicon Melt Level

Science.gov (United States)

Duncan, C. S.; Stickel, W. B.

1982-01-01

A new circuit, when combined with melt-replenishment system and melt level sensor, offers continuous closed-loop automatic control of melt-level during web growth. Installed on silicon-web furnace, circuit controls melt-level to within 0.1 mm for as long as 8 hours. Circuit affords greater area growth rate and higher web quality, automatic melt-level control also allows semiautomatic growth of web over long periods which can greatly reduce costs.

Eliminating PCR contamination

International Nuclear Information System (INIS)

Fox, J.C.; Ait-Khaled, Mounir; Webster, Alison; Emery, V.C.

1991-01-01

The sensitivity of polymerase chain reaction (PCR) can mean that even very low levels of contamination with the target DNA will result in a positive signal. At present this aspect is a major limitation in the use of PCR as a routine diagnostic method. By exposing PCR reagents to UV light, contaminating DNA can be inactivated, thus providing an opportunity to eradicate false positive reactions. UV irradiation was applied to PCR systems used for detection of human cytomegalovirus CMV and human immunodeficiency virus (HIV) and shown to be effective in eradicating both laboratory encountered contamination and plasmid DNA (below 100 pg) added to PCR systems prior to UV exposure. Sensitivity of a PCR system to amplify the long terminal repeat (LTR) sequence of HIV-1 was not affected by the irradiation procedure; however, ultimate sensitivity of a PCR system for the amplification of an early gene pro-motor sequence of the CMV genome was reduced 1000-fold. UV irradiation did not affect the size of the PCR product as determined by strand separating polyacrylamide gel electrophoresis of a 32 P-labelled amplimer. Thus, a simple pre-exposure to UV light would seem a worth-wile step to incorporate into PCR protocols provided that the effects on sensitivity have been determined empirically for each PCR system. (author). 11 refs.; 3 figs

Melting of polydisperse hard disks

NARCIS (Netherlands)

Pronk, S.; Frenkel, D.

2004-01-01

The melting of a polydisperse hard-disk system is investigated by Monte Carlo simulations in the semigrand canonical ensemble. This is done in the context of possible continuous melting by a dislocation-unbinding mechanism, as an extension of the two-dimensional hard-disk melting problem. We find

Application of reverse transcription-PCR and real-time PCR in nanotoxicity research.

Science.gov (United States)

Mo, Yiqun; Wan, Rong; Zhang, Qunwei

2012-01-01

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.

Non-invasive prenatal diagnosis of multiple endocrine neoplasia type 2A using COLD-PCR combined with HRM genotyping analysis from maternal serum.

Science.gov (United States)

Macher, Hada C; Martinez-Broca, Maria A; Rubio-Calvo, Amalia; Leon-Garcia, Cristina; Conde-Sanchez, Manuel; Costa, Alzenira; Navarro, Elena; Guerrero, Juan M

2012-01-01

The multiple endocrine neoplasia type 2A (MEN2A) is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y) in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.

Development of allele-specific multiplex PCR to determine the length of poly-T in intron 8 of CFTR

Directory of Open Access Journals (Sweden)

Neng Chen

2014-07-01

Full Text Available Cystic fibrosis transmembrane conductance regulator (CFTR gene mutation analysis has been implemented for Cystic Fibrosis (CF carrier screening, and molecular diagnosis of CF and congenital bilateral absence of the vas deferens (CBAVD. Although poly-T allele analysis in intron 8 of CFTR is required when a patient is positive for R117H, it is not recommended for routine carrier screening. Therefore, commercial kits for CFTR mutation analysis were designed either to mask the poly-T allele results, unless a patient is R117H positive, or to have the poly-T analysis as a standalone reflex test using the same commercial platform. There are other standalone assays developed to detect poly-T alleles, such as heteroduplex analysis, High Resolution Melting (HRM curve analysis, allele-specific PCR (AS-PCR and Sanger sequencing. In this report, we developed a simple and easy-to-implement multiplex AS-PCR assay using unlabeled standard length primers, which can be used as a reflex or standalone test for CFTR poly-T track analysis. Out of 115 human gDNA samples tested, results from our new AS-PCR matched to the previous known poly-T results or results from Sanger sequencing.

Melt cooling by bottom flooding: The experiment CometPC-H3. Ex-vessel core melt stabilization research

International Nuclear Information System (INIS)

Alsmeyer, H.; Cron, T.; Merkel, G.; Schmidt-Stiefel, S.; Tromm, W.; Wenz, T.

2003-03-01

The CometPC-H3 experiment was performed to investigate melt cooling by water addition to the bottom of the melt. The experiment was performed with a melt mass of 800 kg, 50% metal and 50% oxide, and 300 kW typical decay heat were simulated in the melt. As this was the first experiment after repair of the induction coil, attention was given to avoid overload of the induction coil and to keep the inductor voltage below critical values. Therefore, the height of the sacrificial concrete layer was reduced to 5 cm only, and the height of the porous concrete layers was also minimized to have a small distance and good coupling between heated melt and induction coil. After quite homogeneous erosion of the upper sacrificial concrete layer, passive bottom flooding started from the porous concrete after 220 s with 1.3 liter water/s. The melt was safely stopped, arrested and cooled. The porous, water filled concrete was only slightly attacked by the hot melt in the upper 25 mm of one sector of the coolant device. The peak cooling rate in the early contact phase of coolant water and melt was 4 MW/m 2 , and exceeded the decay heat by one order of magnitude. The cooling rate remarkably dropped, when the melt was covered by the penetrating water and a surface crust was formed. Volcanic eruptions from the melt during the solidification process were observed from 360 - 510 s and created a volcanic dome some 25 cm high, but had only minor effect on the generation of a porous structure, as the expelled melt solidified mostly with low porosity. Unfortunately, decay heat simulation in the melt was interrupted at 720 s by an incorrect safety signal, which excluded further investigation of the long term cooling processes. At that time, the melt was massively flooded by a layer of water, about 80 cm thick, and coolant water inflow was still 1 l/s. The melt had reached a stable situation: Downward erosion was stopped by the cooling process from the water filled, porous concrete layer. Top

Model of interfacial melting

DEFF Research Database (Denmark)

Mouritsen, Ole G.; Zuckermann, Martin J.

1987-01-01

A two-dimensional model is proposed to describe systems with phase transitions which take place in terms of crystalline as well as internal degrees of freedom. Computer simulation of the model shows that the interplay between the two sets of degrees of freedom permits observation of grain-boundar......-boundary formation and interfacial melting, a nonequilibrium process by which the system melts at the boundaries of a polycrystalline domain structure. Lipid membranes are candidates for systems with pronounced interfacial melting behavior....

Melt migration modeling in partially molten upper mantle

Science.gov (United States)

Ghods, Abdolreza

The objective of this thesis is to investigate the importance of melt migration in shaping major characteristics of geological features associated with the partial melting of the upper mantle, such as sea-floor spreading, continental flood basalts and rifting. The partial melting produces permeable partially molten rocks and a buoyant low viscosity melt. Melt migrates through the partially molten rocks, and transfers mass and heat. Due to its much faster velocity and appreciable buoyancy, melt migration has the potential to modify dynamics of the upwelling partially molten plumes. I develop a 2-D, two-phase flow model and apply it to investigate effects of melt migration on the dynamics and melt generation of upwelling mantle plumes and focusing of melt migration beneath mid-ocean ridges. Melt migration changes distribution of the melt-retention buoyancy force and therefore affects the dynamics of the upwelling plume. This is investigated by modeling a plume with a constant initial melt of 10% where no further melting is considered. Melt migration polarizes melt-retention buoyancy force into high and low melt fraction regions at the top and bottom portions of the plume and therefore results in formation of a more slender and faster upwelling plume. Allowing the plume to melt as it ascends through the upper mantle also produces a slender and faster plume. It is shown that melt produced by decompressional melting of the plume migrates to the upper horizons of the plume, increases the upwelling velocity and thus, the volume of melt generated by the plume. Melt migration produces a plume which lacks the mushroom shape observed for the plume models without melt migration. Melt migration forms a high melt fraction layer beneath the sloping base of the impermeable oceanic lithosphere. Using realistic conditions of melting, freezing and melt extraction, I examine whether the high melt fraction layer is able to focus melt from a wide partial melting zone to a narrow region

Expression profiling identifies genes involved in emphysema severity

Directory of Open Access Journals (Sweden)

Bowman Rayleen V

2009-09-01

Full Text Available Abstract Chronic obstructive pulmonary disease (COPD is a major public health problem. The aim of this study was to identify genes involved in emphysema severity in COPD patients. Gene expression profiling was performed on total RNA extracted from non-tumor lung tissue from 30 smokers with emphysema. Class comparison analysis based on gas transfer measurement was performed to identify differentially expressed genes. Genes were then selected for technical validation by quantitative reverse transcriptase-PCR (qRT-PCR if also represented on microarray platforms used in previously published emphysema studies. Genes technically validated advanced to tests of biological replication by qRT-PCR using an independent test set of 62 lung samples. Class comparison identified 98 differentially expressed genes (p p Gene expression profiling of lung from emphysema patients identified seven candidate genes associated with emphysema severity including COL6A3, SERPINF1, ZNHIT6, NEDD4, CDKN2A, NRN1 and GSTM3.

Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats.

Science.gov (United States)

Liu, Yichun; Lee, May-Ann; Ooi, Eng-Eong; Mavis, Yeo; Tan, Ai-Ling; Quek, Hung-Hiang

2003-09-01

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

Multicomponent diffusion in basaltic melts at 1350 °C

Science.gov (United States)

Guo, Chenghuan; Zhang, Youxue

2018-05-01

Nine successful diffusion couple experiments were conducted in an 8-component SiO2-TiO2-Al2O3-FeO-MgO-CaO-Na2O-K2O system at ∼1350 °C and at 1 GPa, to study multicomponent diffusion in basaltic melts. At least 3 traverses were measured to obtain diffusion profiles for each experiment. Multicomponent diffusion matrix at 1350 °C was obtained by simultaneously fitting diffusion profiles of diffusion couple experiments. Furthermore, in order to better constrain the diffusion matrix and reconcile mineral dissolution data, mineral dissolution experiments in the literature and diffusion couple experiments from this study, were fit together. All features of diffusion profiles in both diffusion couple and mineral dissolution experiments were well reproduced by the diffusion matrix. Diffusion mechanism is inferred from eigenvectors of the diffusion matrix, and it shows that the diffusive exchange between network-formers SiO2 and Al2O3 is the slowest, the exchange of SiO2 with other oxide components is the second slowest with an eigenvalue that is only ∼10% larger, then the exchange between divalent oxide components and all the other oxide components is the third slowest with an eigenvalue that is twice the smallest eigenvalue, then the exchange of FeO + K2O with all the other oxide components is the fourth slowest with an eigenvalue that is 5 times the smallest eigenvalue, then the exchange of MgO with FeO + CaO is the third fastest with an eigenvalue that is 6.3 times the smallest eigenvalue, then the exchange of CaO + K2O with all the other oxide components is the second fastest with an eigenvalue that is 7.5 times the smallest eigenvalue, and the exchange of Na2O with all other oxide components is the fastest with an eigenvalue that is 31 times the smallest eigenvalue. The slowest and fastest eigenvectors are consistent with those for simpler systems in most literature. The obtained diffusion matrix was successfully applied to predict diffusion profiles during

Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

Science.gov (United States)

Smith, Cindy J; Osborn, A Mark

2009-01-01

Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

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

2015-01-01

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)

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.

2011-01-01

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

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.

Optical properties of melting first-year Arctic sea ice

Science.gov (United States)

Light, Bonnie; Perovich, Donald K.; Webster, Melinda A.; Polashenski, Christopher; Dadic, Ruzica

2015-11-01

The albedo and transmittance of melting, first-year Arctic sea ice were measured during two cruises of the Impacts of Climate on the Eco-Systems and Chemistry of the Arctic Pacific Environment (ICESCAPE) project during the summers of 2010 and 2011. Spectral measurements were made for both bare and ponded ice types at a total of 19 ice stations in the Chukchi and Beaufort Seas. These data, along with irradiance profiles taken within boreholes, laboratory measurements of the optical properties of core samples, ice physical property observations, and radiative transfer model simulations are employed to describe representative optical properties for melting first-year Arctic sea ice. Ponded ice was found to transmit roughly 4.4 times more total energy into the ocean, relative to nearby bare ice. The ubiquitous surface-scattering layer and drained layer present on bare, melting sea ice are responsible for its relatively high albedo and relatively low transmittance. Light transmittance through ponded ice depends on the physical thickness of the ice and the magnitude of the scattering coefficient in the ice interior. Bare ice reflects nearly three-quarters of the incident sunlight, enhancing its resiliency to absorption by solar insolation. In contrast, ponded ice absorbs or transmits to the ocean more than three-quarters of the incident sunlight. Characterization of the heat balance of a summertime ice cover is largely dictated by its pond coverage, and light transmittance through ponded ice shows strong contrast between first-year and multiyear Arctic ice covers.

Force induced DNA melting

International Nuclear Information System (INIS)

Santosh, Mogurampelly; Maiti, Prabal K

2009-01-01

When pulled along the axis, double-strand DNA undergoes a large conformational change and elongates by roughly twice its initial contour length at a pulling force of about 70 pN. The transition to this highly overstretched form of DNA is very cooperative. Applying a force perpendicular to the DNA axis (unzipping), double-strand DNA can also be separated into two single-stranded DNA, this being a fundamental process in DNA replication. We study the DNA overstretching and unzipping transition using fully atomistic molecular dynamics (MD) simulations and argue that the conformational changes of double-strand DNA associated with either of the above mentioned processes can be viewed as force induced DNA melting. As the force at one end of the DNA is increased the DNA starts melting abruptly/smoothly above a critical force depending on the pulling direction. The critical force f m , at which DNA melts completely decreases as the temperature of the system is increased. The melting force in the case of unzipping is smaller compared to the melting force when the DNA is pulled along the helical axis. In the case of melting through unzipping, the double-strand separation has jumps which correspond to the different energy minima arising due to sequence of different base pairs. The fraction of Watson-Crick base pair hydrogen bond breaking as a function of force does not show smooth and continuous behavior and consists of plateaus followed by sharp jumps.

Applications of the rep-PCR DNA fingerprinting technique to study microbial diversity, ecology and evolution.

Science.gov (United States)

Ishii, Satoshi; Sadowsky, Michael J

2009-04-01

A large number of repetitive DNA sequences are found in multiple sites in the genomes of numerous bacteria, archaea and eukarya. While the functions of many of these repetitive sequence elements are unknown, they have proven to be useful as the basis of several powerful tools for use in molecular diagnostics, medical microbiology, epidemiological analyses and environmental microbiology. The repetitive sequence-based PCR or rep-PCR DNA fingerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA profiles or 'fingerprints' of individual microbial strains. Although this technique has been extensively used to examine diversity among variety of prokaryotic microorganisms, rep-PCR DNA fingerprinting can also be applied to microbial ecology and microbial evolution studies since it has the power to distinguish microbes at the strain or isolate level. Recent advancement in rep-PCR methodology has resulted in increased accuracy, reproducibility and throughput. In this minireview, we summarize recent improvements in rep-PCR DNA fingerprinting methodology, and discuss its applications to address fundamentally important questions in microbial ecology and evolution.

Semi-continuous casting of magnesium alloy AZ91 using a filtered melt delivery system

Directory of Open Access Journals (Sweden)

Mainul Hasan

2015-12-01

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.

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.

2000-01-01

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.)

Particle melting and particle/plasma interactions in DC and RF plasmas: a modeling study. (Volumes I and II)

International Nuclear Information System (INIS)

Wei, D.Y.C.

1987-01-01

Integral process models were developed to predict particle melting in both DC and RF plasmas. Specifically, a numerical model has been developed to predict the temperature history of particles injected in a low pressure DC plasma jet. The temperature and velocity fields of the plasma jet are predicted as a free jet by solving the parabolized Navier-Stokes equations using a spatial marching scheme. Correction factors were introduced to take into account non continuum effects encountered in the low pressure environment. The plasma jet profiles as well as the particle/plasma interactions under different jet pressure ratios (from underexpanded to overexpanded) were investigated. The flow and temperature fields in the RF plasma torch are calculated using the axisymmetric Navier-Stokes equations based on the primitive variables, along with pseudo two-dimensional electromagnetic field equations. Particle trajectories and heat transfer characteristics in both DC and RF plasmas are calculated using predicted plasma jet profiles. Particle melting efficiencies in both DC and RF plasmas are evaluated and compared using model alloy systems. Based on the theoretical considerations, an alternative route of plasma spraying process (hybrid plasma spraying process) is proposed. An evaluation of particle melting in hybrid plasma jets had indicated that further improvement in deposit properties could be made

Melting under shock compression

International Nuclear Information System (INIS)

Bennett, B.I.

1980-10-01

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

Electric melting furnace of solidifying radioactive waste by utilizing magnetic field and melting method

International Nuclear Information System (INIS)

Igarashi, Hiroshi.

1990-01-01

An electric melting furnace for solidification of radioactive wastes utilizing magnetic fields in accordance with the present invention comprises a plurality of electrodes supplying AC current to molten glass in a glass melting furnace and a plurality of magnetic poles for generating AC magnetic fields. Interactions between the current and the magnetic field, generated forces in the identical direction in view of time in the molten glass. That is, forces for promoting the flow of molten glass in the melting furnace are resulted due to the Fleming's left-hand rule. As a result, the following effects can be obtained. (1) The amount of heat ransferred from the molten glass to the starting material layer on the molten surface is increased to improve the melting performance. (2) For an identical melting performance, the size and the weight of the melting furnace can be reduced to decrease the amount of secondary wastes when the apparatus-life is exhausted. (3) Bottom deposits can be suppressed and prevented from settling and depositing to the reactor bottom by the promoted flow in the layer. (4) Further, the size of auxiliary electrodes for directly supplying electric current to heat the molten glass near the reactor bottom can be decreased. (I.S.)

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

International Nuclear Information System (INIS)

Rizzi, Giovanni; Østerberg, Frederik W.; Henriksen, Anders D.; Dufva, Martin; Hansen, Mikkel F.

2015-01-01

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

Laser melting of groove defect repair on high thermal conductivity steel (HTCS-150)

Science.gov (United States)

Norhafzan, B.; Aqida, S. N.; Fazliana, F.; Reza, M. S.; Ismail, I.; Khairil, C. M.

2018-02-01

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.

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

Science.gov (United States)

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

2017-09-06

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

Detection and quantification of Spirocerca lupi by HRM qPCR in fecal samples from dogs with spirocercosis.

Science.gov (United States)

Rojas, Alicia; Segev, Gilad; Markovics, Alex; Aroch, Itamar; Baneth, Gad

2017-09-19

Spirocerca lupi, the dog oesophageal nematode, causes a potentially fatal disease in domestic dogs, and is currently clinically diagnosed by coproscopy and oesophagoscopy. To date, a single molecular method, a semi-nested PCR, targeting the cox1 gene, has been developed to aid in the diagnosis of spirocercosis. The present study describes three novel high-resolution melt (HRM) quantitative PCR (qPCR) assays targeting fragments of the ITS1, 18S and cytb loci of S. lupi. The performance of these molecular assays in feces was compared to fecal flotation and to the previously described cox1 gene semi-nested PCR in 18 fecal samples from dogs with clinical oesophageal spirocercosis diagnosed by oesophagoscopy. The HRM qPCR for ITS1 and 18S were both able to detect 0.2 S. lupi eggs per gram (epg), while the HRM qPCR for the cytb and the semi-nested PCR for the cox1 detected 6 epg and 526 epg, respectively. Spirocerca lupi was detected in 61.1%, 44.4%, 27.8%, 11.1% and 5.6% of the fecal samples of dogs diagnosed with spirocercosis by using the ITS1 and 18S HRM qPCR assays, fecal flotation, cytb HRM qPCR and cox1 semi-nested PCR, respectively. All dogs positive by fecal flotation were also positive by ITS1 and 18S HRM qPCRs. Quantification of S. lupi eggs was successfully achieved in the HRM qPCRs and compared to the fecal flotation with no significant difference in the calculated concentrations between the HRM qPCRs that detected the 18S and ITS1 loci and the fecal flotation. The HRM qPCR for the 18S cross-amplified DNA from Toxocara canis and Toxascaris leonina. In contrast, the HRM qPCR for ITS1 did not cross-amplify DNA from other canine gastrointestinal parasites. This study presents two new molecular assays with significantly increased sensitivity for confirming and quantifying fecal S. lupi eggs. Of these, the HRM qPCR for ITS1 showed the best performance in terms of the limit of detection and absence of cross-amplification with other parasites. These assays will be

Typing of avian pathogenic Escherichia coli strains by REP-PCR Tipificação de amostras aviárias patogênicas de Escherichia coli pela REP-PCR

Directory of Open Access Journals (Sweden)

Marcelo Brocchi

2006-06-01

Full Text Available In the present study the repetitive extragenic palindromic (REP polymerase chain reaction (PCR technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC strains isolated from different outbreak cases of septicemia (n=24, swollen head syndrome (n=14 and omphalitis (n=11. Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.A técnica de REP (Repetitive extragenic palindrome-PCR foi utilizada para avaliar a variabilidade genética de 49 amostras de Escherichia coli patogênicas para aves (APEC, isoladas de aves de corte (frangos em diferentes surtos de septicemia (n=24, síndrome da cabeça inchada (n=14 e onfalite (n=11. Trinta amostras comensais, isoladas de frangos sem sinais de doença, foram utilizadas como controle. A análise do perfil eletroforético obtido por reação de REP-PCR utilizando DNA purificado das amostras evidenciou a amplificação de 0 a 15 bandas de DNA com pesos moleculares

Melting in trivalent metal chlorides

International Nuclear Information System (INIS)

Saboungi, M.L.; Price, D.L.; Scamehorn, C.; Tosi, M.P.

1990-11-01

We report a neutron diffraction study of the liquid structure of YCl 3 and combine the structural data with macroscopic melting and transport data to contrast the behaviour of this molten salt with those of SrCl 2 , ZnCl 2 and AlCl 3 as prototypes of different melting mechanisms for ionic materials. A novel melting mechanism for trivalent metal chlorides, leading to a loose disordered network of edge-sharing octahedral units in the liquid phase, is thereby established. The various melting behaviours are related to bonding character with the help of Pettifor's phenomenological chemical scale. (author). 25 refs, 4 figs, 3 tabs

Melting phenomenon and laser annealing in semiconductors

International Nuclear Information System (INIS)

Narayan, J.

1981-03-01

The work on annealing of displacement damage, dissolution of boron precipitates, and the broadening of dopant profiles in semiconductors after treating with ruby and dye laser pulses is reviewed in order to provide convincing evidence for the melting phenomenon and illustrate the mechanism associated with laser annealing. The nature of the solid-liquid interface and the interface instability during rapid solidification is considered in detail. It is shown that solute concentrations after pulsed laser annealing can far exceed retrograde maxima values. However, there is a critical solute concentration above which a planar solid-liquid interface becomes unstable and breaks into a cellular structure. The solute concentrations and cell sizes associated with this instability are calculated using a perturbation theory, and compared with experimental results

Non-invasive prenatal diagnosis of multiple endocrine neoplasia type 2A using COLD-PCR combined with HRM genotyping analysis from maternal serum.

Directory of Open Access Journals (Sweden)

Hada C Macher

Full Text Available The multiple endocrine neoplasia type 2A (MEN2A is a monogenic disorder characterized by an autosomal dominant pattern of inheritance which is characterized by high risk of medullary thyroid carcinoma in all mutation carriers. Although this disorder is classified as a rare disease, the patients affected have a low life quality and a very expensive and continuous treatment. At present, MEN2A is diagnosed by gene sequencing after birth, thus trying to start an early treatment and by reduction of morbidity and mortality. We first evaluated the presence of MEN2A mutation (C634Y in serum of 25 patients, previously diagnosed by sequencing in peripheral blood leucocytes, using HRM genotyping analysis. In a second step, we used a COLD-PCR approach followed by HRM genotyping analysis for non-invasive prenatal diagnosis of a pregnant woman carrying a fetus with a C634Y mutation. HRM analysis revealed differences in melting curve shapes that correlated with patients diagnosed for MEN2A by gene sequencing analysis with 100% accuracy. Moreover, the pregnant woman carrying the fetus with the C634Y mutation revealed a melting curve shape in agreement with the positive controls in the COLD-PCR study. The mutation was confirmed by sequencing of the COLD-PCR amplification product. In conclusion, we have established a HRM analysis in serum samples as a new primary diagnosis method suitable for the detection of C634Y mutations in MEN2A patients. Simultaneously, we have applied the increase of sensitivity of COLD-PCR assay approach combined with HRM analysis for the non-invasive prenatal diagnosis of C634Y fetal mutations using pregnant women serum.

A simplified strategy for sensitive detection of Rose rosette virus compatible with three RT-PCR chemistries.

Science.gov (United States)

Dobhal, Shefali; Olson, Jennifer D; Arif, Mohammad; Garcia Suarez, Johnny A; Ochoa-Corona, Francisco M

2016-06-01

Rose rosette disease is a disorder associated with infection by Rose rosette virus (RRV), a pathogen of roses that causes devastating effects on most garden cultivated varieties, and the wild invasive rose especially Rosa multiflora. Reliable and sensitive detection of this disease in early phases is needed to implement proper control measures. This study assesses a single primer-set based detection method for RRV and demonstrates its application in three different chemistries: Endpoint RT-PCR, TaqMan-quantitative RT-PCR (RT-qPCR) and SYBR Green RT-qPCR with High Resolution Melting analyses. A primer set (RRV2F/2R) was designed from consensus sequences of the nucleocapsid protein gene p3 located in the RNA 3 region of RRV. The specificity of primer set RRV2F/2R was validated in silico against published GenBank sequences and in-vitro against infected plant samples and an exclusivity panel of near-neighbor and other viruses that commonly infect Rosa spp. The developed assay is sensitive with a detection limit of 1fg from infected plant tissue. Thirty rose samples from 8 different states of the United States were tested using the developed methods. The developed methods are sensitive and reliable, and can be used by diagnostic laboratories for routine testing and disease management decisions. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay.

Science.gov (United States)

de Almeida, Marcos E; Koru, Ozgur; Steurer, Francis; Herwaldt, Barbara L; da Silva, Alexandre J

2017-01-01

Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (T m ) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention for Leishmania diagnostic testing. Specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the T m values of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the Leishmania (Leishmania) donovani complex in aggregate; the species L (L) tropica; and the species L (L) mexicana, L (L) amazonensis, L (L) major, and L (L) aethiopica in aggregate. Copyright © 2016 American Society for Microbiology.

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: pavel@safety.sci.kth.se; Grishchenko, Dmitry, E-mail: dmitry@safety.sci.kth.se; Konovalenko, Alexander, E-mail: kono@kth.se; Karbojian, Aram, E-mail: karbojan@kth.se

2017-04-01

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.

Melting of superheated molecular crystals

Science.gov (United States)

Cubeta, Ulyana; Bhattacharya, Deepanjan; Sadtchenko, Vlad

2017-07-01

Melting dynamics of micrometer scale, polycrystalline samples of isobutane, dimethyl ether, methyl benzene, and 2-propanol were investigated by fast scanning calorimetry. When films are superheated with rates in excess of 105 K s-1, the melting process follows zero-order, Arrhenius-like kinetics until approximately half of the sample has transformed. Such kinetics strongly imply that melting progresses into the bulk via a rapidly moving solid-liquid interface that is likely to originate at the sample's surface. Remarkably, the apparent activation energies for the phase transformation are large; all exceed the enthalpy of vaporization of each compound and some exceed it by an order of magnitude. In fact, we find that the crystalline melting kinetics are comparable to the kinetics of dielectric α-relaxation in deeply supercooled liquids. Based on these observations, we conclude that the rate of non-isothermal melting for superheated, low-molecular-weight crystals is limited by constituent diffusion into an abnormally dense, glass-like, non-crystalline phase.

Advanced One-Dimensional Entrained-Flow Gasifier Model Considering Melting Phenomenon of Ash

Directory of Open Access Journals (Sweden)

Jinsu Kim

2018-04-01

Full Text Available A one-dimensional model is developed to represent the ash-melting phenomenon, which was not considered in the previous one-dimensional (1-D entrained-flow gasifier model. We include sensible heat of slag and the fusion heat of ash in the heat balance equation. To consider the melting of ash, we propose an algorithm that calculates the energy balance for three scenarios based on temperature. We also use the composition and the thermal properties of anorthite mineral to express ash. gPROMS for differential equations is used to solve this algorithm in a simulation; the results include coal conversion, gas composition, and temperature profile. Based on the Texaco pilot plant gasifier, we validate our model. Our results show good agreement with previous experimental data. We conclude that the sensible heat of slag and the fusion heat of ash must be included in the entrained flow gasifier model.

Pressure melting and ice skating

Science.gov (United States)

Colbeck, S. C.

1995-10-01

Pressure melting cannot be responsible for the low friction of ice. The pressure needed to reach the melting temperature is above the compressive failure stress and, if it did occur, high squeeze losses would result in very thin films. Pure liquid water cannot coexist with ice much below -20 °C at any pressure and friction does not increase suddenly in that range. If frictional heating and pressure melting contribute equally, the length of the wetted contact could not exceed 15 μm at a speed of 5 m/s, which seems much too short. If pressure melting is the dominant process, the water films are less than 0.08 μm thick because of the high pressures.

Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons.

Science.gov (United States)

Papadopoulou, Evanthia; Goodchild, Sarah A; Cleary, David W; Weller, Simon A; Gale, Nittaya; Stubberfield, Michael R; Brown, Tom; Bartlett, Philip N

2015-02-03

The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.

Description of the heating and expansion process of a water drop enclosed in a hot melt

International Nuclear Information System (INIS)

Froehlich, G.; Berg, E. von.

1985-11-01

In the present study a simple model for the description of the heating- and expansion-process of a water drop enclosed in hot melt is developed. The model is valid between the first contact of melt and water up to the beginning of evaporation. A possible superheating by retardation of ebullition is disregarded. The balance equations for energy, mass and momentum as well as the equation of state are integrated over the radial space coordinate in both media using appropriate profiles of temperature, pressure and velocity. Thereby a system of coupled ordinary differential equations is formed for the variables of the model which are now time dependent only. The equations are solved numerically by means of a FORTRAN-program. The influence of parameters (melt-temperature, heat-transfer-coefficient between melt and water as well as drop radius) are studied. It is shown that always very rapidly a vapor-layer forms around the water drop, while the inner part of the drop did not yet 'notice' anything of the heating process. An approximation formula for the time-transfer-coefficients between melt and water. Due to this approximation, the time up to incipience of evaporation grows proportional to the drop radius, which means that in the frame of the present model even small droplets won't evaporate as a whole instantaneously. (orig.) [de

Mathematical analysis of the real time array PCR (RTA PCR) process

NARCIS (Netherlands)

Dijksman, Johan Frederik; Pierik, A.

2012-01-01

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

Characterization of bovine ruminal epithelial bacterial communities using 16S rRNA sequencing, PCR-DGGE, and qRT-PCR analysis.

Science.gov (United States)

Li, Meiju; Zhou, Mi; Adamowicz, Elizabeth; Basarab, John A; Guan, Le Luo

2012-02-24

Currently, knowledge regarding the ecology and function of bacteria attached to the epithelial tissue of the rumen wall is limited. In this study, the diversity of the bacterial community attached to the rumen epithelial tissue was compared to the rumen content bacterial community using 16S rRNA gene sequencing, PCR-DGGE, and qRT-PCR analysis. Sequence analysis of 2785 randomly selected clones from six 16S rDNA (∼1.4kb) libraries showed that the community structures of three rumen content libraries clustered together and were separated from the rumen tissue libraries. The diversity index of each library revealed that ruminal content bacterial communities (4.12/4.42/4.88) were higher than ruminal tissue communities (2.90/2.73/3.23), based on 97% similarity. The phylum Firmicutes was predominant in the ruminal tissue communities, while the phylum Bacteroidetes was predominant in the ruminal content communities. The phyla Fibrobacteres, Planctomycetes, and Verrucomicrobia were only detected in the ruminal content communities. PCR-DGGE analysis of the bacterial profiles of the rumen content and ruminal epithelial tissue samples from 22 steers further confirmed that there is a distinct bacterial community that inhibits the rumen epithelium. The distinctive epimural bacterial communities suggest that Firmicutes, together with other epithelial-specific species, may have additional functions other than food digestion. Copyright © 2011 Elsevier B.V. All rights reserved.

Filament stretching rheometry of polymer melts

DEFF Research Database (Denmark)

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

2005-01-01

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...

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

OpenAIRE

Kuppan Gokulan; Katherine Williams; Sangeeta Khare

2017-01-01

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

Melting temperature of graphite

International Nuclear Information System (INIS)

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

2001-01-01

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)

Clinical application of RT-nested PCR integrated with RFLP in Hantavirus detection and genotyping: a prospective study in Shandong Province, PR China.

Science.gov (United States)

Liu, Yun-Xi; Zhao, Zhong-Tang; Cao, Wu-Chun; Xu, Xiao-Qun; Suo, Ji-Jiang; Xing, Yu-Bin; Jia, Ning; Du, Ming-Mei; Liu, Bo-Wei; Yao, Yuan

2013-01-01

The aim of the present study was to evaluate the clinical usefulness of applying RT-nested PCR along with RFLP as a method for diagnosis and genotypic differentiation of Hantavirus in the acute-stage sera of HFRS patients as compared to the ELISA technique. A prospective study of patients with suspected HFRS patients was carried out. Sera were collected for serological evaluation by ELISA and RT-nested PCR testing. Primers were selected from the published sequence of the S segment of HTNV strain 76-118 and SEOV strain SR-11, which made it possible to obtain an amplicon of 403 bp by RT-nested PCR. The genotypic differentiations of the RT-nested PCR amplicons were carried out by RFLP. Sequence analyses of the amplicons were used to confirm the accuracy of the results obtained by RFLP. Of the 48 acute-stage sera from suspected HFRS patients, 35 were ELISA-positive while 41 were positive by RT-nested PCR. With Hind III and Hinf I, RFLP profiles of the RT-nested PCR amplicons of the 41 positive sera exhibited two patterns. 33 had RFLP profiles similar to the reference strain R22, and thus belonged to the SEOV type. The other 8 samples which were collected during October-December had RFLP profiles similar to the reference strain 76-118, and thus belonged to the HTNV type. Sequence phylogenetic analysis of RT-nested PCR amplicons revealed sdp1, sdp2 YXL-2008, and sdp3 as close relatives of HTNV strain 76-118, while sdp22 and sdp37 as close relatives of SEOV strain Z37 and strain R22 located in two separate clusters in the phylogenetic tree. These results were identical to those acquired by RFLP. RT-nested PCR integrated with RFLP was a rapid, simple, accurate method for detecting and differentiating the genotypes of Hantavirus in the acute-stage sera of suspected HFRS patients. In Shandong province, the main genotypes of Hantavirus belonged to the SEOV types, while the HTNV types were observed during the autumn-winter season.

EVA GREEN REAL-TIME PCR USED TO DETECT CELERY AS AN ALLERGEN IN FOOD

Directory of Open Access Journals (Sweden)

Ondrej Škultéty

2011-04-01

Full Text Available EvaGreen®  Real-Time PCR method has been used for celery(Apium graveolens allergen detection. A primer designed in mannitol dehydrogenase gene region has been used for specific celery identification in sample. The results show possibility to create calibration curve using artificially adulterated samples. The increasing variability between parallel calibration of celery samples has been observed from 0.1 % to 100%. Detection limit has been set to value 0.1% in celery representing 1000 ppm. Fluorescent signal has been presented even in samples with lower percentage addition of celery but these samples have been excluded according to unspecific melting curve.doi:10.5219/138

Theoretical melting curve of caesium

International Nuclear Information System (INIS)

Simozar, S.; Girifalco, L.A.; Pennsylvania Univ., Philadelphia

1983-01-01

A statistical-mechanical model is developed to account for the complex melting curve of caesium. The model assumes the existence of three different species of caesium defined by three different electronic states. On the basis of this model, the free energy of melting and the melting curve are computed up to 60 kbar, using the solid-state data and the initial slope of the fusion curve as input parameters. The calculated phase diagram agrees with experiment to within the experimental error. Other thermodynamic properties including the entropy and volume of melting were also computed, and they agree with experiment. Since the theory requires only one adjustable constant, this is taken as strong evidence that the three-species model is satisfactory for caesium. (author)

Mantle melting and melt refertilization beneath the Southwest Indian Ridge: Mineral composition of abyssal peridotites

Science.gov (United States)

Chen, Ling; Zhu, Jihao; Chu, Fengyou; Dong, Yan-hui; Liu, Jiqiang; Li, Zhenggang; Zhu, Zhimin; Tang, Limei

2017-04-01

As one of the slowest spreading ridges of the global ocean ridge system, the Southwest Indian Ridge (SWIR) is characterized by discontinued magmatism. The 53°E segment between the Gallieni fracture zone (FZ) (52°20'E) and the Gazelle FZ (53°30'E) is a typical amagmatic segment (crustal thickness 1cm) Opx, and Mg-rich mineral compositions akin to harzburgite xenoliths that sample old continental lithospheric mantle (Kelemen et al., 1998). Melt refertilization model shows that Group 2 peridotites were affected by an enriched low-degree partial melt from the garnet stability field. These results indicate that depleted mantle which experiences ancient melting event are more sensitive to melt refertilization, thus may reduce the melt flux, leading to extremely thin crust at 53°E segment. This research was granted by the National Basic Research Programme of China (973 programme) (grant No. 2013CB429705) and the Fundamental Research Funds of Second Institute of Oceanography, State Oceanic Administration (JG1603, SZ1507). References: Johnson K T M, Dick H J B, Shimizu N. Melting in the oceanic upper mantle: An ion microprobe study of diopsides in abyssal peridotites[J]. Journal of Geophysical Research, 1990, 95(B3):2661-2678. Kelemen P B, Hart S R, Bernstein S. Silica enrichment in the continental upper mantle via melt/rock reaction[J]. Earth & Planetary Science Letters, 1998, 164(1-2):387-406. Zhou H, Dick H J. Thin crust as evidence for depleted mantle supporting the Marion Rise.[J]. Nature, 2013, 494(7436):195-200.

Melts of garnet lherzolite: experiments, models and comparison to melts of pyroxenite and carbonated lherzolite

Science.gov (United States)

Grove, Timothy L.; Holbig, Eva S.; Barr, Jay A.; Till, Christy B.; Krawczynski, Michael J.

2013-01-01

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.

Authenticity analyses of Rhizoma Paridis using barcoding coupled with high resolution melting (Bar-HRM) analysis to control its quality for medicinal plant product.

Science.gov (United States)

Duan, Bao-Zhong; Wang, Ya-Ping; Fang, Hai-Lan; Xiong, Chao; Li, Xi-Wen; Wang, Ping; Chen, Shi-Lin

2018-01-01

Rhizoma Paridis (Chonglou) is a commonly used and precious traditional Chinese medicine. Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz. and Paris polyphylla Smith var . chinensis (Franch.) Hara are the two main sources of Chonglou under the monograph of Rhizoma Paridis in Chinese Pharmacopoeia. In the local marketplace, however, this medicine is prone to be accidentally contaminated, deliberately substituted or admixed with other species that are similar to Rhizoma Paridis in shape and color. Consequently, these adulterations might compromise quality control and result in considerable health concerns for consumers. This study aims to develop a rapid and sensitive method for accurate identification of Rhizoma Paridis and its common adulterants. DNA barcoding coupled with high resolution melting analysis was applied in this research to distinguish Rhizoma Paridis from its adulteration. The internal transcribed spacer 2 (ITS2) barcode was selected for HRM analysis to produce standard melting profile of the selected species. DNA of the tested herbal medicines was isolated and their melting profiles were generated and compared with the standard melting profile of P. polyphylla var. chinensis . The results indicate that the ITS2 molecular regions coupled with HRM analysis can effectively differentiate nine herbal species, including two authentic origins of Chonglou and their seven common adulterants. Ten herbal medicines labeled "Chonglou" obtained from a local market were collected and identified with our methods, and their sequence information was analyzed to validate the accuracy of HRM analysis. DNA barcoding coupled with HRM analysis is a accurate, reliable, rapid, cost-effective and robust tool, which could contribute to the quality control of Rhizoma Paridis in the supply chain of the natural health product industry (NHP).

The Role of Lipsticks and Blush Sticks in Genetic Profiling for Human Identification

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Nouman Rasool

2016-06-01

Full Text Available The core objectives of the current study are to generate human DNA profiles from used lipsticks and blush sticks of various brands available in Pakistan. A total of 12 international and local brands of lipsticks and blush sticks were selected. The lipsticks and blush sticks were applied by twenty different healthy female volunteers of 21-30 years of age. The heads of used lipsticks and blush sticks were swabbed with dry sterile cotton swabs. The qualitative and quantitative analysis was done by real time polymerase chain reaction (PCR, using a Quantifiler® Duo DNA Quantification Kit on Real Time PCR ABI™ 7500. Samples were amplified for 16 STR loci using an AmpFlSTR® Identifiler® PCR amplification kit on Thermocycler ABI 9700. The amplified product was run on Applied Biosystems 3130™ Genetic Analyzer. The genetic profiles were analyzed on GeneMapper® ID-X software version 1.0. The quantification results showed that the yield of DNA obtained from lipstick samples was greater than that of DNA obtained from blush stick samples. The real-time PCR results showed that only 16% of cosmetic samples had shown inhibition. The DNA profiles obtained from all blush stick samples were of good quality compared to those from lipstick samples. No profile was obtained from one blush stick sample (DNA 0.001 ng/μL and four lipstick samples (DNA 0.001-0.003 ng/μL because the amount of DNA in each of these samples was less than the amount required for successful amplification. DNA profiles were successfully generated from most of the samples of various available brands of lipsticks and blush sticks. This is the first study proving that DNA profiles can be generated from various lip and face cosmetics.

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 http://code.google.com/p/sasqpcr/downloads/list.

Assessment of the real-time PCR and different digital PCR platforms for DNA quantification.

Science.gov (United States)

Pavšič, Jernej; Žel, Jana; Milavec, Mojca

2016-01-01

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.

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

Directory of Open Access Journals (Sweden)

Pereyra Silvana

2012-01-01

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

Material properties influence on steam explosion efficiency. Prototypic versus simulant melts, eutectic versus non-eutectic melts

International Nuclear Information System (INIS)

Leskovar, M.; Mavko, B.

2006-01-01

A steam explosion may occur during a severe nuclear reactor accident if the molten core comes into contact with the coolant water. A strong enough steam explosion in a nuclear power plant could jeopardize the containment integrity and so lead to a direct release of radioactive material to the environment. Details of processes taking place prior and during the steam explosion have been experimentally studied for a number of years with adjunct efforts in modelling these processes to address the scaling of these experiments. Steam explosion experiments have shown that there are important differences of behaviour between simulant and prototypical melts, and that also at prototypical melts the fuel coolant interactions depend on the composition of the corium. In experiments with prototypic materials no spontaneous steam explosions occurred (except with an eutectic composition), whereas with simulant materials the steam explosions were triggered spontaneously. The energy conversion ratio of steam explosions with prototypic melts is at least one order of magnitude lower than the energy conversion ratio of steam explosions with simulant melts. Although the different behaviour of prototypic and simulant melts has been known for a number of years, there is no reliable explanation for these differences. Consequently it is not possible to reliably estimate whether corium would behave so non-explosive also in reactor conditions, where the mass of poured melt is nearly three orders of magnitude larger than in experimental conditions. An even more fascinating material effect was observed recently at corium experiments with eutectic and non-eutectic compositions. It turned out that eutectic corium always exploded spontaneously, whereas non-eutectic corium never exploded spontaneously. In the paper, a possible explanation of both material effects (prototypic/simulant melts, eutectic/non-eutectic corium) on the steam explosion is provided. A model for the calculation of the

Numerical simulation of hot-melt extrusion processes for amorphous solid dispersions using model-based melt viscosity.

Science.gov (United States)

Bochmann, Esther S; Steffens, Kristina E; Gryczke, Andreas; Wagner, Karl G

2018-03-01

Simulation of HME processes is a valuable tool for increased process understanding and ease of scale-up. However, the experimental determination of all required input parameters is tedious, namely the melt rheology of the amorphous solid dispersion (ASD) in question. Hence, a procedure to simplify the application of hot-melt extrusion (HME) simulation for forming amorphous solid dispersions (ASD) is presented. The commercial 1D simulation software Ludovic ® was used to conduct (i) simulations using a full experimental data set of all input variables including melt rheology and (ii) simulations using model-based melt viscosity data based on the ASDs glass transition and the physical properties of polymeric matrix only. Both types of HME computation were further compared to experimental HME results. Variation in physical properties (e.g. heat capacity, density) and several process characteristics of HME (residence time distribution, energy consumption) among the simulations and experiments were evaluated. The model-based melt viscosity was calculated by using the glass transition temperature (T g ) of the investigated blend and the melt viscosity of the polymeric matrix by means of a T g -viscosity correlation. The results of measured melt viscosity and model-based melt viscosity were similar with only few exceptions, leading to similar HME simulation outcomes. At the end, the experimental effort prior to HME simulation could be minimized and the procedure enables a good starting point for rational development of ASDs by means of HME. As model excipients, Vinylpyrrolidone-vinyl acetate copolymer (COP) in combination with various APIs (carbamazepine, dipyridamole, indomethacin, and ibuprofen) or polyethylene glycol (PEG 1500) as plasticizer were used to form the ASDs. Copyright © 2017 Elsevier B.V. All rights reserved.

Melt processed high-temperature superconductors

CERN Document Server

1993-01-01

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

The melting and solidification of nanowires

International Nuclear Information System (INIS)

Florio, B. J.; Myers, T. G.

2016-01-01

A mathematical model is developed to describe the melting of nanowires. The first section of the paper deals with a standard theoretical situation, where the wire melts due to a fixed boundary temperature. This analysis allows us to compare with existing results for the phase change of nanospheres. The equivalent solidification problem is also examined. This shows that solidification is a faster process than melting; this is because the energy transfer occurs primarily through the solid rather than the liquid which is a poorer conductor of heat. This effect competes with the energy required to create new solid surface which acts to slow down the process, but overall conduction dominates. In the second section, we consider a more physically realistic boundary condition, where the phase change occurs due to a heat flux from surrounding material. This removes the singularity in initial melt velocity predicted in previous models of nanoparticle melting. It is shown that even with the highest possible flux the melting time is significantly slower than with a fixed boundary temperature condition.

The melting and solidification of nanowires

Science.gov (United States)

Florio, B. J.; Myers, T. G.

2016-06-01

A mathematical model is developed to describe the melting of nanowires. The first section of the paper deals with a standard theoretical situation, where the wire melts due to a fixed boundary temperature. This analysis allows us to compare with existing results for the phase change of nanospheres. The equivalent solidification problem is also examined. This shows that solidification is a faster process than melting; this is because the energy transfer occurs primarily through the solid rather than the liquid which is a poorer conductor of heat. This effect competes with the energy required to create new solid surface which acts to slow down the process, but overall conduction dominates. In the second section, we consider a more physically realistic boundary condition, where the phase change occurs due to a heat flux from surrounding material. This removes the singularity in initial melt velocity predicted in previous models of nanoparticle melting. It is shown that even with the highest possible flux the melting time is significantly slower than with a fixed boundary temperature condition.

The melting and solidification of nanowires

Energy Technology Data Exchange (ETDEWEB)

Florio, B. J., E-mail: brendan.florio@ul.ie [University of Limerick, Mathematics Applications Consortium for Science and Industry (MACSI), Department of Mathematics and Statistics (Ireland); Myers, T. G., E-mail: tmyers@crm.cat [Centre de Recerca Matemàtica (Spain)

2016-06-15

A mathematical model is developed to describe the melting of nanowires. The first section of the paper deals with a standard theoretical situation, where the wire melts due to a fixed boundary temperature. This analysis allows us to compare with existing results for the phase change of nanospheres. The equivalent solidification problem is also examined. This shows that solidification is a faster process than melting; this is because the energy transfer occurs primarily through the solid rather than the liquid which is a poorer conductor of heat. This effect competes with the energy required to create new solid surface which acts to slow down the process, but overall conduction dominates. In the second section, we consider a more physically realistic boundary condition, where the phase change occurs due to a heat flux from surrounding material. This removes the singularity in initial melt velocity predicted in previous models of nanoparticle melting. It is shown that even with the highest possible flux the melting time is significantly slower than with a fixed boundary temperature condition.

Methods for Melting Temperature Calculation

Science.gov (United States)

Hong, Qi-Jun

Melting temperature calculation has important applications in the theoretical study of phase diagrams and computational materials screenings. In this thesis, we present two new methods, i.e., the improved Widom's particle insertion method and the small-cell coexistence method, which we developed in order to capture melting temperatures both accurately and quickly. We propose a scheme that drastically improves the efficiency of Widom's particle insertion method by efficiently sampling cavities while calculating the integrals providing the chemical potentials of a physical system. This idea enables us to calculate chemical potentials of liquids directly from first-principles without the help of any reference system, which is necessary in the commonly used thermodynamic integration method. As an example, we apply our scheme, combined with the density functional formalism, to the calculation of the chemical potential of liquid copper. The calculated chemical potential is further used to locate the melting temperature. The calculated results closely agree with experiments. We propose the small-cell coexistence method based on the statistical analysis of small-size coexistence MD simulations. It eliminates the risk of a metastable superheated solid in the fast-heating method, while also significantly reducing the computer cost relative to the traditional large-scale coexistence method. Using empirical potentials, we validate the method and systematically study the finite-size effect on the calculated melting points. The method converges to the exact result in the limit of a large system size. An accuracy within 100 K in melting temperature is usually achieved when the simulation contains more than 100 atoms. DFT examples of Tantalum, high-pressure Sodium, and ionic material NaCl are shown to demonstrate the accuracy and flexibility of the method in its practical applications. The method serves as a promising approach for large-scale automated material screening in which

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

African Journals Online (AJOL)

Yomi

2011-12-21

Dec 21, 2011 ... membrane filtration was carried out with a commercial PCR product purification kit (Generay, Shanghai), according to the manufacture's instruction. In brief, 50 µl PCR product was mixed thoroughly with binding buffer, and the resultant mixture was loaded directly onto a silica membrane Gelclean column.

Composition profile determination in isomorphous binary alloys

International Nuclear Information System (INIS)

An, C.Y.; Bandeira, I.N.

1983-07-01

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

Recent Changes in the Arctic Melt Season

Science.gov (United States)

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

2007-01-01

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.

Rapid molecular characterization of Acinetobacter baumannii clones with rep-PCR and evaluation of carbapenemase genes by new multiplex PCR in Hospital District of Helsinki and Uusimaa.

Directory of Open Access Journals (Sweden)

Tanja Pasanen

Full Text Available Multidrug-resistant Acinetobacter baumannii (MDRAB is an increasing problem worldwide. Prevalence of carbapenem resistance in Acinetobacter spp. due to acquired carbapenemase genes is not known in Finland. The purpose of this study was to examine prevalence and clonal spread of multiresistant A. baumannii group species, and their carbapenemase genes. A total of 55 Acinetobacter isolates were evaluated with repetitive PCR (DiversiLab to analyse clonality of isolates, in conjunction with antimicrobial susceptibility profile for ampicillin/sulbactam, colistin, imipenem, meropenem, rifampicin and tigecycline. In addition, a new real-time PCR assay, detecting most clinically important carbapenemase genes just in two multiplex reactions, was developed. The assay detects genes for KPC, VIM, IMP, GES-1/-10, OXA-48, NDM, GIM-1, SPM-1, IMI/NMC-A, SME, CMY-10, SFC-1, SIM-1, OXA-23-like, OXA-24/40-like, OXA-58 and ISAbaI-OXA-51-like junction, and allows confident detection of isolates harbouring acquired carbapenemase genes. There was a time-dependent, clonal spread of multiresistant A. baumannii strongly correlating with carbapenamase gene profile, at least in this geographically restricted study material. The new carbapenemase screening assay was able to detect all the genes correctly suggesting it might be suitable for epidemiologic screening purposes in clinical laboratories.

On melting of boron phosphide under pressure

OpenAIRE

Solozhenko, Vladimir; Mukhanov, V. A.

2015-01-01

Melting of cubic boron phosphide, BP, has been studied at pressures to 9 GPa using synchrotron X-ray diffraction and electrical resistivity measurements. It has been found that above 2.6 GPa BP melts congruently, and the melting curve exhibits negative slope (–60 ± 7 K/GPa), which is indicative of a higher density of the melt as compared to the solid phase.

Melting Can Hinder Impact-Induced Adhesion

Science.gov (United States)

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

2017-10-01

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.

Property Investigation of Laser Cladded, Laser Melted and Electron Beam Melted Ti-Al6-V4

Science.gov (United States)

2006-05-01

UNCLASSIFIED/UNLIMITED UNCLASSIFIED/UNLIMITED Figure 3: Examples of electron beam melted net shape parts; powder bed [3]. 1.4 Laser Cladding ...description, www.arcam.com. [4] K.-H. Hermann, S. Orban, S. Nowotny, Laser Cladding of Titanium Alloy Ti6242 to Restore Damaged Blades, Proceedings...Property Investigation of Laser Cladded , Laser Melted and Electron Beam Melted Ti-Al6-V4 Johannes Vlcek EADS Deutschland GmbH Corporate Research

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

Directory of Open Access Journals (Sweden)

Caligari Peter DS

2010-05-01

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

A miniaturized and integrated gel post platform for multiparameter PCR detection of herpes simplex viruses from raw genital swabs.

Science.gov (United States)

Manage, Dammika P; Lauzon, Jana; Atrazhev, Alexey; Morrissey, Yuen C; Edwards, Ann L; Stickel, Alexander J; Crabtree, H John; Pabbaraju, Kanti; Zahariadis, George; Yanow, Stephanie K; Pilarski, Linda M

2012-05-07

Herpes simplex virus (HSV) is one of the most prevalent viruses, with acute and recurrent infections in humans. The current gold standard for the diagnosis of HSV is viral culture which takes 2-14 days and has low sensitivity. In contrast, DNA amplification by polymerase chain reaction (PCR) can be performed within 1-2 h. We here describe a multiparameter PCR assay to simultaneously detect HSV-1 and HSV-2 DNA templates, together with integrated positive and negative controls, with product detection by melting curve analysis (MCA), in an array of semi-solid polyacrylamide gel posts. Each gel post is 0.67 μL in volume, and polymerized with all the components required for PCR. Both PCR and MCA can currently be performed in one hour and 20 min. Unprocessed genital swabs collected in universal transport medium were directly added to the reagents before or after polymerization, diffusing from atop the gel posts. The gel post platform detects HSV templates in as little as 2.5 nL of raw sample. In this study, 45 genital swab specimens were tested blindly as a preliminary validation of this platform. The concordance of PCR on gel posts with conventional PCR was 91%. The primer sequestration method introduced here (wherein different primers are placed in different sets of posts) enables the simultaneous detection of multiple pathogens for the same sample, together with positive and negative controls, on a single chip. This platform accepts unprocessed samples and is readily adaptable to detection of multiple different pathogens or biomarkers for point-of-care diagnostics.

Characterization of South American Snails of the Genus Biomphalaria (Basommatophora: Planorbidae and Schistosoma mansoni (Platyhelminthes: Trematoda in Molluscs by PCR-RFLP

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Roberta Lima Caldeira

2016-01-01

Full Text Available The identification of snails of the genus Biomphalaria can be done using morphological characteristics which depends on the size of the snails and skill and knowledge of researcher. These methods sometimes are not adequate for identification of species. The PCR-RFLP, using the ITS region of the rDNA, has been used to identify Brazilian species of the genus Biomphalaria. Nevertheless, there is a lack of information about snails from other Latin American countries. In addition, some snails may be infected by Schistosoma mansoni and when submitted to PCR-RFLP they show molecular profiles different from those previously standardized for the other mollusc species. In this work the molecular profiles of 15 species and the subspecies were established by PCR-RFLP of ITS-rDNA with the enzyme DdeI. Moreover, the molecular profiles of host species, B. glabrata, B. straminea, B. tenagophila, and B. prona, infected by S. mansoni were also established. The molluscs were dissected to permit morphological identification. These results contribute to a correct identification of snails of the genus Biomphalaria and detection of these snails infected by S. mansoni.

Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional and real-time PCR.

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Demeler, Janina; Ramünke, Sabrina; Wolken, Sonja; Ianiello, Davide; Rinaldi, Laura; Gahutu, Jean Bosco; Cringoli, Giuseppe; von Samson-Himmelstjerna, Georg; Krücken, Jürgen

2013-01-01

Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg) and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles - only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM) analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP) and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many different post PCR methods can

Discrimination of gastrointestinal nematode eggs from crude fecal egg preparations by inhibitor-resistant conventional and real-time PCR.

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Janina Demeler

Full Text Available Diagnosis of gastrointestinal nematodes relies predominantly on coproscopic methods such as flotation, Kato-Katz, McMaster or FLOTAC. Although FLOTAC allows accurate quantification, many nematode eggs can only be differentiated to genus or family level. Several molecular diagnostic tools discriminating closely related species suffer from high costs for DNA isolation from feces and limited sensitivity since most kits use only small amounts of feces (<1 g. A direct PCR from crude egg preparations was designed for full compatibility with FLOTAC to accurately quantify eggs per gram feces (epg and determine species composition. Eggs were recovered from the flotation solution and concentrated by sieving. Lysis was achieved by repeated boiling and freezing cycles - only Trichuris eggs required additional mechanic disruption. Egg lysates were directly used as template for PCR with Phusion DNA polymerase which is particularly resistant to PCR inhibitors. Qualitative results were obtained with feces of goats, cattle, horses, swine, cats, dogs and mice. The finally established protocol was also compatible with quantitative real-time PCR in the presence of EvaGreen and no PCR inhibition was detectable when extracts were diluted at least fourfold. Sensitivity was comparable to DNA isolation protocols and spiked samples with five epg were reliably detected. For Toxocara cati a detection limit below one epg was demonstrated. It was possible to distinguish T. cati and Toxocara canis using high resolution melt (HRM analysis, a rapid tool for species identification. In human samples, restriction fragment length polymorphism (RFLP and HRM analysis were used to discriminate Necator americanus and Ancylostoma duodenale. The method is able to significantly improve molecular diagnosis of gastrointestinal nematodes by increasing speed and sensitivity while decreasing overall costs. For identification of species or resistance alleles, analysis of PCR products with many

Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in milk.

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Wang, Meng; Yang, Junjie; Gai, Zhongtao; Huo, Shengnan; Zhu, Jianhua; Li, Jun; Wang, Ranran; Xing, Sheng; Shi, Guosheng; Shi, Feng; Zhang, Lei

2018-02-02

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.

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

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

2017-07-17

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

Plasma arc melting of titanium-tantalum alloys

International Nuclear Information System (INIS)

Dunn, P.; Patterson, R.A.; Haun, R.

1994-01-01

Los Alamos has several applications for high temperature, oxidation and liquid-metal corrosion resistant materials. Further, materials property constraints are dictated by a requirement to maintain low density; e.g., less than the density of stainless steel. Liquid metal compatibility and density requirements have driven the research toward the Ti-Ta system with an upper bound of 60 wt% Ta-40 wt% Ti. Initial melting of these materials was performed in a small button arc melter with several hundred grams of material; however, ingot quantities were soon needed. But, refractory metal alloys whose constituents possess very dissimilar densities, melting temperatures and vapor pressures pose significant difficulty and require specialized melting practices. The Ti-Ta alloys fall into this category with the density of tantalum 16.5 g/cc and that of titanium 4.5 g/cc. Melting is further complicated by the high melting point of Ta(3020 C) and the relatively low boiling point of Ti(3287 C). Previous electron beam melting experience with these materials resulted, in extensive vaporization of the titanium and poor chemical homogeneity. Vacuum arc remelting(VAR) was considered as a melting candidate and discarded due to density and vapor pressure issues associated with electron beam. Plasma arc melting offered the ability to supply a cover gas to deal with vapor pressure issues as well as solidification control to help with macrosegregation in the melt and has successfully produced high quality ingots of the Ti-Ta alloys

On melting criteria for complex plasma

International Nuclear Information System (INIS)

Klumov, Boris A

2011-01-01

The present paper considers melting criteria for a plasma crystal discovered in dust plasma in 1994. Separate discussions are devoted to three-dimensional (3D) and two-dimensional (2D) systems. In the 3D case, melting criteria are derived based on the properties of local order in a system of microparticles. The order parameters are constructed from the cumulative distributions of the microparticle probability distributions as functions of various rotational invariants. The melting criteria proposed are constructed using static information on microparticle positions: a few snapshots of the system that allow for the determination of particle coordinates are enough to determine the phase state of the system. It is shown that criteria obtained in this way describe well the melting and premelting of 3D complex plasmas. In 2D systems, a system of microparticles interacting via a screened Coulomb (i.e., Debye-Hueckel or Yukawa) potential is considered as an example, using molecular dynamics simulations. A number of new order parameters characterizing the melting of 2D complex plasmas are proposed. The order parameters and melting criteria proposed for 2D and 3D complex plasmas can be applied to other systems as well. (methodological notes)

Quantitative (real-time) PCR

International Nuclear Information System (INIS)

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

2005-01-01

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

Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

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Alok Arun

Full Text Available Real-time quantitative reverse transcription PCR (qRT-PCR is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae, two developmental stages (pupal and adult and two sexes (male and female, all of which were subjected to two food treatments (food stress and control feeding ad libitum. The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the

Selection and validation of reference genes for qRT-PCR expression analysis of candidate genes involved in olfactory communication in the butterfly Bicyclus anynana.

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Arun, Alok; Baumlé, Véronique; Amelot, Gaël; Nieberding, Caroline M

2015-01-01

Real-time quantitative reverse transcription PCR (qRT-PCR) is a technique widely used to quantify the transcriptional expression level of candidate genes. qRT-PCR requires the selection of one or several suitable reference genes, whose expression profiles remain stable across conditions, to normalize the qRT-PCR expression profiles of candidate genes. Although several butterfly species (Lepidoptera) have become important models in molecular evolutionary ecology, so far no study aimed at identifying reference genes for accurate data normalization for any butterfly is available. The African bush brown butterfly Bicyclus anynana has drawn considerable attention owing to its suitability as a model for evolutionary ecology, and we here provide a maiden extensive study to identify suitable reference gene in this species. We monitored the expression profile of twelve reference genes: eEF-1α, FK506, UBQL40, RpS8, RpS18, HSP, GAPDH, VATPase, ACT3, TBP, eIF2 and G6PD. We tested the stability of their expression profiles in three different tissues (wings, brains, antennae), two developmental stages (pupal and adult) and two sexes (male and female), all of which were subjected to two food treatments (food stress and control feeding ad libitum). The expression stability and ranking of twelve reference genes was assessed using two algorithm-based methods, NormFinder and geNorm. Both methods identified RpS8 as the best suitable reference gene for expression data normalization. We also showed that the use of two reference genes is sufficient to effectively normalize the qRT-PCR data under varying tissues and experimental conditions that we used in B. anynana. Finally, we tested the effect of choosing reference genes with different stability on the normalization of the transcript abundance of a candidate gene involved in olfactory communication in B. anynana, the Fatty Acyl Reductase 2, and we confirmed that using an unstable reference gene can drastically alter the expression

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

2013-01-01

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

Assessing the Accuracy of Quantitative Molecular Microbial Profiling

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Denise M. O'Sullivan

2014-11-01

Full Text Available The application of high-throughput sequencing in profiling microbial communities is providing an unprecedented ability to investigate microbiomes. Such studies typically apply one of two methods: amplicon sequencing using PCR to target a conserved orthologous sequence (typically the 16S ribosomal RNA gene or whole (metagenome sequencing (WGS. Both methods have been used to catalog the microbial taxa present in a sample and quantify their respective abundances. However, a comparison of the inherent precision or bias of the different sequencing approaches has not been performed. We previously developed a metagenomic control material (MCM to investigate error when performing different sequencing strategies. Amplicon sequencing using four different primer strategies and two 16S rRNA regions was examined (Roche 454 Junior and compared to WGS (Illumina HiSeq. All sequencing methods generally performed comparably and in good agreement with organism specific digital PCR (dPCR; WGS notably demonstrated very high precision. Where discrepancies between relative abundances occurred they tended to differ by less than twofold. Our findings suggest that when alternative sequencing approaches are used for microbial molecular profiling they can perform with good reproducibility, but care should be taken when comparing small differences between distinct methods. This work provides a foundation for future work comparing relative differences between samples and the impact of extraction methods. We also highlight the value of control materials when conducting microbial profiling studies to benchmark methods and set appropriate thresholds.

Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis.

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Mou, Yi; Athar, Muhammad Ammar; Wu, Yuzhen; Xu, Ye; Wu, Jianhua; Xu, Zhenxing; Hayder, Zulfiqar; Khan, Saeed; Idrees, Muhammad; Nasir, Muhammad Israr; Liao, Yiqun; Li, Qingge

2016-11-01

Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 10 4 copies/μl. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries. Copyright © 2016, American Society for

A novel RT-PCR for the detection of Helicobacter pylori and identification of clarithromycin resistance mediated by mutations in the 23S rRNA gene.

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Redondo, Javier Jareño; Keller, Peter M; Zbinden, Reinhard; Wagner, Karoline

2018-01-01

In this study we evaluated the commercially available LightMix® RT-PCR assay for Helicobacter pylori detection and identification of clarithromycin (CLR) resistance in culture and clinical specimens (gastric biopsies and stool). The H. pylori LightMix® RT-PCR detects a 97bp long fragment of the 23S rRNA gene and allows the identification of 3 distinct point mutations conferring CLR resistance via melting curve analysis. The performance of the H. pylori LightMix® RT-PCR was evaluated using a set of 60 H. pylori strains showing phenotypical CLR susceptibility or CLR resistance (Minimum inhibitory concentrations from 0.016 to 256mg/L). We found high concordance (95%) between phenotypical CLR resistance screening by E-Test® and the Lightmix® RT-PCR. Discrepant results were verified by sequencing of the 23S rRNA gene that always confirmed the results obtained by Lightmix® RT-PCR. Furthermore, H. pylori was detected in clinical biopsy and stool specimens by Lightmix® RT-PCR that identified the correct H. pylori genotype. The LightMix® RT-PCR is an accurate, sensitive and easy to use test for H. pylori and CLR resistance detection and can therefore be readily implemented in any diagnostic laboratory. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy

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Pesta David

2003-06-01

Full Text Available Abstract Background DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein. We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. Results Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. Conclusions The technique detailed

A real time PCR assay on blood for diagnosis of invasive candidiasis in immunocompromised patient

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Mohsen Ashrafi

2015-01-01

Results: From 2009 to 2011, 72 patients with hematologic malignancies and bone marrow transplant recipients were evaluated for IC. The female to male ratio was 27:45; the mean age was 32.1 years. The most common malignancy in this patient was acute myeloid leukemia (AML (27.8% and acute lymphoblastic leukemia (ALL (26.4%. Out of 72 patients, 11 patients (15.3% had positive real time PCR /probe results. Based on the melting temperature (Tm analysis, 5 (45.4% C. krusei, 3 (27.2% C. tropicalis, 2 (18.1% C. parapsilosis and 1 C. albicans (9% were identified. According to the revised EORTC / MSG, 1 patient (9% and 10 patients (91% were defined as proven and possible groups of IC, respectively. The mortality rate in proven and possible IC patient was found 54.5%. Conclusion: The established Real-time PCR/FRET probe assay is an appropriate diagnostic tool for the detection of Candida species DNA and the management of patients suffering from hematologic malignancies and bone marrow recipient are at risk for IC.

Corium melt researches at VESTA test facility

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Hwan Yeol Kim

2017-10-01

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

COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

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Tülin GÜVEN GÖKMEN

Full Text Available SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM, serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively. The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01% than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis.

Permanganate-assisted removal of PCR inhibitors during the DNA Chelex extraction from stained denim samples.

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Pîrlea, Sorina; Puiu, Mihaela; Răducan, Adina; Oancea, Dumitru

2017-03-01

In this study, it was demonstrated that the DNA Chelex extraction combined with the permanganate assisted-oxidation is highly efficient in removing the PCR inhibitors often found in clothing materials, such as phthalocyanine. The extraction assays were conducted in saliva, blood and epithelial cells samples mixed with three oxidation-resistant dye copper(II) α-phthalocyanine, copper(II) β-phthalocyanine and tetrasulfonated copper(II) β-phthalocyanine. After DNA amplification, all samples were able to provide full DNA profiles. The permanganate/Chelex system was tested further on denim-stained samples and displayed the same ability to remove the PCR inhibitors from the commercial textile materials.

A Feasibility Study on UO2/ZrO2 Mixture Melting using Induction Skull Melting Method

International Nuclear Information System (INIS)

Hong, S. W.; Kim, J. H.; Kim, H. D.

1998-01-01

Using ISM(Induction Skull Melting) method, which is usually used for the crystallization of refractory materials, a feasibility study on melting of the UO 2 /ZrO 2 mixture(w/o 8:2) is carried out. Frequency, one of main design parameters for ISM, is determined from electrical resistance of UO 2 /ZrO 2 mixture. Heat loss from the crucible for UO 2 /ZrO 2 20kg melting is predicted by comparison with the existing experimental data for UO , ZrO 2 , and ThO 2 . The analysis shows that melting and superheating of the UO 2 /ZrO 2 mixture using induction skull melting method is possible. To attain the superheat of 300K for 20 kg of UO 2 /ZrO 2 , 100kHz, 100 kW power input for induction coil, and 570L/min coolant flow rate are found to be required. The results of this feasibility study will be adopted for designing UO 2 /ZrO 2 furnace using actual corium material at KAERI

Quantitative PCR for the specific quantification of Lactococcus lactis and Lactobacillus paracasei and its interest for Lactococcus lactis in cheese samples.

Science.gov (United States)

Achilleos, Christine; Berthier, Françoise

2013-12-01

The first objective of this work was to develop real-time quantitative PCR (qPCR) assays to quantify two species of mesophilic lactic acid bacteria technologically active in food fermentation, including cheese making: Lactococcus lactis and Lactobacillus paracasei. The second objective was to compare qPCR and plate counts of these two species in cheese samples. Newly designed primers efficiently amplified a region of the tuf gene from the target species. Sixty-three DNA samples from twenty different bacterial species, phylogenetically related or commonly found in raw milk and dairy products, were selected as positive and negative controls. Target DNA was successfully amplified showing a single peak on the amplicon melting curve; non-target DNA was not amplified. Quantification was linear over 5 log units (R(2) > 0.990), down to 22 gene copies/μL per well for Lc. lactis and 73 gene copies/μL per well for Lb. paracasei. qPCR efficiency ranged from 82.9% to 93.7% for Lc. lactis and from 81.1% to 99.5% for Lb. paracasei. At two stages of growth, Lc. lactis was quantified in 12 soft cheeses and Lb. paracasei in 24 hard cooked cheeses. qPCR proved to be useful for quantifying Lc. lactis, but not Lb. paracasei. Copyright © 2013 Elsevier Ltd. All rights reserved.

Optimization of analytical parameters for inferring relationships among Escherichia coli isolates from repetitive-element PCR by maximizing correspondence with multilocus sequence typing data.

Science.gov (United States)

Goldberg, Tony L; Gillespie, Thomas R; Singer, Randall S

2006-09-01

Repetitive-element PCR (rep-PCR) is a method for genotyping bacteria based on the selective amplification of repetitive genetic elements dispersed throughout bacterial chromosomes. The method has great potential for large-scale epidemiological studies because of its speed and simplicity; however, objective guidelines for inferring relationships among bacterial isolates from rep-PCR data are lacking. We used multilocus sequence typing (MLST) as a "gold standard" to optimize the analytical parameters for inferring relationships among Escherichia coli isolates from rep-PCR data. We chose 12 isolates from a large database to represent a wide range of pairwise genetic distances, based on the initial evaluation of their rep-PCR fingerprints. We conducted MLST with these same isolates and systematically varied the analytical parameters to maximize the correspondence between the relationships inferred from rep-PCR and those inferred from MLST. Methods that compared the shapes of densitometric profiles ("curve-based" methods) yielded consistently higher correspondence values between data types than did methods that calculated indices of similarity based on shared and different bands (maximum correspondences of 84.5% and 80.3%, respectively). Curve-based methods were also markedly more robust in accommodating variations in user-specified analytical parameter values than were "band-sharing coefficient" methods, and they enhanced the reproducibility of rep-PCR. Phylogenetic analyses of rep-PCR data yielded trees with high topological correspondence to trees based on MLST and high statistical support for major clades. These results indicate that rep-PCR yields accurate information for inferring relationships among E. coli isolates and that accuracy can be enhanced with the use of analytical methods that consider the shapes of densitometric profiles.

Features of melting of indium monohalides

Energy Technology Data Exchange (ETDEWEB)

Dmitriev, V S; Smirniv, V A [AN SSSR, Chernogolovka. Inst. Fiziki Tverdogo Tela

1980-12-01

The character of InCl, InBr and InI melting is investigated by the methods of DTA, calorimetry, conductometry and chemical analysis. Partial decomposition of monohalogenides during melting according to the reactions of disproportionation is shown. The presence of disproportionation products (In/sup 0/ and In/sup 3 +/) is manifested in the properties of solid monohalogenides, prepared by the crystallization from melt, in their photosensitivity and electroconductivity.

The coupled response to slope-dependent basal melting

Science.gov (United States)

Little, C. M.; Goldberg, D. N.; Sergienko, O. V.; Gnanadesikan, A.

2009-12-01

Ice shelf basal melting is likely to be strongly controlled by basal slope. If ice shelves steepen in response to intensified melting, it suggests instability in the coupled ice-ocean system. The dynamic response of ice shelves governs what stable morphologies are possible, and thus the influence of melting on buttressing and grounding line migration. Simulations performed using a 3-D ocean model indicate that a simple form of slope-dependent melting is robust under more complex oceanographic conditions. Here we utilize this parameterization to investigate the shape and grounding line evolution of ice shelves, using a shallow-shelf approximation-based model that includes lateral drag. The distribution of melting substantially affects the shape and aspect ratio of unbuttressed ice shelves. Slope-dependent melting thins the ice shelf near the grounding line, reducing velocities throughout the shelf. Sharp ice thickness gradients evolve at high melting rates, yet grounding lines remain static. In foredeepened, buttressed ice shelves, changes in grounding line flux allow two additional options: stable or unstable retreat. Under some conditions, slope-dependent melting results in stable configurations even at high melt rates.

From the 'PCR' function to the 'PCR' profession; de la fonction 'PCR' au metier 'PCR'

Energy Technology Data Exchange (ETDEWEB)

Perrin, L. [CERAP, 91 - Gif sur Yvette (France)

2008-07-01

After having recalled the legal context concerning the appointment and training of a radiation protection expert (PCR for 'personne competente en radioprotection'), the author outlines that the PCR's role has notably evolved: his function is now of primary importance in the company and his activity does not correspond to the legal framework any longer. Moreover, with the application of a European directive, some small establishments possessing ionizing radiation sources are disadvantaged, and the PCR is now facing an increasing number of missions and tasks. The author gives a list of them and assesses a needed time of 146 days per year: this means PCRs cannot have an other activity within their company

Network topology of olivine-basalt partial melts

Science.gov (United States)

Skemer, Philip; Chaney, Molly M.; Emmerich, Adrienne L.; Miller, Kevin J.; Zhu, Wen-lu

2017-07-01

The microstructural relationship between melt and solid grains in partially molten rocks influences many physical properties, including permeability, rheology, electrical conductivity and seismic wave speeds. In this study, the connectivity of melt networks in the olivine-basalt system is explored using a systematic survey of 3-D X-ray microtomographic data. Experimentally synthesized samples with 2 and 5 vol.% melt are analysed as a series of melt tubules intersecting at nodes. Each node is characterized by a coordination number (CN), which is the number of melt tubules that intersect at that location. Statistically representative volumes are described by coordination number distributions (CND). Polyhedral grains can be packed in many configurations yielding different CNDs, however widely accepted theory predicts that systems with small dihedral angles, such as olivine-basalt, should exhibit a predominant CN of four. In this study, melt objects are identified with CN = 2-8, however more than 50 per cent are CN = 4, providing experimental verification of this theoretical prediction. A conceptual model that considers the role of heterogeneity in local grain size and melt fraction is proposed to explain the formation of nodes with CN ≠ 4. Correctly identifying the melt network topology is essential to understanding the relationship between permeability and porosity, and hence the transport properties of partial molten mantle rocks.

Melt Fragmentation Characteristics of Metal Fuel with Melt Injection Mass during Initiating Phase of SFR Severe Accidents

Energy Technology Data Exchange (ETDEWEB)

Heo, Hyo; Lee, Min Ho; Bang, In Cheol [Ulsan National Institute of Science and Technology, Ulsan (Korea, Republic of); Jerng, Dong Wook [Chung-Ang Univ., Seoul (Korea, Republic of)

2016-05-15

The PGSFR has adopted the metal fuel for its inherent safety under severe accident conditions. However, this fuel type is not demonstrated clearly yet under the such severe accident conditions. Additional experiments for examining these issues should be performed to support its licensing activities. Under initiating phase of hypothetic core disruptive accident (HCDA) conditions, the molten metal could be better dispersed and fragmented into the coolant channel than in the case of using oxide fuel. This safety strategy provides negative reactivity driven by a good dispersion of melt. If the coolant channel does not sufficient coolability, the severe recriticality would occur within the core region. Thus, it is important to examine the extent of melt fragmentation. The fragmentation behaviors of melt are closely related to a formation of debris shape. Once the debris shape is formed through the fragmentation process, its coolability is determined by the porosity or thermal conductivity of the melt. There were very limited studies for transient irradiation experiments of the metal fuel. These studies were performed by Transient Reactor Test Facility (TREAT) M series tests in U.S. The TREAT M series tests provided basic information of metal fuel performance under transient conditions. The effect of melt injection mass was evaluated in terms of the fragmentation behaviors of melt. These behaviors seemed to be similar between single-pin and multi-pins failure condition. However, the more melt was agglomerated in case of multi-pins failure.

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)

1990-06-01

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.

Hydrogen study in melt inclusions trapped in quartz with nuclear microprobe

International Nuclear Information System (INIS)

Mosbah, M.; Tirira, J.; Gosset, J.; Massiot, P.

1990-01-01

Elastic recoil spectrometry induced by 3 MeV helium-4 microbeam has been used to determine hydrogen distribution within melt inclusions trapped in quartz. These minerals were selected from different geological environments: Guadeloupe (West Indies), Pantelleria Island (South Sicily - Italy) and San Pietro (South Sardinia - Italy). Bulk hydrogen contents are calculated (H assumed to be in H 2 O form). The knowledge of hydrogen distribution assists both in a better understanding and in the establishment of volcanic dynamism hypotheses. Finally, fluid hydrogen rich inclusions are evidenced and H concentration profile is simulated and reported for the first time in glass inclusion

An investigation of the subtype diversity of clinical isolates of Irish Clostridium difficile ribotypes 027 and 078 by repetitive-extragenic palindromic PCR.

LENUS (Irish Health Repository)

Solomon, K

2011-08-01

A repetitive-extragenic palindromic PCR (rep-PCR) subtyping method (DiversiLab) in conjunction with ribotyping, toxinotyping and antimicrobial-susceptibility testing was used to detect subtypes within Clostridium difficile ribotypes 027 and 078. Clinical isolates of ribotypes 027 (toxinotype III) (n = 30) and 078 (toxinotype V) (n = 23) were provided by health-care facilities across the Republic of Ireland over 2 months in 2006 and 1 month in 2009. Ribotype 027 isolates were significantly more related to each other (9 different subtype profiles) when compared to ribotype 078 isolates (14 different profiles) (P = 0.001; cut-off >90 % similarity). Almost half of ribotype 078 isolates (45.5 %) showed no relationship to each other. The clonality of ribotype 027 isolates suggests effective adaptation to the human niche, whereas the considerable genetic diversity within ribotype 078 isolates suggests that they may have originated from a variety of sources. Subtyping correlated well with antimicrobial susceptibility, in particular clindamycin susceptibility for ribotype 027, but diverse antimicrobial-susceptibility profiles were seen in ribotype 078 isolates, even within a single health-care facility. Between 2006 and 2009, a change in the predominant subtype of ribotype 027 was seen, with the recent clone representing half of all ribotype 027 isolates studied. This strain exhibited 89 % similarity to a rep-PCR profile of the North American NAP-1 strain.

Nitrogen Control in VIM Melts

Science.gov (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.

Additive Manufacturing Processes: Selective Laser Melting, Electron Beam Melting and Binder Jetting-Selection Guidelines.

Science.gov (United States)

Gokuldoss, Prashanth Konda; Kolla, Sri; Eckert, Jürgen

2017-06-19

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.

Additive Manufacturing Processes: Selective Laser Melting, Electron Beam Melting and Binder Jetting—Selection Guidelines

Science.gov (United States)

Konda Gokuldoss, Prashanth; Kolla, Sri; Eckert, Jürgen

2017-01-01

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

Dynamics of upper mantle rocks decompression melting above hot spots under continental plates

Science.gov (United States)

Perepechko, Yury; Sorokin, Konstantin; Sharapov, Victor

2014-05-01

temperature (THS) > 1900oC asthenolens size ~700 km. When THS = of 2000oC the maximum melting degree of the primitive mantle is near 40%. An increase in the TB > 1900oC the maximum degree of melting could rich 100% with the same size of decompression melting zone (700 km). We examined decompression melting above the HS having LHS = 100 km - 780 km at a TB 1850- 2100oC with the thickness of lithosphere = 100 km.It is shown that asthenolens size (Lln) does not change substantially: Lln=700 km at LHS = of 100 km; Lln= 800 km at LHS = of 780 km. In presence of asymmetry of large HS the region of advection is developed above the HS maximum with the formation of asymmetrical cell. Influence of lithospheric plate thicknesses on appearance and evolution of asthenolens above the HS were investigated for the model stepped profile for the TB ≤ of 1750oS with Lhs = 100km and maximum of THS =2350oC. With an increase of TB the Lln difference beneath lithospheric steps is leveled with retention of a certain difference to melting degrees and time of the melting appearance a top of the HS. RFBR grant 12-05-00625.

Modeling the summertime evolution of sea-ice melt ponds

DEFF Research Database (Denmark)

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

2006-01-01

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...

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

Science.gov (United States)

Ogrean, Christy; Jackson, Ben; Covino, James

2010-01-01

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

Insight into the genomic diversity and relationship of Astragalus glycyphyllos symbionts by RAPD, ERIC-PCR, and AFLP fingerprinting.

Science.gov (United States)

Gnat, Sebastian; Małek, Wanda; Oleńska, Ewa; Trościańczyk, Aleksandra; Wdowiak-Wróbel, Sylwia; Kalita, Michał; Wójcik, Magdalena

2015-11-01

We assessed the genomic diversity and genomic relationship of 28 Astragalus glycyphyllos symbionts by three methodologies based on PCR reaction, i.e., RAPD, ERIC-PCR, and AFLP. The AFLP method with one PstI restriction enzyme and selective PstI-GC primer pair had a comparable discriminatory power as ERIC-PCR one and these fingerprinting techniques distinguished among the studied 28 A. glycyphyllos symbionts 18 and 17 genomotypes, respectively. RAPD method was less discriminatory in the genomotyping of rhizobia analyzed and it efficiently resolved nine genomotypes. The cluster analysis of RAPD, ERIC-PCR, and AFLP profiles resulted in a generally similar grouping of the test strains on generated dendrograms supporting a great potential of these DNA fingerprinting techniques for study of genomic polymorphism and evolutionary relationship of A. glycyphyllos nodulators. The RAPD, ERIC-PCR, and AFLP pattern similarity coefficients between A. glycyphyllos symbionts studied was in the ranges 8-100, 18-100, and 23-100%, respectively.

The Laser Damage Threshold for Materials and the Relation Between Solid-Melt and Melt-Vapor Interface Velocities

International Nuclear Information System (INIS)

Khalil, Osama Mostafa

2010-01-01

Numerous experiments have demonstrated and analytic theories have predicted that there is a threshold for pulsed laser ablation of a wide range of materials. Optical surface damage threshold is a very complex and important application of high-power lasers. Optical damage may also be considered to be the initial phase of laser ablation. In this work it was determined the time required and the threshold energy of a layer of thickness to heat up. We used the Finite Difference method to simulate the process of laser-target interaction in three cases. Namely, the case before melting begins using a continuous wave (c.w) laser source and a pulsed laser source, the case after the first change of state (from solid to melt), and the case after the second change of state (from melt to vapor). And also study the relation between the solid-melt and melt-vapor interface velocities to have a commonsense of the laser ablation process.

ICG: a wiki-driven knowledgebase of internal control genes for RT-qPCR normalization.

Science.gov (United States)

Sang, Jian; Wang, Zhennan; Li, Man; Cao, Jiabao; Niu, Guangyi; Xia, Lin; Zou, Dong; Wang, Fan; Xu, Xingjian; Han, Xiaojiao; Fan, Jinqi; Yang, Ye; Zuo, Wanzhu; Zhang, Yang; Zhao, Wenming; Bao, Yiming; Xiao, Jingfa; Hu, Songnian; Hao, Lili; Zhang, Zhang

2018-01-04

Real-time quantitative PCR (RT-qPCR) has become a widely used method for accurate expression profiling of targeted mRNA and ncRNA. Selection of appropriate internal control genes for RT-qPCR normalization is an elementary prerequisite for reliable expression measurement. Here, we present ICG (http://icg.big.ac.cn), a wiki-driven knowledgebase for community curation of experimentally validated internal control genes as well as their associated experimental conditions. Unlike extant related databases that focus on qPCR primers in model organisms (mainly human and mouse), ICG features harnessing collective intelligence in community integration of internal control genes for a variety of species. Specifically, it integrates a comprehensive collection of more than 750 internal control genes for 73 animals, 115 plants, 12 fungi and 9 bacteria, and incorporates detailed information on recommended application scenarios corresponding to specific experimental conditions, which, collectively, are of great help for researchers to adopt appropriate internal control genes for their own experiments. Taken together, ICG serves as a publicly editable and open-content encyclopaedia of internal control genes and accordingly bears broad utility for reliable RT-qPCR normalization and gene expression characterization in both model and non-model organisms. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Melt-processing method for radioactive solid wastes

International Nuclear Information System (INIS)

Kobayashi, Hiroaki

1998-01-01

Radioactive solid wastes are charged into a water-cooled type cold crucible induction melting furnace disposed in high frequency coils, and high frequency currents are supplied to high frequency coils which surround the melting furnace to melt the solid wastes by induction-heating. In this case, heat plasmas are jetted from above the solid wastes to the solid wastes to conduct initial heating to melt a portion of the solid wastes. Then, high frequency currents are supplied to the high frequency coils to conduct induction heating. According to this method, even when waste components of various kinds of materials are mixed, a portion of the solid wastes in the induction melting furnace can be melted by the initial heating by jetting heat plasmas irrespective of the kinds and the electroconductivity of the materials of the solid wastes. With such procedures, entire solid wastes in the furnace can be formed into a molten state uniformly and rapidly. (T.M.)

Development of a multiplex real time PCR to detect thermophilic lactic acid bacteria in natural whey starters.

Science.gov (United States)

Bottari, Benedetta; Agrimonti, Caterina; Gatti, Monica; Neviani, Erasmo; Marmiroli, Nelson

2013-01-01

A multiplex real time PCR (mRealT-PCR) useful to rapidly screen microbial composition of thermophilic starter cultures for hard cooked cheeses and to compare samples with potentially different technological properties was developed. Novel primers directed toward pheS gene were designed and optimized for multiple detection of Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus thermophilus and Lactobacillus fermentum. The assay was based on SYBR Green chemistry followed by melting curves analysis. The method was then evaluated for applications in the specific detection of the 4 lactic acid bacteria (LAB) in 29 different natural whey starters for Parmigiano Reggiano cheese production. The results obtained by mRealT-PCR were also compared with those obtained on the same samples by Fluorescence in Situ Hybridization (FISH) and Length-Heterogeneity PCR (LH-PCR). The mRealT-PCR developed in this study, was found to be effective for analyzing species present in the samples with an average sensitivity down to less than 600 copies of DNA and therefore sensitive enough to detect even minor LAB community members of thermophilic starter cultures. The assay was able to describe the microbial population of all the different natural whey starter samples analyzed, despite their natural variability. A higher number of whey starter samples with S. thermophilus and L. fermentum present in their microbial community were revealed, suggesting that these species could be more frequent in Parmigiano Reggiano natural whey starter samples than previously shown. The method was more effective than LH-PCR and FISH and, considering that these two techniques have to be used in combination to detect the less abundant species, the mRealT-PCR was also faster. Providing a single step sensitive detection of L. helveticus, L. delbrueckii, S. thermophilus and L. fermentum, the developed mRealT-PCR could be used for screening thermophilic starter cultures and to follow the presence of

Realization of Copper Melting Point for Thermocouple Calibrations

Directory of Open Access Journals (Sweden)

Y. A. ABDELAZIZ

2011-08-01

Full Text Available Although the temperature stability and uncertainty of the freezing plateau is better than that of the melting plateau in most of the thermometry fixed points, but realization of melting plateaus are easier than that of freezing plateaus for metal fixed points. It will be convenient if the melting points can be used instead of the freezing points in calibration of standard noble metal thermocouples because of easier realization and longer plateau duration of melting plateaus. In this work a comparison between the melting and freezing points of copper (Cu was carried out using standard noble metal thermocouples. Platinum - platinum 10 % rhodium (type S, platinum – 30 % rhodium / platinum 6 % rhodium (type B and platinum - palladium (Pt/Pd thermocouples are used in this study. Uncertainty budget analysis of the melting points and freezing points is presented. The experimental results show that it is possible to replace the freezing point with the melting point of copper cell in the calibration of standard noble metal thermocouples in secondary-level laboratories if the optimal methods of realization of melting points are used.

Partial enthalpies of Bi and Te in Bi-Te melts and of In and Te in In-Te melts

International Nuclear Information System (INIS)

Yassin, Abeer; Amzil, Abdelhamid; Castanet, Robert

2000-01-01

Full text.Calorimetric measurement are reported which allow the enthalpic behaviour of Bi-Te melts to be established. Further work is required, however, to supplement results obtained for In-Te melts. The partial enthalpies of bismuth and tellurium in the Bi-Te melts at 755K and those of indium and tellurium in the In-Te melts at 1010 and 987K were measured at high dilution by direct reaction calorimetry (drop method) with the help of a Tian-Calvet calorimeter. The limiting partial enthalpies of the components were deduced by extrapolation at infinite dilution: Δh f,∞ B i(755K)/KJ.mol -1 = -34.0 and Δh f,∞ Te(755K) /KJ·mol -1 = -24.1 in the Bi-Te melts Δh f,∞ In(1010K) /KJ·mol -1 = -75.9 and Δh f,∞ Te(1010K) /KJ·mol -1 = -47.8 in the In-Te melts Δh f,∞ In(987K) /KJ·mol -1 = -75.2 and Δh f,∞ Te(987K) /KJ·mol -1 = -48.0 in the In-Te melts

Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM.

Science.gov (United States)

Kristensen, Lasse S; Wojdacz, Tomasz K; Thestrup, Britta B; Wiuf, Carsten; Hager, Henrik; Hansen, Lise Lotte

2009-12-21

The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample. CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP. MS-HRM and SMART-MSP can be successfully used for single locus

Single-cell gene-expression profiling and its potential diagnostic applications

Czech Academy of Sciences Publication Activity Database

Stahlberg, A.; Kubista, Mikael; Aman, P.

2011-01-01

Roč. 11, č. 7 (2011), s. 735-740 ISSN 1473-7159 R&D Projects: GA ČR(CZ) GAP303/10/1338; GA ČR(CZ) GA301/09/1752 Institutional research plan: CEZ:AV0Z50520701 Keywords : gene-expression profiling * RT-qPCR * single-cell gene-expression profiling Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.859, year: 2011

ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq data

Directory of Open Access Journals (Sweden)

Perkins James R

2012-07-01

Full Text Available Abstract Background Measuring gene transcription using real-time reverse transcription polymerase chain reaction (RT-qPCR technology is a mainstay of molecular biology. Technologies now exist to measure the abundance of many transcripts in parallel. The selection of the optimal reference gene for the normalisation of this data is a recurring problem, and several algorithms have been developed in order to solve it. So far nothing in R exists to unite these methods, together with other functions to read in and normalise the data using the chosen reference gene(s. Results We have developed two R/Bioconductor packages, ReadqPCR and NormqPCR, intended for a user with some experience with high-throughput data analysis using R, who wishes to use R to analyse RT-qPCR data. We illustrate their potential use in a workflow analysing a generic RT-qPCR experiment, and apply this to a real dataset. Packages are available from http://www.bioconductor.org/packages/release/bioc/html/ReadqPCR.htmland http://www.bioconductor.org/packages/release/bioc/html/NormqPCR.html Conclusions These packages increase the repetoire of RT-qPCR analysis tools available to the R user and allow them to (amongst other things read their data into R, hold it in an ExpressionSet compatible R object, choose appropriate reference genes, normalise the data and look for differential expression between samples.

Rapid detection and typing of pathogenic nonpneumophila Legionella spp. isolates using a multiplex real-time PCR assay.

Science.gov (United States)

Benitez, Alvaro J; Winchell, Jonas M

2016-04-01

We developed a single tube multiplex real-time PCR assay that allows for the rapid detection and typing of 9 nonpneumophila Legionella spp. isolates that are clinically relevant. The multiplex assay is capable of simultaneously detecting and discriminating L. micdadei, L. bozemanii, L. dumoffii, L. longbeachae, L. feeleii, L. anisa, L. parisiensis, L. tucsonensis serogroup (sg) 1 and 3, and L. sainthelensis sg 1 and 2 isolates. Evaluation of the assay with nucleic acid from each of these species derived from both clinical and environmental isolates and typing strains demonstrated 100% sensitivity and 100% specificity when tested against 43 other Legionella spp. Typing of L. anisa, L. parisiensis, and L. tucsonensis sg 1 and 3 isolates was accomplished by developing a real-time PCR assay followed by high-resolution melt (HRM) analysis targeting the ssrA gene. Further typing of L. bozemanii, L. longbeachae, and L. feeleii isolates to the serogroup level was accomplished by developing a real-time PCR assay followed by HRM analysis targeting the mip gene. When used in conjunction with other currently available diagnostic tests, these assays may aid in rapidly identifying specific etiologies associated with Legionella outbreaks, clusters, sporadic cases, and potential environmental sources. Published by Elsevier Inc.

Ferric iron partitioning between pyroxene and melt during partial melting of the Earth's upper mantle

Science.gov (United States)

Rudra, A.; Hirschmann, M. M.

2017-12-01

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.

Plasma arc melting of zirconium

International Nuclear Information System (INIS)

Tubesing, P.K.; Korzekwa, D.R.; Dunn, P.S.

1997-01-01

Zirconium, like some other refractory metals, has an undesirable sensitivity to interstitials such as oxygen. Traditionally, zirconium is processed by electron beam melting to maintain minimum interstitial contamination. Electron beam melted zirconium, however, does not respond positively to mechanical processing due to its large grain size. The authors undertook a study to determine if plasma arc melting (PAM) technology could be utilized to maintain low interstitial concentrations and improve the response of zirconium to subsequent mechanical processing. The PAM process enabled them to control and maintain low interstitial levels of oxygen and carbon, produce a more favorable grain structure, and with supplementary off-gassing, improve the response to mechanical forming

Effects of surface shape on the geometry and surface topography of the melt pool in low-power density laser melting

KAUST Repository

Kim, Youngdeuk; Kim, Wooseung

2011-01-01

The quantitative correlations between workpiece volume and melt pool geometry, as well as the flow and thermal features of the melt pool are established. Thermocapillary convections in melt pool with a deformable free surface are investigated

External PCR, ASN's decision

International Nuclear Information System (INIS)

Anon.

2012-01-01

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.)

MELTS_Excel: A Microsoft Excel-based MELTS interface for research and teaching of magma properties and evolution

Science.gov (United States)

Gualda, Guilherme A. R.; Ghiorso, Mark S.

2015-01-01

thermodynamic modeling software MELTS is a powerful tool for investigating crystallization and melting in natural magmatic systems. Rhyolite-MELTS is a recalibration of MELTS that better captures the evolution of silicic magmas in the upper crust. The current interface of rhyolite-MELTS, while flexible, can be somewhat cumbersome for the novice. We present a new interface that uses web services consumed by a VBA backend in Microsoft Excel©. The interface is contained within a macro-enabled workbook, where the user can insert the model input information and initiate computations that are executed on a central server at OFM Research. Results of simple calculations are shown immediately within the interface itself. It is also possible to combine a sequence of calculations into an evolutionary path; the user can input starting and ending temperatures and pressures, temperature and pressure steps, and the prevailing oxidation conditions. The program shows partial updates at every step of the computations; at the conclusion of the calculations, a series of data sheets and diagrams are created in a separate workbook, which can be saved independently of the interface. Additionally, the user can specify a grid of temperatures and pressures and calculate a phase diagram showing the conditions at which different phases are present. The interface can be used to apply the rhyolite-MELTS geobarometer. We demonstrate applications of the interface using an example early-erupted Bishop Tuff composition. The interface is simple to use and flexible, but it requires an internet connection. The interface is distributed for free from http://melts.ofm-research.org.

Melting technique for vanadium containing steels

Energy Technology Data Exchange (ETDEWEB)

Grishanov, M P; Gutovskij, I B; Vakhrushev, A S

1980-04-28

To descrease cost price of high-quality vanadium steels a method of their melting in open-hearth furnaces with acid lining using slag-metal fraction of vanadium, which is loaded in the content of 2.1-4.7% of melting mass, is suggested. Introduction of slag-metal fraction of vanadium ensures the formation of slag with composition that guarantees the necessary content of vanadium in steel and does not require introduction of expensive vanadium-containing ferroalloys into the melt.

Tracking the Depleted Mantle Signature in Melt Inclusions and Residual Glass of Basaltic Martian Shergottites using Secondary Ionization Mass Spectrometry

Science.gov (United States)

Peters, Timothy J.; Simon, Justin I.; Jones, John H.; Usui, Tomohiro; Economos, Rita C.; Schmitt, Axel K.; McKeegan, Kevin D.

2013-01-01

Trace element abundances of depleted shergottite magmas recorded by olivine-hosted melt inclusions (MI) and interstitial mesostasis glass were measured using the Cameca ims-1270 ion microprobe. Two meteorites: Tissint, an olivine-­phyric basaltic shergottite which fell over Morocco July 18th 2001; and the Antarctic meteorite Yamato 980459 (Y98), an olivine-phyric basaltic shergottite with abundant glassy mesostasis have been studied. Chondrite-­normalized REE patterns for MI in Tissint and Y98 are characteristically LREE depleted and, within analytical uncertainty, parallel those of their respective whole rock composition; supporting each meteorite to represent a melt composition that has experienced closed-­system crystallization. REE profiles for mesostasis glass in Y98 lie about an order of magnitude higher than those from the MI; with REE profiles for Tissint MI falling in between. Y98 MI have the highest average Sm/Nd and Y/Ce ratios, reflecting their LREE depletion and further supporting Y98 as one of our best samples to probe the depleted shergotitte mantle. In general, Zr/Nb ratios overlap between Y98 and Tissint MI, Ce/Nb ratios overlap between Y98 MI and mesostasis glass, and Sm/Nd ratios overlap between Y98 mesostasis glass and Tissint MI. These features support similar sources for both, but with subtle geochemical differences that may reflect different melting conditions or fractionation paths during ascent from the mantle. Interestingly, the REE patterns for both Y98 bulk and MI analyses display a flattening of the LREE that suggests a crustal contribution to the Y98 parent melt. This observation has important implications for the origins of depleted and enriched shergottites.

PCR Assays for Identification of Coccidioides posadasii Based on the Nucleotide Sequence of the Antigen 2/Proline-Rich Antigen

Science.gov (United States)

Bialek, Ralf; Kern, Jan; Herrmann, Tanja; Tijerina, Rolando; Ceceñas, Luis; Reischl, Udo; González, Gloria M.

2004-01-01

A conventional nested PCR and a real-time LightCycler PCR assay for detection of Coccidioides posadasii DNA were designed and tested in 120 clinical strains. These had been isolated from 114 patients within 10 years in Monterrey, Nuevo Leon, Mexico, known to be endemic for coccidioidomycosis. The gene encoding the specific antigen 2/proline-rich antigen (Ag2/PRA) was used as a target. All strains were correctly identified, whereas DNA from related members of the family Onygenaceae remained negative. Melting curve analysis by LightCycler and sequencing of the 526-bp product of the first PCR demonstrated either 100% identity to the GenBank sequence of the Silveira strain, now known to be C. posadasii (accession number AF013256), or a single silent mutation at position 1228. Length determination of two microsatellite-containing loci (GAC and 621) identified all 120 isolates as C. posadasii. Specific DNA was amplified by conventional nested PCR from three microscopically spherule-positive paraffin-embedded tissue samples, whereas 20 human tissue samples positive for other dimorphic fungi remained negative. Additionally, the safety of each step of a modified commercially available DNA extraction procedure was evaluated by using 10 strains. At least three steps of the protocol were demonstrated to sufficiently kill arthroconidia. This safe procedure is applicable to cultures and to clinical specimens. PMID:14766853

Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR.

Directory of Open Access Journals (Sweden)

Yogita Maheshwari

Full Text Available Droplet digital polymerase chain reaction (ddPCR is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative PCR (qPCR for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.

Influence of gas-generation on melt/concrete interaction

International Nuclear Information System (INIS)

Powers, D.A.

1979-01-01

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

Rhenium corrosion in chloride melts

International Nuclear Information System (INIS)

Stepanov, A.D.; Shkol'nikov, S.N.; Vetyukov, M.M.

1989-01-01

The results investigating rhenium corrosion in chloride melts containing sodium, potassium and chromium ions by a gravimetry potentials in argon atmosphere in a sealing quarth cell are described. Rhenium corrosion is shown to be rather considerable in melts containing CrCl 2 . The value of corrosion rate depending on temperature is determined

UNCONSTRAINED MELTING AND SOLIDIFICATION INSIDE ...

African Journals Online (AJOL)

2015-09-01

Sep 1, 2015 ... There is a large number of experimental and numerical works on melting and solidification of PCM[6-10], and also its usage as thermal management in building [11-14], electronic devices [15-16] and solar energy. [17-20].Most investigated geometries in melting and freezing process are sphere (spherical.

Identification of genus Acinetobacter: Standardization of in-house PCR and its comparison with conventional phenotypic methods.

Science.gov (United States)

Kulkarni, Sughosh S; Madalgi, Radhika; Ajantha, Ganavalli S; Kulkarni, Raghavendra D

2017-01-01

Acinetobacter is grouped under nonfermenting Gram-negative bacilli. It is increasingly isolated from pathological samples. The ability of this genus to acquire drug resistance and spread in the hospital settings is posing a grave problem in healthcare. Specific treatment protocols are advocated for Acinetobacter infections. Hence, rapid identification and drug susceptibility profiling are critical in the management of these infections. To standardize an in-house polymerase chain reaction (PCR) for identification of genus Acinetobacter and to compare PCR with two protocols for its phenotypic identification. A total of 96 clinical isolates of Acinetobacter were included in the study. An in-house PCR for genus level identification of Acinetobacter was standardized. All the isolates were phenotypically identified by two protocols. The results of PCR and phenotypic identification protocols were compared. The in-house PCR standardized was highly sensitive and specific for the genus Acinetobacter . There was 100% agreement between the phenotypic and molecular identification of the genus. The preliminary identification tests routinely used in clinical laboratories were also in complete agreement with phenotypic and molecular identification. The in-house PCR for genus level identification is specific and sensitive. However, it may not be essential for routine identification as the preliminary phenotypic identification tests used in the clinical laboratory reliably identify the genus Acinetobacter .

Shape evolution of a melting nonspherical particle

Science.gov (United States)

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

2015-09-01

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.

Melt-quenched glasses of metal-organic frameworks

DEFF Research Database (Denmark)

Bennett, T.D.; Yue, Yuanzheng; Li, P.

2016-01-01

Crystalline solids dominate the field of metal−organic frameworks (MOFs), with access to the liquid and glass states of matter usually prohibited by relatively low temperatures of thermal decomposition. In this work, we give due consideration to framework chemistry and topology to expand...... of other MOFs. The glasses formed upon vitrification are chemically and structurally distinct from the three other existing categories of melt-quenched glasses (inorganic nonmetallic, organic, and metallic), and retain the basic metal−ligand connectivity of crystalline MOFs, which connects their mechanical...... the phenomenon of the melting of 3D MOFs, linking crystal chemistry to framework melting temperature and kinetic fragility of the glass-forming liquids. Here we show that melting temperatures can be lowered by altering the chemistry of the crystalline MOF state, which provides a route to facilitate the melting...

PENGARUH LAJU PENDINGINAN, SUHU, DAN LAMA KRISTALISASI PADA PROFIL TRIASILGLISEROL DAN SIFAT PELELEHAN PRODUK FRAKSIONASI MINYAK KELAPA

Directory of Open Access Journals (Sweden)

Mursalin

2013-04-01

Full Text Available Dry fractionation will produce a fraction of olein and stearin with different composition of high melting and low melting as well the distribution of triacylglycerol (TAG in the form of trisaturated (St3, disaturated (St2U and monosaturated (StU2. This research aimed to study the effect of critical cooling rate and crystallization temperature on the composition and profile of TAG changes as well the melting properties of the fractionated coconut oil products. Coconut oilwas heated at 70°C then cooledat differentcooling rate to reach various crystalization temperatures. The oil was then stirred at 15rpm and allowed to crystallize at different period of time, and finally fractionated by vacuum filtration using Whatman #40 paper. Fractionation temperatures were the same as crystalization temperatures. The results showed that the critical cooling rate was proportional to the solid-like to liquid-like ratio, the content of St3 and SFC profile of olein fraction but inversely proportional to the content of St2U and StU2 TAG. Interval crystallization temperature between 21.30 and 21.73 °C produced the S/L ratio, the content of St3 TAG and SFC profiles of olein fractions lower and the content of St2U and StU2 TAG higher than the temperature interval below or above it. Coconut oil fractionation more effective in higher crystallization temperature or lower critical cooling rate. In these cooling treatments, St3 TAG which has high melting pointwould be concentrated at stearin fraction, while St2U and StU2 TAG and MCTwould be at olein fraction. Therefore, it will increase melting properties of stearin fraction and decrease olein fraction.

Melt inclusion: methods, applications and problem: Silica-rich melts in quartz xenoliths from Vulcano islands and their bearing on processes of crustal melting and crust-magma interaction in the Aeolian Arc, Italy

NARCIS (Netherlands)

Frezzotti, M.L.; Zavon, V.; Peccerillo, A.; Nikogosian, I.

2002-01-01

Silica-rich melts in quartz xenoliths from Vulcano islands and their bearing on processes of crustal melting and crust-magma interaction in the Aeolian Arc, Italy Proceedings of workshop Melt inclusion: methods, applications and problem. Napoli, Italy, September 2002, p. 71-73

Study on superheat of TiAl melt during cold crucible levitation melting. TiAl no cold crucible levitation yokai ni okeru yoto kanetsudo no kenkyu

Energy Technology Data Exchange (ETDEWEB)

Miwa, K.; Kobayashi, K.; Ninomiya, M. (Government Industrial Research Institute, Nagoya, Nagoya (Japan))

1992-06-20

Investigations were given on effects of test sample weights and sample positions in cold crucibles on superheat of melts when the intermetallic compound TiAl is melted using cold crucible levitation melting process, one of noncontaminated melting processes. The cold crucibles used in the experiment are a water-cooled copper crucible with an inner diameter of 42 mm and a length of 140 mm, into which a column-like ingot sample with an outer diameter of 32 mm (Al containing Ti at 33.5% by mass) was put and melted using the levitation melting. Comparisons and discussions were given on the relationship between sample weights and melt temperatures, the relationship between positions of the inserted samples and melt temperatures, and the state of contamination at melting of casts obtained from the melts resulted from the levitation melting and high-frequency melting poured into respective ceramic dies. Elevating the superheat temperature of the melts requires optimizing the sample weights and positions. Melt temperatures were measured using a radiation thermometer and a thermocouple, and the respective measured values were compared. 7 refs., 4 figs., 1 tab.

Differentiation between Aspergillus flavus and Aspergillus parasiticus from Pure Culture and Aflatoxin-Contaminated Grapes Using PCR-RFLP Analysis of aflR-aflJ Intergenic Spacer

International Nuclear Information System (INIS)

El Khoury, A.; Atoui, A.; Lebrihi, A.; Rizk, T.; Lteif, R.; Kallassy, M.

2011-01-01

Aflatoxins (AFs) represent the most important single mycotoxin-related food safety problem in developed and developing countries as they have adverse effects on human and animal health. They are produced mainly by Aspergillus flavus and A. parasiticus. Both species have different aflatoxinogenic profile. In order to distinguish between A. flavus and A. parasiticus, gene-specific primers were designed to target the intergenic spacer (IGS) for the AF biosynthesis genes, aflJ and aflR. Polymerase chain reaction (PCR) products were subjected to restriction endonuclease analysis using BglII to look for restriction fragment length polymorphisms (RFLPs). Our result showed that both species displayed different PCR-based RFLP (PCR-RFLP) profile. PCR products from A. flavus cleaved into 3 fragments of 362, 210, and 102 bp. However, there is only one restriction site for this enzyme in the sequence of A. parasiticus that produced only 2 fragments of 363 and 311 bp. The method was successfully applied to contaminated grapes samples. This approach of differentiating these 2 species would be simpler, less costly, and quicker than conventional sequencing of PCR products and/or morphological identification. (author)

Molecular diagnostic PCR handbook

International Nuclear Information System (INIS)

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

2005-01-01

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

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

2010-01-01

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.

Simulation of melt spreading in consideration of phase transitions

Energy Technology Data Exchange (ETDEWEB)

Spengler, C. [Gesellschaft fuer Anlagen- und Reaktorsicherheit (GRS) mbH, Koeln (Germany)

2002-07-01

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.)

Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection.

Science.gov (United States)

Hernández, Marta; Rodríguez-Lázaro, David; Esteve, Teresa; Prat, Salomé; Pla, Maria

2003-12-15

Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.

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

Directory of Open Access Journals (Sweden)

Kuppan Gokulan

2017-04-01

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

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

Science.gov (United States)

Gokulan, Kuppan; Williams, Katherine; Khare, Sangeeta

2017-04-01

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

Magnetic susceptibility of semiconductor melts

International Nuclear Information System (INIS)

Kutvitskij, V.A.; Shurygin, P.M.

1975-01-01

The temperature dependences chi of various alloys confirm the existence of cluster formations in molten semiconductors, the stability of these formations in melts being considerably affected by the anion nature. The concentrational dependences of the magnetic susceptibility for all the investigated systems exhibit the diamagnetism maxima corresponding to the compound compositions. Heating the melt causes ''smearing'' the maxima, which is related with the cluster structure dissociation. The existence of the maxima concentrational dependence chi corresponding to BiTe and BiSe is found in the isotherms. The non-linear dependence of chi on the composition shows the absence of a single-valued relation between the phase diagram and the chi-diagram for melts

Vacancies and a generalised melting curve of metals

International Nuclear Information System (INIS)

Gorecki, T.

1979-01-01

The vacancy mechanism of the melting process is used as a starting point for deriving an expression for the pressure dependence of the melting temperature of metals. The results obtained for the initial slope of the melting curve are compared with experimental data for 45 metals and in most cases the agreement is very good. The nonlinearity of the melting curve and the appearance of a maximum on the melting curve at a pressure approximately equal to the bulk modules is also predicted, with qualitative agreement with experimental data. A relation between bonding energy, atomic volume, and bulk modulus of metals is established. On the basis of this relation and the proposed vacancy mechanism, a generalised equation for the pressure dependence of the melting temperature of metals is derived. (author)

Molecular, Serological And Microbiological Profiling Evidence Of ...

African Journals Online (AJOL)

All items that the boy had contact with including a laboratory coat, bunch of keys and shoes were swabbed. Finally samples of all the boy's food and drinks were taken. Microbiological, Serological and Polymerase Chain Reaction (PCR) Profiling Assays. l the samples were cultured on Sorbitol - MacConkey (SMAC) agar, ...

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

1998-01-01

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)

Pneumocystis PCR: It Is Time to Make PCR the Test of Choice.

Science.gov (United States)

Doyle, Laura; Vogel, Sherilynn; Procop, Gary W

2017-01-01

The testing strategy for Pneumocystis at the Cleveland Clinic changed from toluidine blue staining to polymerase chain reaction (PCR). We studied the differences in positivity rates for these assays and compared each with the detection of Pneumocystis in companion specimens by cytology and surgical pathology. We reviewed the results of all Pneumocystis test orders 1 year before and 1 year after the implementation of a Pneumocystis -specific PCR. We also reviewed the corresponding cytology and surgical pathology results, if performed. Finally, we reviewed the medical records of patients with rare Pneumocystis detected by PCR in an effort to differentiate colonization vs true disease. Toluidine blue staining and surgical pathology had similar sensitivities and negative predictive values, both of which were superior to cytology. There was a >4-fold increase in the annual detection of Pneumocystis by PCR compared with toluidine blue staining (toluidine blue staining: 11/1583 [0.69%] vs PCR: 44/1457 [3.0%]; chi-square P < .001). PCR detected 1 more case than surgical pathology and was far more sensitive than cytology. Chart review demonstrated that the vast majority of patients with rare Pneumocystis detected were immunosuppressed, had radiologic findings supportive of this infection, had no other pathogens detected, and were treated for pneumocystosis by the clinical team. PCR was the most sensitive method for the detection of Pneumocystis and should be considered the diagnostic test of choice. Correlation with clinical and radiologic findings affords discrimination of early true disease from the far rarer instances of colonization.

A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR

OpenAIRE

Sakalar, Erg?n; Erg?n, Seyma ?z?irak; Akar, Emine

2015-01-01

A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN? Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks...

Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates.

Science.gov (United States)

Smiljanic, M; Kaase, M; Ahmad-Nejad, P; Ghebremedhin, B

2017-07-10

Carbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE). 116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes. Among the carbapenem-resistant isolates the genes bla KPC , bla VIM , bla NDM or bla OXA were detected. Both RT-PCR assays detected all bla KPC , bla VIM and bla NDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials. The Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.

Comparison of allele-specific PCR, created restriction-site PCR, and PCR with primer-introduced restriction analysis methods used for screening complex vertebral malformation carriers in Holstein cattle

Science.gov (United States)

Altınel, Ahmet

2017-01-01

Complex vertebral malformation (CVM) is an inherited, autosomal recessive disorder of Holstein cattle. The aim of this study was to compare sensitivity, specificity, positive and negative predictive values, accuracy, and rapidity of allele-specific polymerase chain reaction (AS-PCR), created restriction-site PCR (CRS-PCR), and PCR with primer-introduced restriction analysis (PCR-PIRA), three methods used in identification of CVM carriers in a Holstein cattle population. In order to screen for the G>T mutation in the solute carrier family 35 member A3 (SLC35A3) gene, DNA sequencing as the gold standard method was used. The prevalence of carriers and the mutant allele frequency were 3.2% and 0.016, respectively, among Holstein cattle in the Thrace region of Turkey. Among the three methods, the fastest but least accurate was AS-PCR. Although the rapidity of CRS-PCR and PCR-PIRA were nearly equal, the accuracy of PCR-PIRA was higher than that of CRS-PCR. Therefore, among the three methods, PCR-PIRA appears to be the most efficacious for screening of mutant alleles when identifying CVM carriers in a Holstein cattle population. PMID:28927256

Analytical Performance of Four Polymerase Chain Reaction (PCR and Real Time PCR (qPCR Assays for the Detection of Six Leishmania Species DNA in Colombia

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Cielo M. León

2017-10-01

Full Text Available Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR, limit of detection (LoD and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia. Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

Science.gov (United States)

León, Cielo M.; Muñoz, Marina; Hernández, Carolina; Ayala, Martha S.; Flórez, Carolina; Teherán, Aníbal; Cubides, Juan R.; Ramírez, Juan D.

2017-01-01

Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America. PMID:29046670

Melting of gold microclusters

International Nuclear Information System (INIS)

Garzon, I.L.; Jellinek, J.

1991-01-01

The transition from solid-like to liquid-like behavior in Au n , n=6, 7, 13, clusters is studied using molecular dynamics simulations. A Gupta-type potential with all-neighbour interactions is employed to incorporate n-body effects. The melting-like transition is described in terms of short-time averages of the kinetic energy per particle, root-mean-square bond length fluctuations and mean square displacements. A comparison between melting temperatures of Au n and Ni n clusters is presented. (orig.)

Statistical modelling of transcript profiles of differentially regulated genes

Directory of Open Access Journals (Sweden)

Sergeant Martin J

2008-07-01

Full Text Available Abstract Background The vast quantities of gene expression profiling data produced in microarray studies, and the more precise quantitative PCR, are often not statistically analysed to their full potential. Previous studies have summarised gene expression profiles using simple descriptive statistics, basic analysis of variance (ANOVA and the clustering of genes based on simple models fitted to their expression profiles over time. We report the novel application of statistical non-linear regression modelling techniques to describe the shapes of expression profiles for the fungus Agaricus bisporus, quantified by PCR, and for E. coli and Rattus norvegicus, using microarray technology. The use of parametric non-linear regression models provides a more precise description of expression profiles, reducing the "noise" of the raw data to produce a clear "signal" given by the fitted curve, and describing each profile with a small number of biologically interpretable parameters. This approach then allows the direct comparison and clustering of the shapes of response patterns between genes and potentially enables a greater exploration and interpretation of the biological processes driving gene expression. Results Quantitative reverse transcriptase PCR-derived time-course data of genes were modelled. "Split-line" or "broken-stick" regression identified the initial time of gene up-regulation, enabling the classification of genes into those with primary and secondary responses. Five-day profiles were modelled using the biologically-oriented, critical exponential curve, y(t = A + (B + CtRt + ε. This non-linear regression approach allowed the expression patterns for different genes to be compared in terms of curve shape, time of maximal transcript level and the decline and asymptotic response levels. Three distinct regulatory patterns were identified for the five genes studied. Applying the regression modelling approach to microarray-derived time course data

A rapid two-step algorithm detects and identifies clinical macrolide and beta-lactam antibiotic resistance in clinical bacterial isolates.

Science.gov (United States)

Lu, Xuedong; Nie, Shuping; Xia, Chengjing; Huang, Lie; He, Ying; Wu, Runxiang; Zhang, Li

2014-07-01

Aiming to identify macrolide and beta-lactam resistance in clinical bacterial isolates rapidly and accurately, a two-step algorithm was developed based on detection of eight antibiotic resistance genes. Targeting at genes linked to bacterial macrolide (msrA, ermA, ermB, and ermC) and beta-lactam (blaTEM, blaSHV, blaCTX-M-1, blaCTX-M-9) antibiotic resistances, this method includes a multiplex real-time PCR, a melting temperature profile analysis as well as a liquid bead microarray assay. Liquid bead microarray assay is applied only when indistinguishable Tm profile is observed. The clinical validity of this method was assessed on clinical bacterial isolates. Among the total 580 isolates that were determined by our diagnostic method, 75% of them were identified by the multiplex real-time PCR with melting temperature analysis alone, while the remaining 25% required both multiplex real-time PCR with melting temperature analysis and liquid bead microarray assay for identification. Compared with the traditional phenotypic antibiotic susceptibility test, an overall agreement of 81.2% (kappa=0.614, 95% CI=0.550-0.679) was observed, with a sensitivity and specificity of 87.7% and 73% respectively. Besides, the average test turnaround time is 3.9h, which is much shorter in comparison with more than 24h for the traditional phenotypic tests. Having the advantages of the shorter operating time and comparable high sensitivity and specificity with the traditional phenotypic test, our two-step algorithm provides an efficient tool for rapid determination of macrolide and beta-lactam antibiotic resistances in clinical bacterial isolates. Copyright © 2014 Elsevier B.V. All rights reserved.

IDENTIFIKASI DAGING BABI MENGGUNAKAN METODE PCR-RFLP GEN Cytochrome b DAN PCR PRIMER SPESIFIK GEN AMELOGENIN (Pork Identification Using PCR-RFLP of Cytochrome b Gene and Species Specific PCR of Amelogenin Gene

Directory of Open Access Journals (Sweden)

Yuny Erwanto

2013-03-01

Full Text Available A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP and species specific PCR methods had been applied for identifying pork in mixture of meat. Pork sample in various levels (1, 3, 5 and 10% was prepared in mixture with beef, chicken and mutton. The primary CYTb1 and CYTb2 were designed in the mitochondrial cytochrome b b (cytochrome b gene and PCR successfully amplified fragments of 359 bp. To distinguish pig species existence, the amplified PCR products of mitochondrial DNA were cut by BseDI restriction enzyme. The result showed that pig mitochondrial DNA was cut into 131 and 228 bp fragments. A polymerase chain reaction (PCR method based on the nucleotide sequence variation in the amelogenin gene has been chosen for the specific identification of pork DNAs in mixture meat. The primers designed generated specific fragments of 353 and 312 bp length for pork. The specificity of the primary designed was tested on 4 animal species including pig, cattle, chicken and goat species. Analysis of experimental mixture meat demonstrated that 1% of raw pork tissues could be detected using PCR-RFLP with BseDI restriction enzyme but detection using species-specific PCR showed the cross reactivity to beef, chicken and mutton. The cytochrome b PCR-RFLP species identification assay yielded excellent results for identification of pig species. PCR-RFLP is a potentially reliable technique for detection of the existence of pork in animal food product for Halal authentication. Keywords: Pork identification, cytochrome b, amelogenin, polymerase chain reaction   ABSTRAK   Penelitian ini dilakukan untuk mengaplikasikan metode deteksi daging babi dalam campuan daging dengan sapi, kambing dan ayam melalui PCR-RFLP dan PCR dengan primer spesifik untuk babi. Level kontaminasi daging babi dibuat sebesar 1, 3, 5 dan 10% dari total daging dalam campuran. Metode PCR-RFLP menggunakan sepasang primer yaitu gen cytochrome b dari mitokondria yang

Improved capacitive melting curve measurements

International Nuclear Information System (INIS)

Sebedash, Alexander; Tuoriniemi, Juha; Pentti, Elias; Salmela, Anssi

2009-01-01

Sensitivity of the capacitive method for determining the melting pressure of helium can be enhanced by loading the empty side of the capacitor with helium at a pressure nearly equal to that desired to be measured and by using a relatively thin and flexible membrane in between. This way one can achieve a nanobar resolution at the level of 30 bar, which is two orders of magnitude better than that of the best gauges with vacuum reference. This extends the applicability of melting curve thermometry to lower temperatures and would allow detecting tiny anomalies in the melting pressure, which must be associated with any phenomena contributing to the entropy of the liquid or solid phases. We demonstrated this principle in measurements of the crystallization pressure of isotopic helium mixtures at millikelvin temperatures by using partly solid pure 4 He as the reference substance providing the best possible universal reference pressure. The achieved sensitivity was good enough for melting curve thermometry on mixtures down to 100 μK. Similar system can be used on pure isotopes by virtue of a blocked capillary giving a stable reference condition with liquid slightly below the melting pressure in the reference volume. This was tested with pure 4 He at temperatures 0.08-0.3 K. To avoid spurious heating effects, one must carefully choose and arrange any dielectric materials close to the active capacitor. We observed some 100 pW loading at moderate excitation voltages.

Detection of sdhB Gene Mutations in SDHI-Resistant Isolates of Botrytis cinerea Using High Resolution Melting (HRM) Analysis.

Science.gov (United States)

Samaras, Anastasios; Madesis, Panagiotis; Karaoglanidis, George S

2016-01-01

Botrytis cinerea , is a high risk pathogen for fungicide resistance development. Pathogen' resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM) analysis for the identification of P225H/F/L//T, N230I, and H272L/R/Y mutations. Based on the sequence of sdh B subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant's DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the four mutations located at codon 225, the N230I mutation, the three mutations located in codon 272, and the non-mutated isolates (isolates of wild-type sensitivity). Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdh B mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping-off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I, and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate, and rapid identification of several sdh B mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of anti-resistance strategies implemented in

Detection of sdhB gene mutations in SDHI-resistant isolates of Botrytis cinerea using high resolution melting (HRM analysis

Directory of Open Access Journals (Sweden)

Anastasios Samaras

2016-11-01

Full Text Available Botrytis cinerea, is a high-risk pathogen for fungicide resistance development. Pathogen` resistance to SDHIs is associated with several mutations in sdh gene. The diversity of mutations and their differential effect on cross-resistance patterns among SDHIs and the fitness of resistant strains necessitate the availability of a tool for their rapid identification. This study was initiated to develop and validate a high-resolution melting (HRM analysis for the identification of P225H/F/L//T, N230I and H272L/R/Y mutations. Based on the sequence of sdhB subunit of resistant and sensitive isolates, a universal primer pair was designed. The specificity of the HRM analysis primers was verified to ensure against the cross-reaction with other fungal species and its sensitivity was evaluated using concentrations of known amounts of mutant`s DNA. The melting curve analysis generated nine distinct curve profiles, enabling the discrimination of all the 4 mutations located at codon 225, the N230I mutation, the 3 mutations located in codon 272 and the non mutated isolates (isolates of wild type sensitivity. Similar results were obtained when DNA was extracted directly from artificially inoculated strawberry fruit. The method was validated by monitoring the presence of sdhB mutations in samples of naturally infected strawberry fruits and stone fruit rootstock seedling plants showing damping off symptoms. HRM analysis data were compared with a standard PIRA-PCR technique and an absolute agreement was observed suggesting that in both populations the H272R mutation was the predominant one, while H272Y, N230I and P225H were detected in lower frequencies. The results of the study suggest that HRM analysis can be a useful tool for sensate, accurate and rapid identification of several sdhB mutations in B. cinerea and it is expected to contribute in routine fungicide resistance monitoring or assessments of the effectiveness of antiresistance strategies implemented in

Differential melt scaling for oblique impacts on terrestrial planets

Science.gov (United States)

Abramov, Oleg; Wong, Stephanie M. Wong; Kring, David A. Kring

2012-01-01

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.