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Sample records for one-step pcr sequencing

  1. One-Step PCR Sequencing. Final Technical Progress Report for February 15, 1997 - November 30, 2001

    Energy Technology Data Exchange (ETDEWEB)

    Shaw, B. R.

    2004-04-16

    We investigated new chemistries and alternate approaches for direct gene sequencing and detection based on the properties of boron-substituted nucleotides as chain delimiters in lieu of conventional chain terminators. Chain terminators, such as the widely used Sanger dideoxynucleotide truncators, stop DNA synthesis during replication and hence are incompatible with further PCR amplification. Chain delimiters, on the other hand, are chemically-modified, ''stealth'' nucleotides that act like normal nucleotides in DNA synthesis and PCR amplification, but can be unmasked following chain extension and exponential amplification. Specifically, chain delimiters give rise to an alternative sequencing strategy based on selective degradation of DNA chains generated by PCR amplification with modified nucleotides. The method as originally devised employed template-directed enzymatic, random incorporation of small amounts of boron-modified nucleotides (e.g., 2'-deoxynucleoside 5'-alpha-[P-borano]- triphosphates) during PCR amplification. Rather than incorporation of dideoxy chain terminators, which are less efficiently incorporated in PCR-based amplification than natural deoxynucleotides, our method is based on selective incorporation and exonuclease degradation of DNA chains generated by efficient PCR amplification of chemically-modified ''stealth'' nucleotides. The stealth nucleotides have a boranophosphate group instead of a normal phosphate, yet behave like normal nucleotides during PCR-amplification. The unique feature of our method is that the position of the stealth nucleotide, and hence DNA sequencing fragments, are revealed at the desired, appropriate moment following PCR amplification. During the current grant period, a variety of new boron-modified nucleotides were synthesized, and new chemistries and enzymatic methods and combinations thereof were explored to improve the method and study the effects of borane modified

  2. A one-step, real-time PCR assay for rapid detection of rhinovirus.

    Science.gov (United States)

    Do, Duc H; Laus, Stella; Leber, Amy; Marcon, Mario J; Jordan, Jeanne A; Martin, Judith M; Wadowsky, Robert M

    2010-01-01

    One-step, real-time PCR assays for rhinovirus have been developed for a limited number of PCR amplification platforms and chemistries, and some exhibit cross-reactivity with genetically similar enteroviruses. We developed a one-step, real-time PCR assay for rhinovirus by using a sequence detection system (Applied Biosystems; Foster City, CA). The primers were designed to amplify a 120-base target in the noncoding region of picornavirus RNA, and a TaqMan (Applied Biosystems) degenerate probe was designed for the specific detection of rhinovirus amplicons. The PCR assay had no cross-reactivity with a panel of 76 nontarget nucleic acids, which included RNAs from 43 enterovirus strains. Excellent lower limits of detection relative to viral culture were observed for the PCR assay by using 38 of 40 rhinovirus reference strains representing different serotypes, which could reproducibly detect rhinovirus serotype 2 in viral transport medium containing 10 to 10,000 TCID(50) (50% tissue culture infectious dose endpoint) units/ml of the virus. However, for rhinovirus serotypes 59 and 69, the PCR assay was less sensitive than culture. Testing of 48 clinical specimens from children with cold-like illnesses for rhinovirus by the PCR and culture assays yielded detection rates of 16.7% and 6.3%, respectively. For a batch of 10 specimens, the entire assay was completed in 4.5 hours. This real-time PCR assay enables detection of many rhinovirus serotypes with the Applied Biosystems reagent-instrument platform.

  3. Detection of Tomato black ring virus by real-time one-step RT-PCR.

    Science.gov (United States)

    Harper, Scott J; Delmiglio, Catia; Ward, Lisa I; Clover, Gerard R G

    2011-01-01

    A TaqMan-based real-time one-step RT-PCR assay was developed for the rapid detection of Tomato black ring virus (TBRV), a significant plant pathogen which infects a wide range of economically important crops. Primers and a probe were designed against existing genomic sequences to amplify a 72 bp fragment from RNA-2. The assay amplified all isolates of TBRV tested, but no amplification was observed from the RNA of other nepovirus species or healthy host plants. The detection limit of the assay was estimated to be around nine copies of the TBRV target region in total RNA. A comparison with conventional RT-PCR and ELISA, indicated that ELISA, the current standard test method, lacked specificity and reacted to all nepovirus species tested, while conventional RT-PCR was approximately ten-fold less sensitive than the real-time RT-PCR assay. Finally, the real-time RT-PCR assay was tested using five different RT-PCR reagent kits and was found to be robust and reliable, with no significant differences in sensitivity being found. The development of this rapid assay should aid in quarantine and post-border surveys for regulatory agencies. Copyright © 2010 Elsevier B.V. All rights reserved.

  4. Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species.

    Science.gov (United States)

    Chae, Hansong; Han, Seung Jung; Kim, Su-Young; Ki, Chang-Seok; Huh, Hee Jae; Yong, Dongeun; Koh, Won-Jung; Shin, Sung Jae

    2017-09-01

    The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis ( M. tuberculosis ) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680 , and (iv) simultaneously detect five clinically important NTM ( M. avium , M. intracellulare , M. abscessus , M. massiliense , and M. kansasii ) by targeting IS 1311 , DT1, mass_3210 , and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 10 3 and 10 4 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis , M. tuberculosis Beijing genotype, and major NTM species. Copyright © 2017 American Society for Microbiology.

  5. Canine distemper virus detection by different methods of One-Step RT-qPCR

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    Claudia de Camargo Tozato

    2016-01-01

    Full Text Available ABSTRACT: Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A and a One-Step RT-qPCR combined with NESTED-qPCR (system B. Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100% urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specific method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology.

  6. One-step triplex PCR/RT-PCR to detect canine distemper virus, canine parvovirus, and canine kobuvirus.

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    Liu, Dafei; Liu, Fei; Guo, Dongchun; Hu, Xiaoliang; Li, Zhijie; Li, Zhigang; Ma, Jianzhang; Liu, Chunguo

    2018-01-23

    To rapidly distinguish Canine distemper virus (CDV), canine parvovirus (CPV), and canine kobuvirus (CaKoV) in practice, a one-step multiplex PCR/RT-PCR assay was developed, with detection limits of 10 2.1 TCID 50 for CDV, 10 1.9 TCID 50 for CPV and 10 3 copies for CaKoV. This method did not amplify nonspecific DNA or RNA from other canine viruses. Therefore, the assay provides a sensitive tool for the rapid clinical detection and epidemiological surveillance of CDV, CPV and CaKoV in dogs.

  7. Simple, specific molecular typing of dengue virus isolates using one-step RT-PCR and restriction fragment length polymorphism.

    Science.gov (United States)

    Ortiz, Alma; Capitan, Zeuz; Mendoza, Yaxelis; Cisneros, Julio; Moreno, Brechla; Zaldivar, Yamitzel; Garcia, Mariana; Smith, Rebecca E; Motta, Jorge; Pascale, Juan Miguel

    2012-10-01

    A one-step RT-PCR and one-enzyme RFLP was used to detect and distinguish among flaviviruses, including the four serotypes of dengue and the St. Louis Encephalitis, West Nile and Yellow Fever viruses in cultured virus samples or acute-phase human serum. Using a previously described RT-PCR, but novel RFLP procedure, results are obtained in 24 h with basic PCR and electrophoresis equipment. There is 95% agreement between RT-PCR/RFLP results and those achieved by indirect immunofluorescence assays, and 100% agreement between RT-PCR/RFLP results and gene sequencing. This method is more rapid than tests of cytopathic effect based on virus isolation in tissue culture, and simpler than real-time PCR. It does not require specialized equipment, radioisotopes or computer analysis and is a method that can be applied widely in the developing world. It allows for prompt determination of whether a flavivirus is the cause of illness in a febrile patient, rapid identification of dengue serotypes in circulation, and improved patient management in cases where prior dengue exposure make dengue hemorrhagic fever or dengue shock syndrome a risk. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. One-step multiplex PCR method for the determination of pecan and Brazil nut allergens in food products.

    Science.gov (United States)

    Hubalkova, Zora; Rencova, Eva

    2011-10-01

    A one-step polymerase chain reaction (PCR) method for the simultaneous detection of the major allergens of pecan and Brazil nuts was developed. Primer pairs for the amplification of partial sequences of genes encoding the allergens were designed and tested for their specificity on a range of food components. The targeted amplicon size was 173 bp of Ber e 1 gene of Brazil nuts and 72 bp of vicilin-like seed storage protein gene in pecan nuts. The primer pair detecting the noncoding region of the chloroplast DNA was used as the internal control of amplification. The intrinsic detection limit of the PCR method was 100 pg mL(-1) pecan or Brazil nuts DNA. The practical detection limit was 0.1% w/w (1 g kg(-1)). The method was applied for the investigation of 63 samples with the declaration of pecans, Brazil nuts, other different nut species or nuts generally. In 15 food samples pecans and Brazil nuts allergens were identified in the conformity with the food declaration. The presented multiplex PCR method is specific enough and can be used as a fast approach for the detection of major allergens of pecan or Brazil nuts in food. Copyright © 2011 Society of Chemical Industry.

  9. Development and evaluation of a real-time one step Reverse-Transcriptase PCR for quantitation of Chandipura Virus

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    Tandale Babasaheb V

    2008-12-01

    Full Text Available Abstract Background Chandipura virus (CHPV, a member of family Rhabdoviridae was attributed to an explosive outbreak of acute encephalitis in children in Andhra Pradesh, India in 2003 and a small outbreak among tribal children from Gujarat, Western India in 2004. The case-fatality rate ranged from 55–75%. Considering the rapid progression of the disease and high mortality, a highly sensitive method for quantifying CHPV RNA by real-time one step reverse transcriptase PCR (real-time one step RT-PCR using TaqMan technology was developed for rapid diagnosis. Methods Primers and probe for P gene were designed and used to standardize real-time one step RT-PCR assay for CHPV RNA quantitation. Standard RNA was prepared by PCR amplification, TA cloning and run off transcription. The optimized real-time one step RT-PCR assay was compared with the diagnostic nested RT-PCR and different virus isolation systems [in vivo (mice in ovo (eggs, in vitro (Vero E6, PS, RD and Sand fly cell line] for the detection of CHPV. Sensitivity and specificity of real-time one step RT-PCR assay was evaluated with diagnostic nested RT-PCR, which is considered as a gold standard. Results Real-time one step RT-PCR was optimized using in vitro transcribed (IVT RNA. Standard curve showed linear relationship for wide range of 102-1010 (r2 = 0.99 with maximum Coefficient of variation (CV = 5.91% for IVT RNA. The newly developed real-time RT-PCR was at par with nested RT-PCR in sensitivity and superior to cell lines and other living systems (embryonated eggs and infant mice used for the isolation of the virus. Detection limit of real-time one step RT-PCR and nested RT-PCR was found to be 1.2 × 100 PFU/ml. RD cells, sand fly cells, infant mice, and embryonated eggs showed almost equal sensitivity (1.2 × 102 PFU/ml. Vero and PS cell-lines (1.2 × 103 PFU/ml were least sensitive to CHPV infection. Specificity of the assay was found to be 100% when RNA from other viruses or healthy

  10. Enhanced capillary electrophoretic screening of Alzheimer based on direct apolipoprotein E genotyping and one-step multiplex PCR.

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    Woo, Nain; Kim, Su-Kang; Sun, Yucheng; Kang, Seong Ho

    2018-01-01

    Human apolipoprotein E (ApoE) is associated with high cholesterol levels, coronary artery disease, and especially Alzheimer's disease. In this study, we developed an ApoE genotyping and one-step multiplex polymerase chain reaction (PCR) based-capillary electrophoresis (CE) method for the enhanced diagnosis of Alzheimer's. The primer mixture of ApoE genes enabled the performance of direct one-step multiplex PCR from whole blood without DNA purification. The combination of direct ApoE genotyping and one-step multiplex PCR minimized the risk of DNA loss or contamination due to the process of DNA purification. All amplified PCR products with different DNA lengths (112-, 253-, 308-, 444-, and 514-bp DNA) of the ApoE genes were analyzed within 2min by an extended voltage programming (VP)-based CE under the optimal conditions. The extended VP-based CE method was at least 120-180 times faster than conventional slab gel electrophoresis methods In particular, all amplified DNA fragments were detected in less than 10 PCR cycles using a laser-induced fluorescence detector. The detection limits of the ApoE genes were 6.4-62.0pM, which were approximately 100-100,000 times more sensitive than previous Alzheimer's diagnosis methods In addition, the combined one-step multiplex PCR and extended VP-based CE method was also successfully applied to the analysis of ApoE genotypes in Alzheimer's patients and normal samples and confirmed the distribution probability of allele frequencies. This combination of direct one-step multiplex PCR and an extended VP-based CE method should increase the diagnostic reliability of Alzheimer's with high sensitivity and short analysis time even with direct use of whole blood. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. A novel enterovirus and parechovirus multiplex one-step real-time PCR-validation and clinical experience.

    Science.gov (United States)

    Nielsen, Alex Christian Yde; Böttiger, Blenda; Midgley, Sofie Elisabeth; Nielsen, Lars Peter

    2013-11-01

    As the number of new enteroviruses and human parechoviruses seems ever growing, the necessity for updated diagnostics is relevant. We have updated an enterovirus assay and combined it with a previously published assay for human parechovirus resulting in a multiplex one-step RT-PCR assay. The multiplex assay was validated by analysing the sensitivity and specificity of the assay compared to the respective monoplex assays, and a good concordance was found. Furthermore, the enterovirus assay was able to detect 42 reference strains from all 4 species, and an additional 9 genotypes during panel testing and routine usage. During 15 months of routine use, from October 2008 to December 2009, we received and analysed 2187 samples (stool samples, cerebrospinal fluids, blood samples, respiratory samples and autopsy samples) were tested, from 1546 patients and detected enteroviruses and parechoviruses in 171 (8%) and 66 (3%) of the samples, respectively. 180 of the positive samples could be genotyped by PCR and sequencing and the most common genotypes found were human parechovirus type 3, echovirus 9, enterovirus 71, Coxsackievirus A16, and echovirus 25. During 2009 in Denmark, both enterovirus and human parechovirus type 3 had a similar seasonal pattern with a peak during the summer and autumn. Human parechovirus type 3 was almost invariably found in children less than 4 months of age. In conclusion, a multiplex assay was developed allowing simultaneous detection of 2 viruses, which can cause similar clinical symptoms. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Integration of nanoparticle cell lysis and microchip PCR for one-step rapid detection of bacteria.

    Science.gov (United States)

    Wan, Weijie; Yeow, John T W

    2012-04-01

    This paper describes an integrated microchip system as an efficient and cost-effective solution involving Nanotechnology and Lab-on-a-Chip technology for the rapid detection of bacteria. The system is based on using surface-modified gold nanoparticles for efficient cell lysis followed by microchip PCR without having to remove the nanoparticles from the PCR solution. Poly(quaternary ammonium) modified gold nanoparticles are used to provide a novel and efficient cell lysis method without the need to go through time-consuming, expensive and complicated microfabrication processes as most of current cell lysis methods for Lab-on-a-Chip applications do. It also facilitates the integration of cell lysis and PCR by sharing the same reaction chamber as PCR uses. It is integrated with a prototype microchip PCR system consisting of a physical microchip PCR device and an automated temperature control mechanism. The research work explores solutions for the problem of PCR inhibition caused by gold nanoparticles as well as for the problem of non-specific PCR amplification in the integrated microchip system. It also explores the possibility of greatly reducing PCR cycling time to achieve the same result compared to the protocol for a regular PCR machine. The simplicity of the setup makes it easy to be integrated with other Lab-on-a-Chip functional modules to create customized solutions for target applications.

  13. Identification of Dobrava, Hantaan, Seoul, and Puumala viruses by one-step real-time RT-PCR.

    Science.gov (United States)

    Aitichou, Mohamed; Saleh, Sharron S; McElroy, Anita K; Schmaljohn, C; Ibrahim, M Sofi

    2005-03-01

    We developed four assays for specifically identifying Dobrava (DOB), Hantaan (HTN), Puumala (PUU), and Seoul (SEO) viruses. The assays are based on the real-time one-step reverse transcriptase polymerase chain reaction (RT-PCR) with the small segment used as the target sequence. The detection limits of DOB, HTN, PUU, and SEO assays were 25, 25, 25, and 12.5 plaque-forming units, respectively. The assays were evaluated in blinded experiments, each with 100 samples that contained Andes, Black Creek Canal, Crimean-Congo hemorrhagic fever, Rift Valley fever and Sin Nombre viruses in addition to DOB, HTN, PUU and SEO viruses. The sensitivity levels of the DOB, HTN, PUU, and SEO assays were 98%, 96%, 92% and 94%, respectively. The specificity of DOB, HTN and SEO assays was 100% and the specificity of the PUU assay was 98%. Because of the high levels of sensitivity, specificity, and reproducibility, we believe that these assays can be useful for diagnosing and differentiating these four Old-World hantaviruses.

  14. Simultaneous Detection of Four Foodborne Viruses in Food Samples Using a One-Step Multiplex Reverse Transcription PCR.

    Science.gov (United States)

    Lee, Shin-Young; Kim, Mi-Ju; Kim, Hyun-Joong; Jeong, KwangCheol Casey; Kim, Hae-Yeong

    2018-02-28

    A one-step multiplex reverse transcription PCR (RT-PCR) method comprising six primer sets (for the detection of norovirus GI and GII, hepatitis A virus, rotavirus, and astrovirus) was developed to simultaneously detect four kinds of pathogenic viruses. The size of the PCR products for norovirus GI and GII, hepatitis A virus (VP3/VP1 and P2A regions), rotavirus, and astrovirus were 330, 164, 244, 198, 629, and 449 bp, respectively. The RT-PCR with the six primer sets showed specificity for the pathogenic viruses. The detection limit of the developed multiplex RT-PCR, as evaluated using serially diluted viral RNAs, was comparable to that of one-step single RT-PCR. Moreover, this multiplex RT-PCR was evaluated using food samples such as water, oysters, lettuce, and vegetable product. These food samples were artificially spiked with the four kinds of viruses in diverse combinations, and the spiked viruses in all food samples were detected successfully.

  15. Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

    OpenAIRE

    Parida Manmohan; Shrivastava Ambuj; Santhosh SR; Dash Paban; Saxena Parag; Rao PV

    2008-01-01

    Abstract Background Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR) for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported. Results An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Jap...

  16. Development of the Multiple Gene Knockout System with One-Step PCR in Thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

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    Shoji Suzuki

    2017-01-01

    Full Text Available Multiple gene knockout systems developed in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius are powerful genetic tools. However, plasmid construction typically requires several steps. Alternatively, PCR tailing for high-throughput gene disruption was also developed in S. acidocaldarius, but repeated gene knockout based on PCR tailing has been limited due to lack of a genetic marker system. In this study, we demonstrated efficient homologous recombination frequency (2.8 × 104 ± 6.9 × 103 colonies/μg DNA by optimizing the transformation conditions. This optimized protocol allowed to develop reliable gene knockout via double crossover using short homologous arms and to establish the multiple gene knockout system with one-step PCR (MONSTER. In the MONSTER, a multiple gene knockout cassette was simply and rapidly constructed by one-step PCR without plasmid construction, and the PCR product can be immediately used for target gene deletion. As an example of the applications of this strategy, we successfully made a DNA photolyase- (phr- and arginine decarboxylase- (argD- deficient strain of S. acidocaldarius. In addition, an agmatine selection system consisting of an agmatine-auxotrophic strain and argD marker was also established. The MONSTER provides an alternative strategy that enables the very simple construction of multiple gene knockout cassettes for genetic studies in S. acidocaldarius.

  17. Development and evaluation of one step single tube multiplex RT-PCR for rapid detection and typing of dengue viruses

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    Parida Manmohan

    2008-01-01

    Full Text Available Abstract Background Dengue is emerging as a major public health concern in many parts of the world. The development of a one-step, single tube, rapid, and multiplex reverse transcription polymerase chain reaction (M-RT-PCR for simultaneous detection and typing of dengue virus using serotype specific primers during acute phase of illness is reported. Results An optimal assay condition with zero background was established having no cross-reaction with closely related members of flavivirus (Japanese encephalitis, West Nile, Yellow fever and alphavirus (Chikungunya. The feasibility of M-RT-PCR assay for clinical diagnosis was validated with 620 acute phase dengue patient sera samples of recent epidemics in India. The comparative evaluation vis a vis conventional virus isolation revealed higher sensitivity. None of the forty healthy serum samples screened in the present study revealed any amplification, thereby establishing specificity of the reported assay for dengue virus only. Conclusion These findings clearly suggested that M-RT-PCR assay reported in the present study is the rapid and cost-effective method for simultaneous detection as well as typing of the dengue virus in acute phase patient serum samples. Thus, the M-RT-PCR assay developed in this study will serve as a very useful tool for rapid diagnosis and typing of dengue infections in endemic areas.

  18. Novel One-Step Multiplex PCR-Based Method for HLA Typing and Preimplantational Genetic Diagnosis of -Thalassemia

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    Raquel M. Fernández

    2013-01-01

    Full Text Available Preimplantation genetic diagnosis (PGD of single gene disorders, combined with HLA matching (PGD-HLA, has emerged as a tool for couples at risk of transmitting a genetic disease to select unaffected embryos of an HLA tissue type compatible with that of an existing affected child. Here, we present a novel one-step multiplex PCR to genotype a spectrum of STRs to simultaneously perform HLA typing and PGD for -thalassemia. This method is being routinely used for PGD-HLA cycles in our department, with a genotyping success rate of 100%. As an example, we present the first successful PGD-HLA typing in Spain, which resulted in the birth of a boy and subsequent successful HSC transplantation to his affected brother, who is doing well 4 years following transplantation. The advantage of our method is that it involves only a round of single PCR for multiple markers amplification (up to 10 markers within the HLA and 6 markers at the -globin loci. This strategy has allowed us to considerably reduce the optimization of the PCR method for each specific PGD-HLA family as well as the time to obtain molecular results in each cycle.

  19. A one-step multiplex RT-PCR assay for simultaneous detection of four viruses that infect peach.

    Science.gov (United States)

    Yu, Y; Zhao, Z; Jiang, D; Wu, Z; Li, S

    2013-10-01

    A multiplex reverse transcription polymerase chain reaction (mRT-PCR) assay was developed to enable the simultaneous detection and differentiation of four viruses that infect peach, namely Apple chlorotic leaf spot virus (ACLSV), Cherry green ring mottle virus (CGRMV), Prunus necrotic ringspot virus (PNRSV) and Apricot pseudo-chlorotic leaf spot virus (APCLSV). In this study, four pairs of primers, one specific for each virus, were designed; the corresponding PCR products were 632, 439, 346 and 282 bp in length for ACLSV, CGRMV, PNRSV and APCLSV, respectively, and the fragments could be distinguished clearly by agarose gel electrophoresis. The sensitivity and specificity of the method were tested using individual RT-PCR and enzyme-linked immunosorbent assay (ELISA), and the identity of the RT-PCR amplification products was also confirmed by DNA sequencing. The results of RT-PCR and ELISA, along with batch detection using samples collected from peach orchards, revealed that this rapid and simple technique is an effective way to identify the four viruses simultaneously. The mRT-PCR assay described in this study was developed for the simultaneous detection of four peach viruses from infected peach samples is reliable and sensitive. In contrast to conventional uniplex RT-PCR, mRT-PCR is more efficient, reducing costs, time and handling when testing large numbers of samples. This rapid and simple method is useful for large-scale surveys of viruses that infect peach. © 2013 The Society for Applied Microbiology.

  20. A novel enterovirus and parechovirus multiplex one-step real-time PCR-validation and clinical experience

    DEFF Research Database (Denmark)

    Nielsen, A. C. Y.; Bottiger, B.; Midgley, S. E.

    2013-01-01

    As the number of new enteroviruses and human parechoviruses seems ever growing, the necessity for updated diagnostics is relevant. We have updated an enterovirus assay and combined it with a previously published assay for human parechovirus resulting in a multiplex one-step RT-PCR assay....... The multiplex assay was validated by analysing the sensitivity and specificity of the assay compared to the respective monoplex assays, and a good concordance was found. Furthermore, the enterovirus assay was able to detect 42 reference strains from all 4 species, and an additional 9 genotypes during panel...... testing and routine usage. During 15 months of routine use, from October 2008 to December 2009, we received and analysed 2187 samples (stool samples, cerebrospinal fluids, blood samples, respiratory samples and autopsy samples) were tested, from 1546 patients and detected enteroviruses and parechoviruses...

  1. Development and evaluation of novel one-step TaqMan realtime RT-PCR assays for the detection and direct genotyping of genogroup I and II noroviruses

    DEFF Research Database (Denmark)

    Schultz, Anna Charlotte; Vega, Everado; Dalsgaard, Anders

    2011-01-01

    BackgroundCurrent detection and genotyping methods of genogroup (G) I and II noroviruses (NoVs) consist of a 2-step approach including detection of viral RNA by TaqMan realtime RT-PCR (RT-qPCR) followed by conventional RT-PCR and sequencing of partial regions of ORF1 or ORF2. ObjectiveTo develop ......Man RT-qPCR assays for the sensitive detection and direct genotyping of GI and GII NoVs from clinical and environmental matrices...... novel long-template one-step TaqMan assays (L-RT-qPCR) for the rapid detection and direct genotyping of GI and GII NoVs and to evaluate the sensitivity and specificity of the assays. Study designGI and GII-specific broadly reactive L-RT-qPCR assays were developed by combining existing NoV primers...... and probes targeting the open reading frame (ORF)1–ORF2 junction as well as region C at the 5′–ORF2. The assays were validated using GI and GII RNA transcripts and a coded panel of 75 stool samples containing NoV strains representing 9 GI genotypes and 12 GII genotypes, as well as sapoviruses, astroviruses...

  2. Simultaneous detection of three pome fruit tree viruses by one-step multiplex quantitative RT-PCR.

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    Malandraki, Ioanna; Beris, Despoina; Isaioglou, Ioannis; Olmos, Antonio; Varveri, Christina; Vassilakos, Nikon

    2017-01-01

    A one-step multiplex real-time reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan probes was developed for the simultaneous detection of Apple mosaic virus (ApMV), Apple stem pitting virus (ASPV) and Apple stem grooving virus (ASGV) in total RNA of pome trees extracted with a CTAB method. The sensitivity of the method was established using in vitro synthesized viral transcripts serially diluted in RNA from healthy, virus-tested (negative) pome trees. The three viruses were simultaneously detected up to a 10-4 dilution of total RNA from a naturally triple-infected apple tree prepared in total RNA of healthy apple tissue. The newly developed RT-qPCR assay was at least one hundred times more sensitive than conventional single RT-PCRs. The assay was validated with 36 field samples for which nine triple and 11 double infections were detected. All viruses were detected simultaneously in composite samples at least up to the ratio of 1:150 triple-infected to healthy pear tissue, suggesting the assay has the capacity to examine rapidly a large number of samples in pome tree certification programs and surveys for virus presence.

  3. Detection of SYT-SSX mutant transcripts in formalin-fixed paraffin-embedded sarcoma tissues using one-step reverse transcriptase real-time PCR.

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    Norlelawati, A T; Mohd Danial, G; Nora, H; Nadia, O; Zatur Rawihah, K; Nor Zamzila, A; Naznin, M

    2016-04-01

    Synovial sarcoma (SS) is a rare cancer and accounts for 5-10% of adult soft tissue sarcomas. Making an accurate diagnosis is difficult due to the overlapping histological features of SS with other types of sarcomas and the non-specific immunohistochemistry profile findings. Molecular testing is thus considered necessary to confirm the diagnosis since more than 90% of SS cases carry the transcript of t(X;18)(p11.2;q11.2). The purpose of this study is to diagnose SS at molecular level by testing for t(X;18) fusion-transcript expression through One-step reverse transcriptase real-time Polymerase Chain Reaction (PCR). Formalin-fixed paraffin-embedded tissue blocks of 23 cases of soft tissue sarcomas, which included 5 and 8 cases reported as SS as the primary diagnosis and differential diagnosis respectively, were retrieved from the Department of Pathology, Tengku Ampuan Afzan Hospital, Kuantan, Pahang. RNA was purified from the tissue block sections and then subjected to One-step reverse transcriptase real-time PCR using sequence specific hydrolysis probes for simultaneous detection of either SYT-SSX1 or SYT-SSX2 fusion transcript. Of the 23 cases, 4 cases were found to be positive for SYT-SSX fusion transcript in which 2 were diagnosed as SS whereas in the 2 other cases, SS was the differential diagnosis. Three cases were excluded due to failure of both amplification assays SYT-SSX and control β-2-microglobulin. The remaining 16 cases were negative for the fusion transcript. This study has shown that the application of One-Step reverse transcriptase real time PCR for the detection SYT-SSX transcript is feasible as an aid in confirming the diagnosis of synovial sarcoma.

  4. Development of a one-step duplex RT-qPCR for the quantification of phocine distemper virus.

    Science.gov (United States)

    Bogomolni, Andrea L; Frasca, Salvatore; Matassa, Keith A; Nielsen, Ole; Rogers, Kara; De Guise, Sylvain

    2015-04-01

    Worldwide, stranded marine mammals and the network personnel who respond to marine mammal mortality have provided much of the information regarding marine morbillivirus infections. An assay to determine the amount of virus present in tissue samples would be useful to assist in routine surveying of animal health and for monitoring large-scale die-off events. False negatives from poor-quality samples prevent determination of the true extent of infection, while only small amounts of tissue samples or archived RNA may be available at the time of collection for future retrospective analysis. We developed a one-step duplex real-time reverse transcriptase-quantitative-PCR assay (RT-qPCR) based on Taqman probe technology to quantify phocine distemper virus (PDV) isolated from an outbreak in harbor (Phoca vitulina concolor) and gray seals (Halichoerus grypus) along the northeast US coast in 2006. The glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene was selected to assess RNA quality. This duplex assay is specific for PDV and sensitive through a range of 10(0) to 10(9) copies ds-plasmid DNA. For the GAPDH target, the reaction in duplex amplified 10(0) to 10(9) copies of ds-plasmid DNA and was detectable in multiple seal species. This assay reduced the likelihood of false negative results due to degradation of tissues and well-to-well variability while providing sensitive and specific detection of PDV, which would be applicable in molecular epidemiologic studies and pathogen detection in field and laboratory investigations involving a variety of seal species.

  5. One-step multiplex quantitative RT-PCR for the simultaneous detection of viroids and phytoplasmas of pome fruit trees.

    Science.gov (United States)

    Malandraki, Ioanna; Varveri, Christina; Olmos, Antonio; Vassilakos, Nikon

    2015-03-01

    A one-step multiplex real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) based on TaqMan chemistry was developed for the simultaneous detection of Pear blister canker viroid and Apple scar skin viroid along with universal detection of phytoplasmas, in pome trees. Total nucleic acids (TNAs) extraction was performed according to a modified CTAB protocol. Primers and TaqMan MGB probes for specific detection of the two viroids were designed in this study, whereas for phytoplasma detection published universal primers and probe were used, with the difference that the later was modified to carry a MGB quencher. The pathogens were detected simultaneously in 10-fold serial dilutions of TNAs from infected plant material into TNAs of healthy plant up to dilutions 10(-5) for viroids and 10(-4) for phytoplasmas. The multiplex real-time assay was at least 10 times more sensitive than conventional protocols for viroid and phytoplasma detection. Simultaneous detection of the three targets was achieved in composite samples at least up to a ratio of 1:100 triple-infected to healthy tissue, demonstrating that the developed assay has the potential to be used for rapid and massive screening of viroids and phytoplasmas of pome fruit trees in the frame of certification schemes and surveys. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. A sensitive one-step real-time PCR for detection of avian influenza viruses using a MGB probe and an internal positive control

    Directory of Open Access Journals (Sweden)

    Delogu Mauro

    2006-05-01

    Full Text Available Abstract Background Avian influenza viruses (AIVs are endemic in wild birds and their introduction and conversion to highly pathogenic avian influenza virus in domestic poultry is a cause of serious economic losses as well as a risk for potential transmission to humans. The ability to rapidly recognise AIVs in biological specimens is critical for limiting further spread of the disease in poultry. The advent of molecular methods such as real time polymerase chain reaction has allowed improvement of detection methods currently used in laboratories, although not all of these methods include an Internal Positive Control (IPC to monitor for false negative results. Therefore we developed a one-step reverse transcription real time PCR (RRT-PCR with a Minor Groove Binder (MGB probe for the detection of different subtypes of AIVs. This technique also includes an IPC. Methods RRT-PCR was developed using an improved TaqMan technology with a MGB probe to detect AI from reference viruses. Primers and probe were designed based on the matrix gene sequences from most animal and human A influenza virus subtypes. The specificity of RRT-PCR was assessed by detecting influenza A virus isolates belonging to subtypes from H1–H13 isolated in avian, human, swine and equine hosts. The analytical sensitivity of the RRT-PCR assay was determined using serial dilutions of in vitro transcribed matrix gene RNA. The use of a rodent RNA as an IPC in order not to reduce the efficiency of the assay was adopted. Results The RRT-PCR assay is capable to detect all tested influenza A viruses. The detection limit of the assay was shown to be between 5 and 50 RNA copies per reaction and the standard curve demonstrated a linear range from 5 to 5 × 108 copies as well as excellent reproducibility. The analytical sensitivity of the assay is 10–100 times higher than conventional RT-PCR. Conclusion The high sensitivity, rapidity, reproducibility and specificity of the AIV RRT-PCR with

  7. High sensitive RNA detection by one-step RT-PCR using the genetically engineered variant of DNA polymerase with reverse transcriptase activity from hyperthermophilies.

    Science.gov (United States)

    Okano, Hiroyuki; Baba, Misato; Kawato, Katsuhiro; Hidese, Ryota; Yanagihara, Itaru; Kojima, Kenji; Takita, Teisuke; Fujiwara, Shinsuke; Yasukawa, Kiyoshi

    2018-03-01

    One-step RT-PCR has not been widely used even though some thermostable DNA polymerases with reverse transcriptase (RT) activity were developed from bacterial and archaeal polymerases, which is owing to low cDNA synthesis activity from RNA. In the present study, we developed highly-sensitive one-step RT-PCR using the single variant of family A DNA polymerase with RT activity, K4pol L329A (L329A), from the hyperthermophilic bacterium Thermotoga petrophila K4 or the 16-tuple variant of family B DNA polymerase with RT activity, RTX, from the hyperthermophilic archaeon Thermococcus kodakarensis. Optimization of reaction condition revealed that the activities for cDNA synthesis and PCR of K4pol L329A and RTX were highly affected by the concentrations of MgCl 2 and Mn(OCOCH 3 ) 2 as well as those of K4pol L329A or RTX. Under the optimized condition, 300 copies/μl of target RNA in 10 μl reaction volumes were successfully detected by the one-step RT-PCR with K4pol L329A or RTX, which was almost equally sensitive enough compared with the current RT-PCR condition using retroviral RT and thermostable DNA polymerase. Considering that K4pol L329A and RTX are stable even at 90-100°C, our results suggest that the one-step RT-PCR with K4pol L329A or RTX is more advantageous than the current one. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  8. Efficient One-Step Fusion PCR Based on Dual-Asymmetric Primers and Two-Step Annealing

    DEFF Research Database (Denmark)

    Liu, Yilan; Chen, Jinjin; Thygesen, Anders

    2018-01-01

    Gene splicing by fusion PCR is a versatile and widely used methodology, especially in synthetic biology. We here describe a rapid method for splicing two fragments by one-round fusion PCR with a dual-asymmetric primers and two-step annealing (ODT) method. During the process, the asymmetric...... intermediate fragments were generated in the early stage. Thereafter, they were hybridized in the subsequent cycles to serve as template for the target full-length product. The process parameters such as primer ratio, elongation temperature and cycle numbers were optimized. In addition, the fusion products...

  9. Detection of live Salmonella enterica in fresh-cut vegetables by a TaqMan-based one-step reverse transcription real-time PCR.

    Science.gov (United States)

    Miao, Y J; Xiong, G T; Bai, M Y; Ge, Y; Wu, Z F

    2018-05-01

    Fresh-cut produce is at greater risk of Salmonella contamination. Detection and early warning systems play an important role in reducing the dissemination of contaminated products. One-step Reverse Transcription Polymerase Chain Reaction (RT-qPCR) targeting Salmonella tmRNA with or without a 6-h enrichment was evaluated for the detection of Salmonella in fresh-cut vegetables after 6-h storage. LOD of one-step RT-qPCR was 1·0 CFU per ml (about 100 copies tmRNA per ml) by assessed 10-fold serially diluted RNA from 10 6 CFU per ml bacteria culture. Then, one-step RT-qPCR assay was applied to detect viable Salmonella cells in 14 fresh-cut vegetables after 6-h storage. Without enrichment, this assay could detect 10 CFU per g for fresh-cut lettuce, cilantro, spinach, cabbage, Chinese cabbage and bell pepper, and 10 2 CFU per g for other vegetables. With a 6-h enrichment, this assay could detect 10 CFU per g for all fresh-cut vegetables used in this study. Moreover, this assay was able to discriminate viable cells from dead cells. This rapid detection assay may provide potential processing control and early warning method in fresh-cut vegetable processing to strengthen food safety assurance. Significance and Impact of the Study: Fresh-cut produce is at greater risk of Salmonella contamination. Rapid detection methods play an important role in reducing the dissemination of contaminated products. One-step RT-qPCR assay used in this study could detect 10 CFU per g Salmonella for 14 fresh-cut vegetables with a 6-h short enrichment. Moreover, this assay was able to discriminate viable cells from dead cells. This rapid detection assay may provide potential processing control and early warning method in fresh-cut vegetable processing to strengthen food safety assurance. © 2018 The Society for Applied Microbiology.

  10. Rapid detection of Enterovirus and Coxsackievirus A10 by a TaqMan based duplex one-step real time RT-PCR assay.

    Science.gov (United States)

    Chen, Jingfang; Zhang, Rusheng; Ou, Xinhua; Yao, Dong; Huang, Zheng; Li, Linzhi; Sun, Biancheng

    2017-06-01

    A TaqMan based duplex one-step real time RT-PCR (rRT-PCR) assay was developed for the rapid detection of Coxsackievirus A10 (CV-A10) and other enterovirus (EVs) in clinical samples. The assay was fully evaluated and found to be specific and sensitive. When applied in 115 clinical samples, a 100% diagnostic sensitivity in CV-A10 detection and 97.4% diagnostic sensitivity in other EVs were found. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. One-step cross-genogroup multiplex RT-qPCR with an internal control system for the detection of infectious pancreatic necrosis virus (IPNV).

    Science.gov (United States)

    Hoferer, Marc; Braun, Anne; Skrypski, Julia; Bock, Sabine; Thalheim, Sabine; Sting, Reinhard

    2017-09-01

    Infectious pancreatic necrosis virus (IPNV) causes great losses in fish hatcheries world-wide. The detection of IPNV can be challenging in certain circumstances, particularly due to low viral load and the genetic variability of this RNA virus. For the first time, this project created a quantitative triplex real-time reverse transcription PCR (RT-qPCR), including an endogenous control system, for specific, sensitive and rapid detection of IPNV in routine diagnostics. Multiple sequence alignment of 46 nucleotide sequences of the segment A genome obtained from the NCBI database allowed the design of two RT-qPCR systems covering the IPNV genogroup 1 and genogroups 2-5, respectively. The completed triplex RT-qPCR including a salmonid-specific endogenous control showed high specificity and an analytical sensitivity of 20-40 oligonucleotide copies. Testing of dilution series of virus-loaded cell culture suspensions proved equality of the triplex RT-qPCR with virus detection in cell culture and a higher sensitivity than conventional RT-PCR in field samples. In comparative studies of a total of 77 field samples tested, 51 showed identical positive and 19 identical negative results in cell culture and the triplex RT-qPCR. However, seven other samples yielded positive results in the triplex RT-qPCR, but negative results in cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. One-step simultaneous detection of Ureaplasma parvum and genotypes SV1, SV3 and SV6 from clinical samples using PlexPCR technology.

    Science.gov (United States)

    Payne, M S; Furfaro, L L; Tucker, R; Tan, L Y; Mokany, E

    2017-08-01

    Ureaplasma spp. are associated with preterm birth. In recent times, it has become apparent that Ureaplasma parvum, but not Ureaplasma urealyticum, is of most relevance. We recently demonstrated this in Australian pregnant women and using high-resolution melt (HRM) PCR, further showed that U. parvum genotype SV6 was of particular significance. However, our assay was unable to identify multiple genotypes in the same sample, required a separate species-level qPCR for low titre samples and was not ideal for diagnostic laboratories due to the nature of HRM PCR result interpretation. Consequently, our current study developed a novel, one-step PlexPCR assay capable of detecting U. parvum and genotypes SV1, SV3 and SV6 in a single reaction directly from clinical samples. We then validated this using vaginal swab DNA from our Australian cohort of pregnant women. The PlexPCR was highly sensitive, detecting all targets to between 0.4 × 10 -5  ng DNA (SV3) and 0.4 × 10 -6  ng DNA (U. parvum, SV1 and SV6). Compared to our HRM PCR, the PlexPCR defined genotype distribution in all seven cases previously reported as 'mixed', and detected another eight cases where multiple genotypes (two) were present in samples previously reported as single genotypes using HRM PCR. Ureaplasma spp. have been associated with prematurity for decades, however, only a minority of studies have examined this beyond the genus level. In those that have, Ureaplasma parvum has been strongly associated with preterm birth. We recently demonstrated this in Australian women and further showed that U. parvum genotype SV6 was of particular significance. Our PlexPCR assay allows rapid detection and concurrent genotyping of U. parvum in clinical samples and may be of particular interest to obstetricians, particularly those caring for women at a high risk of preterm birth, and any other disease phenotypes where U. parvum is of interest. © 2017 The Authors. Letters in Applied Microbiology published by John

  13. Development of a one-step RT-PCR assay for detection of pancoronaviruses (α-, β-, γ-, and δ-coronaviruses) using newly designed degenerate primers for porcine and avian `fecal samples.

    Science.gov (United States)

    Hu, Hui; Jung, Kwonil; Wang, Qiuhong; Saif, Linda J; Vlasova, Anastasia N

    2018-06-01

    Coronaviruses (CoVs) are critical human and animal pathogens because of their potential to cause severe epidemics of respiratory or enteric diseases. In pigs, the newly emerged porcine deltacoronavirus (PDCoV) and re-emerged porcine epidemic diarrhea virus (PEDV) reported in the US and Asia, as well as the discovery of novel CoVs in wild bats or birds, has necessitated development of improved detection and control measures for these CoVs. Because the previous pancoronavirus (panCoV) RT-PCR established in our laboratory in 2007-2011 did not detect deltacoronaviruses (δ-CoVs) in swine fecal and serum samples, our goal was to develop a new panCoV RT-PCR assay to detect known human and animal CoVs, including δ-CoVs. In this study, we designed a new primer set to amplify a 668 bp-region within the RNA-dependent RNA polymerase (RdRP) gene that encodes the most conserved protein domain of α-, β-, γ-, and δ-CoVs. We established a one-step panCoV RT-PCR assay and standardized the assay conditions. The newly established panCoV RT-PCR assay was demonstrated to have a high sensitivity and specificity. Using a panel of 60 swine biological samples (feces, intestinal contents, and sera) characterized by PEDV, PDCoV and transmissible gastroenteritis virus-specific RT-PCR assays, we demonstrated that sensitivity and specificity of the newly established panCoV RT-PCR assay were 100%. 400 avian fecal (RNA) samples were further tested simultaneously for CoV by the new panCoV RT-PCR and a one-step RT-PCR assay with the δ-CoV nucleocapsid-specific universal primers. Four of 400 avian samples were positive for CoV, three of which were positive for δ-CoV by the conventional RT-PCR. PanCoV RT-PCR fragments for 3 of the 4 CoVs were sequenced. Phylogenetic analysis revealed the presence of one γ-CoV and two δ-CoV in the sequenced samples. The newly designed panCoV RT-PCR assay should be useful for the detection of currently known CoVs in animal biological samples. Copyright © 2018

  14. Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis

    Science.gov (United States)

    Umhang, Gérald; Poulle, Marie-Lazarine; Millon, Laurence

    2016-01-01

    Studying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes, Canis lupus familiaris, Canis lupus, Felis silvestris catus, Meles meles, Martes foina, and Martes martes. With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens. PMID:26969697

  15. Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis.

    Science.gov (United States)

    Knapp, Jenny; Umhang, Gérald; Poulle, Marie-Lazarine; Millon, Laurence

    2016-05-15

    Studying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes, Canis lupus familiaris, Canis lupus, Felis silvestris catus, Meles meles, Martes foina, and Martes martes With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  16. One-Step Multiplex RT-qPCR Assay for the detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

    International Nuclear Information System (INIS)

    Settypalli, T.B.K.; Lamien, C.; Spergser, J.; Lelenta, M.; Wade, A.; Gelaye, E.; Loitsch, A.; Minoungou, G.; Thiaucourt, F.; Diallo, A.

    2016-01-01

    Full text: Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%–4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in

  17. One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp. capripneumoniae.

    Directory of Open Access Journals (Sweden)

    Tirumala Bharani Kumar Settypalli

    Full Text Available Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV, Peste de petits ruminants virus (PPRV, Pasteurella multocida (PM and Mycoplasma capricolum ssp. capripneumonia (Mccp in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%-4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in

  18. Evaluation of a Rapid One-step Real-time PCR Method as a High-throughput Screening for Quantification of Hepatitis B Virus DNA in a Resource-limited Setting.

    Science.gov (United States)

    Rashed-Ul Islam, S M; Jahan, Munira; Tabassum, Shahina

    2015-01-01

    Virological monitoring is the best predictor for the management of chronic hepatitis B virus (HBV) infections. Consequently, it is important to use the most efficient, rapid and cost-effective testing systems for HBV DNA quantification. The present study compared the performance characteristics of a one-step HBV polymerase chain reaction (PCR) vs the two-step HBV PCR method for quantification of HBV DNA from clinical samples. A total of 100 samples consisting of 85 randomly selected samples from patients with chronic hepatitis B (CHB) and 15 samples from apparently healthy individuals were enrolled in this study. Of the 85 CHB clinical samples tested, HBV DNA was detected from 81% samples by one-step PCR method with median HBV DNA viral load (VL) of 7.50 × 10 3 lU/ml. In contrast, 72% samples were detected by the two-step PCR system with median HBV DNA of 3.71 × 10 3 lU/ml. The one-step method showed strong linear correlation with two-step PCR method (r = 0.89; p Tabassum S. Evaluation of a Rapid One-step Real-time PCR Method as a High-throughput Screening for Quantification of Hepatitis B Virus DNA in a Resource-limited Setting. Euroasian J Hepato-Gastroenterol 2015;5(1):11-15.

  19. One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

    Science.gov (United States)

    Mijatovic-Rustempasic, Slavica; Esona, Mathew D.; Tam, Ka Ian; Quaye, Osbourne; Bowen, Michael D.

    2016-01-01

    Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8

  20. One-step Multiplex RT-PCR Method for Simultaneous Detection of Seed Transmissible Bacterium and Virus Occurring on Brassicaceae Crop Seeds

    Directory of Open Access Journals (Sweden)

    Kyusik Jeong

    2011-04-01

    Full Text Available The aim of this research was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant pathogenic bacteria and seed borne virus in commercial Brassicaceae crop seeds, Xanthomonns campestris pv. campestris (Xcc and Lettuce Mosaic Virus (LMV. Bacterial and virus diseases of Brassicaceae leaves are responsible for heavy losses. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcc and LMV in commercial Brassicaceae crop seeds (lettuce, kohlrabi, radish, chinese cabbage and cabbage, two pairs of specific primer (LMV-F/R, Xcc-F/R were synthesized by using primer-blast program (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/. The multiplex PCR for the two pathogens in Brassicaceae crop seeds could detect specifically without interference among primers and/or cDNA of other plant pathogens. The pathogen detection limit was determined at 1 ng of RNA extracted from pathogens. In the total PCR results for pathogen detection using commercial kohlrabi (10 varieties, lettuce (50 varieties, radish (20 varieties, chinese cabbage (20 varieties and cabbage (20 varieties, LMV and Xcc were detected from 39 and 2 varieties, respectively. In the PCR result of lettuce, LMV and Xcc were simultaneously detected in 8 varieties.

  1. One-step microlithography

    Science.gov (United States)

    Kahlen, Franz-Josef; Sankaranarayanan, Srikanth; Kar, Aravinda

    1997-09-01

    Subject of this investigation is a one-step rapid machining process to create miniaturized 3D parts, using the original sample material. An experimental setup where metal powder is fed to the laser beam-material interaction region has been built. The powder is melted and forms planar, 2D geometries as the substrate is moved under the laser beam in XY- direction. After completing the geometry in the plane, the substrate is displaced in Z-direction, and a new layer of material is placed on top of the just completed deposit. By continuous repetition of this process, 3D parts wee created. In particular, the impact of the focal spot size of the high power laser beam on the smallest achievable structures was investigated. At a translation speed of 51 mm/s a minimum material thickness of 590 micrometers was achieved. Also, it was shown that a small Z-displacement has a negligible influence on the continuity of the material deposition over this power range. A high power CO2 laser was used as energy source, the material powder under investigation was stainless steel SS304L. Helium was used as shield gas at a flow rate of 15 1/min. The incident CO2 laser beam power was varied between 300 W and 400 W, with the laser beam intensity distribute in a donut mode. The laser beam was focused to a focal diameter of 600 (Mu) m.

  2. One-step Multiplex RT-PCR Method for Simultaneous Detection of Seed Transmissible Bacteria and Viruses in Pepper and Tomato Seeds

    Directory of Open Access Journals (Sweden)

    Kyusik Jeong

    2011-04-01

    Full Text Available The aim of this study was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant seed infection pathogenic bacteria and virus, Xanthomonns campestris pv. vesicatoria (Xcv, Clavibacter michiganensis subsp. michiganensis (Cmm, Erwinia carotovora subsp. carotovora (Ecc, Pepper mild mottle virus (PMMoV and Tobacco mild green mosaic virus (TMGMV in pepper and tomato seeds. Most of pepper and tomato bacterial and virus diseases are responsible for germination and growth obstruction. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcv, Cmm, Ecc, PMMoV and TMGMV in pepper and tomato seeds, five pairs (Cmm-F/R, Ecc-F/R, Xcv-F/R, PMMoV-F/R, TMGMV-F/R of specific primer were synthesized by primer-blast program. The multiplex PCR for the five pathogens in pepper and tomato seeds could detect specially without interference among primers and/or cDNA of plant seeds and other plant pathogens. The PCR result for pathogen detection using 20 commercial pepper and 10 tomato seed samples, Ecc was detected from 4 pepper and 2 tomato seed samples, PMMoV was detected from 1 pepper seed sample, and PMMoV and TMGMV were simultaneously detected from 1 pepper seed sample.

  3. Establishment of thermophilic anaerobic terephthalic acid degradation system through one-step temperature increase startup strategy - Revealed by Illumina Miseq Sequencing.

    Science.gov (United States)

    Ma, Kai-Li; Li, Xiang-Kun; Wang, Ke; Meng, Ling-Wei; Liu, Gai-Ge; Zhang, Jie

    2017-10-01

    Over recent years, thermophilic digestion was constantly focused owing to its various advantage over mesophilic digestion. Notably, the startup approach of thermophilic digester needs to be seriously considered as unsuitable startup ways may result in system inefficiency. In this study, one-step temperature increase startup strategy from 37 °C to 55 °C was applied to establish a thermophilic anaerobic system treating terephthalic acid (TA) contained wastewater, meanwhile, the archaeal and bacterial community compositions at steady periods of 37 °C and 55 °C during the experimental process was also compared using Illumina Miseq Sequencing. The process operation demonstrated that the thermophilic TA degradation system was successfully established at 55 °C with over 95% COD reduction. For archaea community, the elevation of operational temperature from 37 °C to 55 °C accordingly increase the enrichment of hydrogenotrophic methanogens but decrease the abundance of the acetotrophic ones. While for bacterial community, the taxonomic analysis suggested that Syntrophorhabdus (27.40%) was the dominant genus promoting the efficient TA degradation under mesophilic condition, whereas OPB95 (24.99%) and TA06 (14.01%) related populations were largely observed and probably take some crucial role in TA degradation under thermophilic condition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Comparison of the genexpert enterovirus assay (GXEA) with real-time one step RT-PCR for the detection of enteroviral RNA in the cerebrospinal fluid of patients with meningitis.

    Science.gov (United States)

    Hong, JiYoung; Kim, Ahyoun; Hwang, Seoyeon; Cheon, Doo-Sung; Kim, Jong-Hyen; Lee, June-Woo; Park, Jae-Hak; Kang, Byunghak

    2015-02-13

    Enteroviruses (EVs) are the leading cause of aseptic meningitis worldwide. Detection of enteroviral RNA in clinical specimens has been demonstrated to improve the management of patient care, especially that of neonates and young children. To establish a sensitive and reliable assay for routine laboratory diagnosis, we compared the sensitivity and specificity of the GeneXpert Enterovirus Assay (GXEA) with that of the reverse transcription polymerase chain reaction (RT-PCR) based assay referred to as real-time one step RT-PCR (RTo-PCR). The sensitivity/specificity produced by GXEA and RTo-PCR were 100%/100% and 65%/100%, respectively. Both methods evaluated in this article can be used for detection of enterovirus in clinical specimens and these nucleic acid amplification methods are useful assays for the diagnosis of enteroviral infection.

  5. A novel one-step real-time multiplex PCR assay to detect Streptococcus agalactiae presence and serotypes Ia, Ib, and III.

    Science.gov (United States)

    Furfaro, Lucy L; Chang, Barbara J; Payne, Matthew S

    2017-09-01

    Streptococcus agalactiae is the leading cause of early-onset neonatal sepsis. Culture-based screening methods lack the sensitivity of molecular assays and do not indicate serotype; a potentially important virulence marker. We aimed to develop a multiplex PCR to detect S. agalactiae while simultaneously identifying serotypes Ia, Ib, and III; commonly associated with infant disease. Primers were designed to target S. agalactiae serotype-specific cps genes and the dltS gene. The assay was validated with 512 vaginal specimens from pregnant women. 112 (21.9%) were dltS positive, with 14.3%, 0.9%, and 6.3% of these identified as cps Ia, Ib, and III, respectively. Our assay is a specific and sensitive method to simultaneously detect S. agalactiae and serotypes Ia, Ib, and III in a single step. It is of high significance for clinical diagnostic applications and also provides epidemiological data on serotype, information that may be important for vaccine development and other targeted non-antibiotic therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Pressure-driven one-step solid phase-based on-chip sample preparation on a microfabricated plastic device and integration with flow-through polymerase chain reaction (PCR).

    Science.gov (United States)

    Tran, Hong Hanh; Trinh, Kieu The Loan; Lee, Nae Yoon

    2013-10-01

    In this study, we fabricate a monolithic poly(methylmethacrylate) (PMMA) microdevice on which solid phase-based DNA preparation and flow-through polymerase chain reaction (PCR) units were functionally integrated for one-step sample preparation and amplification operated by pressure. Chelex resin, which is used as a solid support for DNA preparation, can capture denatured proteins but releases DNA, and the purified DNA can then be used as a template in a subsequent amplification process. Using the PMMA microdevices, DNA was successfully purified from both Escherichia coli and human hair sample, and the plasmid vector inserted in E. coli and the D1S80 locus in human genomic DNA were successfully amplified from on-chip purified E. coli and human hair samples. Furthermore, the integration potential of the proposed sample preparation and flow-through PCR units was successfully demonstrate on a monolithic PMMA microdevice with a seamless flow, which could pave the way for a pressure-driven, simple one-step sample preparation and amplification with greatly decreased manufacture cost and enhanced device disposability. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. A one-step, triplex, real-time RT-PCR assay for the simultaneous detection of enterovirus 71, coxsackie A16 and pan-enterovirus in a single tube.

    Directory of Open Access Journals (Sweden)

    Shiyin Zhang

    Full Text Available The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD, which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71 and coxsackie A16 (CA16 are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001-0.04 TCID50/ml, 0.02 TCID50/ml, and 0.001 TCID50/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.

  8. A one-step, triplex, real-time RT-PCR assay for the simultaneous detection of enterovirus 71, coxsackie A16 and pan-enterovirus in a single tube.

    Science.gov (United States)

    Zhang, Shiyin; Wang, Jin; Yan, Qiang; He, Shuizhen; Zhou, Wenbin; Ge, Shengxiang; Xia, Ningshao

    2014-01-01

    The recent, ongoing epidemic of hand, foot, and mouth disease (HFMD), which is caused by enterovirus infection, has affected millions of children and resulted in thousands of deaths in China. Enterovirus 71 (EV71) and coxsackie A16 (CA16) are the two major distinct pathogens for HFMD. However, EV71 is more commonly associated with neurologic complications and even fatalities. Therefore, simultaneously detecting and differentiating EV71 and CA16 specifically from other enteroviruses for diagnosing HFMD is important. Here, we developed a one-step, triplex, real-time RT-PCR assay for the simultaneous detection of EV71, CA16, and pan-enterovirus (EVs) in a single tube with an internal amplification control. The detection results for the serially diluted viruses indicate that the lower limit of detection for this assay is 0.001-0.04 TCID50/ml, 0.02 TCID50/ml, and 0.001 TCID50/ml for EVs, EV71, and CA16, respectively. After evaluating known HFMD virus stocks of 17 strains of 16 different serotypes, this assay showed a favorable detection spectrum and no obvious cross-reactivity. The results for 141 clinical throat swabs from HFMD-suspected patients demonstrated sensitivities of 98.4%, 98.7%, and 100% for EVs, EV71, and CA16, respectively, and 100% specificity for each virus. The application of this one-step, triplex, real-time RT-PCR assay in clinical units will contribute to HFMD surveillance and help to identify causative pathogen in patients with suspected HFMD.

  9. Molecular characterization of two rocio flavivirus strains isolated during the encephalitis epidemic in são paulo state, brazil and the development of a one-step rt-pcr assay for diagnosis Caracterização molecular de duas cepas do flavivírus Rocio, isoladas durante a epidemia de encefalite no Estado de São Paulo, Brasil e desenvolvimento do teste one-step RT-PCR para diagnóstico

    Directory of Open Access Journals (Sweden)

    Terezinha Lisieux Moraes Coimbra

    2008-04-01

    Full Text Available Rocio virus (ROCV was responsible for an explosive encephalitis epidemic in the 1970s affecting about 1,000 residents of 20 coastland counties in São Paulo State, Brazil. ROCV was first isolated in 1975 from the cerebellum of a fatal human case of encephalitis. Clinical manifestations of the illness are similar to those described for St. Louis encephalitis. ROCV shows intense antigenic cross-reactivity with Japanese encephalitis complex (JEC viruses, particularly with Ilheus (ILHV, St. Louis encephalitis, Murray Valley and West Nile viruses. In this study, we report a specific RT-PCR assay for ROCV diagnosis and the molecular characterization of the SPAn37630 and SPH37623 strains. Partial nucleotide sequences of NS5 and E genes determined from both strains were used in phylogenetic analysis. The results indicated that these strains are closely related to JEC viruses, but forming a distinct subclade together with ILHV, in accordance with results recently reported by Medeiros et al. (2007.O vírus Rocio (ROCV foi responsável por uma explosiva epidemia de encefalite que ocorreu nos anos 70 afetando cerca de 1.000 habitantes de 20 municípios litorâneos do Estado de São Paulo, Brasil. ROCV foi isolado em 1975 de cerebelo de caso humano fatal de encefalite. As manifestações clínicas da doença são semelhantes àquelas descritas para encefalite St. Louis. ROCV apresenta intensa reatividade cruzada com os vírus do Complexo da Encefalite Japonesa (JEV, particularmente com o vírus Ilhéus (ILHV e com os vírus das encefalites St. Louis, Murray Valley e West Nile. Neste estudo, relatamos o desenvolvimento de um teste de RT-PCR específico para diagnóstico de ROCV e a caracterização molecular das cepas SPAn37630 e SPH37623. Foi realizada a análise filogenética das seqüências parciais dos genes NS5 e E, de ambas as cepas. Os resultados indicaram que essas cepas são intimamente relacionadas ao complexo JEV, mas formando um subgrupo com o

  10. A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences.

    Science.gov (United States)

    Ferchichi, M; Valcheva, R; Prévost, H; Onno, B; Dousset, X

    2008-06-01

    Species-specific primers targeting the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough. The 16S-23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388-1406 of the 16S rRNA gene and to positions 207-189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331). Clone libraries of the resulting amplicons were constructed using a pCR2.1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S-23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA(Ile) and tRNA(Ala) genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested. Designed species-specific primers enable a rapid and accurate identification of L. mindensis, L. paralimentarius, L. panis, L. pontis and L. frumenti species among other lactobacilli. The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.

  11. Droplet Digital™ PCR Next-Generation Sequencing Library QC Assay.

    Science.gov (United States)

    Heredia, Nicholas J

    2018-01-01

    Digital PCR is a valuable tool to quantify next-generation sequencing (NGS) libraries precisely and accurately. Accurately quantifying NGS libraries enable accurate loading of the libraries on to the sequencer and thus improve sequencing performance by reducing under and overloading error. Accurate quantification also benefits users by enabling uniform loading of indexed/barcoded libraries which in turn greatly improves sequencing uniformity of the indexed/barcoded samples. The advantages gained by employing the Droplet Digital PCR (ddPCR™) library QC assay includes the precise and accurate quantification in addition to size quality assessment, enabling users to QC their sequencing libraries with confidence.

  12. A rapid, one step molecular identification of Trichoderma citrinoviride and Trichoderma reesei.

    Science.gov (United States)

    Saroj, Dina B; Dengeti, Shrinivas N; Aher, Supriya; Gupta, Anil K

    2015-06-01

    Trichoderma species are widely used as production hosts for industrial enzymes. Identification of Trichoderma species requires a complex molecular biology based identification involving amplification and sequencing of multiple genes. Industrial laboratories are required to run identification tests repeatedly in cell banking procedures and also to prove absence of production host in the product. Such demands can be fulfilled by a brief method which enables confirmation of strain identity. This communication describes one step identification method for two common Trichoderma species; T. citrinoviride and T. reesei, based on identification of polymorphic region in the nucleotide sequence of translation elongation factor 1 alpha. A unique forward primer and common reverse primer resulted in 153 and 139 bp amplicon for T. citrinoviride and T. reesei, respectively. Simplification was further introduced by using mycelium as template for PCR amplification. Method described in this communication allows rapid, one step identification of two Trichoderma species.

  13. RUCS: Rapid identification of PCR primers for unique core sequences

    DEFF Research Database (Denmark)

    Thomsen, Martin Christen Frølund; Hasman, Henrik; Westh, Henrik

    2017-01-01

    Designing PCR primers to target a specific selection of whole genome sequenced strains can be a long, arduous, and sometimes impractical task. Such tasks would benefit greatly from an automated tool to both identify unique targets, and to validate the vast number of potential primer pairs...... for the targets in silico . Here we present RUCS, a program that will find PCR primer pairs and probes for the unique core sequences of a positive genome dataset complement to a negative genome dataset. The resulting primer pairs and probes are in addition to simple selection also validated through a complex...... in silico PCR simulation. We compared our method, which identifies the unique core sequences, against an existing tool called ssGeneFinder, and found that our method was 6.5-20 times more sensitive. We used RUCS to design primer pairs that would target a set of genomes known to contain the mcr-1 colistin...

  14. One-step fabrication of multifunctional micromotors

    Science.gov (United States)

    Gao, Wenlong; Liu, Mei; Liu, Limei; Zhang, Hui; Dong, Bin; Li, Christopher Y.

    2015-08-01

    Although artificial micromotors have undergone tremendous progress in recent years, their fabrication normally requires complex steps or expensive equipment. In this paper, we report a facile one-step method based on an emulsion solvent evaporation process to fabricate multifunctional micromotors. By simultaneously incorporating various components into an oil-in-water droplet, upon emulsification and solidification, a sphere-shaped, asymmetric, and multifunctional micromotor is formed. Some of the attractive functions of this model micromotor include autonomous movement in high ionic strength solution, remote control, enzymatic disassembly and sustained release. This one-step, versatile fabrication method can be easily scaled up and therefore may have great potential in mass production of multifunctional micromotors for a wide range of practical applications.Although artificial micromotors have undergone tremendous progress in recent years, their fabrication normally requires complex steps or expensive equipment. In this paper, we report a facile one-step method based on an emulsion solvent evaporation process to fabricate multifunctional micromotors. By simultaneously incorporating various components into an oil-in-water droplet, upon emulsification and solidification, a sphere-shaped, asymmetric, and multifunctional micromotor is formed. Some of the attractive functions of this model micromotor include autonomous movement in high ionic strength solution, remote control, enzymatic disassembly and sustained release. This one-step, versatile fabrication method can be easily scaled up and therefore may have great potential in mass production of multifunctional micromotors for a wide range of practical applications. Electronic supplementary information (ESI) available: Videos S1-S4 and Fig. S1-S3. See DOI: 10.1039/c5nr03574k

  15. Directed PCR-free engineering of highly repetitive DNA sequences

    Directory of Open Access Journals (Sweden)

    Preissler Steffen

    2011-09-01

    Full Text Available Abstract Background Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products. Results For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays. Conclusion Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.

  16. Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

    Science.gov (United States)

    Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

    2016-12-01

    Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. One-step lowrank wave extrapolation

    KAUST Repository

    Sindi, Ghada Atif

    2014-01-01

    Wavefield extrapolation is at the heart of modeling, imaging, and Full waveform inversion. Spectral methods gained well deserved attention due to their dispersion free solutions and their natural handling of anisotropic media. We propose a scheme a modified one-step lowrank wave extrapolation using Shanks transform in isotropic, and anisotropic media. Specifically, we utilize a velocity gradient term to add to the accuracy of the phase approximation function in the spectral implementation. With the higher accuracy, we can utilize larger time steps and make the extrapolation more efficient. Applications to models with strong inhomogeneity and considerable anisotropy demonstrates the utility of the approach.

  18. Stability study in one step steam generators

    International Nuclear Information System (INIS)

    Abbate, P.

    1990-01-01

    The TWO program is presented developed for the behaviour limit calculation stable in one step steam generators for the case of Density Waves phenomenom. The program is based on a nodal model which, using Laplace transformation equations, allows to study the system's transfer functions and foresee the beginning of the unstable behaviour. This program has been satisfactorily validated against channels data uniformly heated in the range from 4.0 to 6.0 Mpa. Results on the CAREM reactor's steam generator analysis are presented. (Author) [es

  19. Lyondell develops one step isobutylene process

    International Nuclear Information System (INIS)

    Anon.

    1992-01-01

    This paper reports that Lyondell Petrochemical Co., Houston, has developed a one step process to convert normal butylenes to isobutylene, a key component of methyl tertiary butyl ether (MTBE). MTBE is expected to become the additive of choice among U.S. refiners to blend oxygenated gasolines required by 1990 amendments to the Clean Air Act. Lyondell Pres. and Chief Executive Officer Bob Gower the the new process could help assure adequate supplies of MTBE to meet U.S. demand for cleaner burning fuels. Lyondell estimates the capital cost of building a grassroots plant to produce isobutylene with the new process would be less than half the cost of a grassroot plant to produce isobutylene with existing technology starting with normal butane

  20. Automated degenerate PCR primer design for high-throughput sequencing improves efficiency of viral sequencing

    Directory of Open Access Journals (Sweden)

    Li Kelvin

    2012-11-01

    Full Text Available Abstract Background In a high-throughput environment, to PCR amplify and sequence a large set of viral isolates from populations that are potentially heterogeneous and continuously evolving, the use of degenerate PCR primers is an important strategy. Degenerate primers allow for the PCR amplification of a wider range of viral isolates with only one set of pre-mixed primers, thus increasing amplification success rates and minimizing the necessity for genome finishing activities. To successfully select a large set of degenerate PCR primers necessary to tile across an entire viral genome and maximize their success, this process is best performed computationally. Results We have developed a fully automated degenerate PCR primer design system that plays a key role in the J. Craig Venter Institute’s (JCVI high-throughput viral sequencing pipeline. A consensus viral genome, or a set of consensus segment sequences in the case of a segmented virus, is specified using IUPAC ambiguity codes in the consensus template sequence to represent the allelic diversity of the target population. PCR primer pairs are then selected computationally to produce a minimal amplicon set capable of tiling across the full length of the specified target region. As part of the tiling process, primer pairs are computationally screened to meet the criteria for successful PCR with one of two described amplification protocols. The actual sequencing success rates for designed primers for measles virus, mumps virus, human parainfluenza virus 1 and 3, human respiratory syncytial virus A and B and human metapneumovirus are described, where >90% of designed primer pairs were able to consistently successfully amplify >75% of the isolates. Conclusions Augmenting our previously developed and published JCVI Primer Design Pipeline, we achieved similarly high sequencing success rates with only minor software modifications. The recommended methodology for the construction of the consensus

  1. Data Analysis of Sequences and qPCR for Microbial Communities during Algal Blooms

    Science.gov (United States)

    A training opportunity is open to a highly microbial-research-motivated student to conduct sequence analysis, explore novel genes and metabolic pathways, validate resultant findings using qPCR/RT-qPCR and summarize the findings

  2. Polyadenylated Sequencing Primers Enable Complete Readability of PCR Amplicons Analyzed by Dideoxynucleotide Sequencing

    Directory of Open Access Journals (Sweden)

    Martin Beránek

    2012-01-01

    Full Text Available Dideoxynucleotide DNA sequencing is one of the principal procedures in molecular biology. Loss of an initial part of nucleotides behind the 3' end of the sequencing primer limits the readability of sequenced amplicons. We present a method which extends the readability by using sequencing primers modified by polyadenylated tails attached to their 5' ends. Performing a polymerase chain reaction, we amplified eight amplicons of six human genes (AMELX, APOE, HFE, MBL2, SERPINA1 and TGFB1 ranging from 106 bp to 680 bp. Polyadenylation of the sequencing primers minimized the loss of bases in all amplicons. Complete sequences of shorter products (AMELX 106 bp, SERPINA1 121 bp, HFE 208 bp, APOE 244 bp, MBL2 317 bp were obtained. In addition, in the case of TGFB1 products (366 bp, 432 bp, and 680 bp, respectively, the lengths of sequencing readings were significantly longer if adenylated primers were used. Thus, single strand dideoxynucleotide sequencing with adenylated primers enables complete or near complete readability of short PCR amplicons.

  3. Comparison of ELISA, nested PCR and sequencing and a novel qPCR for detection of Giardia isolates from Jordan.

    Science.gov (United States)

    Hijjawi, Nawal; Yang, Rongchang; Hatmal, Ma'mon; Yassin, Yasmeen; Mharib, Taghrid; Mukbel, Rami; Mahmoud, Sameer Alhaj; Al-Shudifat, Abdel-Ellah; Ryan, Una

    2018-02-01

    Little is known about the prevalence of Giardia duodenalis in human patients in Jordan and all previous studies have used direct microscopy, which lacks sensitivity. The present study developed a novel quantitative PCR (qPCR) assay at the β-giardin (bg) locus and evaluated its use as a frontline test for the diagnosis of giardiasis in comparison with a commercially available ELISA using nested PCR and sequencing of the glutamate dehydrogenase (gdh) locus (gdh nPCR) as the gold standard. A total of 96 human faecal samples were collected from 96 patients suffering from diarrhoea from 5 regions of Jordan and were screened using the ELISA and qPCR. The analytical specificity of the bg qPCR assay revealed no cross-reactions with other genera and detected all the Giardia isolates tested. Analytical sensitivity was 1 Giardia cyst per μl of DNA extract. The overall prevalence of Giardia was 64.6%. The clinical sensitivity and specificity of the bg qPCR was 89.9% and 82.9% respectively compared to 76.5 and 68.0% for the ELISA. This study is the first to compare three different methods (ELISA, bg qPCR, nested PCR and sequencing at the gdh locus) to diagnose Jordanian patients suffering from giardiasis and to analyze their demographic data. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. Sources of PCR-induced distortions in high-throughput sequencing data sets

    Science.gov (United States)

    Kebschull, Justus M.; Zador, Anthony M.

    2015-01-01

    PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error—bias, stochasticity, template switches and polymerase errors—on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules. PMID:26187991

  5. The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing

    DEFF Research Database (Denmark)

    Binladen, Jonas; Gilbert, M Thomas P; Bollback, Jonathan P

    2007-01-01

    BACKGROUND: The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine...... primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution...

  6. The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing.

    Directory of Open Access Journals (Sweden)

    Jonas Binladen

    2007-02-01

    Full Text Available The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR reactions and subsequent sequencing runs have been unable to combine template DNA from multiple individuals, as homologous sequences cannot be subsequently assigned to their original sources.We use conventional PCR with 5'-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through the high-throughput Genome Sequence 20 DNA Sequencing System (GS20, Roche/454 Life Sciences. Each DNA sequence is subsequently traced back to its individual source through 5'tag-analysis.We demonstrate that this new approach enables the assignment of virtually all the generated DNA sequences to the correct source once sequencing anomalies are accounted for (miss-assignment rate<0.4%. Therefore, the method enables accurate sequencing and assignment of homologous DNA sequences from multiple sources in single high-throughput GS20 run. We observe a bias in the distribution of the differently tagged primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution of the sequences as sorted by the second nucleotide of the dinucleotide tags. As the results are based on a single GS20 run, the general applicability of the approach requires confirmation. However, our experiments demonstrate that 5'primer tagging is a useful method in which the sequencing power of the GS20 can be applied to PCR-based assays of multiple homologous PCR products. The new approach will be of value to a broad range of research areas, such as those of comparative genomics, complete mitochondrial

  7. Sequence characterisation of deletion breakpoints in the dystrophin gene by PCR

    Energy Technology Data Exchange (ETDEWEB)

    Abbs, S.; Sandhu, S.; Bobrow, M. [Guy`s Hospital, London (United Kingdom)

    1994-09-01

    Partial deletions of the dystrophin gene account for 65% of cases of Duchenne muscular dystrophy. A high proportion of these structural changes are generated by new mutational events, and lie predominantly within two `hotspot` regions, yet the underlying reasons for this are not known. We are characterizing and sequencing the regions surrounding deletion breakpoints in order to: (i) investigate the mechanisms of deletion mutation, and (ii) enable the design of PCR assays to specifically amplify mutant and normal sequences, allowing us to search for the presence of somatic mosaicism in appropriate family members. Using this approach we have been able to demonstrate the presence of somatic mosaicism in a maternal grandfather of a DMD-affected male, deleted for exons 49-50. Three deletions, namely of exons 48-49, 49-50, and 50, have been characterized using a PCR approach that avoids any cloning procedures. Breakpoints were initially localized to within regions of a few kilobases using Southern blot restriction analyses with exon-specific probes and PCR amplification of exonic and intronic loci. Sequencing was performed directly on PCR products: (i) mutant sequences were obtained from long-range or inverse-PCR across the deletion junction fragments, and (ii) normal sequences were obtained from the products of standard PCR, vectorette PCR, or inverse-PCR performed on YACs. Further characterization of intronic sequences will allow us to amplify and sequence across other deletion breakpoints and increase our knowledge of the mechanisms of mutation in the dystophin gene.

  8. An enhanced method for sequence walking and paralog mining: TOPO® Vector-Ligation PCR

    Directory of Open Access Journals (Sweden)

    Davis Thomas M

    2010-03-01

    Full Text Available Abstract Background Although technological advances allow for the economical acquisition of whole genome sequences, many organisms' genomes remain unsequenced, and fully sequenced genomes may contain gaps. Researchers reliant upon partial genomic or heterologous sequence information require methods for obtaining unknown sequences from loci of interest. Various PCR based techniques are available for sequence walking - i.e., the acquisition of unknown DNA sequence adjacent to known sequence. Many such methods require rigid, elaborate protocols and/or impose narrowly confined options in the choice of restriction enzymes for necessary genomic digests. We describe a new method, TOPO® Vector-Ligation PCR (or TVL-PCR that innovatively integrates available tools and familiar concepts to offer advantages as a means of both targeted sequence walking and paralog mining. Findings TVL-PCR exploits the ligation efficiency of the pCR®4-TOPO® (Invitrogen, Carlsbad, California vector system to capture fragments of unknown sequence by creating chimeric molecules containing defined priming sites at both ends. Initially, restriction enzyme-digested genomic DNA is end-repaired to create 3' adenosine overhangs and is then ligated to pCR4-TOPO vectors. The ligation product pool is used directly as a template for nested PCR, using specific primers to target orthologous sequences, or degenerate primers to enable capture of paralogous gene family members. We demonstrated the efficacy of this method by capturing entire coding and partial promoter sequences of several strawberry Superman-like genes. Conclusions TVL-PCR is a convenient and efficient method for DNA sequence walking and paralog mining that is applicable to any organism for which relevant DNA sequence is available as a basis for primer design.

  9. Ensemble Kalman filtering with one-step-ahead smoothing

    KAUST Repository

    Raboudi, Naila F.; Ait-El-Fquih, Boujemaa; Hoteit, Ibrahim

    2018-01-01

    error statistics. This limits their representativeness of the background error covariances and, thus, their performance. This work explores the efficiency of the one-step-ahead (OSA) smoothing formulation of the Bayesian filtering problem to enhance

  10. Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR

    Energy Technology Data Exchange (ETDEWEB)

    D`Souza, T.M.; Boominathan, K.; Reddy, C.A. [Michigan State Univ., East Lansing, MI (United States)

    1996-10-01

    Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequences of each of the PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum, Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases. 36 refs., 6 figs., 2 tabs.

  11. Rapid one-step selection method for generating nucleic acid aptamers: development of a DNA aptamer against α-bungarotoxin.

    Directory of Open Access Journals (Sweden)

    Lasse H Lauridsen

    Full Text Available BACKGROUND: Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen®, an inhibitor of vascular endothelial growth factor (VEGF for the treatment of age related macular degeneration (AMD. Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly in one-step, technique is required for developing aptamers in limited time period. PRINCIPAL FINDINGS: Herein, we present a simple one-step selection of DNA aptamers against α-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed by PCR enrichment of the selected aptamers. One round of selection successfully identified a DNA aptamer sequence with a binding affinity of 7.58 µM. CONCLUSION: We have demonstrated a one-step method for rapid production of nucleic acid aptamers. Although the reported binding affinity is in the low micromolar range, we believe that this could be further improved by using larger targets, increasing the stringency of selection and also by combining a capillary electrophoresis separation prior to the one-step selection. Furthermore, the method presented here is a user-friendly, cheap and an easy way of deriving an aptamer unlike the time consuming conventional SELEX-based approach. The most important application of this method is that chemically-modified nucleic acid libraries can also be used for aptamer selection as it requires only one enzymatic step. This method could equally be suitable for developing RNA aptamers.

  12. Evaluation of one-step luminescent cyanoacrylate fuming.

    Science.gov (United States)

    Khuu, Alicia; Chadwick, Scott; Spindler, Xanthe; Lam, Rolanda; Moret, Sébastien; Roux, Claude

    2016-06-01

    One-step luminescent cyanoacrylates have recently been introduced as an alternative to the conventional cyanoacrylate fuming methods. These new techniques do not require the application of a luminescent post-treatment in order to enhance cyanoacrylate-developed fingermarks. In this study, three one-step polymer cyanoacrylates: CN Yellow Crystals (Aneval Inc.), PolyCyano UV (Foster+Freeman Ltd.) and PECA Multiband (BVDA), and one monomer cyanoacrylate: Lumikit™ (Crime Scene Technology), were evaluated against a conventional two-step cyanoacrylate fuming method (Cyanobloom (Foster+Freeman Ltd.) with rhodamine 6G stain). The manufacturers' recommended conditions or conditions compatible with the MVC™ 1000/D (Foster+Freeman Ltd.) were assessed with fingermarks aged for up to 8 weeks on non-porous and semi-porous substrates. Under white light, Cyanobloom generally gave better development than the one-step treatments across the substrates. Similarly when viewed under the respective luminescent conditions, Cyanobloom with rhodamine 6G stain resulted in improved contrast against the one-step treatments except on polystyrene, where PolyCyano UV and PECA Multiband gave better visualisation. Rhodamine 6G post-treatment of one-step samples did not significantly enhance the contrast of any of the one-step treatments against Cyanobloom/rhodamine 6G-treated samples. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  13. International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

    Science.gov (United States)

    Whale, Alexandra S; Devonshire, Alison S; Karlin-Neumann, George; Regan, Jack; Javier, Leanne; Cowen, Simon; Fernandez-Gonzalez, Ana; Jones, Gerwyn M; Redshaw, Nicholas; Beck, Julia; Berger, Andreas W; Combaret, Valérie; Dahl Kjersgaard, Nina; Davis, Lisa; Fina, Frederic; Forshew, Tim; Fredslund Andersen, Rikke; Galbiati, Silvia; González Hernández, Álvaro; Haynes, Charles A; Janku, Filip; Lacave, Roger; Lee, Justin; Mistry, Vilas; Pender, Alexandra; Pradines, Anne; Proudhon, Charlotte; Saal, Lao H; Stieglitz, Elliot; Ulrich, Bryan; Foy, Carole A; Parkes, Helen; Tzonev, Svilen; Huggett, Jim F

    2017-02-07

    This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

  14. Digital PCR provides sensitive and absolute calibration for high throughput sequencing

    Directory of Open Access Journals (Sweden)

    Fan H Christina

    2009-03-01

    Full Text Available Abstract Background Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing. Results We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth. Conclusion The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.

  15. 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.

  16. Ribosomal PCR and DNA sequencing for detection and identification of bacteria

    DEFF Research Database (Denmark)

    Jensen, Kristine Helander; Dargis, Rimtas; Christensen, Jens Jørgen

    2014-01-01

    -haemolytic streptococci, especially within the mitis group. The data show that ribosomal PCR with subsequent DNA sequencing of the PCR product is a most valuable supplement to culture for identifying bacterial agents of both acute and prolonged infections. However, some bacteria, including non-haemolytic streptococci...

  17. Comparison of three human papillomavirus DNA detection methods: Next generation sequencing, multiplex-PCR and nested-PCR followed by Sanger based sequencing.

    Science.gov (United States)

    da Fonseca, Allex Jardim; Galvão, Renata Silva; Miranda, Angelica Espinosa; Ferreira, Luiz Carlos de Lima; Chen, Zigui

    2016-05-01

    To compare the diagnostic performance for HPV infection using three laboratorial techniques. Ninty-five cervicovaginal samples were randomly selected; each was tested for HPV DNA and genotypes using 3 methods in parallel: Multiplex-PCR, the Nested PCR followed by Sanger sequencing, and the Next_Gen Sequencing (NGS) with two assays (NGS-A1, NGS-A2). The study was approved by the Brazilian National IRB (CONEP protocol 16,800). The prevalence of HPV by the NGS assays was higher than that using the Multiplex-PCR (64.2% vs. 45.2%, respectively; P = 0.001) and the Nested-PCR (64.2% vs. 49.5%, respectively; P = 0.003). NGS also showed better performance in detecting high-risk HPV (HR-HPV) and HPV16. There was a weak interobservers agreement between the results of Multiplex-PCR and Nested-PCR in relation to NGS for the diagnosis of HPV infection, and a moderate correlation for HR-HPV detection. Both NGS assays showed a strong correlation for detection of HPVs (k = 0.86), HR-HPVs (k = 0.91), HPV16 (k = 0.92) and HPV18 (k = 0.91). NGS is more sensitive than the traditional Sanger sequencing and the Multiplex PCR to genotype HPVs, with promising ability to detect multiple infections, and may have the potential to establish an alternative method for the diagnosis and genotyping of HPV. © 2015 Wiley Periodicals, Inc.

  18. Convenient one-step synthesis of 5-carboxy-seminaphthofluoresceins

    DEFF Research Database (Denmark)

    Hammershøj, Peter; Thyhaug, Erling; Harris, Pernille

    2017-01-01

    The one-step synthesis and characterization of a series of regioisomerically pure 5-carboxy-seminaphthofluoresceins (5-carboxy-SNAFLs) is reported. The optical properties were determined in aqueous buffer at around biological pH, and highly pH sensitive, large Stokes-shift fluorophores with emiss......The one-step synthesis and characterization of a series of regioisomerically pure 5-carboxy-seminaphthofluoresceins (5-carboxy-SNAFLs) is reported. The optical properties were determined in aqueous buffer at around biological pH, and highly pH sensitive, large Stokes-shift fluorophores...

  19. PCR-Internal Transcribed Spacer (ITS) genes sequencing and ...

    African Journals Online (AJOL)

    Methods: DNA extraction, purification, amplification and sequencing of Internal Transcribed Spacer (ITS) genes were per- formed using ... Keywords: Internal transcribed spacer genes, phylogenetic, genetic relationship, clinical and environmental fungi, HIV-TB. ... Nigeria. An Ethical clearance was obtained from the Eth-.

  20. Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR.

    Science.gov (United States)

    D'Souza, T M; Boominathan, K; Reddy, C A

    1996-01-01

    Degenerate primers corresponding to the consensus sequences of the copper-binding regions in the N-terminal domains of known basidiomycete laccases were used to isolate laccase gene-specific sequences from strains representing nine genera of wood rot fungi. All except three gave the expected PCR product of about 200 bp. Computer searches of the databases identified the sequence of each of the PCR products analyzed as a laccase gene sequence, suggesting the specificity of the primers. PCR products of the white rot fungi Ganoderma lucidum, Phlebia brevispora, and Trametes versicolor showed 65 to 74% nucleotide sequence similarity to each other; the similarity in deduced amino acid sequences was 83 to 91%. The PCR products of Lentinula edodes and Lentinus tigrinus, on the other hand, showed relatively low nucleotide and amino acid similarities (58 to 64 and 62 to 81%, respectively); however, these similarities were still much higher than when compared with the corresponding regions in the laccases of the ascomycete fungi Aspergillus nidulans and Neurospora crassa. A few of the white rot fungi, as well as Gloeophyllum trabeum, a brown rot fungus, gave a 144-bp PCR fragment which had a nucleotide sequence similarity of 60 to 71%. Demonstration of laccase activity in G. trabeum and several other brown rot fungi was of particular interest because these organisms were not previously shown to produce laccases. PMID:8837429

  1. Molecular Properties of Poliovirus Isolates: Nucleotide Sequence Analysis, Typing by PCR and Real-Time RT-PCR.

    Science.gov (United States)

    Burns, Cara C; Kilpatrick, David R; Iber, Jane C; Chen, Qi; Kew, Olen M

    2016-01-01

    Virologic surveillance is essential to the success of the World Health Organization initiative to eradicate poliomyelitis. Molecular methods have been used to detect polioviruses in tissue culture isolates derived from stool samples obtained through surveillance for acute flaccid paralysis. This chapter describes the use of realtime PCR assays to identify and serotype polioviruses. In particular, a degenerate, inosine-containing, panpoliovirus (panPV) PCR primer set is used to distinguish polioviruses from NPEVs. The high degree of nucleotide sequence diversity among polioviruses presents a challenge to the systematic design of nucleic acid-based reagents. To accommodate the wide variability and rapid evolution of poliovirus genomes, degenerate codon positions on the template were matched to mixed-base or deoxyinosine residues on both the primers and the TaqMan™ probes. Additional assays distinguish between Sabin vaccine strains and non-Sabin strains. This chapter also describes the use of generic poliovirus specific primers, along with degenerate and inosine-containing primers, for routine VP1 sequencing of poliovirus isolates. These primers, along with nondegenerate serotype-specific Sabin primers, can also be used to sequence individual polioviruses in mixtures.

  2. One-step deterministic multipartite entanglement purification with linear optics

    Energy Technology Data Exchange (ETDEWEB)

    Sheng, Yu-Bo [Department of Physics, Tsinghua University, Beijing 100084 (China); Long, Gui Lu, E-mail: gllong@tsinghua.edu.cn [Department of Physics, Tsinghua University, Beijing 100084 (China); Center for Atomic and Molecular NanoSciences, Tsinghua University, Beijing 100084 (China); Key Laboratory for Quantum Information and Measurements, Beijing 100084 (China); Deng, Fu-Guo [Department of Physics, Applied Optics Beijing Area Major Laboratory, Beijing Normal University, Beijing 100875 (China)

    2012-01-09

    We present a one-step deterministic multipartite entanglement purification scheme for an N-photon system in a Greenberger–Horne–Zeilinger state with linear optical elements. The parties in quantum communication can in principle obtain a maximally entangled state from each N-photon system with a success probability of 100%. That is, it does not consume the less-entangled photon systems largely, which is far different from other multipartite entanglement purification schemes. This feature maybe make this scheme more feasible in practical applications. -- Highlights: ► We proposed a deterministic entanglement purification scheme for GHZ states. ► The scheme uses only linear optical elements and has a success probability of 100%. ► The scheme gives a purified GHZ state in just one-step.

  3. House passes energy bill with one-step plant licensing

    International Nuclear Information System (INIS)

    Anon.

    1992-01-01

    The US House of Representatives which has traditionally been wary of measures that would allow nuclear power to expand, came down strongly on the side of nuclear when it approved a much-amended omnibus energy bill on May 27 by a vote of 381 to 37. The key for the nuclear industry is the presence in the bill (H.R. 776) of language on one-step power plant licensing that was taken directly from the Senate energy bill (S. 2166) that passed in February. This means that when the House and Senate work out a compromise version of the legislation, one-step licensing is almost certain to be carried through--and become law once the final bill is signed by President George Bush, which is expected later this year. The House's endorsement of nuclear power--both as it exists now, and as it could be with the introduction of new plant designs and an end to the long hiatus in plant orders by utilities--went beyond one-step licensing. Debate on the House floor prior to Memorial Day totally transformed the nuclear-related part of the energy bill. H.R. 776 was reported to the floor by the Rules Committee with language by the Interior and Insular Affairs Committee that would have created a nominal one-step system, with a full evidentiary hearing prior to plant construction but also allowing an intervenor who later presents new information on the plant to get another full evidentiary hearing after construction but before operation. This would effectively duplicate the two-step process that existed for all plants now in service, and which utilities no longer want to endure

  4. A novel one-step Helicobacter pylori saliva antigen test

    Directory of Open Access Journals (Sweden)

    Bi-Ling Yang

    2015-02-01

    Conclusion: The one-step HPS test exhibited a high sensitivity and low specificity compared with the other tests, indicating that it is not sufficiently accurate for use in a clinical setting for diagnosing H. pylori infection. However, the test is simple to use (requiring only a saliva sample, inexpensive, and noninvasive in its application, and thus appealing for use in population-based prevalence surveys of the epidemiology of H. pylori infection.

  5. Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

    Science.gov (United States)

    Ulrich, Alexander; Andersen, Kasper R.; Schwartz, Thomas U.

    2012-01-01

    We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts. PMID:23300917

  6. Exponential megapriming PCR (EMP cloning--seamless DNA insertion into any target plasmid without sequence constraints.

    Directory of Open Access Journals (Sweden)

    Alexander Ulrich

    Full Text Available We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.

  7. External and semi-internal controls for PCR amplification of homologous sequences in mixed templates

    DEFF Research Database (Denmark)

    Kalle, Elena; Gulevich, Alexander; Rensing, Christopher Günther T

    2013-01-01

    as an acceptable alternative. In order to evaluate the effects of inhibitors, a model multi-template mix was amplified in a mixture with DNAse-treated sample. Semi-internal control allowed establishment of intervals for robust PCR performance for different samples, thus enabling correct comparison of the samples......In a mixed template, the presence of homologous target DNA sequences creates environments that almost inevitably give rise to artifacts and biases during PCR. Heteroduplexes, chimeras, and skewed template-to-product ratios are the exclusive attributes of mixed template PCR and never occur....... This study demonstrated the efficiency of a model mixed template as an adequate external amplification control for a particular PCR application. The conditions of multi-template PCR do not allow implementation of a classic internal control; therefore we developed a convenient semi-internal control...

  8. A novel one-step Helicobacter pylori saliva antigen test.

    Science.gov (United States)

    Yang, Bi-Ling; Yeh, Chun; Kwong, Wei-Gang; Lee, Shou-Dong

    2015-02-01

    A rapid, reliable, and sufficiently accurate test for diagnosing Helicobacter pylori infection is required for screening dyspeptic patients before a referral for endoscopy. The purpose of this article is two-fold: first, to evaluate the accuracy of a one-step H. pylori saliva antigen (HPS) test; and second, to compare noninvasive and invasive H. pylori tests in Taiwanese population. A total of 104 consecutive dyspeptic patients admitted for gastroenterology into the outpatient department underwent a one-step HPS test, rapid urease test, histology, and (13)C-urea breath test (13)C-UBT (proto C-13 urea kit). The accuracy of the HPS test was compared with a gold standard defined by at least two positive H. pylori test results from three H. pylori tests (histology, rapid urease test, and (13)C-UBT). The 104 patients eligible for analysis (mean age: 58 years, range 22-87 years), 21 (20%) were gold standard positive. Among them, the positive of the one-step H. pylori saliva Ag test, rapid urease test, (13)C-UBT, histology were (52; 50%), (17; 16%), (27; 25%) and (22; 21%) respectively. The sensitivity and specificity of the HPS tests, rapid urease test, (13)C-UBTs, and histology were 71.43% and 55.42%, 76.19% and 98.80%, 100% and 92.77%, and 85.71% and 95.18%, respectively, relative to the gold standard. The one-step HPS test exhibited a sensitivity of 71.43%, nearly equivalent to that of the rapid urea test. The one-step HPS test exhibited a high sensitivity and low specificity compared with the other tests, indicating that it is not sufficiently accurate for use in a clinical setting for diagnosing H. pylori infection. However, the test is simple to use (requiring only a saliva sample), inexpensive, and noninvasive in its application, and thus appealing for use in population-based prevalence surveys of the epidemiology of H. pylori infection. Copyright © 2014. Published by Elsevier Taiwan.

  9. Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR.

    Science.gov (United States)

    Osimani, Andrea; Milanović, Vesna; Garofalo, Cristiana; Cardinali, Federica; Roncolini, Andrea; Sabbatini, Riccardo; De Filippis, Francesca; Ercolini, Danilo; Gabucci, Claudia; Petruzzelli, Annalisa; Tonucci, Franco; Clementi, Francesca; Aquilanti, Lucia

    2018-07-02

    The present study aimed to identify the microbiota present in six species of processed edible insects produced in Thailand and marketed worldwide via the internet, namely, giant water bugs (Belostoma lutarium), black ants (Polyrhachis), winged termites (alates, Termitoidae), rhino beetles (Hyboschema contractum), mole crickets (Gryllotalpidae), and silkworm pupae (Bombyx mori). For each species, two samples of boiled, dried and salted insects were purchased. The microbial DNA was extracted from the insect samples and subjected to polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing and qualitative real-time PCR assays. The microbiota of the analyzed samples were widely characterized by the presence of spore-forming bacteria mainly represented by the genera Bacillus and Clostridium. Moreover, the genera Anaerobacillus, Paenibacillus, Geobacillus, Pseudomonas, Stenotrophomonas, Massilia, Delftia, Lactobacillus, Staphylococcus, Streptococcus, Vagococcus, and Vibrio were also detected. Real-time PCR allowed for ascertainment of the absence of Coxiella burnetii, Shiga toxin-producing E. coli (STEC), and Pseudomonas aeruginosa in all samples. The results of this study confirm the importance of combining different molecular techniques to characterize the biodiversity of complex ecosystems such as edible insects. The presence of potential human pathogens suggests the need for a careful application of good manufacturing practices during insect processing. This study provides further data that will be useful in risk analyses of edible insects as a novel food source. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. One Step Quantum Key Distribution Based on EPR Entanglement.

    Science.gov (United States)

    Li, Jian; Li, Na; Li, Lei-Lei; Wang, Tao

    2016-06-30

    A novel quantum key distribution protocol is presented, based on entanglement and dense coding and allowing asymptotically secure key distribution. Considering the storage time limit of quantum bits, a grouping quantum key distribution protocol is proposed, which overcomes the vulnerability of first protocol and improves the maneuverability. Moreover, a security analysis is given and a simple type of eavesdropper's attack would introduce at least an error rate of 46.875%. Compared with the "Ping-pong" protocol involving two steps, the proposed protocol does not need to store the qubit and only involves one step.

  11. One-step Ge/Si epitaxial growth.

    Science.gov (United States)

    Wu, Hung-Chi; Lin, Bi-Hsuan; Chen, Huang-Chin; Chen, Po-Chin; Sheu, Hwo-Shuenn; Lin, I-Nan; Chiu, Hsin-Tien; Lee, Chi-Young

    2011-07-01

    Fabricating a low-cost virtual germanium (Ge) template by epitaxial growth of Ge films on silicon wafer with a Ge(x)Si(1-x) (0 deposition method in one step by decomposing a hazardousless GeO(2) powder under hydrogen atmosphere without ultra-high vacuum condition and then depositing in a low-temperature region. X-ray diffraction analysis shows that the Ge film with an epitaxial relationship is along the in-plane direction of Si. The successful growth of epitaxial Ge films on Si substrate demonstrates the feasibility of integrating various functional devices on the Ge/Si substrates.

  12. ONE STEP SYNTHESIS OF MAGNETIC PARTICLES COVERED WITH CASEIN SURFACTANT

    OpenAIRE

    Urquijo Morales, Jeaneth Patricia; Casanova Yepes, Herley; Morales Aramburo, Álvaro Luis; Zysler, Roberto Daniel

    2014-01-01

    The one-step coprecipitation method is used to obtain magnetic nanoparticles controlling the pH (10 and 12), and casein surfactant (CS) concentrations (1 % and 3 % (m/m)). CS has not been used so far for stabilizing magnetic iron oxide ferrofluids. The magnetic nanoparticles have a magnetite core with maghemite in surface, and a shell of polymer. The transmission electron images confirm the crystallinity, particle size distribution in the range of 5-10 nm, and the spinel structure of the nano...

  13. One-Step-Ahead Predictive Control for Hydroturbine Governor

    Directory of Open Access Journals (Sweden)

    Zhihuai Xiao

    2015-01-01

    Full Text Available The hydroturbine generator regulating system can be considered as one system synthetically integrating water, machine, and electricity. It is a complex and nonlinear system, and its configuration and parameters are time-dependent. A one-step-ahead predictive control based on on-line trained neural networks (NNs for hydroturbine governor with variation in gate position is described in this paper. The proposed control algorithm consists of a one-step-ahead neuropredictor that tracks the dynamic characteristics of the plant and predicts its output and a neurocontroller to generate the optimal control signal. The weights of two NNs, initially trained off-line, are updated on-line according to the scalar error. The proposed controller can thus track operating conditions in real-time and produce the optimal control signal over the wide operating range. Only the inputs and outputs of the generator are measured and there is no need to determine the other states of the generator. Simulations have been performed with varying operating conditions and different disturbances to compare the performance of the proposed controller with that of a conventional PID controller and validate the feasibility of the proposed approach.

  14. External and semi-internal controls for PCR amplification of homologous sequences in mixed templates.

    Science.gov (United States)

    Kalle, Elena; Gulevich, Alexander; Rensing, Christopher

    2013-11-01

    In a mixed template, the presence of homologous target DNA sequences creates environments that almost inevitably give rise to artifacts and biases during PCR. Heteroduplexes, chimeras, and skewed template-to-product ratios are the exclusive attributes of mixed template PCR and never occur in a single template assay. Yet, multi-template PCR has been used without appropriate attention to quality control and assay validation, in spite of the fact that such practice diminishes the reliability of results. External and internal amplification controls became obligatory elements of good laboratory practice in different PCR assays. We propose the inclusion of an analogous approach as a quality control system for multi-template PCR applications. The amplification controls must take into account the characteristics of multi-template PCR and be able to effectively monitor particular assay performance. This study demonstrated the efficiency of a model mixed template as an adequate external amplification control for a particular PCR application. The conditions of multi-template PCR do not allow implementation of a classic internal control; therefore we developed a convenient semi-internal control as an acceptable alternative. In order to evaluate the effects of inhibitors, a model multi-template mix was amplified in a mixture with DNAse-treated sample. Semi-internal control allowed establishment of intervals for robust PCR performance for different samples, thus enabling correct comparison of the samples. The complexity of the external and semi-internal amplification controls must be comparable with the assumed complexity of the samples. We also emphasize that amplification controls should be applied in multi-template PCR regardless of the post-assay method used to analyze products. © 2013 Elsevier B.V. All rights reserved.

  15. A new trilocus sequence-based multiplex-PCR to detect major Acinetobacter baumannii clones.

    Science.gov (United States)

    Martins, Natacha; Picão, Renata Cristina; Cerqueira-Alves, Morgana; Uehara, Aline; Barbosa, Lívia Carvalho; Riley, Lee W; Moreira, Beatriz Meurer

    2016-08-01

    A collection of 163 Acinetobacter baumannii isolates detected in a large Brazilian hospital, was potentially related with the dissemination of four clonal complexes (CC): 113/79, 103/15, 109/1 and 110/25, defined by University of Oxford/Institut Pasteur multilocus sequence typing (MLST) schemes. The urge of a simple multiplex-PCR scheme to specify these clones has motivated the present study. The established trilocus sequence-based typing (3LST, for ompA, csuE and blaOXA-51-like genes) multiplex-PCR rapidly identifies international clones I (CC109/1), II (CC118/2) and III (CC187/3). Thus, the system detects only one (CC109/1) out of four main CC in Brazil. We aimed to develop an alternative multiplex-PCR scheme to detect these clones, known to be present additionally in Africa, Asia, Europe, USA and South America. MLST, performed in the present study to complement typing our whole collection of isolates, confirmed that all isolates belonged to the same four CC detected previously. When typed by 3LST-based multiplex-PCR, only 12% of the 163 isolates were classified into groups. By comparative sequence analysis of ompA, csuE and blaOXA-51-like genes, a set of eight primers was designed for an alternative multiplex-PCR to distinguish the five CC 113/79, 103/15, 109/1, 110/25 and 118/2. Study isolates and one CC118/2 isolate were blind-tested with the new alternative PCR scheme; all were correctly clustered in groups of the corresponding CC. The new multiplex-PCR, with the advantage of fitting in a single reaction, detects five leading A. baumannii clones and could help preventing the spread in healthcare settings. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Fusion primer and nested integrated PCR (FPNI-PCR: a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning

    Directory of Open Access Journals (Sweden)

    Wang Zhen

    2011-11-01

    Full Text Available Abstract Background The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and Arabidopsis. The currently available methods for flanking sequence cloning, including the popular TAIL-PCR technique, are relatively laborious and slow. Results Here, we report a simple and effective fusion primer and nested integrated PCR method (FPNI-PCR for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs. These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall FPNI-PCR protocol. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 MYB genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and Arabidopsis. Conclusions The successful amplification of target products through FPNI-PCR verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, FPNI-PCR represents a more sensitive, rapid and accurate technique than the established TAIL-PCR and hiTAIL-PCR procedures.

  17. Formation of organoclays by a one step synthesis

    Science.gov (United States)

    Jaber, Maguy; Miéhé-Brendlé, Jocelyne; Delmotte, Luc; Le Dred, Ronan

    2005-05-01

    Different lamellar hybrid inorganic-organic materials having as inorganic parent 2:1 (T.O.T.) phyllosilicates such as talc, saponite, pyrophyllite, beidellite and montmorillonite were prepared by a one step synthesis. The solids were characterized by X-ray diffraction, solid state 29Si, 27Al, and 19F nuclear magnetic resonance and transmission electron microscopy. XRD patterns show that solids with inorganic parents having octahedral sheet based on aluminium exhibit a lamellar structure similar to MCM-50, whereas those with magnesium have an organophyllosilicate structure. In the first case, the absence of hexacoordinated aluminium was confirmed by 27Al NMR and an ordered stacking of the layers is observed on TEM micrographs. In opposite, a disorder is observed on the TEM images of organophyllosilicates. The formation of the 2:1 structure was found to be controlled mainly by the insertion of silicic species in the interlamellar space of brucite like layers.

  18. One-step method for the production of nanofluids

    Science.gov (United States)

    Kostic, Milivoje [Chicago, IL; Golubovic, Mihajlo [Chicago, IL; Hull, John R [Downers Grove, IL; Choi, Stephen U. S. [Napersville, IL

    2010-05-18

    A one step method and system for producing nanofluids by a particle-source evaporation and deposition of the evaporant into a base fluid. The base fluid such (i.e. ethylene glycol) is placed in a rotating cylindrical drum having an adjustable heater-boat-evaporator and heat exchanger-cooler apparatus. As the drum rotates, a thin liquid layer is formed on the inside surface of the drum. A heater-boat-evaporator having an evaporant material (particle-source) placed within its boat evaporator is adjustably positioned near a portion of the rotating thin liquid layer, the evaporant material being heated thereby evaporating a portion of the evaporant material, the evaporated material absorbed by the liquid film to form nanofluid.

  19. One-step Fabrication of Cellulose/Graphene Conductive Paper

    Institute of Scientific and Technical Information of China (English)

    KaiWen Mou; LuMing Yang; HuangWei Xiong; RuiTao Cha

    2017-01-01

    In this study,a straightforward,one-step wet-end formation process was employed to prepare cellulose/graphene conductive paper for antistatic packing materials.Cationic polyacrylamide was introduced into the cellulose/graphene slurry to improve the graphene loading on the surfaces of the cellulose fibers.The effect of the super calender process on the properties of the cellulose/graphene conductive paper was investigated.When 55 wt% graphene was added,the volume resistivity of the cellulose/graphene conductive paper was 94.70 Ω·cm,decreasing to 35.46 Ω·cm after the super calender process.The cellulose/graphene conductive paper possessed excellent anti-static ability and could be used as an anti-static material.

  20. One step geometrical calibration method for optical coherence tomography

    International Nuclear Information System (INIS)

    Díaz, Jesús Díaz; Ortmaier, Tobias; Stritzel, Jenny; Rahlves, Maik; Reithmeier, Eduard; Roth, Bernhard; Majdani, Omid

    2016-01-01

    We present a novel one-step calibration methodology for geometrical distortion correction for optical coherence tomography (OCT). A calibration standard especially designed for OCT is introduced, which consists of an array of inverse pyramidal structures. The use of multiple landmarks situated on four different height levels on the pyramids allow performing a 3D geometrical calibration. The calibration procedure itself is based on a parametric model of the OCT beam propagation. It is validated by experimental results and enables the reduction of systematic errors by more than one order of magnitude. In future, our results can improve OCT image reconstruction and interpretation for medical applications such as real time monitoring of surgery. (paper)

  1. Yeast identification by sequencing, biochemical kits, MALDI-TOF MS and rep-PCR DNA fingerprinting.

    Science.gov (United States)

    Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Chan, Jasper F W; Lau, Susanna K P; Kong, Fanrong; Xu, Yingchun; Woo, Patrick C Y

    2017-12-08

    No study has comprehensively evaluated the performance of 28S nrDNA and ITS sequencing, commercial biochemical test kits, MALDI-TOF MS platforms, and the emerging rep-PCR DNA fingerprinting technology using a cohort of yeast strains collected from a clinical microbiology laboratory. In this study, using 71 clinically important yeast isolates (excluding Candida albicans) collected from a single centre, we determined the concordance of 28S nrDNA and ITS sequencing and evaluated the performance of two commercial test kits, two MALDI-TOF MS platforms, and rep-PCR DNA fingerprinting. 28S nrDNA and ITS sequencing showed complete agreement on the identities of the 71 isolates. Using sequencing results as the standard, 78.9% and 71.8% isolates were correctly identified using the API 20C AUX and Vitek 2 YST ID Card systems, respectively; and 90.1% and 80.3% isolates were correctly identified using the Bruker and Vitek MALDI-TOF MS platforms, respectively. Of the 18 strains belonging to the Candida parapsilosis species complex tested by DiversiLab automated rep-PCR DNA fingerprinting, all were identified only as Candida parapsilosis with similarities ≥93.2%, indicating the misidentification of Candida metapsilosis and Candida orthopsilosis. However, hierarchical cluster analysis of the rep-PCR DNA fingerprints of these three species within this species complex formed three different discrete clusters, indicating that this technology can potentially differentiate the three species. To achieve higher accuracies of identification, the databases of commercial biochemical test kits, MALDI-TOF MS platforms, and DiversiLab automated rep-PCR DNA fingerprinting needs further enrichment, particularly for uncommonly encountered yeast species. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Synthetic internal control sequences to increase negative call veracity in multiplexed, quantitative PCR assays for Phakopsora pachyrhizi

    Science.gov (United States)

    Quantitative PCR (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5’-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi, the soybean rust pathogen. Because of the extreme sensitivity of Q-PCR, the DNA of a single u...

  3. Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing

    DEFF Research Database (Denmark)

    Wu, Jia Qian; Shteynberg, David; Arumugam, Manimozhiyan

    2004-01-01

    an alternative approach: reverse transcription-polymerase chain reaction (RT-PCR) and direct sequencing based on dual-genome de novo predictions from TWINSCAN. We tested 444 TWINSCAN-predicted rat genes that showed significant homology to known human genes implicated in disease but that were partially...... in the single-intron experiment. Spliced sequences were amplified in 46 cases (34%). We conclude that this procedure for elucidating gene structures with native cDNA sequences is cost-effective and will become even more so as it is further optimized.......The publication of a draft sequence of a third mammalian genome--that of the rat--suggests a need to rethink genome annotation. New mammalian sequences will not receive the kind of labor-intensive annotation efforts that are currently being devoted to human. In this paper, we demonstrate...

  4. Ensemble Kalman filtering with one-step-ahead smoothing

    KAUST Repository

    Raboudi, Naila F.

    2018-01-11

    The ensemble Kalman filter (EnKF) is widely used for sequential data assimilation. It operates as a succession of forecast and analysis steps. In realistic large-scale applications, EnKFs are implemented with small ensembles and poorly known model error statistics. This limits their representativeness of the background error covariances and, thus, their performance. This work explores the efficiency of the one-step-ahead (OSA) smoothing formulation of the Bayesian filtering problem to enhance the data assimilation performance of EnKFs. Filtering with OSA smoothing introduces an updated step with future observations, conditioning the ensemble sampling with more information. This should provide an improved background ensemble in the analysis step, which may help to mitigate the suboptimal character of EnKF-based methods. Here, the authors demonstrate the efficiency of a stochastic EnKF with OSA smoothing for state estimation. They then introduce a deterministic-like EnKF-OSA based on the singular evolutive interpolated ensemble Kalman (SEIK) filter. The authors show that the proposed SEIK-OSA outperforms both SEIK, as it efficiently exploits the data twice, and the stochastic EnKF-OSA, as it avoids observational error undersampling. They present extensive assimilation results from numerical experiments conducted with the Lorenz-96 model to demonstrate SEIK-OSA’s capabilities.

  5. One-Step Protein Conjugation to Upconversion Nanoparticles.

    Science.gov (United States)

    Lu, Jie; Chen, Yinghui; Liu, Deming; Ren, Wei; Lu, Yiqing; Shi, Yu; Piper, James; Paulsen, Ian; Jin, Dayong

    2015-10-20

    The emerging upconversion nanoparticles offer a fascinating library of ultrasensitive luminescent probes for a range of biotechnology applications from biomarker discovery to single molecule tracking, early disease diagnosis, deep tissue imaging, and drug delivery and therapies. The effective bioconjugation of inorganic nanoparticles to the molecule-specific proteins, free of agglomeration, nonspecific binding, or biomolecule deactivation, is crucial for molecular recognition of target molecules or cells. The current available protocols require multiple steps which can lead to low probe stability, specificity, and reproducibility. Here we report a simple and rapid protein bioconjugation method based on a one-step ligand exchange using the DNAs as the linker. Our method benefits from the robust DNA-protein conjugates as well as from multiple ions binding capability. Protein can be preconjugated via an amino group at the 3' end of a synthetic DNA molecule, so that the 5' end phosphoric acid group and multiple phosphate oxygen atoms in the phosphodiester bonds are exposed to replace the oleic acid ligands on the surface of upconversion nanoparticles due to their stronger chelating capability to lanthanides. We demonstrated that our method can efficiently pull out the upconversion nanoparticles from organic solvent into an aqueous phase. The upconversion nanoparticles then become hydrophilic, stable, and specific biomolecules recognition. This allows us to successfully functionalize the upconversion nanoparticles with horseradish peroxidise (HRP) for catalytic colorimetric assay and for streptavidin (SA)-biotin immunoassays.

  6. ONE STEP SYNTHESIS OF MAGNETIC PARTICLES COVERED WITH CASEIN SURFACTANT

    Directory of Open Access Journals (Sweden)

    Jeaneth Patricia Urquijo Morales

    Full Text Available The one-step coprecipitation method is used to obtain magnetic nanoparticles controlling the pH (10 and 12, and casein surfactant (CS concentrations (1 % and 3 % (m/m. CS has not been used so far for stabilizing magnetic iron oxide ferrofluids. The magnetic nanoparticles have a magnetite core with maghemite in surface, and a shell of polymer. The transmission electron images confirm the crystallinity, particle size distribution in the range of 5-10 nm, and the spinel structure of the nanoparticles. Mössbauer results at 80 K showed line shapes dominated by magnetic relaxation effects with sextets and combinations of sextets and doublets. The interactions of the surfactant with the nanoparticle surface are strong showing at least two surfactant layers. The magnetic behavior was evaluated by moment versus temperature and magnetic field measurements. The nanoparticles showed superparamagnetic behavior at room temperature and blocked (irreversible behavior at 5 K. The saturation magnetization presented lower values than reported bulk systems due to the presence of a large layer of maghemite. The FC/ZFC magnetization vs. temperature curves confirmed the superparamagnetic nature of the iron oxide particles and the strong interactions for pH 12 samples and weak interactions for pH 10 samples. The particle growth was dominated by the surface properties of the nanoparticles.

  7. One Step Hydrogen Generation Through Sorption Enhanced Reforming

    Energy Technology Data Exchange (ETDEWEB)

    Mays, Jeff [Gas Technology Inst., Des Plaines, IL (United States)

    2017-08-03

    One-step hydrogen generation, using Sorption Enhanced Reforming (SER) technology, is an innovative means of providing critical energy and environmental improvements to US manufacturing processes. The Gas Technology Institute (GTI) is developing a Compact Hydrogen Generator (CHG) process, based on SER technology, which successfully integrates previously independent process steps, achieves superior energy efficiency by lowering reaction temperatures, and provides pathways to doubling energy productivity with less environmental pollution. GTI’s prior CHG process development efforts have culminated in an operational pilot plant. During the initial pilot testing, GTI identified two operating risks- 1) catalyst coating with calcium aluminate compounds, 2) limited solids handling of the sorbent. Under this contract GTI evaluated alternative materials (one catalyst and two sorbents) to mitigate both risks. The alternate catalyst met performance targets and did not experience coating with calcium aluminate compounds of any kind. The alternate sorbent materials demonstrated viable operation, with one material enabling a three-fold increase in sorbent flow. The testing also demonstrated operation at 90% of its rated capacity. Lastly, a carbon dioxide co-production study was performed to assess the advantage of the solid phase separation of carbon dioxide- inherent in the CHG process. Approximately 70% lower capital cost is achievable compared to SMR-based hydrogen production with CO2 capture, as well as improved operating costs.

  8. A Comparison of Molecular Typing Methods Applied to Enterobacter cloacae complex: hsp60 Sequencing, Rep-PCR, and MLST

    Directory of Open Access Journals (Sweden)

    Roberto Viau

    2017-02-01

    Full Text Available Molecular typing using repetitive sequenced-based PCR (rep-PCR and hsp60 sequencing were applied to a collection of diverse Enterobacter cloacae complex isolates. To determine the most practical method for reference laboratories, we analyzed 71 E. cloacae complex isolates from sporadic and outbreak occurrences originating from 4 geographic areas. While rep-PCR was more discriminating, hsp60 sequencing provided a broader and a more objective geographical tracking method similar to multilocus sequence typing (MLST. In addition, we suggest that MLST may have higher discriminative power compared to hsp60 sequencing, although rep-PCR remains the most discriminative method for local outbreak investigations. In addition, rep-PCR can be an effective and inexpensive method for local outbreak investigation.

  9. [Influence of PCR cycle number on microbial diversity analysis through next generation sequencing].

    Science.gov (United States)

    An, Yunhe; Gao, Lijuan; Li, Junbo; Tian, Yanjie; Wang, Jinlong; Zheng, Xuejuan; Wu, Huijuan

    2016-08-25

    Using of high throughput sequencing technology to study the microbial diversity in complex samples has become one of the hottest issues in the field of microbial diversity research. In this study, the soil and sheep rumen chyme samples were used to extract DNA, respectively. Then the 25 ng total DNA was used to amplify the 16S rRNA V3 region with 20, 25, 30 PCR cycles, and the final sequencing library was constructed by mixing equal amounts of purified PCR products. Finally, the operational taxonomic unit (OUT) amount, rarefaction curve, microbial number and species were compared through data analysis. It was found that at the same amount of DNA template, the proportion of the community composition was not the best with more numbers of PCR cycle, although the species number was much more. In all, when the PCR cycle number is 25, the number of species and proportion of the community composition were the most optimal both in soil or chyme samples.

  10. Sequence diversity in haloalkane dehalogenases, as revealed by PCR using family-specific primers

    Czech Academy of Sciences Publication Activity Database

    Kotík, Michael; Faměrová, Veronika

    2012-01-01

    Roč. 88, č. 2 (2012), s. 212-217 ISSN 0167-7012 R&D Projects: GA ČR GAP504/10/0137; GA ČR GAP207/10/0135 Institutional research plan: CEZ:AV0Z50200510 Keywords : Dehalogenation * Consensus sequence * Degenerate PCR primer Subject RIV: EE - Microbiology, Virology Impact factor: 2.161, year: 2012

  11. PCR amplification of repetitive sequences as a possible approach in relative species quantification

    DEFF Research Database (Denmark)

    Ballin, Nicolai Zederkopff; Vogensen, Finn Kvist; Karlsson, Anders H

    2012-01-01

    Abstract Both relative and absolute quantifications are possible in species quantification when single copy genomic DNA is used. However, amplification of single copy genomic DNA does not allow a limit of detection as low as one obtained from amplification of repetitive sequences. Amplification...... of repetitive sequences is therefore frequently used in absolute quantification but problems occur in relative quantification as the number of repetitive sequences is unknown. A promising approach was developed where data from amplification of repetitive sequences were used in relative quantification of species...... to relatively quantify the amount of chicken DNA in a binary mixture of chicken DNA and pig DNA. However, the designed PCR primers lack the specificity required for regulatory species control....

  12. One step facile synthesis of ferromagnetic magnetite nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Suppiah, Durga Devi; Abd Hamid, Sharifah Bee, E-mail: sharifahbee@um.edu.my

    2016-09-15

    The ferromagnetic properties of magnetite (Fe{sub 3}O{sub 4}) were influenced by the nanoparticle size, hence importance were given to the synthesis method. This paper clearly shows that magnetite nanoparticles were successfully synthesized by employing one step controlled precipitation method using a single salt (Iron(II) sulfate) iron precursor. The acquired titration curve from this method provides vital information on the possible reaction mechanism leading to the magnetite (Fe{sub 3}O{sub 4}) nanoparticles formation. Goethite (α-FeOOH) was obtained at pH 4, while the continuous addition of hydroxyl ions (OH{sup −}) forms iron hydroxides (Fe(OH){sub 2}). This subsequently reacts with the goethite, producing magnetite (Fe{sub 3}O{sub 4}) at pH 10. Spectroscopy studies validate the magnetite phase existence while structural and morphology analysis illustrates cubic shaped magnetite with an average size of 35 nm was obtained. The synthesized magnetite might be superparamagnetic though lower saturation magnetization (67.5 emu/g) measured at room temperature as compared to bulk magnetite. However the nanoparticles surface anisotropy leads to higher remanence (12 emu/g) and coercivity (117.7 G) making the synthesized magnetite an excellent candidate to be utilized in recording devices. The understanding of the magnetite synthesis mechanism can not only be used to achieve even smaller magnetite nanoparticles but also to prepare different types of iron oxides hydroxides using different iron precursor source. - Highlights: • Magnetite strong magnetism properties make it versatile in various applications including biomedical and electromagnetic materials. • Sulfate (SO{sub 4}{sup 2−}) anion plays a major role in the structure control of iron oxide during synthesis. • Phase pure magnetite nanoparticles with high magnetism properties can be obtained using a single salt (SO{sub 4}{sup 2−}) method.

  13. PCR amplification and DNA sequencing of Demodex injai from otic secretions of a dog.

    Science.gov (United States)

    Milosevic, Milivoj A; Frank, Linda A; Brahmbhatt, Rupal A; Kania, Stephen A

    2013-04-01

    The identification of Demodex mites from dogs is usually based on morphology and location. Mites with uncharacteristic features or from unusual locations, hosts or disease manifestations could represent new species not previously described; however, this is difficult to determine based on morphology alone. The goal of this study was to identify and confirm Demodex injai in association with otitis externa in a dog using PCR amplification and DNA sequencing. Otic samples were obtained from a beagle in which a long-bodied Demodex mite was identified. For comparison, Demodex mite samples were collected from a swab and scraping of the dorsal skin of a wire-haired fox terrier and an otic sample from a dog with generalized and otic demodicosis. To identify the Demodex mite, DNA was extracted, and 16S rRNA was amplified by PCR, sequenced and compared with Demodex sequences available in public databases and from separate samples morphologically diagnosed as D. injai and Demodex canis. PCR amplification of the long-bodied mite rRNA DNA obtained from otic samples was approximately 330 bp and was identical to that from the mite morphologically identified as D. injai obtained from the dorsal skin of a dog. Furthermore, the examined mite did not have any significant homology to any of the reported genes from Demodex spp. These results confirmed that the demodex mites in this case were D. injai. © 2013 The Authors. Veterinary Dermatology © 2013 ESVD and ACVD.

  14. Nested PCR Biases in Interpreting Microbial Community Structure in 16S rRNA Gene Sequence Datasets.

    Science.gov (United States)

    Yu, Guoqin; Fadrosh, Doug; Goedert, James J; Ravel, Jacques; Goldstein, Alisa M

    2015-01-01

    Sequencing of the PCR-amplified 16S rRNA gene has become a common approach to microbial community investigations in the fields of human health and environmental sciences. This approach, however, is difficult when the amount of DNA is too low to be amplified by standard PCR. Nested PCR can be employed as it can amplify samples with DNA concentration several-fold lower than standard PCR. However, potential biases with nested PCRs that could affect measurement of community structure have received little attention. In this study, we used 17 DNAs extracted from vaginal swabs and 12 DNAs extracted from stool samples to study the influence of nested PCR amplification of the 16S rRNA gene on the estimation of microbial community structure using Illumina MiSeq sequencing. Nested and standard PCR methods were compared on alpha- and beta-diversity metrics and relative abundances of bacterial genera. The effects of number of cycles in the first round of PCR (10 vs. 20) and microbial diversity (relatively low in vagina vs. high in stool) were also investigated. Vaginal swab samples showed no significant difference in alpha diversity or community structure between nested PCR and standard PCR (one round of 40 cycles). Stool samples showed significant differences in alpha diversity (except Shannon's index) and relative abundance of 13 genera between nested PCR with 20 cycles in the first round and standard PCR (Pnested PCR with 10 cycles in the first round and standard PCR. Operational taxonomic units (OTUs) that had low relative abundance (sum of relative abundance 27% of total OTUs in stool). Nested PCR introduced bias in estimated diversity and community structure. The bias was more significant for communities with relatively higher diversity and when more cycles were applied in the first round of PCR. We conclude that nested PCR could be used when standard PCR does not work. However, rare taxa detected by nested PCR should be validated by other technologies.

  15. Advantages and Limitations of Ribosomal RNA PCR and DNA Sequencing for Identification of Bacteria in Cardiac Valves of Danish Patients

    DEFF Research Database (Denmark)

    Kemp, Michael; Bangsborg, Jette; Kjerulf, Anne

    2013-01-01

    of direct molecular identification should also address weaknesses, their relevance in the given setting, and possible improvements. In this study cardiac valves from 56 Danish patients referred for surgery for infective endocarditis were analysed by microscopy and culture as well as by PCR targeting part...... of the bacterial 16S rRNA gene followed by DNA sequencing of the PCR product. PCR and DNA sequencing identified significant bacteria in 49 samples from 43 patients, including five out of 13 culture-negative cases. No rare, exotic, or intracellular bacteria were identified. There was a general agreement between...... bacterial identity obtained by ribosomal PCR and DNA sequencing from the valves and bacterial isolates from blood culture. However, DNA sequencing of the 16S rRNA gene did not discriminate well among non-haemolytic streptococci, especially within the Streptococcus mitis group. Ribosomal PCR with subsequent...

  16. Use of amplicon sequencing to improve sensitivity in PCR-based detection of microbial pathogen in environmental samples.

    Science.gov (United States)

    Saingam, Prakit; Li, Bo; Yan, Tao

    2018-06-01

    DNA-based molecular detection of microbial pathogens in complex environments is still plagued by sensitivity, specificity and robustness issues. We propose to address these issues by viewing them as inadvertent consequences of requiring specific and adequate amplification (SAA) of target DNA molecules by current PCR methods. Using the invA gene of Salmonella as the model system, we investigated if next generation sequencing (NGS) can be used to directly detect target sequences in false-negative PCR reaction (PCR-NGS) in order to remove the SAA requirement from PCR. False-negative PCR and qPCR reactions were first created using serial dilutions of laboratory-prepared Salmonella genomic DNA and then analyzed directly by NGS. Target invA sequences were detected in all false-negative PCR and qPCR reactions, which lowered the method detection limits near the theoretical minimum of single gene copy detection. The capability of the PCR-NGS approach in correcting false negativity was further tested and confirmed under more environmentally relevant conditions using Salmonella-spiked stream water and sediment samples. Finally, the PCR-NGS approach was applied to ten urban stream water samples and detected invA sequences in eight samples that would be otherwise deemed Salmonella negative. Analysis of the non-target sequences in the false-negative reactions helped to identify primer dime-like short sequences as the main cause of the false negativity. Together, the results demonstrated that the PCR-NGS approach can significantly improve method sensitivity, correct false-negative detections, and enable sequence-based analysis for failure diagnostics in complex environmental samples. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Flanking sequence determination and specific PCR identification of transgenic wheat B102-1-2.

    Science.gov (United States)

    Cao, Jijuan; Xu, Junyi; Zhao, Tongtong; Cao, Dongmei; Huang, Xin; Zhang, Piqiao; Luan, Fengxia

    2014-01-01

    The exogenous fragment sequence and flanking sequence between the exogenous fragment and recombinant chromosome of transgenic wheat B102-1-2 were successfully acquired using genome walking technology. The newly acquired exogenous fragment encoded the full-length sequence of transformed genes with transformed plasmid and corresponding functional genes including ubi, vector pBANF-bar, vector pUbiGUSPlus, vector HSP, reporter vector pUbiGUSPlus, promoter ubiquitin, and coli DH1. A specific polymerase chain reaction (PCR) identification method for transgenic wheat B102-1-2 was established on the basis of designed primers according to flanking sequence. This established specific PCR strategy was validated by using transgenic wheat, transgenic corn, transgenic soybean, transgenic rice, and non-transgenic wheat. A specifically amplified target band was observed only in transgenic wheat B102-1-2. Therefore, this method is characterized by high specificity, high reproducibility, rapid identification, and excellent accuracy for the identification of transgenic wheat B102-1-2.

  18. Next-generation sequencing can reveal in vitro-generated PCR crossover products: some artifactual sequences correspond to HLA alleles in the IMGT/HLA database.

    Science.gov (United States)

    Holcomb, C L; Rastrou, M; Williams, T C; Goodridge, D; Lazaro, A M; Tilanus, M; Erlich, H A

    2014-01-01

    The high-resolution human leukocyte antigen (HLA) genotyping assay that we developed using 454 sequencing and Conexio software uses generic polymerase chain reaction (PCR) primers for DRB exon 2. Occasionally, we observed low abundance DRB amplicon sequences that resulted from in vitro PCR 'crossing over' between DRB1 and DRB3/4/5. These hybrid sequences, revealed by the clonal sequencing property of the 454 system, were generally observed at a read depth of 5%-10% of the true alleles. They usually contained at least one mismatch with the IMGT/HLA database, and consequently, were easily recognizable and did not cause a problem for HLA genotyping. Sometimes, however, these artifactual sequences matched a rare allele and the automatic genotype assignment was incorrect. These observations raised two issues: (1) could PCR conditions be modified to reduce such artifacts? and (2) could some of the rare alleles listed in the IMGT/HLA database be artifacts rather than true alleles? Because PCR crossing over occurs during late cycles of PCR, we compared DRB genotypes resulting from 28 and (our standard) 35 cycles of PCR. For all 21 cell line DNAs amplified for 35 cycles, crossover products were detected. In 33% of the cases, these hybrid sequences corresponded to named alleles. With amplification for only 28 cycles, these artifactual sequences were not detectable. To investigate whether some rare alleles in the IMGT/HLA database might be due to PCR artifacts, we analyzed four samples obtained from the investigators who submitted the sequences. In three cases, the sequences were generated from true alleles. In one case, our 454 sequencing revealed an error in the previously submitted sequence. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. A one-step miniprep for the isolation of plasmid DNA and lambda phage particles.

    Directory of Open Access Journals (Sweden)

    George Lezin

    Full Text Available Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments E. coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.

  20. Automated purification of Borrelia burgdorferi s.l. PCR products with KingFisherTM magnetic particle processor prior to genome sequencing

    International Nuclear Information System (INIS)

    Maekinen, Johanna; Marttila, Harri; Viljanen, Matti K.

    2001-01-01

    Borrelia burgdorferi sensu lato genospecies were differentiated by PCR-based sequencing of the borrelial flagellin gene. To evaluate the usefulness of KingFisher TM magnetic particle processor in PCR product purification, borrelia PCR products were purified with KingFisher TM magnetic particle processor prior to cycle sequencing and the quality of the sequence data received was analyzed. KingFisher was found to offer a rapid and reliable alternative for borrelial PCR product purification

  1. A Bac Library and Paired-PCR Approach to Mapping and Completing the Genome Sequence of Sulfolobus Solfataricus P2

    DEFF Research Database (Denmark)

    She, Qunxin; Confalonieri, F.; Zivanovic, Y.

    2000-01-01

    The original strategy used in the Sulfolobus solfatnricus genome project was to sequence non overlapping, or minimally overlapping, cosmid or lambda inserts without constructing a physical map. However, after only about two thirds of the genome sequence was completed, this approach became counter......-productive because there was a high sequence bias in the cosmid and lambda libraries. Therefore, a new approach was devised for linking the sequenced regions which may be generally applicable. BAC libraries were constructed and terminal sequences of the clones were determined and used for both end mapping and PCR...

  2. A comparison of EGFR mutation testing methods in lung carcinoma: direct sequencing, real-time PCR and immunohistochemistry.

    Directory of Open Access Journals (Sweden)

    Bárbara Angulo

    Full Text Available The objective of this study is to compare two EGFR testing methodologies (a commercial real-time PCR kit and a specific EGFR mutant immunohistochemistry, with direct sequencing and to investigate the limit of detection (LOD of both PCR-based methods. We identified EGFR mutations in 21 (16% of the 136 tumours analyzed by direct sequencing. Interestingly, the Therascreen EGFR Mutation Test kit was able to characterize as wild-type one tumour that could not be analyzed by direct sequencing of the PCR product. We then compared the LOD of the kit and that of direct sequencing using the available mutant tumours. The kit was able to detect the presence of a mutation in a 1% dilution of the total DNA in nine of the 18 tumours (50%, which tested positive with the real-time quantitative PCR method. In all cases, EGFR mutation was identified at a dilution of 5%. Where the mutant DNA represented 30% of the total DNA, sequencing was able to detect mutations in 12 out of 19 cases (63%. Additional experiments with genetically defined standards (EGFR ΔE746-A750/+ and EGFR L858R/+ yielded similar results. Immunohistochemistry (IHC staining with exon 19-specific antibody was seen in eight out of nine cases with E746-A750del detected by direct sequencing. Neither of the two tumours with complex deletions were positive. Of the five L858R-mutated tumours detected by the PCR methods, only two were positive for the exon 21-specific antibody. The specificity was 100% for both antibodies. The LOD of the real-time PCR method was lower than that of direct sequencing. The mutation specific IHC produced excellent specificity.

  3. Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

    OpenAIRE

    Ram, S.; Vimalin, J.M.; Jambulingam, M.; Tiru, V.; Gopalakrishnan, R.K.; Madhavan, H.N.

    2012-01-01

    Aim: Isolation, dark field detection and microscopic agglutination test (MAT) are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR) - based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septi...

  4. Genotyping by PCR and High-Throughput Sequencing of Commercial Probiotic Products Reveals Composition Biases.

    Directory of Open Access Journals (Sweden)

    Wesley Morovic

    2016-11-01

    Full Text Available Recent advances in microbiome research have brought renewed focus on beneficial bacteria, many of which are available in food and dietary supplements. Although probiotics have historically been defined as microorganisms that convey health benefits when ingested in sufficient viable amounts, this description now includes the stipulation well defined strains, encompassing definitive taxonomy for consumer consideration and regulatory oversight. Here, we evaluated 52 commercial dietary supplements covering a range of labeled species, and determined their content using plate counting, targeted genotyping. Additionally, strain identities were assessed using methods recently published by the United States Pharmacopeial Convention. We also determined the relative abundance of individual bacteria by high-throughput sequencing (HTS of the 16S rRNA sequence using paired-end 2x250bp Illumina MiSeq technology. Using multiple methods, we tested the hypothesis that products do contain the quantitative amount of labeled bacteria, and qualitative list of labeled microbial species. We found that 17 samples (33% were below label claim for CFU prior to their expiration dates. A multiplexed-PCR scheme showed that only 30/52 (58% of the products contained a correctly labeled classification, with issues encompassing incorrect taxonomy, missing species and un-labeled species. The HTS revealed that many blended products consisted predominantly of Lactobacillus acidophilus and Bifidobacterium animalis subsp. lactis. These results highlight the need for reliable methods to qualitatively determine the correct taxonomy and quantitatively ascertain the relative amounts of mixed microbial populations in commercial probiotic products.

  5. Quantitation of next generation sequencing library preparation protocol efficiencies using droplet digital PCR assays - a systematic comparison of DNA library preparation kits for Illumina sequencing.

    Science.gov (United States)

    Aigrain, Louise; Gu, Yong; Quail, Michael A

    2016-06-13

    The emergence of next-generation sequencing (NGS) technologies in the past decade has allowed the democratization of DNA sequencing both in terms of price per sequenced bases and ease to produce DNA libraries. When it comes to preparing DNA sequencing libraries for Illumina, the current market leader, a plethora of kits are available and it can be difficult for the users to determine which kit is the most appropriate and efficient for their applications; the main concerns being not only cost but also minimal bias, yield and time efficiency. We compared 9 commercially available library preparation kits in a systematic manner using the same DNA sample by probing the amount of DNA remaining after each protocol steps using a new droplet digital PCR (ddPCR) assay. This method allows the precise quantification of fragments bearing either adaptors or P5/P7 sequences on both ends just after ligation or PCR enrichment. We also investigated the potential influence of DNA input and DNA fragment size on the final library preparation efficiency. The overall library preparations efficiencies of the libraries show important variations between the different kits with the ones combining several steps into a single one exhibiting some final yields 4 to 7 times higher than the other kits. Detailed ddPCR data also reveal that the adaptor ligation yield itself varies by more than a factor of 10 between kits, certain ligation efficiencies being so low that it could impair the original library complexity and impoverish the sequencing results. When a PCR enrichment step is necessary, lower adaptor-ligated DNA inputs leads to greater amplification yields, hiding the latent disparity between kits. We describe a ddPCR assay that allows us to probe the efficiency of the most critical step in the library preparation, ligation, and to draw conclusion on which kits is more likely to preserve the sample heterogeneity and reduce the need of amplification.

  6. Non PCR-amplified Transcripts and AFLP fragments as reduced representations of the quail genome for 454 Titanium sequencing

    Directory of Open Access Journals (Sweden)

    Leterrier Christine

    2010-07-01

    Full Text Available Abstract Background SNP (Single Nucleotide Polymorphism discovery is now routinely performed using high-throughput sequencing of reduced representation libraries. Our objective was to adapt 454 GS FLX based sequencing methodologies in order to obtain the largest possible dataset from two reduced representations libraries, produced by AFLP (Amplified Fragment Length Polymorphism for genomic DNA, and EST (Expressed Sequence Tag for the transcribed fraction of the genome. Findings The expressed fraction was obtained by preparing cDNA libraries without PCR amplification from quail embryo and brain. To optimize the information content for SNP analyses, libraries were prepared from individuals selected in three quail lines and each individual in the AFLP library was tagged. Sequencing runs produced 399,189 sequence reads from cDNA and 373,484 from genomic fragments, covering close to 250 Mb of sequence in total. Conclusions Both methods used to obtain reduced representations for high-throughput sequencing were successful after several improvements. The protocols may be used for several sequencing applications, such as de novo sequencing, tagged PCR fragments or long fragment sequencing of cDNA.

  7. 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.

  8. Pyrosequencing™ : A one-step method for high resolution HLA typing

    Directory of Open Access Journals (Sweden)

    Marincola Francesco M

    2003-11-01

    Full Text Available Abstract While the use of high-resolution molecular typing in routine matching of human leukocyte antigens (HLA is expected to improve unrelated donor selection and transplant outcome, the genetic complexity of HLA still makes the current methodology limited and laborious. Pyrosequencing™ is a gel-free, sequencing-by-synthesis method. In a Pyrosequencing reaction, nucleotide incorporation proceeds sequentially along each DNA template at a given nucleotide dispensation order (NDO that is programmed into a pyrosequencer. Here we describe the design of a NDO that generates a pyrogram unique for any given allele or combination of alleles. We present examples of unique pyrograms generated from each of two heterozygous HLA templates, which would otherwise remain cis/trans ambiguous using standard sequencing based typing (SBT method. In addition, we display representative data that demonstrate long read and linear signal generation. These features are prerequisite of high-resolution typing and automated data analysis. In conclusion Pyrosequencing is a one-step method for high resolution DNA typing.

  9. Comparison of PCR-RFLP pattern with sequencing analysis of the ITS region of Hyrcanain\\'s Tilia

    Directory of Open Access Journals (Sweden)

    Hamed Yousefzadeh

    2014-01-01

    T. hyrcana and T. rubra from Hyrcanian's origin, but it could not separate T. begonifloia from the other hyrcanian species. In this respect, derived results were similar to sequencing one. In conclusion, with regard to less expensive and less time consuming PCR-RFLP technique and high similarity between its result with sequencing, we recommend this method as a simple and economical method with relatively high efficiency studding plant phylogeny.

  10. Application of PCR-based DNA sequencing technique for the detection of Leptospira in peripheral blood of septicemia patients

    Directory of Open Access Journals (Sweden)

    Ram, S.

    2012-01-01

    Full Text Available Aim: Isolation, dark field detection and microscopic agglutination test (MAT are considered ―gold standard‖ tests for diagnosis of Leptospirosis. Several PCR assays are reported but very few have been evaluated for detection of Leptospirosis. Therefore, this study was undertaken. This study aims to design and standardize polymerase chain reaction (PCR - based DNA sequencing technique for the detection of pathogenic Leptospira from peripheral blood of patients clinically diagnosed with septicemia. Methodology and Results: Two hundred and seven (207 blood samples from patients were diagnosed with septicemia which includes 100 bacterial (other than Leptospira culture positive and 107 bacterial culture negative samples were studied. Primers for Nested PCR targeting LipL32 gene of Leptospira interrogans were designed and the specificity of primers was tested against serum samples positive/negative by either MAT or dark field microscopy. PCR amplified products were further confirmed by DNA sequencing. The standardized nPCR was sensitive and specific to Leptospira interrogans. Twenty-one (21% out of 100 culture positive blood samples, three (2.8% out of 107 culture negative samples showed nPCR positivity and were confirmed as Leptospira interrogans by DNA sequencing (p<0.001. A sensitive nPCR specific to Leptospira interrogans was developed. Conclusion, significance and impact of study: The p value (<0.001 signifies that Leptospira is commonly associated with other bacteria circulating in blood indicating that a decreased immune status is created primarily by a bacterium with enhanced possibility of development of Leptospiral infection probably be of an endogenous origin.

  11. Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons

    Science.gov (United States)

    Haas, Brian J.; Gevers, Dirk; Earl, Ashlee M.; Feldgarden, Mike; Ward, Doyle V.; Giannoukos, Georgia; Ciulla, Dawn; Tabbaa, Diana; Highlander, Sarah K.; Sodergren, Erica; Methé, Barbara; DeSantis, Todd Z.; Petrosino, Joseph F.; Knight, Rob; Birren, Bruce W.

    2011-01-01

    Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products. Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions. Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys. PMID:21212162

  12. Long-PCR based next generation sequencing of the whole mitochondrial genome of the peacock skate Pavoraja nitida (Elasmobranchii: Arhynchobatidae).

    Science.gov (United States)

    Yang, Lei; Naylor, Gavin J P

    2016-01-01

    We determined the complete mitochondrial genome sequence (16,760 bp) of the peacock skate Pavoraja nitida using a long-PCR based next generation sequencing method. It has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 1 control region in the typical vertebrate arrangement. Primers, protocols, and procedures used to obtain this mitogenome are provided. We anticipate that this approach will facilitate rapid collection of mitogenome sequences for studies on phylogenetic relationships, population genetics, and conservation of cartilaginous fishes.

  13. One Step Closer to the Marketplace for State-of-the-Art Wind Turbine

    Science.gov (United States)

    Drivetrain | News | NREL One Step Closer to the Marketplace for State-of-the-Art Wind Turbine Drivetrain One Step Closer to the Marketplace for State-of-the-Art Wind Turbine Drivetrain April 1, 2016 modeling, and testing in state-of-the-art facilities designed to put the drivetrain through its paces

  14. One-step production of biodiesel from Nannochloropsis sp. on solid base Mg-Zr catalyst

    International Nuclear Information System (INIS)

    Li, Yuesong; Lian, Shuang; Tong, Dongmei; Song, Ruili; Yang, Wenyan; Fan, Yong; Qing, Renwei; Hu, Changwei

    2011-01-01

    Nannochloropsis sp., one kind of green microalgae cultivated autotrophically and axenically in laboratory, is used as raw material to produce biodiesel by one-step method in an amended reactor. The effects of several reaction parameters on transesterification over Mg-Zr solid base catalyst were investigated through both conventional method and one-step method. One-step method could give a higher yield of methyl ester than conventional two-step method, which demonstrates that the present one-step method is suitable for biodiesel production from the microalgae Nannochloropsis sp. Moreover, the present one-step method realizes the convenient in situ separation of catalyst from microalgae residue which can be easily used consequently, reducing the procedure units as well as the overall costs.

  15. One step replica symmetry breaking and extreme order statistics of logarithmic REMs

    Directory of Open Access Journals (Sweden)

    Xiangyu Cao, Yan V. Fyodorov, Pierre Le Doussal

    2016-12-01

    Full Text Available Building upon the one-step replica symmetry breaking formalism, duly understood and ramified, we show that the sequence of ordered extreme values of a general class of Euclidean-space logarithmically correlated random energy models (logREMs behave in the thermodynamic limit as a randomly shifted decorated exponential Poisson point process. The distribution of the random shift is determined solely by the large-distance ("infra-red", IR limit of the model, and is equal to the free energy distribution at the critical temperature up to a translation. the decoration process is determined solely by the small-distance ("ultraviolet", UV limit, in terms of the biased minimal process. Our approach provides connections of the replica framework to results in the probability literature and sheds further light on the freezing/duality conjecture which was the source of many previous results for log-REMs. In this way we derive the general and explicit formulae for the joint probability density of depths of the first and second minima (as well its higher-order generalizations in terms of model-specific contributions from UV as well as IR limits. In particular, we show that the second min statistics is largely independent of details of UV data, whose influence is seen only through the mean value of the gap. For a given log-correlated field this parameter can be evaluated numerically, and we provide several numerical tests of our theory using the circular model of $1/f$-noise.

  16. Development of Prevotella intermedia-specific PCR primers based on the nucleotide sequences of a DNA probe Pig27.

    Science.gov (United States)

    Kim, Min Jung; Hwang, Kyung Hwan; Lee, Young-Seok; Park, Jae-Yoon; Kook, Joong-Ki

    2011-03-01

    The aim of this study was to develop Prevotella intermedia-specific PCR primers based on the P. intermedia-specific DNA probe. The P. intermedia-specific DNA probe was screened by inverted dot blot hybridization and confirmed by Southern blot hybridization. The nucleotide sequences of the species-specific DNA probes were determined using a chain termination method. Southern blot analysis showed that the DNA probe, Pig27, detected only the genomic DNA of P. intermedia strains. PCR showed that the PCR primers, Pin-F1/Pin-R1, had species-specificity for P. intermedia. The detection limits of the PCR primer sets were 0.4pg of the purified genomic DNA of P. intermedia ATCC 49046. These results suggest that the PCR primers, Pin-F1/Pin-R1, could be useful in the detection of P. intermedia as well as in the development of a PCR kit in epidemiological studies related to periodontal diseases. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

  17. 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

  18. High Throughput Sample Preparation and Analysis for DNA Sequencing, PCR and Combinatorial Screening of Catalysis Based on Capillary Array Technique

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yonghua [Iowa State Univ., Ames, IA (United States)

    2000-01-01

    Sample preparation has been one of the major bottlenecks for many high throughput analyses. The purpose of this research was to develop new sample preparation and integration approach for DNA sequencing, PCR based DNA analysis and combinatorial screening of homogeneous catalysis based on multiplexed capillary electrophoresis with laser induced fluorescence or imaging UV absorption detection. The author first introduced a method to integrate the front-end tasks to DNA capillary-array sequencers. protocols for directly sequencing the plasmids from a single bacterial colony in fused-silica capillaries were developed. After the colony was picked, lysis was accomplished in situ in the plastic sample tube using either a thermocycler or heating block. Upon heating, the plasmids were released while chromsomal DNA and membrane proteins were denatured and precipitated to the bottom of the tube. After adding enzyme and Sanger reagents, the resulting solution was aspirated into the reaction capillaries by a syringe pump, and cycle sequencing was initiated. No deleterious effect upon the reaction efficiency, the on-line purification system, or the capillary electrophoresis separation was observed, even though the crude lysate was used as the template. Multiplexed on-line DNA sequencing data from 8 parallel channels allowed base calling up to 620 bp with an accuracy of 98%. The entire system can be automatically regenerated for repeated operation. For PCR based DNA analysis, they demonstrated that capillary electrophoresis with UV detection can be used for DNA analysis starting from clinical sample without purification. After PCR reaction using cheek cell, blood or HIV-1 gag DNA, the reaction mixtures was injected into the capillary either on-line or off-line by base stacking. The protocol was also applied to capillary array electrophoresis. The use of cheaper detection, and the elimination of purification of DNA sample before or after PCR reaction, will make this approach an

  19. 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

  20. A dual PCR-based sequencing approach for the identification and discrimination of Echinococcus and Taenia taxa.

    Science.gov (United States)

    Boubaker, Ghalia; Marinova, Irina; Gori, Francesca; Hizem, Amani; Müller, Norbert; Casulli, Adriano; Jerez Puebla, Luis Enrique; Babba, Hamouda; Gottstein, Bruno; Spiliotis, Markus

    2016-08-01

    Reliable and rapid molecular tools for the genetic identification and differentiation of Echinococcus species and/or genotypes are crucial for studying spatial and temporal transmission dynamics. Here, we describe a novel dual PCR targeting regions in the small (rrnS) and large (rrnL) subunits of mitochondrial ribosomal RNA (rRNA) genes, which enables (i) the specific identification of species and genotypes of Echinococcus (rrnS + L-PCR) and/or (ii) the identification of a range of taeniid cestodes, including different species of Echinococcus, Taenia and some others (17 species of diphyllidean helminths). This dual PCR approach was highly sensitive, with an analytical detection limit of 1 pg for genomic DNA of Echinococcus. Using concatenated sequence data derived from the two gene markers (1225 bp), we identified five unique and geographically informative single nucleotide polymorphisms (SNPs) that allowed genotypes (G1 and G3) of Echinococcus granulosus sensu stricto to be distinguished, and 25 SNPs that allowed differentiation within Echinococcus canadensis (G6/7/8/10). In conclusion, we propose that this dual PCR-based sequencing approach can be used for molecular epidemiological studies of Echinococcus and other taeniid cestodes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Combined use of real-time PCR and nested sequence-based typing in survey of human Legionella infection.

    Science.gov (United States)

    Qin, T; Zhou, H; Ren, H; Shi, W; Jin, H; Jiang, X; Xu, Y; Zhou, M; Li, J; Wang, J; Shao, Z; Xu, X

    2016-07-01

    Legionnaires' disease (LD) is a globally distributed systemic infectious disease. The burden of LD in many regions is still unclear, especially in Asian countries including China. A survey of Legionella infection using real-time PCR and nested sequence-based typing (SBT) was performed in two hospitals in Shanghai, China. A total of 265 bronchoalveolar lavage fluid (BALF) specimens were collected from hospital A between January 2012 and December 2013, and 359 sputum specimens were collected from hospital B throughout 2012. A total of 71 specimens were positive for Legionella according to real-time PCR focusing on the 5S rRNA gene. Seventy of these specimens were identified as Legionella pneumophila as a result of real-time PCR amplification of the dotA gene. Results of nested SBT revealed high genetic polymorphism in these L. pneumophila and ST1 was the predominant sequence type. These data revealed that the burden of LD in China is much greater than that recognized previously, and real-time PCR may be a suitable monitoring technology for LD in large sample surveys in regions lacking the economic and technical resources to perform other methods, such as urinary antigen tests and culture methods.

  2. Generic detection of poleroviruses using an RT-PCR assay targeting the RdRp coding sequence.

    Science.gov (United States)

    Lotos, Leonidas; Efthimiou, Konstantinos; Maliogka, Varvara I; Katis, Nikolaos I

    2014-03-01

    In this study a two-step RT-PCR assay was developed for the generic detection of poleroviruses. The RdRp coding region was selected as the primers' target, since it differs significantly from that of other members in the family Luteoviridae and its sequence can be more informative than other regions in the viral genome. Species specific RT-PCR assays targeting the same region were also developed for the detection of the six most widespread poleroviral species (Beet mild yellowing virus, Beet western yellows virus, Cucurbit aphid-borne virus, Carrot red leaf virus, Potato leafroll virus and Turnip yellows virus) in Greece and the collection of isolates. These isolates along with other characterized ones were used for the evaluation of the generic PCR's detection range. The developed assay efficiently amplified a 593bp RdRp fragment from 46 isolates of 10 different Polerovirus species. Phylogenetic analysis using the generic PCR's amplicon sequence showed that although it cannot accurately infer evolutionary relationships within the genus it can differentiate poleroviruses at the species level. Overall, the described generic assay could be applied for the reliable detection of Polerovirus infections and, in combination with the specific PCRs, for the identification of new and uncharacterized species in the genus. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. One-step electrodeposition process of CuInSe2: Deposition time effect

    Indian Academy of Sciences (India)

    Administrator

    CuInSe2 thin films were prepared by one-step electrodeposition process using a simplified two- electrodes system. ... homojunctions or heterojunctions (Rincon et al 1983). Efficiency of ... deposition times onto indium thin oxide (ITO)-covered.

  4. Effect of alternative modes of application on microleakage of one-step self-etch adhesives

    Directory of Open Access Journals (Sweden)

    Mustafa Ulker

    2013-12-01

    Conclusion: Alternative modes of application did not adversely affect the sealing ability of one-step self-etch adhesives. Depending on the type of adhesive, alternative modes of application may contribute to low microleakage values.

  5. One-Step Leapfrog LOD-BOR-FDTD Algorithm with CPML Implementation

    Directory of Open Access Journals (Sweden)

    Yi-Gang Wang

    2016-01-01

    Full Text Available An unconditionally stable one-step leapfrog locally one-dimensional finite-difference time-domain (LOD-FDTD algorithm towards body of revolution (BOR is presented. The equations of the proposed algorithm are obtained by the algebraic manipulation of those used in the conventional LOD-BOR-FDTD algorithm. The equations for z-direction electric and magnetic fields in the proposed algorithm should be treated specially. The new algorithm obtains a higher computational efficiency while preserving the properties of the conventional LOD-BOR-FDTD algorithm. Moreover, the convolutional perfectly matched layer (CPML is introduced into the one-step leapfrog LOD-BOR-FDTD algorithm. The equation of the one-step leapfrog CPML is concise. Numerical results show that its reflection error is small. It can be concluded that the similar CPML scheme can also be easily applied to the one-step leapfrog LOD-FDTD algorithm in the Cartesian coordinate system.

  6. Ultrasensitive quantitation of human papillomavirus type 16 E6 oncogene sequences by nested real time PCR

    Directory of Open Access Journals (Sweden)

    López-Revilla Rubén

    2010-05-01

    Full Text Available Abstract Background We have developed an ultrasensitive method based on conventional PCR preamplification followed by nested amplification through real time PCR (qPCR in the presence of the DNA intercalating agent EvaGreen. Results Amplification mixtures calibrated with a known number of pHV101 copies carrying a 645 base pair (bp-long insert of the human papillomavirus type 16 (HPV16 E6 oncogene were used to generate the E6-1 amplicon of 645 bp by conventional PCR and then the E6-2 amplicon of 237 bp by nested qPCR. Direct and nested qPCR mixtures for E6-2 amplification corresponding to 2.5 × 102-2.5 × 106 initial pHV101 copies had threshold cycle (Ct values in the ranges of 18.7-29.0 and 10.0-25.0, respectively. The Ct of qPCR mixtures prepared with 1/50 volumes of preamplified mixtures containing 50 ng of DNA of the SiHa cell line (derived from an invasive cervical cancer with one HPV16 genome per cell was 19.9. Thermal fluorescence extinction profiles of E6-2 amplicons generated from pHV101 and SiHa DNA were identical, with a peak at 85.5°C. Conclusions Our method based on conventional preamplification for 15 cycles increased 10,750 times the sensitivity of nested qPCR for the quantitation of the E6 viral oncogene and confirmed that the SiHa cell line contains one E6-HPV16 copy per cell.

  7. Genomic Variability of Haemophilus influenzae Isolated from Mexican Children Determined by Using Enterobacterial Repetitive Intergenic Consensus Sequences and PCR

    OpenAIRE

    Gomez-De-Leon, Patricia; Santos, Jose I.; Caballero, Javier; Gomez, Demostenes; Espinosa, Luz E.; Moreno, Isabel; Piñero, Daniel; Cravioto, Alejandro

    2000-01-01

    Genomic fingerprints from 92 capsulated and noncapsulated strains of Haemophilus influenzae from Mexican children with different diseases and healthy carriers were generated by PCR using the enterobacterial repetitive intergenic consensus (ERIC) sequences. A cluster analysis by the unweighted pair-group method with arithmetic averages based on the overall similarity as estimated from the characteristics of the genomic fingerprints, was conducted to group the strains. A total of 69 fingerprint...

  8. A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

    DEFF Research Database (Denmark)

    Uren, Anthony G; Mikkers, Harald; Kool, Jaap

    2009-01-01

    sites has been a major limitation to performing screens on this scale. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA......Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice; however, until recently, the cost-effective isolation and sequencing of insertion...

  9. Simple One-Step Method to Synthesize Polypyrrole-Indigo Carmine-Silver Nanocomposite

    OpenAIRE

    Loguercio, Lara Fernandes; Demingos, Pedro; Manica, Luiza de Mattos; Griep, Jordana Borges; Santos, Marcos José Leite; Ferreira, Jacqueline

    2016-01-01

    A nanocomposite of indigo carmine doped polypyrrole/silver nanoparticles was obtained by a one-step electrochemical process. The nanocomposite was characterized by scanning electron microscopy, infrared spectroscopy, ultraviolet-visible-near infrared spectroscopy, and cyclic voltammetry. The simple one-step process allowed the growth of silver nanoparticles during the polymerization of polypyrrole, resulting in films with electrochromic behavior and improved electroactivity. In addition, poly...

  10. Phylogenetic analysis of Fusobacterium prausnitzii based upon the 16S rRNA gene sequence and PCR confirmation.

    Science.gov (United States)

    Wang, R F; Cao, W W; Cerniglia, C E

    1996-01-01

    In order to develop a PCR method to detect Fusobacterium prausnitzii in human feces and to clarify the phylogenetic position of this species, its 16S rRNA gene sequence was determined. The sequence described in this paper is different from the 16S rRNA gene sequence is specific for F. prausnitzii, and the results of this assay confirmed that F. prausnitzii is the most common species in human feces. However, a PCR assay based on the original GenBank sequence was negative when it was performed with two strains of F. prausnitzii obtained from the American Type Culture Collection. A phylogenetic tree based on the new 16S rRNA gene sequence was constructed. On this tree F. prausnitzii was not a member of the Fusobacterium group but was closer to some Eubacterium spp. and located between Clostridium "clusters III and IV" (M.D. Collins, P.A. Lawson, A. Willems, J.J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J.A.E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994).

  11. A Lab on a chip device for rapid identification of Avian Influenza virus by on-chip sample preparation and solid phase PCR

    DEFF Research Database (Denmark)

    Yi, Sun; Dhumpa, Raghuram; Bang, Dang Duong

    2009-01-01

    In this paper, we describe a novel lab-on-a-chip device for fast AIV screening by integrating DNA microarray-based solid phase PCR on microchip. The device can handle viral samples in an automatic way. Moreover, multiplex PCR and sequence detection are done in one-step, which greatly simplifies...

  12. FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    Directory of Open Access Journals (Sweden)

    Lu Jia

    2011-10-01

    Full Text Available Abstract Background Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. Results Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. Conclusion Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.

  13. Differentiation of sheep pox and goat poxviruses by sequence analysis and PCR-RFLP of P32 gene.

    Science.gov (United States)

    Hosamani, Madhusudan; Mondal, Bimalendu; Tembhurne, Prabhakar A; Bandyopadhyay, Santanu Kumar; Singh, Raj Kumar; Rasool, Thaha Jamal

    2004-08-01

    Sheep pox and Goat pox are highly contagious viral diseases of small ruminants. These diseases were earlier thought to be caused by a single species of virus, as they are serologically indistinguishable. P32, one of the major immunogenic genes of Capripoxvirus, was isolated and Sequenced from two Indian isolates of goat poxvirus (GPV) and a vaccine strain of sheep poxvirus (SPV). The sequences were compared with other P32 sequences of capripoxviruses available in the database. Sequence analysis revealed that sheep pox and goat poxviruses share 97.5 and 94.7% homology at nucleotide and amino acid level, respectively. A major difference between them is the presence of an additional aspartic acid at 55th position of P32 of sheep poxvirus that is absent in both goat poxvirus and lumpy skin disease virus. Further, six unique neutral nucleotide substitutions were observed at positions 77, 275, 403, 552, 867 and 964 in the sequence of goat poxvirus, which can be taken as GPV signature residues. Similar unique nucleotide signatures could be identified in SPV and LSDV sequences also. Phylogenetic analysis showed that members of the Capripoxvirus could be delineated into three distinct clusters of GPV, SPV and LSDV based on the P32 genomic sequence. Using this information, a PCR-RFLP method has been developed for unequivocal genomic differentiation of SPV and GPV.

  14. Effect of One-Step and Multi-Steps Polishing System on Enamel Roughness

    Directory of Open Access Journals (Sweden)

    Cynthia Sumali

    2013-07-01

    Full Text Available Normal 0 false false false MicrosoftInternetExplorer4 The final procedures of orthodontic treatment are bracket debonding and cleaning the remaining adhesive. Multi-step polishing system is the most common method used. The disadvantage of that system is long working time, because of the stages that should be done. Therefore, dental material manufacturer make an improvement to the system, to reduce several stages into one stage only. This new system is known as one-step polishing system. Objective: To compare the effect of one-step and multi-step polishing system on enamel roughness after orthodontic bracket debonding. Methods: Randomized control trial was conducted included twenty-eight maxillary premolar randomized into two polishing system; one-step OptraPol (Ivoclar, Vivadent and multi-step AstroPol (Ivoclar, Vivadent. After bracket debonding, the remaining adhesive on each group was cleaned by subjective polishing system for ninety seconds using low speed handpiece. The enamel roughness was subjected to profilometer, registering two roughness parameters (Ra, Rz. Independent t-test was used to analyze the mean score of enamel roughness in each group. Results: There was no significant difference of enamel roughness between one-step and multi-step polishing system (p>0.005. Conclusion: One-step polishing system can produce a similar enamel roughness to multi-step polishing system after bracket debonding and adhesive cleaning.DOI: 10.14693/jdi.v19i3.136

  15. Evaluation of one-step micro polishers for residual resin removal after debonding on fluorosed teeth

    Directory of Open Access Journals (Sweden)

    Padmalatha Challa

    2014-01-01

    Full Text Available Aim and objectives: To evaluate the effectiveness of one step micro polishers for residual resin removal on fluorosed teeth using scanning electron microscope (SEM. Methods and Material: 55 teeth with mild to moderate fluorosis were selected with five teeth as control. Metal brackets were bonded onto 50 teeth which were divided into 5 groups. The finishing and polishing methods which were tested include tungsten carbide burs (TCB, multistep finishing system (Sof-Lex, one step polishers (PoGo and combination of TCB with multistep and one step polishing systems. After resin removal, all the samples were examined under SEM for assessment of the enamel surface. Results: The enamel surface was closest to untouched enamel in samples finished with the PoGo one step polishers followed by Sof-Lex multistep finishing system. However, they took the longest time to finish. TCB required the shortest time for residual resin removal. Conclusions: All polishing systems produce a certain degree of damage to the enamel surface with the smoothest surface being produced by one step polishers on fluorosed teeth.

  16. Table 1. Primer sequences used for real-time qRT-PCR analysis of ...

    Indian Academy of Sciences (India)

    User

    TGTCCCAGTAAACCGCTC. GAATCCAGCACGATACCAGT. Figure 1. Expression analysis of candidate CsActin and CsFbox genes by qRT-PCR in response to 4°C treatment. The y-axis indicates Cq values, and error bars represent standard deviations of the mean values of four replicates. Rt, roots; St, stems; Le, leaves; Fl ...

  17. PCR Cloning of Partial "nbs" Sequences from Grape ("Vitis aestivalis" Michx)

    Science.gov (United States)

    Chang, Ming-Mei; DiGennaro, Peter; Macula, Anthony

    2009-01-01

    Plants defend themselves against pathogens via the expressions of disease resistance (R) genes. Many plant R gene products contain the characteristic nucleotide-binding site (NBS) and leucine-rich repeat (LRR) domains. There are highly conserved motifs within the NBS domain which could be targeted for polymerase chain reaction (PCR) cloning of R…

  18. Hybridization Capture Using Short PCR Products Enriches Small Genomes by Capturing Flanking Sequences (CapFlank)

    DEFF Research Database (Denmark)

    Tsangaras, Kyriakos; Wales, Nathan; Sicheritz-Pontén, Thomas

    2014-01-01

    , a non-negligible fraction of the resulting sequence reads are not homologous to the bait. We demonstrate that during capture, the bait-hybridized library molecules add additional flanking library sequences iteratively, such that baits limited to targeting relatively short regions (e.g. few hundred...... nucleotides) can result in enrichment across entire mitochondrial and bacterial genomes. Our findings suggest that some of the off-target sequences derived in capture experiments are non-randomly enriched, and that CapFlank will facilitate targeted enrichment of large contiguous sequences with minimal prior...

  19. Identification of a novel Plasmopara halstedii elicitor protein combining de novo peptide sequencing algorithms and RACE-PCR

    Directory of Open Access Journals (Sweden)

    Madlung Johannes

    2010-05-01

    Full Text Available Abstract Background Often high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i. Results The performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set. All three de novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of P. halstedii. We found ten de novo sequenced peptides that showed homology to a Phytophthora infestans protein, a closely related organism of P. halstedii. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase. Conclusions Our study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different de novo peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in P. halstedii.

  20. Reconstruction of networks from one-step data by matching positions

    Science.gov (United States)

    Wu, Jianshe; Dang, Ni; Jiao, Yang

    2018-05-01

    It is a challenge in estimating the topology of a network from short time series data. In this paper, matching positions is developed to reconstruct the topology of a network from only one-step data. We consider a general network model of coupled agents, in which the phase transformation of each node is determined by its neighbors. From the phase transformation information from one step to the next, the connections of the tail vertices are reconstructed firstly by the matching positions. Removing the already reconstructed vertices, and repeatedly reconstructing the connections of tail vertices, the topology of the entire network is reconstructed. For sparse scale-free networks with more than ten thousands nodes, we almost obtain the actual topology using only the one-step data in simulations.

  1. Effect of evaporation of solvents from one-step, self-etching adhesives

    DEFF Research Database (Denmark)

    Furuse, Adilson Yoshio; Peutzfeldt, Anne; Asmussen, Erik

    2008-01-01

    PURPOSE: To investigate whether and to what extent the bonding capacity of one-step, self-etching adhesives is influenced by the degree to which solvent is evaporated. MATERIALS AND METHODS: Seven one-step, self-etching adhesives were tested (Adper Prompt L-Pop, Clearfil S3 Bond, Futurabond NR, G....... After being stored in water at 37 degrees C for 1 week, the bonded specimens were broken in shear. Failure modes were evaluated under stereomicroscope. RESULTS: Air-blowing duration and brand of adhesive both had an effect on shear bond strength. An interaction was found between adhesive and air...... failures were observed with shorter air-blowing durations. A significant negative correlation between number of adhesive failures and bond strength was found. CONCLUSION: On the basis of this in vitro study, it may be concluded that the one-step, self-etching adhesives evaluated were sensitive to degree...

  2. Hybridization Capture Using Short PCR Products Enriches Small Genomes by Capturing Flanking Sequences (CapFlank)

    DEFF Research Database (Denmark)

    Tsangaras, Kyriakos; Wales, Nathan; Sicheritz-Pontén, Thomas

    2014-01-01

    nucleotides) can result in enrichment across entire mitochondrial and bacterial genomes. Our findings suggest that some of the off-target sequences derived in capture experiments are non-randomly enriched, and that CapFlank will facilitate targeted enrichment of large contiguous sequences with minimal prior...

  3. Multisegment one-step RT-PCR fluorescent labeling of influenza A virus genome for use in diagnostic microarray applications

    Energy Technology Data Exchange (ETDEWEB)

    Vasin, A V; Plotnikova, M A; Klotchenko, S A; Elpaeva, E A; Komissarov, A B; Egorov, V V; Kiselev, O I [Research Institute of Influenza of the Ministry of Health and Social Development of the Russian Federation, 15/17 Prof. Popova St., St. Petersburg (Russian Federation); Sandybaev, N T; Chervyakova, O V; Strochkov, V M; Taylakova, E T; Koshemetov, J K; Mamadaliev, S M, E-mail: vasin@influenza.spb.ru [Research Institute for Biological Safety Problems of the RK NBC/SC ME and S RK, Gvardeiskiy (Kazakhstan)

    2011-04-01

    Microarray technology is one of the most challenging methods of influenza A virus subtyping, which is based on the antigenic properties of viral surface glycoproteins - hemagglutinin and neuraminidase. On the example of biochip for detection of influenza A/H5N1 virus we showed the possibility of using multisegment RTPCR method for amplification of fluorescently labeled cDNA of all possible influenza A virus subtypes with a single pair of primers in influenza diagnostic microarrays.

  4. Absence of Mycoplasma-specific DNA sequence in brain, blood and CSF of patients with multiple sclerosis (MS): a study by PCR and real-time PCR.

    Science.gov (United States)

    Casserly, Georgina; Barry, Thomas; Tourtellotte, Wallace W; Hogan, Edward L

    2007-02-15

    Mycoplasmas are the smallest of the known self-replicating organisms. They lack cell walls and are associated with numerous diseases in humans and animals. We are exploring the possibility that infection by Mycoplasma may induce the inflammatory demyelinating disease of the central nervous system (CNS) that is MS. The presence of specific Mycoplasma species DNA was sought in brain, serum and cerebrospinal fluid (CSF) of patients diagnosed with multiple sclerosis (MS) and other neurological diseases (OND) including inflammatory disorders. The MS samples from patients with active and progressive MS, as well as in remission, a variety of other neurological disease controls, including inflammatory CNS diseases such as meningitis, cryptococcal meningitis and encephalitis and other neurological disorders such as migraine were also examined. Clinical samples were provided by the National Neurological Research Specimen Bank and the Human Brain and Spinal Fluid Resource Centre, Los Angeles. Analysis was carried out by conventional PCR using Mycoplasma-specific primers (McAuliffe et al., 2005) that target the 16S rDNA gene in Mycoplasma species. The Mycoplasma-specific primers could detect 102 Mycoplasma species. In this study, 30 samples of human brain and 57 pairs of serum and CSF and were examined. No Mycoplasma-specific nucleic acid sequence was detected, and the consistent observation of an endogenous gene, human serum albumin (HSA), as a positive control documented the adequacy of the method. Real-time PCR analysis of serum and CSF was done also targeting utilizing the Mycoplasma 16S rDNA gene, and this also demonstrated the lack of Mycoplasma in these samples. The presence of Mycoplasma at extraneural sites in MS patients is now being explored.

  5. Simple One-Step Method to Synthesize Polypyrrole-Indigo Carmine-Silver Nanocomposite

    Directory of Open Access Journals (Sweden)

    Lara Fernandes Loguercio

    2016-01-01

    Full Text Available A nanocomposite of indigo carmine doped polypyrrole/silver nanoparticles was obtained by a one-step electrochemical process. The nanocomposite was characterized by scanning electron microscopy, infrared spectroscopy, ultraviolet-visible-near infrared spectroscopy, and cyclic voltammetry. The simple one-step process allowed the growth of silver nanoparticles during the polymerization of polypyrrole, resulting in films with electrochromic behavior and improved electroactivity. In addition, polypyrrole chains in the nanocomposite were found to present longer conjugation length than pristine polypyrrole films.

  6. Synthesis and characterization of copper nanofluid by a novel one-step method

    International Nuclear Information System (INIS)

    Kumar, S. Ananda; Meenakshi, K. Shree; Narashimhan, B.R.V.; Srikanth, S.; Arthanareeswaran, G.

    2009-01-01

    This paper presents a novel one-step method for the preparation of stable, non-agglomerated copper nanofluids by reducing copper sulphate pentahydrate with sodium hypophosphite as reducing agent in ethylene glycol as base fluid by means of conventional heating. This is an in situ, one-step method which gives high yield of product with less time consumption. The characterization of the nanofluid is done by particle size analyzer, X-ray diffraction topography, UV-vis analysis and Fourier transform infrared spectroscopy (FT-IR) followed by the study of thermal conductivity of nanofluid by the transient hot wire method

  7. The symmetric MSD encoder for one-step adder of ternary optical computer

    Science.gov (United States)

    Kai, Song; LiPing, Yan

    2016-08-01

    The symmetric Modified Signed-Digit (MSD) encoding is important for achieving the one-step MSD adder of Ternary Optical Computer (TOC). The paper described the symmetric MSD encoding algorithm in detail, and developed its truth table which has nine rows and nine columns. According to the truth table, the state table was developed, and the optical-path structure and circuit-implementation scheme of the symmetric MSD encoder (SME) for one-step adder of TOC were proposed. Finally, a series of experiments were designed and performed. The observed results of the experiments showed that the scheme to implement SME was correct, feasible and efficient.

  8. Legionella confirmation in cooling tower water. Comparison of culture, real-time PCR and next generation sequencing.

    Science.gov (United States)

    Farhat, Maha; Shaheed, Raja A; Al-Ali, Haider H; Al-Ghamdi, Abdullah S; Al-Hamaqi, Ghadeer M; Maan, Hawraa S; Al-Mahfoodh, Zainab A; Al-Seba, Hussain Z

    2018-02-01

    To investigate the presence of Legionella spp in cooling tower water. Legionella proliferation in cooling tower water has serious public health implications as it can be transmitted to humans via aerosols and cause Legionnaires' disease. Samples of cooling tower water were collected from King Fahd Hospital of the University (KFHU) (Imam Abdulrahman Bin Faisal University, 2015/2016). The water samples were analyzed by a standard Legionella culture method, real-time polymerase chain reaction (RT-PCR), and 16S rRNA next-generation sequencing. In addition, the bacterial community composition was evaluated. All samples were negative by conventional Legionella culture. In contrast, all water samples yielded positive results by real-time PCR (105 to 106 GU/L). The results of 16S rRNA next generation sequencing showed high similarity and reproducibility among the water samples. The majority of sequences were Alpha-, Beta-, and Gamma-proteobacteria, and Legionella was the predominant genus. The hydrogen-oxidizing gram-negative bacterium Hydrogenophaga was present at high abundance, indicating high metabolic activity. Sphingopyxis, which is known for its resistance to antimicrobials and as a pioneer in biofilm formation, was also detected. Our findings indicate that monitoring of Legionella in cooling tower water would be enhanced by use of both conventional culturing and molecular methods.

  9. Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences.

    Directory of Open Access Journals (Sweden)

    Koen M Verstappen

    Full Text Available Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and cats and occasionally causes infections in humans. S. pseudintermedius is often resistant to multiple classes of antimicrobials. It requires a reliable detection so that it is not misidentified as S. aureus. Phenotypic and currently-used molecular-based diagnostic assays lack specificity or are labour-intensive using multiplex PCR or nucleic acid sequencing. The aim of this study was to identify a specific target for real-time PCR by comparing whole genome sequences of S. pseudintermedius and non-pseudintermedius.Genome sequences were downloaded from public repositories and supplemented by isolates that were sequenced in this study. A Perl-script was written that analysed 300-nt fragments from a reference genome sequence of S. pseudintermedius and checked if this sequence was present in other S. pseudintermedius genomes (n = 74 and non-pseudintermedius genomes (n = 138. Six sequences specific for S. pseudintermedius were identified (sequence length between 300-500 nt. One sequence, which was located in the spsJ gene, was used to develop primers and a probe. The real-time PCR showed 100% specificity when testing for S. pseudintermedius isolates (n = 54, and eight other staphylococcal species (n = 43. In conclusion, a novel approach by comparing whole genome sequences identified a sequence that is specific for S. pseudintermedius and provided a real-time PCR target for rapid and reliable detection of S. pseudintermedius.

  10. Diversity analysis of Bemisia tabaci biotypes: RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region

    OpenAIRE

    Rabello, Aline R.; Queiroz, Paulo R.; Simões, Kenya C.C.; Hiragi, Cássia O.; Lima, Luzia H.C.; Oliveira, Maria Regina V.; Mehta, Angela

    2008-01-01

    The Bemisia tabaci complex is formed by approximately 41 biotypes, two of which (B and BR) occur in Brazil. In this work we aimed at obtaining genetic markers to assess the genetic diversity of the different biotypes. In order to do that we analyzed Bemisia tabaci biotypes B, BR, Q and Cassava using molecular techniques including RAPD, PCR-RFLP and sequencing of the ITS1 rDNA region. The analyses revealed a high similarity between the individuals of the B and Q biotypes, which could be distin...

  11. An integrated PCR colony hybridization approach to screen cDNA libraries for full-length coding sequences.

    Science.gov (United States)

    Pollier, Jacob; González-Guzmán, Miguel; Ardiles-Diaz, Wilson; Geelen, Danny; Goossens, Alain

    2011-01-01

    cDNA-Amplified Fragment Length Polymorphism (cDNA-AFLP) is a commonly used technique for genome-wide expression analysis that does not require prior sequence knowledge. Typically, quantitative expression data and sequence information are obtained for a large number of differentially expressed gene tags. However, most of the gene tags do not correspond to full-length (FL) coding sequences, which is a prerequisite for subsequent functional analysis. A medium-throughput screening strategy, based on integration of polymerase chain reaction (PCR) and colony hybridization, was developed that allows in parallel screening of a cDNA library for FL clones corresponding to incomplete cDNAs. The method was applied to screen for the FL open reading frames of a selection of 163 cDNA-AFLP tags from three different medicinal plants, leading to the identification of 109 (67%) FL clones. Furthermore, the protocol allows for the use of multiple probes in a single hybridization event, thus significantly increasing the throughput when screening for rare transcripts. The presented strategy offers an efficient method for the conversion of incomplete expressed sequence tags (ESTs), such as cDNA-AFLP tags, to FL-coding sequences.

  12. Variable order one-step methods for initial value problems I ...

    African Journals Online (AJOL)

    A class of variable order one-step integrators is proposed for Initial Value Problems (IVPs) in Ordinary Differential Equations (ODEs). It is based on a rational interpolant. Journal of the Nigerian Association of Mathematical Physics Vol. 10 2006: pp. 91-96 ...

  13. One-step implementation of the Toffoli gate via quantum Zeno dynamics

    International Nuclear Information System (INIS)

    Shao Xiaoqiang; Wang Hongfu; Chen Li; Zhang Shou; Yeon, Kyu-Hwang

    2009-01-01

    Based on the quantum Zeno dynamics, we present a scheme for one-step implementation of a Toffoli gate via manipulating three rf superconducting quantum interference device (SQUID) qubits to resonantly interact with a superconducting cavity. The effects of decoherence such as spontaneous emission and the loss of cavity are also considered.

  14. Fast nanostructured carbon microparticle synthesis by one-step high-flux plasma processing

    NARCIS (Netherlands)

    Aussems, D. U. B.; Bystrov, K.; Dogan, I.; Arnas, C.; Cabié, M.; Neisius, T.; Rasinski, M.; Zoethout, E.; Lipman, P.; van de Sanden, M. C. M.; Morgan, T. W.

    2017-01-01

    This study demonstrates a fast one-step synthesis method for nanostructured carbon microparticles on graphite samples using high-flux plasma exposure. These structures are considered as potential candidates for energy applications such as Li-ion batteries and supercapacitors. The samples were

  15. Fast nanostructured carbon microparticle synthesis by one-step high-flux plasma processing

    NARCIS (Netherlands)

    Aussems, D.U.B.; Bystrov, K.E.; Doǧan, I.; Arnas, C.; Cabié, M.; Neisius, T.; Rasinski, M.; Lipman, P.J.L.; van de Sanden, M.C.M.; Morgan, T.W.

    This study demonstrates a fast one-step synthesis method for nanostructured carbon microparticles on graphite samples using high-flux plasma exposure. These structures are considered as potential candidates for energy applications such as Li-ion batteries and supercapacitors. The samples were

  16. One-step polymer surface modification for minimizing drug, protein, and DNA adsorption in microanalytical systems

    DEFF Research Database (Denmark)

    Larsen, Esben Kjær Unmack; Larsen, Niels Bent

    2013-01-01

    The non-specific adsorption of dissolved analytes strongly reduces the sensitivity and reliability in polymer microanalytical systems. Here, a one-step aqueous phase procedure modifies polymer material surfaces to strongly reduce their non-specific adsorption of a broad range of organic analytes ...

  17. Fast-ion energy resolution by one-step reaction gamma-ray spectrometry

    DEFF Research Database (Denmark)

    Salewski, Mirko; Nocente, M.; Gorini, G.

    2016-01-01

    The spectral broadening of γ-rays from fusion plasmas can be measured in high-resolution gamma-ray spectrometry (GRS). We derive weight functions that determine the observable velocity space and quantify the velocity-space sensitivity of one-step reaction high-resolution GRS measurements in magne...

  18. Changes in human fecal microbiota due to chemotherapy analyzed by TaqMan-PCR, 454 sequencing and PCR-DGGE fingerprinting.

    Directory of Open Access Journals (Sweden)

    Jutta Zwielehner

    Full Text Available BACKGROUND: We investigated whether chemotherapy with the presence or absence of antibiotics against different kinds of cancer changed the gastrointestinal microbiota. METHODOLOGY/PRINCIPAL FINDINGS: Feces of 17 ambulant patients receiving chemotherapy with or without concomitant antibiotics were analyzed before and after the chemotherapy cycle at four time points in comparison to 17 gender-, age- and lifestyle-matched healthy controls. We targeted 16S rRNA genes of all bacteria, Bacteroides, bifidobacteria, Clostridium cluster IV and XIVa as well as C. difficile with TaqMan qPCR, denaturing gradient gel electrophoresis (DGGE fingerprinting and high-throughput sequencing. After a significant drop in the abundance of microbiota (p = 0.037 following a single treatment the microbiota recovered within a few days. The chemotherapeutical treatment marginally affected the Bacteroides while the Clostridium cluster IV and XIVa were significantly more sensitive to chemotherapy and antibiotic treatment. DGGE fingerprinting showed decreased diversity of Clostridium cluster IV and XIVa in response to chemotherapy with cluster IV diversity being particularly affected by antibiotics. The occurrence of C. difficile in three out of seventeen subjects was accompanied by a decrease in the genera Bifidobacterium, Lactobacillus, Veillonella and Faecalibacterium prausnitzii. Enterococcus faecium increased following chemotherapy. CONCLUSIONS/SIGNIFICANCE: Despite high individual variations, these results suggest that the observed changes in the human gut microbiota may favor colonization with C. difficile and Enterococcus faecium. Perturbed microbiota may be a target for specific mitigation with safe pre- and probiotics.

  19. Verification of Frequency in Species of Nontuberculous Mycobacteria in Kermanshah Drinking Water Supplies Using the PCR-Sequencing Method.

    Science.gov (United States)

    Mohajeri, Parviz; Yazdani, Laya; Shahraki, Abdolrazagh Hashemi; Alvandi, Amirhoshang; Atashi, Sara; Farahani, Abbas; Almasi, Ali; Rezaei, Mansour

    2017-04-01

    Nontuberculous mycobacteria are habitants of environment, especially in aquatic systems. Some of them cause problems in immunodeficient patients. Over the last decade, 16S rRNA gene sequencing was established in 45 novel species of nontuberculous mycobacteria. Experiences revealed that this method underestimates the diversity, but does not distinguish between some of mycobacterium subsp. To recognize emerging rapidly growing mycobacteria and identify their subsp, rpoB gene sequencing has been developed. To better understand the transmission of nontuberculous mycobacterial species from drinking water and preventing the spread of illness with these bacteria, the aim of this study was to detect the presence of bacteria by PCR-sequencing techniques. Drinking water samples were collected from different areas of Kermanshah city in west of IRAN. After decontamination with cetylpyridinium chloride, samples were filtered with 0.45-micron filters, the filter transferred directly on growth medium waiting to appear in colonies, then DNA extraction and PCR were performed, and products were sent to sequencing. We found 35/110 (32%) nontuberculous mycobacterial species in drinking water samples, isolates included Mycobacterium goodii, Mycobacterium aurum, and Mycobacterium gastri with the most abundance (11.5%), followed by Mycobacterium smegmatis, Mycobacterium porcinum, Mycobacterium peregrinum, Mycobacterium mucogenicum, and Mycobacterium chelonae (8%). In this study, we recognized the evidence of contamination by nontuberculous mycobacteria in corroded water pipes. As a result of the high prevalence of these bacteria in drinking water in Kermanshah, this is important evidence of transmission through drinking water. This finding can also help public health policy makers control these isolates in drinking water supplies in Kermanshah.

  20. Use of PCR with Sequence-specific Primers for High-Resolution Human Leukocyte Antigen Typing of Patients with Narcolepsy

    Science.gov (United States)

    Woo, Hye In; Joo, Eun Yeon; Lee, Kyung Wha

    2012-01-01

    Background Narcolepsy is a neurologic disorder characterized by excessive daytime sleepiness, symptoms of abnormal rapid eye movement (REM) sleep, and a strong association with HLA-DRB1*1501, -DQA1*0102, and -DQB1*0602. Here, we investigated the clinico-physical characteristics of Korean patients with narcolepsy, their HLA types, and the clinical utility of high-resolution PCR with sequence-specific primers (PCR-SSP) as a simple typing method for identifying DRB1*15/16, DQA1, and DQB1 alleles. Methods The study population consisted of 67 consecutively enrolled patients having unexplained daytime sleepiness and diagnosed narcolepsy based on clinical and neurological findings. Clinical data and the results of the multiple sleep latency test and polysomnography were reviewed, and HLA typing was performed using both high-resolution PCR-SSP and sequence-based typing (SBT). Results The 44 narcolepsy patients with cataplexy displayed significantly higher frequencies of DRB1*1501 (Pc= 0.003), DQA1*0102 (Pc=0.001), and DQB1*0602 (Pc=0.014) than the patients without cataplexy. Among patients carrying DRB1*1501-DQB1*0602 or DQA1*0102, the frequencies of a mean REM sleep latency of less than 20 min in nocturnal polysomnography and clinical findings, including sleep paralysis and hypnagogic hallucination were significantly higher. SBT and PCR-SSP showed 100% concordance for high-resolution typing of DRB1*15/16 alleles and DQA1 and DQB1 loci. Conclusions The clinical characteristics and somnographic findings of narcolepsy patients were associated with specific HLA alleles, including DRB1*1501, DQA1*0102, and DQB1*0602. Application of high-resolution PCR-SSP, a reliable and simple method, for both allele- and locus-specific HLA typing of DRB1*15/16, DQA1, and DQB1 would be useful for characterizing clinical status among subjects with narcolepsy. PMID:22259780

  1. A thaumatin-like genomic sequence identification in Vitis vinifera l., stormy wines and musts based on direct pcr

    Directory of Open Access Journals (Sweden)

    Jana Žiarovská

    2018-03-01

    Full Text Available Normal 0 21 false false false MicrosoftInternetExplorer4 Direct polymerase chain reaction method was use to amplify a thaumatin-like sequence of Vitis vinifera L. in grapes as well as in stormy wines and musts. Thaumatin-like proteins (TLPs of Vitis vinifera possess beside its function in abiotic and biotic stress response another one - they are able to cause protein haze in wine unless removed prior to bottling. Direct PCR is an approach where omission of DNA extraction is typical prior the amplification of the target site of plant genome. Crude extract or small pieces of plant tissues are used in the analysis directly without steps of extraction and purification of gDNA. The biological material that was used in analysis was collected during August - October 2017 in local stores and winery Sabo and comprises from cultivars Iršai, Muškát, Savignon Blanc, Svätovavrinecké, Dornfelder and Pálava. Direct PCR was performed by a cutted piece of grape tissue and a dilution buffer was use in 1:2 for stormy wine or must, respectively. Direct amplification of thaumatin-like protein sequence of Vitis vinifera was performed along with the control reactions with the primers for conserved region of plant chloroplast. Possitive amplification of thaumatin-like allergen sequence resulted in 570 bp amplicon. The most abundant amplicons were amplified in stormy wines, followed by musts and the amplicons from grapes were weaker when comparing them to others. The amplicon specificity checking of obtained PCR product of thaumatin-like allergen was performed by restriction cleavage by Psi I and resulted in restriction amplicons of the 80 bp, 81 bp, 94 bp and 315 bp in length. Confirmation of the amplicon specificity by restriction cleavage support the potential of direct PCR to become a reproducible method that will be fully applicable in routine analysis of not only plant genomes in the future, but it was demonstrated, that it works in liquids, too.  

  2. IDENTIFICATION OF PUTATIVE SEQUENCE SPECIFIC PCR PRIMERS FOR DETECTION OF THE TOXIGENIC FUNGAL SPECIES STACHYBOTRYS CHARTARUM

    Science.gov (United States)

    The nucleotide sequence of a c 936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal species Stachybotrys chartarum and for other species of Stachbotrys and the related genus Memnoniella. This information was used to infer the phylogenetic relatio...

  3. IDENTIFICATION OF SEQUENCE SPECIFIC PCR PRIMERS FOR DETECTION OF THE TOXIGENIC FUNGAL SPECIES STACHYBOTRYS CHARTARUM

    Science.gov (United States)

    The nucleotide sequence of a 936 bp segment of the nuclear rRNA gene operon was determined for the toxigenic fungal species Stachybotrys chartarum and for other species of Stachybotrys and the related genus Memnoniella. This information was used to infer the phylogenitic relati...

  4. Real-Time PCR Assay for a Unique Chromosomal Sequence of Bacillus anthracis

    Science.gov (United States)

    2004-12-01

    13061 Neisseria lactamica .............................................................. 23970 Bacillus coagulans ...NEG Bacillus coagulane 7050 NEG NEG Bacillus cereus 13472 NEG NEG Bacillus licheniforms 12759 NEG NEG Bacillus cereus 13824 NEG NEG Bacillus ...Assay for a Unique Chromosomal Sequence of Bacillus anthracis Elizabeth Bode,1 William Hurtle,2† and David Norwood1* United States Army Medical

  5. Combining real-time PCR and next-generation DNA sequencing to provide quantitative comparisons of fungal aerosol populations

    Science.gov (United States)

    Dannemiller, Karen C.; Lang-Yona, Naama; Yamamoto, Naomichi; Rudich, Yinon; Peccia, Jordan

    2014-02-01

    We examined fungal communities associated with the PM10 mass of Rehovot, Israel outdoor air samples collected in the spring and fall seasons. Fungal communities were described by 454 pyrosequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal RNA encoding gene. To allow for a more quantitative comparison of fungal exposure in humans, the relative abundance values of specific taxa were transformed to absolute concentrations through multiplying these values by the sample's total fungal spore concentration (derived from universal fungal qPCR). Next, the sequencing-based absolute concentrations for Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, and Penicillium/Aspergillus spp. were compared to taxon-specific qPCR concentrations for A. alternata, C. cladosporioides, E. nigrum, and Penicillium/Aspergillus spp. derived from the same spring and fall aerosol samples. Results of these comparisons showed that the absolute concentration values generated from pyrosequencing were strongly associated with the concentration values derived from taxon-specific qPCR (for all four species, p 0.70). The correlation coefficients were greater for species present in higher concentrations. Our microbial aerosol population analyses demonstrated that fungal diversity (number of fungal operational taxonomic units) was higher in the spring compared to the fall (p = 0.02), and principal coordinate analysis showed distinct seasonal differences in taxa distribution (ANOSIM p = 0.004). Among genera containing allergenic and/or pathogenic species, the absolute concentrations of Alternaria, Aspergillus, Fusarium, and Cladosporium were greater in the fall, while Cryptococcus, Penicillium, and Ulocladium concentrations were greater in the spring. The transformation of pyrosequencing fungal population relative abundance data to absolute concentrations can improve next-generation DNA sequencing-based quantitative aerosol exposure assessment.

  6. FDSTools: A software package for analysis of massively parallel sequencing data with the ability to recognise and correct STR stutter and other PCR or sequencing noise.

    Science.gov (United States)

    Hoogenboom, Jerry; van der Gaag, Kristiaan J; de Leeuw, Rick H; Sijen, Titia; de Knijff, Peter; Laros, Jeroen F J

    2017-03-01

    Massively parallel sequencing (MPS) is on the advent of a broad scale application in forensic research and casework. The improved capabilities to analyse evidentiary traces representing unbalanced mixtures is often mentioned as one of the major advantages of this technique. However, most of the available software packages that analyse forensic short tandem repeat (STR) sequencing data are not well suited for high throughput analysis of such mixed traces. The largest challenge is the presence of stutter artefacts in STR amplifications, which are not readily discerned from minor contributions. FDSTools is an open-source software solution developed for this purpose. The level of stutter formation is influenced by various aspects of the sequence, such as the length of the longest uninterrupted stretch occurring in an STR. When MPS is used, STRs are evaluated as sequence variants that each have particular stutter characteristics which can be precisely determined. FDSTools uses a database of reference samples to determine stutter and other systemic PCR or sequencing artefacts for each individual allele. In addition, stutter models are created for each repeating element in order to predict stutter artefacts for alleles that are not included in the reference set. This information is subsequently used to recognise and compensate for the noise in a sequence profile. The result is a better representation of the true composition of a sample. Using Promega Powerseq™ Auto System data from 450 reference samples and 31 two-person mixtures, we show that the FDSTools correction module decreases stutter ratios above 20% to below 3%. Consequently, much lower levels of contributions in the mixed traces are detected. FDSTools contains modules to visualise the data in an interactive format allowing users to filter data with their own preferred thresholds. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  7. RT-PCR and sequence analysis of the full-length fusion protein of Canine Distemper Virus from domestic dogs.

    Science.gov (United States)

    Romanutti, Carina; Gallo Calderón, Marina; Keller, Leticia; Mattion, Nora; La Torre, José

    2016-02-01

    During 2007-2014, 84 out of 236 (35.6%) samples from domestic dogs submitted to our laboratory for diagnostic purposes were positive for Canine Distemper Virus (CDV), as analyzed by RT-PCR amplification of a fragment of the nucleoprotein gene. Fifty-nine of them (70.2%) were from dogs that had been vaccinated against CDV. The full-length gene encoding the Fusion (F) protein of fifteen isolates was sequenced and compared with that of those of other CDVs, including wild-type and vaccine strains. Phylogenetic analysis using the F gene full-length sequences grouped all the Argentinean CDV strains in the SA2 clade. Sequence identity with the Onderstepoort vaccine strain was 89.0-90.6%, and the highest divergence was found in the 135 amino acids corresponding to the F protein signal-peptide, Fsp (64.4-66.7% identity). In contrast, this region was highly conserved among the local strains (94.1-100% identity). One extra putative N-glycosylation site was identified in the F gene of CDV Argentinean strains with respect to the vaccine strain. The present report is the first to analyze full-length F protein sequences of CDV strains circulating in Argentina, and contributes to the knowledge of molecular epidemiology of CDV, which may help in understanding future disease outbreaks. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. One-step electrochemical synthesis of a graphene–ZnO hybrid for improved photocatalytic activity

    International Nuclear Information System (INIS)

    Wei, Ang; Xiong, Li; Sun, Li; Liu, Yanjun; Li, Weiwei; Lai, Wenyong; Liu, Xiangmei; Wang, Lianhui; Huang, Wei; Dong, Xiaochen

    2013-01-01

    Graphical abstract: - Highlights: • Graphene–ZnO hybrid was synthesized by one-step electrochemical deposition. • Graphene–ZnO hybrid presents a special structure and wide UV–vis absorption spectra. • Graphene–ZnO hybrid exhibits an exceptionally higher photocatalytic activity for the degradation of dye methylene blue. - Abstract: A graphene–ZnO (G-ZnO) hybrid was synthesized by one-step electrochemical deposition. During the formation of ZnO nanostructure by cathodic electrochemical deposition, the graphene oxide was electrochemically reduced to graphene simultaneously. Scanning electron microscope images, X-ray photoelectron spectroscopy, X-ray diffraction, Raman spectra, and UV–vis absorption spectra indicate the resulting G-ZnO hybrid presents a special structure and wide UV–vis absorption spectra. More importantly, it exhibits an exceptionally higher photocatalytic activity for the degradation of dye methylene blue than that of pure ZnO nanostructure under both ultraviolet and sunlight irradiation

  9. One-Step Dynamic Classifier Ensemble Model for Customer Value Segmentation with Missing Values

    Directory of Open Access Journals (Sweden)

    Jin Xiao

    2014-01-01

    Full Text Available Scientific customer value segmentation (CVS is the base of efficient customer relationship management, and customer credit scoring, fraud detection, and churn prediction all belong to CVS. In real CVS, the customer data usually include lots of missing values, which may affect the performance of CVS model greatly. This study proposes a one-step dynamic classifier ensemble model for missing values (ODCEM model. On the one hand, ODCEM integrates the preprocess of missing values and the classification modeling into one step; on the other hand, it utilizes multiple classifiers ensemble technology in constructing the classification models. The empirical results in credit scoring dataset “German” from UCI and the real customer churn prediction dataset “China churn” show that the ODCEM outperforms four commonly used “two-step” models and the ensemble based model LMF and can provide better decision support for market managers.

  10. The One-Step Pickering Emulsion Polymerization Route for Synthesizing Organic-Inorganic Nanocomposite Particles

    Directory of Open Access Journals (Sweden)

    Kaushal Rege

    2010-02-01

    Full Text Available Polystyrene-silica core-shell nanocomposite particles are successfully prepared via one-step Pickering emulsion polymerization. Possible mechanisms of Pickering emulsion polymerization are addressed in the synthesis of polystyrene-silica nanocomposite particles using 2,2-azobis(2-methyl-N-(2-hydroxyethylpropionamide (VA-086 and potassium persulfate (KPS as the initiator. Motivated by potential applications of “smart” composite particles in controlled drug delivery, the one-step Pickering emulsion polymerization route is further applied to synthesize polystyrene/poly(N-isopropylacrylamide (PNIPAAm-silica core-shell nanoparticles with N-isopropylacrylamide incorporated into the core as a co-monomer. The polystyrene/PNIPAAm-silica composite nanoparticles are temperature sensitive and can be taken up by human prostate cancer (PC3-PSMA cells.

  11. Genomic prediction for Nordic Red Cattle using one-step and selection index blending

    DEFF Research Database (Denmark)

    Guosheng, Su; Madsen, Per; Nielsen, Ulrik Sander

    2012-01-01

    This study investigated the accuracy of direct genomic breeding values (DGV) using a genomic BLUP model, genomic enhanced breeding values (GEBV) using a one-step blending approach, and GEBV using a selection index blending approach for 15 traits of Nordic Red Cattle. The data comprised 6,631 bulls...... genotyped and nongenotyped bulls for one-step blending, and to scale DGV and its expected reliability in the selection index blending. Weighting (scaling) factors had a small influence on reliabilities of GEBV, but a large influence on the variation of GEBV. Based on the validation analyses, averaged over...... the 15 traits, the reliability of DGV for bulls without daughter records was 11.0 percentage points higher than the reliability of conventional pedigree index. Further gain of 0.9 percentage points was achieved by combining information from conventional pedigree index using the selection index blending...

  12. One-step generation of continuous-variable quadripartite cluster states in a circuit QED system

    Science.gov (United States)

    Yang, Zhi-peng; Li, Zhen; Ma, Sheng-li; Li, Fu-li

    2017-07-01

    We propose a dissipative scheme for one-step generation of continuous-variable quadripartite cluster states in a circuit QED setup consisting of four superconducting coplanar waveguide resonators and a gap-tunable superconducting flux qubit. With external driving fields to adjust the desired qubit-resonator and resonator-resonator interactions, we show that continuous-variable quadripartite cluster states of the four resonators can be generated with the assistance of energy relaxation of the qubit. By comparison with the previous proposals, the distinct advantage of our scheme is that only one step of quantum operation is needed to realize the quantum state engineering. This makes our scheme simpler and more feasible in experiment. Our result may have useful application for implementing quantum computation in solid-state circuit QED systems.

  13. Chitosan-Coated Magnetic Nanoparticles Prepared in One Step by Reverse Microemulsion Precipitation

    OpenAIRE

    Hened Saade; Salvador Fernández; Ramón Díaz de León; Gilberto Hurtado; María G. Pineda; Raúl G. López; Darío Bueno

    2013-01-01

    Chitosan-coated magnetic nanoparticles (CMNP) were obtained at 70 ?C and 80 ?C in a one-step method, which comprises precipitation in reverse microemulsion in the presence of low chitosan concentration in the aqueous phase. X-ray diffractometry showed that CMNP obtained at both temperatures contain a mixture of magnetite and maghemite nanoparticles with ?4.5 nm in average diameter, determined by electron microscopy, which suggests that precipitation temperature does not affect the particle si...

  14. Design and implementation of the one-step MSD adder of optical computer.

    Science.gov (United States)

    Song, Kai; Yan, Liping

    2012-03-01

    On the basis of the symmetric encoding algorithm for the modified signed-digit (MSD), a 7*7 truth table that can be realized with optical methods was developed. And based on the truth table, the optical path structures and circuit implementations of the one-step MSD adder of ternary optical computer (TOC) were designed. Experiments show that the scheme is correct, feasible, and efficient. © 2012 Optical Society of America

  15. [Bacterial diversity in sequencing batch biofilm reactor (SBBR) for landfill leachate treatment using PCR-DGGE].

    Science.gov (United States)

    Xiao, Yong; Yang, Zhao-hui; Zeng, Guang-ming; Ma, Yan-he; Liu, You-sheng; Wang, Rong-juan; Xu, Zheng-yong

    2007-05-01

    For studying the bacterial diversity and the mechanism of denitrification in sequencing bath biofilm reactor (SBBR) treating landfill leachate to provide microbial evidence for technique improvements, total microbial DNA was extracted from samples which were collected from natural landfill leachate and biofilm of a SBBR that could efficiently remove NH4+ -N and COD of high concentration. 16S rDNA fragments were amplified from the total DNA successfully using a pair of universal bacterial 16S rDNA primer, GC341F and 907R, and then were used for denaturing gradient gel electrophoresis (DGGE) analysis. The bands in the gel were analyzed by statistical methods and excided from the gel for sequencing, and the sequences were used for homology analysis and then two phylogenetic trees were constructed using DNAStar software. Results indicated that the bacterial diversity of the biofilm in SBBR and the landfill leachate was abundant, and no obvious change of community structure happened during running in the biofilm, in which most bacteria came from the landfill leachate. There may be three different modes of denitrification in the reactor because several different nitrifying bacteria, denitrifying bacteria and anaerobic ammonia oxidation bacteria coexisted in it. The results provided some valuable references for studying microbiological mechanism of denitrification in SBBR.

  16. Will learning to solve one-step equations pose a challenge to 8th grade students?

    Science.gov (United States)

    Ngu, Bing Hiong; Phan, Huy P.

    2017-08-01

    Assimilating multiple interactive elements simultaneously in working memory to allow understanding to occur, while solving an equation, would impose a high cognitive load. Element interactivity arises from the interaction between elements within and across operational and relational lines. Moreover, operating with special features (e.g. negative pronumeral) poses additional challenge to master equation solving skills. In an experiment, 41 8th grade students (girls = 16, boys = 25) sat for a pre-test, attended a session about equation solving, completed an acquisition phase which constituted the main intervention and were tested again in a post-test. The results showed that at post-test, students performed better on one-step equations tapping low rather than high element interactivity knowledge. In addition, students performed better on those one-step equations that contained no special features. Thus, both the degree of element interactivity and the operation with special features affect the challenge posed to 8th grade students on learning how to solve one-step equations.

  17. One step synthesis of 6-oxo-cholestan-3β,5α-diol

    International Nuclear Information System (INIS)

    Voisin, Maud; Silvente-Poirot, Sandrine; Poirot, Marc

    2014-01-01

    Highlights: • Cholesterol-5,6-epoxides are metabolized into cholestane-3β,5α,6β-triol (CT) in cancer cells. • 6-Oxo-cholestan-3β,5α-diol (OCDO) is a putative metabolite of CT. • The one step syntheses of CT and OCDO from cholesterol are reported. • The one step syntheses of labelled CT and OCDO are reported. - Abstract: Cholesterol metabolism has been recently linked to cancer, highlighting the importance of the characterization of new metabolic pathways in the sterol series. One of these pathways is centered on cholesterol-5,6-epoxides (5,6-ECs). 5,6-ECs can either generate dendrogenin A, a tumor suppressor present in healthy mammalian tissues, or the carcinogenic cholestane-3β,5α,6β-triol (CT) and its putative metabolite 6-oxo-cholestan-3β,5α-diol (OCDO) in tumor cells. We are currently investigating the identification of the enzyme involved in OCDO biosynthesis, which would be highly facilitated by the use of commercially unavailable [ 14 C]-cholestane-3β,5α,6β-triol and [ 14 C]-6-oxo-cholestan-3β,5α-diol. In the present study we report the one-step synthesis of [ 14 C]-cholestane-3β,5α,6β-triol and [ 14 C]-6-oxo-cholestan-3β,5α-diol by oxidation of [ 14 C]-cholesterol with iodide metaperiodate (HIO 4 )

  18. One-step purification and characterization of alginate lyase from a clinical Pseudomonas aeruginosa with destructive activity on bacterial biofilm

    Directory of Open Access Journals (Sweden)

    Parinaz Ghadam

    2017-05-01

    Full Text Available Objective(s: Pseudomonas aeruginosais a Gram-negative and aerobic rod bacterium that displays mucoid and non-mucoid phenotype. Mucoid strains secrete alginate, which is the main agent of biofilms in chronic P. aeruginosa infections, show high resistance to antibiotics; consequently, the biological disruption of mucoid P. aeruginosa biofilms is an attractive area of study for researchers. Alginate lyase gene (algl is a member of alginate producing operon which by glycosidase activity produces primer for other enzymes in this cluster. Also this activity can destroy the extracellular alginate; therefore this enzyme participates in alginate production and destruction pathway. Alginate lyase causes detachment of a biofilm by reducing its adhesion to the surfaces, and increases phagocytosis and antibiotic susceptibility. In this study, alginate lyase was purified in just one step and its properties were investigated. Materials and Methods: The purification was done by affinity chromatography, analysed by SDS-PAGE, and its effect on P. aeruginosa biofilms was surveyed by micro titer plate assay and SEM. The substrate specificity of the enzyme was determined by PCR. Results: Alginate lyase from isolate 48 was purified in one step. It is more thermally resistant than alginate lyase from Pseudomonas aeruginosa PAO1 and poly M, poly G and poly MG alginate were the substrate of this enzyme. Moreover, it has an eradication effect on biofilms from P. aeruginosa 48 and PAO1. Conclusion: In this study an alginate lyase with many characteristics suitable in medicine such as thermal stability, effective on poly M alginate, and bacterial biofilm destructive was introduced and purified.

  19. Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in Dogs in a Village of Eastern Sudan by Using a Screening PCR and Sequencing Methodologies

    Science.gov (United States)

    Oyamada, Maremichi; Davoust, Bernard; Boni, Mickaël; Dereure, Jacques; Bucheton, Bruno; Hammad, Awad; Itamoto, Kazuhito; Okuda, Masaru; Inokuma, Hisashi

    2005-01-01

    Babesia and Hepatozoon infections of dogs in a village of eastern Sudan were analyzed by using a single PCR and sequencing. Among 78 dogs, 5 were infected with Babesia canis rossi and 2 others were infected with B. canis vogeli. Thirty-three dogs were positive for Hepatozoon. Hepatozoon canis was detected by sequence analysis. PMID:16275954

  20. Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in dogs in a village of eastern Sudan by using a screening PCR and sequencing methodologies.

    Science.gov (United States)

    Oyamada, Maremichi; Davoust, Bernard; Boni, Mickaël; Dereure, Jacques; Bucheton, Bruno; Hammad, Awad; Itamoto, Kazuhito; Okuda, Masaru; Inokuma, Hisashi

    2005-11-01

    Babesia and Hepatozoon infections of dogs in a village of eastern Sudan were analyzed by using a single PCR and sequencing. Among 78 dogs, 5 were infected with Babesia canis rossi and 2 others were infected with B. canis vogeli. Thirty-three dogs were positive for Hepatozoon. Hepatozoon canis was detected by sequence analysis.

  1. Detection of Babesia canis rossi, B. canis vogeli, and Hepatozoon canis in Dogs in a Village of Eastern Sudan by Using a Screening PCR and Sequencing Methodologies

    OpenAIRE

    Oyamada, Maremichi; Davoust, Bernard; Boni, Mickaël; Dereure, Jacques; Bucheton, Bruno; Hammad, Awad; Itamoto, Kazuhito; Okuda, Masaru; Inokuma, Hisashi

    2005-01-01

    Babesia and Hepatozoon infections of dogs in a village of eastern Sudan were analyzed by using a single PCR and sequencing. Among 78 dogs, 5 were infected with Babesia canis rossi and 2 others were infected with B. canis vogeli. Thirty-three dogs were positive for Hepatozoon. Hepatozoon canis was detected by sequence analysis.

  2. A seminested PCR assay for detection and typing of human papillomavirus based on E1 gene sequences.

    Science.gov (United States)

    Cavalcante, Gustavo Henrique O; de Araújo, Josélio M G; Fernandes, José Veríssimo; Lanza, Daniel C F

    2018-05-01

    HPV infection is considered one of the leading causes of cervical cancer in the world. To date, more than 180 types of HPV have been described and viral typing is critical for defining the prognosis of cancer. In this work, a seminested PCR which allow fast and inexpensively detection and typing of HPV is presented. The system is based on the amplification of a variable length region within the viral gene E1, using three primers that potentially anneal in all HPV genomes. The amplicons produced in the first step can be identified by high resolution electrophoresis or direct sequencing. The seminested step includes nine specific primers which can be used in multiplex or individual reactions to discriminate the main types of HPV by amplicon size differentiation using agarose electrophoresis, reducing the time spent and cost per analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. 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.

  4. One-step synthesis of Zn doped titania nanotubes and investigation of their visible photocatalytic activity

    Energy Technology Data Exchange (ETDEWEB)

    Benjwal, Poonam [Advanced Nanoengineering Materials Laboratory, Materials Science Programme, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Kar, Kamal K., E-mail: kamalkk@iitk.ac.in [Advanced Nanoengineering Materials Laboratory, Materials Science Programme, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Advanced Nanoengineering Materials Laboratory, Department of Mechanical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India)

    2015-06-15

    Highly oriented undoped and Zn-doped titania (TiO{sub 2}) nanotubes were electrochemically fabricated by one-step anodization of titanium foil in a freshly prepared aqueous solution of zinc fluoride (ZnF{sub 2}) and ethylene glycol (EG). XRD and Raman spectroscopy unveiled the typical characteristic of anatase phase of TiO{sub 2} nanotube without any distinct dopant related peaks. SEM and AFM observation confirmed the formation of nanotubes and revealed that the Zn doping did not distort the tube morphology of TiO{sub 2}. The doping of Zn was confirmed by energy dispersive X-ray as well as X-ray photospectroscopy. Due to one-step anodization process, instead of surface doping, the Zn{sup 2+} ions were incorporated into the bulk of TiO{sub 2} nanotubes. With increasing Zn doping in nanotubes, a gradual decrease in the band gap of TiO{sub 2} (2.84 eV) was observed. Photoluminescence measurements revealed that the doping of Zn enhanced the number of charge carriers, which eventually boosted the photocatalytic activity of TiO{sub 2} nanotubes. Compared to undoped nanotubes, the as prepared Zn-doped TiO{sub 2}-nanotubes showed excellent photocatalytic activity for methylene blue degradation (reaction rate constant k = 0.19 min{sup −1}) under visible light irradiation. - Highlights: • A facile one-step anodization method is used for Zn doped TiO{sub 2}-nanotubes synthesis. • Zn{sup 2+} ions are doped into the bulk of TiO{sub 2} nanotubes. • Doped TiO{sub 2}-nanotubes unveil pure anatase phase and reduced band gap. • Compared to undoped, doped TiO{sub 2} nanotubes exhibit enhanced photocatalytic activity.

  5. ONE-STEP AND TWO-STEP CALIBRATION OF A PORTABLE PANORAMIC IMAGE MAPPING SYSTEM

    Directory of Open Access Journals (Sweden)

    P.-C. Wang

    2012-07-01

    Full Text Available A Portable Panoramic Image Mapping System (PPIMS is proposed for rapid acquisition of three-dimensional spatial information. By considering the convenience of use, cost, weight of equipment, precision, and power supply, the designed PPIMS is equipped with 6 circularly arranged cameras to capture panoramic images and a GPS receiver for positioning. The motivation for this design is to develop a hand-held Mobile Mapping System (MMS for some difficult accessing areas by vehicle MMS, such as rugged terrains, forest areas, heavily damaged disaster areas, and crowed places etc. This PPIMS is in fact a GPS assisted close-range photogrammetric system. Compared with the traditional close-range photogrammetry, PPIMS can reduce the need of ground control points significantly. Under the condition of knowing the relative geometric relationships of the equipped sensors, the elements of exterior orientation of each captured image can be solved. However, the procedure of a system calibration should be done accurately to determine the relative geometric relationships of multi-cameras and the GPS antenna center, before the PPIMS can be applied for geo-referenced mapping. In this paper, both of one-step and two-step calibration procedures for PPIMS are performed to determine the lever-arm offsets and boresight angles among cameras and GPS. The performance of the one-step and two-step calibration is evaluated through the analysis of the experimental results. The comparison between these two calibration procedures was also conducted. The two-step calibration method outperforms the one-step calibration method in terms of calibration accuracy and operation convenience. We expect that the proposed two-step calibration procedure can also be applied to other platform-based MMSs.

  6. One step synthesis of 6-oxo-cholestan-3β,5α-diol

    Energy Technology Data Exchange (ETDEWEB)

    Voisin, Maud; Silvente-Poirot, Sandrine; Poirot, Marc, E-mail: marc.poirot@inserm.fr

    2014-04-11

    Highlights: • Cholesterol-5,6-epoxides are metabolized into cholestane-3β,5α,6β-triol (CT) in cancer cells. • 6-Oxo-cholestan-3β,5α-diol (OCDO) is a putative metabolite of CT. • The one step syntheses of CT and OCDO from cholesterol are reported. • The one step syntheses of labelled CT and OCDO are reported. - Abstract: Cholesterol metabolism has been recently linked to cancer, highlighting the importance of the characterization of new metabolic pathways in the sterol series. One of these pathways is centered on cholesterol-5,6-epoxides (5,6-ECs). 5,6-ECs can either generate dendrogenin A, a tumor suppressor present in healthy mammalian tissues, or the carcinogenic cholestane-3β,5α,6β-triol (CT) and its putative metabolite 6-oxo-cholestan-3β,5α-diol (OCDO) in tumor cells. We are currently investigating the identification of the enzyme involved in OCDO biosynthesis, which would be highly facilitated by the use of commercially unavailable [{sup 14}C]-cholestane-3β,5α,6β-triol and [{sup 14}C]-6-oxo-cholestan-3β,5α-diol. In the present study we report the one-step synthesis of [{sup 14}C]-cholestane-3β,5α,6β-triol and [{sup 14}C]-6-oxo-cholestan-3β,5α-diol by oxidation of [{sup 14}C]-cholesterol with iodide metaperiodate (HIO{sub 4})

  7. Smear layer-deproteinizing improves bonding of one-step self-etch adhesives to dentin.

    Science.gov (United States)

    Thanatvarakorn, Ornnicha; Prasansuttiporn, Taweesak; Thittaweerat, Suppason; Foxton, Richard M; Ichinose, Shizuko; Tagami, Junji; Hosaka, Keiichi; Nakajima, Masatoshi

    2018-03-01

    Smear layer deproteinizing was proved to reduce the organic phase of smear layer covered on dentin surface. It was shown to eliminate hybridized smear layer and nanoleakage expression in resin-dentin bonding interface of two-step self-etch adhesive. This study aimed to investigate those effects on various one-step self-etch adhesives. Four different one-step self-etch adhesives were used in this study; SE One (SE), Scotchbond™ Universal (SU), BeautiBond Multi (BB), and Bond Force (BF). Flat human dentin surfaces with standardized smear layer were prepared. Smear layer deproteinizing was carried out by the application of 50ppm hypochlorous acid (HOCl) on dentin surface for 15s followed by Accel ® (p-toluenesulfinic acid salt) for 5s prior to adhesive application. No surface pretreatment was used as control. Microtensile bond strength (μTBS) and nanoleakage under TEM observation were investigated. The data were analyzed by two-way ANOVA and Tukey's post-hoc test and t-test at the significant level of 0.05. Smear layer deproteinizing significantly improved μTBS of SE, SU, and BB (player observed in control groups of SE, BB, and BF, and reticular nanoleakage presented throughout the hybridized complex in control groups of BB and BF were eliminated upon the smear layer deproteinizing. Smear layer deproteinizing by HOCl and Accel ® application could enhance the quality of dentin for bonding to one-step self-etch adhesives, resulting in the improving μTBS, eliminating hybridized smear layer and preventing reticular nanoleakage formation in resin-dentin bonding interface. Copyright © 2018 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  8. Effect of one-step polishing system on the color stability of nanocomposites.

    Science.gov (United States)

    Alawjali, S S; Lui, J L

    2013-08-01

    This study was to compare the effect of three different one-step polishing systems on the color stability of three different types of nanocomposites after immersion in coffee for one day and seven days and determine which nanocomposite material has the best color stability following polishing with each of the one-step polishing system. The nanocomposites tested were Tetric EvoCeram, Grandio and Herculite Précis. A total of 120 discs (40/nanocomposite, 8mm×2mm) were fabricated. Ten specimens for each nanocomposite cured under Mylar strips served as the control. The other specimens were polished with OptraPol, OneGloss and Occlubrush immersed in coffee (Nescafé) up to seven days. Color measurements were made with a spectrophotometer at baseline and after one and seven days. Two way repeated measure ANOVA, two way ANOVA and Bonferroni tests were used for statistical analyses (P<0.05). The immersion time was a significant factor in the discoloration of the nanocomposites. The effect of three one-step polishing systems on the color stability was also significant. The color change values of the materials cured against Mylar strips were the greatest. The lowest mean color change values were from the Occlubrush polished groups. The effect of the three different types of nanocomposite on the color change was significant. The highest color change values were with Tetric EvoCeram groups. The lowest color change values were with Herculite Précis groups. The color change of nanocomposite resins is affected by the type of composite, polishing procedure and the period of immersion in the staining agent. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Monoclonal antibodies to human factor VII: a one step immunoradiometric assay for VII:Ag.

    OpenAIRE

    Takase, T; Tuddenham, E G; Chand, S; Goodall, A H

    1988-01-01

    Three mouse monoclonal antibodies (RFF-VII/1, RFF-VII/2, and RFF-VII/3) which bind specifically to different epitopes on human factor VII antigen were raised. Two of the antibodies, RFF-VII/1 and RFF-VII/2, bound strongly to factor VII antigen (VII:Ag), but only RFF-VII/1 and RFF-VII/3 were potent inhibitors of factor VII coagulation activity (VII:C). RFF-VII/1 and RFF-VII/2 were used in a one step, double monoclonal immunoradiometric assay for VII:Ag. This was highly reproducible and detecte...

  10. Influence of the dopant during the one step mechano-chemical synthesis of sodium alanate

    International Nuclear Information System (INIS)

    Rongeat, C; Geipel, C; Llamas-Jansa, I; Schultz, L; Gutfleisch, O

    2009-01-01

    High-pressure reactive milling under hydrogen atmosphere is used for the one-step synthesis of doped sodium alanate. In-situ monitoring of the pressure and the temperature inside the vial gives a direct feedback about the reactions occurring during the milling. This information is used to study the influence of the dopant during synthesis, e.g. the amount of dopant added. The study of the pressure variations during milling is a reliable tool for screening the efficiency of different dopants.

  11. One-Step Hydrothermal-Electrochemical Route to Carbon-Stabilized Anatase Powders

    Science.gov (United States)

    Tao, Ying; Yi, Danqing; Zhu, Baojun

    2013-04-01

    Black carbon-stabilized anatase particles were prepared by a simple one-step hydrothermal-electrochemical method using glucose and titanium citrate as the carbon and titanium source, respectively. Morphological, chemical, structural, and electrochemical characterizations of these powders were carried out by Raman spectroscopy, Fourier-transform infrared spectroscopy, x-ray diffraction, scanning electron microscopy, and cyclic voltammetry. It was revealed that 200-nm carbon/anatase TiO2 was homogeneously dispersed, and the powders exhibited excellent cyclic performance at high current rates of 0.05 V/s. The powders are interesting potential materials that could be used as anodes for lithium-ion batteries.

  12. One-step synthesis of gold-polyaniline core-shell particles

    International Nuclear Information System (INIS)

    Wang Zhijuan; Yuan Junhua; Han Dongxue; Niu Li; Ivaska, Ari

    2007-01-01

    A one-step method has been developed for synthesizing gold-polyaniline (Au-PANI) core-shell particles by using chlorauric acid (HAuCl 4 ) to oxidize aniline in the presence of acetic acid and Tween 40 at room temperature. SEM images indicated that the resulting core-shell particles were composed of submicrometre-scale Au particles and PANI shells with an average thickness of 25 nm. Furthermore, a possible mechanism concerning the growth of Au-PANI particles was also proposed based on the results of control experiments

  13. An infinite-order two-component relativistic Hamiltonian by a simple one-step transformation.

    Science.gov (United States)

    Ilias, Miroslav; Saue, Trond

    2007-02-14

    The authors report the implementation of a simple one-step method for obtaining an infinite-order two-component (IOTC) relativistic Hamiltonian using matrix algebra. They apply the IOTC Hamiltonian to calculations of excitation and ionization energies as well as electric and magnetic properties of the radon atom. The results are compared to corresponding calculations using identical basis sets and based on the four-component Dirac-Coulomb Hamiltonian as well as Douglas-Kroll-Hess and zeroth-order regular approximation Hamiltonians, all implemented in the DIRAC program package, thus allowing a comprehensive comparison of relativistic Hamiltonians within the finite basis approximation.

  14. One-step trinary signed-digit arithmetic using an efficient encoding scheme

    Science.gov (United States)

    Salim, W. Y.; Fyath, R. S.; Ali, S. A.; Alam, Mohammad S.

    2000-11-01

    The trinary signed-digit (TSD) number system is of interest for ultra fast optoelectronic computing systems since it permits parallel carry-free addition and borrow-free subtraction of two arbitrary length numbers in constant time. In this paper, a simple coding scheme is proposed to encode the decimal number directly into the TSD form. The coding scheme enables one to perform parallel one-step TSD arithmetic operation. The proposed coding scheme uses only a 5-combination coding table instead of the 625-combination table reported recently for recoded TSD arithmetic technique.

  15. Noise Suppression in ECG Signals through Efficient One-Step Wavelet Processing Techniques

    Directory of Open Access Journals (Sweden)

    E. Castillo

    2013-01-01

    Full Text Available This paper illustrates the application of the discrete wavelet transform (DWT for wandering and noise suppression in electrocardiographic (ECG signals. A novel one-step implementation is presented, which allows improving the overall denoising process. In addition an exhaustive study is carried out, defining threshold limits and thresholding rules for optimal wavelet denoising using this presented technique. The system has been tested using synthetic ECG signals, which allow accurately measuring the effect of the proposed processing. Moreover, results from real abdominal ECG signals acquired from pregnant women are presented in order to validate the presented approach.

  16. Field theoretical approach to proton-nucleus reactions. I - One step inelastic scattering

    International Nuclear Information System (INIS)

    Eiras, A.; Kodama, T.; Nemes, M.C.

    1988-01-01

    In this work we obtain a closed form expression to the double differential cross section for one step proton-nucleus reaction within a field theoretical framework. Energy and momentum conservation as well as nuclear structure effects are consistently taken into account within the field theoretical eikonal approximation. In our formulation the kinematics of such reaction is not dominated by the free nucleon-nucleon cross section but a new factor which we call relativistic differential cross section in a Born Approximation. (author) [pt

  17. Influence of application methods of one-step self-etching adhesives on microtensile bond strength

    OpenAIRE

    Chul-Kyu Choi,; Sung-Ae Son; Jin-Hee Ha; Bock Hur; Hyeon-Cheol Kim; Yong-Hun Kwon; Jeong-Kil Park

    2011-01-01

    Objectives The purpose of this study was to evaluate the effect of various application methods of one-step self-etch adhesives to microtensile resin-dentin bond strength. Materials and Methods Thirty-six extracted human molars were used. The teeth were assigned randomly to twelve groups (n = 15), according to the three different adhesive systems (Clearfil Tri-S Bond, Adper Prompt L-Pop, G-Bond) and application methods. The adhesive systems were applied on the dentin as follows: 1) T...

  18. Efficient one-step direct labelling of recombinant antibodies with technetium-99m

    International Nuclear Information System (INIS)

    Liberatore, M.; Neri, D.; Neri, G.; Pini, A.; Lurilli, A.P.; Ponzo, F.; Spampinato, G.; Padula, F.; Pala, A.; Colella, A.C.

    1995-01-01

    High-affinity bacterially expressed antibody fragments can nowadays be cloned from established hybridomas or, more conveniently, isolated directly from antibody libraries displayed on filamentous phage. Such antibodies can be tagged with C-terminal peptide tags containing one cysteine residue, which represents a convenient functionalisation site for a number of applications, including technetium-99m labelling. Here we describe a simple one-step method for 99m Tc labelling of cysteine-tagged recombinant antibodies with more than 50% radionuclide incorporation. The labelled antibodies displayed full retention of immuoreactivity and good stability. (orig.)

  19. Efficient one-step direct labelling of recombinant antibodies with technetium-99m

    Energy Technology Data Exchange (ETDEWEB)

    Liberatore, M. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Neri, D. [Cambridge Centre for Protein Engineering - MRC Centre (United Kingdom); Neri, G. [Dipartimento di Biologia Molecolare, Universita di Siena (Italy); Pini, A. [Dipartimento di Biologia Molecolare, Universita di Siena (Italy); Lurilli, A.P. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Ponzo, F. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy); Spampinato, G. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Padula, F. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Pala, A. [Laboratorio di Biochimica degli Ormoni Sessuali, Il Instituto di Clinica Ostetrica e Ginecologica, Universita di Roma `La Sapienza` (Italy); Colella, A.C. [Dipartimento di Medicina Sperimentale, Sezione di Medicina Nucleare, Policlinico Umberto I, Universita di Roma `La Sapienza` (Italy)

    1995-11-01

    High-affinity bacterially expressed antibody fragments can nowadays be cloned from established hybridomas or, more conveniently, isolated directly from antibody libraries displayed on filamentous phage. Such antibodies can be tagged with C-terminal peptide tags containing one cysteine residue, which represents a convenient functionalisation site for a number of applications, including technetium-99m labelling. Here we describe a simple one-step method for {sup 99m}Tc labelling of cysteine-tagged recombinant antibodies with more than 50% radionuclide incorporation. The labelled antibodies displayed full retention of immuoreactivity and good stability. (orig.)

  20. One-step microwave synthesis of photoluminescent carbon nanoparticles from sodium dextran sulfate water solution

    Science.gov (United States)

    Kokorina, Alina A.; Goryacheva, Irina Y.; Sapelkin, Andrei V.; Sukhorukov, Gleb B.

    2018-04-01

    Photoluminescent (PL) carbon nanoparticles (CNPs) have been synthesized by one-step microwave irradiation from water solution of sodium dextran sulfate (DSS) as the sole carbon source. Microwave (MW) method is very simple and cheap and it provides fast synthesis of CNPs. We have varied synthesis time for obtaining high luminescent CNPs. The synthesized CNPs exhibit excitation-dependent photoluminescent. Final CNPs water solution has a blue- green luminescence. CNPs have low cytotoxicity, good photostability and can be potentially suitable candidates for bioimaging, analysis or analytical tests.

  1. Optimized spray drying process for preparation of one-step calcium-alginate gel microspheres

    Energy Technology Data Exchange (ETDEWEB)

    Popeski-Dimovski, Riste [Department of physic, Faculty of Natural Sciences and Mathematics, “ss. Cyril and Methodius” University, Arhimedova 3, 1000 Skopje, R. Macedonia (Macedonia, The Former Yugoslav Republic of)

    2016-03-25

    Calcium-alginate micro particles have been used extensively in drug delivery systems. Therefore we establish a one-step method for preparation of internally gelated micro particles with spherical shape and narrow size distribution. We use four types of alginate with different G/M ratio and molar weight. The size of the particles is measured using light diffraction and scanning electron microscopy. Measurements showed that with this method, micro particles with size distribution around 4 micrometers can be prepared, and SEM imaging showed that those particles are spherical in shape.

  2. Optimized PCR with sequence specific primers (PCR-SSP for fast and efficient determination of Interleukin-6 Promoter -597/-572/-174Haplotypes

    Directory of Open Access Journals (Sweden)

    Bugert Peter

    2009-12-01

    Full Text Available Abstract Background Interleukin-6 (IL-6 promoter polymorphisms at positions -597(G→A, -572(G→C and -174(G→C were shown to have a clinical impact on different major diseases. At present PCR-SSP protocols for IL-6 -597/-572/-174haplotyping are elaborate and require large amounts of genomic DNA. Findings We describe an improved typing technique requiring a decreased number of PCR-reactions and a reduced PCR-runtime due to optimized PCR-conditions. Conclusion This enables a fast and efficient determination of IL-6 -597/-572/-174haplotypes in clinical diagnosis and further evaluation of IL-6 promoter polymorphisms in larger patient cohorts.

  3. 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...

  4. DNA Fingerprinting of Lactobacillus crispatus Strain CTV-05 by Repetitive Element Sequence-Based PCR Analysis in a Pilot Study of Vaginal Colonization

    OpenAIRE

    Antonio, May A. D.; Hillier, Sharon L.

    2003-01-01

    Lactobacillus crispatus is one of the predominant hydrogen peroxide (H2O2)-producing species found in the vagina and is under development as a probiotic for the treatment of bacterial vaginosis. In this study, we assessed whether DNA fingerprinting by repetitive element sequence-based PCR (rep-PCR) can be used to distinguish the capsule strain of L. crispatus (CTV-05) from other endogenous strains as well as other species of vaginal lactobacilli. Vaginal and rectal lactobacilli were identifie...

  5. One Step Solution to the Local Tie Vectors of GNSS/SLR in ITRF

    Directory of Open Access Journals (Sweden)

    MA Xiaping

    2018-01-01

    Full Text Available This paper proposes a one-step solution of local tie vectors,the solution takes RP (Reference Point and axes offsets of SLR telescope as unknown parameters,GNSS baselines network and conventional terrestrial observation (horizontal,vertical angle and distance are combined in ITRF (International Terrestrial Reference Frame,and multiply constraint conditions are established between RP and observation marks and two axes offsets.Local tie vectors and related covariance matrices of the 3 co-located sites in Beijing,Kunming,Xi'an of CMONOC (Crustal Movement Observation Network of China are solved with the proposed one step solution.The results show that the RMS (root mean square error of local tie vectors is less than 2.0 mm.Besides,the differences are less than 2.0 mm compared with the traditional step by step solution,and the offsets and corresponding RMS between the horizontal axis and vertical axis are 3.8 mm,0.7 mm,3.6 mm and 1.3 mm,1.2 mm,1.3 mm,respectively.

  6. Stability of one-step methods in transient nonlinear heat conduction

    International Nuclear Information System (INIS)

    Hughes, J.R.

    1977-01-01

    The purpose of the present work is to ascertain practical stability conditions for one-step methods commonly used in transient nonlinear heat conduction analyses. The class of problems considered is governed by a temporally continuous, spatially discrete system involving the capacity matrix C, conductivity matrix K, heat supply vector, temperature vector and time differenciation. In the linear case, in which K and C are constant, the stability behavior of one-step methods is well known. But in this paper the concepts of stability, appropriate to the nonlinear problem, are thoroughly discussed. They of course reduce to the usual stability criterion for the linear, constant coefficient case. However, for nonlinear problems there are differences and these ideas are of key importance in obtaining practical stability conditions. Of particular importance is a recent result which indicates that, in a sense, the trapezoidal and midpoint families are quivalent. Thus, stability results for one family may be translated into a result for the other. The main results obtained are summarized as follows. The stability behavior of the explicit Euler method in the nonlinear regime is analogous to that for linear problems. In particular, an a priori step size restriction may be determined for each time step. The precise time step restriction on implicit conditionally stable members of the trapezoidal and midpoint families is shown not to be determinable a priori. Of considerable practical significance, unconditionally stable members of the trapezoidal and midpoint families are identified

  7. One-step synthesis of hybrid nanocrystals with rational tuning of the morphology.

    Science.gov (United States)

    Sang, Wei; Zheng, Tingting; Wang, Youcheng; Li, Xu; Zhao, Xu; Zeng, Jie; Hou, J G

    2014-11-12

    Metal-sulfide hybrid nanocrystals (HNCs) have been of great interest for their distinguished interfacial effect, which gives rise to unique catalytic properties. However, most of the reported metal-sulfide HNCs were synthesized via two-step approaches and few were fabricated based on the one-step strategies. Herein, we report a facile one-pot synthesis of CuPt-Cu2S, Pt-Cu2S HNCs, and CuPt nanocubes by simply changing the Pt precursor types. 1-Hexadecanethiol (HDT) was employed in this system to mediate the reduction of metal precursors and also as capping agent and sulfur source. Moreover, CuPd-Cu2S and Au-Cu2S HNCs were successfully prepared by using this one-step method. The catalytic properties of the obtained three nanocrystals were investigated in hydrogenation of cinnamaldehyde. Results show that CuPt-Cu2S HNCs exhibited the highest conversion rate and the highest selectivity toward hydrocinnamaldehyde while 3-phenyl-1-propanol was the only product over Pt-Cu2S HNCs.

  8. One-step preparation of antimicrobial silver nanoparticles in polymer matrix

    International Nuclear Information System (INIS)

    Lyutakov, O.; Kalachyova, Y.; Solovyev, A.; Vytykacova, S.; Svanda, J.; Siegel, J.; Ulbrich, P.; Svorcik, V.

    2015-01-01

    Simple one-step procedure for in situ preparation of silver nanoparticles (AgNPs) in the polymer thin films is described. Nanoparticles (NPs) were prepared by reaction of N-methyl pyrrolidone with silver salt in semi-dry polymer film and characterized by transmission electron microscopy, XPS, and UV–Vis spectroscopy techniques. Direct synthesis of NPs in polymer has several advantages; even though it avoids time-consuming NPs mixing with polymer matrix, uniform silver distribution in polymethylmethacrylate (PMMA) films is achieved without necessity of additional stabilization. The influence of the silver concentration, reaction temperature and time on reaction conversion rate, and the size and size-distribution of the AgNPs was investigated. Polymer films doped with AgNPs were tested for their antibacterial activity on Gram-negative bacteria. Antimicrobial properties of AgNPs/PMMA films were found to be depended on NPs concentration, their size and distribution. Proposed one-step synthesis of functional polymer containing AgNPs is environmentally friendly, experimentally simple and extremely quick. It opens up new possibilities in development of antimicrobial coatings with medical and sanitation applications

  9. Classified one-step high-radix signed-digit arithmetic units

    Science.gov (United States)

    Cherri, Abdallah K.

    1998-08-01

    High-radix number systems enable higher information storage density, less complexity, fewer system components, and fewer cascaded gates and operations. A simple one-step fully parallel high-radix signed-digit arithmetic is proposed for parallel optical computing based on new joint spatial encodings. This reduces hardware requirements and improves throughput by reducing the space-bandwidth produce needed. The high-radix signed-digit arithmetic operations are based on classifying the neighboring input digit pairs into various groups to reduce the computation rules. A new joint spatial encoding technique is developed to present both the operands and the computation rules. This technique increases the spatial bandwidth product of the spatial light modulators of the system. An optical implementation of the proposed high-radix signed-digit arithmetic operations is also presented. It is shown that our one-step trinary signed-digit and quaternary signed-digit arithmetic units are much simpler and better than all previously reported high-radix signed-digit techniques.

  10. IPMP Global Fit - A one-step direct data analysis tool for predictive microbiology.

    Science.gov (United States)

    Huang, Lihan

    2017-12-04

    The objective of this work is to develop and validate a unified optimization algorithm for performing one-step global regression analysis of isothermal growth and survival curves for determination of kinetic parameters in predictive microbiology. The algorithm is incorporated with user-friendly graphical interfaces (GUIs) to develop a data analysis tool, the USDA IPMP-Global Fit. The GUIs are designed to guide the users to easily navigate through the data analysis process and properly select the initial parameters for different combinations of mathematical models. The software is developed for one-step kinetic analysis to directly construct tertiary models by minimizing the global error between the experimental observations and mathematical models. The current version of the software is specifically designed for constructing tertiary models with time and temperature as the independent model parameters in the package. The software is tested with a total of 9 different combinations of primary and secondary models for growth and survival of various microorganisms. The results of data analysis show that this software provides accurate estimates of kinetic parameters. In addition, it can be used to improve the experimental design and data collection for more accurate estimation of kinetic parameters. IPMP-Global Fit can be used in combination with the regular USDA-IPMP for solving the inverse problems and developing tertiary models in predictive microbiology. Published by Elsevier B.V.

  11. One-step preparation of antimicrobial silver nanoparticles in polymer matrix

    Energy Technology Data Exchange (ETDEWEB)

    Lyutakov, O., E-mail: lyutakoo@vscht.cz; Kalachyova, Y. [Institute of Chemical Technology, Department of Solid State Engineering (Czech Republic); Solovyev, A. [Institute of Chemical Process Fundamentals of the ASCR (Czech Republic); Vytykacova, S. [Institute of Chemical Technology, Department of Glass and Ceramics (Czech Republic); Svanda, J.; Siegel, J. [Institute of Chemical Technology, Department of Solid State Engineering (Czech Republic); Ulbrich, P. [Institute of Chemical Technology, Department of Biochemistry and Microbiology (Czech Republic); Svorcik, V. [Institute of Chemical Technology, Department of Solid State Engineering (Czech Republic)

    2015-03-15

    Simple one-step procedure for in situ preparation of silver nanoparticles (AgNPs) in the polymer thin films is described. Nanoparticles (NPs) were prepared by reaction of N-methyl pyrrolidone with silver salt in semi-dry polymer film and characterized by transmission electron microscopy, XPS, and UV–Vis spectroscopy techniques. Direct synthesis of NPs in polymer has several advantages; even though it avoids time-consuming NPs mixing with polymer matrix, uniform silver distribution in polymethylmethacrylate (PMMA) films is achieved without necessity of additional stabilization. The influence of the silver concentration, reaction temperature and time on reaction conversion rate, and the size and size-distribution of the AgNPs was investigated. Polymer films doped with AgNPs were tested for their antibacterial activity on Gram-negative bacteria. Antimicrobial properties of AgNPs/PMMA films were found to be depended on NPs concentration, their size and distribution. Proposed one-step synthesis of functional polymer containing AgNPs is environmentally friendly, experimentally simple and extremely quick. It opens up new possibilities in development of antimicrobial coatings with medical and sanitation applications.

  12. Influence of application methods of one-step self-etching adhesives on microtensile bond strength

    Directory of Open Access Journals (Sweden)

    Chul-Kyu Choi,

    2011-05-01

    Full Text Available Objectives The purpose of this study was to evaluate the effect of various application methods of one-step self-etch adhesives to microtensile resin-dentin bond strength. Materials and Methods Thirty-six extracted human molars were used. The teeth were assigned randomly to twelve groups (n = 15, according to the three different adhesive systems (Clearfil Tri-S Bond, Adper Prompt L-Pop, G-Bond and application methods. The adhesive systems were applied on the dentin as follows: 1 The single coating, 2 The double coating, 3 Manual agitation, 4 Ultrasonic agitation. Following the adhesive application, light-cure composite resin was constructed. The restored teeth were stored in distilled water at room temperature for 24 hours, and prepared 15 specimens per groups. Then microtensile bond strength was measured and the failure mode was examined. Results Manual agitation and ultrasonic agitation of adhesive significantly increased the microtensile bond strength than single coating and double coating did. Double coating of adhesive significantly increased the microtensile bond strength than single coating did and there was no significant difference between the manual agitation and ultrasonic agitation group. There was significant difference in microtensile bonding strength among all adhesives and Clearfil Tri-S Bond showed the highest bond strength. Conclusions In one-step self-etching adhesives, there was significant difference according to application methods and type of adhesives. No matter of the material, the manual or ultrasonic agitation of the adhesive showed significantly higher microtensile bond strength.

  13. One step linear reconstruction method for continuous wave diffuse optical tomography

    Science.gov (United States)

    Ukhrowiyah, N.; Yasin, M.

    2017-09-01

    The method one step linear reconstruction method for continuous wave diffuse optical tomography is proposed and demonstrated for polyvinyl chloride based material and breast phantom. Approximation which used in this method is selecting regulation coefficient and evaluating the difference between two states that corresponding to the data acquired without and with a change in optical properties. This method is used to recovery of optical parameters from measured boundary data of light propagation in the object. The research is demonstrated by simulation and experimental data. Numerical object is used to produce simulation data. Chloride based material and breast phantom sample is used to produce experimental data. Comparisons of results between experiment and simulation data are conducted to validate the proposed method. The results of the reconstruction image which is produced by the one step linear reconstruction method show that the image reconstruction almost same as the original object. This approach provides a means of imaging that is sensitive to changes in optical properties, which may be particularly useful for functional imaging used continuous wave diffuse optical tomography of early diagnosis of breast cancer.

  14. One-step synthesis of chlorinated graphene by plasma enhanced chemical vapor deposition

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Liwei; Zhang, Hui; Zhang, Pingping; Sun, Xuhui, E-mail: xhsun@suda.edu.cn

    2015-08-30

    Highlights: • We developed a simple approach to synthesize the single layer chlorinated graphene. • CuCl{sub 2} on Cu surface is used as Cl source under the plasma treatment. • The formation of covalent C−Cl bond has been investigated by Raman and XPS. • Raman results indicate the p-type doping effect of chlorination. - Abstract: We developed an approach to synthesize the chlorinated single layer graphene (Cl-G) by one-step plasma enhanced chemical vapor deposition. Copper foil was simply treated with hydrochloric acid and then CuCl{sub 2} formed on the surface was used as Cl source under the assistance of plasma treatment. Compared with other two-step methods by post plasma/photochemical treatment of CVD-grown single layer graphene (SLG), one-step Cl-G synthesis approach is quite straightforward and effective. X-ray photoelectron spectroscopy (XPS) revealed that ∼2.45 atom% Cl remained in SLG. Compared with the pristine SLG, the obvious blue shifts of G band and 2D band along with the appearance of D’ band and D + G band in the Raman spectra indicate p-type doping of Cl-G.

  15. One-step synthesis of magnetic chitosan for controlled release of 5-hydroxytryptophan

    Energy Technology Data Exchange (ETDEWEB)

    Santos Menegucci, Jucély dos; Santos, Mac-Kedson Medeiros Salviano; Dias, Diego Juscelino Santos; Chaker, Juliano Alexandre; Sousa, Marcelo Henrique, E-mail: mhsqui@gmail.com

    2015-04-15

    In this work, nanoparticles of chitosan embedded with 25% (w/w) of iron oxide magnetic nanoparticles (magnetite/maghemite) with narrow size-distribution and with a loading efficiency of about 80% for 5-hydroxytryptophan (5-HTP), which is a chemical precursor in the biosynthesis of important neurotransmitters as serotonin, were synthesized with an initial mass ratio of 5-HTP/magnetic chitosan=1.2, using homogeneous precipitation by urea decomposition, in an efficient one-step procedure. Characterization of morphology, structure and surface were performed by XRD, TEM, FTIR, TGA, magnetization and zeta potential measurements, while drug loading and drug releasing were investigated using UV–vis spectroscopy. Kinetic drug release experiments under different pH conditions revealed a pH-sensitivecontrolled-release system, ruled by polymer swelling and/or particle dissolution. - Highlights: • One-step synthesis and incorporation of drug in magnetic chitosan. • Synthesis utilizes a cost-effective and environmentally friendly procedure. • Narrow size distribution of magnetic nanoparticles in the composite. • Composite is a basis for a magnetic pH triggered drug release system.

  16. A simple one-step chemistry model for partially premixed hydrocarbon combustion

    Energy Technology Data Exchange (ETDEWEB)

    Fernandez-Tarrazo, Eduardo [Instituto Nacional de Tecnica Aeroespacial, Madrid (Spain); Sanchez, Antonio L. [Area de Mecanica de Fluidos, Universidad Carlos III de Madrid, Leganes 28911 (Spain); Linan, Amable [ETSI Aeronauticos, Pl. Cardenal Cisneros 3, Madrid 28040 (Spain); Williams, Forman A. [Department of Mechanical and Aerospace Engineering, University of California San Diego, La Jolla, CA 92093-0411 (United States)

    2006-10-15

    This work explores the applicability of one-step irreversible Arrhenius kinetics with unity reaction order to the numerical description of partially premixed hydrocarbon combustion. Computations of planar premixed flames are used in the selection of the three model parameters: the heat of reaction q, the activation temperature T{sub a}, and the preexponential factor B. It is seen that changes in q with equivalence ratio f need to be introduced in fuel-rich combustion to describe the effect of partial fuel oxidation on the amount of heat released, leading to a universal linear variation q(f) for f>1 for all hydrocarbons. The model also employs a variable activation temperature T{sub a}(f) to mimic changes in the underlying chemistry in rich and very lean flames. The resulting chemistry description is able to reproduce propagation velocities of diluted and undiluted flames accurately over the whole flammability limit. Furthermore, computations of methane-air counterflow diffusion flames are used to test the proposed chemistry under nonpremixed conditions. The model not only predicts the critical strain rate at extinction accurately but also gives near-extinction flames with oxygen leakage, thereby overcoming known predictive limitations of one-step Arrhenius kinetics. (author)

  17. Shear bond strength of one-step self-etch adhesives: pH influence

    Science.gov (United States)

    Poggio, Claudio; Beltrami, Riccardo; Scribante, Andrea; Colombo, Marco; Chiesa, Marco

    2015-01-01

    Background: The aim of this study was to compare the shear bond strength of four one-step self-etch adhesives with different pH values to enamel and dentin. Materials and Methods: In this in vitro study, 200 bovine permanent mandibular incisors were used. Four one-step self-etch adhesives with different pH values were tested both on enamel and on dentin: Adper™ Easy Bond Self-Etch Adhesive (pH = 0.8-1), Futurabond NR (pH=2), G-aenial Bond (pH = 1.5), Clearfil S3 Bond (pH = 2.7). After adhesive systems application, a nanohybrid composite resin was inserted into the bonded surface. The specimens were placed in a universal testing machine. The shear bond strength was performed at a cross-head speed of 1 mm/min until the sample rupture. The shear bond strength values (MPa) of the different groups were compared with analysis of variance after that Kolmogorov and Smirnov tests were applied to assess normality of distributions. P enamel shear bond strength, the highest shear bond strength values were reported with Futurabond NR (P adhesive systems showed lower shear bond strength values with significant differences between them (P 0.05). Conclusion: The pH values of adhesive systems did not influence significantly their shear bond strength to enamel or dentin. PMID:26005459

  18. Effect of one step KOH activation and CaO modified carbon in transesterification reaction

    Science.gov (United States)

    Yacob, Abd Rahim; Zaki, Muhammad Azam Muhammad

    2017-11-01

    In this work, one step activation was introduced using potassium hydroxide (KOH) and calcium oxide (CaO) modified palm kernel shells. Various concentration of calcium oxide was used as catalyst while maintaining the same concentration of potassium hydroxide to activate and impregnate the palm kernel shell before calcined at 500°C for 5 hours. All the prepared samples were characterized using Fourier Transform Infrared (FTIR) and Field Emission Scanning Electron Microscope (FESEM). FTIR analysis of raw palm kernel shell showed the presence of various functional groups. However, after activation, most of the functional groups were eliminated. The basic strength of the prepared samples were determined using back titration method. The samples were then used as base heterogeneous catalyst for the transesterification reaction of rice bran oil with methanol. Analysis of the products were performed using Gas Chromatography Flame Ionization Detector (GC-FID) to calculate the percentage conversion of the biodiesel products. This study shows, as the percentage of one step activation potassium and calcium oxide doped carbon increases thus, the basic strength also increases followed by the increase in biodiesel production. Optimization study shows that the optimum biodiesel production was at 8 wt% catalyst loading, 9:1 methanol: oil molar ratio at 65°C and 6 hours which gives a conversion up to 95%.

  19. One-Step Printable Perovskite Films Fabricated under Ambient Conditions for Efficient and Reproducible Solar Cells.

    Science.gov (United States)

    Jung, Yen-Sook; Hwang, Kyeongil; Heo, Youn-Jung; Kim, Jueng-Eun; Lee, Donmin; Lee, Cheol-Ho; Joh, Han-Ik; Yeo, Jun-Seok; Kim, Dong-Yu

    2017-08-23

    Despite the potential of roll-to-roll processing for the fabrication of perovskite films, the realization of highly efficient and reproducible perovskite solar cells (PeSCs) through continuous coating techniques and low-temperature processing is still challenging. Here, we demonstrate that efficient and reliable CH 3 NH 3 PbI 3 (MAPbI 3 ) films fabricated by a printing process can be achieved through synergetic effects of binary processing additives, N-cyclohexyl-2-pyrrolidone (CHP) and dimethyl sulfoxide (DMSO). Notably, these perovskite films are deposited from premixed perovskite solutions for facile one-step processing under a room-temperature and ambient atmosphere. The CHP molecules result in the uniform and homogeneous perovskite films even in the one-step slot-die system, which originate from the high boiling point and low vapor pressure of CHP. Meanwhile, the DMSO molecules facilitate the growth of perovskite grains by forming intermediate states with the perovskite precursor molecules. Consequently, fully printed PeSC based on the binary additive system exhibits a high PCE of 12.56% with a high reproducibility.

  20. Endophytic bacterial diversity in grapevine (Vitis vinifera L.) leaves described by 16S rRNA gene sequence analysis and length heterogeneity-PCR.

    Science.gov (United States)

    Bulgari, Daniela; Casati, Paola; Brusetti, Lorenzo; Quaglino, Fabio; Brasca, Milena; Daffonchio, Daniele; Bianco, Piero Attilio

    2009-08-01

    Diversity of bacterial endophytes associated with grapevine leaf tissues was analyzed by cultivation and cultivation-independent methods. In order to identify bacterial endophytes directly from metagenome, a protocol for bacteria enrichment and DNA extraction was optimized. Sequence analysis of 16S rRNA gene libraries underscored five diverse Operational Taxonomic Units (OTUs), showing best sequence matches with gamma-Proteobacteria, family Enterobacteriaceae, with a dominance of the genus Pantoea. Bacteria isolation through cultivation revealed the presence of six OTUs, showing best sequence matches with Actinobacteria, genus Curtobacterium, and with Firmicutes genera Bacillus and Enterococcus. Length Heterogeneity-PCR (LH-PCR) electrophoretic peaks from single bacterial clones were used to setup a database representing the bacterial endophytes identified in association with grapevine tissues. Analysis of healthy and phytoplasma-infected grapevine plants showed that LH-PCR could be a useful complementary tool for examining the diversity of bacterial endophytes especially for diversity survey on a large number of samples.

  1. Use of mariner transposases for one-step delivery and integration of DNA in prokaryotes and eukaryotes by transfection.

    Science.gov (United States)

    Trubitsyna, Maryia; Michlewski, Gracjan; Finnegan, David J; Elfick, Alistair; Rosser, Susan J; Richardson, Julia M; French, Christopher E

    2017-06-02

    Delivery of DNA to cells and its subsequent integration into the host genome is a fundamental task in molecular biology, biotechnology and gene therapy. Here we describe an IP-free one-step method that enables stable genome integration into either prokaryotic or eukaryotic cells. A synthetic mariner transposon is generated by flanking a DNA sequence with short inverted repeats. When purified recombinant Mos1 or Mboumar-9 transposase is co-transfected with transposon-containing plasmid DNA, it penetrates prokaryotic or eukaryotic cells and integrates the target DNA into the genome. In vivo integrations by purified transposase can be achieved by electroporation, chemical transfection or Lipofection of the transposase:DNA mixture, in contrast to other published transposon-based protocols which require electroporation or microinjection. As in other transposome systems, no helper plasmids are required since transposases are not expressed inside the host cells, thus leading to generation of stable cell lines. Since it does not require electroporation or microinjection, this tool has the potential to be applied for automated high-throughput creation of libraries of random integrants for purposes including gene knock-out libraries, screening for optimal integration positions or safe genome locations in different organisms, selection of the highest production of valuable compounds for biotechnology, and sequencing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Identification and molecular epidemiology of dermatophyte isolates by repetitive-sequence-PCR-based DNA fingerprinting using the DiversiLab system in Turkey.

    Science.gov (United States)

    Koc, A Nedret; Atalay, Mustafa A; Inci, Melek; Sariguzel, Fatma M; Sav, Hafize

    2017-05-01

    Dermatophyte species, isolation and identification in clinical samples are still difficult and take a long time. The identification and molecular epidemiology of dermatophytes commonly isolated in a clinical laboratory in Turkey by repetitive sequence-based PCR (rep-PCR) were assessed by comparing the results with those of reference identification. A total of 44 dermatophytes isolated from various clinical specimens of 20 patients with superficial mycoses in Kayseri and 24 patients in Hatay were studied. The identification of dermatophyte isolates was based on the reference identification and rep-PCR using the DiversiLab System (BioMerieux). The genotyping of dermatophyte isolates from different patients was determined by rep-PCR. In the identification of dermatophyte isolates, agreement between rep-PCR and conventional methods was 87.8 % ( 36 of 41). The dermatophyte strains belonged to four clones (A -D) which were determined by the use of rep-PCR. The dermatophyte strains in Clone B, D showed identical patterns with respect to the region. In conclusion, rep-PCR appears to be useful for evaluation of the identification and clonal relationships between Trichophyton rubrum species complex and Trichophyton mentagrophytes species complex isolates. The similarity and diversity of these isolates may be assessed according to different regions by rep-PCR. © 2017 Blackwell Verlag GmbH.

  3. Examples of Nearly Net Zero Energy Buildings Through One-Step and Stepwise Retrofits

    DEFF Research Database (Denmark)

    Galiotto, Nicolas; Heiselberg, Per; Knudstrup, Mary-Ann

    2012-01-01

    This paper presents the review of eight single-family house retrofit projects. The main objective is to collect and classify several approaches to nearly net zero energy building retrofitting. The selection has been made on the capacity of reaching a nearly net zero energy level via a one......-step or stepwise retrofit process. The review work is part of a more global Ph.D. project and is used as one of the basement of the future research work. The considered approaches have been sorted in two categories. The first approach has a very high use of energy conservation measures and low use of renewable...... energy production measures. The second approach has a lower use of energy conservation measures (but still high compared to a traditional renovation) and a higher use of renewable energy production measures. A third approach to nearly net zero energy building renovation exists but has not been considered...

  4. One-Step Electrochemical Polymerization of Polyaniline Flexible Counter Electrode Doped by Graphene

    Directory of Open Access Journals (Sweden)

    Qi Qin

    2016-01-01

    Full Text Available To improve the photoelectric property of polyaniline (PANI counter electrode using for flexible dye-sensitized solar cell (DSSC, graphene (GN was doped in PANI films covered on flexible conducting substrate by one-step electrochemical method, and then GN/PANI composites are characterized by scanning electron microscope (SEM, fourier transform infrared spectroscopy (FTIR, four probe instrument, and so on. The results show that PANI particles can be electrodeposited on the surface of GN sheets as the potential rising to 2.0 V. This formed unique PANI-GN-PANI lamellar structure owing to the strong interaction of conjugated π electron between GN and PANI results in the superior conductivity and catalytic performance of GN/PANI electrode. The maximum conversion efficiency of dye-sensitized solar cell with this counter electrode reaches 4.31%, which is much higher than that of GN-free PANI counter electrode.

  5. One-Step Facile Synthesis of a Simple Hole Transport Material for Efficient Perovskite Solar Cells

    KAUST Repository

    Chen, Hu

    2016-04-04

    A hole transporting material was designed for use in perovskite solar cells, with a facile one-step synthesis from inexpensive, com-mercially available reagents. The molecule comprises a central fluorinated phenyl core with pendant aryl amines, namely, 3,6-difluoro-N1,N1,N2,N2,N4,N4,N5,N5-octakis(4-methoxyphenyl)benzene-1,2,4,5-tetraamine (DFTAB). A power conversion efficiency of up to 10.4% was achieved in a mesoporous perovskite device architecture. The merits of a simple and potentially low cost syn-thetic route as well as promising performance in perovskite devices, encourages further development of this materials class as new low-cost hole transporting materials for the scale up of perovskite solar cells.

  6. One-step isolation of plasma membrane proteins using magnetic beads with immobilized concanavalin A

    DEFF Research Database (Denmark)

    Lee, Yu-Chen; Block, Gregory; Chen, Huiwen

    2008-01-01

    We have developed a simple method for isolating and purifying plasma membrane proteins from various cell types. This one-step affinity-chromatography method uses the property of the lectin concanavalin A (ConA) and the technique of magnetic bead separation to obtain highly purified plasma membrane...... proteins from crude membrane preparations or cell lines. ConA is immobilized onto magnetic beads by binding biotinylated ConA to streptavidin magnetic beads. When these ConA magnetic beads were used to enrich plasma membranes from a crude membrane preparation, this procedure resulted in 3.7-fold enrichment...... of plasma membrane marker 5'-nucleotidase activity with 70% recovery of the activity in the crude membrane fraction of rat liver. In agreement with the results of 5'-nucleotidase activity, immunoblotting with antibodies specific for a rat liver plasma membrane protein, CEACAM1, indicated that CEACAM1...

  7. One step paired electrochemical synthesis of iron and iron oxide nanoparticles

    Directory of Open Access Journals (Sweden)

    Ordoukhanian Juliet

    2016-09-01

    Full Text Available In this study, a new one step paired electrochemical method is developed for simultaneous synthesis of iron and iron oxide nanoparticles. iron and iron oxide are prepared as cathodic and anodic products from iron (ii sulfate aqueous solution in a membrane divided electrolytic cell by the pulsed current electrosynthesis. Because of organic solvent-free and electrochemical nature of the synthesis, the process could be considered as green and environmentally friendly. The reduction of energy consumption and low cost are the other significant advantages of this new method that would have a great application potential in the chemical industry. The nanostructure of prepared samples was characterized by Fourier transform infrared spectroscopy (FT-IR, X-ray diffraction (XRD, scanning electron microscopy (SEM and transmission electron microscopy (TEM. The magnetic properties were studied by vibrating sample magnetometer (VsM.

  8. Production of biovanillin by one-step biotransformation using fungus Pycnoporous cinnabarinus.

    Science.gov (United States)

    Tilay, Ashwini; Bule, Mahesh; Annapure, Uday

    2010-04-14

    The current study proposes a one-step biotransformation process for vanillin production from ferulic acid using the wild fungal strain Pycnoporous cinnabarinus belonging to the family Basidiomycete. Improvement of biotransformation conditions was performed in two steps; initially a one factor at a time method was used to investigate effects of medium composition variables (i.e., carbon, nitrogen) and environmental factors such as pH on vanillin production. Subsequently, concentrations of medium components were optimized using an orthogonal matrix method. After primary screening, glucose as carbon source and corn steep liquor and ammonium chloride as organic and inorganic nitrogen source, respectively, supported maximum biotransformation of ferulic acid to vanillin. Under statistically optimum conditions vanillin production from ferulic acid by P. cinnabarinus was 126 mg/L with a molar yield of 54%. The overall molar yield of vanillin production increased by 4 times.

  9. Hydrophobic lappaconitine loaded into iota-carrageenan by one step self-assembly.

    Science.gov (United States)

    Sun, Wenxiu; Saldaña, Marleny D A; Zhao, Yujia; Wu, Lingling; Dong, Tungalag; Jin, Ye; Zhang, Ji

    2016-02-10

    New data on the loading of pH-sensitive lappaconitine loaded into iota-carrageenan (LA-ICG) is provided. This LA-ICG ionic biopolymer was prepared by one step self-assembly. The LA-ICG was characterized in terms of the loading capacity, lappaconitine (LA) releasing behavior, pH-sensitivity, and analgesic properties. Iota-carrageenan (ICG) high loading capacity reached up to 26.18% (w/w). Also, the LA, loaded with ICG, was released faster in an acidic environment than that in neutral or alkaline environments. Animal analgesic experiments showed that the LA-ICG of low molecular weight had earlier onset time and longer duration than the LA. These results suggest that the ICG of low molecular weight has great potential to achieve the synergistic effect of LA. In addition, the ICG can be used as a novel natural polymeric carrier for loading a hydrophobic alkaloid. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Thread-like supercapacitors based on one-step spun nanocomposite yarns.

    Science.gov (United States)

    Meng, Qinghai; Wang, Kai; Guo, Wei; Fang, Jin; Wei, Zhixiang; She, Xilin

    2014-08-13

    Thread-like electronic devices have attracted great interest because of their potential applications in wearable electronics. To produce high-performance, thread-like supercapacitors, a mixture of stable dispersions of single-walled carbon nanotubes and conducting polyaniline nanowires are prepared. Then, the mixture is spun into flexible yarns with a polyvinyl alcohol outer sheath by a one-step spinning process. The composite yarns show excellent mechanical properties and high electrical conductivities after sufficient washing to remove surfactants. After applying a further coating layer of gel electrolyte, two flexible yarns are twisted together to form a thread-like supercapacitor. The supercapacitor based on these two yarns (SWCNTs and PAniNWs) possesses a much higher specific capacitance than that based only on pure SWCNTs yarns, making it an ideal energy-storage device for wearable electronics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Centrifuge modeling of one-step outflow tests for unsaturated parameter estimations

    Directory of Open Access Journals (Sweden)

    H. Nakajima

    2006-01-01

    Full Text Available Centrifuge modeling of one-step outflow tests were carried out using a 2-m radius geotechnical centrifuge, and the cumulative outflow and transient pore water pressure were measured during the tests at multiple gravity levels. Based on the scaling laws of centrifuge modeling, the measurements generally showed reasonable agreement with prototype data calculated from forward simulations with input parameters determined from standard laboratory tests. The parameter optimizations were examined for three different combinations of input data sets using the test measurements. Within the gravity level examined in this study up to 40g, the optimized unsaturated parameters compared well when accurate pore water pressure measurements were included along with cumulative outflow as input data. With its capability to implement variety of instrumentations under well controlled initial and boundary conditions and to shorten testing time, the centrifuge modeling technique is attractive as an alternative experimental method that provides more freedom to set inverse problem conditions for the parameter estimation.

  12. Centrifuge modeling of one-step outflow tests for unsaturated parameter estimations

    Science.gov (United States)

    Nakajima, H.; Stadler, A. T.

    2006-10-01

    Centrifuge modeling of one-step outflow tests were carried out using a 2-m radius geotechnical centrifuge, and the cumulative outflow and transient pore water pressure were measured during the tests at multiple gravity levels. Based on the scaling laws of centrifuge modeling, the measurements generally showed reasonable agreement with prototype data calculated from forward simulations with input parameters determined from standard laboratory tests. The parameter optimizations were examined for three different combinations of input data sets using the test measurements. Within the gravity level examined in this study up to 40g, the optimized unsaturated parameters compared well when accurate pore water pressure measurements were included along with cumulative outflow as input data. With its capability to implement variety of instrumentations under well controlled initial and boundary conditions and to shorten testing time, the centrifuge modeling technique is attractive as an alternative experimental method that provides more freedom to set inverse problem conditions for the parameter estimation.

  13. One step synthesis of porous graphene by laser ablation: A new and facile approach

    Science.gov (United States)

    Kazemizadeh, Fatemeh; Malekfar, Rasoul

    2018-02-01

    Porous graphene (PG) was obtained using one step laser process. Synthesis was carried out by laser ablation of nickel-graphite target under ultra-high flow of argon gas. The field emission scanning electron microscopy (FE-SEM) results showed the formation of a porous structure and the transmission electron microscopy (TEM) revealed that the porosity of PGs increase under intense laser irradiation. Structural characterization study using Raman spectroscopy, X-ray powder diffraction (XRD) and selected area electron diffraction (SAED) technique showed that the obtained PGs display high crystalline structure in the form of few layer rhombohedral graphitic arrangement that can be interpreted as the phase prior to the formation of other carbon nanostructures.

  14. A rapid one-step radiometric assay for hepatitis B surface antigen utilising monoclonal antibodies

    International Nuclear Information System (INIS)

    Goodall, A.H.; Meek, F.L.; Waters, J.A.; Miescher, G.C.; Janossy, G.; Thomas, H.C.

    1982-01-01

    A two-site antigen assay for HBsAg has been developed that employs 3 monoclonal antibodies. The antibodies were selected for their high affinity and their particular epitope specificity to establish an assay with a sensitivity for the antigen comparable with that of a conventional assay with heterologous antisera. In addition, by selecting a monoclonal antibody for use as a tracer which does not compete for antigenic binding sites with the solid-phase monoclonal antibodies, it has been possible to perform a two-site assay in a single 1 h incubation step, achieving the same degree of sensitivity. This principle of using monoclonal antibodies in a one-step assay therefore gives advantages of speed and simplicity over assays using heterologous antisera and would be applicable to a variety of antigen assays for which appropriate monoclonal antibodies are available. (Auth.)

  15. Fibrillar polyaniline/diatomite composite synthesized by one-step in situ polymerization method

    International Nuclear Information System (INIS)

    Li Xingwei; Li Xiaoxuan; Wang Gengchao

    2005-01-01

    A fibrillar polyaniline/diatomite composite was prepared by one-step in situ polymerization of aniline in the dispersed system of diatomite, and was characterized via Fourier-transform infrared spectra (FT-IR), UV-vis-NIR spectra, wide-angle X-ray diffraction (WXRD), thermogravimetric analysis (TGA) and transmission electron microscopy (TEM), as well as conductivity. Morphology of the composite is uniform nanofibers, which the diameters of nanofibers are about 50-80 nm. The conductivity of polyaniline/diatomite composite contained 28% polyaniline is 0.29 S cm -1 at 25 deg. C, and temperature of thermal degradation has reached 493 deg. C in air. The composite has potential commercial applications as fillers for electromagnetic shielding materials and conductive coatings

  16. Solution Mask Liquid Lithography (SMaLL) for One-Step, Multimaterial 3D Printing.

    Science.gov (United States)

    Dolinski, Neil D; Page, Zachariah A; Callaway, E Benjamin; Eisenreich, Fabian; Garcia, Ronnie V; Chavez, Roberto; Bothman, David P; Hecht, Stefan; Zok, Frank W; Hawker, Craig J

    2018-06-21

    A novel methodology for printing 3D objects with spatially resolved mechanical and chemical properties is reported. Photochromic molecules are used to control polymerization through coherent bleaching fronts, providing large depths of cure and rapid build rates without the need for moving parts. The coupling of these photoswitches with resin mixtures containing orthogonal photo-crosslinking systems allows simultaneous and selective curing of multiple networks, providing access to 3D objects with chemically and mechanically distinct domains. The power of this approach is showcased through the one-step fabrication of bioinspired soft joints and mechanically reinforced "brick-and-mortar" structures. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. One-step preparation of zeolite silicalite-1 microspheres with adjustable macroporosity

    KAUST Repository

    Hua, Jia

    2009-06-23

    A facile one-step method was developed for the preparation of zeolite silicalite-1 (or ZSM-5) microspheres without the need of pre-synthesizing zeolite nanocrystals. In this method, Pluronic triblock copolymer F127 was added into a homogeneous silicalite-1 synthesis solution. During the hydrothermal synthesis, zeolite nanocrystals (∼ 100 nm) were formed and spontaneously assembled into uniform micrometer-sized (3-5 μm) spheres due to the presence of F127. The obtained microspheres possess significant textual porosity (up to 0.24 cm 3/g), which can be adjusted by simply adding different amounts of styrene in the synthesis. The zeolite microspheres are useful for enzyme immobilization, and the enzyme loading is proportional to the textual porosity. © 2009 American Chemical Society.

  18. Facile fabrication of HDPE-g-MA/nanodiamond nanocomposites via one-step reactive blending.

    Science.gov (United States)

    Song, Ping'an; Yu, Youming; Wu, Qiang; Fu, Shenyuan

    2012-06-29

    In this letter, nanocomposites based on maleic anhydride grafted high density polyethylene (HDPE-g-MA) and amine-functionalized nanodiamond (ND) were fabricated via one-step reactive melt-blending, generating a homogeneous dispersion of ND, as evidenced by transmission electron microscope observations. Thermal analysis results suggest that addition of ND does not affect significantly thermal stability of polymer matrix in nitrogen. However, it was interestingly found that incorporating pure ND decreases the thermal oxidation degradation stability temperature, but blending amino-functionalized ND via reactive processing significantly enhances it of HDPE in air condition. Most importantly, cone tests revealed that both ND additives and reactive blending greatly reduce the heat release rate of HDPE. The results suggest that ND has a potential application as flame retardant alternative for polymers. Tensile results show that adding ND considerably enhances Young's modulus, and reactive blending leads to further improvement in Young's modulus while hardly reducing the elongation at break of HDPE.

  19. The measurement of unsaturated hydraulic conductivity from one-step outflow method

    International Nuclear Information System (INIS)

    Lee, S. H.; Hwang, J. H.; Lee, J. M.; Kim, C. R.

    2003-01-01

    One of the most important parts in constructing radioactive waste repository may be its safety aspect. The fundamental function of the repository is to isolate completely and forever the radioactive wastes disposed of in it. However, since either normally or abnormally nuclides are to be released from the repository with a certain causes. The hydraulic conductivity is related to transportation of nuclide in soil. However, hydraulic characteristics research in unsaturated soil is not enough at present time. A fast and easy procedure for estimating unsaturated flow parameters is presented. The estimation is based on direct measurement of the retention characteristics combined with inverse estimation of the hydraulic conductivity characteristics from one-step outflow experiment

  20. One-step solution fabrication of magnetic chains consisting of jingle-bell-shaped cobalt mesospheres

    Science.gov (United States)

    Liang, Fang; Guo, Lin; Zhong, QunPeng; Wen, Xiaogang; Yang, Shihe; Zheng, Wangzhi; Chen, Chinping; Zhang, Nina; Chu, Weiguo

    2006-09-01

    Using a one-step solution phase approach, the authors have synthesized uniform jingle bell-shaped cobalt mesopheres (550-750nm) and assembled the mesospheres into long magnetic chains (20-30μm). All of the cobalt spheres are hollow with ˜40nm thick shells but each contains an ˜200nm diameter solid ball. The nano- to mesoscale structures were realized via reaction of CoCl2•6H2O and N2H4•H2O in the presence of polyvinylpyrrolidone (PVP) in an ethylene glycol solution. Magnetic measurements show a coercivity of about 75Oe with a remnance of 9.6emu /g at 300K. We propose a possible mechanism for the formation of the nanoto mesoscale structures.

  1. One-Step Method for Preparation of Magnetic Nanoparticles Coated with Chitosan

    Directory of Open Access Journals (Sweden)

    Karla M. Gregorio-Jauregui

    2012-01-01

    Full Text Available Preparation of magnetic nanoparticles coated with chitosan in one step by the coprecipitation method in the presence of different chitosan concentrations is reported here. Obtaining of magnetic superparamagnetic nanoparticles was confirmed by X-ray diffraction and magnetic measurements. Scanning transmission electron microscopy allowed to identify spheroidal nanoparticles with around 10-11 nm in average diameter. Characterization of the products by Fourier transform infrared spectroscopy demonstrated that composite chitosan-magnetic nanoparticles were obtained. Chitosan content in obtained nanocomposites was estimated by thermogravimetric analysis. The nanocomposites were tested in Pb2+ removal from a PbCl2 aqueous solution, showing a removal efficacy up to 53.6%. This work provides a simple method for chitosan-coated nanoparticles obtaining, which could be useful for heavy metal ions removal from water.

  2. Chitosan-Coated Magnetic Nanoparticles with Low Chitosan Content Prepared in One-Step

    Directory of Open Access Journals (Sweden)

    Yolanda Osuna

    2012-01-01

    Full Text Available Chitosan-coated magnetic nanoparticles (CMNP were obtained at 50°C in a one-step method comprising coprecipitation in the presence of low chitosan content. CMNP showed high magnetization and superparamagnetism. They were composed of a core of 9.5 nm in average diameter and a very thin chitosan layer in accordance with electron microscopy measurements. The results from Fourier transform infrared spectrometry demonstrated that CMNP were obtained and those from thermogravimetric analysis allowed to determine that they were composed of 95 wt% of magnetic nanoparticles and 5 wt% of chitosan. 67% efficacy in the Pb+2 removal test indicated that only 60% of amino groups on CMNP surface bound to Pb, probably due to some degree of nanoparticle flocculation during the redispersion. The very low weight ratio chitosan to magnetic nanoparticles obtained in this study, 0.053, and the high yield of the precipitation reactions (≈97% are noticeable.

  3. Preparation of one-step NiO/Ni-CGO composites using factorial design

    International Nuclear Information System (INIS)

    Araujo, A.J.M. de; Sousa, A R.O. de; Camposa, L.F.A.; Macedo, D.A.; Loureiro, F. J.A.; Fagg, D.P.

    2016-01-01

    This work deals with the synthesis, processing and characterization of NiO/Ni- CGO composite materials as potential solid oxide fuel cell (SOFC) anodes. The particulate materials were obtained by a one-step synthesis method and characterized by thermal analysis (prior to calcination) and X-ray diffraction (calcined powder). The ceramic processing of samples containing from 30 to 70 wt.% NiO was carried out by factorial design. Besides the NiO content controlled during the chemical synthesis, the impacts of the pore-former content (citric acid, used in proportions of 0, 7.5 and 15 wt.%) and the sintering temperature (1300, 1350 and 1400 °C) were also investigated. The open porosity of NiO-CGO composites and reduced Ni-CGO cermets was modeled as a function of factors (NiO content, citric acid content and sintering temperature) and interaction of factors. (author)

  4. One step sintering of homogenized bauxite raw material and kinetic study

    Science.gov (United States)

    Gao, Chang-he; Jiang, Peng; Li, Yong; Sun, Jia-lin; Zhang, Jun-jie; Yang, Huan-ying

    2016-10-01

    A one-step sintering process of bauxite raw material from direct mining was completed, and the kinetics of this process was analyzed thoroughly. The results show that the sintering kinetics of bauxite raw material exhibits the liquid-phase sintering behavior. A small portion of impurities existed in the raw material act as a liquid phase. After X-ray diffraction analyses, scanning electron microscopy observations, and kinetics calculations, sintering temperature and heating duration were determined as the two major factors contributing to the sintering process and densification of bauxite ore. An elevated heating temperature and longer duration favor the densification process. The major obstacle for the densification of bauxite material is attributed to the formation of the enclosed blowhole during liquid-phase sintering.

  5. Biodiesel Production from Wet Spirulina sp. by One-Step Extraction-Transesterification

    Directory of Open Access Journals (Sweden)

    Surya Pradana Yano

    2018-01-01

    Full Text Available Microalgae has gained immense interests as the raw material for biofuel production. The lipid content in microalgae can be converted into biodiesel through conventional method which involves separated process of extraction and transesterification. In this study, the production of biodiesel from Spirulina sp. was performed through one-step extraction-transesterification using KOH as base catalyst to simplify the production of biodiesel. The mixture of methanol-hexane was employed as both solvent and reactant in the process. The resulting biodiesel was found to be mainly composed of methyl oleate and methyl palmitate. On the other hand, increasing the reaction temperature and reducing the quantity of methanol in solvent mixture would also increase the yield of biodiesel. The optimum methanol-hexane volumetric ratio and temperature which gave the highest biodiesel yield were 3:7 and 50°C, respectively.

  6. One-Step Facile Synthesis of a Simple Hole Transport Material for Efficient Perovskite Solar Cells

    KAUST Repository

    Chen, Hu; Bryant, Daniel; Troughton, Joel; Kirkus, Mindaugas; Neophytou, Marios; Miao, Xiaohe; Durrant, James R.; McCulloch, Iain

    2016-01-01

    A hole transporting material was designed for use in perovskite solar cells, with a facile one-step synthesis from inexpensive, com-mercially available reagents. The molecule comprises a central fluorinated phenyl core with pendant aryl amines, namely, 3,6-difluoro-N1,N1,N2,N2,N4,N4,N5,N5-octakis(4-methoxyphenyl)benzene-1,2,4,5-tetraamine (DFTAB). A power conversion efficiency of up to 10.4% was achieved in a mesoporous perovskite device architecture. The merits of a simple and potentially low cost syn-thetic route as well as promising performance in perovskite devices, encourages further development of this materials class as new low-cost hole transporting materials for the scale up of perovskite solar cells.

  7. Linear and exponential TAIL-PCR: a method for efficient and quick amplification of flanking sequences adjacent to Tn5 transposon insertion sites.

    Science.gov (United States)

    Jia, Xianbo; Lin, Xinjian; Chen, Jichen

    2017-11-02

    Current genome walking methods are very time consuming, and many produce non-specific amplification products. To amplify the flanking sequences that are adjacent to Tn5 transposon insertion sites in Serratia marcescens FZSF02, we developed a genome walking method based on TAIL-PCR. This PCR method added a 20-cycle linear amplification step before the exponential amplification step to increase the concentration of the target sequences. Products of the linear amplification and the exponential amplification were diluted 100-fold to decrease the concentration of the templates that cause non-specific amplification. Fast DNA polymerase with a high extension speed was used in this method, and an amplification program was used to rapidly amplify long specific sequences. With this linear and exponential TAIL-PCR (LETAIL-PCR), we successfully obtained products larger than 2 kb from Tn5 transposon insertion mutant strains within 3 h. This method can be widely used in genome walking studies to amplify unknown sequences that are adjacent to known sequences.

  8. Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning ▿

    Science.gov (United States)

    Ruecker, Norma J.; Hoffman, Rebecca M.; Chalmers, Rachel M.; Neumann, Norman F.

    2011-01-01

    Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis, C. parvum, C. felis, C. meleagridis, C. ubiquitum, C. muris, and C. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common. PMID:21498746

  9. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography.

    Science.gov (United States)

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin

    2015-01-01

    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  10. One-step green synthesis of bimetallic Fe/Pd nanoparticles used to degrade Orange II

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Fang; Yang, Die; Chen, Zuliang, E-mail: zuliang.chen@newcastle.edu.au; Megharaj, Mallavarapu; Naidu, Ravendra

    2016-02-13

    Highlights: • Green synthesis of bimetallic Fe/Pd NPs was firstly reported using the one-step method. • 98.0% of Orange II was removed by Fe/Pd NPs, but only 16.0% by Fe NPs. • Fe/Pd NPs with a diameter ranging from 10 to 100 nm were observed. • Removing Orange II using Fe/Pd NPs involved both adsorption and catalytic degradation. - Abstract: To reduce cost and enhance reactivity, bimetallic Fe/Pd nanoparticles (NPs) were firstly synthesized using grape leaf aqueous extract to remove Orange II. Green synthesized bimetallic Fe/Pd NPs (98.0%) demonstrated a far higher ability to remove Orange II in 12 h compared to Fe NPs (16.0%). Meanwhile, all precursors, e.g., grape leaf extract, Fe{sup 2+} and Pd{sup 2+}, had no obvious effect on removing Orange II since less than 2.0% was removed. Kinetics study revealed that the removal rate fitted well to the pseudo-first-order reduction and pseudo-second-order adsorption model, meaning that removing Orange II via Fe/Pd NPs involved both adsorption and catalytic reduction. The remarkable stability of Fe/Pd NPs showed the potential application for removing azo dyes. Furthermore, Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FTIR) confirmed the changes in Fe/Pd NPs before and after reaction with Orange II. High Performance Liquid Chromatography–Mass Spectrum (HPLC–MS) identified the degraded products in the removal of Orange II, and finally a removal mechanism was proposed. This one-step strategy using grape leaf aqueous extract to synthesize Fe/Pd NPs is simple, cost-effective and environmentally benign, making possible the large-scale production of Fe/Pd NPs for field remediation.

  11. One-step reduced kinetics for lean hydrogen-air deflagration

    Energy Technology Data Exchange (ETDEWEB)

    Fernandez-Galisteo, D.; Sanchez, A.L. [Area de Mecanica de Fluidos, Univ. Carlos III de Madrid, Leganes 28911 (Spain); Linan, A. [ETSI Aeronauticos, Pl. Cardenal Cisneros 3, Madrid 28040 (Spain); Williams, F.A. [Dept. of Mechanical and Aerospace Engineering, University of California San Diego, La Jolla, CA 92093-0411 (United States)

    2009-05-15

    A short mechanism consisting of seven elementary reactions, of which only three are reversible, is shown to provide good predictions of hydrogen-air lean-flame burning velocities. This mechanism is further simplified by noting that over a range of conditions of practical interest, near the lean flammability limit all reaction intermediaries have small concentrations in the important thin reaction zone that controls the hydrogen-air laminar burning velocity and therefore follow a steady state approximation, while the main species react according to the global irreversible reaction 2H{sub 2} + O{sub 2} {yields} 2H{sub 2}O. An explicit expression for the non-Arrhenius rate of this one-step overall reaction for hydrogen oxidation is derived from the seven-step detailed mechanism, for application near the flammability limit. The one-step results are used to calculate flammability limits and burning velocities of planar deflagrations. Furthermore, implications concerning radical profiles in the deflagration and reasons for the success of the approximations are clarified. It is also demonstrated that adding only two irreversible direct recombination steps to the seven-step mechanism accurately reproduces burning velocities of the full detailed mechanism for all equivalence ratios at normal atmospheric conditions and that an eight-step detailed mechanism, constructed from the seven-step mechanism by adding to it the fourth reversible shuffle reaction, improves predictions of O and OH profiles. The new reduced-chemistry descriptions can be useful for both analytical and computational studies of lean hydrogen-air flames, decreasing required computation times. (author)

  12. Improved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nucleotide sequence of an outer membrane lipoprotein

    DEFF Research Database (Denmark)

    Gram, Trine; Ahrens, Peter

    1998-01-01

    species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR, as were tonsil cultures from 50 pigs of an A. pleuropneumoniae-negative herd. The sensitivity assessed by agarose gel analysis of the PCR product was 10(2) CFU/PCR test tube. The specificity...

  13. Conserved PCR primer set designing for closely-related species to complete mitochondrial genome sequencing using a sliding window-based PSO algorithm.

    Directory of Open Access Journals (Sweden)

    Cheng-Hong Yang

    Full Text Available BACKGROUND: Complete mitochondrial (mt genome sequencing is becoming increasingly common for phylogenetic reconstruction and as a model for genome evolution. For long template sequencing, i.e., like the entire mtDNA, it is essential to design primers for Polymerase Chain Reaction (PCR amplicons which are partly overlapping each other. The presented chromosome walking strategy provides the overlapping design to solve the problem for unreliable sequencing data at the 5' end and provides the effective sequencing. However, current algorithms and tools are mostly focused on the primer design for a local region in the genomic sequence. Accordingly, it is still challenging to provide the primer sets for the entire mtDNA. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of this study is to develop an integrated primer design algorithm for entire mt genome in general, and for the common primer sets for closely-related species in particular. We introduce ClustalW to generate the multiple sequence alignment needed to find the conserved sequences in closely-related species. These conserved sequences are suitable for designing the common primers for the entire mtDNA. Using a heuristic algorithm particle swarm optimization (PSO, all the designed primers were computationally validated to fit the common primer design constraints, such as the melting temperature, primer length and GC content, PCR product length, secondary structure, specificity, and terminal limitation. The overlap requirement for PCR amplicons in the entire mtDNA is satisfied by defining the overlapping region with the sliding window technology. Finally, primer sets were designed within the overlapping region. The primer sets for the entire mtDNA sequences were successfully demonstrated in the example of two closely-related fish species. The pseudo code for the primer design algorithm is provided. CONCLUSIONS/SIGNIFICANCE: In conclusion, it can be said that our proposed sliding window-based PSO

  14. Rapid establishment of thermophilic anaerobic microbial community during the one-step startup of thermophilic anaerobic digestion from a mesophilic digester.

    Science.gov (United States)

    Tian, Zhe; Zhang, Yu; Li, Yuyou; Chi, Yongzhi; Yang, Min

    2015-02-01

    The purpose of this study was to explore how fast the thermophilic anaerobic microbial community could be established during the one-step startup of thermophilic anaerobic digestion from a mesophilic digester. Stable thermophilic anaerobic digestion was achieved within 20 days from a mesophilic digester treating sewage sludge by adopting the one-step startup strategy. The succession of archaeal and bacterial populations over a period of 60 days after the temperature increment was followed by using 454-pyrosequencing and quantitative PCR. After the increase of temperature, thermophilic methanogenic community was established within 11 days, which was characterized by the fast colonization of Methanosarcina thermophila and two hydrogenotrophic methanogens (Methanothermobacter spp. and Methanoculleus spp.). At the same time, the bacterial community was dominated by Fervidobacterium, whose relative abundance rapidly increased from 0 to 28.52 % in 18 days, followed by other potential thermophilic genera, such as Clostridium, Coprothermobacter, Anaerobaculum and EM3. The above result demonstrated that the one-step startup strategy could allow the rapid establishment of the thermophilic anaerobic microbial community. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Legionella species and serogroups in Malaysian water cooling towers: identification by latex agglutination and PCR-DNA sequencing of isolates.

    Science.gov (United States)

    Yong, Stacey Foong Yee; Goh, Fen-Ning; Ngeow, Yun Fong

    2010-03-01

    In this study, we investigated the distribution of Legionella species in water cooling towers located in different parts of Malaysia to obtain information that may inform public health policies for the prevention of legionellosis. A total of 20 water samples were collected from 11 cooling towers located in three different states in east, west and south Malaysia. The samples were concentrated by filtration and treated with an acid buffer before plating on to BCYE agar. Legionella viable counts in these samples ranged from 100 to 2,000 CFU ml(-1); 28 isolates from the 24 samples were examined by latex agglutination as well as 16S rRNA and rpoB PCR-DNA sequencing. These isolates were identified as Legionella pneumophila serogroup 1 (35.7%), L. pneumophila serogroup 2-14 (39%), L. pneumophila non-groupable (10.7%), L. busanensis, L. gormanii, L. anisa and L. gresilensis. L. pneumophila was clearly the predominant species at all sampling sites. Repeat sampling from the same cooling tower and testing different colonies from the same water sample showed concurrent colonization by different serogroups and different species of Legionella in some of the cooling towers.

  16. Toxic Cyanobacterial Bloom Triggers in Missisquoi Bay, Lake Champlain, as Determined by Next-Generation Sequencing and Quantitative PCR

    Directory of Open Access Journals (Sweden)

    Nathalie Fortin

    2015-05-01

    Full Text Available Missisquoi Bay (MB is a temperate eutrophic freshwater lake that frequently experiences toxic Microcystis-dominated cyanobacterial blooms. Non-point sources are responsible for the high concentrations of phosphorus and nitrogen in the bay. This study combined data from environmental parameters, E. coli counts, high-throughput sequencing of 16S rRNA gene amplicons, quantitative PCR (16S rRNA and mcyD genes and toxin analyses to identify the main bloom-promoting factors. In 2009, nutrient concentrations correlated with E. coli counts, abundance of total cyanobacterial cells, Microcystis 16S rRNA and mcyD genes and intracellular microcystin. Total and dissolved phosphorus also correlated significantly with rainfall. The major cyanobacterial taxa were members of the orders Chroococcales and Nostocales. The genus Microcystis was the main mcyD-carrier and main microcystin producer. Our results suggested that increasing nutrient concentrations and total nitrogen:total phosphorus (TN:TP ratios approaching 11:1, coupled with an increase in temperature, promoted Microcystis-dominated toxic blooms. Although the importance of nutrient ratios and absolute concentrations on cyanobacterial and Microcystis dynamics have been documented in other laboratories, an optimum TN:TP ratio for Microcystis dominance has not been previously observed in situ. This observation provides further support that nutrient ratios are an important determinant of species composition in natural phytoplankton assemblages.

  17. 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...

  18. Efficacy of Pulsed-Field Gel Electrophoresis and Repetitive Element Sequence-Based PCR in Typing of Salmonella Isolates from Assam, India.

    Science.gov (United States)

    Gogoi, Purnima; Borah, Probodh; Hussain, Iftikar; Das, Leena; Hazarika, Girin; Tamuly, Shantanu; Barkalita, Luit Moni

    2018-05-01

    A total of 12 Salmonella isolates belonging to different serovars, viz , Salmonella enterica serovar Enteritidis ( n = 4), Salmonella enterica serovar Weltevreden ( n = 4), Salmonella enterica serovar Newport ( n = 1), Salmonella enterica serovar Litchifield ( n = 1), and untypeable strains ( n = 2) were isolated from 332 diarrheic fecal samples collected from animals, birds, and humans. Of the two molecular typing methods applied, viz , repetitive element sequence-based PCR (REP-PCR) and pulsed-field gel electrophoresis (PFGE), PFGE could clearly differentiate the strains belonging to different serovars as well as differentiate between strains of the same serovar with respect to their source of isolation, whereas REP-PCR could not differentiate between strains of the same serovar. Thus, it can be suggested that PFGE is more useful and appropriate for molecular typing of Salmonella isolates during epidemiological investigations than REP-PCR. Copyright © 2018 American Society for Microbiology.

  19. Characterisation of Toxoplasma gondii isolates using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the non-coding Toxoplasma gondii (TGR)-gene sequences

    DEFF Research Database (Denmark)

    Høgdall, Estrid; Vuust, Jens; Lind, Peter

    2000-01-01

    of using TGR gene variants as markers to distinguish among T. gondii isolates from different animals and different geographical sources. Based on the band patterns obtained by restriction fragment length polymorphism (RFLP) analysis of the polymerase chain reaction (PCR) amplified TGR sequences, the T...

  20. Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency

    International Nuclear Information System (INIS)

    Ruijter, Jan M.; Hoff, Maurice J. B. van den; Lorenz, Peter; Tuomi, Jari M.; Hecker, Michael

    2014-01-01

    The analysis of quantitative PCR data usually does not take into account the fact that the increase in fluorescence depends on the monitoring chemistry, the input of ds-DNA or ss-cDNA, and the directionality of the targeting of probes or primers. The monitoring chemistries currently available can be categorized into six groups: (A) DNA-binding dyes; (B) hybridization probes; (C) hydrolysis probes; (D) LUX primers; (E) hairpin primers; and (F) the QZyme system. We have determined the kinetics of the increase in fluorescence for each of these groups with respect to the input of both ds-DNA and ss-cDNA. For the latter, we also evaluated mRNA and cDNA targeting probes or primers. This analysis revealed three situations. Hydrolysis probes and LUX primers, compared to DNA-binding dyes, do not require a correction of the observed quantification cycle. Hybridization probes and hairpin primers require a correction of −1 cycle (dubbed C-lag), while the QZyme system requires the C-lag correction and an efficiency-dependent C-shift correction. A PCR efficiency value can be derived from the relative increase in fluorescence in the exponential phase of the amplification curve for all monitoring chemistries. In case of hydrolysis probes, LUX primers and hairpin primers, however, this should be performed after cycle 12, and for the QZyme system after cycle 19, to keep the overestimation of the PCR efficiency below 0.5 %. (author)

  1. One-step preparation and photocatalytic performance of vanadium doped TiO{sub 2} coatings

    Energy Technology Data Exchange (ETDEWEB)

    Vasilić, R., E-mail: rastko.vasilic@ff.bg.ac.rs [University of Belgrade, Faculty of Physics, Studentski trg 12-16, 11000 Belgrade (Serbia); Stojadinović, S. [University of Belgrade, Faculty of Physics, Studentski trg 12-16, 11000 Belgrade (Serbia); Radić, N. [University of Belgrade, Institute of Chemistry, Technology and Metallurgy, Department of Catalysis and Chemical Engineering, Njegoševa 12, 11000 Belgrade (Serbia); Stefanov, P. [Institute of General and Inorganic Chemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Block 11, Sofia 1113 (Bulgaria); Dohčević-Mitrović, Z. [University of Belgrade, Institute of Physics, Pregrevica 118, 11080 Belgrade (Serbia); Grbić, B. [University of Belgrade, Institute of Chemistry, Technology and Metallurgy, Department of Catalysis and Chemical Engineering, Njegoševa 12, 11000 Belgrade (Serbia)

    2015-02-01

    In this paper, we have investigated one-step preparation of vanadium doped TiO{sub 2} coatings formed by plasma electrolytic oxidation (PEO) of titanium in electrolyte containing 10 g/L Na{sub 3}PO{sub 4}·12H{sub 2}O + 0.5 g/L NH{sub 4}VO{sub 3}. The morphology, phase structure, and elemental composition of the formed coatings were characterized by atomic force microscopy (AFM), x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) techniques. Ultraviolet–visible diffuse reflectance spectroscopy (UV–Vis DRS) was employed to evaluate the band gap energy of obtained coatings. Vanadium doped TiO{sub 2} coatings are partly crystallized and mainly composed of anatase phase TiO{sub 2}, with up to about 2 wt% of vanadium present in the surface layer of the oxide. The valence band photoelectron spectra and UV–Vis DRS showed that vanadium doped TiO{sub 2} coatings exhibit notable red shift with respect to the pure TiO{sub 2} coatings. The photocatalytic activity was evaluated by monitoring the degradation of methyl orange under simulated sunlight conditions. Photocatalytic activity of vanadium doped TiO{sub 2} coatings increases with PEO time. Prolonged PEO times result in higher roughness of obtained coatings, thus increasing surface area available for methyl orange degradation. Vanadium doped TiO{sub 2} coatings obtained after 180 s of PEO time exhibit the best photocatalytic activity and about 67% of methyl orange is degraded after 12 h of irradiation under simulated sunlight. - Highlights: • One-step preparation of V-doped TiO{sub 2} coatings in 10 g/L Na{sub 3}PO{sub 4}·12H{sub 2}O + 0.5 g/L NH{sub 4}VO{sub 3}. • Properties of obtained coatings strongly depend on microdischarge characteristics. • Band gap of V-doped TiO{sub 2} coatings is shifted towards red side of the spectrum. • V-doped TiO{sub 2} coatings have better photocatalytic activity than pure TiO{sub 2}. • After 12 h of simulated sunlight irradiation, 67% of

  2. One-step preparation and photocatalytic performance of vanadium doped TiO2 coatings

    International Nuclear Information System (INIS)

    Vasilić, R.; Stojadinović, S.; Radić, N.; Stefanov, P.; Dohčević-Mitrović, Z.; Grbić, B.

    2015-01-01

    In this paper, we have investigated one-step preparation of vanadium doped TiO 2 coatings formed by plasma electrolytic oxidation (PEO) of titanium in electrolyte containing 10 g/L Na 3 PO 4 ·12H 2 O + 0.5 g/L NH 4 VO 3 . The morphology, phase structure, and elemental composition of the formed coatings were characterized by atomic force microscopy (AFM), x-ray diffraction (XRD), and x-ray photoelectron spectroscopy (XPS) techniques. Ultraviolet–visible diffuse reflectance spectroscopy (UV–Vis DRS) was employed to evaluate the band gap energy of obtained coatings. Vanadium doped TiO 2 coatings are partly crystallized and mainly composed of anatase phase TiO 2 , with up to about 2 wt% of vanadium present in the surface layer of the oxide. The valence band photoelectron spectra and UV–Vis DRS showed that vanadium doped TiO 2 coatings exhibit notable red shift with respect to the pure TiO 2 coatings. The photocatalytic activity was evaluated by monitoring the degradation of methyl orange under simulated sunlight conditions. Photocatalytic activity of vanadium doped TiO 2 coatings increases with PEO time. Prolonged PEO times result in higher roughness of obtained coatings, thus increasing surface area available for methyl orange degradation. Vanadium doped TiO 2 coatings obtained after 180 s of PEO time exhibit the best photocatalytic activity and about 67% of methyl orange is degraded after 12 h of irradiation under simulated sunlight. - Highlights: • One-step preparation of V-doped TiO 2 coatings in 10 g/L Na 3 PO 4 ·12H 2 O + 0.5 g/L NH 4 VO 3 . • Properties of obtained coatings strongly depend on microdischarge characteristics. • Band gap of V-doped TiO 2 coatings is shifted towards red side of the spectrum. • V-doped TiO 2 coatings have better photocatalytic activity than pure TiO 2 . • After 12 h of simulated sunlight irradiation, 67% of methyl orange was decomposed

  3. One-step breast reconstruction with polyurethane-covered implants after skin-sparing mastectomy.

    Science.gov (United States)

    Rancati, Alberto; Soderini, Alejandro; Dorr, Julio; Gercovich, Gustavo; Tessari, Luciano; Gonzalez, Eduardo

    2013-12-01

    Skin-sparing mastectomy (SSM) and immediate one-step breast reconstruction with implants has become an increasingly popular, effective treatment for selected patients with breast carcinoma. However, it is associated with high complication rates. Breast augmentation with polyurethane-covered implants (PCIs) has consistently had optimal short-term and long-term results with low rates of capsular contracture. The aim of this study was to evaluate the clinical and aesthetic outcomes of immediate one-step breast reconstruction with PCI after SSM in early breast cancer patients at a single institution. We reviewed the records of 221 consecutive breast cancer patients who underwent one-stage immediate reconstruction with PCI after SSM from 1995 through 2005. Patient and tumour characteristics, type of reconstruction, postoperative complications, aesthetic results and recurrence rate were analysed. The mean age of the patients was 52±11 years (range, 30-76; standard deviation (SD), 11). The American Joint Committee on Cancer (AJCC) pathologic stages were 0 (10%), I (63.3%) and II (26.7%). Thirty-nine (17.65%; confidence interval (CI)=13.04-23.1) of the 221 patients had complications; seven had prosthesis extrusion requiring an implant (five due to skin necrosis, one due to infection and one due to late haematoma). In six of these seven cases, the procedure was indicated for local recurrence after conservative breast surgery with adjunctive radiation therapy (rescue procedure). Thirty-two (14.4%) patients had minor complications: 12 had cutaneous rash, four had malpositioned implants and 16 had inadequate implant projection. At long-term follow-up, four (1.8%) patients had developed grade IV capsular contracture associated with postoperative radiation therapy. At a median follow-up of 98 months (range, 36-156), 14 (6.3%) patients had tumour recurrence and 12.2% had distant metastasis. Nineteen patients had died of cancer, and 192 (86.8%) remained disease free. One

  4. Conventional Morphology Versus PCR Sequencing, rep-PCR, and MALDI-TOF-MS for Identification of Clinical Aspergillus Isolates Collected Over a 2-Year Period in a University Hospital at Kayseri, Turkey.

    Science.gov (United States)

    Atalay, Altay; Koc, Ayse Nedret; Suel, Ahmet; Sav, Hafize; Demir, Gonca; Elmali, Ferhan; Cakir, Nuri; Seyedmousavi, Seyedmojtaba

    2016-09-01

    Aspergillus species cause a wide range of diseases in humans, including allergies, localized infections, or fatal disseminated diseases. Rapid detection and identification of Aspergillus spp. facilitate effective patient management. In the current study we compared conventional morphological methods with PCR sequencing, rep-PCR, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for the identification of Aspergillus strains. A total of 24 consecutive clinical isolates of Aspergillus were collected during 2012-2014. Conventional morphology and rep-PCR were performed in our Mycology Laboratory. The identification, evaluation, and reporting of strains using MALDI-TOF-MS were performed by BioMérieux Diagnostic, Inc. in Istanbul. DNA sequence analysis of the clinical isolates was performed by the BMLabosis laboratory in Ankara. Samples consisted of 18 (75%) lower respiratory tract specimens, 3 otomycosis (12.5%) ear tissues, 1 sample from keratitis, and 1 sample from a cutaneous wound. According to DNA sequence analysis, 12 (50%) specimens were identified as A. fumigatus, 8 (33.3%) as A. flavus, 3 (12.5%) as A. niger, and 1 (4.2%) as A. terreus. Statistically, there was good agreement between the conventional morphology and rep-PCR and MALDI-TOF methods; kappa values were κ = 0.869, 0.871, and 0.916, respectively (P < 0.001). The good level of agreement between the methods included in the present study and sequence method could be due to the identification of Aspergillus strains that were commonly encountered. Therefore, it was concluded that studies conducted with a higher number of isolates, which include other Aspergillus strains, are required. © 2016 Wiley Periodicals, Inc.

  5. A Cancer Cell-Activatable Aptamer-Reporter System for One-Step Assay of Circulating Tumor Cells

    Directory of Open Access Journals (Sweden)

    Zihua Zeng

    2014-01-01

    Full Text Available The current antibody-mediated numeration assays of circulating tumor cells (CTCs require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

  6. Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae.

    Science.gov (United States)

    Bao, Zehua; Xiao, Han; Liang, Jing; Zhang, Lu; Xiong, Xiong; Sun, Ning; Si, Tong; Zhao, Huimin

    2015-05-15

    One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

  7. Stability of one-step methods in transient nonlinear heat conduction

    International Nuclear Information System (INIS)

    Hughes, J.R.

    1977-01-01

    The purpose of the present work is to ascertain practical stability conditions for one-step methods commonly used in transient nonlinear heat conduction analyses. In this paper the concepts of stability, appropriate to the nonlinear problem, are thoroughly discussed. They of course reduce to the usual stability critierion for the linear, constant coefficient case. However, for nonlinear problems there are differences and theses ideas are of key importance in obtaining practical stability conditions. Of particular importance is a recent result which indicates that, in a sense, the trapezoidal and midpoint families are equivalent. Thus, stability results for one family may be translated into a result for the other. The main results obtained are: The stability behaviour of the explicit Euler method in the nonlinear regime is analogous to that for linear problems. In particular, an a priori step size restriction may be determined for each time step. The precise time step restriction on implicit conditionally stable members of the trapezoidal and midpoint families is shown not to be determinable a priori. Of considerable practical significance, unconditionally stable members of the trapezoidal and midpoint families are identified. All notions of stability employed are motivated and defined, and their interpretations in practical computing are indicated. (Auth.)

  8. InGaN directional coupler made with a one-step etching technique

    Science.gov (United States)

    Gao, Xumin; Yuan, Jialei; Yang, Yongchao; Zhang, Shuai; Shi, Zheng; Li, Xin; Wang, Yongjin

    2017-06-01

    We propose, fabricate and characterize an on-chip integration of light source, InGaN waveguide, directional coupler and photodiode, in which AlGaN layers are used as top and bottom optical claddings to form an InGaN waveguide for guiding the in-plane emitted light from the InGaN/GaN multiple-quantum-well light-emitting diode (MQW-LED). The difference in etch rate caused by different exposure windows leads to an etching depth discrepancy using the one-step etching technique, which forms the InGaN directional coupler with the overlapped underlying slab. Light propagation results directly confirm effective light coupling in the InGaN directional coupler, which is achieved through high-order guided modes. The InGaN waveguide couples the modulated light from the InGaN/GaN MQW-LED and transfers part of light to the coupled waveguide via the InGaN directional coupler. The in-plane InGaN/GaN MQW-photodiode absorbs the guided light by the coupled InGaN waveguide and induces the photocurrent. The on-chip InGaN photonic integration experimentally demonstrates an in-plane light communication with a data transmission of 50 Mbps.

  9. Analyses of one-step liquid hydrogen production from methane and landfill gas

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Cunping; T-Raissi, Ali [University of Central Florida, Florida Solar Energy Center, 1679 Clearlake Road, Cocoa, FL 32922-5703 (United States)

    2007-11-15

    Conventional liquid hydrogen (LH{sub 2}) production consists of two basic steps: (1) gaseous hydrogen (GH{sub 2}) production via steam methane reformation followed by purification by means of pressure swing adsorption (PSA), and (2) GH{sub 2} liquefaction. LH{sub 2} produced by the conventional processes is not carbon neutral because of the carbon dioxide (CO{sub 2}) emission from PSA operation. A novel concept is herein presented and flowsheeted for LH{sub 2} production with zero carbon emission using methane (CH{sub 4}) or landfill gas as feedstock. A cryogenic process is used for both H{sub 2} separation/purification and liquefaction. This one-step process can substantially increase the efficiency and reduce costs because no PSA step is required. Furthermore, the integrated process results in no CO{sub 2} emissions and minimal H{sub 2} losses. Of the five flowsheets presented, one that combines low and high temperature CO/CH{sub 4} reforming reactions in a single reactor shows the highest overall efficiency with the first and second law efficiencies of 85% and 56%, respectively. The latter figure assumes 10% overall energy loss and 30% efficiency for the cryogenic process. (author)

  10. [Exploration of one-step preparation of Ganoderma lucidum multicomponent microemulsion].

    Science.gov (United States)

    He, Jun-Jie; Chen, Yan; Du, Meng; Cao, Wei; Yuan, Ling; Zheng, Li-Yan

    2013-03-01

    To explore one-step method for the preparation of Ganoderma lucidum multicomponent microemulsion, according to the dissolution characteristics of triterpenes and polysaccharides in Ganoderma lucidum, formulation of the microemulsion was optimized. The optimal blank microemulsion was used as a solvent to sonicate the Ganoderma lucidum powder to prepare the multicomponent microemulsion, besides, its physicochemical properties were compared with the microemulsion made by conventional method. The results showed that the multicomponent microemulsion was characterized as (43.32 +/- 6.82) nm in size, 0.173 +/- 0.025 in polydispersity index (PDI) and -(3.98 +/- 0.82) mV in zeta potential. The contents of Ganoderma lucidum triterpenes and polysaccharides were (5.95 +/- 0.32) and (7.58 +/- 0.44) mg x mL(-1), respectively. Sonicating Ganoderma lucidum powder by blank microemulsion could prepare the multicomponent microemulsion. Compared with the conventional method, this method is simple and low cost, which is suitable for industrial production.

  11. One-Step Electrodeposited Nickel Cobalt Sulfide Nanosheet Arrays for High-Performance Asymmetric Supercapacitors

    KAUST Repository

    Chen, Wei

    2014-09-23

    A facile one-step electrodeposition method is developed to prepare ternary nickel cobalt sulfide interconnected nanosheet arrays on conductive carbon substrates as electrodes for supercapacitors, resulting in exceptional energy storage performance. Taking advantages of the highly conductive, mesoporous nature of the nanosheets and open framework of the three-dimensional nanoarchitectures, the ternary sulfide electrodes exhibit high specific capacitance (1418 F g(-1) at 5 A g(-1) and 1285 F g(-1) at 100 A g(-1)) with excellent rate capability. An asymmetric supercapacitor fabricated by the ternary sulfide nanosheet arrays as positive electrode and porous graphene film as negative electrode demonstrates outstanding electrochemical performance for practical energy storage applications. Our asymmetric supercapacitors show a high energy density of 60 Wh kg(-1) at a power density of 1.8 kW kg(-1). Even when charging the cell within 4.5 s, the energy density is still as high as 33 Wh kg(-1) at an outstanding power density of 28.8 kW kg(-1) with robust long-term cycling stability up to 50 000 cycles.

  12. One-step sol-gel imprint lithography for guided-mode resonance structures.

    Science.gov (United States)

    Huang, Yin; Liu, Longju; Johnson, Michael; C Hillier, Andrew; Lu, Meng

    2016-03-04

    Guided-mode resonance (GMR) structures consisting of sub-wavelength periodic gratings are capable of producing narrow-linewidth optical resonances. This paper describes a sol-gel-based imprint lithography method for the fabrication of submicron 1D and 2D GMR structures. This method utilizes a patterned polydimethylsiloxane (PDMS) mold to fabricate the grating coupler and waveguide for a GMR device using a sol-gel thin film in a single step. An organic-inorganic hybrid sol-gel film was selected as the imprint material because of its relatively high refractive index. The optical responses of several sol-gel GMR devices were characterized, and the experimental results were in good agreement with the results of electromagnetic simulations. The influence of processing parameters was investigated in order to determine how finely the spectral response and resonant wavelength of the GMR devices could be tuned. As an example potential application, refractometric sensing experiments were performed using a 1D sol-gel device. The results demonstrated a refractive index sensitivity of 50 nm/refractive index unit. This one-step fabrication process offers a simple, rapid, and low-cost means of fabricating GMR structures. We anticipate that this method can be valuable in the development of various GMR-based devices as it can readily enable the fabrication of complex shapes and allow the doping of optically active materials into sol-gel thin film.

  13. Microwave-assisted one-step synthesis of white light-emitting carbon dot suspensions

    Science.gov (United States)

    Vanessa, Hinterberger; Wenshuo, Wang; Cornelia, Damm; Simon, Wawra; Martin, Thoma; Wolfgang, Peukert

    2018-06-01

    In this contribution, we demonstrate that an aqueous solution with adjustable fluorescent color, including white light emission, can be achieved by a rapid one-step microwave synthesis method resulting in a mixture of blue-emitting carbon dots (CDs) and the yellow-emitting 2,3-diaminophenazine (DAP). Aqueous mixtures of o-phenylene-diamine (oPD) and citric acid (CA) are used as precursors. The resulting product structures are analyzed by FT-IR and NMR spectroscopy and the size of the resulting CDs is determined by atomic force microscopy to be 1.1 ± 0.3 nm. The synthesized solution exhibits two fluorescence emission peaks at 430 and 560 nm, which were found to originate from the CDs and DAP, respectively. The intensity ratio of both fluorescence peaks depends on pH, which is driven by the protonation state of DAP. In consequence, the fluorescence emission color of the CD solution can be tuned precisely and reproducibly from blue to white to yellow by careful control of the pH. Finally, at a pH level of 5.4, at which there is equal blue and yellow emission intensity, a white light emitting solution can be successfully produced in a very fast and simple synthesis procedure.

  14. One-step exfoliation and surface modification of lamellar hydroxyapatite by intercalation of glucosamine

    Energy Technology Data Exchange (ETDEWEB)

    Luo, Honglin [Research Institute for Biomaterials and Transportation, East China Jiaotong University, Nanchang, 330013 (China); School of Materials Science and Engineering, Tianjin University, Tianjin, 300072 (China); Li, Wei; Ji, Dehui [Research Institute for Biomaterials and Transportation, East China Jiaotong University, Nanchang, 330013 (China); Zuo, Guifu [Hebei Provincial Key Laboratory of Inorganic Nonmetallic Materials, College of Materials Science and Engineering, Hebei United University, Tangshan, 063009 (China); Xiong, Guangyao, E-mail: xiongguangyao@163.com [Research Institute for Biomaterials and Transportation, East China Jiaotong University, Nanchang, 330013 (China); Zhu, Yong [School of Chemical Engineering, Tianjin University, Tianjin, 300072 (China); Li, Lili; Han, Ming [Research Institute for Biomaterials and Transportation, East China Jiaotong University, Nanchang, 330013 (China); Wu, Caoqun [School of Materials Science and Engineering, Tianjin University, Tianjin, 300072 (China); Wan, Yizao, E-mail: yzwantju@126.com [Research Institute for Biomaterials and Transportation, East China Jiaotong University, Nanchang, 330013 (China); School of Materials Science and Engineering, Tianjin University, Tianjin, 300072 (China)

    2016-04-15

    Effective exfoliation is crucial to the application of layered materials in many fields. Herein, we report a novel effective, scalable, and ecofriendly method for the exfoliation of lamellar HAp by glucosamine intercalation such that individual HAp nanoplates can be obtained. The as-exfoliated HAp nanoplates were characterized by Fourier transform infrared (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), and thermogravimetric (TG) analysis. It is found that the glucosamine intercalation not only results in complete exfoliation of lamellar HAp but also introduces the glucosamine molecules onto the surface of individual HAp nanoplates, thus obtaining separated glucosamine-grafted HAp nanoplates (Glu-HAps). Results from MTT assay demonstrate that glucosamine grafting on HAp nanoplates greatly improves the cell growth and proliferation as compared to nongrafted HAp counterparts. - Highlights: • Glucosamine was used as intercalation agent to exfoliate lamellar hydroxyapatite. • Glucosamine was grafted onto the as-exfoliated nanoplate-like hydroxyapatite. • Exfoliation and surface grafting were accomplished in one step. • Glucosamine-grafted HAp showed improved biocompatibility over nongrafted one.

  15. One-step electrodeposited nickel cobalt sulfide nanosheet arrays for high-performance asymmetric supercapacitors.

    Science.gov (United States)

    Chen, Wei; Xia, Chuan; Alshareef, Husam N

    2014-09-23

    A facile one-step electrodeposition method is developed to prepare ternary nickel cobalt sulfide interconnected nanosheet arrays on conductive carbon substrates as electrodes for supercapacitors, resulting in exceptional energy storage performance. Taking advantages of the highly conductive, mesoporous nature of the nanosheets and open framework of the three-dimensional nanoarchitectures, the ternary sulfide electrodes exhibit high specific capacitance (1418 F g(-1) at 5 A g(-1) and 1285 F g(-1) at 100 A g(-1)) with excellent rate capability. An asymmetric supercapacitor fabricated by the ternary sulfide nanosheet arrays as positive electrode and porous graphene film as negative electrode demonstrates outstanding electrochemical performance for practical energy storage applications. Our asymmetric supercapacitors show a high energy density of 60 Wh kg(-1) at a power density of 1.8 kW kg(-1). Even when charging the cell within 4.5 s, the energy density is still as high as 33 Wh kg(-1) at an outstanding power density of 28.8 kW kg(-1) with robust long-term cycling stability up to 50,000 cycles.

  16. Factors associated with one step surgery in case of non-palpable breast cancer

    International Nuclear Information System (INIS)

    Schaefer, Fritz K.; Eden, I.; Schaefer, P.J.; Peter, D.; Jonat, W.; Heller, M.; Schreer, I.

    2007-01-01

    Purpose: To examine factors associated with one step surgery in case of non-palpable breast cancer. Materials and methods: Clinical data of 152 consecutively diagnosed patients with breast cancer were analyzed retrospectively. Preoperative diagnostic findings were divided in subgroups: mammographically visible mass/microcalcifications/sonographically visible mass/sonographically visible architectural distortion. Correlation between tumor-size, radiologic tumor morphology, quality of localization and number of operation was evulated. For localization exact wire position was defined less than 3 mm apart from the lesion. Results: One hundred and thirty-six patients attempted breast conservation and underwent preoperative tumor localization. Fourteen of 16 patients had mastectomy without preoperative localization. Average tumor size was 12 mm for one-operation, and 17 mm for re-operation. Significant correlation (p < 0.001) was found between one operation and masses visible in mammograms (55/62 (89%) patients) or sonography (53/64 (83%) patients). Significant correlation was found (p < 0.001) between more re-operation and microcalcifications in mammograms (33/89 (37% patients). In 123/138 (89%) cases wire position was central, in 15/138 (11%) cases distance was maximally 10 mm. No significant correlation was found between number of operation and wire position. Re-operation was required in 38 cases. Conclusion: Mammographically or sonographically visible mass, small size of tumors, preoperative percutaneous biopsy and exact preoperative localization are important for a single step procedure for definite surgical treatment, that we found in 74% of the patients

  17. Engineering more stable, selectable marker-free autoluminescent mycobacteria by one step.

    Science.gov (United States)

    Yang, Feng; Njire, Moses M; Liu, Jia; Wu, Tian; Wang, Bangxing; Liu, Tianzhou; Cao, Yuanyuan; Liu, Zhiyong; Wan, Junting; Tu, Zhengchao; Tan, Yaoju; Tan, Shouyong; Zhang, Tianyu

    2015-01-01

    In our previous study, we demonstrated that the use of the autoluminescent Mycobacterium tuberculosis as a reporter strain had the potential to drastically reduce the time, effort, animals and costs consumed in evaluation of the activities of drugs and vaccines in live mice. However, the strains were relatively unstable and lost reporter with time without selection. The kanamycin selection marker used wasn't the best choice as it provides resistance to amino glycosides which are an important class of second line drugs used in tuberculosis treatment. In addition, the marker could limit utility of the strains for screening of new potential drugs or evaluating drug combinations for tuberculosis treatment. Limited selection marker genes for mycobacterial genetic manipulation is a major drawback for such a marker-containing strain in many research fields. Therefore, selectable marker-free, more stable autoluminescent mycobacteria are highly needed. After trying several strategies, we created such mycobacterial strains successfully by using an integrative vector and removing both the resistance maker and integrase genes by Xer site-specific recombination in one step. The corresponding plasmid vectors developed in this study could be very convenient in constructing other selectable marker-free, more stable reporter mycobacteria with diverse applications.

  18. Engineering more stable, selectable marker-free autoluminescent mycobacteria by one step.

    Directory of Open Access Journals (Sweden)

    Feng Yang

    Full Text Available In our previous study, we demonstrated that the use of the autoluminescent Mycobacterium tuberculosis as a reporter strain had the potential to drastically reduce the time, effort, animals and costs consumed in evaluation of the activities of drugs and vaccines in live mice. However, the strains were relatively unstable and lost reporter with time without selection. The kanamycin selection marker used wasn't the best choice as it provides resistance to amino glycosides which are an important class of second line drugs used in tuberculosis treatment. In addition, the marker could limit utility of the strains for screening of new potential drugs or evaluating drug combinations for tuberculosis treatment. Limited selection marker genes for mycobacterial genetic manipulation is a major drawback for such a marker-containing strain in many research fields. Therefore, selectable marker-free, more stable autoluminescent mycobacteria are highly needed. After trying several strategies, we created such mycobacterial strains successfully by using an integrative vector and removing both the resistance maker and integrase genes by Xer site-specific recombination in one step. The corresponding plasmid vectors developed in this study could be very convenient in constructing other selectable marker-free, more stable reporter mycobacteria with diverse applications.

  19. Preparation and characterization of dialdehyde starch by one-step acid hydrolysis and oxidation.

    Science.gov (United States)

    Zuo, Yingfeng; Liu, Wenjie; Xiao, Junhua; Zhao, Xing; Zhu, Ying; Wu, Yiqiang

    2017-10-01

    Dialdehyde starch was prepared by one-step synthesis of acid hydrolysis and oxidation, using corn starch as the raw material, sodium periodate (NaIO 4 ) as the oxidant, and hydrochloric acid (HCl) as the acid solution. The prepared dialdehyde starch was characterized by Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and gel permeation chromatography (GPC). The results confirmed that oxidation occurred between the starch and NaIO 4 . The acid hydrolysis reaction reduced the molecular weight of starch and effectively improved the aldehyde group contents (92.7%). Scanning electron microscope (SEM) analysis indicated that the average particle size decreased after acid hydrolysis and oxidation reaction. X-ray diffraction (XRD) and thermal gravimetric analyzer (TGA) analysis demonstrated that the crystallinity of the obtained dialdehyde starch showed a downward trend and a decelerated thermal decomposition rate. The starch after acid hydrolysis and oxidation exhibited lower hot paste viscosity and higher reactivity. Copyright © 2017. Published by Elsevier B.V.

  20. A Facile One Step Solution Route to Synthesize Cuprous Oxide Nanofluid

    Directory of Open Access Journals (Sweden)

    Shenoy U. Sandhya

    2013-05-01

    Full Text Available A cuprous oxide nanofluid stabilized by sodium lauryl sulfate, synthesized by using the one step method, has been reported. Nanofluids were synthesized by using a well‐ controlled surfactant‐assisted solution phase synthesis. The method involved reduction of copper acetate by glucose in a mixture of water and ethylene glycol serving as the base fluid. The synthesized fluid was characterized by X‐ray and electron diffraction techniques, in addition, transmission and field emission microscopic techniques and Fourier transform infra red spectroscopic analysis was undertaken. The rheological property, as well as the thermal conductivity of the fluid, were measured. The variation of reaction parameters considerably affected the size of the particles as well as the reaction rate. The uniform dispersion of the particles in the base fluid led to a stability period of three months under stationary state, augmenting the thermal conductivity of the nanofluid. The method is found to be simple, reliable and fast for the synthesis of Newtonian nanofluids containing cuprous oxide nanoparticles.

  1. The study of heavy metals leaching from waste foundry sands using a one-step extraction

    Science.gov (United States)

    Bożym, Marta

    2017-10-01

    There are a number of leaching test which are used to evaluate the effect of foundry waste disposal on the environment (TCLP, SPLP, ASTM at al.). Because the spent foundry sand are usually deposited at the ground level and they have a similar structure to the soil, survey mobility of metals using the same methods seems appropriate. One-step extraction allows for the evaluation of the mobility and bioavailability of metals in soil and waste. Waste foundry sands have been successfully used as a component in manufactured soils in U.S., but concern over metal contamination must be eliminated before considering this direction of use. The study evaluated the leaching of heavy metals (Cd, Pb, Cu, Zn, Cr, Ni) from deposited waste foundry sands. The overall, as well as heavy metals were extracted by different type of extractants: H2O, CH3COOH, HCl, EDTA, MgCl2 and NaCOOH. These extractants are most commonly used to study the mobility and bioavailability of metals in soil and waste. In the present study applicable standards and methodology described in the literature in analysis were used. The results allowed to evaluate the bioavailability of metals leached from those wastes.

  2. Effect of saliva decontamination procedures on shear bond strength of a one-step adhesive system.

    Science.gov (United States)

    Ülker, E; Bilgin, S; Kahvecioğlu, F; Erkan, A I

    2017-09-01

    To evaluate the effect of different saliva decontamination procedures on the shear bond strength of a one-step universal adhesive system (Single Bond™ Universal Adhesive, 3M ESPE, St. Paul, MN, USA). The occlusal surfaces of 75 human third molars were ground to expose dentin. The teeth were divided into the following groups: Group 1 (control group): Single Bond™ Universal Adhesive was applied to the prepared tooth according to the manufacturer's recommendations and light cured; no contamination procedure was performed. Group 2: Bonding, light curing, saliva contamination, and dry. Group 3: Bonding, light curing, saliva contamination, rinse, and dry. Group 4: After the procedure performed in Group 2, reapplication of bonding. Group 5: After the procedure performed in Group 3, reapplication of bonding. Then, composite resins were applied with cylindrical-shaped plastic matrixes and light cured. For shear bond testing, a notch-shaped force transducer apparatus was applied to each specimen at the interface between the tooth and composite until failure occurred. The data were statistically analyzed using one-way ANOVA. One-way ANOVA revealed significant differences in shear bond strength between the control group and experimental Groups 2 and 4 (P 0.05). The present in vitro study showed that water rinsing is necessary if cured adhesive resin is contaminated with saliva to ensure adequate bond strength.

  3. Accelerated solvent extraction method with one-step clean-up for hydrocarbons in soil

    International Nuclear Information System (INIS)

    Nurul Huda Mamat Ghani; Norashikin Sain; Rozita Osman; Zuraidah Abdullah Munir

    2007-01-01

    The application of accelerated solvent extraction (ASE) using hexane combined with neutral silica gel and sulfuric acid/ silica gel (SA/ SG) to remove impurities prior to analysis by gas chromatograph with flame ionization detector (GC-FID) was studied. The efficiency of extraction was evaluated based on the three hydrocarbons; dodecane, tetradecane and pentadecane spiked to soil sample. The effect of ASE operating conditions (extraction temperature, extraction pressure, static time) was evaluated and the optimized condition obtained from the study was extraction temperature of 160 degree Celsius, extraction pressure of 2000 psi with 5 minutes static extraction time. The developed ASE with one-step clean-up method was applied in the extraction of hydrocarbons from spiked soil and the amount extracted was comparable to ASE extraction without clean-up step with the advantage of obtaining cleaner extract with reduced interferences. Therefore in the developed method, extraction and clean-up for hydrocarbons in soil can be achieved rapidly and efficiently with reduced solvent usage. (author)

  4. [Choledocholithiasis and pregnancy. Hybrid laparo-endoscopic treatment in one step].

    Science.gov (United States)

    Valadez-Caballero, David; González-Santamaría, Roberto; Soto-Mendoza, Héctor; Alberto Blanco-Figueroa, Jorge; Flores-Pantoja, Juan Manuel

    2014-01-01

    Complications associated with choledocholithiasis are uncommon during pregnancy. However, when it occurs, the morbidity and mortality related for the product and the mother increases, so a proper treatment is imperative in these patients. A 25-year-old pregnant woman on her second trimester. The current condition was started four days prior to the hospital admission with abdominal pain in the right upper quadrant with nausea and vomit.Twenty-four hours later jaundice and dark urine is observed. Physical examination shows scleral jaundice, right upper quadrant abdominal pain without peritoneal irritation. Uterus at umbilicus level without uterine activity. Uterus at umbilicus level without uterine activity. Laboratory tests showed elevated bilirubin and transaminases without leukocytosis; ultrasound reported live intrauterine unique product, with a heart rate of 128 beats per minute, gallbladder with multiple images inside that cast acoustic shadowing, and a 10 mm common bile duct dilated bile duct with dilatation of intrahepatic bile ducts. It was perform laparoscopic cholecystectomy with intraoperative cholangiography and placement of a transcystic jaguar guide for selective cannulation of the common bile duct. Intraoperative endoscopic retrograde cholangiopancreatography was performed with endoscopic stone extraction. The outcome was satisfactory with a hospital discharge at 48 hours. Current evidence has shown that the combined use of laparoscopy and therapeutic endoscopic cholangiography in one step is effective for the treatment of choledocholithiasis, decreasing the risk of complications associated with cannulation of the bile duct.

  5. One-step aqueous synthesis of fluorescent copper nanoclusters by direct metal reduction

    International Nuclear Information System (INIS)

    Fernández-Ujados, Mónica; Trapiella-Alfonso, Laura; Costa-Fernández, José M; Pereiro, Rosario; Sanz-Medel, Alfredo

    2013-01-01

    A one-step aqueous synthesis of highly fluorescent water-soluble copper nanoclusters (CuNCs) is here described, based on direct reduction of the metal precursor with NaBH 4 in the presence of bidentate ligands (made of lipoic acid anchoring groups, appended with a poly(ethylene glycol) short chain). A complete optical and structural characterization was carried out: the optical emission was centred at 416 nm, with a luminescence quantum yield in water of 3.6% (the highest one reported so far in water for this kind of nanocluster). The structural characterization reveals a homogeneous size distribution (of 2.5 nm diameter) with spherical shape. The CuNCs obtained offer long-term stability (the luminescence emission remained unaltered after more than two months) under a broad range of chemical conditions (e.g. stored at pH 3–12 or even in a high ionic strength medium such as 1 M NaCl) and high photostability, keeping their fluorescence emission intact after more than 2 h of daylight and UV-light exposition. All those advantageous features warrant synthesized CuNCs being promising fluorescent nanoprobes for further developments including (bio)applications. (paper)

  6. One-step large-scale deposition of salt-free DNA origami nanostructures

    Science.gov (United States)

    Linko, Veikko; Shen, Boxuan; Tapio, Kosti; Toppari, J. Jussi; Kostiainen, Mauri A.; Tuukkanen, Sampo

    2015-01-01

    DNA origami nanostructures have tremendous potential to serve as versatile platforms in self-assembly -based nanofabrication and in highly parallel nanoscale patterning. However, uniform deposition and reliable anchoring of DNA nanostructures often requires specific conditions, such as pre-treatment of the chosen substrate or a fine-tuned salt concentration for the deposition buffer. In addition, currently available deposition techniques are suitable merely for small scales. In this article, we exploit a spray-coating technique in order to resolve the aforementioned issues in the deposition of different 2D and 3D DNA origami nanostructures. We show that purified DNA origamis can be controllably deposited on silicon and glass substrates by the proposed method. The results are verified using either atomic force microscopy or fluorescence microscopy depending on the shape of the DNA origami. DNA origamis are successfully deposited onto untreated substrates with surface coverage of about 4 objects/mm2. Further, the DNA nanostructures maintain their shape even if the salt residues are removed from the DNA origami fabrication buffer after the folding procedure. We believe that the presented one-step spray-coating method will find use in various fields of material sciences, especially in the development of DNA biochips and in the fabrication of metamaterials and plasmonic devices through DNA metallisation. PMID:26492833

  7. One-Step Electrodeposited Nickel Cobalt Sulfide Nanosheet Arrays for High-Performance Asymmetric Supercapacitors

    KAUST Repository

    Chen, Wei; Xia, Chuan; Alshareef, Husam N.

    2014-01-01

    A facile one-step electrodeposition method is developed to prepare ternary nickel cobalt sulfide interconnected nanosheet arrays on conductive carbon substrates as electrodes for supercapacitors, resulting in exceptional energy storage performance. Taking advantages of the highly conductive, mesoporous nature of the nanosheets and open framework of the three-dimensional nanoarchitectures, the ternary sulfide electrodes exhibit high specific capacitance (1418 F g(-1) at 5 A g(-1) and 1285 F g(-1) at 100 A g(-1)) with excellent rate capability. An asymmetric supercapacitor fabricated by the ternary sulfide nanosheet arrays as positive electrode and porous graphene film as negative electrode demonstrates outstanding electrochemical performance for practical energy storage applications. Our asymmetric supercapacitors show a high energy density of 60 Wh kg(-1) at a power density of 1.8 kW kg(-1). Even when charging the cell within 4.5 s, the energy density is still as high as 33 Wh kg(-1) at an outstanding power density of 28.8 kW kg(-1) with robust long-term cycling stability up to 50 000 cycles.

  8. One-Step Solvent Evaporation-Assisted 3D Printing of Piezoelectric PVDF Nanocomposite Structures.

    Science.gov (United States)

    Bodkhe, Sampada; Turcot, Gabrielle; Gosselin, Frederick P; Therriault, Daniel

    2017-06-21

    Development of a 3D printable material system possessing inherent piezoelectric properties to fabricate integrable sensors in a single-step printing process without poling is of importance to the creation of a wide variety of smart structures. Here, we study the effect of addition of barium titanate nanoparticles in nucleating piezoelectric β-polymorph in 3D printable polyvinylidene fluoride (PVDF) and fabrication of the layer-by-layer and self-supporting piezoelectric structures on a micro- to millimeter scale by solvent evaporation-assisted 3D printing at room temperature. The nanocomposite formulation obtained after a comprehensive investigation of composition and processing techniques possesses a piezoelectric coefficient, d 31 , of 18 pC N -1 , which is comparable to that of typical poled and stretched commercial PVDF film sensors. A 3D contact sensor that generates up to 4 V upon gentle finger taps demonstrates the efficacy of the fabrication technique. Our one-step 3D printing of piezoelectric nanocomposites can form ready-to-use, complex-shaped, flexible, and lightweight piezoelectric devices. When combined with other 3D printable materials, they could serve as stand-alone or embedded sensors in aerospace, biomedicine, and robotic applications.

  9. Efficient Preparation of Super Antifouling PVDF Ultrafiltration Membrane with One Step Fabricated Zwitterionic Surface.

    Science.gov (United States)

    Zhao, Xinzhen; He, Chunju

    2015-08-19

    On the basis of the excellent fouling resistance of zwitterionic materials, the super antifouling polyvinylidene fluoride (PVDF) membrane was efficiently prepared though one-step sulfonation of PVDF and polyaniline blend membrane in situ. The self-doped sulfonated polyaniline (SPANI) was generated as a novel zwitterionic polymer to improve the antifouling property of PVDF ultrafiltration membrane used in sewage treatment. Surface attenuated total reflection Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, surface zeta potential, and water contact angle demonstrated the successful fabrication of zwitterionic interface by convenient sulfonation modification. The static adsorption fouling test showed the quantified adsorption mass of bovine serum albumin (BSA) pollutant on the PVDF/SPANI membrane surface decreases to 3(±2) μg/cm(2), and the water flux recovery ratio (FRR) values were no less than 95% for the three model pollutants of BSA, sodium alginate (SA), and humic acid (HA), which were corresponding hydrophobic, hydrophilic, and natural pollutants in sewage, respectively. This Research Article demonstrated the antifouling advantages of zwitterionic SPANI and aimed to provide a simple method for the large scale preparation of zwitterionic antifouling ultrafiltration membranes.

  10. One-Step Synthesis of PEGylated Gold Nanoparticles with Tunable Surface Charge

    Directory of Open Access Journals (Sweden)

    Rares Stiufiuc

    2013-01-01

    Full Text Available The present work reports a rapid, simple and efficient one-step synthesis and detailed characterisation of stable aqueous colloids of gold nanoparticles (AuNPs coated with unmodified poly(ethyleneglycol (PEG molecules of different molecular weights and surface charges. By mixing and heating aqueous solutions of PEG with variable molecular chain and gold(III chloride hydrate (HAuCl4 in the presence of NaOH, we have successfully produced uniform colloidal 5 nm PEG coated AuNPs of spherical shape with tunable surface charge and an average diameter of 30 nm within a few minutes. It has been found out that PEGylated AuNPs provide optical enhancement of the characteristic vibrational bands of PEG molecules attached to the gold surface when they are excited with both visible (532 nm and NIR (785 nm laser lines. The surface enhanced Raman scattering (SERS signal does not depend on the length of the PEG molecular chain enveloping the AuNPs, and the stability of the colloid is not affected by the addition of concentrated salt solution (0.1 M NaCl, thus suggesting their potential use for in vitro and in vivo applications. Moreover, by gradually changing the chain length of the biopolymer, we were able to control nanoparticles’ surface charge from −28 to −2 mV, without any modification of the Raman enhancement properties and of the colloidal stability.

  11. One-step femtosecond laser welding and internal machining of three glass substrates

    Science.gov (United States)

    Tan, Hua; Duan, Ji'an

    2017-05-01

    In this paper, it demonstrated one-step femtosecond laser welding and internal machining of three fused silica substrates in the optical- and non-optical-contact regimes by focusing 1030-nm laser pulses at the middle of the second substrate. Focusing laser pulses within the second glass in optical-contact and non-optical-contact samples induces permanent internal structural modification, leading to the three glass substrates bonding together simultaneously. The bonding mechanism is based on the internal modification of glass, and this mechanism is different from that of ordinary glass welding at the interface. Welding-spot size is affected by not only the gap distance (ablation effect) and heat transmission, but also by gravity through examining the sizes of the welding spots on the four contact welding surfaces. The maximum bonding strength of the lower interface (56.2 MPa) in the optical-contact regime is more than double that (27.6 MPa) in the non-optical-contact regime.

  12. Production and characterization of carbon nano colloid via one-step electrochemical method

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Doohyun; Hwang, Yujin; Cheong, Seong Ir; Lee, Jae Keun [Pusan National University, Department of Mechanical Engineering (Korea, Republic of); Hong, Daeseung; Moon, Seongyong [N-BARO TECH CO., LTD, Institute of SamchangTsinghua Nano Application (Korea, Republic of); Lee, Jung Eun [Pusan National University, Industrial Liaison Innovation Cluster (Korea, Republic of); Kim, Soo H., E-mail: sookim@pusan.ac.k [Pusan National University, Department of Nanosystem and Nanoprocess Engineering (Korea, Republic of)

    2008-10-15

    We present a one-step electrochemical method to produce water-based stable carbon nano colloid (CNC) without adding any surfactants at the room temperature. The physical, chemical, and thermal properties of CNC prepared were characterized by using various techniques, such as particle size analyzer, zeta potential meter, TEM, XRD, FT-IR, turbidity meter, viscometer, and transient hot-wire method. The average primary size of the suspended spherical-shaped nanoparticles in the CNC was found to be {approx}15 nm in diameter. The thermal conductivity of CNC compared with that of water was observed to increase up to {approx}14% with the CNC concentration of {approx}4.2 wt%. The CNC prepared in this study was considerably stable over the period of 600 h. With the assistance of FT-IR spectroscopy analysis, we confirmed the presence of carboxyl group (i.e., O-H stretching (3,458 cm{sup -1}) and C=O stretching (1,712 cm{sup -1})) formed in the outer atomic layer of carbon nanoparticles, which (i) made the carbon particles hydrophilic and (ii) prevented the aggregation among primary nanoparticles by increasing the magnitude of zeta potential over the long period.

  13. One-step synthesis and microstructure of CuO-SDC composites

    Energy Technology Data Exchange (ETDEWEB)

    Firmino, H.C.T.; Araujo, A.J.M.; Dutra, R.P.S.; Macedo, D.A., E-mail: hellentorrano@hotmail.com, E-mail: allanjp1993@hotmail.com, E-mail: ricardopsd@gmail.com, E-mail: damaced@gmail.com [Universidade Federal da Paraiba (UFPB), Joao Pessoa, PB (Brazil); Nascimento, R.M., E-mail: rmaribondo@ufrnet.br [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil); Rajesh, S., E-mail: rajeshayr@gmail.com [University of Aveiro (Portugal)

    2017-01-15

    An in situ one step synthesis route based on the polymeric precursor method was used to produce dual phase CuO-samaria doped ceria (SDC) nanocomposite powders. This chemical route allowed to obtain composite powders with reduced particle size and uniform distribution of Cu, Ce and Sm elements. The particulate material was characterized by powder X-ray diffraction (XRD) combined with Rietveld refinement. CuO-SDC sintered in air between 950 to 1050 °C and subsequently reduced to Cu-SDC cermets were further characterized by XRD and scanning electron microscopy. The open porosity was measured using the Archimedes' principle. Suitable microstructures for both charge transfer and mass transport processes (30 to 45% porosity) were attained in Cu-SDC cermets previously fired at 1000 to 1050 °C. Overall results indicated that CuO-SDC composites and Cu-SDC cermets with potential application as anodes for solid oxide fuel cells (SOFCs) can be obtained by microstructural design. An anode supported half-cell was prepared by co-pressing and co-firing gadolinia doped ceria (CGO) and the herein synthesized CuO-SDC nanocomposite powder. (author)

  14. One-step engineered self-assembly Co3O4 nanoparticles to nanocubes for supercapacitors

    Science.gov (United States)

    Nagajyothi, P. C.; Pandurangan, M.; Sreekanth, T. V. M.; Shim, Jaesool

    2018-02-01

    Tricobalt tetraoxide or cobalt oxide (Co3O4) nanocubes (NCs) were synthesized from the self-assemblies of Co3O4 nanoparticles (NPs) via a simple one-step hydrothermal method. X-ray diffraction analysis confirmed the cubic crystal structure of Co3O4 NCs. The surface properties were investigated by x-ray photoelectron spectroscopy, which suggests the co-existence of Co in +2 and +3 states. The self-assemblies of aggregation of NPs to NCs were inspected using scanning electron microscopy, which is supported by transmission electron microscopy. The electrochemical properties of Co3O4 NCs were carried out by cyclic voltammetry (CV), galvanostatic charge-discharge (GCD) curves and impedance analysis. The areal capacitance of 3.04 mF cm-2 was obtained at current density of 10 μA cm-2. The Co3O4 NCs electrode exhibits good long-cyclic stability with 92.1% capacitance retention over 3000 cycles. The CV, GCD and impedance curves of Co3O4 NCs were recorded after cyclic test, which are similar to the curves recorded before the test. Therefore, the Co3O4 NCs serves good candidate as positive electrode materials for asymmetric supercapacitors.

  15. Fluorinated alkyne-derived monolayers on oxide-free silicon nanowires via one-step hydrosilylation

    International Nuclear Information System (INIS)

    Nguyen Minh, Quyen; Pujari, Sidharam P.; Wang, Bin; Wang, Zhanhua; Haick, Hossam; Zuilhof, Han; Rijn, Cees J.M. van

    2016-01-01

    Highlights: • Oxide-free H-terminated silicon nanowires undergo efficient surface modification by reaction with fluorinated 1-alkynes (HC≡C−(CH 2 ) 6 C 8 H 17−x F x ; x = 0–17). • These surface-modified Si NWs are chemically stable under range of conditions (including acid, base). • The surface coating yields efficient electrical passivation as demonstrated by a near-zero electrochemical activity of the surface. - Abstract: Passivation of oxide-free silicon nanowires (Si NWs) by the formation of high-quality fluorinated 1-hexadecyne-derived monolayers with varying fluorine content has been investigated. Alkyl chain monolayers (C 16 H 30−x F x ) with a varying number of fluorine substituents (x = 0, 1, 3, 9, 17) were attached onto hydrogen-terminated silicon (Si−H) surfaces with an effective one-step hydrosilylation. This surface chemistry gives well-defined monolayers on nanowires that have a cylindrical core–shell structure, as characterized by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR) and static contact angle (SCA) analysis. The monolayers were stable under acidic and basic conditions, as well as under extreme conditions (such as UV exposure), and provide excellent surface passivation, which opens up applications in the fields of field effect transistors, optoelectronics and especially for disease diagnosis.

  16. One-step hydrothermal synthesis of carboxyl-functionalized upconversion phosphors for bioapplications.

    Science.gov (United States)

    Yang, Jianping; Shen, Dengke; Li, Xiaomin; Li, Wei; Fang, Yin; Wei, Yong; Yao, Chi; Tu, Bo; Zhang, Fan; Zhao, Dongyuan

    2012-10-22

    In this paper, we report a facile one-step hydrothermal method to synthesize phase-, size-, and shape-controlled carboxyl-functionalized rare-earth fluorescence upconversion phosphors by using a small-molecule binary acid, such as malonic acid, oxalic acid, succinic acid, or tartaric acid as capping agent. The crystals, from nano- to microstructures with diverse shapes that include nanospheres, microrods, hexagonal prisms, microtubes, microdisks, polygonal columns, and hexagonal tablets, can be obtained with different reaction times, reaction temperatures, molar ratios of capping agent to sodium hydroxide, and by varying the binary acids. Fourier transform infrared, thermogravimetric analysis, and upconversion luminescence spectra measurements indicate that the synthesized NaYF(4):Yb/Er products with hydrophilic carboxyl-functionalized surface offer efficient upconversion luminescent performance. Furthermore, the antibody/secondary antibody conjugation can be realized by the carboxyl-functionalized surfaces of the upconversion phosphors, thus indicating the potential bioapplications of these kinds of materials. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants

    International Nuclear Information System (INIS)

    Kelly, R.; Miller, S.M.; Kurtz, M.B.; Kirsch, D.R.

    1987-01-01

    A method for introducing specific mutations into the diploid Candida albicans by one-step gene disruption and subsequent UV-induced recombination was developed. The cloned C. albicans URA3 gene was disrupted with the C. albicans ADE2 gene, and the linearized DNA was used for transformation of two ade2 mutants, SGY-129 and A81-Pu. Both an insertional inactivation of the URA3 gene and a disruption which results in a 4.0-kilobase deletion were made. Southern hybridization analyses demonstrated that the URA3 gene was disrupted on one of the chromosomal homologs in 15 of the 18 transformants analyzed. These analyses also revealed restriction site dimorphism of EcoRI at the URA3 locus which provides a unique marker to distinguish between chromosomal homologs. This enabled us to show that either homolog could be disrupted and that disrupted transformants of SGY-129 contained more than two copies of the URA3 locus. The A81-Pu transformants heterozygous for the ura3 mutations were rendered homozygous and Ura- by UV-induced recombination. The homozygosity of a deletion mutant and an insertion mutant was confirmed by Southern hybridization. Both mutants were transformed to Ura+ with plasmids containing the URA3 gene and in addition, were resistant to 5-fluoro-orotic acid, a characteristic of Saccharomyces cerevisiae ura3 mutants as well as of orotidine-5'-phosphate decarboxylase mutants of other organisms

  18. One-step synthesis of graphene-Pt nanocomposites by gamma-ray irradiation

    International Nuclear Information System (INIS)

    Tokai, Akihiro; Okitsu, Kenji; Hori, Fuminobu; Mizukoshi, Yoshiteru; Iwase, Akihiro

    2016-01-01

    We developed a one-step gamma-ray irradiation method to synthesize nanocomposites composed of graphene and Pt nanoparticles from aqueous solution containing graphene and Pt(IV) complex ions in the presence of 2-propanol (IPA) or sodium dodecyl sulfate (SDS). It was confirmed that gamma-ray irradiation provided carbonyl groups on graphene and Pt nanoparticles formed from the radiolytic reduction of Pt(IV) complex ions were deposited onto the carbonyl modified graphene. In the presence of IPA, small Pt nanoparticles were deposited on graphene, but large Pt nanoparticles were deposited in the presence of SDS: the size of Pt nanoparticles formed was larger in the presence of SDS than IPA. Based on the results, formation and deposition mechanisms of Pt nanoparticles were proposed. - Highlights: • Graphene-Pt nanocomposites were synthesized by gamma-ray irradiation. • Reduction of Pt(IV) complex ions and oxidation of graphene occurred simultaneously. • Smaller Pt nanoparticles were formed in the presence of IPA than SDS. • Mechanism for formation of graphene-Pt nanocomposites was proposed.

  19. One-step synthesis of multi-emission carbon nanodots for ratiometric temperature sensing

    Science.gov (United States)

    Nguyen, Vanthan; Yan, Lihe; Xu, Huanhuan; Yue, Mengmeng

    2018-01-01

    Measuring temperature with greater precision at localized small length scales or in a nonperturbative manner is a necessity in widespread applications, such as integrated photonic devices, micro/nano electronics, biology, and medical diagnostics. To this context, use of nanoscale fluorescent temperature probes is regarded as the most promising method for temperature sensing because they are noninvasive, accurate, and enable remote micro/nanoscale imaging. Here, we propose a novel ratiometric fluorescent sensor for nanothermometry using carbon nanodots (C-dots). The C-dots were synthesized by one-step method using femtosecond laser ablation and exhibit unique multi-emission property due to emissions from abundant functional groups on its surface. The as-prepared C-dots demonstrate excellent ratiometric temperature sensing under single wavelength excitation that achieves high temperature sensitivity with a 1.48% change per °C ratiometric response over wide-ranging temperature (5-85 °C) in aqueous buffer. The ratiometric sensor shows excellent reversibility and stability, holding great promise for the accurate measurement of temperature in many practical applications.

  20. One-Step 18F-Labeling of Estradiol Derivative for PET Imaging of Breast Cancer

    Directory of Open Access Journals (Sweden)

    Hongbo Huang

    2018-01-01

    Full Text Available Positron emission tomography (PET imaging is a useful method to evaluate in situ estrogen receptor (ER status for the early diagnosis of breast cancer and optimization of the appropriate treatment strategy. The 18F-labeled estradiol derivative has been successfully used to clinically assess the ER level of breast cancer. In order to simplify the radiosynthesis process, one-step 18F-19F isotope exchange reaction was employed for the 18F-fluorination of the tracer of [18F]AmBF3-TEG-ES. The radiotracer was obtained with the radiochemical yield (RCY of ~61% and the radiochemical purity (RCP of >98% within 40 min. Cell uptake and blocking assays indicated that the tracer could selectively accumulate in the ER-positive human breast cancer cell lines MCF-7 and T47D. In vivo PET imaging on the MCF-7 tumor-bearing mice showed relatively high tumor uptake (1.4~2.3 %D/g and tumor/muscle uptake ratio (4~6. These results indicated that the tracer is a promising PET imaging agent for ER-positive breast cancers.

  1. Chitosan-coated magnetic nanoparticles prepared in one step by reverse microemulsion precipitation.

    Science.gov (United States)

    López, Raúl G; Pineda, María G; Hurtado, Gilberto; León, Ramón Díaz de; Fernández, Salvador; Saade, Hened; Bueno, Darío

    2013-09-27

    Chitosan-coated magnetic nanoparticles (CMNP) were obtained at 70 °C and 80 °C in a one-step method, which comprises precipitation in reverse microemulsion in the presence of low chitosan concentration in the aqueous phase. X-ray diffractometry showed that CMNP obtained at both temperatures contain a mixture of magnetite and maghemite nanoparticles with ≈4.5 nm in average diameter, determined by electron microscopy, which suggests that precipitation temperature does not affect the particle size. The chitosan coating on nanoparticles was inferred from Fourier transform infrared spectrometry measurements; furthermore, the carbon concentration in the nanoparticles allowed an estimation of chitosan content in CMNP of 6%-7%. CMNP exhibit a superparamagnetic behavior with relatively high final magnetization values (≈49-53 emu/g) at 20 kOe and room temperature, probably due to a higher magnetite content in the mixture of magnetic nanoparticles. In addition, a slight direct effect of precipitation temperature on magnetization was identified, which was ascribed to a possible higher degree of nanoparticles crystallinity as temperature at which they are obtained increases. Tested for Pb2+ removal from a Pb(NO3)2 aqueous solution, CMNP showed a recovery efficacy of 100%, which makes them attractive for using in heavy metals ion removal from waste water.

  2. One-step sol–gel imprint lithography for guided-mode resonance structures

    International Nuclear Information System (INIS)

    Huang, Yin; Liu, Longju; Lu, Meng; Johnson, Michael; C Hillier, Andrew

    2016-01-01

    Guided-mode resonance (GMR) structures consisting of sub-wavelength periodic gratings are capable of producing narrow-linewidth optical resonances. This paper describes a sol–gel-based imprint lithography method for the fabrication of submicron 1D and 2D GMR structures. This method utilizes a patterned polydimethylsiloxane (PDMS) mold to fabricate the grating coupler and waveguide for a GMR device using a sol–gel thin film in a single step. An organic–inorganic hybrid sol–gel film was selected as the imprint material because of its relatively high refractive index. The optical responses of several sol–gel GMR devices were characterized, and the experimental results were in good agreement with the results of electromagnetic simulations. The influence of processing parameters was investigated in order to determine how finely the spectral response and resonant wavelength of the GMR devices could be tuned. As an example potential application, refractometric sensing experiments were performed using a 1D sol–gel device. The results demonstrated a refractive index sensitivity of 50 nm/refractive index unit. This one-step fabrication process offers a simple, rapid, and low-cost means of fabricating GMR structures. We anticipate that this method can be valuable in the development of various GMR-based devices as it can readily enable the fabrication of complex shapes and allow the doping of optically active materials into sol–gel thin film. (paper)

  3. One-step aerosol synthesis of nanoparticle agglomerate films: simulation of film porosity and thickness

    International Nuclear Information System (INIS)

    Maedler, Lutz; Lall, Anshuman A; Friedlander, Sheldon K

    2006-01-01

    A method is described for designing nanoparticle agglomerate films with desired film porosity and film thickness. Nanoparticle agglomerates generated in aerosol reactors can be directly deposited on substrates to form uniform porous films in one step, a significant advance over existing technologies. The effect of agglomerate morphology and deposition mechanism on film porosity and thickness are discussed. Film porosity was calculated for a given number and size of primary particles that compose the agglomerates, and fractal dimension. Agglomerate transport was described by the Langevin equation of motion. Deposition enhancing forces such as thermophoresis are incorporated in the model. The method was validated for single spherical particles using previous theoretical studies. An S-shape film porosity dependence on the particle Peclet number typical for spherical particles was also observed for agglomerates, but films formed from agglomerates had much higher porosities than films from spherical particles. Predicted film porosities compared well with measurements reported in the literature. Film porosities increased with the number of primary particles that compose an agglomerate and higher fractal dimension agglomerates resulted in denser films. Film thickness as a function of agglomerate deposition time was calculated from the agglomerate deposition flux in the presence of thermophoresis. The calculated film thickness was in good agreement with measured literature values. Thermophoresis can be used to reduce deposition time without affecting the film porosity

  4. Production and characterization of carbon nano colloid via one-step electrochemical method

    International Nuclear Information System (INIS)

    Kim, Doohyun; Hwang, Yujin; Cheong, Seong Ir; Lee, Jae Keun; Hong, Daeseung; Moon, Seongyong; Lee, Jung Eun; Kim, Soo H.

    2008-01-01

    We present a one-step electrochemical method to produce water-based stable carbon nano colloid (CNC) without adding any surfactants at the room temperature. The physical, chemical, and thermal properties of CNC prepared were characterized by using various techniques, such as particle size analyzer, zeta potential meter, TEM, XRD, FT-IR, turbidity meter, viscometer, and transient hot-wire method. The average primary size of the suspended spherical-shaped nanoparticles in the CNC was found to be ∼15 nm in diameter. The thermal conductivity of CNC compared with that of water was observed to increase up to ∼14% with the CNC concentration of ∼4.2 wt%. The CNC prepared in this study was considerably stable over the period of 600 h. With the assistance of FT-IR spectroscopy analysis, we confirmed the presence of carboxyl group (i.e., O-H stretching (3,458 cm -1 ) and C=O stretching (1,712 cm -1 )) formed in the outer atomic layer of carbon nanoparticles, which (i) made the carbon particles hydrophilic and (ii) prevented the aggregation among primary nanoparticles by increasing the magnitude of zeta potential over the long period.

  5. The study of heavy metals leaching from waste foundry sands using a one-step extraction

    Directory of Open Access Journals (Sweden)

    Bożym Marta

    2017-01-01

    Full Text Available There are a number of leaching test which are used to evaluate the effect of foundry waste disposal on the environment (TCLP, SPLP, ASTM at al.. Because the spent foundry sand are usually deposited at the ground level and they have a similar structure to the soil, survey mobility of metals using the same methods seems appropriate. One-step extraction allows for the evaluation of the mobility and bioavailability of metals in soil and waste. Waste foundry sands have been successfully used as a component in manufactured soils in U.S., but concern over metal contamination must be eliminated before considering this direction of use. The study evaluated the leaching of heavy metals (Cd, Pb, Cu, Zn, Cr, Ni from deposited waste foundry sands. The overall, as well as heavy metals were extracted by different type of extractants: H2O, CH3COOH, HCl, EDTA, MgCl2 and NaCOOH. These extractants are most commonly used to study the mobility and bioavailability of metals in soil and waste. In the present study applicable standards and methodology described in the literature in analysis were used. The results allowed to evaluate the bioavailability of metals leached from those wastes.

  6. Chiroplasmonic magnetic gold nanocomposites produced by one-step aqueous method using κ-carrageenan.

    Science.gov (United States)

    Lesnichaya, Marina V; Sukhov, Boris G; Aleksandrova, Galina P; Gasilova, Ekaterina R; Vakul'skaya, Tamara I; Khutsishvili, Spartak S; Sapozhnikov, Anatoliy N; Klimenkov, Igor V; Trofimov, Boris A

    2017-11-01

    Novel water-soluble chiroplasmonic nanobiocomposites with directly varied gold content were synthesized by a one-step redox method in water using a biocompatible polysaccharide κ-carrageenan (industrial product from algae) as both reducing and stabilizing matrix. The influence of the reactants ratio, temperature, and pH on the reaction was studied and the optimal reaction parameters were found. The structure and the properties of composite nanomaterials were examined in solid state and aqueous solutions by using complementary physical-chemical methods X-ray diffraction analysis, transmission electron microscopy, spectroscopy of electron paramagnetic resonance, atomic absorption and optical spectroscopy, polarimetry including optical rotatory dispersion with registration of interphase-crossbred Cotton effect of a chiral polysaccharide matrix on plasmonic chromophore of gold nanoparticles, dynamic and static light scattering. The new perspective multi-purpose nanocomposites demonstrate a complex of chiroplasmonic and magnetic properties, imparted by both nanoparticles and radicals enriched chiral polysaccharide matrix. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Fluorinated alkyne-derived monolayers on oxide-free silicon nanowires via one-step hydrosilylation

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen Minh, Quyen [Laboratory of Organic Chemistry, Wageningen University, Stippeneng 4, 6708 WE Wageningen (Netherlands); Nanosens, IJsselkade 7, 7201 HB Zutphen (Netherlands); Pujari, Sidharam P. [Laboratory of Organic Chemistry, Wageningen University, Stippeneng 4, 6708 WE Wageningen (Netherlands); Wang, Bin [The Department of Chemical Engineering and Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa 3200003 (Israel); Wang, Zhanhua [Laboratory of Organic Chemistry, Wageningen University, Stippeneng 4, 6708 WE Wageningen (Netherlands); Haick, Hossam [The Department of Chemical Engineering and Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa 3200003 (Israel); Zuilhof, Han [Laboratory of Organic Chemistry, Wageningen University, Stippeneng 4, 6708 WE Wageningen (Netherlands); Rijn, Cees J.M. van, E-mail: cees.vanrijn@wur.nl [Laboratory of Organic Chemistry, Wageningen University, Stippeneng 4, 6708 WE Wageningen (Netherlands)

    2016-11-30

    Highlights: • Oxide-free H-terminated silicon nanowires undergo efficient surface modification by reaction with fluorinated 1-alkynes (HC≡C−(CH{sub 2}){sub 6}C{sub 8}H{sub 17−x}F{sub x}; x = 0–17). • These surface-modified Si NWs are chemically stable under range of conditions (including acid, base). • The surface coating yields efficient electrical passivation as demonstrated by a near-zero electrochemical activity of the surface. - Abstract: Passivation of oxide-free silicon nanowires (Si NWs) by the formation of high-quality fluorinated 1-hexadecyne-derived monolayers with varying fluorine content has been investigated. Alkyl chain monolayers (C{sub 16}H{sub 30−x}F{sub x}) with a varying number of fluorine substituents (x = 0, 1, 3, 9, 17) were attached onto hydrogen-terminated silicon (Si−H) surfaces with an effective one-step hydrosilylation. This surface chemistry gives well-defined monolayers on nanowires that have a cylindrical core–shell structure, as characterized by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR) and static contact angle (SCA) analysis. The monolayers were stable under acidic and basic conditions, as well as under extreme conditions (such as UV exposure), and provide excellent surface passivation, which opens up applications in the fields of field effect transistors, optoelectronics and especially for disease diagnosis.

  8. Controllable Organic Resistive Switching Achieved by One-Step Integration of Cone-Shaped Contact.

    Science.gov (United States)

    Ling, Haifeng; Yi, Mingdong; Nagai, Masaru; Xie, Linghai; Wang, Laiyuan; Hu, Bo; Huang, Wei

    2017-09-01

    Conductive filaments (CFs)-based resistive random access memory possesses the ability of scaling down to sub-nanoscale with high-density integration architecture, making it the most promising nanoelectronic technology for reclaiming Moore's law. Compared with the extensive study in inorganic switching medium, the scientific challenge now is to understand the growth kinetics of nanoscale CFs in organic polymers, aiming to achieve controllable switching characteristics toward flexible and reliable nonvolatile organic memory. Here, this paper systematically investigates the resistive switching (RS) behaviors based on a widely adopted vertical architecture of Al/organic/indium-tin-oxide (ITO), with poly(9-vinylcarbazole) as the case study. A nanoscale Al filament with a dynamic-gap zone (DGZ) is directly observed using in situ scanning transmission electron microscopy (STEM) , which demonstrates that the RS behaviors are related to the random formation of spliced filaments consisting of Al and oxygen vacancy dual conductive channels growing through carbazole groups. The randomicity of the filament formation can be depressed by introducing a cone-shaped contact via a one-step integration method. The conical electrode can effectively shorten the DGZ and enhance the localized electric field, thus reducing the switching voltage and improving the RS uniformity. This study provides a deeper insight of the multiple filamentary mechanisms for organic RS effect. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. One-step synthesis of silver nanoparticles at the air-water interface using different methods

    International Nuclear Information System (INIS)

    Liu Hongguo; Xiao Fei; Wang Changwei; Lee, Yong-Ill; Xue Qingbin; Chen Xiao; Qian Dongjin; Hao Jingcheng; Jiang Jianzhuang

    2008-01-01

    Silver nanoparticles were synthesized in a one-step process at the air-AgNO 3 aqueous solution interface under Langmuir monolayers of 5,10,15,20-tetra-4-oxy(2-stearic acid) phenyl porphyrin (TSPP) at room temperature by using different methods including UV-light irradiation, ambient light irradiation, and formaldehyde gas reduction. It was found that parallel aligned one-dimensional (1D) chains composed of discrete silver nanoparticles with the size of 3-5 nm were formed under UV-light irradiation for a short time, while large areas of uniform silver spherical nanoparticles were formed under natural daylight illumination for several days or by formaldehyde gas treatment for several hours. The average size of the spherical nanoparticles ranges from 6.88 ± 0.46 to 11.10 ± 1.47 nm, depending on the experimental conditions. The 1D chains formed under UV-light irradiation result from the templating effect of parallel aligned linear supramolecular arrays formed by TSPP at the air-water interface, and rapid nucleation and growth of the nanoparticles. The formation of the uniform silver nanoparticles under daylight illumination or by formaldehyde gas treatment, however, should be ascribed to a kinetically controlled growth process of the nanoparticles

  10. One-step synthesis of gold bimetallic nanoparticles with various metal-compositions

    International Nuclear Information System (INIS)

    Bratescu, Maria Antoaneta; Takai, Osamu; Saito, Nagahiro

    2013-01-01

    Highlights: ► Synthesis of bimetallic nanoparticles in an aqueous solution discharge. ► Alloying gold with divalent sp metals, trivalent sp metals, 3d or 4d metals. ► Formation mechanism of bimetallic nanoparticles by metal reduction and gold erosion. ► Blue and red shift of surface plasmon resonance. -- Abstract: A rapid, one-step process for the synthesis of bimetallic nanoparticles by simultaneous metal reduction and gold erosion in an aqueous solution discharge was investigated. Gold bimetallic nanoparticles were obtained by alloying gold with various types of metals belonging to one of the following categories: divalent sp metals, trivalent sp metals, 3d or 4d metals. The composition of the various gold bimetallic nanoparticles obtained depends on electrochemical factors, charge transfer between gold and other metal, and initial concentration of metal in solution. Transmission electron microscopy and energy dispersive spectroscopy show that the gold bimetallic nanoparticles were of mixed pattern, with sizes of between 5 and 20 nm. A red-shift of the surface plasmon resonance band in the case of the bimetallic nanoparticles Au–Fe, Au–Ga, and Au–In, and a blue-shift of the plasmon band of the Au–Ag nanoparticles was observed. In addition, the interaction of gold bimetallic nanoparticles with unpaired electrons, provided by a stable free radical molecule, was highest for those NPs obtained by alloying gold with a 3d metal

  11. In situ biosynthesis of bacterial nanocellulose-CaCO3 hybrid bionanocomposite: One-step process.

    Science.gov (United States)

    Mohammadkazemi, Faranak; Faria, Marisa; Cordeiro, Nereida

    2016-08-01

    In this work, a simple and green route to the synthesis of the bacterial nanocellulose-calcium carbonate (BNC/CaCO3) hybrid bionanocomposites using one-step in situ biosynthesis was studied. The CaCO3 was incorporated in the bacterial nanocellulose structure during the cellulose biosynthesis by Gluconacetobacter xylinus PTCC 1734 bacteria. Hestrin-Schramm (HS) and Zhou (Z) culture media were used to the hybrid bionanocomposites production and the effect of ethanol addition was investigated. Attenuated total reflection Fourier transform infrared spectroscopy, field emission scanning electron microscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, inverse gas chromatography and thermogravimetric analysis were used to characterize the samples. The experimental results demonstrated that the ethanol and culture medium play an important role in the BNC/CaCO3 hybrid bionanocomposites production, structure and properties. The BNC/CaCO3 biosynthesized in Z culture medium revealed higher O/C ratio and amphoteric surface character, which justify the highest CaCO3 content incorporation. The CaCO3 was incorporated into the cellulosic matrix decreasing the bacterial nanocellulose crystallinity. This work reveals the high potential of in situ biosynthesis of BNC/CaCO3 hybrid bionanocomposites and opens a new way to the high value-added applications of bacterial nanocellulose. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Technology Advancement and the CTBT: Taking One Step Back from the Nuclear Brink

    Science.gov (United States)

    Perry, W. J.

    2016-12-01

    Technology plays a pivotal role in international nuclear security and technological advancement continues to support a path toward stability. One near-term and readily-obtainable step back from the nuclear brink is the Comprehensive Nuclear-test Ban Treaty (CTBT). The technology to independently verify adherence to the CTBT has matured in the 20 years since the Treaty was opened for signature. Technology has also improved the safety and reliability of the US nuclear stockpile in the absence of testing. Due to these advances over the past two decades neither verification nor stockpiles effectiveness should be an impediment to the Treaty's entry into force. Other technical and geo-political evolution in this same period has changed the perceived benefit of nuclear weapons as instruments of security. Recognizing the change technology has brought to deliberation of nuclear security, nations are encouraged to take this one step away from instability.This presentation will reflect on the history and assumptions that have been used to justify the build-up and configuration of nuclear stockpiles, the changes in technology and conditions that alter the basis of these original assumptions, and the re-analysis of security using current and future assumptions that point to the need for revised nuclear policies. The author has a unique and well informed perspective as both the most senior US Defense Official and a technologist.

  13. One-step controllable fabrication of superhydrophobic surfaces with special composite structure on zinc substrates.

    Science.gov (United States)

    Ning, Tao; Xu, Wenguo; Lu, Shixiang

    2011-09-01

    Stable superhydrophobic platinum surfaces have been effectively fabricated on the zinc substrates through one-step replacement deposition process without further modification or any other post-treatment procedures. The fabrication process was controllable, which could be testified by various morphologies and hydrophobic properties of different prepared samples. By conducting SEM and water CA analysis, the effects of reaction conditions on the surface morphology and hydrophobicity of the resulting surfaces were carefully studied. The results show that the optimum condition of superhydrophobic surface fabrication depends largely on the positioning of zinc plate and the concentrations of reactants. When the zinc plate was placed vertically and the concentration of PtCl(4) solution was 5 mmol/L, the zinc substrate would be covered by a novel and interesting composite structure. The structure was composed by microscale hexagonal cavities, densely packed nanoparticles layer and top micro- and nanoscale flower-like structures, which exhibit great surface roughness and porosity contributing to the superhydrophobicity. The maximal CA value of about 171° was obtained under the same reaction condition. The XRD, XPS and EDX results indicate that crystallite pure platinum nanoparticles were aggregated on the zinc substrates in accordance with a free deposition way. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Nearly Perfect Durable Superhydrophobic Surfaces Fabricated by a Simple One-Step Plasma Treatment.

    Science.gov (United States)

    Ryu, Jeongeun; Kim, Kiwoong; Park, JooYoung; Hwang, Bae Geun; Ko, YoungChul; Kim, HyunJoo; Han, JeongSu; Seo, EungRyeol; Park, YongJong; Lee, Sang Joon

    2017-05-16

    Fabrication of superhydrophobic surfaces is an area of great interest because it can be applicable to various engineering fields. A simple, safe and inexpensive fabrication process is required to fabricate applicable superhydrophobic surfaces. In this study, we developed a facile fabrication method of nearly perfect superhydrophobic surfaces through plasma treatment with argon and oxygen gases. A polytetrafluoroethylene (PTFE) sheet was selected as a substrate material. We optimized the fabrication parameters to produce superhydrophobic surfaces of superior performance using the Taguchi method. The contact angle of the pristine PTFE surface is approximately 111.0° ± 2.4°, with a sliding angle of 12.3° ± 6.4°. After the plasma treatment, nano-sized spherical tips, which looked like crown-structures, were created. This PTFE sheet exhibits the maximum contact angle of 178.9°, with a sliding angle less than 1°. As a result, this superhydrophobic surface requires a small external force to detach water droplets dripped on the surface. The contact angle of the fabricated superhydrophobic surface is almost retained, even after performing an air-aging test for 80 days and a droplet impacting test for 6 h. This fabrication method can provide superb superhydrophobic surface using simple one-step plasma etching.

  15. One-step electrodeposition process to fabricate corrosion-resistant superhydrophobic surface on magnesium alloy.

    Science.gov (United States)

    Liu, Qin; Chen, Dexin; Kang, Zhixin

    2015-01-28

    A simple, one-step method has been developed to construct a superhydrophobic surface by electrodepositing Mg-Mn-Ce magnesium plate in an ethanol solution containing cerium nitrate hexahydrate and myristic acid. Scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy were employed to characterize the surfaces. The shortest electrodeposition time to obtain a superhydrophobic surface was about 1 min, and the as-prepared superhydrophobic surfaces had a maximum contact angle of 159.8° and a sliding angle of less than 2°. Potentiodynamic polarization and electrochemical impedance spectroscopy measurements demonstrated that the superhydrophobic surface greatly improved the corrosion properties of magnesium alloy in 3.5 wt % aqueous solutions of NaCl, Na2SO4, NaClO3, and NaNO3. Besides, the chemical stability and mechanical durability of the as-prepared superhydrophobic surface were also examined. The presented method is rapid, low-cost, and environmentally friendly and thus should be of significant value for the industrial fabrication of anticorrosive superhydrophobic surfaces and should have a promising future in expanding the applications of magnesium alloys.

  16. Dynamic cholesteric liquid crystal superstructures photoaligned by one-step polarization holography

    Science.gov (United States)

    Li, Sen-Sen; Shen, Yuan; Chang, Zhen-Ni; Li, Wen-Song; Xu, Yan-Chao; Fan, Xing-Yu; Chen, Lu-Jian

    2017-12-01

    A convenient approach to modulate the fingerprint textures of methyl red (MR) doped cholesteric liquid crystals by asymmetric photoalignment in the green-light waveband is presented, resulting in the generation of voltage-controllable helical superstructures. The interaction between the MR molecules and the incident light polarization determines the initial twisted planar geometry, providing a multivariant control over the stripe directions of fingerprint textures by applying a proper electric field. The key factors for precise manipulation of fingerprint stripes in a predictable and rewritable manner are analyzed theoretically and investigated experimentally, which involves the alignment asymmetry, the ratio of cell gap to natural pitch length, and the chirality of chiral dopant. Dynamic periodic fingerprint textures in shapes of dashed curve and dashed line are further demonstrated by utilizing a facile one-step polarization holography process using two beams with orthogonal circular and orthogonal linear polarizations, respectively. It is believed that the practical approach described in this study would enrich the research contents of self-assembled hierarchical superstructures using soft liquid crystal building blocks.

  17. One step gold (bio)functionalisation based on CS{sub 2}-amine reaction

    Energy Technology Data Exchange (ETDEWEB)

    Almeida, Ines [Centro de Quimica e Bioquimica, Faculdade de Ciencias da Universidade de Lisboa, Ed. C8, Campo Grande, 1749-016 Lisboa (Portugal); Cascalheira, Antonio C. [Lumisense, Lda, Campus Faculdade de Ciencias da Universidade de Lisboa, Ed. ICAT, Campo Grande, 1749-016 Lisboa (Portugal); Viana, Ana S., E-mail: anaviana@fc.ul.p [Centro de Quimica e Bioquimica, Faculdade de Ciencias da Universidade de Lisboa, Ed. C8, Campo Grande, 1749-016 Lisboa (Portugal)

    2010-12-01

    Dithiocarbamates have been regarded as alternative anchor groups to thiols on gold surfaces, and claimed to be formed in situ through the reaction between secondary amines and carbon disulphide. In this paper, we further exploit this methodology for a convenient one step biomolecule immobilisation onto gold surfaces. First, the reactivity between CS{sub 2} and electroactive compounds containing amines, primary (dopamine), secondary (epinephrine), and an amino acid (tryptophan) has been investigated by electrochemical methods. Cyclic voltammetric characterisation of the modified electrodes confirmed the immobilisation of all the target compounds, allowing the estimation of their surface concentration. The best result was obtained with epinephrine, a secondary amine, for which a typical quasi-reversible behaviour of surface confined electroactive species could be clearly depicted. Electrochemical reductive desorption studies enabled to infer on the extent of the reaction and on the relative stability of the generated monolayers. Bio-functionalisation studies have been accomplished through the reaction of CS{sub 2} with glucose oxidase in aqueous medium, and the catalytic activity of the immobilised enzyme was evaluated towards glucose, by electrochemical methods in the presence of a redox mediator. Scanning tunnelling microscopy (STM) and Atomic force microscopy (AFM) were used respectively, to characterize the gold electrodes and Glucose Oxidase coverage and distribution on the modified surfaces.

  18. One-step synthesis of Ag@PANI nanocomposites and their application to detection of mercury

    International Nuclear Information System (INIS)

    Wang, Xiufang; Shen, Yuhua; Xie, Anjian; Chen, Shaohua

    2013-01-01

    The Ag@Polyaniline (PANI) core–shell nanoparticles were successfully synthesized by one-step radiation of sunlight method using silver nitrate as the oxidant for aniline without any additives or templates. The Ag@PANI core–shell nanocomposites were used as active surface enhanced Raman scattering (SERS) nanoprobes for the detection of some heavy metal ions, such as Pb 2+ , Cu 2+ Hg 2+ , Cd 2+ , and so on, which is based upon the Raman intensity response of PANI to metal ions. It turns out that the nanoprobes represent a rather high selectivity for Hg 2+ detection. It is also important that the detection limit of Hg 2+ concentration is ca. 1 × 10 −12 M in this system, due to the great sensitivity of SERS nanoprobes. The developed nanoprobes could have potential applications in highly sensitive chemical, environmental and biological analysis, as well as medical detection. - Highlights: • The Ag@PANI nanocomposites are fabricated by a simple synthetic route. • Nanocomposites are utilized directly as SERS nanosensors without being additionally functionalized. • Higher selectivity and ultrasensitive sensitivity for Hg 2+ ions detection

  19. Recyclable crosslinked polymer networks with full property recovery made via one-step controlled radical polymerization

    Science.gov (United States)

    Jin, Kailong; Li, Lingqiao; Torkelson, John

    Rubber tires illustrate well the issues ranging from economic loss to environmental problems and sustainability issues that arise with spent, covalently crosslinked polymers. A nitroxide-mediated polymerization (NMP) strategy has been developed that allows for one-step synthesis of recyclable crosslinked polymers from monomers or polymers that contain carbon-carbon double bonds amenable to radical polymerization. Resulting materials possess dynamic alkoxyamine crosslinks that undergo reversible decrosslinking as a function of temperature. Using polybutadiene as starting material, and styrene, an appropriate nitroxide molecule and bifunctional initiator for initial crosslinking, a model for tire rubber can be produced by reaction at temperatures comparable to those employed in tire molding. Upon cooling, the crosslinks are made permanent due to the extraordinarily strong temperature dependence of the reverisible nitroxide capping and uncapping reaction. Based on thermomechanical property characterization, when the original crosslinked model rubber is chopped into bits and remolded in the melt state, a well-consolidated material is obtained which exhibits full recovery of properties reflecting crosslink density after multiple recycling steps.

  20. Identification of Bacillus Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, recA, rpoB Gene Sequencing and RAPD-PCR.

    Science.gov (United States)

    Mohkam, Milad; Nezafat, Navid; Berenjian, Aydin; Mobasher, Mohammad Ali; Ghasemi, Younes

    2016-03-01

    Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.

  1. Microfluidic PCR Amplification and MiSeq Amplicon Sequencing Techniques for High-Throughput Detection and Genotyping of Human Pathogenic RNA Viruses in Human Feces, Sewage, and Oysters

    Directory of Open Access Journals (Sweden)

    Mamoru Oshiki

    2018-04-01

    Full Text Available Detection and genotyping of pathogenic RNA viruses in human and environmental samples are useful for monitoring the circulation and prevalence of these pathogens, whereas a conventional PCR assay followed by Sanger sequencing is time-consuming and laborious. The present study aimed to develop a high-throughput detection-and-genotyping tool for 11 human RNA viruses [Aichi virus; astrovirus; enterovirus; norovirus genogroup I (GI, GII, and GIV; hepatitis A virus; hepatitis E virus; rotavirus; sapovirus; and human parechovirus] using a microfluidic device and next-generation sequencer. Microfluidic nested PCR was carried out on a 48.48 Access Array chip, and the amplicons were recovered and used for MiSeq sequencing (Illumina, Tokyo, Japan; genotyping was conducted by homology searching and phylogenetic analysis of the obtained sequence reads. The detection limit of the 11 tested viruses ranged from 100 to 103 copies/μL in cDNA sample, corresponding to 101–104 copies/mL-sewage, 105–108 copies/g-human feces, and 102–105 copies/g-digestive tissues of oyster. The developed assay was successfully applied for simultaneous detection and genotyping of RNA viruses to samples of human feces, sewage, and artificially contaminated oysters. Microfluidic nested PCR followed by MiSeq sequencing enables efficient tracking of the fate of multiple RNA viruses in various environments, which is essential for a better understanding of the circulation of human pathogenic RNA viruses in the human population.

  2. Simultaneous discrimination of species and strains in Lactobacillus rhamnosus using species-specific PCR combined with multiplex mini-sequencing technology.

    Science.gov (United States)

    Huang, Chien-Hsun; Chang, Mu-Tzu; Huang, Lina; Chu, Wen-Shen

    2015-12-01

    This study described the use of species-specific PCR in combination with SNaPshot mini-sequencing to achieve species identification and strain differentiation in Lactobacillus rhamnosus. To develop species-specific PCR and strain subtyping primers, the dnaJ gene was used as a target, and its corresponding sequences were analyzed both in Lb. rhamnosus and in a subset of its phylogenetically closest species. The results indicated that the species-specific primer pair was indeed specific for Lb. rhamnosus, and the mini-sequencing assay was able to unambiguously distinguish Lb. rhamnosus strains into different haplotypes. In conclusion, we have successfully developed a rapid, accurate and cost-effective assay for inter- and intraspecies discrimination of Lb. rhamnosus, which can be applied to achieve efficient quality control of probiotic products. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. 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.

  4. Molecular discrimination of Echinococcus granulosus and Echinococcus multilocularis by sequencing and a new PCR-RFLP method with the potential use for other Echinococcus species.

    Science.gov (United States)

    Şakalar, Çağrı; Kuk, Salih; Erensoy, Ahmet; Dağli, Adile Ferda; Özercan, İbrahim Hanifi; Çetınkaya, Ülfet; Yazar, Süleyman

    2014-01-01

    To develop a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial genomic marker region was amplified and sequenced using a novel primer pair and a new PCR-RFLP protocol was developed for the detection and discrimination of E. granulosus and E. multilocularis using a set of restriction enzymes including AccI, MboI, MboII, and TsoI. The selected marker region was amplified using DNA isolated from FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis and the discrimination of E. granulosus and E. multilocularis was accomplished by use of the novel PCR-RFLP method. In this PCR-RFLP protocol, use of any single restriction enzyme is enough for the discrimination of E. granulosus and E. multilocularis. The PCR-RFLP protocol can be potentially used for the discrimination of 5 other Echinococcus species: E. oligarthus, E. shiquicus, E. ortleppi, E. canadensis, and E. vogeli.

  5. Vygotsky's Principle "One Step in Learning - One Hundred Steps In Development": From Idea To Practice

    Directory of Open Access Journals (Sweden)

    Zaretsky V.K.,

    2016-12-01

    Full Text Available The article reviews Lev Vygotsky’s published works to trace the evolution of his understanding of child development. The authors believe that his assumption that one step in learning may mean one hundred steps in development, is as important as the two other key postulates of the cultural-historical theory: the principle that learning precedes development and the concept of zone of proximal development. The authors provide a rationale for utilization of these assumptions in the practice of development-facilitating psychological and educational assistance. A mechanism of this learning-development relationship is hypothesized. The article outlines a multidimensional model of the zone of proximal development illustrating the above mechanism. This model is one of the conceptual tools of the Reflection and Activity Approach helping children overcome learning difficulties and promoting their development. Having given the account of how they proceeded “from the idea to the problem” and “from the idea to the mechanism”, the authors provide case studies showing how this mechanism allows working with learning difficulties to trigger simultaneous improvement in multiple developmental dimensions. The article reports on the experience of running special Summer Schools for children with learning difficulties, implementing the “Chess for General Development” Project, and assisting orphaned children with severe somatic conditions. A case study of a female college student displaying signs of the learned helplessness syndrome is presented. The authors infer that Vygotsky’s idea of a specific relationship between learning and development may be of fundamental theoretical and practical value, especially for working with children with special needs.

  6. Synthesis of carbon nanoparticles using one step green approach and their application as mercuric ion sensor

    Energy Technology Data Exchange (ETDEWEB)

    Roshni, V.; Ottoor, Divya, E-mail: divya@chem.unipune.ac.in

    2015-05-15

    Carbon nanoparticles (CNPs) have been evolved as a promising candidate for the metal sensing applications due to their synthesis from naturally occurring and easily available non-toxic molecular precursors by green chemistry. A simple and one step procedure is reported here for the synthesis of CNPs from coconut milk by thermal pyrolysis at a temperature of 120–150 °C for 2–5 min without using any carbonizing or passivating agent. On pyrolysis the coconut oil is separated from the carbon rich residue and the residue when dissolved in water showed blue fluorescence under UV light. The CNPs produced are found to show an emission maximum at 440 nm when excited at 360 nm. Synthesis by green approach makes CNPs a promising substitute for the metal sensing applications. Series of metal ions which have a hazardous impact on the ecological system have been taken for the analysis and it is observed that the fluorescence of CNPs gets remarkably quenched by mercuric ions. Fluorescence quenching was studied using standard Stern–Volmer quenching model. Limit of detection was found to be 16.5 nM Hg{sup 2+} concentration. - Highlights: • Green and economical synthesis of carbon nanoparticles (CNPs) from naturally abundant material. • Coconut milk is used as molecular precursor, which on thermal pyrolysis at 120 °C yielded CNPs. • Highly fluorescent CNPs show an emission maxima of 440 nm when excited at 360 nm. • Application of CNPs for metal ion sensing using fluorescence quenching phenomena. • Hg{sup 2+} is most effectively sensed with a detection limit of 16.5 nM.

  7. One step synthesis of polyacrylamide functionalized graphene and its application in Pb(II) removal

    International Nuclear Information System (INIS)

    Xu, Zhiwei; Zhang, Yaoyao; Qian, Xiaoming; Shi, Jie; Chen, Lei; Li, Baodong; Niu, Jiarong; Liu, Liangsen

    2014-01-01

    Highlights: • PAM-g-graphene is synthesized by the co-irradiation between GO and AM monomers. • PAM graft on GO has led to the exfoliation of GO into individual sheets. • The γ-ray induced reduction of GO. • PAM-g-graphene exhibits high adsorption capacities toward Pb(II) ions. • PAM-g-graphene provides a new idea for heavy metal pollutants’ removal in water. - Abstract: Polyacrylamide grafted graphene (PAM-g-graphene) from graphite oxide (GO) was successfully prepared by γ-ray irradiation with acrylamide monomers in aqueous at room temperature in this paper. Our strategy involves the PAM chains graft on the surface and between the layers of GO by in situ radical polymerization which led to the exfoliation of GO into individual sheets. Results show that the degree of grafting of PAM-g-graphene samples is 24.2%, and the thickness is measured to be 2.59 nm. Moreover, the as-prepared PAM-g-graphene with some amino from PAM and little oxygen functional groups exhibit superior adsorption of Pb(II) ions. The adsorption processes reach equilibrium in just 30 min and the adsorption isotherms are described well by Langmuir and Freundlich classical isotherms models. The determined adsorption capacity of PAM-g-graphene is 819.67 mg g −1 (pH 6) for Pb(II), which is 20 times and 8 times capacities of that for graphene nanosheets and carbon nanotubes according to reports, respectively. This chemically modified graphene synthesized by this fast one-step approach, featuring a good versatility and adaptability, excellent adsorption capacity and rapid extraction, may provide a new idea for the global problem of heavy metal pollutants’ removal in water

  8. In situ biosynthesis of bacterial nanocellulose-CaCO3 hybrid bionanocomposite: One-step process

    International Nuclear Information System (INIS)

    Mohammadkazemi, Faranak; Faria, Marisa; Cordeiro, Nereida

    2016-01-01

    In this work, a simple and green route to the synthesis of the bacterial nanocellulose-calcium carbonate (BNC/CaCO 3 ) hybrid bionanocomposites using one-step in situ biosynthesis was studied. The CaCO 3 was incorporated in the bacterial nanocellulose structure during the cellulose biosynthesis by Gluconacetobacter xylinus PTCC 1734 bacteria. Hestrin-Schramm (HS) and Zhou (Z) culture media were used to the hybrid bionanocomposites production and the effect of ethanol addition was investigated. Attenuated total reflection Fourier transform infrared spectroscopy, field emission scanning electron microscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, inverse gas chromatography and thermogravimetric analysis were used to characterize the samples. The experimental results demonstrated that the ethanol and culture medium play an important role in the BNC/CaCO 3 hybrid bionanocomposites production, structure and properties. The BNC/CaCO 3 biosynthesized in Z culture medium revealed higher O/C ratio and amphoteric surface character, which justify the highest CaCO 3 content incorporation. The CaCO 3 was incorporated into the cellulosic matrix decreasing the bacterial nanocellulose crystallinity. This work reveals the high potential of in situ biosynthesis of BNC/CaCO 3 hybrid bionanocomposites and opens a new way to the high value-added applications of bacterial nanocellulose. - Graphical Abstract: Display Omitted - Highlights: • BNC/CaCO 3 hybrid bionanocomposites were produced using in situ biosynthesis process. • Ethanol and culture medium play an important role in the production and properties. • Z-BNC/CaCO 3 bionanocomposites revealed higher O/C ratio and amphoteric surface character. • CaCO 3 incorporated into the BNC decreased crystallinity.

  9. One-step radiolabelled biotin scintigraphy in patients with suspected vertebral infections

    International Nuclear Information System (INIS)

    Lazzeri, E.; Erba, P.; Chinol, M.; Tascini, C.; Menichetti, F.; Paganelli, G.; Mariani, G.

    2003-01-01

    Full text: Biotin (B), or vitamin H, belonging to the B-complex group is utilized by bacteria for acid synthesis by acetyl-CoA carboxylase. We evaluated the diagnostic potential per se of radiolabeled biotin without avidin pre-targeting, in patients with suspected vertebral bacterial infections. We evaluated 31 patients for suspected spine infection. All patients were selected on clinical ground, blood chemistry findings, back pain and fever. Patients were injected i.v. with 500 μg of DTPA-conjugated Biotin labelled with 111In (110-130 MBq); planar and SPECT scans were recorded starting 90 min post-injection. All patients underwent MR, or CT and some patients were imaged with either 99mTc-HMPAO-WBC and/or 67Ga-citrate. Diagnostic-quality imaging was obtained at 90 min and 4 hr after 111In-DTPA-Biotin injection. We observed only 2 false-negative results, while 24 studies were true-positive (4 performed during follow-up) and 10 true-negative (1 during follow-up) (91% sensitivity, 100% specificity). Either MR, CT or 99mTc-HMPAO-WBC had high proportions of either false-negative, false-positive or equivocal results (sensitivity/specificity around 50%). These preliminary results outline the high diagnostic potential of one-step 111In-DTPA-Biotin scintigraphy without avidin pre-targeting) in patients with suspected vertebral infection. The high true-positive and true-negative rate suggests that this system displays some specificity for bacterial infections. The high diagnostic accuracy of 111In-Biotin scintigraphy seems to be independent from antibiotic therapy, thus making this method very helpful relative to other imaging modalities in the clinical decision on starting proper therapy and for monitoring the efficacy of treatment. (author)

  10. Synthesis of carbon nanoparticles using one step green approach and their application as mercuric ion sensor

    International Nuclear Information System (INIS)

    Roshni, V.; Ottoor, Divya

    2015-01-01

    Carbon nanoparticles (CNPs) have been evolved as a promising candidate for the metal sensing applications due to their synthesis from naturally occurring and easily available non-toxic molecular precursors by green chemistry. A simple and one step procedure is reported here for the synthesis of CNPs from coconut milk by thermal pyrolysis at a temperature of 120–150 °C for 2–5 min without using any carbonizing or passivating agent. On pyrolysis the coconut oil is separated from the carbon rich residue and the residue when dissolved in water showed blue fluorescence under UV light. The CNPs produced are found to show an emission maximum at 440 nm when excited at 360 nm. Synthesis by green approach makes CNPs a promising substitute for the metal sensing applications. Series of metal ions which have a hazardous impact on the ecological system have been taken for the analysis and it is observed that the fluorescence of CNPs gets remarkably quenched by mercuric ions. Fluorescence quenching was studied using standard Stern–Volmer quenching model. Limit of detection was found to be 16.5 nM Hg 2+ concentration. - Highlights: • Green and economical synthesis of carbon nanoparticles (CNPs) from naturally abundant material. • Coconut milk is used as molecular precursor, which on thermal pyrolysis at 120 °C yielded CNPs. • Highly fluorescent CNPs show an emission maxima of 440 nm when excited at 360 nm. • Application of CNPs for metal ion sensing using fluorescence quenching phenomena. • Hg 2+ is most effectively sensed with a detection limit of 16.5 nM

  11. Surface roughness of novel resin composites polished with one-step systems.

    Science.gov (United States)

    Ergücü, Z; Türkün, L S

    2007-01-01

    This study: 1) analyzed the surface roughness of five novel resin composites that contain nanoparticles after polishing with three different one-step systems and 2) evaluated the effectiveness of these polishers and their possible surface damage using scanning electron microscope (SEM) analysis. The resin composites evaluated in this study include CeramX, Filtek Supreme XT, Grandio, Premise and Tetric EvoCeram. A total of 100 discs (20/resin composites, 10 x 2 mm) were fabricated. Five specimens/resin composites cured under Mylar strips served as the control. The samples were polished for 30 seconds with PoGo, OptraPol and One Gloss discs at 15,000 rpm using a slow speed handpiece. The surfaces were tested for roughness (Ra) with a surface roughness tester and examined with SEM. One-way ANOVA was used for statistical analysis (p = 0.05). For all the composites tested, differences between the polishing systems were found to be significant (p One Gloss applications. For Grandio, Mylar and PoGo created equally smooth surfaces, while OptraPol and One Gloss produced equally rougher surfaces. Tetric EvoCeram exhibited the roughest surface with OptraPol, while no significant differences were found between Premise and CeramX. According to SEM images, OptraPol and One Gloss scratched and plucked the particles away from the surface, while PoGo created a uniform finish, although the roughness values were not the same for each composite. Effectiveness of the polishers seems to be material dependent.

  12. Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5'-transgene integration sequence.

    Science.gov (United States)

    Li, P; Jia, J W; Jiang, L X; Zhu, H; Bai, L; Wang, J B; Tang, X M; Pan, A H

    2012-04-27

    To ensure the implementation of genetically modified organism (GMO)-labeling regulations, an event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic carnation variety Moonlite. The 5'-transgene integration sequence was isolated by thermal asymmetric interlaced PCR. Based upon the 5'-transgene integration sequence, the event-specific primers and TaqMan probe were designed to amplify the fragments, which spanned the exogenous DNA and carnation genomic DNA. Qualitative and quantitative PCR assays were developed employing the designed primers and probe. The detection limit of the qualitative PCR assay was 0.05% for Moonlite in 100 ng total carnation genomic DNA, corresponding to about 79 copies of the carnation haploid genome; the limit of detection and quantification of the quantitative PCR assay were estimated to be 38 and 190 copies of haploid carnation genomic DNA, respectively. Carnation samples with different contents of genetically modified components were quantified and the bias between the observed and true values of three samples were lower than the acceptance criterion (GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite.

  13. Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples.

    Science.gov (United States)

    Borsu, Laetitia; Intrieri, Julie; Thampi, Linta; Yu, Helena; Riely, Gregory; Nafa, Khedoudja; Chandramohan, Raghu; Ladanyi, Marc; Arcila, Maria E

    2016-11-01

    Although next-generation sequencing (NGS) is a robust technology for comprehensive assessment of EGFR-mutant lung adenocarcinomas with acquired resistance to tyrosine kinase inhibitors, it may not provide sufficiently rapid and sensitive detection of the EGFR T790M mutation, the most clinically relevant resistance biomarker. Here, we describe a digital PCR (dPCR) assay for rapid T790M detection on aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples. Tumor DNAs from patients with EGFR-mutant lung adenocarcinomas and acquired resistance to epidermal growth factor receptor inhibitors were prepared for Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets sequencing, a hybrid capture-based assay interrogating 410 cancer-related genes. Precapture library aliquots were used for rapid EGFR T790M testing by dPCR, and results were compared with NGS and locked nucleic acid-PCR Sanger sequencing (reference high sensitivity method). Seventy resistance samples showed 99% concordance with the reference high sensitivity method in accuracy studies. Input as low as 2.5 ng provided a sensitivity of 1% and improved further with increasing DNA input. dPCR on libraries required less DNA and showed better performance than direct genomic DNA. dPCR on NGS libraries is a robust and rapid approach to EGFR T790M testing, allowing most economical utilization of limited material for comprehensive assessment. The same assay can also be performed directly on any limited DNA source and cell-free DNA. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  14. Two unusual hepatitis C virus subtypes, 2j and 2q, in Spain: Identification by nested-PCR and sequencing of a NS5B region.

    Science.gov (United States)

    Margall, N; March, F; Español, M; Torras, X; Gallego, A; Coll, P

    2015-10-01

    Many studies have reported the use of the NS5B gene to subtype hepatitis C virus (HCV). Other HCV genes, such as HCV-5' UTR, Core (C) and E1, have also been used. In some studies, NS5B have been used together with 5'-UTR or C genes to improve genotyping results obtained using commercial procedures. Only two studies in Spain have compared molecular techniques versus commercial procedures regarding the efficacy of HCV subtyping. The aim of this study was to determine whether nested PCR and sequencing of a NS5B region was more reliable than commercial procedures to subtype HCV. We analyzed the results of HCV genotyping in [726] serum specimens collected from 2001 to 2013. From 2001 to 2011, we used PCR and INNO-LiPA hybridization or its new version Versant HCV Genotype 2.0 assay (471 samples). From 2012 to 2013, we used nested PCR and sequencing of a NS5B region (255 cases). This method used two pairs of primers to amplify the RNA of the sample converted to DNA by retrotranscription. The amplification product of 270 base pairs was further sequenced. To identify the subtype, the sequences obtained were compared to those in the international database: http://hcv.lanl.gov./content/sequence/, HCV/ToolsOutline.html and Geno2pheno[hcv] http://hcv.bioinf.mpi-inf.mpg.de/index.php. Nested PCR of a NS5B region and sequencing identified all but one subtype (0.4%, 1/255), differentiated all 1a subtypes from 1b subtypes, and characterized all HCV 2-4 subtypes. This approach also distinguished two subtypes, 2j and 2q, that had rarely been detected previously in Spain. However, commercial procedures failed to subtype 12.7% (60/471) of samples and to genotype 0.6% of specimens (3/471). Nested PCR and sequencing of a NS5B region improved the subtyping of HCV in comparison with classical procedures and identified two rare subtypes in Spain: 2j and 2q. However, full length genome sequencing is recommended to confirm HCV 2j and 2q subtypes. Copyright © 2015. Published by Elsevier B.V.

  15. A versatile one-step CRISPR-Cas9 based approach to plasmid-curing

    DEFF Research Database (Denmark)

    Lauritsen, Ida; Porse, Andreas; Sommer, Morten Otto Alexander

    2017-01-01

    tool enabling rapid removal of plasmids from bacterial cells is lacking. Results Based on replicon abundance and sequence conservation analysis, we show that the vast majority of bacterial cloning and expression vectors share sequence similarities that allow for broad CRISPR-Cas9 targeting. We have...... widely used for expression and engineering purposes. By virtue of the CRISPR-Cas9 targeting, our platform is highly expandable and can be applied in a broad host context. We exemplify the wide applicability of our system in Gram-negative bacteria by demonstrating the successful application in both...

  16. Lactobacillus strain diversity based on partial hsp60 gene sequences and design of PCR-restriction fragment length polymorphism assays for species identification and differentiation.

    Science.gov (United States)

    Blaiotta, Giuseppe; Fusco, Vincenzina; Ercolini, Danilo; Aponte, Maria; Pepe, Olimpia; Villani, Francesco

    2008-01-01

    A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.

  17. Genetic Diversity and Phylogenetic Analysis of the Iranian Leishmania Parasites Based on HSP70 Gene PCR-RFLP and Sequence Analysis.

    Science.gov (United States)

    Nemati, Sara; Fazaeli, Asghar; Hajjaran, Homa; Khamesipour, Ali; Anbaran, Mohsen Falahati; Bozorgomid, Arezoo; Zarei, Fatah

    2017-08-01

    Despite the broad distribution of leishmaniasis among Iranians and animals across the country, little is known about the genetic characteristics of the causative agents. Applying both HSP70 PCR-RFLP and sequence analyses, this study aimed to evaluate the genetic diversity and phylogenetic relationships among Leishmania spp. isolated from Iranian endemic foci and available reference strains. A total of 36 Leishmania isolates from almost all districts across the country were genetically analyzed for the HSP70 gene using both PCR-RFLP and sequence analysis. The original HSP70 gene sequences were aligned along with homologous Leishmania sequences retrieved from NCBI, and subjected to the phylogenetic analysis. Basic parameters of genetic diversity were also estimated. The HSP70 PCR-RFLP presented 3 different electrophoretic patterns, with no further intraspecific variation, corresponding to 3 Leishmania species available in the country, L. tropica, L. major, and L. infantum. Phylogenetic analyses presented 5 major clades, corresponding to 5 species complexes. Iranian lineages, including L. major, L. tropica, and L. infantum, were distributed among 3 complexes L. major, L. tropica, and L. donovani. However, within the L. major and L. donovani species complexes, the HSP70 phylogeny was not able to distinguish clearly between the L. major and L. turanica isolates, and between the L. infantum, L. donovani, and L. chagasi isolates, respectively. Our results indicated that both HSP70 PCR-RFLP and sequence analyses are medically applicable tools for identification of Leishmania species in Iranian patients. However, the reduced genetic diversity of the target gene makes it inevitable that its phylogeny only resolves the major groups, namely, the species complexes.

  18. One step synthesis of chlorine-free Pt/Nitrogen-doped graphene composite for oxygen reduction reaction

    KAUST Repository

    Varga, Tamá s; Varga, Á gnes Tí mea; Ballai, Gergő; Haspel, Henrik; Kukovecz, Á kos; Kó nya, Z.

    2018-01-01

    Chlorine-free Platinum/nitrogen-doped graphene oxygen reduction reaction catalysts were synthesized by a one step method of annealing a mixture of platinum acetylacetonate and graphene oxide under ammonia atmosphere. Nanoparticles with close

  19. Patient satisfaction with a novel one-step hydrogen peroxide solution

    Directory of Open Access Journals (Sweden)

    Schafer J

    2014-10-01

    Full Text Available Jeffery Schafer, Robert Steffen, Marjorie J Rah Bausch & Lomb Incorporated, Rochester, NY, USA Purpose: We aimed to evaluate the product performance of a novel one-step hydrogen ­peroxide cleaning and disinfecting solution, PeroxiClear (“Test” solution, when used by habitual Clear Care users to bilaterally clean and disinfect their soft contact lenses, for approximately 2 weeks.Methods: This was a 2-week, open-label, bilateral eye study designed to include subjects ­ranging in age from 18 to 55 years, inclusive. All subjects were habitual users of the Clear Care peroxide regimen for cleaning, disinfecting, and storage of their soft contact lenses, for at least 6 months prior to enrolling in the study. Subjects were examined at two study visits: a screening/­dispensing visit and a 2-week follow-up visit. The primary end point, patient preference for the Test solution, was evaluated with an online survey administered after 7 days of using the Test cleaning and disinfecting solution. Respondents could answer questions with neutral or nonneutral responses (better or worse. Statistical analyses were conducted to compare differences for nonneutral responses.Results: Of the 299 eligible subjects enrolled, 297 completed the study, conducted at 21 sites by 21 investigators in the United States. A significantly higher proportion of nonneutral respondents reported the Test solution was better overall (85.9% than their habitual contact lens solution (14.1% (P<0.001. The proportion of subjects who preferred the Test solution over their habitual solution was significantly higher for each of the preference questions regarding comfort (85.4% vs 14.6%, moistness (90.0% vs 10.0%, cleanness (91.6% vs 8.4%, and clarity of vision (85.8% vs 14.2%.Conclusion: After 7 days of using the Test cleaning and disinfecting solution, survey results indicated high levels of patient satisfaction and preference over the habitual solution, particularly in the areas of

  20. A new deterministic Ensemble Kalman Filter with one-step-ahead smoothing for storm surge forecasting

    KAUST Repository

    Raboudi, Naila

    2016-11-01

    The Ensemble Kalman Filter (EnKF) is a popular data assimilation method for state-parameter estimation. Following a sequential assimilation strategy, it breaks the problem into alternating cycles of forecast and analysis steps. In the forecast step, the dynamical model is used to integrate a stochastic sample approximating the state analysis distribution (called analysis ensemble) to obtain a forecast ensemble. In the analysis step, the forecast ensemble is updated with the incoming observation using a Kalman-like correction, which is then used for the next forecast step. In realistic large-scale applications, EnKFs are implemented with limited ensembles, and often poorly known model errors statistics, leading to a crude approximation of the forecast covariance. This strongly limits the filter performance. Recently, a new EnKF was proposed in [1] following a one-step-ahead smoothing strategy (EnKF-OSA), which involves an OSA smoothing of the state between two successive analysis. At each time step, EnKF-OSA exploits the observation twice. The incoming observation is first used to smooth the ensemble at the previous time step. The resulting smoothed ensemble is then integrated forward to compute a "pseudo forecast" ensemble, which is again updated with the same observation. The idea of constraining the state with future observations is to add more information in the estimation process in order to mitigate for the sub-optimal character of EnKF-like methods. The second EnKF-OSA "forecast" is computed from the smoothed ensemble and should therefore provide an improved background. In this work, we propose a deterministic variant of the EnKF-OSA, based on the Singular Evolutive Interpolated Ensemble Kalman (SEIK) filter. The motivation behind this is to avoid the observations perturbations of the EnKF in order to improve the scheme\\'s behavior when assimilating big data sets with small ensembles. The new SEIK-OSA scheme is implemented and its efficiency is demonstrated

  1. Dose Rate Experiment at JET for Benchmarking the Calculation Direct One Step Method

    International Nuclear Information System (INIS)

    Angelone, M.; Petrizzi, L.; Pillon, M.; Villari, R.; Popovichev, S.

    2006-01-01

    Neutrons produced by D-D and D-T plasmas induce the activation of tokamak materials and of components. The development of reliable methods to assess dose rates is a key issue for maintenance and operating nuclear machines, in normal and off-normal conditions. In the frame of the EFDA Fusion Technology work programme, a computational tool based upon MCNP Monte Carlo code has been developed to predict the dose rate after shutdown: it is called Direct One Step Method (D1S). The D1S is an innovative approach in which the decay gammas are coupled to the neutrons as in the prompt case and they are transported in one single step in the same run. Benchmarking of this new tool with experimental data taken in a complex geometry like that of a tokamak is a fundamental step to test the reliability of the D1S method. A dedicated benchmark experiment was proposed for the 2005-2006 experimental campaign of JET. Two irradiation positions have been selected for the benchmark: one inner position inside the vessel, not far from the plasma, called the 2 upper irradiation end (IE2), where neutron fluence is relatively high. The second position is just outside a vertical port in an external position (EX). Here the neutron flux is lower and the dose rate to be measured is not very far from the residual background. Passive detectors are used for in-vessel measurements: the high sensitivity Thermo Luminescent Dosimeters (TLDs) GR-200A (natural LiF), which ensure measurements down to environmental dose level. An active detector of Geiger-Muller (GM) type is used for out of vessel dose rate measurement. Before their use the detectors were calibrated in a secondary gamma-ray standard (Cs-137 and Co-60) facility in term of air-kerma. The background measurement was carried-out in the period July -September 2005 in the outside position EX using the GM tube and in September 2005 inside the vacuum vessel using TLD detectors located in the 2 Upper irradiation end IE2. In the present work

  2. Lipoprotein metabolism in familial hypercholesterolemia: Serial assessment using a one-step ultracentrifugation method

    Directory of Open Access Journals (Sweden)

    Hayato Tada

    2015-04-01

    Full Text Available Objectives: It is well known that familial hypercholesterolemia (FH is a common inherited disorder that can markedly elevate the level of plasma LDL cholesterol. However, little data exists regarding the clinical impact of the plasma triglyceride (TG-rich lipoprotein fraction, including VLDL and IDL, in FH. Thus, we assessed the hypothesis that the mutations in the LDL receptor modulate lipoprotein metabolism other than the LDL fraction. Design and methods: We investigated plasma lipoprotein with a one-step ultracentrifugation method for 146 controls (mean age=61.4±17.1 yr, mean LDL cholesterol=92.7±61.2 mg/dl, 213 heterozygous mutation-determined FH subjects (mean age=46.0±18.0 yr, mean LDL cholesterol=225.1±61.2 mg/dl, and 16 homozygous/compound heterozygous mutation-determined FH subjects (mean age=26.9±17.1 yr, mean LDL cholesterol=428.6±86.1 mg/dl. In addition, we evaluated cholesterol/TG ratio in each lipoprotein fraction separated by ultracentrifugation. Results: In addition to total cholesterol and LDL cholesterol levels, VLDL cholesterol (19.5±10.4, 25.2±19.3, 29.5±21.4 mg/dl, respectively and IDL cholesterol (8.3±3.7, 16.8±11.5, 40.0±37.3 mg/dl, respectively exhibited a tri-model distribution according to their status in LDL receptor mutation(s. Moreover, the ratios of cholesterol/TG of each lipoprotein fraction increased significantly in heterozygous FH and homozygous/compound heterozygous FH groups, compared with that of controls, suggesting that the abnormality in LDL receptor modulates the quality as well as the quantity of each lipoprotein fraction. Conclusions: Our results indicate that cholesterol in TG-rich lipoproteins, including VLDL and IDL, are significantly higher in FH subjects, revealing a tri-modal distribution according to the number of LDL receptor mutations. Keywords: LDL cholesterol, Familial hypercholesterolemia, Ultracentrifugation, Lipoprotein

  3. Detecting authorized and unauthorized genetically modified organisms containing vip3A by real-time PCR and next-generation sequencing.

    Science.gov (United States)

    Liang, Chanjuan; van Dijk, Jeroen P; Scholtens, Ingrid M J; Staats, Martijn; Prins, Theo W; Voorhuijzen, Marleen M; da Silva, Andrea M; Arisi, Ana Carolina Maisonnave; den Dunnen, Johan T; Kok, Esther J

    2014-04-01

    The growing number of biotech crops with novel genetic elements increasingly complicates the detection of genetically modified organisms (GMOs) in food and feed samples using conventional screening methods. Unauthorized GMOs (UGMOs) in food and feed are currently identified through combining GMO element screening with sequencing the DNA flanking these elements. In this study, a specific and sensitive qPCR assay was developed for vip3A element detection based on the vip3Aa20 coding sequences of the recently marketed MIR162 maize and COT102 cotton. Furthermore, SiteFinding-PCR in combination with Sanger, Illumina or Pacific BioSciences (PacBio) sequencing was performed targeting the flanking DNA of the vip3Aa20 element in MIR162. De novo assembly and Basic Local Alignment Search Tool searches were used to mimic UGMO identification. PacBio data resulted in relatively long contigs in the upstream (1,326 nucleotides (nt); 95 % identity) and downstream (1,135 nt; 92 % identity) regions, whereas Illumina data resulted in two smaller contigs of 858 and 1,038 nt with higher sequence identity (>99 % identity). Both approaches outperformed Sanger sequencing, underlining the potential for next-generation sequencing in UGMO identification.

  4. Validation of a One-Step Method for Extracting Fatty Acids from Salmon, Chicken and Beef Samples.

    Science.gov (United States)

    Zhang, Zhichao; Richardson, Christine E; Hennebelle, Marie; Taha, Ameer Y

    2017-10-01

    Fatty acid extraction methods are time-consuming and expensive because they involve multiple steps and copious amounts of extraction solvents. In an effort to streamline the fatty acid extraction process, this study compared the standard Folch lipid extraction method to a one-step method involving a column that selectively elutes the lipid phase. The methods were tested on raw beef, salmon, and chicken. Compared to the standard Folch method, the one-step extraction process generally yielded statistically insignificant differences in chicken and salmon fatty acid concentrations, percent composition and weight percent. Initial testing showed that beef stearic, oleic and total fatty acid concentrations were significantly lower by 9-11% with the one-step method as compared to the Folch method, but retesting on a different batch of samples showed a significant 4-8% increase in several omega-3 and omega-6 fatty acid concentrations with the one-step method relative to the Folch. Overall, the findings reflect the utility of a one-step extraction method for routine and rapid monitoring of fatty acids in chicken and salmon. Inconsistencies in beef concentrations, although minor (within 11%), may be due to matrix effects. A one-step fatty acid extraction method has broad applications for rapidly and routinely monitoring fatty acids in the food supply and formulating controlled dietary interventions. © 2017 Institute of Food Technologists®.

  5. Comparison of Four Human Papillomavirus Genotyping Methods: Next-generation Sequencing, INNO-LiPA, Electrochemical DNA Chip, and Nested-PCR.

    Science.gov (United States)

    Nilyanimit, Pornjarim; Chansaenroj, Jira; Poomipak, Witthaya; Praianantathavorn, Kesmanee; Payungporn, Sunchai; Poovorawan, Yong

    2018-03-01

    Human papillomavirus (HPV) infection causes cervical cancer, thus necessitating early detection by screening. Rapid and accurate HPV genotyping is crucial both for the assessment of patients with HPV infection and for surveillance studies. Fifty-eight cervicovaginal samples were tested for HPV genotypes using four methods in parallel: nested-PCR followed by conventional sequencing, INNO-LiPA, electrochemical DNA chip, and next-generation sequencing (NGS). Seven HPV genotypes (16, 18, 31, 33, 45, 56, and 58) were identified by all four methods. Nineteen HPV genotypes were detected by NGS, but not by nested-PCR, INNO-LiPA, or electrochemical DNA chip. Although NGS is relatively expensive and complex, it may serve as a sensitive HPV genotyping method. Because of its highly sensitive detection of multiple HPV genotypes, NGS may serve as an alternative for diagnostic HPV genotyping in certain situations. © The Korean Society for Laboratory Medicine

  6. IS1111 insertion sequences of Coxiella burnetii: characterization and use for repetitive element PCR-based differentiation of Coxiella burnetii isolates

    Directory of Open Access Journals (Sweden)

    Massung Robert F

    2007-10-01

    Full Text Available Abstract Background Coxiella burnetii contains the IS1111 transposase which is present 20 times in the Nine Mile phase I (9Mi/I genome. A single PCR primer that binds to each IS element, and primers specific to a region ~500-bp upstream of each of the 20 IS1111 elements were designed. The amplified products were characterized and used to develop a repetitive element PCR genotyping method. Results Isolates Nine Mile phase II, Nine Mile RSA 514, Nine Mile Baca, Scottish, Ohio, Australian QD, Henzerling phase I, Henzerling phase II, M44, KAV, PAV, Q238, Q195 and WAV were tested by PCR and compared to 9Mi/I. Sequencing was used to determine the exact differences in isolates which lacked specific IS elements or produced PCR products of differing size. From this data, an algorithm was created utilizing four primer pairs that allows for differentiation of unknown isolates into five genomic groups. Additional isolates (Priscilla Q177, Idaho Q, Qiyi, Poker Cat, Q229 and Q172 and nine veterinary samples were characterized using the algorithm which resulted in their placement into three distinct genomic groups. Conclusion Through this study significant differences, including missing elements and sequence alterations within and near IS element coding regions, were found between the isolates tested. Further, a method for differentiation of C. burnetii isolates into one of five genomic groups was created. This algorithm may ultimately help to determine the relatedness between known and unknown isolates of C. burnetii.

  7. Development of a Species-specific PCR Assay for Three Xanthomonas Species, Causing Bulb and Flower Diseases, Based on Their Genome Sequences

    Directory of Open Access Journals (Sweden)

    Chang-Gi Back

    2015-09-01

    Full Text Available In this study, we developed a species-specific PCR assay for rapid and accurate detection of three Xanthomonas species, X. axonopodis pv. poinsettiicola (XAP, X. hyacinthi (XH and X. campestris pv. zantedeschiae (XCZ, based on their draft genome sequences. XAP, XH and XCZ genomes consist of single chromosomes that contain 5,221, 4,395 and 7,986 protein coding genes, respectively. Species-specific primers were designed from variable regions of the draft genome sequence data and assessed by a PCR-based detection method. These primers were also tested for specificity against 17 allied Xanthomonas species as well as against the host DNA and the microbial community of the host surface. Three primer sets were found to be very specific and no amplification product was obtained with the host DNA and the microbial community of the host surface. In addition, a detection limit of 1 pg/μl per PCR reaction was detected when these primer sets were used to amplify corresponding bacterial DNAs. Therefore, these primer sets and the developed species-specific PCR assay represent a valuable, sensitive, and rapid diagnostic tool that can be used to detect three specific pathogens at early stages of infection and may help control diseases.

  8. The possibility of discriminating within the Bacillus cereus group using gyrB sequencing and PCR-RFLP

    DEFF Research Database (Denmark)

    Jensen, Gert B; Fisker, Niels; Sparsø, Thomas

    2005-01-01

    Based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion), we have examined the possibility of differentiating members of the Bacillus cereus group. Fragments of the gyrB gene (362 bp) from pure cultures of 12 B. cereus, 25 B. thuringiensis, 25 B. mycoides and two......, it was not possible to discriminate between the B. cereus and the B. thuringiensis strains using the methods described....

  9. Molecular discrimination of Echinococcus granulosus and Echinococcus multilocularis by sequencing and a new PCR-RFLP method with the potential use for other Echinococcus species

    OpenAIRE

    ŞAKALAR, Çağrı; KUK, Salih; ERENSOY, Ahmet; DAĞLI, Adile Ferda; ÖZERCAN, İbrahim Hanifi

    2015-01-01

    To develop a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. Materials and methods: DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial...

  10. Enterobacterial repetitive intergenic consensus sequences and the PCR to generate fingerprints of genomic DNAs from Vibrio cholerae O1, O139, and non-O1 strains.

    OpenAIRE

    Rivera, I G; Chowdhury, M A; Huq, A; Jacobs, D; Martins, M T; Colwell, R R

    1995-01-01

    Enterobacterial repetitive intergenic consensus (ERIC) sequence polymorphism was studied in Vibrio Cholerae strains isolated before and after the cholera epidemic in Brazil (in 1991), along with epidemic strains from Peru, Mexico, and India, by PCR. A total of 17 fingerprint patterns (FPs) were detected in the V. cholerae strains examined; 96.7% of the toxigenic V. cholerae O1 strains and 100% of the O139 serogroup strains were found to belong to the same FP group comprising four fragments (F...

  11. A Simple Method for the Extraction, PCR-amplification, Cloning, and Sequencing of Pasteuria 16S rDNA from Small Numbers of Endospores.

    Science.gov (United States)

    Atibalentja, N; Noel, G R; Ciancio, A

    2004-03-01

    For many years the taxonomy of the genus Pasteuria has been marred with confusion because the bacterium could not be cultured in vitro and, therefore, descriptions were based solely on morphological, developmental, and pathological characteristics. The current study sought to devise a simple method for PCR-amplification, cloning, and sequencing of Pasteuria 16S rDNA from small numbers of endospores, with no need for prior DNA purification. Results show that DNA extracts from plain glass bead-beating of crude suspensions containing 10,000 endospores at 0.2 x 10 endospores ml(-1) were sufficient for PCR-amplification of Pasteuria 16S rDNA, when used in conjunction with specific primers. These results imply that for P. penetrans and P. nishizawae only one parasitized female of Meloidogyne spp. and Heterodera glycines, respectively, should be sufficient, and as few as eight cadavers of Belonolaimus longicaudatus with an average number of 1,250 endospores of "Candidatus Pasteuria usgae" are needed for PCR-amplification of Pasteuria 16S rDNA. The method described in this paper should facilitate the sequencing of the 16S rDNA of the many Pasteuria isolates that have been reported on nematodes and, consequently, expedite the classification of those isolates through comparative sequence analysis.

  12. Pseudogene of dihydrolipoyl succinyltransferase (E2k) found by PCR amplification and direct sequencing of rodent-human cell hybrid DNAs

    Energy Technology Data Exchange (ETDEWEB)

    Cai, X.; Ali, G.; Blass, J.P. [Cornell Univ. Medical College, White Plains, NY (United States); Szabo, P. [Cornell Univ. Medical College, New York, NY (United States); Tanzi, R.E. [Massachusetts General Hospital, Boston, MA (United States)

    1994-07-01

    Previous studies have indicated that the cDNA for the E2k component of the human {alpha}-ketoglutarate dehydrogenase complex (KGDHC) hybridized not only to a major locus on chromosome 14q24.3 in a region associated with familial Alzheimer`s disease and with Joseph-Machado disease, but also to another locus on chromosome 1p31. The authors now report that PCR of genomic DNA and direct sequencing indicated that the chromosome 1 locus is an intronless pseudogene. PCR of genomic DNA amplified E2k fragments from mouse-human cell hybrids containing human chromosome 1 DNA but not from hybrids containing human chromosome 14 DNA. The resulting amplicons were of comparable sizes to those when the cDNA was used to template. The direct sequencing of these amplicons confirmed the lack of introns and indicated a frame shift, which led to the presence of four termination codons early in the coding region. PCR followed by direct sequencing of the amplicons appears to be a convenient method for identifying intronless pseudogenes.

  13. One Step Closer to Mars with Aquaponics: Cultivating Citizen Science in K12 Schools

    Science.gov (United States)

    Kolattukudy, Maria; Puranik, Niyati; Sane, Nishant; Bisht, Kritika; Saffat, Nabeeha; Gupta, Anika; McHugh, Anne; Detweiler, Angela; Bebout, Brad; Everroad, R. Craig

    2017-01-01

    The Microbial Ecology and Biogeochemistry Research Laboratory at NASA Ames Research Center focuses primarily on the nutrient cycling and diversity of complex microbial communities. NASA is interested in the composition and functioning of microbial mat communities as these processes fundamentally shape the form and function of these analogs for the earliest forms of life on Earth (3.6 billion years ago), and likely will on other planets as well. Aquaponics systems are supported by microbial communities who perform many complex ecosystem services, including cycling nitrogen. Microbes are integral to the stability and productivity of aquaponics systems, which are analogous to microbial communities in food production systems that are essential for building efficient life support systems for long-distance space travel. Students at Meadow Park Middle School created 10 parallel aquaponics systems and took temporal microbial samples to characterize whether any macro-ecology variables impacted or changed the microbial diversity of these systems. Students additionally created a website so that other classrooms can pursue similar projects in their own schools (https://go.nasa.gov/2uJhxmF). Our lab at NASA Ames has sequenced water samples from each of the 10 tanks at 3 timepoints using a MinION sequencer. MPMS students will be involved in the analysis of the bioinformatics data generated through this collaboration. Our ongoing collaboration aims to collect and analyze data in the classroom setting that has utility for research scientists, while involving students as collaborators in the research process.

  14. Enhanced planar perovskite solar cell efficiency and stability using a perovskite/PCBM heterojunction formed in one step.

    Science.gov (United States)

    Zhou, Long; Chang, Jingjing; Liu, Ziye; Sun, Xu; Lin, Zhenhua; Chen, Dazheng; Zhang, Chunfu; Zhang, Jincheng; Hao, Yue

    2018-02-08

    Perovskite/PCBM heterojunctions are efficient for fabricating perovskite solar cells with high performance and long-term stability. In this study, an efficient perovskite/PCBM heterojunction was formed via conventional sequential deposition and one-step formation processes. Compared with conventional deposition, the one-step process was more facile, and produced a perovskite thin film of substantially improved quality due to fullerene passivation. Moreover, the resulting perovskite/PCBM heterojunction exhibited more efficient carrier transfer and extraction, and reduced carrier recombination. The perovskite solar cell device based on one-step perovskite/PCBM heterojunction formation exhibited a higher maximum PCE of 17.8% compared with that from the conventional method (13.7%). The device also showed exceptional stability, retaining 83% of initial PCE after 60 days of storage under ambient conditions.

  15. A simple, flexible and efficient PCR-fusion/Gateway cloning procedure for gene fusion, site-directed mutagenesis, short sequence insertion and domain deletions and swaps

    Directory of Open Access Journals (Sweden)

    Etchells J Peter

    2009-10-01

    Full Text Available Abstract Background The progress and completion of various plant genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating binary vectors containing: gene fusions, variants from site-directed mutagenesis, addition of protein tags together with domain swaps and deletions. Furthermore, efficient cloning procedures, ideally high throughput, are essential for pyramiding of multiple gene constructs. Results Here, we present a simple, flexible and efficient PCR-fusion/Gateway cloning procedure for construction of binary vectors for a range of gene fusions or variants with single or multiple nucleotide substitutions, short sequence insertions, domain deletions and swaps. Results from selected applications of the procedure which include ORF fusion, introduction of Cys>Ser mutations, insertion of StrepII tag sequence and domain swaps for Arabidopsis secondary cell wall AtCesA genes are demonstrated. Conclusion The PCR-fusion/Gateway cloning procedure described provides an elegant, simple and efficient solution for a wide range of diverse and complicated cloning tasks. Through streamlined cloning of sets of gene fusions and modification variants into binary vectors for systematic functional studies of gene families, our method allows for efficient utilization of the growing sequence and expression data.

  16. One step versus two step approach for gestational diabetes screening: systematic review and meta-analysis of the randomized trials.

    Science.gov (United States)

    Saccone, Gabriele; Caissutti, Claudia; Khalifeh, Adeeb; Meltzer, Sara; Scifres, Christina; Simhan, Hyagriv N; Kelekci, Sefa; Sevket, Osman; Berghella, Vincenzo

    2017-12-03

    To compare both the prevalence of gestational diabetes mellitus (GDM) as well as maternal and neonatal outcomes by either the one-step or the two-step approaches. Electronic databases were searched from their inception until June 2017. We included all randomized controlled trials (RCTs) comparing the one-step with the two-step approaches for the screening and diagnosis of GDM. The primary outcome was the incidence of GDM. Three RCTs (n = 2333 participants) were included in the meta-analysis. 910 were randomized to the one step approach (75 g, 2 hrs), and 1423 to the two step approach. No significant difference in the incidence of GDM was found comparing the one step versus the two step approaches (8.4 versus 4.3%; relative risk (RR) 1.64, 95%CI 0.77-3.48). Women screened with the one step approach had a significantly lower risk of preterm birth (PTB) (3.7 versus 7.6%; RR 0.49, 95%CI 0.27-0.88), cesarean delivery (16.3 versus 22.0%; RR 0.74, 95%CI 0.56-0.99), macrosomia (2.9 versus 6.9%; RR 0.43, 95%CI 0.22-0.82), neonatal hypoglycemia (1.7 versus 4.5%; RR 0.38, 95%CI 0.16-0.90), and admission to neonatal intensive care unit (NICU) (4.4 versus 9.0%; RR 0.49, 95%CI 0.29-0.84), compared to those randomized to screening with the two step approach. The one and the two step approaches were not associated with a significant difference in the incidence of GDM. However, the one step approach was associated with better maternal and perinatal outcomes.

  17. Shear bond strength of one-step self-etch adhesives to dentin : evaluation of NaOCl pretreatment

    OpenAIRE

    Colombo, Marco; Beltrami, Riccardo; Chiesa, Marco; Poggio, Claudio; Scribante, Andrea

    2018-01-01

    The aim of this study was to evaluate the influence of dentin pretreatment with NaOCl on shear bond strength of four one-step self-etch adhesives with different pH values. Bovine permanent incisors were used. Four one-step self-etch adhesives were tested: Adper? Easy Bond, Futurabond NR, G-aenial Bond, Clearfil S3 Bond. One two-step self-etch adhesive (Clearfil SE Bond) was used as control. Group 1- no pretreatment; group 2- pretratment with 5,25 % NaOCl; group 3- pretreatment with 37 % H3PO4...

  18. One-Step Borylation of 1,3-Diaryloxybenzenes Towards Efficient Materials for Organic Light-Emitting Diodes.

    Science.gov (United States)

    Hirai, Hiroki; Nakajima, Kiichi; Nakatsuka, Soichiro; Shiren, Kazushi; Ni, Jingping; Nomura, Shintaro; Ikuta, Toshiaki; Hatakeyama, Takuji

    2015-11-09

    The development of a one-step borylation of 1,3-diaryloxybenzenes, yielding novel boron-containing polycyclic aromatic compounds, is reported. The resulting boron-containing compounds possess high singlet-triplet excitation energies as a result of localized frontier molecular orbitals induced by boron and oxygen. Using these compounds as a host material, we successfully prepared phosphorescent organic light-emitting diodes exhibiting high efficiency and adequate lifetimes. Moreover, using the present one-step borylation, we succeeded in the synthesis of an efficient, thermally activated delayed fluorescence emitter and boron-fused benzo[6]helicene. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Combination of real-time PCR and sequencing to detect multiple clinically relevant genetic variations in the lactase gene

    DEFF Research Database (Denmark)

    Brasen, Claus Lohman; Frischknecht, Lone; Ørnskov, Dorthe

    2017-01-01

    in the probe-binding site may cause errors in analysis. The aim of this study was to determine the prevalence of the variants in a Danish cohort examined for lactose intolerance as well as to improve the real-time PCR analysis for detection of the different variants. METHODS: We genotyped 3395 routine samples...

  20. PCR detection of ansA from marine bacteria and its sequence characteristics from Bacillus tequilensis NIOS4

    Digital Repository Service at National Institute of Oceanography (India)

    Nayak, S.; Porob, S.; Fernandes, Areena; Meena, R.M.; Ramaiah, N.

    As many as 71 marine bacterial DNA extracts were PCR screened for L-asparaginase (ansA), a key gene in anti-cancer molecular-searches. Over 62% (44) of them were positive for ansA gene. The positive cultures were from genera Bacillus...

  1. PCR amplification and sequences of cDNA clones for the small and large subunits of ADP-glucose pyrophosphorylase from barley tissues.

    Science.gov (United States)

    Villand, P; Aalen, R; Olsen, O A; Lüthi, E; Lönneborg, A; Kleczkowski, L A

    1992-06-01

    Several cDNAs encoding the small and large subunit of ADP-glucose pyrophosphorylase (AGP) were isolated from total RNA of the starchy endosperm, roots and leaves of barley by polymerase chain reaction (PCR). Sets of degenerate oligonucleotide primers, based on previously published conserved amino acid sequences of plant AGP, were used for synthesis and amplification of the cDNAs. For either the endosperm, roots and leaves, the restriction analysis of PCR products (ca. 550 nucleotides each) has revealed heterogeneity, suggesting presence of three transcripts for AGP in the endosperm and roots, and up to two AGP transcripts in the leaf tissue. Based on the derived amino acid sequences, two clones from the endosperm, beps and bepl, were identified as coding for the small and large subunit of AGP, respectively, while a leaf transcript (blpl) encoded the putative large subunit of AGP. There was about 50% identity between the endosperm clones, and both of them were about 60% identical to the leaf cDNA. Northern blot analysis has indicated that beps and bepl are expressed in both the endosperm and roots, while blpl is detectable only in leaves. Application of the PCR technique in studies on gene structure and gene expression of plant AGP is discussed.

  2. Iteratively improving Hi-C experiments one step at a time.

    Science.gov (United States)

    Golloshi, Rosela; Sanders, Jacob T; McCord, Rachel Patton

    2018-04-30

    The 3D organization of eukaryotic chromosomes affects key processes such as gene expression, DNA replication, cell division, and response to DNA damage. The genome-wide chromosome conformation capture (Hi-C) approach can characterize the landscape of 3D genome organization by measuring interaction frequencies between all genomic regions. Hi-C protocol improvements and rapid advances in DNA sequencing power have made Hi-C useful to study diverse biological systems, not only to elucidate the role of 3D genome structure in proper cellular function, but also to characterize genomic rearrangements, assemble new genomes, and consider chromatin interactions as potential biomarkers for diseases. Yet, the Hi-C protocol is still complex and subject to variations at numerous steps that can affect the resulting data. Thus, there is still a need for better understanding and control of factors that contribute to Hi-C experiment success and data quality. Here, we evaluate recently proposed Hi-C protocol modifications as well as often overlooked variables in sample preparation and examine their effects on Hi-C data quality. We examine artifacts that can occur during Hi-C library preparation, including microhomology-based artificial template copying and chimera formation that can add noise to the downstream data. Exploring the mechanisms underlying Hi-C artifacts pinpoints steps that should be further optimized in the future. To improve the utility of Hi-C in characterizing the 3D genome of specialized populations of cells or small samples of primary tissue, we identify steps prone to DNA loss which should be considered to adapt Hi-C to lower cell numbers. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. One Step Forward, Two Steps Back; Xeno-MicroRNAs Reported in Breast Milk Are Artifacts.

    Directory of Open Access Journals (Sweden)

    Caner Bağcı

    Full Text Available MicroRNAs (miRNAs are short RNA sequences that guide post-transcriptional regulation of gene expression via complementarity to their target mRNAs. Discovered only recently, miRNAs have drawn a lot of attention. Multiple protein complexes interact to first cleave a hairpin from nascent RNA, export it into the cytosol, trim its loop, and incorporate it into the RISC complex which is important for binding its target mRNA. This process works within one cell, but circulating miRNAs have been described suggesting a role in cell-cell communication.Viruses and intracellular parasites like Toxoplasma gondii use miRNAs to manipulate host gene expression from within the cellular environment. However, recent research has claimed that a rice miRNA may regulate human gene expression. Despite ongoing debates about these findings and general reluctance to accept them, a recent report claimed that foodborne plant miRNAs pass through the digestive tract, travel through blood to be incorporated by alveolar cells excreting milk. The miRNAs are then said to have some immune-related function in the newborn.We acquired the data that supports their claim and performed further analyses. In addition to the reported miRNAs, we were able to detect almost complete mRNAs and found that the foreign RNA expression profiles among samples are exceedingly similar. Inspecting the source of the data helped understand how RNAs could contaminate the samples.Viewing these findings in context with the difficulties foreign RNAs face on their route into breast milk and the fact that many identified foodborne miRNAs are not from actual food sources, we can conclude beyond reasonable doubt that the original claims and evidence presented may be due to artifacts. We report that the study claiming their existence is more likely to have detected RNA contamination than miRNAs.

  4. Detection of hepatitis A virus by the nucleic acid sequence-based amplification technique and comparison with reverse transcription-PCR.

    Science.gov (United States)

    Jean, J; Blais, B; Darveau, A; Fliss, I

    2001-12-01

    A nucleic acid sequence-based amplification (NASBA) technique for the detection of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide primers targeting the VP1 and VP2 genes encoding the major HAV capsid proteins were used for the amplification of viral RNA in an isothermal process resulting in the accumulation of RNA amplicons. Amplicons were detected by hybridization with a digoxigenin-labeled oligonucleotide probe in a dot blot assay format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detected per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was used, a detection limit of 2 PFU (4 x 10(2) PFU/ml) was obtained with NASBA, compared to 50 PFU (1 x 10(4) PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess nontarget RNA or DNA. The NASBA system successfully detected HAV recovered from experimentally inoculated samples of waste water, lettuce, and blueberries. Compared to RT-PCR and other amplification techniques, the NASBA system offers several advantages in terms of sensitivity, rapidity, and simplicity. This technique should be readily adaptable for detection of other RNA viruses in both foods and clinical samples.

  5. PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing.

    Directory of Open Access Journals (Sweden)

    Meetha P Gould

    Full Text Available Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear DNA. Reduction in nuclear DNA (nDNA content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts. We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.

  6. Use of next generation sequencing data to develop a qPCR method for specific detection of EU-unauthorized genetically modified Bacillus subtilis overproducing riboflavin.

    Science.gov (United States)

    Barbau-Piednoir, Elodie; De Keersmaecker, Sigrid C J; Delvoye, Maud; Gau, Céline; Philipp, Patrick; Roosens, Nancy H

    2015-11-11

    Recently, the presence of an unauthorized genetically modified (GM) Bacillus subtilis bacterium overproducing vitamin B2 in a feed additive was notified by the Rapid Alert System for Food and Feed (RASFF). This has demonstrated that a contamination by a GM micro-organism (GMM) may occur in feed additives and has confronted for the first time,the enforcement laboratories with this type of RASFF. As no sequence information of this GMM nor any specific detection or identification method was available, Next GenerationSequencing (NGS) was used to generate sequence information. However, NGS data analysis often requires appropriate tools, involving bioinformatics expertise which is not alwayspresent in the average enforcement laboratory. This hampers the use of this technology to rapidly obtain critical sequence information in order to be able to develop a specific qPCRdetection method. Data generated by NGS were exploited using a simple BLAST approach. A TaqMan® qPCR method was developed and tested on isolated bacterial strains and on the feed additive directly. In this study, a very simple strategy based on the common BLAST tools that can be used by any enforcement lab without profound bioinformatics expertise, was successfully used toanalyse the B. subtilis data generated by NGS. The results were used to design and assess a new TaqMan® qPCR method, specifically detecting this GM vitamin B2 overproducing bacterium. The method complies with EU critical performance parameters for specificity, sensitivity, PCR efficiency and repeatability. The VitB2-UGM method also could detect the B. subtilis strain in genomic DNA extracted from the feed additive, without prior culturing step. The proposed method, provides a crucial tool for specifically and rapidly identifying this unauthorized GM bacterium in food and feed additives by enforcement laboratories. Moreover, this work can be seen as a case study to substantiate how the use of NGS data can offer an added value to easily

  7. One-step isothermal detection of multiple KRAS mutations by forming SNP specific hairpins on a gold nanoshell.

    Science.gov (United States)

    Chung, Chan Ho; Kim, Joong Hyun

    2018-04-24

    We developed a one-step isothermal method for typing multiple KRAS mutations using a designed set of primers to form a hairpin on a gold nanoshell upon being ligated by a SNP specific DNA ligase after binding of targets. As a result, we could detect as low as 20 attomoles of KRAS mutations within 1 h.

  8. One-step Maskless Fabrication and Optical Characterization of Silicon Surfaces with Antireflective Properties and a White Color Appearance

    DEFF Research Database (Denmark)

    Sun, Ling; Feidenhans'l, Nikolaj Agentoft; Telecka, Agnieszka

    2016-01-01

    We report a simple one-step maskless fabrication of inverted pyramids on silicon wafers by reactive ion etching. The fabricated surface structures exhibit excellent anti-reflective properties: The total reflectance of the nano inverted pyramids fabricated by our method can be as low as 12% withou...... milky white color....

  9. One-step synthesis and characterization of CoS-Nanoparticles decorated multi-walled carbon nanotubes

    CSIR Research Space (South Africa)

    Kesavan Pillai, Sreejarani

    2009-02-01

    Full Text Available of applications. It is vital to develop efficient methods to attach quantum dots onto the sidewalls of CNTs whose structures have been preserved intact. In this study, a simple and facile one-step synthesis approach for the in situ mineralisation of Co...

  10. Catalysis engineering of bifunctional solids for the one-step synthesis of liquid fuels from syngas : A review

    NARCIS (Netherlands)

    Sartipi, S.; Makkee, M.; Kapteijn, F.; Gascon, J.

    2014-01-01

    The combination of acidic zeolites and Fischer–Tropsch synthesis (FTS) catalysts for one-step production of liquid fuels from syngas is critically reviewed. Bifunctional systems are classified by the proximity between FTS and acid functionalities on three levels: reactor, catalyst particle, and

  11. In silico and in vitro evaluation of PCR-based assays for the detection of Bacillus anthracis chromosomal signature sequences

    DEFF Research Database (Denmark)

    Ågren, Joakim; Hamidjaja, Raditijo A.; Hansen, Trine

    2013-01-01

    Bacillus anthracis, the causative agent of anthrax, is a zoonotic pathogen that is relatively common throughout the world and may cause life threatening diseases in animals and humans. There are many PCR-based assays in use for the detection of B. anthracis. While most of the developed assays rely...... on unique markers present on virulence plasmids pXO1 and pXO2, relatively few assays incorporate chromosomal DNA markers due to the close relatedness of B. anthracis to the B. cereus group strains. For the detection of chromosomal DNA, different genes have been used, such as BA813, rpoB, gyrA, plcR, S...... targets evaluated are claimed to be specific to B. anthracis, cross-reactions with closely related B. cereus and B. thuringiensis strains were often observed. Of the 35 investigated PCR assays, only 4 were 100% specific for the B. anthracis chromosome. An interlaboratory ring trial among five European...

  12. Discrimination of the Lactobacillus acidophilus group using sequencing, species-specific PCR and SNaPshot mini-sequencing technology based on the recA gene.

    Science.gov (United States)

    Huang, Chien-Hsun; Chang, Mu-Tzu; Huang, Mu-Chiou; Wang, Li-Tin; Huang, Lina; Lee, Fwu-Ling

    2012-10-01

    To clearly identify specific species and subspecies of the Lactobacillus acidophilus group using phenotypic and genotypic (16S rDNA sequence analysis) techniques alone is difficult. The aim of this study was to use the recA gene for species discrimination in the L. acidophilus group, as well as to develop a species-specific primer and single nucleotide polymorphism primer based on the recA gene sequence for species and subspecies identification. The average sequence similarity for the recA gene among type strains was 80.0%, and most members of the L. acidophilus group could be clearly distinguished. The species-specific primer was designed according to the recA gene sequencing, which was employed for polymerase chain reaction with the template DNA of Lactobacillus strains. A single 231-bp species-specific band was found only in L. delbrueckii. A SNaPshot mini-sequencing assay using recA as a target gene was also developed. The specificity of the mini-sequencing assay was evaluated using 31 strains of L. delbrueckii species and was able to unambiguously discriminate strains belonging to the subspecies L. delbrueckii subsp. bulgaricus. The phylogenetic relationships of most strains in the L. acidophilus group can be resolved using recA gene sequencing, and a novel method to identify the species and subspecies of the L. delbrueckii and L. delbrueckii subsp. bulgaricus was developed by species-specific polymerase chain reaction combined with SNaPshot mini-sequencing. Copyright © 2012 Society of Chemical Industry.

  13. One-step process of hydrothermal and alkaline treatment of wheat straw for improving the enzymatic saccharification.

    Science.gov (United States)

    Sun, Shaolong; Zhang, Lidan; Liu, Fang; Fan, Xiaolin; Sun, Run-Cang

    2018-01-01

    To increase the production of bioethanol, a two-step process based on hydrothermal and dilute alkaline treatment was applied to reduce the natural resistance of biomass. However, the process required a large amount of water and a long operation time due to the solid/liquid separation before the alkaline treatment, which led to decrease the pure economic profit for production of bioethanol. Therefore, four one-step processes based on order of hydrothermal and alkaline treatment have been developed to enhance concentration of glucose of wheat straw by enzymatic saccharification. The aim of the present study was to systematically evaluated effect for different one-step processes by analyzing the physicochemical properties (composition, structural change, crystallinity, surface morphology, and BET surface area) and enzymatic saccharification of the treated substrates. In this study, hemicelluloses and lignins were removed from wheat straw and the morphologic structures were destroyed to various extents during the four one-step processes, which were favorable for cellulase absorption on cellulose. A positive correlation was also observed between the crystallinity and enzymatic saccharification rate of the substrate under the conditions given. The surface area of the substrate was positively related to the concentration of glucose in this study. As compared to the control (3.0 g/L) and treated substrates (11.2-14.6 g/L) obtained by the other three one-step processes, the substrate treated by one-step process based on successively hydrothermal and alkaline treatment had a maximum glucose concentration of 18.6 g/L, which was due to the high cellulose concentration and surface area for the substrate, accompanying with removal of large amounts of lignins and hemicelluloses. The present study demonstrated that the order of hydrothermal and alkaline treatment had significant effects on the physicochemical properties and enzymatic saccharification of wheat straw. The one-step

  14. Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

    Science.gov (United States)

    Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

    2011-09-01

    The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

  15. Generation of human scFv antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

    Science.gov (United States)

    Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

    2011-09-01

    The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human scFv (single chain antibody fragment) libraries using a short linker (GGSSRSS) or a long linker (GGSSRSSSSGGGGSGGGG). In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final scFv products that are used for cloning.

  16. Differentiation of mycoplasmalike organisms (MLOs) in European fruit trees by PCR using specific primers derived from the sequence of a chromosomal fragment of the apple proliferation MLO.

    Science.gov (United States)

    Jarausch, W; Saillard, C; Dosba, F; Bové, J M

    1994-01-01

    A 1.8-kb chromosomal DNA fragment of the mycoplasmalike organism (MLO) associated with apple proliferation was sequenced. Three putative open reading frames were observed on this fragment. The protein encoded by open reading frame 2 shows significant homologies with bacterial nitroreductases. From the nucleotide sequence four primer pairs for PCR were chosen to specifically amplify DNA from MLOs associated with European diseases of fruit trees. Primer pairs specific for (i) Malus-affecting MLOs, (ii) Malus- and Prunus-affecting MLOs, and (iii) Malus-, Prunus-, and Pyrus-affecting MLOs were obtained. Restriction enzyme analysis of the amplification products revealed restriction fragment length polymorphisms between Malus-, Prunus, and Pyrus-affecting MLOs as well as between different isolates of the apple proliferation MLO. No amplification with either primer pair could be obtained with DNA from 12 different MLOs experimentally maintained in periwinkle. Images PMID:7916180

  17. Large-area gold nanohole arrays fabricated by one-step method for surface plasmon resonance biochemical sensing.

    Science.gov (United States)

    Qi, Huijie; Niu, Lihong; Zhang, Jie; Chen, Jian; Wang, Shujie; Yang, Jingjing; Guo, Siyi; Lawson, Tom; Shi, Bingyang; Song, Chunpeng

    2018-04-01

    Surface plasmon resonance (SPR) nanosensors based on metallic nanohole arrays have been widely reported to detect binding interactions in biological specimens. A simple and effective method for constructing nanoscale arrays is essential for the development of SPR nanosensors. In this work, we report a one-step method to fabricate nanohole arrays by thermal nanoimprinting in the matrix of IPS (Intermediate Polymer Stamp). No additional etching process or supporting substrate is required. The preparation process is simple, time-saving and compatible for roll-to-roll process, potentially allowing mass production. Moreover, the nanohole arrays were integrated into detection platform as SPR sensors to investigate different types of biological binding interactions. The results demonstrate that our one-step method can be used to efficiently fabricate large-area and uniform nanohole arrays for biochemical sensing.

  18. Amperometric sensor for ethanol based on one-step electropolymerization of thionine-carbon nanofiber nanocomposite containing alcohol oxidase.

    Science.gov (United States)

    Wu, Lina; McIntosh, Mike; Zhang, Xueji; Ju, Huangxian

    2007-12-15

    Thionine had strong interaction with carbon nanofiber (CNF) and was used in the non-covalent functionalization of carbon nanofiber for the preparation of stable thionine-CNF nanocomposite with good dispersion. With a simple one-step electrochemical polymerization of thionine-CNF nanocomposite and alcohol oxidase (AOD), a stable poly(thionine)-CNF/AOD biocomposite film was formed on electrode surface. Based on the excellent catalytic activity of the biocomposite film toward reduction of dissolved oxygen, a sensitive ethanol biosensor was proposed. The ethanol biosensor could monitor ethanol ranging from 2.0 to 252 microM with a detection limit of 1.7 microM. It displayed a rapid response, an expanded linear response range as well as excellent reproducibility and stability. The combination of catalytic activity of CNF and the promising feature of the biocomposite with one-step non-manual technique favored the sensitive determination of ethanol with improved analytical capabilities.

  19. Rapid One-Step Selection Method for Generating Nucleic Acid Aptamers: Development of a DNA Aptamer against alpha-Bungarotoxin

    DEFF Research Database (Denmark)

    Lauridsen, Lasse Holm; Shamaileh, Hadi A.; Edwards, Stacey L.

    2012-01-01

    Background: Nucleic acids based therapeutic approaches have gained significant interest in recent years towards the development of therapeutics against many diseases. Recently, research on aptamers led to the marketing of Macugen (R), an inhibitor of vascular endothelial growth factor (VEGF......) for the treatment of age related macular degeneration (AMD). Aptamer technology may prove useful as a therapeutic alternative against an array of human maladies. Considering the increased interest in aptamer technology globally that rival antibody mediated therapeutic approaches, a simplified selection, possibly...... in one-step, technique is required for developing aptamers in limited time period. Principal Findings: Herein, we present a simple one-step selection of DNA aptamers against alpha-bungarotoxin. A toxin immobilized glass coverslip was subjected to nucleic acid pool binding and extensive washing followed...

  20. A new one-step procedure for pulmonary valve implantation of the melody valve: Simultaneous prestenting and valve implantation.

    Science.gov (United States)

    Boudjemline, Younes

    2018-01-01

    To describe a new modification, the one-step procedure, that allows interventionists to pre-stent and implant a Melody valve simultaneously. Percutaneous pulmonary valve implantation (PPVI) is the standard of care for managing patients with dysfunctional right ventricular outflow tract, and the approach is standardized. Patients undergoing PPVI using the one-step procedure were identified in our database. Procedural data and radiation exposure were compared to those in a matched group of patients who underwent PPVI using the conventional two-step procedure. Between January 2016 and January 2017, PPVI was performed in 27 patients (median age/range, 19.1/10-55 years) using the one-step procedure involving manual crimping of one to three bare metal stents over the Melody valve. The stent and Melody valve were delivered successfully using the Ensemble delivery system. No complications occurred. All patients had excellent hemodynamic results (median/range post-PPVI right ventricular to pulmonary artery gradient, 9/0-20 mmHg). Valve function was excellent. Median procedural and fluoroscopic times were 56 and 10.2 min, respectively, which significantly differed from those of the two-step procedure group. Similarly, the dose area product (DAP), and radiation time were statistically lower in the one-step group than in the two-step group (P step procedure is a safe modification that allows interventionists to prestent and implants the Melody valve simultaneously. It significantly reduces procedural and fluoroscopic times, and radiation exposure. © 2017 Wiley Periodicals, Inc.

  1. Kinetic study of solid phase demineralization by weak acids in one-step enzymatic bio-refinery of shrimp cuticles

    OpenAIRE

    Baron, Regis; Socol, Marius; Arhaliass, A.; Bruzac, Sandrine; Le Roux, Karine; Del Pino, J. Rodriguez; Berge, Jean-pascal; Kaas, Raymond

    2015-01-01

    We describe a one-step bio-refinery process for shrimp composites by-products. Its originality lies in a simple rapid (6 h) biotechnological cuticle fragmentation process that recovers all major compounds (chitins, peptides and minerals in particular calcium). The process consists of a controlled exogenous enzymatic proteolysis in a food-grade acidic medium allowing chitin purification (solid phase), and recovery of peptides and minerals (liquid phase). At a pH of between 3.5 and 4, protease ...

  2. [Evaluation of the effect of one-step self etching adhesives applied in pit and fissure sealing].

    Science.gov (United States)

    Su, Hong-Ru; Xu, Pei-Cheng; Qian, Wen-Hao

    2016-06-01

    To observe the effect of three one-step self etching adhesive systems used in fit and fissure sealant and explore the feasibility of application in caries prevention in school. Seven hundred and twenty completely erupted mandibular first molars in 360 children aged 7 to 9 years old were chosen. The split-mouth design was used to select one side as the experimental group, divided into A1(Easy One Adper), B1(Adper Easy One), and C1(iBond SE).The contra lateral teeth served as A2,B2 and C2 groups (phosphoric acid). The retention and caries status were regularly reviewed .The clinical effect of the two groups was compared using SPSS19.0 software package for Chi - square test. At 3 and 6 months, pit and fissure sealant retention rate in A1 and A2, B1 and B2,C1 and C2 group had no significant difference. At 12 months, sealant retention in A1 and B1 group was significantly lower than A2 and B2 group (P0.05). At 24 months, sealant retention rate in A1, B1 and C1 group was significantly lower than A2, B2 and C2 group (P0.05). The clinical anticariogenic effect of three kinds of one-step etching adhesives and phosphoric acid etching sealant was similar .One-step self etching adhesive system was recommended for pit and fissure sealant to improve the students' oral health. The long-term retention rate of one-step self etching adhesive system was lower than the phosphoric acid method to long term observation is needed.

  3. One-step preparation of [18F]FPBM for PET imaging of serotonin transporter (SERT) in the brain

    International Nuclear Information System (INIS)

    Qiao, Hongwen; Zhang, Yan; Wu, Zehui; Zhu, Lin; Choi, Seok Rye; Ploessl, Karl; Kung, Hank F.

    2016-01-01

    Serotonin transporters (SERT) in the brain play an important role in normal brain function. Selective serotonin reuptake inhibitors such as fluoxetine, sertraline, paroxetine, escitalopram, etc., specifically target SERT binding in the brain. Development of SERT imaging agents may be useful for studying the function of SERT by in vivo imaging. A one-step preparation of [ 18 F]FPBM, 2-(2′-(dimethylamino)methyl)-4′-(3-([ 18 F]fluoropropoxy)phenylthio) benzenamine, for positron emission tomography (PET) imaging of SERT binding in the brain was achieved. An active OTs intermediate, 9, was reacted with [ 18 F]F − /K 222 to produce [ 18 F]FPBM in one step and in high radiochemical yield. This labeling reaction was evaluated and optimized under different temperatures, bases, solvents, and varying amounts of precursor 9. The radiolabeling reaction led to the desired [ 18 F]FPBM in one step and the crude product was purified by HPLC purification to give no-carrier-added [ 18 F]FPBM (radiochemical yield, 24–33%, decay corrected; radiochemical purity > 99%). PET imaging studies in normal monkeys (n = 4) showed fast, pronounced uptakes in the midbrain and thalamus, regions known to be rich in SERT binding sites. A displacement experiment with escitalopram (5 mg/kg iv injection at 30 min after [ 18 F]FPBM injection) showed a rapid and complete reversal of SERT binding, suggesting that binding by [ 18 F]FPBM was highly specific and reversible. A one-step radiolabeling method coupled with HPLC purification for preparation of [ 18 F]FPBM was developed. Imaging studies suggest that it is feasible to use this method to prepare [ 18 F]FPBM for in vivo PET imaging of SERT binding in the brain.

  4. Catalysis engineering of bifunctional solids for the one-step synthesis of liquid fuels from syngas: A review

    OpenAIRE

    Sartipi, S.; Makkee, M.; Kapteijn, F.; Gascon, J.

    2014-01-01

    The combination of acidic zeolites and Fischer–Tropsch synthesis (FTS) catalysts for one-step production of liquid fuels from syngas is critically reviewed. Bifunctional systems are classified by the proximity between FTS and acid functionalities on three levels: reactor, catalyst particle, and active phase. A thorough analysis of the published literature on this topic reveals that efficiency in the production of liquid fuels correlates well with the proximity of FTS and acid sites. Moreover,...

  5. Balanced Photodetection in One-Step Liquid-Phase-Synthesized CsPbBr3 Micro-/Nanoflake Single Crystals.

    Science.gov (United States)

    Zheng, Wei; Xiong, Xufan; Lin, Richeng; Zhang, Zhaojun; Xu, Cunhua; Huang, Feng

    2018-01-17

    Here, we reported a low-cost and high-compatibility one-step liquid-phase synthesis method for synthesizing high-purity CsPbBr 3 micro-/nanoflake single crystals. On the basis of the high-purity CsPbBr 3 , we further prepared a low-dimensional photodetector capable of balanced photodetection, involving both high external quantum efficiency and rapid temporal response, which is barely realized in previously reported low-dimensional photodetectors.

  6. One Step Further

    DEFF Research Database (Denmark)

    Hansen, Finn Thorbjørn

    2012-01-01

    The phenomenological attitude is essential for practising phenomenology. Many refer to wonder and wonderment as basic attitudes and ways of being present with and listening to phenomena. In this article a critical view is placed on the typically psychologically-loaded language and tonality that i...

  7. One-step preparation of N-doped graphene/Co nanocomposite as an advanced oxygen reduction electrocatalyst

    International Nuclear Information System (INIS)

    Bai, Fo; Huang, Hao; Tan, Yanlei; Hou, Changmin; Zhang, Ping

    2015-01-01

    Graphical abstract: N-doped graphene/Co nanocomposites were synthesized through one-step pyrolysis process and the product exhibits high performance for ORR and excellent stability in alkaline medium. - Highlights: • N-doped graphene/Co nano-composite is directly synthesized by a one-step method from Co(NO3)2∙6H2O, glucose and dicyandiamide (DCDA). • The electrocatalytic performance of as-prepared NG/Co-0.5 shows the peak potential positively shifts about 10 mV than commercial Pt/C electrode. • The material shows an excellent stability and tolerance to methanol poisoning effects in alkaline medium. - Abstract: N-doped graphene/Co nanocomposites (NG/Co NPs) have been prepared by a simple one-step pyrolysis of Co(NO 3 ) 2 ∙6H 2 O, glucose and dicyandiamide (DCDA). The products with nitrogen doped and suitable graphitic degree perform high electrocatalytic activity (with the reduction peak at −0.099 V vs Ag/AgCl) and near four-electron selectivity for the oxygen reduction reaction (ORR), with excellent stability and durability in alkaline medium comparable to a commercial Pt/C catalyst. Owing to the superb ORR performance, low cost and facile preparation, the catalysts of NG/Co NPs have great potential applications in fuel cells, metal-air batteries and ORR-related electrochemical industries

  8. Numerical characterisation of one-step and three-step solar air heating collectors used for cocoa bean solar drying.

    Science.gov (United States)

    Orbegoso, Elder Mendoza; Saavedra, Rafael; Marcelo, Daniel; La Madrid, Raúl

    2017-12-01

    In the northern coastal and jungle areas of Peru, cocoa beans are dried using artisan methods, such as direct exposure to sunlight. This traditional process is time intensive, leading to a reduction in productivity and, therefore, delays in delivery times. The present study was intended to numerically characterise the thermal behaviour of three configurations of solar air heating collectors in order to determine which demonstrated the best thermal performance under several controlled operating conditions. For this purpose, a computational fluid dynamics model was developed to describe the simultaneous convective and radiative heat transfer phenomena under several operation conditions. The constructed computational fluid dynamics model was firstly validated through comparison with the data measurements of a one-step solar air heating collector. We then simulated two further three-step solar air heating collectors in order to identify which demonstrated the best thermal performance in terms of outlet air temperature and thermal efficiency. The numerical results show that under the same solar irradiation area of exposition and operating conditions, the three-step solar air heating collector with the collector plate mounted between the second and third channels was 67% more thermally efficient compared to the one-step solar air heating collector. This is because the air exposition with the surface of the collector plate for the three-step solar air heating collector former device was twice than the one-step solar air heating collector. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. One-step leapfrog ADI-FDTD method for simulating electromagnetic wave propagation in general dispersive media.

    Science.gov (United States)

    Wang, Xiang-Hua; Yin, Wen-Yan; Chen, Zhi Zhang David

    2013-09-09

    The one-step leapfrog alternating-direction-implicit finite-difference time-domain (ADI-FDTD) method is reformulated for simulating general electrically dispersive media. It models material dispersive properties with equivalent polarization currents. These currents are then solved with the auxiliary differential equation (ADE) and then incorporated into the one-step leapfrog ADI-FDTD method. The final equations are presented in the form similar to that of the conventional FDTD method but with second-order perturbation. The adapted method is then applied to characterize (a) electromagnetic wave propagation in a rectangular waveguide loaded with a magnetized plasma slab, (b) transmission coefficient of a plane wave normally incident on a monolayer graphene sheet biased by a magnetostatic field, and (c) surface plasmon polaritons (SPPs) propagation along a monolayer graphene sheet biased by an electrostatic field. The numerical results verify the stability, accuracy and computational efficiency of the proposed one-step leapfrog ADI-FDTD algorithm in comparison with analytical results and the results obtained with the other methods.

  10. 16S-23S rDNA intergenic spacer region polymorphism of Lactococcus garvieae, Lactococcus raffinolactis and Lactococcus lactis as revealed by PCR and nucleotide sequence analysis.

    Science.gov (United States)

    Blaiotta, Giuseppe; Pepe, Olimpia; Mauriello, Gianluigi; Villani, Francesco; Andolfi, Rosamaria; Moschetti, Giancarlo

    2002-12-01

    The intergenic spacer region (ISR) between the 16S and 23S rRNA genes was tested as a tool for differentiating lactococci commonly isolated in a dairy environment. 17 reference strains, representing 11 different species belonging to the genera Lactococcus, Streptococcus, Lactobacillus, Enterococcus and Leuconostoc, and 127 wild streptococcal strains isolated during the whole fermentation process of "Fior di Latte" cheese were analyzed. After 16S-23S rDNA ISR amplification by PCR, species or genus-specific patterns were obtained for most of the reference strains tested. Moreover, results obtained after nucleotide analysis show that the 16S-23S rDNA ISR sequences vary greatly, in size and sequence, among Lactococcus garvieae, Lactococcus raffinolactis, Lactococcus lactis as well as other streptococci from dairy environments. Because of the high degree of inter-specific polymorphism observed, 16S-23S rDNA ISR can be considered a good potential target for selecting species-specific molecular assays, such as PCR primer or probes, for a rapid and extremely reliable differentiation of dairy lactococcal isolates.

  11. The gut microbiotassay – a high-throughput real-time PCR chip combined with next generation sequencing

    DEFF Research Database (Denmark)

    Hermann-Bank, Marie Louise; Skovgaard, Kerstin; Mølbak, Lars

    it demonstrated distinct quantities of bacteria in the different gut sections, with the highest number found in colon as expected. From the sequence data it was evident that primer systems targeting lower taxonomical levels, contributed with a higher resolution, revealing species that primer system targeting...

  12. Unexpected Diagnosis of Cerebral Toxoplasmosis by 16S and D2 Large-Subunit Ribosomal DNA PCR and Sequencing

    DEFF Research Database (Denmark)

    Kruse, Alexandra Yasmin Collin; Kvich, Lasse Andersson; Eickhardt-Dalbøge, Steffen Robert

    2015-01-01

    The protozoan parasite Toxoplasma gondii causes severe opportunistic infections. Here, we report an unexpected diagnosis of cerebral toxoplasmosis. T. gondii was diagnosed by 16S and D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing of a cerebral biopsy specimen and confirmed by T. gondii...

  13. Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray.

    Science.gov (United States)

    El-Ashker, Maged; Hotzel, Helmut; Gwida, Mayada; El-Beskawy, Mohamed; Silaghi, Cornelia; Tomaso, Herbert

    2015-01-30

    In this preliminary study, a novel DNA microarray system was tested for the diagnosis of bovine piroplasmosis and anaplasmosis in comparison with microscopy and PCR assay results. In the Dakahlia Governorate, Egypt, 164 cattle were investigated for the presence of piroplasms and Anaplasma species. All investigated cattle were clinically examined. Blood samples were screened for the presence of blood parasites using microscopy and PCR assays. Seventy-one animals were acutely ill, whereas 93 were apparently healthy. In acutely ill cattle, Babesia/Theileria species (n=11) and Anaplasma marginale (n=10) were detected. Mixed infections with Babesia/Theileria spp. and A. marginale were present in two further cases. A. marginale infections were also detected in apparently healthy subjects (n=23). The results of PCR assays were confirmed by DNA sequencing. All samples that were positive by PCR for Babesia/Theileria spp. gave also positive results in the microarray analysis. The microarray chips identified Babesia bovis (n=12) and Babesia bigemina (n=2). Cattle with babesiosis were likely to have hemoglobinuria and nervous signs when compared to those with anaplasmosis that frequently had bloody feces. We conclude that clinical examination in combination with microscopy are still very useful in diagnosing acute cases of babesiosis and anaplasmosis, but a combination of molecular biological diagnostic assays will detect even asymptomatic carriers. In perspective, parallel detection of Babesia/Theileria spp. and A. marginale infections using a single microarray system will be a valuable improvement. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Two-stage clustering (TSC: a pipeline for selecting operational taxonomic units for the high-throughput sequencing of PCR amplicons.

    Directory of Open Access Journals (Sweden)

    Xiao-Tao Jiang

    Full Text Available Clustering 16S/18S rRNA amplicon sequences into operational taxonomic units (OTUs is a critical step for the bioinformatic analysis of microbial diversity. Here, we report a pipeline for selecting OTUs with a relatively low computational demand and a high degree of accuracy. This pipeline is referred to as two-stage clustering (TSC because it divides tags into two groups according to their abundance and clusters them sequentially. The more abundant group is clustered using a hierarchical algorithm similar to that in ESPRIT, which has a high degree of accuracy but is computationally costly for large datasets. The rarer group, which includes the majority of tags, is then heuristically clustered to improve efficiency. To further improve the computational efficiency and accuracy, two preclustering steps are implemented. To maintain clustering accuracy, all tags are grouped into an OTU depending on their pairwise Needleman-Wunsch distance. This method not only improved the computational efficiency but also mitigated the spurious OTU estimation from 'noise' sequences. In addition, OTUs clustered using TSC showed comparable or improved performance in beta-diversity comparisons compared to existing OTU selection methods. This study suggests that the distribution of sequencing datasets is a useful property for improving the computational efficiency and increasing the clustering accuracy of the high-throughput sequencing of PCR amplicons. The software and user guide are freely available at http://hwzhoulab.smu.edu.cn/paperdata/.

  15. A newly constructed primer pair for the PCR amplification, cloning and sequencing of the flagellin (flaA) gene from isolatesof urease-negative Campylobacter lari.

    Science.gov (United States)

    Sekizuka, Tsuyoshi; Yokoi, Taeko; Murayama, Ohoshi; Millar, B Cherie; Moore, Johne; Matsuda, Motoo

    2005-08-01

    A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564-572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70-75% sequence similarities to those of Campylobacter jejuni isolates. The approximate Mr of the flagellin purified from some of the isolates of urease-negative C. lari was estimated to range from 59.6 to 61.8 kDa. Thus, flagellin from the isolates of urease-negative C. lari was shown for the first time to have a molecular size similar to those of C. jejuni and Campylobacter coli isolates, but to be different from the shorter flaA and smaller flagellin of urease-positive thermophilic Campylobacter (UPTC) isolates. Flagellins from C. lari spp., consisting of the two representative taxa of urease-negative C. lari and UPTC, thus show genotypic and phenotypic diversity.

  16. A facile one-step fluorescence method for the quantitation of low-content single base deamination impurity in synthetic oligonucleotides.

    Science.gov (United States)

    Su, Xiaoye; Liang, Ruiting; Stolee, Jessica A

    2018-06-05

    Oligonucleotides are being researched and developed as potential drug candidates for the treatment of a broad spectrum of diseases. The characterization of antisense oligonucleotide (ASO) impurities caused by base mutations (e.g. deamination) which are closely related to the target ASO is a significant analytical challenge. Herein, we describe a novel one-step method, utilizing a strategy that combines fluorescence-ON detection with competitive hybridization, to achieve single base mutation quantitation in extensively modified synthetic ASOs. Given that this method is highly specific and sensitive (LoQ = 4 nM), we envision that it will find utility for screening other impurities as well as sequencing modified oligonucleotides. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Molecular characterization of the rDNA-ITS sequence and a PCR diagnostic technique for Pileolaria terebinthi, the cause of pistachio rust

    Directory of Open Access Journals (Sweden)

    Hossein ALAEI

    2013-01-01

    Full Text Available Eleven samples of the most important pistachio rust (caused by Pileolaria terebinthi (DC. Cast.,, which causes disease on Beneh (Pistacia atlantica Desf. subsp. mutica (Fisch. & Mey. Rech. F and Kasoor (Pistacia khinjuk Stocks., were collected from herbarium specimens and pistachio fields at the Pistachio Research Institute in Rafsanjan, Iran. The complete sequences of ribosomal DNA internal transcribed spacers ITS1 and ITS2 (rDNA ITS from the samples were determined and analysed. In general, very little rDNA ITS sequence variation was observed between rDNA ITS sequences of P. terebinthi samples. The length of the PCR fragments was 621 bp (for ITS1F-ITS4 and 1177 bp (for ITS1F-rust1, and consisted of 67 bp at the 3 ́ end of 18S rDNA, 93 bp of ITS1 region, 154 bp of 5.8S rDNA, 246 bp of the ITS2 region, 57 bp (for ITS1F-ITS4 and 613 bp (for ITS1F-rust1 at the 5 ́ end of the 28S rDNA. Restriction fragment length polymorphisms (RFLPs of the rDNA-ITS region were used to identify Pileolaria terebinthi. Three strong bands of 105, 134 and 381 bp and five bands of 105, 134, 200, 301 and 437 bp are observed for the fragment of “ITS1F-ITS4” and “ITS1F-rust1”, respectively. A PCR-RFLP diagnostic technique provided effective identification of the species by a unique pattern with the specific restriction enzyme XapI (ApoI.

  18. Effect of double-layer application on dentin bond durability of one-step self-etch adhesives.

    Science.gov (United States)

    Taschner, M; Kümmerling, M; Lohbauer, U; Breschi, L; Petschelt, A; Frankenberger, R

    2014-01-01

    The aim of this in vitro study was 1) to analyze the influence of a double-layer application technique of four one-step self-etch adhesive systems on dentin and 2) to determine its effect on the stability of the adhesive interfaces stored under different conditions. Four different one-step self-etch adhesives were selected for the study (iBondSE, Clearfil S(3) Bond, XenoV(+), and Scotchbond Universal). Adhesives were applied according to manufacturers' instructions or with a double-layer application technique (without light curing of the first layer). After bonding, resin-dentin specimens were sectioned for microtensile bond strength testing in accordance with the nontrimming technique and divided into 3 subgroups of storage: a) 24 hours (immediate bond strength, T0), b) six months (T6) in artificial saliva at 37°C, or c) five hours in 10 % NaOCl at room temperature. After storage, specimens were stressed to failure. Fracture mode was assessed under a light microscope. At T0, iBond SE showed a significant increase in microtensile bond strength when the double-application technique was applied. All adhesive systems showed reduced bond strengths after six months of storage in artificial saliva and after storage in 10% NaOCl for five hours; however at T6, iBond SE, Clearfil S(3) Bond, and XenoV(+) showed significantly higher microtensile bond strength results for the double-application technique compared with the single-application technique. Scotchbond Universal showed no difference between single- or double-application, irrespective of the storage conditions. The results of this study show that improvements in bond strength of one-step self-etch adhesives by using the double-application technique are adhesive dependent.

  19. Bond strength of one-step self-etch adhesives and their predecessors to ground versus unground enamel.

    Science.gov (United States)

    Yazici, A Ruya; Yildirim, Zeren; Ertan, Atila; Ozgunaltay, Gül; Dayangac, Berrin; Antonson, Sibel A; Antonson, Donald E

    2012-07-01

    The aim of this study was to compare the shear bond strength of several self-etch adhesives to their two-step predecessors to ground and unground enamel. Seventy-five extracted, non-carious human third molar teeth were selected for this study. The buccal surfaces of each tooth were mechanically ground to obtain flat enamel surfaces (ground enamel), while the lingual surfaces were left intact (unground enamel). The teeth were randomly divided into five groups according to the adhesive systems (n=15): one-step self-etch adhesive - Clearfil S3 Bond, its two-step predecessor - Clearfil SE Bond, one-step self-etch adhesive - AdheSE One, and its two-step predecessor - AdheSE, and a two-step etch-and-rinse adhesive - Adper Single Bond 2(control). After application of the adhesives to the buccal and lingual enamel surfaces of each tooth, a cylindrical capsule filled with a hybrid composite resin (TPH) was seated against the surfaces. The specimens were stored in distilled water at 37°C for 24 hours, followed by thermocy-cling (5°C-55°C/500 cycles). They were subjected to shear bond strength test in a universal testing machine at a crosshead speed of 1.0 mm/minute. The data were compared using a two-way ANOVA, followed by Bonferroni test at Padhesives exhibited statistically similar bond strengths to ground and unground enamel except for the etch-and-rinse adhesive that showed significantly higher bond strengths than the self-etch adhesives (Padhesives tested (P=.17). Similar bond strengths to ground and unground enamel were achieved with one-step self-etch adhesives and their predecessors. Enamel preparation did not influence the bonding performance of the adhesives tested.

  20. Bond strength of one-step self-etch adhesives and their predecessors to ground versus unground enamel

    Science.gov (United States)

    Yazici, A. Ruya; Yildirim, Zeren; Ertan, Atila; Ozgunaltay, Gül; Dayangac, Berrin; Antonson, Sibel A; Antonson, Donald E

    2012-01-01

    Objective The aim of this study was to compare the shear bond strength of several self-etch adhesives to their two-step predecessors to ground and unground enamel. Methods: Seventy-five extracted, non-carious human third molar teeth were selected for this study. The buccal surfaces of each tooth were mechanically ground to obtain flat enamel surfaces (ground enamel), while the lingual surfaces were left intact (unground enamel). The teeth were randomly divided into five groups according to the adhesive systems (n=15): one-step self-etch adhesive - Clearfil S3 Bond, its two-step predecessor - Clearfil SE Bond, one-step self-etch adhesive - AdheSE One, and its two-step predecessor - AdheSE, and a two-step etch-and-rinse adhesive - Adper Single Bond 2(control). After application of the adhesives to the buccal and lingual enamel surfaces of each tooth, a cylindrical capsule filled with a hybrid composite resin (TPH) was seated against the surfaces. The specimens were stored in distilled water at 37°C for 24 hours, followed by thermocy-cling (5°C–55°C/500 cycles). They were subjected to shear bond strength test in a universal testing machine at a crosshead speed of 1.0 mm/minute. The data were compared using a two-way ANOVA, followed by Bonferroni test at Padhesives exhibited statistically similar bond strengths to ground and unground enamel except for the etch-and-rinse adhesive that showed significantly higher bond strengths than the self-etch adhesives (Penamel for any of the adhesives tested (P=.17). Conclusion: Similar bond strengths to ground and unground enamel were achieved with one-step self-etch adhesives and their predecessors. Enamel preparation did not influence the bonding performance of the adhesives tested. PMID:22904656

  1. Shear bond strength of one-step self-etch adhesives to enamel: effect of acid pretreatment.

    Science.gov (United States)

    Poggio, Claudio; Scribante, Andrea; Della Zoppa, Federica; Colombo, Marco; Beltrami, Riccardo; Chiesa, Marco

    2014-02-01

    The purposes of this study were to evaluate the effect of surface pretreatment with phosphoric acid on the enamel bond strength of four-one-step self-etch adhesives with different pH values. One hundred bovine permanent mandibular incisors were used. The materials used in this study included four-one-step self-etch adhesives with different pH values: Adper(™) Easy Bond Self-Etch Adhesive (ph = 0,8-1), Futurabond NR (ph = 1,4), G-aenial Bond (ph = 1,5), Clearfil(3) S Bond (ph = 2,7). One two-step self-etch adhesive (Clearfil SE Bond/ph = 0,8-1) was used as control. The teeth were assigned into two subgroups according to bonding procedure. In the first subgroup (n = 50), no pretreatment agent was applied. In the second subgroup (n = 50), etching was performed using 37% phosphoric acid for 30 s. After adhesive systems application, a nanohybrid composite resin was inserted into the enamel surface. The specimens were placed in a universal testing machine (Model 3343, Instron Corp., Canton, Mass., USA). After the testing procedure, the fractured surfaces were examined with an optical microscope at a magnification of 10× to determine failure modes. The adhesive remnant index (ARI) was used to assess the amount of adhesive left on the enamel surface. Descriptive statistics of the shear bond strength and frequency distribution of ARI scores were calculated. Enamel pretreatment with phosphoric acid significantly increased bond strength values of all the adhesives tested. No significant differences in bond strength were detected among the four different one-step self-etch adhesives with different pH. Two-step self-etch adhesive showed the highest bond strength. © 2013 John Wiley & Sons A/S.

  2. Characterization of relative abundance of lactic acid bacteria species in French organic sourdough by cultural, qPCR and MiSeq high-throughput sequencing methods.

    Science.gov (United States)

    Michel, Elisa; Monfort, Clarisse; Deffrasnes, Marion; Guezenec, Stéphane; Lhomme, Emilie; Barret, Matthieu; Sicard, Delphine; Dousset, Xavier; Onno, Bernard

    2016-12-19

    In order to contribute to the description of sourdough LAB composition, MiSeq sequencing and qPCR methods were performed in association with cultural methods. A panel of 16 French organic bakers and farmer-bakers were selected for this work. The lactic acid bacteria (LAB) diversity of their organic sourdoughs was investigated quantitatively and qualitatively combining (i) Lactobacillus sanfranciscensis-specific qPCR, (ii) global sequencing with MiSeq Illumina technology and (iii) molecular isolates identification. In addition, LAB and yeast enumeration, pH, Total Titratable Acidity, organic acids and bread specific volume were analyzed. Microbial and physico-chemical data were statistically treated by Principal Component Analysis (PCA) and Hierarchical Ascendant Classification (HAC). Total yeast counts were 6 log 10 to 7.6 log 10 CFU/g while LAB counts varied from 7.2 log 10 to 9.6 log 10 CFU/g. Values obtained by L. sanfranciscensis-specific qPCR were estimated between 7.2 and 10.3 log 10 CFU/g, except for one sample at 4.4 log 10 CFU/g. HAC and PCA clustered the sixteen sourdoughs into three classes described by their variables but without links to bakers' practices. L. sanfranciscensis was the dominant species in 13 of the 16 sourdoughs analyzed by Next Generation Sequencing (NGS), by the culture dependent method this species was dominant only in only 10 samples. Based on isolates identification, LAB diversity was higher for 7 sourdoughs with the recovery of L. curvatus, L. brevis, L. heilongjiangensis, L. xiangfangensis, L. koreensis, L. pontis, Weissella sp. and Pediococcus pentosaceus, as the most representative species. L. koreensis, L. heilongjiangensis and L. xiangfangensis were identified in traditional Asian food and here for the first time as dominant in organic sourdough. This study highlighted that L. sanfranciscensis was not the major species in 6/16 sourdough samples and that a relatively high LAB diversity can be observed in French organic

  3. One-Step Generation of Multi-Qubit GHZ and W States in Superconducting Transmon Qubit System

    International Nuclear Information System (INIS)

    Gao Guilong; Huang Shousheng; Wang Mingfeng; Jiang Nianquan; Cai Genchang

    2012-01-01

    We propose a one-step method to prepare multi-qubit GHZ and W states with transmon qubits capacitively coupled to a superconducting transmission line resonator (TLR). Compared with the scheme firstly introduced by Wang et al. [Phys. Rev. B 81 (2010) 104524], our schemes have longer dephasing time and much shorter operation time because the transmon qubits we used are not only more robust to the decoherence and the unavoidable parameter variations, but also have much stronger coupling constant with TLR. Based on the favourable properties of transmons and TLR, our method is more feasible in experiment. (general)

  4. One-Step UV-Induced Synthesis of Polypyrrole/Ag Nanocomposites at the Water/Ionic Liquid Interface

    Science.gov (United States)

    Wei, Yuyan; Li, Liang; Yang, Xiaoming; Pan, Guoliang; Yan, Guoping; Yu, Xianghua

    2010-02-01

    Polpyrrole (PPy)/Ag nanocomposites were successfully synthesized at the interface of water and ionic liquid by one-step UV-induced polymerization. Highly dispersed PPy/Ag nanoparticles were obtained by controlling the experimental conditions. The results of Fourier-transform infrared spectroscopy, X-ray diffraction, transmission electron microscopy and X-ray photoelectron spectroscopy revealed that the UV-induced interface polymerization leaded to the formation of PPy incorporating silver nanoparticles. It was also found that the electrical conductivity of PPy/Ag nanocomposite was about 100 times higher than that of pure PPy.

  5. One-Step UV-Induced Synthesis of Polypyrrole/Ag Nanocomposites at the Water/Ionic Liquid Interface

    Directory of Open Access Journals (Sweden)

    Yang Xiaoming

    2009-01-01

    Full Text Available Abstract Polpyrrole (PPy/Ag nanocomposites were successfully synthesized at the interface of water and ionic liquid by one-step UV-induced polymerization. Highly dispersed PPy/Ag nanoparticles were obtained by controlling the experimental conditions. The results of Fourier-transform infrared spectroscopy, X-ray diffraction, transmission electron microscopy and X-ray photoelectron spectroscopy revealed that the UV-induced interface polymerization leaded to the formation of PPy incorporating silver nanoparticles. It was also found that the electrical conductivity of PPy/Ag nanocomposite was about 100 times higher than that of pure PPy.

  6. One-step hydrothermal synthesis of hexangular starfruit-like vanadium oxide for high power aqueous supercapacitors

    Science.gov (United States)

    Shao, Jie; Li, Xinyong; Qu, Qunting; Zheng, Honghe

    2012-12-01

    Homogenous hexangular starfruit-like vanadium oxide was prepared for the first time by a one-step hydrothermal method. The assembly process of hexangular starfruit-like structure was observed from TEM images. The electrochemical performance of starfruit-like vanadium oxide was examined by cyclic voltammetry and galvanostatic charge/discharge. The obtained starfruit-like vanadium oxide exhibits a high power capability (19 Wh kg-1 at the specific power of 3.4 kW kg-1) and good cycling stability for supercapacitors application.

  7. Fabrication of an all-polymer electrochemical sensor by using a one-step hot embossing procedure

    DEFF Research Database (Denmark)

    Kafka, Jan Robert; Larsen, Niels Bent; Skaarup, Steen

    2010-01-01

    We present a fast one-step hot embossing procedure for fabricating an all-polymer electrochemical sen¬sor based on a thin, conductive film of poly(3,4-ethylenedioxythiophene) (PEDOT), a few 100s of nano¬meters in thickness, polymerised on top of a non-conductive TOPAS® (Cyclic Olefin Copolymer) car...... of electrodes was achieved by spatial separation (Fig. 1). Functionality of the electrochemical system was shown by amperometric detection of physiologically relevant glucose concentrations (0–10 mM)....

  8. One-step flame synthesis of an active Pt/TiO2 catalyst for SO2 oxidation

    DEFF Research Database (Denmark)

    Johannessen, Tue; Koutsopoulos, Sotiris

    2002-01-01

    Flame synthesis as a route for production of composite metal oxides has been employed for the one-step synthesis of a supported noble metal catalyst, i.e. a Pt/TiO2 catalyst, by simultaneous combustion of Ti-isopropoxide and platinum acetylacetonate in a quench-cooled flame reactor. The average...... size of the platinum particles supported on aggregated nano-particles of TiO2 is approximately 2 nm. The high SO2-oxidation activity of the catalyst proves that platinum is not hidden in the titania matrix. The flame-produced catalyst showed catalytic activity similar to samples prepared by wet...

  9. Rapid Synthesis of Lead Oxide Nanorods by One-step Solid-state Chemical Reaction at Room Temperature

    Institute of Scientific and Technical Information of China (English)

    CAO, Ya-Li(曹亚丽); JIA, Dian-Zeng(贾殿赠); LIU, Lang(刘浪); LUO, Jian-Min(骆建敏)

    2004-01-01

    A simple and facile method was reported to synthesize lead oxide nanorods. Nanorods of lead oxide were obtained directly from grinding solid metal salt and sodium hydroxide in agate mortar with the assistance of a suitable nonionic surfactant in only one step, which is different from the result of hydroxide in solution. The product has been characterized by XRD, TEM and SEM. The formation mechanism of rod-like morphology is discussed and the surfactant plays an important soft-template role in modifying the interface of solid-state reaction and according process of rod-formation.

  10. One-Step Nucleic Acid Amplification in Breast Cancer Sentinel Lymph Node: A Single Institutional Experience and a Short Review

    OpenAIRE

    Brambilla, Tatiana; Fiamengo, Barbara; Tinterri, Corrado; Testori, Alberto; Grassi, Massimo Maria; Sciarra, Amedeo; Abbate, Tommaso; Gatzemeier, Wolfgang; Roncalli, Massimo; Di Tommaso, Luca

    2015-01-01

    Sentinel lymph node (SLN) examination is a standard in breast cancer patients, with several methods employed along its 20 years history, the last one represented by one-step nucleic acid amplification (OSNA). The latter is a intra-operative molecular assay searching for CK19 mRNA as a surrogate of metastatic cells. Our 3 years experience with OSNA (1122 patients) showed results overlapping those recorded in the same institution with a morphological evaluation (930 patients) of SLN. In detail,...

  11. One-step oxidation preparation of unfolded and good soluble graphene nanoribbons by longitudinal unzipping of carbon nanotubes

    Science.gov (United States)

    Hu, Xiaolin; Hu, Yizhen; Huang, Jindan; Zhou, Ning; Liu, Yuhan; Wei, Lin; Chen, Xin; Zhuang, Naifeng

    2018-04-01

    A simple one-step method to prepare graphene nanoribbon (GNR) is reported in this paper. Compared with water steam etching, the oxidation and co-etching of dilute sulfuric acid can result in the more complete longitudinal unzipping of carbon nanotube, although there is no other strong oxidant. As-prepared GNRs are more flat and have more oxygenated functional groups along the edge. Moreover, they can steadily disperse in a water system. These make them suitable as a carrier for supporting palladium (Pd) nanoparticles. The Pd/GNR composite exhibits a superior electrocatalytic activity for ethanol oxidation.

  12. One-Step Generation of Multiqubit Greenberger-Horne-Zeilinger States in a Driven Circuit QED System

    International Nuclear Information System (INIS)

    Huang Jinsong; Nie Wei; Wei Lianfu

    2011-01-01

    We propose an efficient scheme to generate multiqubit Greenberger-Horne-Zeilinger (GHZ) states by one-step quantum operation in a driven circuit quantum electrodynamics (QED) system. Our proposal is based on a unitary evolution exp[-iλS 2 x ], with S x being the collective spin operator in x direction and λ a controllable parameter, induced by driving the resonator. The quantum operation avoids resonator-field decay and may achieve the GHZ states with ideal success probability. The feasibility with the experimentally-demonstrated circuit QED system is also discussed. (general)

  13. A One-Step-Ahead Smoothing-Based Joint Ensemble Kalman Filter for State-Parameter Estimation of Hydrological Models

    KAUST Repository

    El Gharamti, Mohamad

    2015-11-26

    The ensemble Kalman filter (EnKF) recursively integrates field data into simulation models to obtain a better characterization of the model’s state and parameters. These are generally estimated following a state-parameters joint augmentation strategy. In this study, we introduce a new smoothing-based joint EnKF scheme, in which we introduce a one-step-ahead smoothing of the state before updating the parameters. Numerical experiments are performed with a two-dimensional synthetic subsurface contaminant transport model. The improved performance of the proposed joint EnKF scheme compared to the standard joint EnKF compensates for the modest increase in the computational cost.

  14. Simple, quick and cost-efficient: A universal RT-PCR and sequencing strategy for genomic characterisation of foot-and-mouth disease viruses.

    Science.gov (United States)

    Dill, V; Beer, M; Hoffmann, B

    2017-08-01

    Foot-and-mouth disease (FMD) is a major contributor to poverty and food insecurity in Africa and Asia, and it is one of the biggest threats to agriculture in highly developed countries. As FMD is extremely contagious, strategies for its prevention, early detection, and the immediate characterisation of outbreak strains are of great importance. The generation of whole-genome sequences enables phylogenetic characterisation, the epidemiological tracing of virus transmission pathways and is supportive in disease control strategies. This study describes the development and validation of a rapid, universal and cost-efficient RT-PCR system to generate genome sequences of FMDV, reaching from the IRES to the end of the open reading frame. The method was evaluated using twelve different virus strains covering all seven serotypes of FMDV. Additionally, samples from experimentally infected animals were tested to mimic diagnostic field samples. All primer pairs showed a robust amplification with a high sensitivity for all serotypes. In summary, the described assay is suitable for the generation of FMDV sequences from all serotypes to allow immediate phylogenetic analysis, detailed genotyping and molecular epidemiology. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

    Science.gov (United States)

    Gille, Johan J. P.; Floor, Karijn; Kerkhoven, Lianne; Ameziane, Najim; Joenje, Hans; de Winter, Johan P.

    2012-01-01

    Fanconi anemia (FA) is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD) and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening of FANCA, FANCB, FANCC, FANCE, FANCF, and FANCG and present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed. PMID:22778927

  16. Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

    Directory of Open Access Journals (Sweden)

    Johan J. P. Gille

    2012-01-01

    Full Text Available Fanconi anemia (FA is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening of FANCA, FANCB, FANCC, FANCE, FANCF, and FANCG and present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed.

  17. Whole Genome Sequencing and Multiplex qPCR Methods to Identify Campylobacter jejuni Encoding cst-II or cst-III Sialyltransferase

    Directory of Open Access Journals (Sweden)

    Jason M. Neal-McKinney

    2018-03-01

    Full Text Available Campylobacter jejuni causes more than 2 million cases of gastroenteritis annually in the United States, and is also linked to the autoimmune sequelae Guillan–Barre syndrome (GBS. GBS often results in flaccid paralysis, as the myelin sheaths of nerve cells are degraded by the adaptive immune response. Certain strains of C. jejuni modify their lipooligosaccharide (LOS with the addition of neuraminic acid, resulting in LOS moieties that are structurally similar to gangliosides present on nerve cells. This can trigger GBS in a susceptible host, as antibodies generated against C. jejuni can cross-react with gangliosides, leading to demyelination of nerves and a loss of signal transduction. The goal of this study was to develop a quantitative PCR (qPCR method and use whole genome sequencing data to detect the Campylobactersialyltransferase (cst genes responsible for the addition of neuraminic acid to LOS. The qPCR method was used to screen a library of 89 C. jejuni field samples collected by the Food and Drug Administration Pacific Northwest Lab (PNL as well as clinical isolates transferred to PNL. In silico analysis was used to screen 827 C. jejuni genomes in the FDA GenomeTrakr SRA database. The results indicate that a majority of C. jejuni strains could produce LOS with ganglioside mimicry, as 43.8% of PNL isolates and 46.9% of the GenomeTrakr isolates lacked the cst genes. The methods described in this study can be used by public health laboratories to rapidly determine whether a C. jejuni isolate has the potential to induce GBS. Based on these results, a majority of C. jejuni in the PNL collection and submitted to GenomeTrakr have the potential to produce LOS that mimics human gangliosides.

  18. Detection of a putative novel adenovirus by PCR amplification, sequencing and phylogenetic characterisation of two gene fragments from formalin-fixed paraffin-embedded tissues of a cat diagnosed with disseminated adenovirus disease.

    Science.gov (United States)

    Lakatos, Béla; Hornyák, Ákos; Demeter, Zoltán; Forgách, Petra; Kennedy, Frances; Rusvai, Miklós

    2017-12-01

    Adenoviral nucleic acid was detected by polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissue samples of a cat that had suffered from disseminated adenovirus infection. The identity of the amplified products from the hexon and DNA-dependent DNA polymerase genes was confirmed by DNA sequencing. The sequences were clearly distinguishable from corresponding hexon and polymerase sequences of other mastadenoviruses, including human adenoviruses. These results suggest the possible existence of a distinct feline adenovirus.

  19. Correlation, temperature and disorder: Recent developments in the one-step description of angle-resolved photoemission

    Science.gov (United States)

    Braun, Jürgen; Minár, Ján; Ebert, Hubert

    2018-04-01

    Various apparative developments extended the potential of angle-resolved photoemission spectroscopy tremendously during the last two decades. Modern experimental arrangements consisting of new photon sources, analyzers and detectors supply not only extremely high angle and energy resolution but also spin resolution. This provides an adequate platform to study in detail new materials like low-dimensional magnetic structures, Rashba systems, topological insulator materials or high TC superconductors. The interest in such systems has grown enormously not only because of their technological relevance but even more because of exciting new physics. Furthermore, the use of photon energies from few eV up to several keV makes this experimental technique a rather unique tool to investigate the electronic properties of solids and surfaces. The following article reviews the corresponding recent theoretical developments in the field of angle-resolved photoemission with a special emphasis on correlation effects, temperature and relativistic aspects. The most successful theoretical approach to deal with angle-resolved photoemission is the so-called spectral function or one-step formulation of the photoemission process. Nowadays, the one-step model allows for photocurrent calculations for photon energies ranging from a few eV to more than 10 keV, to deal with arbitrarily ordered and disordered systems, to account for finite temperatures, and considering in addition strong correlation effects within the dynamical mean-field theory or similar advanced approaches.

  20. Biomass-based magnetic fluorescent nanoparticles: One-step scalable synthesis, application as drug carriers and mechanism study.

    Science.gov (United States)

    Li, Lei; Wang, Feijun; Shao, Ziqiang

    2018-03-15

    A biomass-based magnetic fluorescent nanoparticle (MFNPs) was successively in situ synthesized via a one-step high-gravity approach, which constructed by a magnetic core of Fe 3 O 4 nanoparticles, the fluorescent marker of carbon dots (CDs), and shells of chitosan (CS). The obtained MFNPs had a 10 nm average diameter and narrow particle size distribution, low cytotoxicity, superior fluorescent emission and superparamagnetic properties. The encapsulating and release 5-fluorouracil experiments confirmed that the introduction of CS/CDs effectively improved the drug loading capacity. Mechanism and kinetic studies proved that: (i) the monolayer adsorption was the main sorption mode under the studied conditions; (ii) the whole adsorption process was controlled by intra-liquid diffusion mass transfer and governed by chemisorption; and (iii) the release process was controlled by Fickian diffusion. These results demonstrated this method to one-step continuously produce MFNPs and the construction of non-toxic nanostructure possessed great superiority in currently Nano-delivery systems, which would show high application value in targeted drug delivery, magnetic fluid hyperthermia treatment, magnetic resonance imaging (MRI), in vitro testing and relative research. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. One-Step Purification of Human Skeletal Muscle Myoblasts and Subsequent Expansion Using Laminin-Coated Surface.

    Science.gov (United States)

    Chowdhury, Shiplu Roy; binti Ismail, Annis; Chee, Sia Chye; bin Laupa, Mohd Suffian; binti Jaffri, Fadhlun; Saberi, Salfarina Ezrina Mohmad; Idrus, Ruszymah Bt Hj

    2015-11-01

    Skeletal myoblasts have been extensively used to study muscle growth and differentiation, and were recently tested for their application as cell therapy and as a gene delivery system to treat muscle and nonmuscle diseases. However, contamination of fibroblasts in isolated cells from skeletal muscle is one of the long-standing problems for routine expansion. This study aimed to establish a simple one-step process to purify myoblasts and maintain their purity during expansion. Mixed cells were preplated serially on laminin- and collagen type I-coated surfaces in a different array for 5, 10, and 15 min. Immunocytochemical staining with antibodies specific to myoblasts was performed to evaluate myoblast attachment efficiency, purity, and yield. It was found that laminin-coated surface favors the attachment of myoblasts. Highest myoblast purity of 78.9% ± 6.8% was achieved by 5 min of preplating only on the laminin-coated surface with a yield of 56.9% ± 3.3%. Primary cells, isolated from skeletal muscle (n = 4), confirm the enhancement of purity through preplating on laminin-coated surface for 5 min. Subsequent expansion after preplating enhanced myoblast purity due to an increase in myoblast growth than fibroblasts. Myoblast purity of ∼ 98% was achieved when another preplating was performed during passaging. In conclusion, myoblasts can be purified and efficiently expanded in one step by preplating on laminin-coated surface, which is a simple and robust technique.

  2. A one-step bioprocess for production of high-content fructo-oligosaccharides from inulin by yeast.

    Science.gov (United States)

    Wang, Da; Li, Fu-Li; Wang, Shi-An

    2016-10-20

    Commercial fructo-oligosaccharides (FOS) are predominantly produced from sucrose by transfructosylation process that presents a maximum theoretical yield below 0.60gFOSgSucrose(-1). To obtain high-content FOS, costly purification is generally employed. Additionally, high-content FOS can be produced from inulin by using endo-inulinases. However, commercial endo-inulinases have not been extensively used in scale-up production of FOS. In the present study, a one-step bioprocess that integrated endo-inulinase production, FOS fermentation, and non-FOS sugars removal into one reactor was proposed to produce high-content FOS from inulin. The bioprocess was implemented by a recombinant yeast strain JZHΔS-TSC, in which a heterologous endo-inulinase gene was expressed and the inherent invertase gene SUC2 was disrupted. FOS fermentation at 40°C from 200g/L chicory inulin presented the maximun titer, yield, and productivity of 180.2±0.8g/L, 0.9gFOSgInulin(-1), and 7.51±0.03g/L/h, respectively. This study demonstrated that the one-step bioprocess was simple and highly efficient. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Natural printed silk substrate circuit fabricated via surface modification using one step thermal transfer and reduction graphene oxide

    Science.gov (United States)

    Cao, Jiliang; Huang, Zhan; Wang, Chaoxia

    2018-05-01

    Graphene conductive silk substrate is a preferred material because of its biocompatibility, flexibility and comfort. A flexible natural printed silk substrate circuit was fabricated by one step transfer of graphene oxide (GO) paste from transfer paper to the surface of silk fabric and reduction of the GO to reduced graphene oxide (RGO) using a simple hot press treatment. The GO paste was obtained through ultrasonic stirring exfoliation under low temperature, and presented excellent printing rheological properties at high concentration. The silk fabric was obtained a surface electric resistance as low as 12.15 KΩ cm-1, in the concentration of GO 50 g L-1 and hot press at 220 °C for 120 s. Though the whiteness and strength decreased with the increasing of hot press temperature and time slowly, the electric conductivity of RGO surface modification silk substrate improved obviously. The surface electric resistance of RGO/silk fabrics increased from 12.15 KΩ cm-1 to 18.05 KΩ cm-1, 28.54 KΩ cm-1 and 32.53 KΩ cm-1 after 10, 20 and 30 washing cycles, respectively. The results showed that the printed silk substrate circuit has excellent washability. This process requires no chemical reductant, and the reduction efficiency and reduction degree of GO is high. This time-effective and environmentally-friendly one step thermal transfer and reduction graphene oxide onto natural silk substrate method can be easily used to production of reduced graphene oxide (RGO) based flexible printed circuit.

  4. Effects of Sheet Resistance on mc-Si Selective Emitter Solar Cells Using Laser Opening and One-Step Diffusion

    Directory of Open Access Journals (Sweden)

    Sheng-Shih Wang

    2015-01-01

    Full Text Available In order to simplify process procedure and improve conversion efficiency (η, we present new steps of laser opening and one-step POCl3 diffusion to fabricate selective emitter (SE solar cells, in which heavily doped regions (HDR and lightly doped regions (LDR were formed simultaneously. For HDR, we divided six cells into two groups for POCl3 diffusion with sheet resistance (RS of 40 Ω/sq (for group A and 50 Ω/sq (for group B. The dry oxidation duration at a temperature of 850°C was 18, 25, and 35 min for the 3 different cells in each group. This created six SE samples with different RS pairings for the HDR and LDR. The optimal cell (sample SE2 with RS values of 40/81 Ω/Sq in HDR/LDR showed the best η of 16.20%, open circuit voltage (VOC of 612.52 mV, and fill factor (FF of 75.83%. The improvement ratios are 1.57% for η and 14.32% for external quantum efficiency (EQE as compared with those of the two-step diffusion process of our previous study. Moreover, the one-step laser opening process and omitting the step of removing the damage caused by laser ablation especially reduce chemistry pollution, thus showing ecofriendly process for use in industrial-scale production.

  5. One-step green synthesis of cuprous oxide crystals with truncated octahedra shapes via a high pressure flux approach

    International Nuclear Information System (INIS)

    Li Benxian; Wang Xiaofeng; Xia Dandan; Chu Qingxin; Liu Xiaoyang; Lu Fengguo; Zhao Xudong

    2011-01-01

    Cuprous oxide (Cu 2 O) was synthesized via reactions between cupric oxide (CuO) and copper metal (Cu) at a low temperature of 300 deg. C. This progress is green, environmentally friendly and energy efficient. Cu 2 O crystals with truncated octahedra morphology were grown under high pressure using sodium hydroxide (NaOH) and potassium hydroxide (KOH) with a molar ratio of 1:1 as a flux. The growth mechanism of Cu 2 O polyhedral microcrystals are proposed and discussed. - Graphical Abstract: The Cu 2 O crystals with truncated octahedral shape were one-step synthesized in high yield via high pressure flux method for the first time, which is green and environmentally friendly. The mechanisms of synthesis and crystal growth were discussed in this paper. Highlights: → Cuprous oxide was one-step green synthesized by high pressure flux method. → The approach was based on the reverse dismutation reactions between cupric oxide and copper metal. → This progress is green, environmentally friendly and energy efficient. → The synthesized Cu2O crystals were of truncated octahedra morphology.

  6. One-step synthesis of graphene-Au nanoparticle hybrid materials from metal salt-loaded micelles

    International Nuclear Information System (INIS)

    Liu, X; Zhang, X W; Meng, J H; Wang, H L; Yin, Z G; Wu, J L; Gao, H L

    2014-01-01

    In this study, we present a facile one-step method to synthesize graphene-Au nanoparticle (NP) hybrid materials by using HAuCl 4 -loaded poly(styrene)-block-poly(2-vinylpyridine) (PS-P2VP) micelles as solid carbon sources. N-doped graphene with controllable thickness can be grown from PS-P2VP micelles covered by a Ni capping layer by an annealing process; simultaneously, the HAuCl 4 in the micelles were reduced into Au NPs under a reductive atmosphere to form Au NPs on graphene. The decoration of Au NPs leads to an obviously enhanced electrical conductivity and a slightly increased work function of graphene due to the electron transfer effect. The graphene-Au NP hybrid materials also exhibit a localized surface plasmon resonance feature of Au NPs. This work provides a novel and accessible route for the one-step synthesis of graphene-Au NP hybrid materials with high quality, which might be useful for future applications in optoelectronic devices. (paper)

  7. Optimization of One-Step In Situ Transesterification Method for Accurate Quantification of EPA in Nannochloropsis gaditana

    Directory of Open Access Journals (Sweden)

    Yuting Tang

    2016-11-01

    Full Text Available Microalgae are a valuable source of lipid feedstocks for biodiesel and valuable omega-3 fatty acids. Nannochloropsis gaditana has emerged as a promising producer of eicosapentaenoic acid (EPA due to its fast growth rate and high EPA content. In the present study, the fatty acid profile of Nannochloropsis gaditana was found to be naturally high in EPA and devoid of docosahexaenoic acid (DHA, thereby providing an opportunity to maximize the efficacy of EPA production. Using an optimized one-step in situ transesterification method (methanol:biomass = 90 mL/g; HCl 5% by vol.; 70 °C; 1.5 h, the maximum fatty acid methyl ester (FAME yield of Nannochloropsis gaditana cultivated under rich condition was quantified as 10.04% ± 0.08% by weight with EPA-yields as high as 4.02% ± 0.17% based on dry biomass. The total FAME and EPA yields were 1.58- and 1.23-fold higher separately than that obtained using conventional two-step method (solvent system: methanol and chloroform. This one-step in situ method provides a fast and simple method to measure fatty acid methyl ester (FAME yields and could serve as a promising method to generate eicosapentaenoic acid methyl ester from microalgae.

  8. One-step method for the fabrication of superhydrophobic surface on magnesium alloy and its corrosion protection, antifouling performance

    International Nuclear Information System (INIS)

    Zhao, Lin; Liu, Qi; Gao, Rui; Wang, Jun; Yang, Wanlu; Liu, Lianhe

    2014-01-01

    Highlights: •The myristic acid iron superhydrophobic surface was formatted on AZ31. •Two procedures to build a super-hydrophobic were simplified to one step. •The superhydrophobic surface shows good anticorrosion, antifouling properties. •We report a new approach for the superhydrophobic surface protection on AZ31. -- Abstract: Inspired by the lotus leaf, various methods to fabricate artificial superhydrophobic surfaces have been developed. Our purpose is to create a simple, one-step and environment-friendly method to construct a superhydrophobic surface on a magnesium alloy substrate. The substrate was immersed in a solution containing ferric chloride (FeCl 3 ·6H 2 O), deionized water, tetradecanoic acid (CH 3 (CH 2 ) 12 COOH) and ethanol. Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and Fourier transformed infrared (FT-IR) were employed to characterize the substrate surface. The obtained surface showed a micron rough structure, a high contact angle (CA) of 165° ± 2° and desirable corrosion protection and antifouling properties

  9. Synthesis, nanostructure and magnetic properties of FeCo-reduced graphene oxide composite films by one-step electrodeposition

    International Nuclear Information System (INIS)

    Cao, Derang; Li, Hao; Wang, Zhenkun; Wei, Jinwu; Wang, Jianbo; Liu, Qingfang

    2015-01-01

    FeCo-reduced graphene oxide (FeCo-RGO) composite film was fabricated on indium tin oxide substrate using one-step electrodeposition method. Raman spectroscopy and field emission scanning electron microscope results show that the reduced graphene oxide is coprecipitated with the FeCo film. The energy-dispersive spectrometer results demonstrate that the atomic ratio of Fe/Co in FeCo-RGO composite film is larger than that of the FeCo film under the same fabrication condition. As a result, the FeCo-RGO composite film exhibits good soft magnetic properties and high-frequency properties as well as the FeCo film. The magnetic anisotropy field and saturation magnetization of FeCo-RGO composite film are increased when compared with FeCo film. Furthermore, the ferromagnetic resonance frequency is improved from 2.15 GHz for the FeCo film to 3.9 GHz for the FeCo-RGO composite film. - Highlights: • FeCo-reduced graphene oxide composite film was fabricated on indium tin oxide substrate. • One step electrodeposition method was used. • Good soft magnetic properties were exhibited by the composite films. • Increase of resonance frequency from 2.15 GHz for FeCo film to 3.9 GHz for composite film

  10. Facile one-step synthesis and photoluminescence properties of Ag–ZnO core–shell structure

    International Nuclear Information System (INIS)

    Zhai, HongJu; Wang, LiJing; Han, DongLai; Wang, Huan; Wang, Jian; Liu, XiaoYan; Lin, Xue; Li, XiuYan; Gao, Ming; Yang, JingHai

    2014-01-01

    Graphical abstract: The PL of the Ag–ZnO core-shell nanostructure showed obvious increase of UV emission and slight decrease of visible light emission compared to that of the pure ZnO. With the calcination temperature increasing from 300 to 600 °C, the primary peak located at 380 nm became stronger and sharper, indicating that the increasing calcination temperature made the samples crystallize better. - Highlights: • Ag-ZnO core-shell structure was obtained via a simple one-step solvothermal process. • The approach was simple, mild, low cost, reproducible and easy-to-handle. • The obvious enhancement of UV luminescent has been observed. • Effects of the calcining temperature to luminescence were investigated in detail. - Abstract: Ag–ZnO core–shell structures were gained via one-step solvothermal process. The products were characterized by means of X-ray diffraction (XRD), transmission electron microscopy (TEM), Raman spectroscopy, photoluminescence (PL) and UV–vis spectroscopy, respectively. It was shown that the properties were greatly changed compared to pure ZnO from the PL and Raman spectra, which indicated the strong interfacial interaction between ZnO and Ag. The work provides a feasible method to synthesize Ag–ZnO core–shell structure photocatalyst, which is promising in the further practical application of ZnO-based photocatalytic materials

  11. Effect of moisture and drying time on the bond strength of the one-step self-etching adhesive system

    Directory of Open Access Journals (Sweden)

    Yoon Lee

    2012-08-01

    Full Text Available Objectives To investigate the effect of dentin moisture degree and air-drying time on dentin-bond strength of two different one-step self-etching adhesive systems. Materials and Methods Twenty-four human third molars were used for microtensile bond strength testing of G-Bond and Clearfil S3 Bond. The dentin surface was either blot-dried or air-dried before applying these adhesive agents. After application of the adhesive agent, three different air drying times were evaluated: 1, 5, and 10 sec. Composite resin was build up to 4 mm thickness and light cured for 40 sec with 2 separate layers. Then the tooth was sectioned and trimmed to measure the microtensile bond strength using a universal testing machine. The measured bond strengths were analyzed with three-way ANOVA and regression analysis was done (p = 0.05. Results All three factors, materials, dentin wetness and air drying time, showed significant effect on the microtensile bond strength. Clearfil S3 Bond, dry dentin surface and 10 sec air drying time showed higher bond strength. Conclusions Within the limitation of this experiment, air drying time after the application of the one-step self-etching adhesive agent was the most significant factor affecting the bond strength, followed by the material difference and dentin moisture before applying the adhesive agent.

  12. One-step synthesis and characterization of polyaniline nanofiber/silver nanoparticle composite networks as antibacterial agents.

    Science.gov (United States)

    Poyraz, Selcuk; Cerkez, Idris; Huang, Tung Shi; Liu, Zhen; Kang, Litao; Luo, Jujie; Zhang, Xinyu

    2014-11-26

    Through a facile and effective seeding polymerization reaction via a one-step redox/complexation process, which took place in aqueous medium at ambient temperature, silver nanoparticles (Ag NPs) embedded polyaniline nanofiber (PANI NF) networks were synthesized as antibacterial agents. During the reaction, not only NF morphology formation of the resulting conducting polymers (CPs) but also amplification of the aqueous silver nitrate (AgNO3) solutions' oxidative potentials were managed by vanadium pentoxide (V2O5) sol-gel nanofibers, which acted as well-known nanofibrous seeding agents and the auxiliary oxidative agent at the same time. The PANI/Ag nanocomposites were proven to exhibit excellent antibacterial property against both Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Antibacterial property performance and average life span of the nanocomposite network were optimized through the homogeneous distribution/embedment of Ag NPs within one-dimensional (1-D) PANI NF matrix. The antibacterial efficacy tests and nanocomposite material characterization results further indicated that the sole components of PANI/Ag have a synergistic effect to each other in terms of antibacterial property. Thus, this well-known catalytic seeding approach via a one-step oxidative polymerization reaction can be considered as a general methodology and a substantial fabrication tool to synthesize Ag NP decorated nanofibrillar PANI networks as advanced antibacterial agents.

  13. Fabrication and characterization of metal-packaged fiber Bragg grating sensor by one-step ultrasonic welding

    Science.gov (United States)

    Zhang, Yumin; Zhu, Lianqing; Luo, Fei; Dong, Mingli; Ding, Xiangdong; He, Wei

    2016-06-01

    A metallic packaging technique of fiber Bragg grating (FBG) sensors is developed for measurement of strain and temperature, and it can be simply achieved via one-step ultrasonic welding. The average strain transfer rate of the metal-packaged sensor is theoretically evaluated by a proposed model aiming at surface-bonded metallic packaging FBG. According to analytical results, the metallic packaging shows higher average strain transfer rate compared with traditional adhesive packaging under the same packaging conditions. Strain tests are performed on an elaborate uniform strength beam for both tensile and compressive strains; strain sensitivities of approximately 1.16 and 1.30 pm/μɛ are obtained for the tensile and compressive situations, respectively. Temperature rising and cooling tests are also executed from 50°C to 200°C, and the sensitivity of temperature is 36.59 pm/°C. All the measurements of strain and temperature exhibit good linearity and stability. These results demonstrate that the metal-packaged sensors can be successfully fabricated by one-step welding technique and provide great promise for long-term and high-precision structural health monitoring.

  14. One-step green synthesis of non-hazardous dicarboxyl cellulose flocculant and its flocculation activity evaluation

    International Nuclear Information System (INIS)

    Zhu, Hangcheng; Zhang, Yong; Yang, Xiaogang; Liu, Hongyi; Shao, Lan; Zhang, Xiumei; Yao, Juming

    2015-01-01

    The waste management of used flocculants is a thorny issue in the field of wastewater treatment. To natural cellulose based flocculants, utilization of hazardous cellulose solvent and simplification of synthetic procedure are the two urgent problems needing to be further improved. In this work, a series of natural dicarboxyl cellulose flocculants (DCCs) were one-step synthesized via Schiff-base route. The cellulose solvent (NaOH/Urea solution) was utilized during the synthesis process. The full-biodegradable flocculants avoid causing secondary pollution to environment. The chemical structure and solution property of the DCC products were characterized by FT-IR, 1 H NMR, 13 C NMR, TGA, FESEM, charge density and ζ-potential. Kaolin suspension and effluent from paper mill were selected to evaluate the flocculation activity of the DCCs. Their flocculation performance was compared with that of commercial cationic polyacrylamide and poly aluminium chloride flocculants. The positive results showed that the NaOH/Urea solvent effectively promoted the dialdehyde cellulose (DAC) conversion to DCC in the one-step synthesis reaction. The DCCs with the carboxylate content more than 1 mmol/g exhibited steady flocculation performance to kaolin suspension in the broad pH range from 4 to 10. Its flocculation capacity to the effluent from paper mill also showed excellent

  15. Functionalization of monolithic and porous three-dimensional graphene by one-step chitosan electrodeposition for enzymatic biosensor.

    Science.gov (United States)

    Liu, Jiyang; Wang, Xiaohui; Wang, Tianshu; Li, Dan; Xi, Fengna; Wang, Jin; Wang, Erkang

    2014-11-26

    Biological modification of monolithic and porous 3D graphene is of great significance for extending its application in fabricating highly sensitive biosensors. The present work reports on the first biofunctionalization of monolithic and freestanding 3D graphene foam for one-step preparation of reagentless enzymatic biosensors by controllable chitosan (CS) electrodeposition technology. Using a homogeneous three-component electrodeposition solution containing a ferrocene (Fc) grafted CS hybrid (Fc-CS), glucose oxidase (GOD), and single-walled carbon nanotubes (SWNTs), a homogeneous biocomposite film of Fc-CS/SWNTs/GOD was immobilized on the surface of 3D graphene foam by one-step electrodeposition. The Fc groups grafted on chitosan can be stably immobilized on the 3D graphene surface and keep their original electrochemical activity. The SWNTs doped into the Fc-CS matrix act as a nanowire to facilitate electron transfer and improve the conductivity of the biocomposite film. Combined with the extraordinary properties of 3D graphene foam including large active surface area, high conductivity, and fast mass transport dynamics, the 3D graphene based enzymatic biosensor achieved a large linear range (5.0 μM to 19.8 mM), a low detection limit (1.2 μM), and rapid response (reaching the 95% steady-state response within 8 s) for reagentless detection of glucose in the phosphate buffer solution.

  16. Succession of methanogenic archaea in rice straw incorporated into a Japanese rice field: estimation by PCR-DGGE and sequence analyses

    Directory of Open Access Journals (Sweden)

    Atsuo Sugano

    2005-01-01

    Full Text Available The succession and phylogenetic profiles of methanogenic archaeal communities associated with rice straw decomposition in rice-field soil were studied by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE analysis followed by 16S rDNA sequencing. Nylon bags containing either leaf sheaths or blades were buried in the plowed layer of a Japanese rice field under drained conditions during the off-crop season and under flooded conditions after transplanting. In addition, rice straw samples that had been buried in the rice field under drained conditions during the off-crop season were temporarily removed during spring plowing and then re-buried in the same rice field under flooded conditions at transplanting. Populations of methanogenic archaea were examined by amplification of the 16S rRNA genes in the DNA extracted from the rice straw samples. No PCR product was produced for samples of leaf sheath or blade prior to burial or after burial under drained conditions, indicating that the methanogen population was very small during decomposition of rice straw under oxic conditions. Many common bands were observed in rice straw samples of leaf sheath and blade during decomposition of rice straw under flooded conditions. Cluster analysis based on DGGE patterns divided methanogenic archaeal communities into two groups before and after the mid-season drainage. Sequence analysis of DGGE bands that were commonly present were closely related to Methanomicrobiales and Rice cluster I. Methanomicrobiales, Rice cluster I and Methanosarcinales were major members before the mid-season drainage, whereas the DGGE bands that characterized methanogenic archaeal communities after the mid-season drainage were closely related to Methanomicrobiales. These results indicate that mid-season drainage affected the methanogenic archaeal communities irrespective of their location on rice straw (sheath and blade and the previous history of decomposition

  17. Effect of air-blowing duration on the bond strength of current one-step adhesives to dentin.

    Science.gov (United States)

    Fu, Jiale; Saikaew, Pipop; Kawano, Shimpei; Carvalho, Ricardo M; Hannig, Matthias; Sano, Hidehiko; Selimovic, Denis

    2017-08-01

    To evaluate the influence of different air-blowing durations on the micro-tensile bond strength (μTBS) of five current one-step adhesive systems to dentin. One hundred and five caries-free human molars and five current one-step adhesive systems were used: ABU (All Bond Universal, Bisco, Inc.), CUB (CLEARFIL™ Universal Bond, Kuraray), GPB (G-Premio BOND, GC), OBA (OptiBond All-in-one, Kerr) and SBU (Scotchbond Universal, 3M ESPE). The adhesives were applied to 600 SiC paper-flat dentin surfaces according to each manufacturer's instructions and were air-dried with standard, oil-free air pressure of 0.25MPa for either 0s, 5s, 15s or 30s before light-curing. Bond strength to dentin was determined by using μTBS test after 24h of water storage. The fracture pattern on the dentin surface was analyzed by SEM. The resin-dentin interface of untested specimens was visualized by panoramic SEM image. Data from μTBS were analyzed using two-way ANOVA (adhesive vs. air-blowing time), and Games-Howell (a=0.05). Two-way ANOVA revealed a significant effect of materials (p=0.000) and air-blowing time (p=0.000) on bond strength to dentin. The interaction between factors was also significantly different (p=0.000). Maximum bond strength for each system were recorded, OBA/15s (76.34±19.15MPa), SBU/15s (75.18±12.83MPa), CUB/15s (68.23±16.36MPa), GPB/30s (55.82±12.99MPa) and ABU/15s (44.75±8.95MPa). The maximum bond strength of OBA and SUB were significantly higher than that of GPB and ABU (padhesive systems is material-dependent (p=0.000), and was influenced by air-blowing duration (p=0.000). For the current one-step adhesive systems, higher bond strengths could be achieved with prolonged air-blowing duration between 15-30s. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  18. 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

  19. A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches

    Science.gov (United States)

    Zheng, Xiasheng; Zhang, Peng; Liao, Baosheng; Li, Jing; Liu, Xingyun; Shi, Yuhua; Cheng, Jinle; Lai, Zhitian; Xu, Jiang; Chen, Shilin

    2017-01-01

    Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis), to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control. Highlight: We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis) with analytical chemistry approaches to achieve the authentication and quality connotation of the samples. PMID:28955365

  20. A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches

    Directory of Open Access Journals (Sweden)

    Xiasheng Zheng

    2017-09-01

    Full Text Available Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis, to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control.Highlight: We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis with analytical chemistry approaches to achieve the authentication and quality connotation of the samples.

  1. A Comprehensive Quality Evaluation System for Complex Herbal Medicine Using PacBio Sequencing, PCR-Denaturing Gradient Gel Electrophoresis, and Several Chemical Approaches.

    Science.gov (United States)

    Zheng, Xiasheng; Zhang, Peng; Liao, Baosheng; Li, Jing; Liu, Xingyun; Shi, Yuhua; Cheng, Jinle; Lai, Zhitian; Xu, Jiang; Chen, Shilin

    2017-01-01

    Herbal medicine is a major component of complementary and alternative medicine, contributing significantly to the health of many people and communities. Quality control of herbal medicine is crucial to ensure that it is safe and sound for use. Here, we investigated a comprehensive quality evaluation system for a classic herbal medicine, Danggui Buxue Formula, by applying genetic-based and analytical chemistry approaches to authenticate and evaluate the quality of its samples. For authenticity, we successfully applied two novel technologies, third-generation sequencing and PCR-DGGE (denaturing gradient gel electrophoresis), to analyze the ingredient composition of the tested samples. For quality evaluation, we used high performance liquid chromatography assays to determine the content of chemical markers to help estimate the dosage relationship between its two raw materials, plant roots of Huangqi and Danggui. A series of surveys were then conducted against several exogenous contaminations, aiming to further access the efficacy and safety of the samples. In conclusion, the quality evaluation system demonstrated here can potentially address the authenticity, quality, and safety of herbal medicines, thus providing novel insight for enhancing their overall quality control. Highlight : We established a comprehensive quality evaluation system for herbal medicine, by combining two genetic-based approaches third-generation sequencing and DGGE (denaturing gradient gel electrophoresis) with analytical chemistry approaches to achieve the authentication and quality connotation of the samples.

  2. Color-sensitive photoconductivity of nanostructured ZnO/dye hybrid films prepared by one-step electrodeposition

    Energy Technology Data Exchange (ETDEWEB)

    Oekermann, T. [Gifu University, Graduate School of Engineering, Yanagido 1-1, Gifu 501-1193 (Japan) and University of Hannover, Institute of Physical Chemistry and Electrochemistry, Callinstrasse 3-3A, 30167 Hannover (Germany)]. E-mail: torsten.oekermann@pci.uni-hannover.de; Yoshida, T. [Gifu University, Graduate School of Engineering, Yanagido 1-1, Gifu 501-1193 (Japan)]. E-mail: yoshida@apchem.gifu-u.ac.jp; Tada, H. [Institute for Molecular Science, Okazaki National Research Institutes, Myodaiji, Okazaki 444-8585 (Japan); Minoura, H. [Gifu University, Graduate School of Engineering, Yanagido 1-1, Gifu 501-1193 (Japan)

    2006-07-26

    Nanostructured ZnO/dye hybrid films prepared by one-step electrodeposition have been investigated in conductivity and photoconductivity measurements in view of applications in dye-sensitized solar cells (DSSC) and in optoelectronics. Highly porous ZnO/eosin Y films, which were obtained at potentials < - 0.9 V vs. SCE, were found to have a very high conductivity already in the dark, probably because of a higher n-doping, which is due to a higher concentration of Zn atoms in the film. On the other hand, less porous or non-porous films, which were obtained at more positive potentials and in which the dye molecules are located within the ZnO crystals, were found to show a much higher sensitivity to illumination with visible light in photoconductivity measurements due to a higher absolute photoconductivity and a lower conductivity in the dark.

  3. One-step synthesis of graphene/SnO2 nanocomposites and its application in electrochemical supercapacitors.

    Science.gov (United States)

    Li, Fenghua; Song, Jiangfeng; Yang, Huafeng; Gan, Shiyu; Zhang, Qixian; Han, Dongxue; Ivaska, Ari; Niu, Li

    2009-11-11

    A one-step method was developed to fabricate conductive graphene/SnO2 (GS) nanocomposites in acidic solution. Graphite oxides were reduced by SnCl2 to graphene sheets in the presence of HCl and urea. The reducing process was accompanied by generation of SnO2 nanoparticles. The structure and composition of GS nanocomposites were confirmed by means of transmission electron microscopy, x-ray photoelectron and Raman spectroscopy. Moreover, the ultracapacitor characteristics of GS nanocomposites were studied by cyclic voltammograms (CVs) and electrical impedance spectroscopy (EIS). The CVs of GS nanocomposites are nearly rectangular in shape and the specific capacitance degrades slightly as the voltage scan rate is increased. The EIS of GS nanocomposites presents a phase angle close to pi/2 at low frequency, indicating a good capacitive behavior. In addition, the GS nanocomposites could be promisingly applied in many fields such as nanoelectronics, ultracapacitors, sensors, nanocomposites, batteries and gas storage.

  4. One-step synthesis of Zn/ZnO hollow nanoparticles by the laser ablation in liquid technique

    International Nuclear Information System (INIS)

    Desarkar, H S; Kumbhakar, P; Mitra, A K

    2013-01-01

    Here, one-step synthesis of Zn/ZnO hollow nanoparticles along with solid nanoparticles is reported using the laser ablation in liquid (LAL) technique. Laser radiation of the 1064 nm wavelength is emitted from a Q-switched Nd:YAG laser and is incident on a solid zinc target kept in a water medium. The as-obtained hollow and solid particles are characterized by transmission electron microscopy (TEM) and UV–visible absorption spectroscopy. Hollow nanoparticles are produced by the laser generated bubbles produced in water. The surface of a hollow nanoparticle is assembled from smaller solid nanoparticles. A strong laser–particle interaction is also observed when laser ablation is carried out for a longer time duration. Photoluminescence (PL) emission measurements at room temperature show that all samples exhibit PL emission in the UV–visible region. A reduction in size and an increase in concentration of the synthesized nanoparticles is observed with increasing laser ablation time. (letter)

  5. Formation of a hydrophobic and corrosion resistant coating on magnesium alloy via a one-step hydrothermal method.

    Science.gov (United States)

    Zheng, Tianxu; Hu, Yaobo; Zhang, Yuxin; Pan, Fusheng

    2017-11-01

    A hydrophobic coating was fabricated on the surface of magnesium alloy using a simple one-step hydrothermal method with the use of environmentally friendly agent. Scanning electron microscopy, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and contact angle test were used to characterize the surfaces. Corrosion behavior in a 3.5wt.% NaCl solution was evaluated using OCP time curves test, potentiodynamic polarization test and EIS analysis. The findings show that the substrate is covered by the coating of magnesium hydroxide and magnesium stearate, reaching a contact angle of around 146°. Corrosion behavior show huge improvement, the progress with increase of treatment time could be related to the increased growth rate of coating. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. One step hydrothermal synthesis of 3D CoS2@MoS2-NG for high performance supercapacitors.

    Science.gov (United States)

    Meng, Qi; Chen, Yizhi; Zhu, Wenkun; Zhang, Ling; Yang, Xiaoyong; Duan, Tao

    2018-05-03

    A three-dimensional (3D) MoS 2 coated CoS 2 -nitrogen doped graphene (NG) (CoS 2 @MoS 2 -NG) hybrid has been synthesized by a one step hydrothermal method as supercapacitor (SC) electrode material for the first time. Such a composite consists of NG embedded with stacked CoS 2 @MoS 2 sheets. With a 3D skeleton, it prevents the agglomeration of CoS 2 @MoS 2 nanoparticles, resulting in sound conductivity, rich porous structures and a large surface area. The results indicate that CoS 2 @MoS 2 -NG has higher specific capacitance (198 F g -1 at 1 A g -1 ), better rate performance (with about 56.57% from 1 to 16 A g -1 ) and an improved cycle stability (with about 96.97% after 1000 cycles). It is an ideal candidate for SC electrode materials.

  7. Color-sensitive photoconductivity of nanostructured ZnO/dye hybrid films prepared by one-step electrodeposition

    International Nuclear Information System (INIS)

    Oekermann, T.; Yoshida, T.; Tada, H.; Minoura, H.

    2006-01-01

    Nanostructured ZnO/dye hybrid films prepared by one-step electrodeposition have been investigated in conductivity and photoconductivity measurements in view of applications in dye-sensitized solar cells (DSSC) and in optoelectronics. Highly porous ZnO/eosin Y films, which were obtained at potentials < - 0.9 V vs. SCE, were found to have a very high conductivity already in the dark, probably because of a higher n-doping, which is due to a higher concentration of Zn atoms in the film. On the other hand, less porous or non-porous films, which were obtained at more positive potentials and in which the dye molecules are located within the ZnO crystals, were found to show a much higher sensitivity to illumination with visible light in photoconductivity measurements due to a higher absolute photoconductivity and a lower conductivity in the dark

  8. The amphiphilic hydrophobin Vmh2 plays a key role in one step synthesis of hybrid protein-gold nanoparticles.

    Science.gov (United States)

    Politi, Jane; De Stefano, Luca; Longobardi, Sara; Giardina, Paola; Rea, Ilaria; Methivier, Christophe; Pradier, Claire-Marie; Casale, Sandra; Spadavecchia, Jolanda

    2015-12-01

    We report a simple and original method to synthesize gold nanoparticles in which a fungal protein, the hydrophobin Vmh2 from Pleurotus ostreatus and dicarboxylic acid-terminated polyethylene-glycol (PEG) has been used as additional components in a one step process, leading to hybrid protein-metal nanoparticles (NPs). The nanoparticles have been characterized by ultra-violet/visible, infrared and X-ray photoelectron spectroscopies, dynamic light scattering and also by electron microscopy imaging. The results of these analytical techniques highlight nanometric sized, stable, hybrid complexes of about 12 nm, with outer surface rich in functional chemical groups. Interaction with protein and antibodies has also been exploited. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Preparation of TiC/W core–shell structured powders by one-step activation and chemical reduction process

    International Nuclear Information System (INIS)

    Ding, Xiao-Yu; Luo, Lai-Ma; Huang, Li-Mei; Luo, Guang-Nan; Zhu, Xiao-Yong; Cheng, Ji-Gui; Wu, Yu-Cheng

    2015-01-01

    Highlights: • A novel wet chemical method was used to prepare TiC/W core–shell structure powders. • TiC nanoparticles were well-encapsulated by W shells. • TiC phase was present in the interior of tungsten grains. - Abstract: In the present study, one-step activation and chemical reduction process as a novel wet-chemical route was performed for the preparation of TiC/W core–shell structured ultra-fine powders. The XRD, FE-SEM, TEM and EDS results demonstrated that the as-synthesized powders are of high purity and uniform with a diameter of approximately 500 nm. It is also found that the TiC nanoparticles were well-encapsulated by W shells. Such a unique process suggests a new method for preparing X/W (X refers the water-insoluble nanoparticles) core–shell nanoparticles with different cores

  10. Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling.

    Science.gov (United States)

    Schumacher, Dominik; Lemke, Oliver; Helma, Jonas; Gerszonowicz, Lena; Waller, Verena; Stoschek, Tina; Durkin, Patrick M; Budisa, Nediljko; Leonhardt, Heinrich; Keller, Bettina G; Hackenberger, Christian P R

    2017-05-01

    The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking experiments and all-atom molecular dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.

  11. One-step synthesis of Pt-reduced graphene oxide composites based on high-energy radiation technique

    International Nuclear Information System (INIS)

    Liu, Xuqiang; Jiang, Shubin; Huang, Wei; Song, Hongtao

    2014-01-01

    In this paper, we introduce a novel 60 Co-ray-irradiation-based one-step synthesis method of Pt-reduced graphene oxide composites (Pt-RGO) in acid aqueous solution. The compositional distribution of the particles in the samples was characterized by transmission electron microscopy. The structure and composition of the nanocomposite has been determined with a scanning electron microscope (SEM) equipped with an energy dispersion X-ray (EDS) analyzer. Surface enhanced Raman scattering (SERS) of graphene deposited by the Pt nanoparticles were investigated with the 514.5 nm excitation. It was found that small-sized and highly-dispersed Pt nanoparticles could easily grow on the RGO surface under acidic conditions. In addition, the obtained homogeneous dispersions exhibit long-term stability, which will facilitate the production of homogeneous composites. (orig.)

  12. Blazed vector grating liquid crystal cells with photocrosslinkable polymeric alignment films fabricated by one-step polarizer rotation method

    Science.gov (United States)

    Kawai, Kotaro; Kuzuwata, Mitsuru; Sasaki, Tomoyuki; Noda, Kohei; Kawatsuki, Nobuhiro; Ono, Hiroshi

    2014-12-01

    Blazed vector grating liquid crystal (LC) cells, in which the directors of low-molar-mass LCs are antisymmetrically distributed, were fabricated by one-step exposure of an empty glass cell inner-coated with a photocrosslinkable polymer LC (PCLC) to UV light. By adopting a LC cell structure, twisted nematic (TN) and homogeneous (HOMO) alignments were obtained in the blazed vector grating LC cells. Moreover, the diffraction efficiency of the blazed vector grating LC cells was greatly improved by increasing the thickness of the device in comparison with that of a blazed vector grating with a thin film structure obtained in our previous study. In addition, the diffraction efficiency and polarization states of ±1st-order diffracted beams from the resultant blazed vector grating LC cells were controlled by designing a blazed pattern in the alignment films, and these diffraction properties were well explained on the basis of Jones calculus and the elastic continuum theory of nematic LCs.

  13. One Step Melanoma Surgery for Patient with Thick Primary Melanomas: "To Break the Rules, You Must First Master Them!"

    Directory of Open Access Journals (Sweden)

    Georgi Tchernev

    2018-02-01

    CONCLUSIONS: In this case remains unclear the following question: For what reason a preoperative high - frequent ultrasonography (HFUS is not recommended to be used as it will allow only one surgical excision with the elimination of a tumour with a safety field of 2cm in all directions? The enigma about the obstacles preventing such a rational optimisation of the current diagnostic and therapeutic algorithm in patients with melanomas remains unresolved. One step surgery for cutaneous melanoma is widely used in many countries although it continues to be considered as a matter of dispute for some experts. Once again, by a clinical case and the following analysis, we would like to focus the attention of the dermatosurgical community on this crucial and highly significant problem. Innovations are very often resulting from the simplicity of logic, which unfortunately is not always accepted appropriately.

  14. One-step sub-10 μm patterning of carbon-nanotube thin films for transparent conductor applications.

    Science.gov (United States)

    Fukaya, Norihiro; Kim, Dong Young; Kishimoto, Shigeru; Noda, Suguru; Ohno, Yutaka

    2014-04-22

    We propose a technique for one-step micropatterning of as-grown carbon-nanotube films on a plastic substrate with sub-10 μm resolution on the basis of the dry transfer process. By utilizing this technique, we demonstrated the novel high-performance flexible carbon-nanotube transparent conductive film with a microgrid structure, which enabled improvement of the performance over the trade-off between the sheet resistance and transmittance of a conventional uniform carbon-nanotube film. The sheet resistance was reduced by 46% at its maximum by adding the microgrid, leading to a value of 53 Ω/sq at a transmittance of 80%. We also demonstrated easy fabrication of multitouch projected capacitive sensors with 12 × 12 electrodes. The technique is quite promising for energy-saving production of transparent conductor devices with 100% material utilization.

  15. One-step spray-coating process for the fabrication of colorful superhydrophobic coatings with excellent corrosion resistance.

    Science.gov (United States)

    Li, Jian; Wu, Runni; Jing, Zhijiao; Yan, Long; Zha, Fei; Lei, Ziqiang

    2015-10-06

    A simple method was used to generate colorful hydrophobic stearate particles via chemical reactions between inorganic salts and sodium stearate. Colored self-cleaning superhydrophobic coatings were prepared through a facile one-step spray-coating process by spraying the stearate particle suspensions onto stainless steel substrates. Furthermore, the colorful superhydrophobic coating maintains excellent chemical stability under both harsh acidic and alkaline circumstances. After being immersed in a 3.5 wt % NaCl aqueous solution for 1 month, the as-prepared coatings remained superhydrophobic; however, they lost their self-cleaning property with a sliding angle of about 46 ± 3°. The corrosion behavior of the superhydrophobic coatings on the Al substrate was characterized by the polarization curve and electrochemical impedance spectroscopy (EIS). The electrochemical corrosion test results indicated that the superhydrophobic coatings possessed excellent corrosion resistance, which could supply efficient and long-term preservation for the bare Al substrate.

  16. Novel one-pot one-step synthesis of 2'-[(18)F]fluoroflumazenil (FFMZ) for benzodiazepine receptor imaging.

    Science.gov (United States)

    Yoon, Young Hyun; Jeong, Jae Min; Kim, Hyung Woo; Hong, Sung Hyun; Lee, Yun-Sang; Kil, Hee Sup; Chi, Dae Yoon; Lee, Dong Soo; Chung, June-Key; Lee, Myung Chul

    2003-07-01

    We describe the synthesis of 2'-[(18)F]fluoroflumazenil (FFMZ), which differs from the typically used [(18)F]fluoroethylflumazenil (FEFMZ) for benzodiazepine receptor imaging. For one-pot one-step labeling, the precursors, 2'-tosyloxyflumazenil (TFMZ) and 2'-mesyloxyflumazenil (MFMZ), were synthesized in three steps. The precursors were successfully labeled with no-carrier-added (18)F-fluoride which was activated by repeated azeotropic distillation with Kryptofix 2.2.2./potassium carbonate in MeCN. An automated system for labeling and purification of [(18)F]FFMZ was developed. Labeling efficiency and radiochemical purity of [(18)F]FFMZ after synthesis by the automated system were 68% and 98%, respectively. Specific binding of [(18)F]FFMZ to central benzodiazepine receptor of rats was demonstrated by phosphoimaging.

  17. Novel one-pot one-step synthesis of 2'-[18F]fluoroflumazenil (FFMZ) for benzodiazepine receptor imaging

    International Nuclear Information System (INIS)

    Young, Hyun Yoon; Jae, Min Jeong; Hyung, Woo Kim; Sung, Hyun Hong; Lee, Yun-Sang; Hee, Sup Kil; Dae, Yoon Chi; Dong, Soo Lee; Chung, June-Key; Myung, Chul Lee

    2003-01-01

    We describe the synthesis of 2'-[ 18 F]fluoroflumazenil (FFMZ), which differs from the typically used [ 18 F]fluoroethylflumazenil (FEFMZ) for benzodiazepine receptor imaging. For one-pot one-step labeling, the precursors, 2'-tosyloxyflumazenil (TFMZ) and 2'-mesyloxyflumazenil (MFMZ), were synthesized in three steps. The precursors were successfully labeled with no-carrier-added 18 F-fluoride which was activated by repeated azeotropic distillation with Kryptofix 2.2.2./potassium carbonate in MeCN. An automated system for labeling and purification of [ 18 F]FFMZ was developed. Labeling efficiency and radiochemical purity of [ 18 F]FFMZ after synthesis by the automated system were 68% and 98%, respectively. Specific binding of [ 18 F]FFMZ to central benzodiazepine receptor of rats was demonstrated by phosphoimaging

  18. Efficiency Improvement of Kalman Filter for GNSS/INS through One-Step Prediction of P Matrix

    Directory of Open Access Journals (Sweden)

    Qingli Li

    2015-01-01

    Full Text Available To meet the real-time and low power consumption demands in MEMS navigation and guidance field, an improved Kalman filter algorithm for GNSS/INS was proposed in this paper named as one-step prediction of P matrix. Quantitative analysis of field test datasets was made to compare the navigation accuracy with the standard algorithm, which indicated that the degradation caused by the simplified algorithm is small enough compared to the navigation errors of the GNSS/INS system itself. Meanwhile, the computation load and time consumption of the algorithm decreased over 50% by the improved algorithm. The work has special significance for navigation applications that request low power consumption and strict real-time response, such as cellphone, wearable devices, and deeply coupled GNSS/INS systems.

  19. One-step hydrothermal synthesis of three-dimensional porous graphene aerogels/sulfur nanocrystals for lithium–sulfur batteries

    International Nuclear Information System (INIS)

    Jiang, Yong; Lu, Mengna; Ling, Xuetao; Jiao, Zheng; Chen, Lingli; Chen, Lu; Hu, Pengfei; Zhao, Bing

    2015-01-01

    Highlights: • 3D porous GA/S nanocrystals are prepared by a one-step hydrothermal method. • The structure is affected by hydrothermal temperature and liquid sulfur’s viscosity. • The hybrid delivers a capacity of 716.2 mA h g −1 after 50 cycles at 100 mA g −1 . • The nanosized S, strong adsorbability and intimate contact of GNS are main factors. - Abstract: Lithium–sulfur (Li–S) batteries are receiving significant attention as a new energy source because of its high theoretical capacity and specific energy. However, the low sulfur loading and large particles (usually in submicron dimension) in the cathode greatly offset its advantage in high energy density and lead to the instability of the cathode and rapid capacity decay. Herein, we introduce a one-step hydrothermal synthesis of three-dimensional porous graphene aerogels/sulfur nanocrystals to suppress the rapid fading of sulfur electrode. It is found that the hydrothermal temperature and viscosity of liquid sulfur have significant effects on particle size and loading mass of sulfur nanocrystals, graphitization degree of graphene and chemical bonding between sulfur and oxygen-containing groups of graphene. The hybrid could deliver a specific capacity of 716.2 mA h g −1 after 50 cycles at a current density of 100 mA g −1 and reversible capacity of 517.9 mA h g −1 at 1 A g −1 . The performance we demonstrate herein suggests that Li–S battery may provide an opportunity for development of rechargeable battery systems

  20. Mixed models for data from thorough QT studies: part 2. One-step assessment of conditional QT prolongation.

    Science.gov (United States)

    Schall, Robert

    2011-01-01

    We investigate mixed analysis of covariance models for the 'one-step' assessment of conditional QT prolongation. Initially, we consider three different covariance structures for the data, where between-treatment covariance of repeated measures is modelled respectively through random effects, random coefficients, and through a combination of random effects and random coefficients. In all three of those models, an unstructured covariance pattern is used to model within-treatment covariance. In a fourth model, proposed earlier in the literature, between-treatment covariance is modelled through random coefficients but the residuals are assumed to be independent identically distributed (i.i.d.). Finally, we consider a mixed model with saturated covariance structure. We investigate the precision and robustness of those models by fitting them to a large group of real data sets from thorough QT studies. Our findings suggest: (i) Point estimates of treatment contrasts from all five models are similar. (ii) The random coefficients model with i.i.d. residuals is not robust; the model potentially leads to both under- and overestimation of standard errors of treatment contrasts and therefore cannot be recommended for the analysis of conditional QT prolongation. (iii) The combined random effects/random coefficients model does not always converge; in the cases where it converges, its precision is generally inferior to the other models considered. (iv) Both the random effects and the random coefficients model are robust. (v) The random effects, the random coefficients, and the saturated model have similar precision and all three models are suitable for the one-step assessment of conditional QT prolongation. Copyright © 2010 John Wiley & Sons, Ltd.

  1. Impact of styrenic polymer one-step hyper-cross-linking on volatile organic compound adsorption and desorption performance.

    Science.gov (United States)

    Ghafari, Mohsen; Atkinson, John D

    2018-06-05

    A novel one-step hyper-cross-linking method, using 1,2-dichloroethane (DCE) and 1,6-dichlorohexane (DCH) cross-linkers, expands the micropore volume of commercial styrenic polymers. Performance of virgin and modified polymers was evaluated by measuring hexane, toluene, and methyl-ethyl-ketone (MEK) adsorption capacity, adsorption/desorption kinetics, and desorption efficiency. Hyper-cross-linked polymers have up to 128% higher adsorption capacity than virgin polymers at P/P 0  = 0.05 due to micropore volume increases up to 330%. Improvements are most pronounced with the DCE cross-linker. Hyper-cross-linking has minimal impact on hexane adsorption kinetics, but adsorption rates for toluene and MEK decrease by 6-41%. Desorption rates decreased (3-36%) for all materials after hyper-cross-linking, with larger decreases for DCE hyper-cross-linked polymers due to smaller average pore widths. For room temperature desorption, 20-220% more adsorbate remains in hyper-cross-linked polymers after regeneration compared to virgin materials. DCE hyper-cross-linked polymers have 13-92% more residual adsorbate than DCH counterparts. Higher temperatures were required for DCE hyper-cross-linked polymers to completely desorb VOCs compared to the DCH hyper-cross-linked and virgin counterparts. Results show that the one-step hyper-cross-linking method for modifying styrenic polymers improves adsorption capacity because of added micropores, but decreases adsorption/desorption kinetics and desorption efficiency for large VOCs due to a decrease in average pore width. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

    Directory of Open Access Journals (Sweden)

    Ashwani Sanghi

    2010-06-01

    Full Text Available The present study describes the one-step purification and characterization of an extracellular cellulase-free xylanase from a newly isolated alkalophilic and moderately thermophilic strain of Bacillus subtilis ASH. Xylanase was purified to homogeneity by 10.5-fold with ~43% recovery using ion-exchange chromatography through CM-Sephadex C-50. The purified enzyme revealed a single band on SDS-PAGE gel with a molecular mass of 23 kDa. It showed an optimum pH at 7.0 and was stable over the pH range 6.0-9.0. The optimum temperature for enzyme activity was 55 ºC. The purified xylanase did not lose any activity up to 45 ºC, however, it retained 80% and 51% of its activity after pre-incubation at 55 ºC and 60 ºC, respectively. The enzyme obeyed Michaelis-Menton kinetics towards birch wood xylan with apparent Km 3.33 mg/ml and Vmax 100 IU/ml. The enzyme was strongly inhibited by Hg2+ and Cu2+ while enhanced by Co2+ and Mn2+. The purified enzyme could be stored at 4 ºC for six weeks without any loss of catalytic activity. The faster and economical purification of the cellulase-free xylanase from B. subtilis ASH by one-step procedure together with its appreciable stability at high temperature and alkaline pH makes it potentially effective for industrial applications.

  3. One-step fabrication of multifunctional composite polyurethane spider-web-like nanofibrous membrane for water purification

    International Nuclear Information System (INIS)

    Pant, Hem Raj; Kim, Han Joo; Joshi, Mahesh Kumar; Pant, Bishweshwar; Park, Chan Hee; Kim, Jeong In; Hui, K.S.; Kim, Cheol Sang

    2014-01-01

    Highlights: • A single mat having varieties of performance for water treatment is simply introduced. • Cost effective Ag-doped fly ash/PU nanofibers are fabricated in one-step. • Solvent reduction of AgNO 3 could produce Ag-loaded spider-web nets. • Size of Ag NPs on fiber surface can be controlled by controlling stirring time. • Fabrication of nanocomposite using pollutant material to control other pollutents. -- Abstract: A stable silver-doped fly ash/polyurathene (Ag-FA/PU) nanocomposite multifunctional membrane is prepared by a facile one-step electrospinning process using fly ash particles (FAPs). Colloidal solution of PU with FAPs and Ag metal precursor was subjected to fabricate nanocomposite spider-web-like membrane using electrospinning process. Presence of N,N-dimethylformamide (solvent of PU) led to reduce silver nitrate into Ag NPs. Incorporation of Ag NPs and FAPs through electrospun PU fibers is proven through electron microscopy and spectroscopic techniques. Presence of these NPs on PU nanofibers introduces several potential physicochemical properties such as spider-web-like nano-neeting for NPs separation, enhanced absorption capacity to remove carcinogenic arsenic (As) and toxic organic dyes, and antibacterial properties with reduce bio-fouling for membrane filter application. Preliminary observations used for above-mentioned applications for water treatment showed that it will be an economically and environmentally friendly nonwoven matrix for water purification. This simple approach highlights new avenues about the utilization of one pollutant material to control other pollutants in scalable and inexpensive ways

  4. One step transesterification process of sludge palm oil (SPO) by using deep eutectic solvent (DES) in biodiesel production

    Science.gov (United States)

    Manurung, Renita; Ramadhani, Debbie Aditia; Maisarah, Siti

    2017-06-01

    Biodiesel production by using sludge palm oil (SPO) as raw material is generally synthesized in two step reactions, namely esterification and transesterification, because the free fatty acid (FFA) content of SPO is relatively high. However, the presence of choline chloride (ChCl), glycerol based deep eutectic solvent (DES), in transesterification may produce biodiesel from SPO in just one step. In this study, DES was produced by the mixture of ChCl and glycerol at molar ratio of 1:2 at a temperature of 80°C and stirring speed of 400 rpm for 1 hour. DES was characterized by its density and viscosity. The transesterification process was performed at reaction temperature of 70 °C, ethanol to oil molar with ratio of 9:1, sodium hydroxide as catalyst concentration of 1 % wt, DES as cosolvent with concentration of 0 to 5 % wt, stirring speed of 400 rpm, and one hour reaction time. The obtained biodiesel was then assessed with density, viscosity, and ester content as the parameters. FFA content of SPO as the raw material was 7.5290 %. In this case, DES as cosolvent in one step transesterification process of low feedstock could reduce the side reaction (saponification), decrease the time reaction, decrease the surface tension between ethanol and oil, and increase the mass transfer that simultaneously simplified the purification process and obtained the highest yield. The esters properties met the international standards of ASTM D 6751, with the highest yield obtained was 83.19% with 99.55% of ester content and the ratio of ethanol:oil of 9:1, concentration of DES of 4%, catalyst amount of 1%, temperature of reaction at 70°C and stirring speed of 400 rpm.

  5. Shear bond strength of one-step self-etch adhesives to dentin: Evaluation of NaOCl pretreatment.

    Science.gov (United States)

    Colombo, Marco; Beltrami, Riccardo; Chiesa, Marco; Poggio, Claudio; Scribante, Andrea

    2018-02-01

    The aim of this study was to evaluate the influence of dentin pretreatment with NaOCl on shear bond strength of four one-step self-etch adhesives with different pH values. Bovine permanent incisors were used. Four one-step self-etch adhesives were tested: Adper™ Easy Bond, Futurabond NR, G-aenial Bond, Clearfil S3 Bond. One two-step self-etch adhesive (Clearfil SE Bond) was used as control. Group 1- no pretreatment; group 2- pretratment with 5,25 % NaOCl; group 3- pretreatment with 37 % H3PO4 etching and 5,25 % NaOCl. A hybrid composite resin was inserted into the dentin surface. The specimens were tested in a universal testing machine. The examiners evaluated the fractured surfaces in optical microscope to determine failure modes, quantified with adhesive remnant index (ARI). Dentin pretreatment variably influenced bond strength values of the different adhesive systems. When no dentin pretreatment was applied, no significant differences were found ( P >.05) among four adhesives tested. No significant differences were recorded when comparing NaOCl pretreatment with H3PO4 + NaOCl pretreatment for all adhesive tested ( P >.05) except Clearfil S3 Bond that showed higher shear bond strength values when H3PO4 was applied. Frequencies of ARI scores were calculated. The influence of dentin pretreatment with NaOCl depends on the composition of each adhesive system used. There was no difference in bond strength values among self-etch adhesives with different pH values. Key words: Dentin, pretreatment, self-etch adhesives.

  6. One-step synthesis of graphene/polyaniline hybrids by in situ intercalation polymerization and their electromagnetic properties

    Science.gov (United States)

    Chen, Xiangnan; Meng, Fanchen; Zhou, Zuowan; Tian, Xin; Shan, Liming; Zhu, Shibu; Xu, Xiaoling; Jiang, Man; Wang, Li; Hui, David; Wang, Yong; Lu, Jun; Gou, Jihua

    2014-06-01

    A new method is introduced for the preparation of graphene/polyaniline hybrids using a one-step intercalation polymerization of aniline inside the expanded graphite. The structural and morphological characterizations were performed by X-ray diffraction analysis, transmission electron microscopy and field emission scanning electron microscopy. Both the experimental and first-principles simulated results show that the aniline cation formed by aniline and H+ tends to be drawn towards the electron-enriched zone and to intercalate into the interlayer of graphite. Subsequently, an in situ polymerization leads to the separation of graphite into graphene sheet, resulting from the exothermic effect and more vigorous movements of the chain molecules of polyaniline. The interactions between polyaniline and graphene were confirmed by Fourier transform infrared spectroscopy and Raman spectra. In addition, the graphene/polyaniline hybrid exhibited a breakthrough in the improvement of microwave absorption.A new method is introduced for the preparation of graphene/polyaniline hybrids using a one-step intercalation polymerization of aniline inside the expanded graphite. The structural and morphological characterizations were performed by X-ray diffraction analysis, transmission electron microscopy and field emission scanning electron microscopy. Both the experimental and first-principles simulated results show that the aniline cation formed by aniline and H+ tends to be drawn towards the electron-enriched zone and to intercalate into the interlayer of graphite. Subsequently, an in situ polymerization leads to the separation of graphite into graphene sheet, resulting from the exothermic effect and more vigorous movements of the chain molecules of polyaniline. The interactions between polyaniline and graphene were confirmed by Fourier transform infrared spectroscopy and Raman spectra. In addition, the graphene/polyaniline hybrid exhibited a breakthrough in the improvement of

  7. Low-temperature synthesis and characterization of helical carbon fibers by one-step chemical vapour deposition

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Yongzhong [Department of Materials and Chemistry Engineering, Sichuan University of Science and Engineering, 643000 Zigong, Sichuan (China); Chen, Jian, E-mail: wuhangzs@163.com [Department of Materials and Chemistry Engineering, Sichuan University of Science and Engineering, 643000 Zigong, Sichuan (China); Fu, Qingshan [Department of Materials and Chemistry Engineering, Sichuan University of Science and Engineering, 643000 Zigong, Sichuan (China); Li, Binghong [China Rubber Group Carbon Black Industry Research and Design Institute, 643000 Zigong, Sichuan (China); Zhang, Huazhi; Gong, Yong [Department of Materials and Chemistry Engineering, Sichuan University of Science and Engineering, 643000 Zigong, Sichuan (China)

    2015-01-01

    Graphical abstract: - Highlights: • HCNFs were synthesized by one-step CVD using cupric tartrate as a catalyst at temperature below 500 °C. • The synthesis of HCNFs is highly temperature-dependent at the synthesis temperature of 280–480 °C. • The addition of HCNFs makes a noticeable contribution to the reinforcement of NR/CB system. - Abstract: Helical carbon fibers (HCNFs) were synthesized by one-step chemical vapour deposition using cupric tartrate as a catalyst at temperature below 500 °C. The bound rubber of natural rubber (NR)/HCNFs were also prepared in this study. The results of thermogravimetry–differential scanning calorimetry (TG/DSC) for cupric tartrate nanoparticles show that the transformation of C{sub 4}H{sub 4}CuO{sub 6} → Cu reaction occurs at ∼250–310 °C. The characterization of scanning electron microscopy (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD) and Raman spectrum for the synthesized products confirms that the synthesis of HCNFs is highly temperature-dependent. The straight fibers with the fiber diameter of 100–400 nm are obtained at 280 °C and HCNFs can be synthesized at higher temperature, with the coil diameter of 0.5–1 μm and fiber diameter of 100–200 nm at 380 °C, and the coil diameter of ∼100 nm and fiber diameter of ∼80 nm at 480 °C. The maximum of the bound-rubber content (37%) can be obtained with the addition of 100 wt.% HCNFs in NR, which indicates that the coiled configuration of HCNFs makes a noticeable contribution to the reinforcement of NR/CB system.

  8. Low-temperature synthesis and characterization of helical carbon fibers by one-step chemical vapour deposition

    International Nuclear Information System (INIS)

    Jin, Yongzhong; Chen, Jian; Fu, Qingshan; Li, Binghong; Zhang, Huazhi; Gong, Yong

    2015-01-01

    Graphical abstract: - Highlights: • HCNFs were synthesized by one-step CVD using cupric tartrate as a catalyst at temperature below 500 °C. • The synthesis of HCNFs is highly temperature-dependent at the synthesis temperature of 280–480 °C. • The addition of HCNFs makes a noticeable contribution to the reinforcement of NR/CB system. - Abstract: Helical carbon fibers (HCNFs) were synthesized by one-step chemical vapour deposition using cupric tartrate as a catalyst at temperature below 500 °C. The bound rubber of natural rubber (NR)/HCNFs were also prepared in this study. The results of thermogravimetry–differential scanning calorimetry (TG/DSC) for cupric tartrate nanoparticles show that the transformation of C 4 H 4 CuO 6 → Cu reaction occurs at ∼250–310 °C. The characterization of scanning electron microscopy (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD) and Raman spectrum for the synthesized products confirms that the synthesis of HCNFs is highly temperature-dependent. The straight fibers with the fiber diameter of 100–400 nm are obtained at 280 °C and HCNFs can be synthesized at higher temperature, with the coil diameter of 0.5–1 μm and fiber diameter of 100–200 nm at 380 °C, and the coil diameter of ∼100 nm and fiber diameter of ∼80 nm at 480 °C. The maximum of the bound-rubber content (37%) can be obtained with the addition of 100 wt.% HCNFs in NR, which indicates that the coiled configuration of HCNFs makes a noticeable contribution to the reinforcement of NR/CB system

  9. One-step fabrication of multifunctional composite polyurethane spider-web-like nanofibrous membrane for water purification

    Energy Technology Data Exchange (ETDEWEB)

    Pant, Hem Raj, E-mail: hempant@jbnu.ac.kr [Department of Bio-nano System Engineering, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Department of Engineering Science and Humanities, Institute of Engineering, Pulchowk Campus, Tribhuvan University, Kathmandu (Nepal); Kim, Han Joo [Division of Mechanical Design Engineering, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Joshi, Mahesh Kumar; Pant, Bishweshwar; Park, Chan Hee; Kim, Jeong In [Department of Bio-nano System Engineering, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Hui, K.S., E-mail: kshui@hanyang.ac.kr [Department of Mechanical Engineering, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Kim, Cheol Sang, E-mail: chskim@jbnu.ac.kr [Department of Bio-nano System Engineering, Chonbuk National University, Jeonju 561-756 (Korea, Republic of); Division of Mechanical Design Engineering, Chonbuk National University, Jeonju 561-756 (Korea, Republic of)

    2014-01-15

    Highlights: • A single mat having varieties of performance for water treatment is simply introduced. • Cost effective Ag-doped fly ash/PU nanofibers are fabricated in one-step. • Solvent reduction of AgNO{sub 3} could produce Ag-loaded spider-web nets. • Size of Ag NPs on fiber surface can be controlled by controlling stirring time. • Fabrication of nanocomposite using pollutant material to control other pollutents. -- Abstract: A stable silver-doped fly ash/polyurathene (Ag-FA/PU) nanocomposite multifunctional membrane is prepared by a facile one-step electrospinning process using fly ash particles (FAPs). Colloidal solution of PU with FAPs and Ag metal precursor was subjected to fabricate nanocomposite spider-web-like membrane using electrospinning process. Presence of N,N-dimethylformamide (solvent of PU) led to reduce silver nitrate into Ag NPs. Incorporation of Ag NPs and FAPs through electrospun PU fibers is proven through electron microscopy and spectroscopic techniques. Presence of these NPs on PU nanofibers introduces several potential physicochemical properties such as spider-web-like nano-neeting for NPs separation, enhanced absorption capacity to remove carcinogenic arsenic (As) and toxic organic dyes, and antibacterial properties with reduce bio-fouling for membrane filter application. Preliminary observations used for above-mentioned applications for water treatment showed that it will be an economically and environmentally friendly nonwoven matrix for water purification. This simple approach highlights new avenues about the utilization of one pollutant material to control other pollutants in scalable and inexpensive ways.

  10. One-step hydrothermal synthesis of three-dimensional porous graphene aerogels/sulfur nanocrystals for lithium–sulfur batteries

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Yong; Lu, Mengna; Ling, Xuetao; Jiao, Zheng; Chen, Lingli; Chen, Lu [School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444 (China); Hu, Pengfei [Instrumental Analysis and Research Center, Shanghai University, Shanghai 200444 (China); Zhao, Bing, E-mail: bzhao@shu.edu.cn [School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444 (China)

    2015-10-05

    Highlights: • 3D porous GA/S nanocrystals are prepared by a one-step hydrothermal method. • The structure is affected by hydrothermal temperature and liquid sulfur’s viscosity. • The hybrid delivers a capacity of 716.2 mA h g{sup −1} after 50 cycles at 100 mA g{sup −1}. • The nanosized S, strong adsorbability and intimate contact of GNS are main factors. - Abstract: Lithium–sulfur (Li–S) batteries are receiving significant attention as a new energy source because of its high theoretical capacity and specific energy. However, the low sulfur loading and large particles (usually in submicron dimension) in the cathode greatly offset its advantage in high energy density and lead to the instability of the cathode and rapid capacity decay. Herein, we introduce a one-step hydrothermal synthesis of three-dimensional porous graphene aerogels/sulfur nanocrystals to suppress the rapid fading of sulfur electrode. It is found that the hydrothermal temperature and viscosity of liquid sulfur have significant effects on particle size and loading mass of sulfur nanocrystals, graphitization degree of graphene and chemical bonding between sulfur and oxygen-containing groups of graphene. The hybrid could deliver a specific capacity of 716.2 mA h g{sup −1} after 50 cycles at a current density of 100 mA g{sup −1} and reversible capacity of 517.9 mA h g{sup −1} at 1 A g{sup −1}. The performance we demonstrate herein suggests that Li–S battery may provide an opportunity for development of rechargeable battery systems.

  11. Structural and optical properties of Co-doped ZnO nanocrystallites prepared by a one-step solution route

    International Nuclear Information System (INIS)

    Li Ping; Wang Sha; Li Jibiao; Wei Yu

    2012-01-01

    Zinc oxide (ZnO) nanocrystallites with different Co-doping levels were successfully synthesized by a simple one-step solution route at low temperature (95 deg. C) in this study. The structure and morphology of the samples thus obtained were characterized by XRD, EDS, XPS and FESEM. Results show that cobalt ions, in the oxidation state of Co 2+ , replace Zn 2+ ions in the ZnO lattice without changing its wurtzite structure. The dopant content varies from 0.59% to 5.39%, based on Co-doping levels. The pure ZnO particles exhibit well-defined 3D flower-like morphology with an average size of 550 nm, while the particles obtained after Co-doping are mostly cauliflower-like nanoclusters with an average size of 120 nm. Both the flower-like pure ZnO and the cauliflower-like Co:ZnO nanoclusters are composed of densely arrayed nanorods. The optical properties of the ZnO nanocrystallites following Co-doping were also investigated by UV-Visible absorption and Photoluminescence spectra. Our results indicate that Co-doping can change the energy-band structure and effectively adjust the luminescence properties of ZnO nanocrystallites. - Highlights: → Co-doped ZnO nanocrystallites were synthesized via a simple one-step solution route. → Co 2+ ions incorporated into the ZnO lattice without changing its wurtzite structure. → Co-doping changed the energy band structure of ZnO. → Co-doping effectively adjusted the luminescence properties of ZnO nanocrystallites.

  12. Structural and optical properties of Co-doped ZnO nanocrystallites prepared by a one-step solution route

    Energy Technology Data Exchange (ETDEWEB)

    Li Ping, E-mail: lipingchina@yahoo.com.cn [Provincial Key Laboratory of Inorganic Nanomaterials, School of Chemistry and Materials Science, Hebei Normal University, 113 Yuhua Road, Shijiazhuang 050016, Hebei (China); Wang Sha; Li Jibiao; Wei Yu [Provincial Key Laboratory of Inorganic Nanomaterials, School of Chemistry and Materials Science, Hebei Normal University, 113 Yuhua Road, Shijiazhuang 050016, Hebei (China)

    2012-01-15

    Zinc oxide (ZnO) nanocrystallites with different Co-doping levels were successfully synthesized by a simple one-step solution route at low temperature (95 deg. C) in this study. The structure and morphology of the samples thus obtained were characterized by XRD, EDS, XPS and FESEM. Results show that cobalt ions, in the oxidation state of Co{sup 2+}, replace Zn{sup 2+} ions in the ZnO lattice without changing its wurtzite structure. The dopant content varies from 0.59% to 5.39%, based on Co-doping levels. The pure ZnO particles exhibit well-defined 3D flower-like morphology with an average size of 550 nm, while the particles obtained after Co-doping are mostly cauliflower-like nanoclusters with an average size of 120 nm. Both the flower-like pure ZnO and the cauliflower-like Co:ZnO nanoclusters are composed of densely arrayed nanorods. The optical properties of the ZnO nanocrystallites following Co-doping were also investigated by UV-Visible absorption and Photoluminescence spectra. Our results indicate that Co-doping can change the energy-band structure and effectively adjust the luminescence properties of ZnO nanocrystallites. - Highlights: > Co-doped ZnO nanocrystallites were synthesized via a simple one-step solution route. > Co{sup 2+} ions incorporated into the ZnO lattice without changing its wurtzite structure. > Co-doping changed the energy band structure of ZnO. > Co-doping effectively adjusted the luminescence properties of ZnO nanocrystallites.

  13. Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant peroxidases.

    Science.gov (United States)

    Kjaersgård, I V; Jespersen, H M; Rasmussen, S K; Welinder, K G

    1997-03-01

    cDNA clones encoding two new Arabidopsis thaliana peroxidases, ATP 1a and ATP 2a, have been identified by searching the Arabidopsis database of expressed sequence tags (dbEST). They represent a novel branch of hitherto uncharacterized plant peroxidases which is only 35% identical in amino acid sequence to the well characterized group of basic plant peroxidases represented by the horseradish (Armoracia rusticana) isoperoxidases HRP C, HRP E5 and the similar Arabidopsis isoperoxidases ATP Ca, ATP Cb, and ATP Ea. However ATP 1a is 87% identical in amino acid sequence to a peroxidase encoded by an mRNA isolated from cotton (Gossypium hirsutum). As cotton and Arabidopsis belong to rather diverse families (Malvaceae and Crucifereae, respectively), in contrast with Arabidopsis and horseradish (both Crucifereae), the high degree of sequence identity indicates that this novel type of peroxidase, albeit of unknown function, is likely to be widespread in plant species. The atp 1 and atp 2 types of cDNA sequences were the most redundant among the 28 different isoperoxidases identified among about 200 peroxidase encoding ESTs. Interestingly, 8 out of totally 38 EST sequences coding for ATP 1 showed three identical nucleotide substitutions. This variant form is designated ATP 1b. Similarly, six out of totally 16 EST sequences coding for ATP 2 showed a number of deletions and nucleotide changes. This variant form is designated ATP 2b. The selected EST clones are full-length and contain coding regions of 993 nucleotides for atp 1a, and 984 nucleotides for atp 2a. These regions show 61% DNA sequence identity. The predicted mature proteins ATP 1a, and ATP 2a are 57% identical in sequence and contain the structurally and functionally important residues, characteristic of the plant peroxidase superfamily. However, they do show two differences of importance to peroxidase catalysis: (1) the asparagine residue linked with the active site distal histidine via hydrogen bonding is absent

  14. Enterobacterial repetitive intergenic consensus sequences and the PCR to generate fingerprints of genomic DNAs from Vibrio cholerae O1, O139, and non-O1 strains.

    Science.gov (United States)

    Rivera, I G; Chowdhury, M A; Huq, A; Jacobs, D; Martins, M T; Colwell, R R

    1995-08-01

    Enterobacterial repetitive intergenic consensus (ERIC) sequence polymorphism was studied in Vibrio Cholerae strains isolated before and after the cholera epidemic in Brazil (in 1991), along with epidemic strains from Peru, Mexico, and India, by PCR. A total of 17 fingerprint patterns (FPs) were detected in the V. cholerae strains examined; 96.7% of the toxigenic V. cholerae O1 strains and 100% of the O139 serogroup strains were found to belong to the same FP group comprising four fragments (FP1). The nontoxigenic V. cholerae O1 also yielded four fragments but constituted a different FP group (FP2). A total of 15 different patterns were observed among the V. cholerae non-O1 strains. Two patterns were observed most frequently for V. cholerae non-01 strains, 25% of which have FP3, with five fragments, and 16.7% of which have FP4, with two fragments. Three fragments, 1.75, 0.79, and 0.5 kb, were found to be common to both toxigenic and nontoxigenic V. cholerae O1 strains as well as to group FP3, containing V. cholerae non-O1 strains. Two fragments of group FP3, 1.3 and 1.0 kb, were present in FP1 and FP2 respectively. The 0.5-kb fragment was common to all strains and serogroups of V. cholerae analyzed. It is concluded from the results of this study, based on DNA FPs of environmental isolates, that it is possible to detect an emerging virulent strain in a cholera-endemic region. ERIC-PCR constitutes a powerful tool for determination of the virulence potential of V. cholerae O1 strains isolated in surveillance programs and for molecular epidemiological investigations.

  15. Analysis of bacterial communities and bacterial pathogens in a biogas plant by the combination of ethidium monoazide, PCR and Ion Torrent sequencing.

    Science.gov (United States)

    Luo, Gang; Angelidaki, Irini

    2014-09-01

    The present study investigated the changes of bacterial community composition including bacterial pathogens along a biogas plant, i.e. from the influent, to the biogas reactor and to the post-digester. The effects of post-digestion temperature and time on the changes of bacterial community composition and bacterial pathogens were also studied. Microbial analysis was made by Ion Torrent sequencing of the PCR amplicons from ethidium monoazide treated samples, and ethidium monoazide was used to cleave DNA from dead cells and exclude it from PCR amplification. Both similarity and taxonomic analysis showed that the bacterial community composition in the influent was changed after anaerobic digestion. Firmicutes were dominant in all the samples, while Proteobacteria decreased in the biogas reactor compared with the influent. Variations of bacterial community composition in the biogas reactor with time were also observed. This could be attributed to varying composition of the influent. Batch experiments showed that the methane recovery from the digested residues (obtained from biogas reactor) was mainly related with post-digestion temperature. However, post-digestion time rather than temperature had a significant effect on the changes of bacterial community composition. The changes of bacterial community composition were also reflected in the changes of relative abundance of bacterial pathogens. The richness and relative abundance of bacterial pathogens were reduced after anaerobic digestion in the biogas reactor. It was found in batch experiments that bacterial pathogens showed the highest relative abundance and richness after 30 days' post-digestion. Streptococcus bovis was found in all the samples. Our results showed that special attention should be paid to the post-digestion since the increase in relative abundance of bacterial pathogens after post-digestion might reflect regrowth of bacterial pathogens and limit biosolids disposal vectors. Copyright © 2014 Elsevier

  16. A facile one-step route to RGO/Ni3S2 for high-performance supercapacitors

    International Nuclear Information System (INIS)

    Zhang, Zhuomin; Zhao, Chongjun; Min, Shudi; Qian, Xiuzhen

    2014-01-01

    Graphical abstract: - Highlights: • A uniform 3D nest-like nanostructure of RGO/Ni 3 S 2 nanocomposite on Ni foam, in-situ synthesized using a simple, green one-pot hydrothermal approach, exhibits superior capacitive performance (7440 mF cm −2 at 10 mA cm −2 , i.e., 2188.8 F g −1 at 2.9 A g −1 and 1016 F g −1 at 29.0 A g −1 ).Highlights. • RGO/Ni 3 S 2 /NF composite was in-situ synthesized through a one-step hydrothermal process. • No other reagent except Ni foam, GO and S powder was added. • RGO/Ni 3 S 2 , RGO/Ni(OH) 2 , and RGO/Ni 3 S 2 /Ni(OH) 2 can be controllably synthesized. • As-prepared RGO/Ni 3 S 2 /NF nanocomposite exhibits high capability and cyclability. - Abstract: A facile one-step solution-phase route to RGO/Ni 3 S 2 on nickel foam (RGO/Ni 3 S 2 /NF) was presented. The RGO/Ni 3 S 2 /NF (RNS) nanocomposites were hydrothermal-assisted synthesized, in which nickel foam acted as an auxiliary reductant of GO and S, a Ni source of Ni 3 S 2 , and a substrate for composite film. RGO/Ni 3 S 2 /NF composites were characterized by X-ray diffraction (XRD), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), field-emission scanning electron microscope (FESEM), and transmission electron microscopy (TEM). The electrochemical performances of the supercapacitor with as-synthesized RGO/Ni 3 S 2 /NF (RNS) electrodes are evaluated using cyclic voltammetry (CV), galvanostatic charge-discharge (GCD), and electrochemical impedance spectrometry (EIS) in 2 M KOH aqueous solution. It is found that the RGO/Ni 3 S 2 /NF electrode exhibits superior supercapacitor performance (7440 mF cm −2 at 10 mA cm −2 , i.e., 2188.8 F g −1 at 2.9 A g −1 ), compared with the Ni 3 S 2 /NF electrode (4360 mF cm −2 at 10 mA cm −2 ) and the RGO/Ni(OH) 2 /NF electrode (3400 mF cm −2 at 10 mA cm −2 ) prepared under identical conditions. Both the temperature and sulfur content play important roles in the controlled synthesis of RNS and its

  17. Multifunctionalized polyethyleneimine-based nanocarriers for gene and chemotherapeutic drug combination therapy through one-step assembly strategy

    Directory of Open Access Journals (Sweden)

    Jiang D

    2017-12-01

    Full Text Available Dandan Jiang,1,* Mingfang Wang,1,* Tianqi Wang,1 Bo Zhang,1 Chunxi Liu,2 Na Zhang1 1Department of Pharmaceutics, Key Laboratory of Chemical Biology (Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan, China; 2Pharmaceutical Department, Qilu Hospital of Shandong University, Jinan, China *These authors contributed equally to this work Abstract: Gene therapy combined with chemotherapy to achieve synergistic therapeutic effects has been a hot topic in recent years. In this project, the human tumor necrosis factor-related apoptosis-inducing ligand-encoding plasmid gene (TRAIL and doxorubicin (Dox-coloaded multifunctional nanocarrier was constructed based on the theory of circulation, accumulation, internalization, and release. Briefly, polyethyleneimine (PEI was selected as skeleton material to synthesize PEI–polyethylene glycol (PEG–TAT (PPT. Dox was conjugated to PEI using C6-succinimidyl 6-hydrazinonicotinate acetone hydrazone (C6-SANH, and a pH-sensitive Dox-PEI (DP conjugate was obtained. Then, intracellular cationic pH-sensitive cellular assistant PPT and DP were mixed to condense TRAIL, and TRAIL–Dox coloaded PPT/DP/TRAIL (PDT nanocarriers were obtained by one-step assembly. TRAIL was completely condensed by DP or PPT when mass ratios (DP/PPT to TRAIL were up to 100:64, which indicated that DP and PPT could be mixed at any ratio for TRAIL condensation. The intracellular uptake rate of PDT was enhanced (P<0.05 when the contents of PPT in PPT+DP increased from 0 to 30%. Free Dox and TRAIL-loaded nanocarriers (PPT/C6-SANH-PEI/TRAIL [PCT] were selected as controls to verify the synergistic antitumor effects of PDT. Compared with free TRAIL, TRAIL-protein expression was upregulated by PDT and PCT on Western blotting assays. The in vitro cytotoxicity of PDT was significantly enhanced compared to free Dox and PCT (P<0.01. Furthermore, murine PDT nanocarriers showed higher in vivo antitumor ability than both the