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Sample records for acid sequence-based amplification

  1. Nucleic acid sequence-based amplification with oligochromatography for detection of Trypanosoma brucei in clinical samples

    NARCIS (Netherlands)

    C.M. Mugasa; T. Laurent; G.J. Schoone; P.A. Kager; G.W. Lubega; H.D.F.H. Schallig

    2009-01-01

    Molecular tools, such as real-time nucleic acid sequence-based amplification (NASBA) and PCR, have been developed to detect Trypanosoma brucei parasites in blood for the diagnosis of human African trypanosomiasis (HAT). Despite good sensitivity, these techniques are not implemented in HAT control pr

  2. Comparison of nucleic acid sequence-based amplification and loop-mediated isothermal amplification for diagnosis of human African trypanosomiasis.

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    Mugasa, Claire M; Katiti, Diana; Boobo, Alex; Lubega, George W; Schallig, Henk D F H; Matovu, Enock

    2014-02-01

    Diagnosis of human African trypanosomiasis (HAT) using molecular tests should ideally achieve high sensitivity without compromising specificity. This study compared 2 simplified tests, nucleic acid sequence-based amplification (NASBA) combined with oligochromatography (OC) and loop-mediated isothermal amplification (LAMP), executed on 181 blood samples from 65 Trypanosoma brucei gambiense HAT patients, 86 controls, and 30 serological suspects from Uganda. Basing on the composite reference standard, the diagnostic sensitivity and specificity of NASBA were 93.9% (95% confidence interval [CI] = 84.9-98.3%) and 100% (95% CI = 94.9-100%), respectively. The same parameters for LAMP were 76.9% (95% CI = 64.8-86.5%) and 100% (95% CI = 91.6-100%), respectively. The level of agreement between LAMP and microscopy was good with a kappa (κ) value of 79.2% (95% CI = 69.4-88.9%), while that of NASBA-OC/microscopy was very good (κ value 94.6%; 95% CI = 89.3-99.8%). The sensitivity of NASBA-OC was significantly higher than that of LAMP (Z = 2.723; P = 0.007). These tests have potential application to HAT surveillance.

  3. Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification

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    Kager Piet A

    2006-10-01

    Full Text Available Abstract Background Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (Plasmodium antigen detection do often not allow for species discrimination as microscopy does, but also become insensitive at Methods This paper reports the development of a sensitive and specific real-time Quantitative Nucleic Acid Sequence Based Amplification (real-time QT-NASBA assays, based on the small-subunit 18S rRNA gene, to identify the four human Plasmodium species. Results The lower detection limit of the assay is 100 – 1000 molecules in vitro RNA for all species, which corresponds to 0.01 – 0.1 parasite per diagnostic sample (i.e. 50 μl of processed blood. The real-time QT-NASBA was further evaluated using 79 clinical samples from malaria patients: i.e. 11 Plasmodium. falciparum, 37 Plasmodium vivax, seven Plasmodium malariae, four Plasmodium ovale and 20 mixed infections. The initial diagnosis of 69 out of the 79 samples was confirmed with the developed real-time QT-NASBA. Re-analysis of seven available original slides resolved five mismatches. Three of those were initially identified as P. malariae mono-infection, but after re-reading the slides P. falciparum was found, confirming the real-time QT-NASBA result. The other two slides were of poor quality not allowing true species identification. The remaining five discordant results could not be explained by microscopy, but may be due to extreme low numbers of parasites present in the samples. In addition, 12 Plasmodium berghei isolates from mice and 20 blood samples from healthy donors did not show any reaction in the assay. Conclusion Real-time QT-NASBA is a very sensitive and specific technique with a detection limit of 0.1 Plasmodium parasite per diagnostic sample (50 μl of blood and can be used for the detection, identification and quantitative measurement of low parasitaemia of Plasmodium species, thus

  4. Detection of Campylobacter jejuni and Campylobacter coli in chicken meat samples by real-time nucleic acid sequence-based amplification with molecular beacons.

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    Churruca, E; Girbau, C; Martínez, I; Mateo, E; Alonso, R; Fernández-Astorga, A

    2007-06-10

    A nucleic acid sequence-based amplification (NASBA) assay based on molecular beacons was used for real-time detection of Campylobacter jejuni and Campylobacter coli in samples of chicken meat. A set of specific primers and beacon probe were designed to target the 16S rRNA of both species. The real-time NASBA protocol including the RNA isolation was valid for both of the cell suspensions in buffered saline and the artificially contaminated chicken meat samples. The presence of rRNA could be correlated with cellular viability, following inactivation of the bacteria by heating, in inoculated chicken meat samples but not in RNase-free cell suspensions.

  5. Visual detection and differentiation of Classic Swine Fever Virus strains using nucleic acid sequence-based amplification (NASBA) and G-quadruplex DNAzyme assay

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    Lu, Xiaolu; Shi, Xueyao; Wu, Gege; Wu, Tiantian; Qin, Rui; Wang, Yi

    2017-01-01

    The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Here, we successfully integrated colorimetric split G-quadruplex DNAzyme assay with nucleic acid sequence-based amplification to generate a novel detection approach, allowing visual and rapid detection for the RNA of Shimen and HCLV strains of Classic Swine Fever Virus (CSFV). CSFV is a RNA virus that causes a highly contagious disease in domestic pigs and wild boar. With this method, we were able to detect as little as 10 copies/ml of CSF viral RNA within 3 h in serum samples taken from the field. No interference was encountered in the amplification and detection of Classic Swine Fever Virus in the presence of non-target RNA or DNA. Moreover, Shimen and HCLV strains of Classic Swine Fever Virus could be easily differentiated using the NASBA-DNAzyme system. These findings indicate the NASBA-DNAzyme system is a rapid and practical technique for detecting and discriminating CSFV strains and may be applied to the detection of other RNA viruses. PMID:28287135

  6. Sensitivity and Specificity of Nucleic Acid Sequence-Based Amplification Method (NASBA for Diagnosis of Cutaneous Leishmaniasis

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    Niazi, A

    2014-05-01

    Full Text Available Background and Objective: Employing advanced diagnostics for molecular identification of the Lishmania parasite is has a more sensitivity and specificity in comparison to the microscopic methods. RT- PCR is also introduced as one of the best known techniques for diagnosis of this parasite; however, the method is not widely used due to its expensive equipments and the time requested.the application of NASBA method is shown high efficient for diagnosis of live parasite. The aim of this study is comparison sensivity and specificity between NASBA isothermal amplification and RT-PCR for molecular detection of lishmania major. Material and Methods: 28 skin biopsy from Oscar of patients was prepared and total RNA was extracted. Then, the using of specific primers designed for 18srRNA region, this region was amplified using NASBA isothemal amplification. The RNA amplicons were produced in less than 90 minutes and then identified via electrophoresed agaros gel after staining with Syber Gold flourecent probes for the purpose increasing sensitivity and specificity Result: In this study, NASBA and RT-PCR method are sensitivity 81%, specificity of 51% and 100% respectively for detection of Leishmania parasites inscars Conclusion: NASBA isothermal method can be applied with high sensitivity and specificity for the identification of cutaneous leishmaniasis, this method can be fed with live microorganisms and response to treatment in patients examined. Keywords: Cutaneous Leishmanisis, NASBA, 18S rRNA

  7. Real-time nucleic acid sequence-based amplification (NASBA) using an adenine-induced quenching probe and an intercalator dye.

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    Kouguchi, Y; Teramoto, M; Kuramoto, M

    2010-11-01

    We found that an adenine base caused fluorescence quenching of a fluorescein (FL)-labelled probe in DNA:RNA hybrid sequences, and applied this finding to a nucleic acid sequence-based amplification (NASBA) method. The present NASBA method employed a probe containing an FL-modified thymine at its 3' end and ethidium bromide (EtBr) on the basis of a combination of adenine-induced quenching and fluorescence resonance energy transfer (FRET) between the FL donor and EtBr acceptor. This NASBA was used to detect Shiga toxin (STX) stx-specific mRNA in STX-producing Escherichia coli, demonstrating rapid quantification of the target gene with high sensitivity. Although the inherent quenching effect of adenine was inferior to that of guanine, FRET between the FL and EtBr moieties enhanced the adenine-induced quenching, allowing rapid and sensitive real-time NASBA detection. This study gives a novel real-time diagnostic system based on NASBA for a sensitive mRNA (or viral RNA) detection. © 2010 The Authors. Journal of Applied Microbiology © 2010 The Society for Applied Microbiology.

  8. Higher specificity of nucleic acid sequence-based amplification isothermal technology than of real-time PCR for quantification of HIV-1 RNA on dried blood spots.

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    Mercier-Delarue, Severine; Vray, Muriel; Plantier, Jean Christophe; Maillard, Theodora; Adjout, Zidan; de Olivera, Fabienne; Schnepf, Nathalie; Maylin, Sarah; Simon, Francois; Delaugerre, Constance

    2014-01-01

    Dried blood spots (DBS) are widely proposed as a plasma surrogate for monitoring antiretroviral treatment efficacy based on the HIV-1 RNA level (viral load [VL]) in resource-limited settings. Interfering coamplification of cell-associated HIV-1 DNA during reverse transcription (RT)-PCR can be avoided by using nucleic acid sequence-based amplification (NASBA) technology, which is based on an RNA template and isothermic conditions. We analyzed VL values obtained with DBS and plasma samples by comparing isothermic NASBA (NucliSENS EasyQ HIV-1 V2.0; bioMérieux) with real-time RT-PCR (Cobas TaqMan HIV-1 V2.0; Roche). Samples from 197 HIV-1-infected patients were tested (non-B subtypes in 51% of the cases). Nucleic acid extractions were performed by use of NucliSENS EasyMAG (bioMérieux) and Cobas AmpliPrep (Roche) before the NASBA and RT-PCR quantifications, respectively. Both quantification assays have lower limits of detection of 20 (1.3) and 800 (2.9) log10 copies/ml (log) in plasma and DBS, respectively. The mean (DBS minus plasma) differences were -0.39 and -0.46 log, respectively, for RT-PCR and NASBA. RT-PCR on DBS identified virological failure in 122 of 126 patients (sensitivity, 97%) and viral suppression in 58 of 70 patients (specificity, 83%), yielding 12 false-positive results (median, 3.2 log). NASBA on DBS identified virological failure in 85 of 96 patients (sensitivity, 89%) and viral suppression in 95 of 97 patients (specificity, 98%) and yielded 2 false-positive results (3.0 log for both). Both technologies detected HIV-1 RNA in DBS at a threshold of 800 copies/ml. This higher specificity of NASBA technology could avoid overestimation of poor compliance or the emergence of resistance when monitoring antiretroviral efficacy with the DBS method.

  9. Establishment of the Nucleic Acid Sequence-based Amplification Method for Detecting Vibio Alginolyticus%溶藻弧菌的依赖于核酸序列恒温扩增检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    秦胜利; 王建广

    2012-01-01

    A new method, based on Nucleic Acid Sequence-based Amplification (NAS-BA) to detect Vibio alginolyticus of samples, was established. A highly specific set of primers was synthesized to target the hsp60 gene of Vibio alginolyticus so as to establish Nucleic Acid Sequence-based Amplification method. Specificity and sensitivity were tested. The results showed that the sensitivity of NASBA was 6. 9×102cfu ? mL-1 which was higher than the result of PCR method. Detecting Vibio alginolyticus with NASBA was more specific and sensitive than PCR method and has lower instrumental requirement. So,there is a broad prospect.%采用自行建立和优化的依赖于核酸序列恒温扩增(NASBA)检测体系,对溶藻弧菌进行检测.采用溶藻弧菌的hsp60基因为目的片段设计特异性引物,建成可快速检测溶藻弧菌的NASBA检测法,并进行了特异性和灵敏度试验.结果表明:所建立起的NASBA检测方法,灵敏度为6.9× 102 cfu·mL-1,高于普通PCR方法.溶藻弧菌的依赖于核酸序列恒温扩增检测方法具有较高灵敏度和和良好特异性,并且对仪器要求更低,用普通恒温设备即可进行反应,具有广阔的推广前景.

  10. Comparison of levels of human immunodeficiency virus type 1 RNA in plasma as measured by the NucliSens nucleic acid sequence-based amplification and Quantiplex branched-DNA assays.

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    Ginocchio, C C; Tetali, S; Washburn, D; Zhang, F; Kaplan, M H

    1999-04-01

    This study compared levels of human immunodeficiency virus type 1 RNA in plasma as measured by the Quantiplex branched-DNA and NucliSens nucleic acid sequence-based amplification assays. RNA was detectable in 118 of 184 samples (64.13%) by the Quantiplex assay and in 171 of 184 samples (92.94%) by the NucliSens assay. Regression analysis indicated that a linear relationship existed between the two sets of values (P < 0.0001), although the Quantiplex and NucliSens values were significantly different (P < 0.001), with the NucliSens values being approximately 0.323 log higher. Spearman correlation analysis indicated that the overall changes in patient viral load patterns were highly correlative between the two assays: r = 0.912, P < 0.0001. The lower limits of sensitivity were determined to be approximately 100 copies/ml and 1,200 to 1,400 copies/ml for the NucliSens and Quantiplex assays, respectively.

  11. Miniaturized isothermal nucleic acid amplification, a review.

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    Asiello, Peter J; Baeumner, Antje J

    2011-04-21

    Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.

  12. Bioanalytical applications of isothermal nucleic acid amplification techniques.

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    Deng, Huimin; Gao, Zhiqiang

    2015-01-01

    The most popular in vitro nucleic acid amplification techniques like polymerase chain reaction (PCR) including real-time PCR are costly and require thermocycling, rendering them unsuitable for uses at point-of-care. Highly efficient in vitro nucleic acid amplification techniques using simple, portable and low-cost instruments are crucial in disease diagnosis, mutation detection and biodefense. Toward this goal, isothermal amplification techniques that represent a group of attractive in vitro nucleic acid amplification techniques for bioanalysis have been developed. Unlike PCR where polymerases are easily deactivated by thermally labile constituents in a sample, some of the isothermal nucleic acid amplification techniques, such as helicase-dependent amplification and nucleic acid sequence-based amplification, enable the detection of bioanalytes with much simplified protocols and with minimal sample preparations since the entire amplification processes are performed isothermally. This review focuses on the isothermal nucleic acid amplification techniques and their applications in bioanalytical chemistry. Starting off from their amplification mechanisms and significant properties, the adoption of isothermal amplification techniques in bioanalytical chemistry and their future perspectives are discussed. Representative examples illustrating the performance and advantages of each isothermal amplification technique are discussed along with some discussion on the advantages and disadvantages of each technique.

  13. Isothermal Amplification of Nucleic Acids.

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    Zhao, Yongxi; Chen, Feng; Li, Qian; Wang, Lihua; Fan, Chunhai

    2015-11-25

    Isothermal amplification of nucleic acids is a simple process that rapidly and efficiently accumulates nucleic acid sequences at constant temperature. Since the early 1990s, various isothermal amplification techniques have been developed as alternatives to polymerase chain reaction (PCR). These isothermal amplification methods have been used for biosensing targets such as DNA, RNA, cells, proteins, small molecules, and ions. The applications of these techniques for in situ or intracellular bioimaging and sequencing have been amply demonstrated. Amplicons produced by isothermal amplification methods have also been utilized to construct versatile nucleic acid nanomaterials for promising applications in biomedicine, bioimaging, and biosensing. The integration of isothermal amplification into microsystems or portable devices improves nucleic acid-based on-site assays and confers high sensitivity. Single-cell and single-molecule analyses have also been implemented based on integrated microfluidic systems. In this review, we provide a comprehensive overview of the isothermal amplification of nucleic acids encompassing work published in the past two decades. First, different isothermal amplification techniques are classified into three types based on reaction kinetics. Then, we summarize the applications of isothermal amplification in bioanalysis, diagnostics, nanotechnology, materials science, and device integration. Finally, several challenges and perspectives in the field are discussed.

  14. Enrichment, Amplification, and Sequence-Based Typing of Salmonella enterica and Other Foodborne Pathogens.

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    Edlind, Tom; Brewster, Jeffrey D; Paoli, George C

    2017-01-01

    Detection of Salmonella enterica in foods typically involves microbiological enrichment, molecular-based assay, and subsequent isolation and identification of a pure culture. This is ideally followed by strain typing, which provides information critical to the investigation of outbreaks and the attribution of their sources. Pulsed-field gel electrophoresis is the "gold standard" for S. enterica strain typing, but its limitations have encouraged the search for alternative methods, including whole genome sequencing. Both methods typically require a pure culture, which adds to the cost and turnaround time. A more rapid and cost-effective method with sufficient discriminatory power would benefit food industries, regulatory agencies, and public health laboratories. To address this need, a novel enrichment, amplification, and sequence-based typing (EAST) approach was developed involving (i) overnight enrichment and total DNA preparation, (ii) amplification of polymorphic tandem repeat-containing loci with electrophoretic detection, and (iii) DNA sequencing and bioinformatic analysis to identify related strains. EAST requires 3 days or less and provides a strain resolution that exceeds serotyping and is comparable to pulsed-field gel electrophoresis. Evaluation with spiked ground turkey demonstrated its sensitivity (with a starting inoculum of ≤1 CFU/g) and specificity (with unique or nearly unique alleles relative to databases of >1,000 strains). In tests with unspiked retail chicken parts, 3 of 11 samples yielded S. enterica -specific PCR products. Sequence analysis of three distinct typing targets (SeMT1, SeCRISPR1, and SeCRISPR2) revealed consistent similarities to specific serotype Schwarzengrund, Montevideo, and Typhimurium strains. EAST provides a time-saving and cost-effective approach for detecting and typing foodborne S. enterica , and postenrichment steps can be commercially outsourced to facilitate its implementation. Initial studies with Listeria

  15. Nucleic acid amplification: Alternative methods of polymerase chain reaction.

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    Fakruddin, Md; Mannan, Khanjada Shahnewaj Bin; Chowdhury, Abhijit; Mazumdar, Reaz Mohammad; Hossain, Md Nur; Islam, Sumaiya; Chowdhury, Md Alimuddin

    2013-10-01

    Nucleic acid amplification is a valuable molecular tool not only in basic research but also in application oriented fields, such as clinical medicine development, infectious diseases diagnosis, gene cloning and industrial quality control. A comperehensive review of the literature on the principles, applications, challenges and prospects of different alternative methods of polymerase chain reaction (PCR) was performed. PCR was the first nucleic acid amplification method. With the advancement of research, a no of alternative nucleic acid amplification methods has been developed such as loop mediated isothermal amplification, nucleic acid sequence based amplification, strand displacement amplification, multiple displacement amplification. Most of the alternative methods are isothermal obviating the need for thermal cyclers. Though principles of most of the alternate methods are relatively complex than that of PCR, they offer better applicability and sensitivity in cases where PCR has limitations. Most of the alternate methods still have to prove themselves through extensive validation studies and are not available in commercial form; they pose the potentiality to be used as replacements of PCR. Continuous research is going on in different parts of the world to make these methods viable technically and economically.

  16. The application of nucleic acid sequence?based amplification,real?time PCR and GM test in invasive aspergillosis diagnosis%核酸序列依赖性扩增、Real?time PCR及GM试验诊断侵袭性曲霉菌感染的临床应用评价

    Institute of Scientific and Technical Information of China (English)

    王立朋; 鲍翠霞; 于丽梅; 张晓录; 于威娟; 张霞; 李玮; 黄葆华; 李杰

    2015-01-01

    Objective To study the diagnostic performance of nucleic acid sequence?based amplification ( NASBA) assay,real?time PCR and GM test in detecting invasive aspergillosis for clinical diagnosis.Methods Blood samples from 80 patients at a high risk for IA were collected during from November 2013 to June 2014.These patients were categorized as 8 proven IA,26 probable IA, and 46 non?IA according to the 2008 revised definitions of EORTC/MSG.Blood samples were tested by NASBA,real?time PCR and GM test and their diagnostic parameters were calculated,respectively.Result The sensitivity of NASBA,real?time PCR and GM test was 76.47%,67.65% and 52.94%,while their specificity was 80.43%,89.13%,80.43%,respectively.The efficiency of various com?binations of tests was also evaluated.Perfect specificity (100%) and positive predictive value (100%) were achieved by combining NASBA and real?time PCR as a serial testing.A combination of NASBA and real?time PCR as a parallel testing was the most sensitive (94.12%).Conclusion The sensitivity and specificity of NASBA and real?time PCR were superior to GM test.Combination of these assays could be particularly useful in specific clinical situations.%目的 核酸序列依赖性扩增 ( nucleic acid sequence?based amplification,NASBA)、Real?time PCR及GM试验在侵袭性曲霉菌感染中的诊断价值. 方法 收集2013年11月~2014年6月临床上曲霉菌感染高危病患的血液标本80例,并根据EORTC/MSG诊断标准分为确诊组8例,拟诊组26例,非感染组46例,分别利用NASBA、real?time PCR及GM试验进行检测,计算3种方法的诊断指标并分析评价. 结果 NASBA、real?time PCR及GM试验3种方法的灵敏度分别为76.47%、67.65%、52.94%,特异度分别为80.43%、89.13%、80.43%. 联合诊断结果显示,NASBA与real?time PCR串联方案有最好的特异度 (100%)及阳性预测值(100%);NASBA与real?time PCR并联方案则最为灵敏(94.12%). 结论 NASBA用于诊断IA最为敏感,而real?time PCR

  17. Detection and quantification of the toxic microalgae Karenia brevis using lab on a chip mRNA sequence-based amplification.

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    Loukas, Christos-Moritz; McQuillan, Jonathan S; Laouenan, Florian; Tsaloglou, Maria-Nefeli; Ruano-Lopez, Jesus M; Mowlem, Matthew C

    2017-08-01

    Now and again, the rapid proliferation of certain species of phytoplankton can give rise to Harmful Algal Blooms, which pose a serious threat to marine life and human health. Current methods of monitoring phytoplankton are limited by poor specificity or by the requirement to return samples to a highly resourced, centralised lab. The Lab Card is a small, microfluidic cassette which, when used in tandem with a portable Lab Card Reader can be used to sensitively and specifically quantify harmful algae in the field, from nucleic acid extracts using RNA amplification; a sensitive and specific method for the enumeration of potentially any species based on their unique genetic signatures. This study reports the culmination of work to develop a Lab Card-based genetic assay to quantify the harmful algae Karenia brevis using mRNA amplification by the Nucleic Acid Sequence Based Amplification (NASBA) method. K. brevis cells were quantified by amplification of the rbcL gene transcript in nucleic acid extracts of K. brevis cell samples. A novel enzyme dehydration and preservation method was combined with a pre-existing reagent Gelification method to prepare fully preserved Lab Cards with a shelf-life of at least six weeks prior to use. Using an internal control (IC), the Lab Card-based rbcL NASBA was demonstrated for the quantification of K. brevis from cell extracts containing between 50 and 5000 cells. This is the first demonstration of quantitation of K. brevis using IC-NASBA on a Lab Card. Copyright © 2017. Published by Elsevier B.V.

  18. A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR.

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    Gao, Song; He, Dan; Li, Guangquan; Zhang, Yanhua; Lv, Huiying; Wang, Li

    2016-09-15

    Thermal asymmetric staggered PCR is the most widely used technique to obtain the flanking sequences. However, it has some limitations, including a low rate of positivity, and complex operation. In this study, a improved method of it was made based on suppression-PCR and touchdown PCR. The PCR fragment obtained by the amplification was used directly for sequencing after gel purification. Using this improved method, the positive rate of amplified flanking sequences of the ATMT mutants reached 99%. In addition, the time from DNA extraction to flanking sequence analysis was shortened to 2 days with about 6 dollars each sample.

  19. Pelacakan Gen Env-TM Virus Penyakit Jembrana Galur Tabanan 1995 dengan Metode Nucleic Acid Sequence Based Amplificaton

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    Asmarani Kusumawati

    2016-01-01

    Full Text Available Jembrana disease is an infectious disease in Bali cattle cause by a member of lentivirus calledjembrana disease virus (JDV. It causes an acute and severe disease syndrome with short incubationperiod. As the disease has spread to several areas in Indonesia, a simple and rapid detection method isrequired. The objective of this study to apply rapid diagnostic method for JDVTabanan 1995 strain basedon Nucleic Acid Sequence Based Amplification (NASBA methods targeting env-tm gene. The steps of thisresearch consisted of viral RNA isolation from organ and blood of cattle experimentaly infected withJDVTabanan 1995 strain . RNA amplification was conducted by NASBA using waterbath. The NASBAproducts were then separated on 2 % agarose gel. Using this technique JDV positive result was obtainedfrom organ samples such as spleen, liver, lung, prefemoralis lymph node, prescapularis lymph node andblood generating a RNA fragment of 207 bp. In this study, diagnosis method for env tm of JDV Tabanan1995 strain can be conducted by isothermal amplification NASBA.

  20. Nucleic acid amplification using microfluidic systems.

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    Chang, Chen-Min; Chang, Wen-Hsin; Wang, Chih-Hung; Wang, Jung-Hao; Mai, John D; Lee, Gwo-Bin

    2013-04-07

    In the post-human-genome-project era, the development of molecular diagnostic techniques has advanced the frontiers of biomedical research. Nucleic-acid-based technology (NAT) plays an especially important role in molecular diagnosis. However, most research and clinical protocols still rely on the manual analysis of individual samples by skilled technicians which is a time-consuming and labor-intensive process. Recently, with advances in microfluidic designs, integrated micro total-analysis-systems have emerged to overcome the limitations of traditional detection assays. These microfluidic systems have the capability to rapidly perform experiments in parallel and with a high-throughput which allows a NAT analysis to be completed in a few hours or even a few minutes. These features have a significant beneficial influence on many aspects of traditional biological or biochemical research and this new technology is promising for improving molecular diagnosis. Thus, in the foreseeable future, microfluidic systems developed for molecular diagnosis using NAT will become an important tool in clinical diagnosis. One of the critical issues for NAT is nucleic acid amplification. In this review article, recent advances in nucleic acid amplification techniques using microfluidic systems will be reviewed. Different approaches for fast amplification of nucleic acids for molecular diagnosis will be highlighted.

  1. Digital Microfluidics for Nucleic Acid Amplification

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    Beatriz Coelho

    2017-06-01

    Full Text Available Digital Microfluidics (DMF has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings.

  2. A multicenter international evaluation of single-tube amplification protocols for sequencing-based typing of HLA-DRB1 and HLA-DRB3,4,5.

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    Sayer, D C; Whidborne, R; De Santis, D; Rozemuller, E H; Christiansen, F T; Tilanus, M G

    2004-05-01

    We have described previously a novel single-tube polymerase chain reaction (PCR) amplification (STAmp) protocol for the efficient sequencing-based typing (SBT) of human leukocyte antigen (HLA)-DRB1. The PCR amplification mix includes primers to each of seven allele group-sequence motifs. We have applied this principle to the simultaneous SBT of HLA-DRB3, -DRB4, and -DRB5 using locus specific primers. We report here a multicenter international evaluation of the STAmp protocols performed as a component of the 13th International Histocompatibility Workshop. Identical amplification primer mixes, sequencing primers, and DNA were sent to participating laboratories. The primer mixes contained the amplification primers and the PCR buffer. Each laboratory was requested to amplify the DNA with the primer mixes and perform SBT on the resulting PCR protocols, using their own protocols, and return the typing results for analysis. The reported results indicated that the expected sequence could be obtained with a variety of PCR amplification and sequencing platforms and protocols. There were difficulties but these seemed unrelated to STAmp reagents and suggest that optimal SBT results can be obtained if bi-directional sequencing is performed and software is used for sequence verification and editing. This indicates that SBT by STAmp can be applied in many laboratories for high-throughput HLA-DRB1 and HLA-DRB3,4,5 SBT.

  3. SNBRFinder: A Sequence-Based Hybrid Algorithm for Enhanced Prediction of Nucleic Acid-Binding Residues.

    Directory of Open Access Journals (Sweden)

    Xiaoxia Yang

    Full Text Available Protein-nucleic acid interactions are central to various fundamental biological processes. Automated methods capable of reliably identifying DNA- and RNA-binding residues in protein sequence are assuming ever-increasing importance. The majority of current algorithms rely on feature-based prediction, but their accuracy remains to be further improved. Here we propose a sequence-based hybrid algorithm SNBRFinder (Sequence-based Nucleic acid-Binding Residue Finder by merging a feature predictor SNBRFinderF and a template predictor SNBRFinderT. SNBRFinderF was established using the support vector machine whose inputs include sequence profile and other complementary sequence descriptors, while SNBRFinderT was implemented with the sequence alignment algorithm based on profile hidden Markov models to capture the weakly homologous template of query sequence. Experimental results show that SNBRFinderF was clearly superior to the commonly used sequence profile-based predictor and SNBRFinderT can achieve comparable performance to the structure-based template methods. Leveraging the complementary relationship between these two predictors, SNBRFinder reasonably improved the performance of both DNA- and RNA-binding residue predictions. More importantly, the sequence-based hybrid prediction reached competitive performance relative to our previous structure-based counterpart. Our extensive and stringent comparisons show that SNBRFinder has obvious advantages over the existing sequence-based prediction algorithms. The value of our algorithm is highlighted by establishing an easy-to-use web server that is freely accessible at http://ibi.hzau.edu.cn/SNBRFinder.

  4. One New Method of Nucleic Acid Amplification-Loop-mediated Isothermal Amplification of DNA

    Institute of Scientific and Technical Information of China (English)

    Xue-en FANG; Jian LI; Qin CHEN

    2008-01-01

    Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method, which amplifies DNA with high specificity, sensitivity, rapidity and efficiency under isothermal conditions using a set of four specially designed primers and a Bst DNA polymerase with strand displacement activity. The basic principle, characteristics, development of LAMP and its applications are summarized in this article.

  5. Next-generation sequencing-based 5' rapid amplification of cDNA ends for alternative promoters.

    Science.gov (United States)

    Perera, Bambarendage P U; Kim, Joomyeong

    2016-02-01

    Mammalian genomes contain many unknown alternative first exons and promoters. Thus, we have modified the existing 5'RACE (5' rapid amplification of cDNA ends) approach into a next-generation sequencing (NGS)-based new protocol that can identify these alternative promoters. This protocol has incorporated two main ideas: (i) 5'RACE starting from the known second exons of genes and (ii) NGS-based sequencing of the subsequent cDNA products. This protocol also provides a bioinformatics strategy that processes the sequence reads from NGS runs. This protocol has successfully identified several alternative promoters for an imprinted gene, PEG3. Overall, this NGS-based 5'RACE protocol is a sensitive and reliable method for detecting low-abundant transcripts and promoters.

  6. Repetitive sequence based polymerase chain reaction to differentiate close bacteria strains in acidic sites

    Institute of Scientific and Technical Information of China (English)

    XIE Ming; YIN Hua-qun; LIU Yi; LIU Jie; LIU Xue-duan

    2008-01-01

    To study the diversity of bacteria strains newly isolated from several acid mine drainage(AMD) sites in China,repetitive sequence based polymerase chain reaction (rep-PCR),a well established technology for diversity analysis of closely related bacteria strains,was conducted on 30 strains of bacteria Leptospirillum ferriphilium,8 strains of bacteria Acidithiobacillus ferrooxidans,as well as the Acidithiobacillus ferrooxidans type strain ATCC (American Type Culture Collection) 23270.The results showed that,using ERIC and BOX primer sets,rep-PCR produced highly discriminatory banding patterns.Phylogenetic analysis based on ERIC-PCR banding types was made and the results indicated that rep-PCR could be used as a rapid and highly discriminatory screening technique in studying bacterial diversity,especially in differentiating bacteria within one species in AMD.

  7. Diagnostic Devices for Isothermal Nucleic Acid Amplification

    Directory of Open Access Journals (Sweden)

    Chia-Chen Chang

    2012-06-01

    Full Text Available Since the development of the polymerase chain reaction (PCR technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

  8. Loop-mediated isothermal amplification for detection of nucleic acids.

    Science.gov (United States)

    Tanner, Nathan A; Evans, Thomas C

    2014-01-06

    Sequence-specific isothermal nucleic acid amplification techniques are ideally suited for use in molecular diagnostic applications because they do not require thermal cycling equipment and the reactions are typically fast. One of the most widely cited isothermal techniques is termed loop-mediated isothermal amplification (LAMP). This protocol allows amplification times as fast as 5 to 10 min. Furthermore, various methodologies to detect amplification have been applied to LAMP to increase its utility for the point-of-care market. Basic LAMP protocols are provided herein for detection of specific DNA and RNA targets, along with a method to perform multiplex LAMP reactions, permitting even greater flexibility from this powerful technique.

  9. Comparison of Three Nucleic Acid Amplification Assays of Cerebrospinal Fluid for Diagnosis of Cytomegalovirus Encephalitis

    Science.gov (United States)

    Bestetti, Arabella; Pierotti, Chiara; Terreni, Mariarosa; Zappa, Alessandra; Vago, Luca; Lazzarin, Adriano; Cinque, Paola

    2001-01-01

    The diagnostic reliabilities of three cytomegalovirus (CMV) nucleic acid amplification assays of cerebrospinal fluid (CSF) were compared by using CSF samples from human immunodeficiency virus-infected patients with a postmortem histopathological diagnosis of CMV encephalitis (n = 15) or other central nervous system conditions (n = 16). By using a nested PCR assay, the quantitative COBAS AMPLICOR CMV MONITOR PCR, and the NucliSens CMV pp67 nucleic acid sequence-based amplification assay, sensitivities were 93.3, 86.6, and 93.3%, respectively, and specificities were 93.7, 93.7, and 87.5%, respectively. The COBAS AMPLICOR assay revealed significantly higher CMV DNA levels in patients with diffuse ventriculoencephalitis than in patients with focal periventricular lesions. PMID:11230445

  10. Prevalence of Plasmodium spp. in malaria asymptomatic African migrants assessed by nucleic acid sequence based amplification

    NARCIS (Netherlands)

    Marangi, M.; Di Tullio, R.; Mens, P.F.; Martinelli, D.; Fazio, V.; Angarano, G.; Schallig, H.D.F.H.; Giangaspero, A.; Scotto, G.

    2009-01-01

    Background: Malaria is one of the most important infectious diseases in the world. Although most cases are found distributed in the tropical regions of Africa, Asia, Central and South Americas, there is in Europe a significant increase in the number of imported cases in non-endemic countries, in

  11. Prevalence of Plasmodium spp. in malaria asymptomatic African migrants assessed by nucleic acid sequence based amplification

    NARCIS (Netherlands)

    M. Marangi; R. Di Tullio; P.F. Mens; D. Martinelli; V. Fazio; G. Angarano; H.D.F.H. Schallig; A. Giangaspero; G. Scotto

    2009-01-01

    Background: Malaria is one of the most important infectious diseases in the world. Although most cases are found distributed in the tropical regions of Africa, Asia, Central and South Americas, there is in Europe a significant increase in the number of imported cases in non-endemic countries, in par

  12. Development and implementation of real-time nucleic acid amplification for the detection of enterovirus infections in comparison to rapid culture of various clinical specimens

    NARCIS (Netherlands)

    van Doornum, G J J; Schutten, Martin; Voermans, J; Guldemeester, G J J; Niesters, H G M

    2007-01-01

    Several real-time PCR and nucleic acid sequence-based amplification (NASBA) primer pairs and a modified real-time PCR primer pair for the detection of enteroviruses were compared. The modified real-time PCR primer pair was evaluated on clinical samples in comparison with cell culture using the Magna

  13. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    OpenAIRE

    Mauk, Michael G.; Changchun Liu; Jinzhao Song; Bau, Haim H.

    2015-01-01

    Microfluidic components and systems for rapid (<60 min), low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs) are described. A microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (poly...

  14. An integrated portable hand-held analyser for real-time isothermal nucleic acid amplification.

    Science.gov (United States)

    Smith, Matthew C; Steimle, George; Ivanov, Stan; Holly, Mark; Fries, David P

    2007-08-29

    A compact hand-held heated fluorometric instrument for performing real-time isothermal nucleic acid amplification and detection is described. The optoelectronic instrument combines a Printed Circuit Board/Micro Electro Mechanical Systems (PCB/MEMS) reaction detection/chamber containing an integrated resistive heater with attached miniature LED light source and photo-detector and a disposable glass waveguide capillary to enable a mini-fluorometer. The fluorometer is fabricated and assembled in planar geometry, rolled into a tubular format and packaged with custom control electronics to form the hand-held reactor. Positive or negative results for each reaction are displayed to the user using an LED interface. Reaction data is stored in FLASH memory for retrieval via an in-built USB connection. Operating on one disposable 3 V lithium battery >12, 60 min reactions can be performed. Maximum dimensions of the system are 150 mm (h) x 48 mm (d) x 40 mm (w), the total instrument weight (with battery) is 140 g. The system produces comparable results to laboratory instrumentation when performing a real-time nucleic acid sequence-based amplification (NASBA) reaction, and also displayed comparable precision, accuracy and resolution to laboratory-based real-time nucleic acid amplification instrumentation. A good linear response (R2 = 0.948) to fluorescein gradients ranging from 0.5 to 10 microM was also obtained from the instrument indicating that it may be utilized for other fluorometric assays. This instrument enables an inexpensive, compact approach to in-field genetic screening, providing results comparable to laboratory equipment with rapid user feedback as to the status of the reaction.

  15. Quantitation of viral load using real-time amplification techniques

    NARCIS (Netherlands)

    Niesters, H G

    2001-01-01

    Real-time PCR amplification techniques are currently used to determine the viral load in clinical samples for an increasing number of targets. Real-time PCR reduces the time necessary to generate results after amplification. In-house developed PCR and nucleic acid sequence-based amplification (NASBA

  16. The structural analysis of protein sequences based on the quasi-amino acids code

    Institute of Scientific and Technical Information of China (English)

    Zhu Ping; Tang Xu-Qing; Xu Zhen-Yuan

    2009-01-01

    Proteomics is the study of proteins and their interactions in a cell. With the successful completion of the Human Genome Project, it comes the postgenome era when the proteomics technology is emerging. This paper studies protein molecule from the algebraic point of view. The algebraic system (∑, +, *) is introduced, where ∑ is the set of 64 codons. According to the characteristics of (∑,+, *), a novel quasi-amino acids code classification method is introduced and the corresponding algebraic operation table over the set ZU of the 16 kinds of quasi-amino acids is established. The internal relation is revealed about quasi-amino acids. The results show that there exist some very close correlations between the properties of the quasi-amino acids and the codon. All these correlation relationships may play an important part in establishing the logic relationship between codons and the quasi-amino acids during the course of life origination. According to Ma F et al (2003 J. Anhui Agricultural University 30 439), the corresponding relation and the excellent properties about amino acids code are very difficult to observe. The present paper shows that (ZU, +, ×) is a field. Furthermore, the operational results display that the codon tga has different property from other stop codons. In fact, in the mitochondrion from human and ox genomic codon, tga is just tryptophane, is not the stop codon like in other genetic code, it is the case of the Chen W C et al (2002 Acta Biophysica Sinica 18(1) 87). The present theory avoids some inexplicable events of the 20 kinds of amino acids code, in other words it solves the problem of 'the 64 codon assignments of mRNA to amino acids is probably completely wrong' proposed by Yang (2006 Progress in Modern Biomedicine 6 3).

  17. Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics.

    Science.gov (United States)

    Linnes, J C; Rodriguez, N M; Liu, L; Klapperich, C M

    2016-04-01

    Devices based on rapid, paper-based, isothermal nucleic acid amplification techniques have recently emerged with the potential to fill a growing need for highly sensitive point-of-care diagnostics throughout the world. As this field develops, such devices will require optimized materials that promote amplification and sample preparation. Herein, we systematically investigated isothermal nucleic acid amplification in materials currently used in rapid diagnostics (cellulose paper, glass fiber, and nitrocellulose) and two additional porous membranes with upstream sample preparation capabilities (polyethersulfone and polycarbonate). We compared amplification efficiency from four separate DNA and RNA targets (Bordetella pertussis, Chlamydia trachomatis, Neisseria gonorrhoeae, and Influenza A H1N1) within these materials using two different isothermal amplification schemes, helicase dependent amplification (tHDA) and loop-mediated isothermal amplification (LAMP), and traditional PCR. We found that the current paper-based diagnostic membranes inhibited nucleic acid amplification when compared to membrane-free controls; however, polyethersulfone allowed for efficient amplification in both LAMP and tHDA reactions. Further, observing the performance of traditional PCR amplification within these membranes was not predicative of their effects on in situ LAMP and tHDA. Polyethersulfone is a new material for paper-based nucleic acid amplification, yet provides an optimal support for rapid molecular diagnostics for point-of-care applications.

  18. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification

    Directory of Open Access Journals (Sweden)

    Michael G. Mauk

    2015-10-01

    Full Text Available Microfluidic components and systems for rapid (<60 min, low-cost, convenient, field-deployable sequence-specific nucleic acid-based amplification tests (NAATs are described. A microfluidic point-of-care (POC diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of “lab on a chip” NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1 nucleic acids (NAs are extracted from relatively large (~mL volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase (“membrane” to capture sample NAs in a flow-through, filtration mode; (2 NAs captured on the membrane are isothermally (~65 °C amplified; (3 amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4 paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD better than 103 virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  19. Shortening distance of forward and reverse primers for nucleic acid isothermal amplification.

    Science.gov (United States)

    Haitao, Qu; Wenchao, Zhang; Xiaohui, Zhang; Xiujun, Wang; Sulong, Li

    2014-06-01

    Existent nucleic acid isothermal detection techniques for clinical diseases are difficult to promote greatly due to limitations in such aspects as methodology, costs of detection, amplification efficiency and conditions for operation. There is therefore an urgent need for a new isothermal amplification method with the characteristics of high accuracy, easy operation, short time of detection and low costs. We have devised a new method of nucleic acid isothermal amplification using Bst DNA polymerase under isothermal conditions (60-65°C). We call this method of amplification by shortening the distance between forward and reverse primers for nucleic acid isothermal amplification SDAMP. The results demonstrated that this technique is highly sensitive, specific and has short reaction times (40-60 min). Results of sequencing show that the products of SDAMP amplification are mainly polymers formed by series connection of monomers formed through linkage of forward primer and complementary sequences in reverse primer via a few bases. The method is different from current methods of nucleic acid amplification. Our study shows, however, that it is a specific method of nucleic acid isothermal amplification depending on interactions between primers and DNA template.

  20. Detection and identification of human Plasmodium species with real-time quantitative nucleic acid sequence-based amplification

    NARCIS (Netherlands)

    P.F. Mens; G.J. Schoone; P.A. Kager; H.D.F.H. Schallig

    2006-01-01

    Background: Decisions concerning malaria treatment depend on species identification causing disease. Microscopy is most frequently used, but at low parasitaemia (< 20 parasites/mu l) the technique becomes less sensitive and time consuming. Rapid diagnostic tests based on Plasmodium antigen detection

  1. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification.

    Science.gov (United States)

    Mauk, Michael G; Liu, Changchun; Song, Jinzhao; Bau, Haim H

    2015-10-20

    Microfluidic components and systems for rapid (microfluidic point-of-care (POC) diagnostics test to quantify HIV viral load from blood samples serves as a representative and instructive example to discuss the technical issues and capabilities of "lab on a chip" NAAT devices. A portable, miniaturized POC NAAT with performance comparable to conventional PCR (polymerase-chain reaction)-based tests in clinical laboratories can be realized with a disposable, palm-sized, plastic microfluidic chip in which: (1) nucleic acids (NAs) are extracted from relatively large (~mL) volume sample lysates using an embedded porous silica glass fiber or cellulose binding phase ("membrane") to capture sample NAs in a flow-through, filtration mode; (2) NAs captured on the membrane are isothermally (~65 °C) amplified; (3) amplicon production is monitored by real-time fluorescence detection, such as with a smartphone CCD camera serving as a low-cost detector; and (4) paraffin-encapsulated, lyophilized reagents for temperature-activated release are pre-stored in the chip. Limits of Detection (LOD) better than 10³ virons/sample can be achieved. A modified chip with conduits hosting a diffusion-mode amplification process provides a simple visual indicator to readily quantify sample NA template. In addition, a companion microfluidic device for extracting plasma from whole blood without a centrifuge, generating cell-free plasma for chip-based molecular diagnostics, is described. Extensions to a myriad of related applications including, for example, food testing, cancer screening, and insect genotyping are briefly surveyed.

  2. Isothermal amplification detection of nucleic acids by a double-nicked beacon.

    Science.gov (United States)

    Shi, Chao; Zhou, Meiling; Pan, Mei; Zhong, Guilin; Ma, Cuiping

    2016-03-01

    Isothermal and rapid amplification detection of nucleic acids is an important technology in environmental monitoring, foodborne pathogen detection, and point-of-care clinical diagnostics. Here we have developed a novel method of isothermal signal amplification for single-stranded DNA (ssDNA) detection. The ssDNA target could be used as an initiator, coupled with a double-nicked molecular beacon, to originate amplification cycles, achieving cascade signal amplification. In addition, the method showed good specificity and strong anti-jamming capability. Overall, it is a one-pot and isothermal strand displacement amplification method without the requirement of a stepwise procedure, which greatly simplifies the experimental procedure and decreases the probability of contamination of samples. With its advantages, the method would be very useful to detect nucleic acids in point-of-care or field use.

  3. Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

    Science.gov (United States)

    Qing, Taiping; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Wen, Li; Shangguan, Jingfang; Mao, Zhengui; Lei, Yanli

    2016-04-01

    Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

  4. Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices

    Directory of Open Access Journals (Sweden)

    Giuseppe Spoto

    2012-12-01

    Full Text Available Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed.

  5. Powerful Amplification Cascades of FRET-Based Two-Layer Nonenzymatic Nucleic Acid Circuits.

    Science.gov (United States)

    Quan, Ke; Huang, Jin; Yang, Xiaohai; Yang, Yanjing; Ying, Le; Wang, He; Xie, Nuli; Ou, Min; Wang, Kemin

    2016-06-07

    Nucleic acid circuits have played important roles in biological engineering and have increasingly attracted researchers' attention. They are primarily based on nucleic acid hybridizations and strand displacement reactions between nucleic acid probes of different lengths. Signal amplification schemes that do not rely on protein enzyme show great potential in analytical applications. While the single amplification circuit often achieves linear amplification that may not meet the need for detection of target in a very small amount, it is very necessary to construct cascade circuits that allow for larger amplification of inputs. Herein, we have successfully engineered powerful amplification cascades of FRET-based two-layer nonenzymatic nucleic acid circuits, in which the outputs of catalyzed hairpin assembly (CHA) activate hybridization chain reactions (HCR) circuits to induce repeated hybridization, allowing real-time monitoring of self-assembly process by FRET signal. The cascades can yield 50000-fold signal amplification with the help of the well-designed and high-quality nucleic acid circuit amplifiers. Subsequently, with coupling of structure-switching aptamer, as low as 200 pM adenosine is detected in buffer, as well as in human serum. To our knowledge, we have for the first time realized real-time monitoring adaptation of HCR to CHA circuits and achieved amplified detection of nucleic acids and small molecules with relatively high sensitivity.

  6. Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids.

    Science.gov (United States)

    Kivlehan, Francine; Mavré, François; Talini, Luc; Limoges, Benoît; Marchal, Damien

    2011-09-21

    We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.

  7. Novel bioluminescent quantitative detection of nucleic acid amplification in real-time.

    Directory of Open Access Journals (Sweden)

    Olga A Gandelman

    Full Text Available BACKGROUND: The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. PRINCIPAL FINDINGS: Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. CONCLUSIONS: The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a

  8. A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

    Directory of Open Access Journals (Sweden)

    Pascal Craw

    2015-09-01

    Full Text Available Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings.

  9. Instrument for Real-Time Digital Nucleic Acid Amplification on Custom Microfluidic Devices.

    Science.gov (United States)

    Selck, David A; Ismagilov, Rustem F

    2016-01-01

    Nucleic acid amplification tests that are coupled with a digital readout enable the absolute quantification of single molecules, even at ultralow concentrations. Digital methods are robust, versatile and compatible with many amplification chemistries including isothermal amplification, making them particularly invaluable to assays that require sensitive detection, such as the quantification of viral load in occult infections or detection of sparse amounts of DNA from forensic samples. A number of microfluidic platforms are being developed for carrying out digital amplification. However, the mechanistic investigation and optimization of digital assays has been limited by the lack of real-time kinetic information about which factors affect the digital efficiency and analytical sensitivity of a reaction. Commercially available instruments that are capable of tracking digital reactions in real-time are restricted to only a small number of device types and sample-preparation strategies. Thus, most researchers who wish to develop, study, or optimize digital assays rely on the rate of the amplification reaction when performed in a bulk experiment, which is now recognized as an unreliable predictor of digital efficiency. To expand our ability to study how digital reactions proceed in real-time and enable us to optimize both the digital efficiency and analytical sensitivity of digital assays, we built a custom large-format digital real-time amplification instrument that can accommodate a wide variety of devices, amplification chemistries and sample-handling conditions. Herein, we validate this instrument, we provide detailed schematics that will enable others to build their own custom instruments, and we include a complete custom software suite to collect and analyze the data retrieved from the instrument. We believe assay optimizations enabled by this instrument will improve the current limits of nucleic acid detection and quantification, improving our fundamental

  10. Amplification of deoxyribonucleic acid (DNA) fragment using two ...

    African Journals Online (AJOL)

    user

    2011-04-11

    Apr 11, 2011 ... 2Deparment of Life Sciences and Technology, Xinxiang Medical University, ... comprises three steps: denaturation at a high temperature, annealing at a low ..... type 1 by an in-house method using locked nucleic acid-based.

  11. On-Chip, Amplification-Free Quantification of Nucleic Acid for Point-of-Care Diagnosis

    OpenAIRE

    Yen, Tony Minghung

    2017-01-01

    This dissertation demonstrates three physical device concepts to overcome limitations in point-of-care quantification of nucleic acids. Enabling sensitive, high throughput nucleic acid quantification on a chip, outside of hospital and centralized laboratory setting, is crucial for improving pathogen detection and cancer diagnosis and prognosis. Among existing platforms, microarray have the advantages of being amplification free, low instrument cost, and high throughput, but are generally le...

  12. Development of Temperature Control Solutions for Non-Instrumented Nucleic Acid Amplification Tests (NINAAT

    Directory of Open Access Journals (Sweden)

    Tamás Pardy

    2017-06-01

    Full Text Available Non-instrumented nucleic acid amplification tests (NINAAT are a novel paradigm in portable molecular diagnostics. They offer the high detection accuracy characteristic of nucleic acid amplification tests (NAAT in a self-contained device, without the need for any external instrumentation. These Point-of-Care tests typically employ a Lab-on-a-Chip for liquid handling functionality, and perform isothermal nucleic acid amplification protocols that require low power but high accuracy temperature control in a single well-defined temperature range. We propose temperature control solutions based on commercially available heating elements capable of meeting these challenges, as well as demonstrate the process by which such elements can be fitted to a NINAAT system. Self-regulated and thermostat-controlled resistive heating elements were evaluated through experimental characterization as well as thermal analysis using the finite element method (FEM. We demonstrate that the proposed solutions can support various NAAT protocols, as well as demonstrate an optimal solution for the loop-mediated isothermal amplification (LAMP protocol. Furthermore, we present an Arduino-compatible open-source thermostat developed for NINAAT applications.

  13. Instrument-free nucleic acid amplification assays for global health settings

    Science.gov (United States)

    LaBarre, Paul; Boyle, David; Hawkins, Kenneth; Weigl, Bernhard

    2011-06-01

    Many infectious diseases that affect global health are most accurately diagnosed through nucleic acid amplification and detection. However, existing nucleic acid amplification tests are too expensive and complex for most low-resource settings. The small numbers of centralized laboratories that exist in developing countries tend to be in urban areas and primarily cater to the affluent. In contrast, rural area health care facilities commonly have only basic equipment and health workers have limited training and little ability to maintain equipment and handle reagents.1 Reliable electric power is a common infrastructure shortfall. In this paper, we discuss a practical approach to the design and development of non-instrumented molecular diagnostic tests that exploit the benefits of isothermal amplification strategies. We identify modular instrument-free technologies for sample collection, sample preparation, amplification, heating, and detection. By appropriately selecting and integrating these instrument-free modules, we envision development of an easy to use, infrastructure independent diagnostic test that will enable increased use of highly accurate molecular diagnostics at the point of care in low-resource settings.

  14. Direct detection of potato leafroll virus in potato tubers by immunocapture and the isothermal nucleic acid amplification method NASBA

    NARCIS (Netherlands)

    Leone, G.; Schijndel, van H.B.; Gemen, van B.; Schoen, C.D.

    1997-01-01

    NASBA, an isothermal amplification method for nucleic acids, was applied to the detection of RNA of potato leafroll virus (PLRV) in a single enzymatic reaction at 41 °C. A set of primers was selected from the coat protein open reading frame sequence of PLRV to allow amplification of viral RNA. The

  15. 核酸扩增技术在病原体检测中的应用%Application of nucleic acid amplification in pathogen detection

    Institute of Scientific and Technical Information of China (English)

    范吉云; 郭晓奎; 李擎天

    2009-01-01

    传统的病原体检测方法在检测敏感性、特异性、应用范围等方面存在许多不足.PCR技术已被用于感染早期的病原体检测,但在应用过程中也面临不少问题.近年来出现的新的核酸扩增技术可弥补普通PCR技术的缺点.此文就其中依赖核酸序列的扩增技术、PCR-胶体金免疫层析技术和多重荧光实时PcR技术进行综述.%The traditional pathogen detection mathods have many disadvantages in the sensitivity, specificity and application range. There are still some problems in PCB, which has been used for pathogen detection in the early stage of infection. New nucleic acid amplification technologies may make up the deficiency of PCR. In this review, three methods including nucleic acid sequence-based amplification (NASBA), PCR-immunochromatography test and multiplex fluorescent real-time PCR technology are described.

  16. Nuclemeter: A Reaction-Diffusion Column for Quantifying Nucleic Acids Undergoing Enzymatic Amplification

    Science.gov (United States)

    Bau, Haim; Liu, Changchun; Killawala, Chitvan; Sadik, Mohamed; Mauk, Michael

    2014-11-01

    Real-time amplification and quantification of specific nucleic acid sequences plays a major role in many medical and biotechnological applications. In the case of infectious diseases, quantification of the pathogen-load in patient specimens is critical to assessing disease progression, effectiveness of drug therapy, and emergence of drug-resistance. Typically, nucleic acid quantification requires sophisticated and expensive instruments, such as real-time PCR machines, which are not appropriate for on-site use and for low resource settings. We describe a simple, low-cost, reactiondiffusion based method for end-point quantification of target nucleic acids undergoing enzymatic amplification. The number of target molecules is inferred from the position of the reaction-diffusion front, analogous to reading temperature in a mercury thermometer. We model the process with the Fisher Kolmogoroff Petrovskii Piscounoff (FKPP) Equation and compare theoretical predictions with experimental observations. The proposed method is suitable for nucleic acid quantification at the point of care, compatible with multiplexing and high-throughput processing, and can function instrument-free. C.L. was supported by NIH/NIAID K25AI099160; M.S. was supported by the Pennsylvania Ben Franklin Technology Development Authority; C.K. and H.B. were funded, in part, by NIH/NIAID 1R41AI104418-01A1.

  17. Effect of nucleic acid binding dyes on DNA extraction, amplification, and STR typing.

    Science.gov (United States)

    Haines, Alicia M; Tobe, Shanan S; Kobus, Hilton J; Linacre, Adrian

    2015-10-01

    We report on the effects of six dyes used in the detection of DNA on the process of DNA extraction, amplification, and detection of STR loci. While dyes can be used to detect the presence of DNA, their use is restricted if they adversely affect subsequent DNA typing processes. Diamond™ Nucleic Acid Dye, GelGreen™, GelRed™, RedSafe™, SYBR(®) Green I, and EvaGreen™ were evaluated in this study. The percentage of dye removed during the extraction process was determined to be: 70.3% for SYBR(®) Green I; 99.6% for RedSafe™; 99.4% for EvaGreen™; 52.7% for Diamond™ Dye; 50.6% for GelRed™, and; could not be determined for GelGreen™. It was then assumed that the amount of dye in the fluorescent quantification assay had no effect on the DNA signal. The presence of all six dyes was then reviewed for their effect on DNA extraction. The t-test showed no significant difference between the dyes and the control. These extracts were then STR profiled and all dyes and control produced full DNA profiles. STR loci in the presence of GelGreen(TM) at 1X concentration showed increased amplification products in comparison to the control samples. Full STR profiles were detected in the presence of EvaGreen™ (1X), although with reduced amplification products. RedSafe™ (1X), Diamond™ Dye (1X), and SYBR(®) Green I (1X) all exhibited varying degrees of locus drop-out with GelRed™ generating no loci at all. We provide recommendations for the best dye to visualize the presence of DNA profile as a biological stain and its subsequent amplification and detection. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. The origin of biased sequence depth in sequence-independent nucleic acid amplification and optimization for efficient massive parallel sequencing.

    Directory of Open Access Journals (Sweden)

    Toon Rosseel

    Full Text Available Sequence Independent Single Primer Amplification is one of the most widely used random amplification approaches in virology for sequencing template preparation. This technique relies on oligonucleotides consisting of a 3' random part used to prime complementary DNA synthesis and a 5' defined tag sequence for subsequent amplification. Recently, this amplification method was combined with next generation sequencing to obtain viral sequences. However, these studies showed a biased distribution of the resulting sequence reads over the analyzed genomes. The aim of this study was to elucidate the mechanisms that lead to biased sequence depth when using random amplification. Avian paramyxovirus type 8 was used as a model RNA virus to investigate these mechanisms. We showed, based on in silico analysis of the sequence depth in relation to GC-content, predicted RNA secondary structure and sequence complementarity to the 3' part of the tag sequence, that the tag sequence has the main contribution to the observed bias in sequence depth. We confirmed this finding experimentally using both fragmented and non-fragmented viral RNAs as well as primers differing in random oligomer length (6 or 12 nucleotides and in the sequence of the amplification tag. The observed oligonucleotide annealing bias can be reduced by extending the random oligomer sequence and by in silico combining sequence data from SISPA experiments using different 5' defined tag sequences. These findings contribute to the optimization of random nucleic acid amplification protocols that are currently required for downstream applications such as viral metagenomics and microarray analysis.

  19. Detection of human cytomegalovirus pp67 late gene transcripts in cerebrospinal fluid of human immunodeficiency virus type 1-infected patients by nucleic acid sequence-based amplification.

    Science.gov (United States)

    Zhang, F; Tetali, S; Wang, X P; Kaplan, M H; Cromme, F V; Ginocchio, C C

    2000-05-01

    This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82. 7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease.

  20. Real-time detection of noroviruses in surface water by use of a broadly reactive nucleic acid sequence-based amplification assay.

    NARCIS (Netherlands)

    Rutjes, Saskia A; Berg, Harold H J L van den; Lodder, Willemijn J; Roda Husman, Ana Maria de

    2006-01-01

    Noroviruses are the most common agents causing outbreaks of viral gastroenteritis. Outbreaks originating from contaminated drinking water and from recreational waters have been described. Due to a lack of cell culture systems, noroviruses are detected mostly by molecular methods. Molecular detection

  1. Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum.

    NARCIS (Netherlands)

    Schneider, P.; Wolters, L.R.; Schoone, G.; Schallig, H.; Sillekens, P.; Hermsen, R.; Sauerwein, R.W.

    2005-01-01

    Determination of the number of malaria parasites by routine or even expert microscopy is not always sufficiently sensitive for detailed quantitative studies on the population dynamics of Plasmodium falciparum, such as intervention or vaccine trials. To circumvent this problem, two more sensitive ass

  2. Application of Legionella pneumophila-specific quantitative real-time PCR combined with direct amplification and sequence-based typing in the diagnosis and epidemiological investigation of Legionnaires' disease.

    Science.gov (United States)

    Mentasti, M; Fry, N K; Afshar, B; Palepou-Foxley, C; Naik, F C; Harrison, T G

    2012-08-01

    The detection of Legionella pneumophila DNA in clinical specimens using quantitative real-time polymerase chain reaction (qPCR) combined with direct sequence-based typing (SBT) offers rapid confirmation and timely intervention in the investigation of cases of Legionnaires' disease (LD). We assessed the utility of a specific L. pneumophila qPCR assay targeting the macrophage infectivity potentiator (mip) gene and internal process control with three clinical specimen types from confirmed LD cases. The assay was completely specific for L. pneumophila, as demonstrated by positive results for 39/39 strains from all subspecies and 16 serogroups. No cross-reaction was observed with any of the 54 Legionella non-pneumophila (0/69 strains) or 21 non-Legionella (0/58 strains). All L. pneumophila culture-positive respiratory samples (81/81) were qPCR-positive. Of 80 culture-negative samples tested, 47 (58.8%) were qPCR-positive and none were inhibitory. PCR was significantly more sensitive than culture for samples taken ≤ 2 days of hospitalisation (94.7% vs. 79.6%), with the difference being even more marked for samples taken between 3 and 14 days (79.3% vs. 47.8%). Overall, the sensitivity of the qPCR was ∼30% greater than that of culture and direct typing on culture-negative PCR-positive samples resulted in full 7-allele profiles from 23/46, 5 to 6 alleles from 8/46 and ≥ 1 allele from 43/46 strains.

  3. Improved sensitivity of nucleic acid amplification for rapid diagnosis of tuberculous meningitis

    DEFF Research Database (Denmark)

    Johansen, Isik Somuncu; Lundgren, Bettina; Tabak, Fehmi

    2004-01-01

    Early diagnosis of tuberculous meningitis (TBM) is essential for a positive outcome; but present microbiological diagnostic techniques are insensitive, slow, or laborious. We evaluated the standard BDProbeTec ET strand displacement amplification method (the standard ProbeTec method) for the detec......Early diagnosis of tuberculous meningitis (TBM) is essential for a positive outcome; but present microbiological diagnostic techniques are insensitive, slow, or laborious. We evaluated the standard BDProbeTec ET strand displacement amplification method (the standard ProbeTec method......) for the detection of Mycobacterium tuberculosis complex organisms in parallel with the ProbeTec method with a modified pretreatment procedure with 101 prospectively collected cerebrospinal fluid specimens from 94 patients with suspected TBM. By the modified method, the sample-washing step was omitted. A definitive...... diagnosis was attained by culture. Thirteen specimens from 12 patients were culture positive for M. tuberculosis complex organisms; three specimens (23%) were microscopy positive for acid-fast bacilli. Among the culture-positive specimens, the standard ProbeTec method was positive for 8 (61...

  4. Ternary surface monolayers for ultrasensitive (zeptomole) amperometric detection of nucleic acid hybridization without signal amplification.

    Science.gov (United States)

    Wu, Jie; Campuzano, Susana; Halford, Colin; Haake, David A; Wang, Joseph

    2010-11-01

    A ternary surface monolayer, consisting of coassembled thiolated capture probe, mercaptohexanol and dithiothreitol, is shown to offer dramatic improvements in the signal-to-noise characteristics of electrochemical DNA hybridization biosensors based on common self-assembled monolayers. Remarkably low detection limits down to 40 zmol (in 4 μL samples) as well as only 1 CFU Escherichia coli per sensor are thus obtained without any additional amplification step in connection to the commonly used horseradish peroxidase/3,3',5,5'-tetramethylbenzidine system. Such dramatic improvements in the detection limits (compared to those of common binary alkanethiol interfaces and to those of most electrochemical DNA sensing strategies without target or signal amplification) are attributed primarily to the remarkably higher resistance to nonspecific adsorption. This reflects the highly compact layer (with lower pinhole density) produced by the coupling of the cyclic- and linear-configuration "backfillers" that leads to a remarkably low background noise even in the presence of complex sample matrixes. A wide range of surface compositions have been investigated, and the ternary mixed monolayer has been systematically optimized. Detailed impedance spectroscopy and cyclic voltammetric studies shed useful insights into the surface coverage. The impressive sensitivity and high specificity of the simple developed methodology indicate great promise for a wide range of nucleic acid testing, including clinical diagnostics, biothreat detection, food safety, and forensic analysis.

  5. Emerging Loop-Mediated Isothermal Amplification-Based Microchip and Microdevice Technologies for Nucleic Acid Detection.

    Science.gov (United States)

    Safavieh, Mohammadali; Kanakasabapathy, Manoj K; Tarlan, Farhang; Ahmed, Minhaz U; Zourob, Mohammed; Asghar, Waseem; Shafiee, Hadi

    2016-03-14

    Rapid, sensitive, and selective pathogen detection is of paramount importance in infectious disease diagnosis and treatment monitoring. Currently available diagnostic assays based on polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) are time-consuming, complex, and relatively expensive, thus limiting their utility in resource-limited settings. Loop-mediated isothermal amplification (LAMP) technique has been used extensively in the development of rapid and sensitive diagnostic assays for pathogen detection and nucleic acid analysis and hold great promise for revolutionizing point-of-care molecular diagnostics. Here, we review novel LAMP-based lab-on-a-chip (LOC) diagnostic assays developed for pathogen detection over the past several years. We review various LOC platforms based on their design strategies for pathogen detection and discuss LAMP-based platforms still in development and already in the commercial pipeline. This review is intended as a guide to the use of LAMP techniques in LOC platforms for molecular diagnostics and genomic amplifications.

  6. Review of 2005 Public Health Laboratory Network Neisseria gonorrhoeae nucleic acid amplification tests guidelines.

    Science.gov (United States)

    Whiley, David M; Lahra, Monica M

    2015-03-31

    At the request of the Public Health Laboratory Network (PHLN), the National Neisseria Network (NNN) met to discuss the 2009 PHLN Neisseria gonorrhoeae nucleic acid amplification test (NAAT) guidelines and the need for supplementary testing. A central point of discussion at this NNN meeting, which took place in May 2013, was the potential for N. gonorrhoeae supplementary testing to lead to false-negative results. Data were presented at the meeting that questioned the sensitivity of commonly used in-house supplementary methods as compared with later generation commercial NAAT systems. It was the opinion of the NNN that supplementary testing remains best practice, but that caution should be used when reporting negative results. The NNN recommends that urogenital samples providing a positive result in a screening method and a negative result by a supplemental method should not be reported as negative for N. gonorrhoeae without an appropriate explanatory comment indicating that gonococcal infection cannot be excluded.

  7. Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato

    NARCIS (Netherlands)

    Bentsink, L.; Leone, G.O.M.; Beckhoven, van J.R.C.M.; Schijndel, van H.B.; Gemen, van B.; Wolf, van der J.M.

    2002-01-01

    Aims: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a usefu

  8. Culture confirmation of gonococcal infection by recall of subjects found to be positive by nucleic acid amplification tests in general practice

    DEFF Research Database (Denmark)

    Møller, Jens Kjølseth

    2010-01-01

    To evaluate a routine notification of general practitioners to recall nucleic acid amplification test (NAAT)-positive subjects for culture of Neisseria gonorrhoeae to confirm gonococcal infection in the community.......To evaluate a routine notification of general practitioners to recall nucleic acid amplification test (NAAT)-positive subjects for culture of Neisseria gonorrhoeae to confirm gonococcal infection in the community....

  9. Immunocytochemistry versus nucleic acid amplification in fine needle aspirates and tissues of extrapulmonary tuberculosis

    Directory of Open Access Journals (Sweden)

    Madhu Mati Goel

    2012-01-01

    Full Text Available Background: Immunocytochemistry (ICC is an established routine diagnostic adjunct to cytology and histology for tumor diagnosis but has received little attention for diagnosis of tuberculosis. Aims: To have an objective method of direct visualization of mycobacteria or their products in clinical extrapulmonary tuberculosis (EPTB specimens, immunocytochemical localization of M. tuberculosis antigen by staining with species specific monoclonal antibody to 38-kDa antigen of Mycobacterium tuberculosis complex. Materials and Methods: Immunostaining with specific monoclonal antibody to 38-kDa antigen of Mycobacterium tuberculosis complex was done in fresh and archival fine needle aspirates and tissue granulomata of 302 cases of extrapulmonary tuberculosis and was compared with the molecular diagnostic i.e., nucleic amplification and conventional [Cytomorphology, Ziehl Neelsen (ZN staining and culture] tests and 386 controls. Results: Diagnostic indices by Bayesian analysis for all types of archival and fresh material varied from 64 to 76% in nucleic acid amplification (NAA and 96 to 98% in ICC. There was no significant difference in the diagnostic indices of ZN staining and/ or ICC in fresh or archival material whereas the sensitivity of NAA differed significantly in fresh versus archival material both in cytology (71.4% vs 52.1% and histology (51.1% vs 38.8%. ICC can be easily used on archival smears and formalin-fixed paraffin-embedded tissue sections with almost equal sensitivity and specificity as with fresh material, in contrast to NAA which showed significant difference in test results on archival and fresh material. Conclusions: Low detection sensitivity of MTB DNA in archival material from known tuberculous cases showed the limitation of in-house NAA-based molecular diagnosis. ICC was found to be sensitive, specific and a better technique than NAA and can be used as an adjunct to conventional morphology and ZN staining for the diagnosis of

  10. Enhanced nucleic acid amplification with blood in situ by wire-guided droplet manipulation (WDM).

    Science.gov (United States)

    Harshman, Dustin K; Reyes, Roberto; Park, Tu San; You, David J; Song, Jae-Young; Yoon, Jeong-Yeol

    2014-03-15

    There are many challenges facing the use of molecular biology to provide pertinent information in a timely, cost effective manner. Wire-guided droplet manipulation (WDM) is an emerging format for conducting molecular biology with unique characteristics to address these challenges. To demonstrate the use of WDM, an apparatus was designed and assembled to automate polymerase chain reaction (PCR) on a reprogrammable platform. WDM minimizes thermal resistance by convective heat transfer to a constantly moving droplet in direct contact with heated silicone oil. PCR amplification of the GAPDH gene was demonstrated at a speed of 8.67 s/cycle. Conventional PCR was shown to be inhibited by the presence of blood. WDM PCR utilizes molecular partitioning of nucleic acids and other PCR reagents from blood components, within the water-in-oil droplet, to increase PCR reaction efficiency with blood in situ. The ability to amplify nucleic acids in the presence of blood simplifies pre-treatment protocols towards true point-of-care diagnostic use. The 16s rRNA hypervariable regions V3 and V6 were amplified from Klebsiella pneumoniae genomic DNA with blood in situ. The detection limit of WDM PCR was 1 ng/μL or 10(5)genomes/μL with blood in situ. The application of WDM for rapid, automated detection of bacterial DNA from whole blood may have an enormous impact on the clinical diagnosis of infections in bloodstream or chronic wound/ulcer, and patient safety and morbidity.

  11. Entropy Beacon: A Hairpin-Free DNA Amplification Strategy for Efficient Detection of Nucleic Acids.

    Science.gov (United States)

    Lv, Yifan; Cui, Liang; Peng, Ruizi; Zhao, Zilong; Qiu, Liping; Chen, Huapei; Jin, Cheng; Zhang, Xiao-Bing; Tan, Weihong

    2015-12-01

    Here, we propose an efficient strategy for enzyme- and hairpin-free nucleic acid detection called an entropy beacon (abbreviated as Ebeacon). Different from previously reported DNA hybridization/displacement-based strategies, Ebeacon is driven forward by increases in the entropy of the system, instead of free energy released from new base-pair formation. Ebeacon shows high sensitivity, with a detection limit of 5 pM target DNA in buffer and 50 pM in cellular homogenate. Ebeacon also benefits from the hairpin-free amplification strategy and zero-background, excellent thermostability from 20 °C to 50 °C, as well as good resistance to complex environments. In particular, based on the huge difference between the breathing rate of a single base pair and two adjacent base pairs, Ebeacon also shows high selectivity toward base mutations, such as substitution, insertion, and deletion and, therefore, is an efficient nucleic acid detection method, comparable to most reported enzyme-free strategies.

  12. Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids.

    Science.gov (United States)

    Li, Zhao; Liu, Yong; Wei, Qingquan; Liu, Yuanjie; Liu, Wenwen; Zhang, Xuelian; Yu, Yude

    2016-01-01

    Absolute, precise quantification methods expand the scope of nucleic acids research and have many practical applications. Digital polymerase chain reaction (dPCR) is a powerful method for nucleic acid detection and absolute quantification. However, it requires thermal cycling and accurate temperature control, which are difficult in resource-limited conditions. Accordingly, isothermal methods, such as recombinase polymerase amplification (RPA), are more attractive. We developed a picoliter well array (PWA) chip with 27,000 consistently sized picoliter reactions (314 pL) for isothermal DNA quantification using digital RPA (dRPA) at 39°C. Sample loading using a scraping liquid blade was simple, fast, and required small reagent volumes (i.e., PEG-silane agent effectively eliminated cross-contamination during dRPA. Our creative optical design enabled wide-field fluorescence imaging in situ and both end-point and real-time analyses of picoliter wells in a 6-cm(2) area. It was not necessary to use scan shooting and stitch serial small images together. Using this method, we quantified serial dilutions of a Listeria monocytogenes gDNA stock solution from 9 × 10(-1) to 4 × 10(-3) copies per well with an average error of less than 11% (N = 15). Overall dRPA-on-chip processing required less than 30 min, which was a 4-fold decrease compared to dPCR, requiring approximately 2 h. dRPA on the PWA chip provides a simple and highly sensitive method to quantify nucleic acids without thermal cycling or precise micropump/microvalve control. It has applications in fast field analysis and critical clinical diagnostics under resource-limited settings.

  13. Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids

    Science.gov (United States)

    Li, Zhao; Liu, Yong; Wei, Qingquan; Liu, Yuanjie; Liu, Wenwen; Zhang, Xuelian; Yu, Yude

    2016-01-01

    Absolute, precise quantification methods expand the scope of nucleic acids research and have many practical applications. Digital polymerase chain reaction (dPCR) is a powerful method for nucleic acid detection and absolute quantification. However, it requires thermal cycling and accurate temperature control, which are difficult in resource-limited conditions. Accordingly, isothermal methods, such as recombinase polymerase amplification (RPA), are more attractive. We developed a picoliter well array (PWA) chip with 27,000 consistently sized picoliter reactions (314 pL) for isothermal DNA quantification using digital RPA (dRPA) at 39°C. Sample loading using a scraping liquid blade was simple, fast, and required small reagent volumes (i.e., <20 μL). Passivating the chip surface using a methoxy-PEG-silane agent effectively eliminated cross-contamination during dRPA. Our creative optical design enabled wide-field fluorescence imaging in situ and both end-point and real-time analyses of picoliter wells in a 6-cm2 area. It was not necessary to use scan shooting and stitch serial small images together. Using this method, we quantified serial dilutions of a Listeria monocytogenes gDNA stock solution from 9 × 10-1 to 4 × 10-3 copies per well with an average error of less than 11% (N = 15). Overall dRPA-on-chip processing required less than 30 min, which was a 4-fold decrease compared to dPCR, requiring approximately 2 h. dRPA on the PWA chip provides a simple and highly sensitive method to quantify nucleic acids without thermal cycling or precise micropump/microvalve control. It has applications in fast field analysis and critical clinical diagnostics under resource-limited settings. PMID:27074005

  14. Automated nucleic acid amplification testing in blood banks: An additional layer of blood safety

    Directory of Open Access Journals (Sweden)

    Pragati Chigurupati

    2015-01-01

    Full Text Available Context: A total of 30 million blood components are transfused each year in India. Blood safety thus becomes a top priority, especially with a population of around 1.23 billion and a high prevalence rate of human immunodeficiency virus (HIV, hepatitis B virus (HBV and hepatitis C virus (HCV in general population. Nucleic acid amplification testing (NAT in blood donor screening has been implemented in many developed countries to reduce the risk of transfusion-transmitted viral infections (TTIs. NAT takes care of the dynamics of window period of viruses and offers the safest blood pack for donation. Aims: The aim of this study is to show the value of NAT in blood screening. Settings and Design: Dhanavantari Blood Bank, Rajahmundry, Andhra Pradesh, India. Subjects and Methods: Over a period of 1 year from January 2012 to December 2012, a total number of 15,000 blood donor samples were subjected to tests for HIV, HBV, and HCV by enzyme-linked immunosorbent assay (ELISA method and 8000 ELISA nonreactive samples were subjected for NAT using multiplex polymerase chain reaction technology. Results: Of the 15,000 donors tested, 525 were seroreactive. In 8000 ELISA negative blood samples subjected to NAT, 4 donor samples were reactive for HBV. The NAT yield was 1 in 2000. Conclusions: NAT could detect HIV, HBV, and HCV cases in blood donor samples those were undetected by serological tests. NAT could interdict 2500 infectious donations among our approximate 5 million annual blood donations.

  15. Commercial nucleic acid amplification tests in tuberculous meningitis--a meta-analysis.

    Science.gov (United States)

    Solomons, Regan S; van Elsland, Sabine L; Visser, Douwe H; Hoek, Kim G P; Marais, Ben J; Schoeman, Johan F; van Furth, Anne M

    2014-04-01

    Although nucleic acid amplification tests (NAATs) promise a rapid, definitive diagnosis of tuberculous meningitis, the performance of first-generation NAATs was suboptimal and variable. We conducted a meta-analysis of studies published between 2003 and 2013, using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool to evaluate methodological quality. The diagnostic accuracy of newer commercial NAATs was assessed. Pooled estimates of diagnostic accuracy for commercial NAATs measured against a cerebrospinal fluid Mycobacterium tuberculosis culture-positive gold standard were sensitivity 0.64, specificity 0.98, and diagnostic odds ratio 64.0. Heterogeneity was limited; P value = 0.147 and I(2) = 33.85%. The Xpert MTB/RIF® test was evaluated in 1 retrospective study and 4 prospective studies, with pooled sensitivity 0.70 and specificity 0.97. The QUADAS-2 tool revealed low risk of bias, as well as low concerns regarding applicability. Heterogeneity was pronounced among studies of in-house tests. Commercial NAATs proved to be highly specific with greatly reduced heterogeneity compared to in-house tests. Sub-optimal sensitivity remains a limitation. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Early detection of Mycobacterium tuberculosis complex in BACTEC MGIT cultures using nucleic acid amplification.

    Science.gov (United States)

    Lin, S Y; Hwang, S C; Yang, Y C; Wang, C F; Chen, Y H; Chen, T C; Lu, P L

    2016-06-01

    We evaluated the application of nucleic acid amplification (NAA) in liquid cultures for the early detection of Mycobacterium tuberculosis. The Cobas TaqMan MTB test, IS6110 real-time PCR, and hsp65 PCR-restriction fragment length polymorphism (RFLP) analysis were used to detect BACTEC MGIT 960 (MGIT) cultures on days 3, 5, 7, and 14. The procedure was initially tested with a reference strain, H37Rv (ATCC 27294). Subsequently, 200 clinical specimens, including 150 Acid Fast bacillus (AFB) smear-positive and 50 AFB smear-negative samples, were examined. The Cobas TaqMan MTB test and IS6110-based PCR analysis were able to detect M. tuberculosis after 1 day when the inoculum of H37Rv was >3 x 10(-2) CFU/ml. After a 5-day incubation in the MGIT system, all three NAA assays had a positive detection regardless of the inoculum size. After a 1-day incubation of the clinical specimens in the MGIT system, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for the Cobas TaqMan MTB assay were 70.2%, 100%, 100%, and 82.3% respectively. For IS6110-based PCR analysis, these values were 63.1%, 100%, 100%, and 78.9%, and were 88.1%, 100%, 100%, and 92.1% respectively for hsp65 PCR-RFLP analysis. After a 3-day incubation, the specificity and PPV were 100% for all three NAA tests; the Cobas TaqMan MTB assay had the best sensitivity (97.6%) and NPV (98.3%). The sensitivity, specificity, PPV, and NPV for conventional culture analysis were 98.8%, 100%, 100%, and 99.1%. Thus, NAA may be useful for the early detection of M. tuberculosis after 3 days in MGIT.

  17. Establishment of the nucleic acid sequence-based amplificationmethod of detecting Vibrio parahaemolyticus%依赖于核酸序列恒温扩增技术快速检测副溶血性弧菌方法的建立

    Institute of Scientific and Technical Information of China (English)

    倪鑫; 王志聪; 雷质文; 梁成珠; 汪东风; 姜英辉; 王建广; 祝素贞

    2011-01-01

    为建立副溶血性弧菌的快速检测方法,本研究采用依赖于核酸序列恒温扩增(NASBA)技术,以副溶血性弧菌的tlh基因为扩增的靶基因设计特异性引物和探针,建立可快速扩增副溶血性弧菌的NASBA方法.特异性和灵敏度试验结果表明:该NASBA方法对副溶血性弧菌的最小检出量为5.1×102 cfu/mL,高于普通PCR方法,而且与其他种属的菌无任何交叉反应.此外,本研究将副溶血弧菌扩增产物采用通用型核酸扩增物快速检测板进行检测,实现了特异性强的快速副溶血弧菌的检测.该方法对仪器要求低,具有良好的应用前景.%In this study, we adopted a technology based on nucleic acid sequence-based amplification (NASBA), to establish a rapid test method of Vibrio parahaemolyticus. Specific primers and probes were designed for the tlh target gene of V. Parahaemolyticus and the detection method of NASBA was established. Its specificity and sensitivity were tested. The results showed that the sensitivity of the NASBA was 5.1 x 102 cfii/mL which was higher than that of PCR method and this method had no cross reaction with other species of bacteria. The amplification products of V. Pamhaemolyticus were rapidly detected by using the universal rapid detection board of nucleic acid amplification product. In conclusion, detecting V. Parahaemolyticus with NASBA method was more specific and sensitive than PCR method and has lower instrumental requirement and a broad application.

  18. Successful Combination of Nucleic Acid Amplification Test Diagnostics and Targeted Deferred Neisseria gonorrhoeae Culture.

    Science.gov (United States)

    Wind, Carolien M; de Vries, Henry J C; Schim van der Loeff, Maarten F; Unemo, Magnus; van Dam, Alje P

    2015-06-01

    Nucleic acid amplification tests (NAATs) are recommended for the diagnosis of N. gonorrhoeae infections because of their superior sensitivity. Increasing NAAT use causes a decline in crucial antimicrobial resistance (AMR) surveillance data, which rely on culture. We analyzed the suitability of the ESwab system for NAAT diagnostics and deferred targeted N. gonorrhoeae culture to allow selective and efficient culture based on NAAT results. We included patients visiting the STI Clinic Amsterdam, The Netherlands, in 2013. Patient characteristics and urogenital and rectal samples for direct N. gonorrhoeae culture, standard NAAT, and ESwab were collected. Standard NAAT and NAAT on ESwab samples were performed using the Aptima Combo 2 assay for N. gonorrhoeae and C. trachomatis. Two deferred N. gonorrhoeae cultures were performed on NAAT-positive ESwab samples after storage at 4°C for 1 to 3 days. We included 2,452 samples from 1,893 patients. In the standard NAAT, 107 samples were N. gonorrhoeae positive and 284 were C. trachomatis positive. The sensitivities of NAAT on ESwab samples were 83% (95% confidence interval [CI], 75 to 90%) and 87% (95% CI, 82 to 90%), respectively. ESwab samples were available for 98 of the gonorrhea-positive samples. Of these, 82% were positive in direct culture and 69% and 56% were positive in the 1st and 2nd deferred cultures, respectively (median storage times, 27 and 48 h, respectively). Deferred culture was more often successful in urogenital samples or when the patient had symptoms at the sampling site. Deferred N. gonorrhoeae culture of stored ESwab samples is feasible and enables AMR surveillance. To limit the loss in NAAT sensitivity, we recommend obtaining separate samples for NAAT and deferred culture.

  19. Impact of nucleic acid amplification test on screening of blood donors in Northern Pakistan.

    Science.gov (United States)

    Niazi, Saifullah Khan; Bhatti, Farhat Abbas; Salamat, Nuzhat; Ghani, Eijaz; Tayyab, Muhammad

    2015-07-01

    The Armed Forces Institute of Transfusion located in Rawalpindi, Northern Pakistan, acts as a regional blood center with more than 50,000 donations collected annually. Nucleic acid amplification testing (NAT) was introduced in our institution in September 2012 for screening all seronegative blood donors. The study was conducted from September 21, 2012, to September 20, 2013. Samples from the seronegative donors were run on cobas s 201 platform (Roche) in pools of six. Reactive donors were followed up for further confirmatory testing to rule out false-positive results. Viral load estimation was done for all NAT-reactive donors. After serologic screening of 56,772 blood donors, 2334 were found to be reactive; 719 (1.27%) were reactive for hepatitis B surface antigen, 1046 (1.84%) for antibody to hepatitis C virus (anti-HCV), 12 (0.02%) for antibody to human immunodeficiency virus, and 557 (0.98%) for syphilis antibodies. A total of 27 NAT-reactive donors were confirmed after testing 54,438 seronegative donors, with an overall NAT yield of one in 2016 donors: 23 for hepatitis B virus (HBV) DNA (HBV NAT yield, 1:2367) and four for HCV RNA (HCV NAT yield, 1:13,609). The residual risk after NAT implementation, calculated for the first-time blood donors, was 62.5 and 4.4 per million donors for HBV and HCV, respectively. NAT has improved the safety of blood products at our transfusion institution. Confirmation of NAT results must always be done either on follow-up samples or on samples from the retrieved frozen plasma bag. © 2015 AABB.

  20. A sensitive SERS assay for detecting proteins and nucleic acids using a triple-helix molecular switch for cascade signal amplification.

    Science.gov (United States)

    Ye, Sujuan; Wu, Yanying; Zhang, Wen; Li, Na; Tang, Bo

    2014-08-25

    A novel surface-enhanced Raman scattering (SERS) detection system is developed for proteins and nucleic acids based on a triple-helix molecular switch for multiple cycle signal amplification, achieving high sensitivity, universality, rapid analysis, and high selectivity.

  1. Culture confirmation of gonococcal infection by recall of subjects found to be positive by nucleic acid amplification tests in general practice

    DEFF Research Database (Denmark)

    Møller, Jens Kjølseth

    2010-01-01

    To evaluate a routine notification of general practitioners to recall nucleic acid amplification test (NAAT)-positive subjects for culture of Neisseria gonorrhoeae to confirm gonococcal infection in the community....

  2. Mobile nucleic acid amplification testing (mobiNAAT) for Chlamydia trachomatis screening in hospital emergency department settings.

    Science.gov (United States)

    Shin, D J; Athamanolap, P; Chen, L; Hardick, J; Lewis, M; Hsieh, Y H; Rothman, R E; Gaydos, C A; Wang, T H

    2017-07-03

    Management of curable sexually-transmitted infections (STI) such as Chlamydia can be revolutionized by highly sensitive nucleic acid testing that is deployable at the point-of-care (POC). Here we report the development of a mobile nucleic acid amplification testing (mobiNAAT) platform utilizing a mobile phone and droplet magnetofluidics to deliver NAAT in a portable and accessible format. By using magnetic particles as a mobile substrate for nucleic acid capture and transport, fluid handling is reduced to particle translocation on a simple magnetofluidic cartridge assembled with reagents for nucleic acid purification and amplification. A mobile phone user interface operating in tandem with a portable Bluetooth-enabled cartridge-processing unit facilitates process integration. We tested 30 potentially Chlamydia trachomatis (CT)-infected patients in a hospital emergency department and confirmed that mobiNAAT showed 100% concordance with laboratory-based NAAT. Concurrent evaluation by a nontechnical study coordinator who received brief training via an embedded mobile app module demonstrated ease of use and reproducibility of the platform. This work demonstrates the potential of mobile nucleic acid testing in bridging the diagnostic gap between centralized laboratories and hospital emergency departments.

  3. A fully integrated paperfluidic molecular diagnostic chip for the extraction, amplification, and detection of nucleic acids from clinical samples.

    Science.gov (United States)

    Rodriguez, Natalia M; Wong, Winnie S; Liu, Lena; Dewar, Rajan; Klapperich, Catherine M

    2016-02-21

    Paper diagnostics have successfully been employed to detect the presence of antigens or small molecules in clinical samples through immunoassays; however, the detection of many disease targets relies on the much higher sensitivity and specificity achieved via nucleic acid amplification tests (NAAT). The steps involved in NAAT have recently begun to be explored in paper matrices, and our group, among others, has reported on paper-based extraction, amplification, and detection of DNA and RNA targets. Here, we integrate these paper-based NAAT steps into a single paperfluidic chip in a modular, foldable system that allows for fully integrated fluidic handling from sample to result. We showcase the functionality of the chip by combining nucleic acid isolation, isothermal amplification, and lateral flow detection of human papillomavirus (HPV) 16 DNA directly from crude cervical specimens in less than 1 hour for rapid, early detection of cervical cancer. The chip is made entirely of paper and adhesive sheets, making it low-cost, portable, and disposable, and offering the potential for a point-of-care molecular diagnostic platform even in remote and resource-limited settings.

  4. Detection of Salmonella invA by isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) in Zambia.

    Science.gov (United States)

    Isogai, Emiko; Makungu, Chitwambi; Yabe, John; Sinkala, Patson; Nambota, Andrew; Isogai, Hiroshi; Fukushi, Hideto; Silungwe, Manda; Mubita, Charles; Syakalima, Michelo; Hang'ombe, Bernard Mudenda; Kozaki, Shunji; Yasuda, Jun

    2005-01-01

    The isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) is a new isothermal DNA amplification method composed of exo Bca DNA polymerase, RNaseH and DNA-RNA chimeric primers. We detected invA of Salmonella from chicken carcasses, egg yolk and cattle fecal samples. Fifty-three of 59 isolates were invA-positive in ICAN-chromatostrip detection. The result was consistent with those obtained by standard PCR. Salmonella invA was detected in 12 of 14 carcass rinses by ICAN, while in 7 of 14 rinses by standard PCR. These results indicate that ICAN is an efficient, sensitive and simple system to detect invA of Salmonella species in developing countries such as Zambia.

  5. Rapid Point-of-Care Isothermal Amplification Assay for the Detection of Malaria without Nucleic Acid Purification.

    Science.gov (United States)

    Modak, Sayli S; Barber, Cheryl A; Geva, Eran; Abrams, William R; Malamud, Daniel; Ongagna, Yhombi Serge Yvon

    2016-01-01

    Malaria remains one of the most prevalent infectious diseases and results in significant mortality. Isothermal amplification (loop-mediated isothermal amplification) is used to detect malarial DNA at levels of ~1 parasite/µL blood in ≤30 minutes without the isolation of parasite nucleic acid from subject's blood or saliva. The technique targets the mitochondrial cytochrome oxidase subunit 1 gene and is capable of distinguishing Plasmodium falciparum from Plasmodium vivax. Malarial diagnosis by the gold standard microscopic examination of blood smears is generally carried out only after moderate-to-severe symptoms appear. Rapid diagnostic antigen tests are available but generally require infection levels in the range of 200-2,000 parasites/µL for a positive diagnosis and cannot distinguish if the disease has been cleared due to the persistence of circulating antigen. This study describes a rapid and simple molecular assay to detect malarial genes directly from whole blood or saliva without DNA isolation.

  6. Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-care diagnosis.

    Science.gov (United States)

    Toley, Bhushan J; Covelli, Isabela; Belousov, Yevgeniy; Ramachandran, Sujatha; Kline, Enos; Scarr, Noah; Vermeulen, Nic; Mahoney, Walt; Lutz, Barry R; Yager, Paul

    2015-11-21

    We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve >10(9)-fold amplification of the target sequence in isothermal DNA amplification methods. iSDA initiates at sites where DNA base pairs spontaneously open or transiently convert into Hoogsteen pairs, i.e. "breathe", and proceeds to exponential amplification by repeated nicking, extension, and displacement of single strands. We demonstrate successful iSDA amplification and lateral flow detection of 10 copies of a Staphylococcus aureus gene, NO.-inducible l-lactate dehydrogenase (ldh1) (Richardson, Libby, and Fang, Science, 2008, 319, 1672-1676), in a clean sample and 50 copies in the presence of high concentrations of genomic DNA and mucins in isothermal amplification reactions. Finally, we demonstrate the multiplexing capability of iSDA by the simultaneous amplification of the target gene and an engineered internal control sequence. The speed, sensitivity, and specificity of iSDA make it a powerful method for point-of-care molecular diagnosis.

  7. Amino Acids Sequence Based in Silico Analysis of RuBisCO (Ribulose-1,5 Bisphosphate Carboxylase Oxygenase Proteins in Some Carthamus L. ssp.

    Directory of Open Access Journals (Sweden)

    Emre SEVİNDİK

    2017-06-01

    Full Text Available RuBisCO is an important enzyme for plants to photosynthesize and balance carbon dioxide in the atmosphere. This study aimed to perform sequence, physicochemical, phylogenetic and 3D (three-dimensional comparative analyses of RuBisCO proteins in the Carthamus ssp. using various bioinformatics tools. The sequence lengths of the RuBisCO proteins were between 166 and 477 amino acids, with an average length of 411.8 amino acids. Their molecular weights (Mw ranged from 18711.47 to 52843.09 Da; the most acidic and basic protein sequences were detected in C. tinctorius (pI = 5.99 and in C. tenuis (pI = 6.92, respectively. The extinction coefficients of RuBisCO proteins at 280 nm ranged from 17,670 to 69,830 M-1 cm-1, the instability index (II values for RuBisCO proteins ranged from 33.31 to 39.39, while the GRAVY values of RuBisCO proteins ranged from -0.313 to -0.250. The most abundant amino acid in the RuBisCO protein was Gly (9.7%, while the least amino acid ratio was Trp (1.6 %. The putative phosphorylation sites of RuBisCO proteins were determined by NetPhos 2.0. Phylogenetic analysis revealed that RuBisCO proteins formed two main clades. A RAMPAGE analysis revealed that 96.3%-97.6% of residues were located in the favoured region of RuBisCO proteins. To predict the three dimensional (3D structure of the RuBisCO proteins PyMOL was used. The results of the current study provide insights into fundamental characteristic of RuBisCO proteins in Carthamus ssp.

  8. Genotyping-by-Sequencing-Based Investigation of the Genetic Architecture Responsible for a ∼Sevenfold Increase in Soybean Seed Stearic Acid.

    Science.gov (United States)

    Heim, Crystal B; Gillman, Jason D

    2017-01-05

    Soybean oil is highly unsaturated but oxidatively unstable, rendering it nonideal for food applications. Until recently, the majority of soybean oil underwent partial chemical hydrogenation, which produces trans fats as an unavoidable consequence. Dietary intake of trans fats and most saturated fats are conclusively linked to negative impacts on cholesterol levels and cardiovascular health. Two major soybean oil breeding targets are: (1) to reduce or eliminate the need for chemical hydrogenation, and (2) to replace the functional properties of partially hydrogenated soybean oil. One potential solution is the elevation of seed stearic acid, a saturated fat which has no negative impacts on cardiovascular health, from 3 to 4% in typical cultivars to > 20% of the seed oil. We performed QTL analysis of a population developed by crossing two mutant lines, one with a missense mutation affecting a stearoyl-acyl-carrier protein desaturase gene resulting in ∼11% seed stearic acid crossed to another mutant, A6, which has 24-28% seed stearic acid. Genotyping-by-sequencing (GBS)-based QTL mapping identified 21 minor and major effect QTL for six seed oil related traits and plant height. The inheritance of a large genomic deletion affecting chromosome 14 is the basis for largest effect QTL, resulting in ∼18% seed stearic acid. This deletion contains SACPD-C and another gene(s); loss of both genes boosts seed stearic acid levels to ≥ 18%. Unfortunately, this genomic deletion has been shown in previous studies to be inextricably correlated with reduced seed yield. Our results will help inform and guide ongoing breeding efforts to improve soybean oil oxidative stability.

  9. Genotyping-by-Sequencing-Based Investigation of the Genetic Architecture Responsible for a ∼Sevenfold Increase in Soybean Seed Stearic Acid

    Directory of Open Access Journals (Sweden)

    Crystal B. Heim

    2017-01-01

    Full Text Available Soybean oil is highly unsaturated but oxidatively unstable, rendering it nonideal for food applications. Until recently, the majority of soybean oil underwent partial chemical hydrogenation, which produces trans fats as an unavoidable consequence. Dietary intake of trans fats and most saturated fats are conclusively linked to negative impacts on cholesterol levels and cardiovascular health. Two major soybean oil breeding targets are: (1 to reduce or eliminate the need for chemical hydrogenation, and (2 to replace the functional properties of partially hydrogenated soybean oil. One potential solution is the elevation of seed stearic acid, a saturated fat which has no negative impacts on cardiovascular health, from 3 to 4% in typical cultivars to > 20% of the seed oil. We performed QTL analysis of a population developed by crossing two mutant lines, one with a missense mutation affecting a stearoyl-acyl-carrier protein desaturase gene resulting in ∼11% seed stearic acid crossed to another mutant, A6, which has 24–28% seed stearic acid. Genotyping-by-sequencing (GBS-based QTL mapping identified 21 minor and major effect QTL for six seed oil related traits and plant height. The inheritance of a large genomic deletion affecting chromosome 14 is the basis for largest effect QTL, resulting in ∼18% seed stearic acid. This deletion contains SACPD-C and another gene(s; loss of both genes boosts seed stearic acid levels to ≥ 18%. Unfortunately, this genomic deletion has been shown in previous studies to be inextricably correlated with reduced seed yield. Our results will help inform and guide ongoing breeding efforts to improve soybean oil oxidative stability.

  10. Diagnostic value of nine nucleic acid amplification test systems for Mycobacterium tuberculosis complex

    Directory of Open Access Journals (Sweden)

    Gülnur Tarhan

    2015-09-01

    Full Text Available Objective: In this study, nine commercial Nucleic Acid Amplification Test Systems (NAATs were evaluated for diagnostic performance of Mycobacterium tuberculosis complex (MTBC from smear positive sputum species (SPss and smear negative sputum specimens (SNss. Methods: Sixty SPss and 55 SNss were examined icroscopically by Ehrlich Ziehl Neelsen (EZN staining method, and also inoculated on Löwenstein Jensen (LJ medium for culture. The sensitivity and specificity of nine NAATs were calculated according to LJ culture method accepted as gold standard. Results: When LJ culture results were taken as gold standard; the sensitivity rates of method COBAS Amplicor MTB (Method A, GenProbe MTD (Method B, Cobas TaqMan MTB PCR Method C, iCycler iQ RT PCR (Method D, TaqMan PCR AB 5700 (Method E, TaqMan PCR AB7700 (Method F, ightCycler® 480 RT PCR (Method G, Rotor Gene RT PCR (Method H and the AdvanSure TB/NTM RT PCR (Method I for SPss were 98.3 %, 93.3 %, 96.7 %, 100 %, 93.3 %, 100 %, 100 %, 100 % and 100 %, respectively. The sensitivity was 53.84% for the methods A, B, D, E, G and I; 38.46% for the method C and H; 61.5% for the method F for the method I in SNss. There were no statistical significant differences between the nine NAATs (p≥0.05. The specificity was 100% for all nine NAATs in SNss. The positivity rates of methods were 53.8% for methods A, B, D, E, G, I; 38.5% for methods C and H, and 61.5% for method F in SNss. These rates were 100% for D, F, G, H and I; 98.3% for method A; 96.7% for method C; 93,3% for methods B and E in SPss. Statistical analysis showed that there was no statistically significant differences among the nine NAATs (p≥0.05. Conclusion: It is concluded that the nine NAATs might be useful for detecting MTBC from SPss, but not effective for SNss. J Microbiol Infect Dis 2015;5(3: 103-109

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

    DEFF Research Database (Denmark)

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

    2005-01-01

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

  12. Two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites.

    Science.gov (United States)

    Liu, Qing; Nam, Jeonghun; Kim, Sangho; Lim, Chwee Teck; Park, Mi Kyoung; Shin, Yong

    2016-08-15

    Rapid, early, and accurate diagnosis of malaria is essential for effective disease management and surveillance, and can reduce morbidity and mortality associated with the disease. Although significant advances have been achieved for the diagnosis of malaria, these technologies are still far from ideal, being time consuming, complex and poorly sensitive as well as requiring separate assays for sample processing and detection. Therefore, the development of a fast and sensitive method that can integrate sample processing with detection of malarial infection is desirable. Here, we report a two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites. It combines the Dimethyl adipimidate (DMA)/Thin film Sample processing (DTS) technique as a first stage and the Mach-Zehnder Interferometer-Isothermal solid-phase DNA Amplification (MZI-IDA) sensing technique as a second stage. The system can extract DNA from malarial parasites using DTS technique in a closed system, not only reducing sample loss and contamination, but also facilitating the multiplexed malarial DNA detection using the fast and accurate MZI-IDA technique. Here, we demonstrated that this system can deliver results within 60min (including sample processing, amplification and detection) with high sensitivity (malaria in low-resource settings.

  13. Development of an electrochemical DNA biosensor for detection of specific Mycobacterium tuberculosis sequence based on poly(L-glutamic acid) modified electrode

    Indian Academy of Sciences (India)

    MERVE YESIL; SONER DONMEZ; FATMA ARSLAN

    2016-11-01

    An electrochemical DNA biosensor was developed by avidin-biotin interaction of a biotinylated probe and avidin-attached, poly(L-glutamic) acid coated pencil graphite electrode (PGA/PGE) for detection of specific Mycobacterium tuberculosis DNA sequence. The discrimination of fully complementary hybridization and mismatch hybridization was carried out by electrochemical reduction current of Meldola’s Blue (MDB) in square wave voltammetry (SWV). The calibration graph of the DNA biosensor was linear between 1.5–12.5 nM and the detection limit was calculated as 1.3 nM. The proposed biosensor successfully discriminated short andlong oligonucleotides related to DNA sequence of Mycobacterium tuberculosis in optimal condition.

  14. Covariance of charged amino acids at positions 322 and 440 of HIV-1 Env contributes to coreceptor specificity of subtype B viruses, and can be used to improve the performance of V3 sequence-based coreceptor usage prediction algorithms.

    Directory of Open Access Journals (Sweden)

    Kieran Cashin

    Full Text Available The ability to determine coreceptor usage of patient-derived human immunodeficiency virus type 1 (HIV-1 strains is clinically important, particularly for the administration of the CCR5 antagonist maraviroc. The envelope glycoprotein (Env determinants of coreceptor specificity lie primarily within the gp120 V3 loop region, although other Env determinants have been shown to influence gp120-coreceptor interactions. Here, we determined whether conserved amino acid alterations outside the V3 loop that contribute to coreceptor usage exist, and whether these alterations improve the performance of V3 sequence-based coreceptor usage prediction algorithms. We demonstrate a significant covariant association between charged amino acids at position 322 in V3 and position 440 in the C4 Env region that contributes to the specificity of HIV-1 subtype B strains for CCR5 or CXCR4. Specifically, positively charged Lys/Arg at position 322 and negatively charged Asp/Glu at position 440 occurred more frequently in CXCR4-using viruses, whereas negatively charged Asp/Glu at position 322 and positively charged Arg at position 440 occurred more frequently in R5 strains. In the context of CD4-bound gp120, structural models suggest that covariation of amino acids at Env positions 322 and 440 has the potential to alter electrostatic interactions that are formed between gp120 and charged amino acids in the CCR5 N-terminus. We further demonstrate that inclusion of a "440 rule" can improve the sensitivity of several V3 sequence-based genotypic algorithms for predicting coreceptor usage of subtype B HIV-1 strains, without compromising specificity, and significantly improves the AUROC of the geno2pheno algorithm when set to its recommended false positive rate of 5.75%. Together, our results provide further mechanistic insights into the intra-molecular interactions within Env that contribute to coreceptor specificity of subtype B HIV-1 strains, and demonstrate that incorporation

  15. Label-free electrochemical nucleic acid biosensing by tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme amplification.

    Science.gov (United States)

    Liu, Shufeng; Gong, Hongwei; Wang, Yanqun; Wang, Li

    2016-03-15

    Owing to the intrinsic importance of nucleic acid as bio-targets, the achievement of its simple and sensitive detection with high confidence is very essential for biological studies and diagnostic purposes. Herein, a label-free, isothermal, and ultrasensitive electrochemical detection of target DNA was developed by using a tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme releasing amplification strategy. Upon sensing of the nucleic acid analyte for the assembled hairpin-like probe DNA on the electrode, the DNA polymerase guided the target recycling and simultaneously triggered the lambda exonuclease cleavage, accompanied by the cascade recycling of the released new complementary strand and the amplified liberation of the G-rich sequence of the HRP-mimicking DNAzyme. The electrocatalytic reduction of H2O2 by the generated hemin/G-quadruplex DNAzyme was used for the signal readout and further amplification toward target response. Such tandem functional operation by DNA polymerase, lambda exonuclease and DNAzyme endows the developed biosensor with a high sensitivity and also a high confidence. A low detection limit of 5 fM with an excellent selectivity toward target DNA could be achieved. It also exhibits the distinct advantages of simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus may offer a promising avenue for the applications in disease diagnosis and clinical biomedicine.

  16. Chip-based device for parallel sorting, amplification, detection, and identification of nucleic acid subsequences

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Colston, Jr, Billy W.

    2016-08-09

    An apparatus for chip-based sorting, amplification, detection, and identification of a sample having a planar substrate. The planar substrate is divided into cells. The cells are arranged on the planar substrate in rows and columns. Electrodes are located in the cells. A micro-reactor maker produces micro-reactors containing the sample. The micro-reactor maker is positioned to deliver the micro-reactors to the planar substrate. A microprocessor is connected to the electrodes for manipulating the micro-reactors on the planar substrate. A detector is positioned to interrogate the sample contained in the micro-reactors.

  17. On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria.

    Science.gov (United States)

    Kunze, A; Dilcher, M; Abd El Wahed, A; Hufert, F; Niessner, R; Seidel, M

    2016-01-01

    This work presents an on-chip isothermal nucleic acid amplification test (iNAAT) for the multiplex amplification and detection of viral and bacterial DNA by a flow-based chemiluminescence microarray. In a principle study, on-chip recombinase polymerase amplification (RPA) on defined spots of a DNA microarray was used to spatially separate the amplification reaction of DNA from two viruses (Human adenovirus 41, Phi X 174) and the bacterium Enterococcus faecalis, which are relevant for water hygiene. By establishing the developed assay on the microarray analysis platform MCR 3, the automation of isothermal multiplex-amplification (39 °C, 40 min) and subsequent detection by chemiluminescence imaging was realized. Within 48 min, the microbes could be identified by the spot position on the microarray while the generated chemiluminescence signal correlated with the amount of applied microbe DNA. The limit of detection (LOD) determined for HAdV 41, Phi X 174, and E. faecalis was 35 GU/μL, 1 GU/μL, and 5 × 10(3) GU/μL (genomic units), which is comparable to the sensitivity reported for qPCR analysis, respectively. Moreover the simultaneous amplification and detection of DNA from all three microbes was possible. The presented assay shows that complex enzymatic reactions like an isothermal amplification can be performed in an easy-to-use experimental setup. Furthermore, iNAATs can be potent candidates for multipathogen detection in clinical, food, or environmental samples in routine or field monitoring approaches.

  18. A sensitive colorimetric assay system for nucleic acid detection based on isothermal signal amplification technology.

    Science.gov (United States)

    Hu, Bo; Guo, Jing; Xu, Ying; Wei, Hua; Zhao, Guojie; Guan, Yifu

    2017-08-01

    Rapid and accurate detection of microRNAs in biological systems is of great importance. Here, we report the development of a visual colorimetric assay which possesses the high amplification capabilities and high selectivity of the rolling circle amplification (RCA) method and the simplicity and convenience of gold nanoparticles used as a signal indicator. The designed padlock probe recognizes the target miRNA and is circularized, and then acts as the template to extend the target miRNA into a long single-stranded nucleotide chain of many tandem repeats of nucleotide sequences. Next, the RCA product is hybridized with oligonucleotides tagged onto gold nanoparticles. This interaction leads to the aggregation of gold nanoparticles, and the color of the system changes from wine red to dark blue according to the abundance of miRNA. A linear correlation between fluorescence and target oligonucleotide content was obtained in the range 0.3-300 pM, along with a detection limit of 0.13 pM (n = 7) and a RSD of 3.9% (30 pM, n = 9). The present approach provides a simple, rapid, and accurate visual colorimetric assay that allows sensitive biodetection and bioanalysis of DNA and RNA nucleotides of interest in biologically important samples. Graphical abstract The colorimetric assay system for analyzing target oligonucleotides.

  19. Nucleic acid amplification tests in the diagnosis of tuberculous pleuritis: a systematic review and meta-analysis

    Directory of Open Access Journals (Sweden)

    Riley Lee W

    2004-02-01

    Full Text Available Abstract Background Conventional tests for tuberculous pleuritis have several limitations. A variety of new, rapid tests such as nucleic acid amplification tests – including polymerase chain reaction – have been evaluated in recent times. We conducted a systematic review to determine the accuracy of nucleic acid amplification (NAA tests in the diagnosis of tuberculous pleuritis. Methods A systematic review and meta-analysis of 38 English and Spanish articles (with 40 studies, identified via searches of six electronic databases, hand searching of selected journals, and contact with authors, experts, and test manufacturers. Sensitivity, specificity, and other measures of accuracy were pooled using random effects models. Summary receiver operating characteristic curves were used to summarize overall test performance. Heterogeneity in study results was formally explored using subgroup analyses. Results Of the 40 studies included, 26 used in-house ("home-brew" tests, and 14 used commercial tests. Commercial tests had a low overall sensitivity (0.62; 95% confidence interval [CI] 0.43, 0.77, and high specificity (0.98; 95% CI 0.96, 0.98. The positive and negative likelihood ratios for commercial tests were 25.4 (95% CI 16.2, 40.0 and 0.40 (95% CI 0.24, 0.67, respectively. All commercial tests had consistently high specificity estimates; the sensitivity estimates, however, were heterogeneous across studies. With the in-house tests, both sensitivity and specificity estimates were significantly heterogeneous. Clinically meaningful summary estimates could not be determined for in-house tests. Conclusions Our results suggest that commercial NAA tests may have a potential role in confirming (ruling in tuberculous pleuritis. However, these tests have low and variable sensitivity and, therefore, may not be useful in excluding (ruling out the disease. NAA test results, therefore, cannot replace conventional tests; they need to be interpreted in parallel

  20. A novel, sensitive and label-free loop-mediated isothermal amplification detection method for nucleic acids using luminophore dyes.

    Science.gov (United States)

    Roy, Sharmili; Wei, Sim Xiao; Ying, Jean Liew Zhi; Safavieh, Mohammadali; Ahmed, Minhaz Uddin

    2016-12-15

    Electrochemiluminescence (ECL) has been widely rendered for nucleic acid testing. Here, we integrate loop-mediated isothermal amplification (LAMP) with ECL technique for DNA detection and quantification. The target LAMP DNA bound electrostatically with [Ru(bpy)3](+2) on the carbon electrode surface, and an ECL reaction was triggered by tripropylamine (TPrA) to yield luminescence. We illustrated this method as a new and highly sensitive strategy for the detection of sequence-specific DNA from different meat species at picogram levels. The proposed strategy renders the signal amplification capacities of TPrA and combines LAMP with inherently high sensitivity of the ECL technique, to facilitate the detection of low quantities of DNA. By leveraging this technique, target DNA of Sus scrofa (pork) meat was detected as low as 1pg/µL (3.43×10(-1)copies/µL). In addition, the proposed technique was applied for detection of Bacillus subtilis DNA samples and detection limit of 10pg/µL (2.2×10(3)copies/µL) was achieved. The advantages of being isothermal, sensitive and robust with ability for multiplex detection of bio-analytes makes this method a facile and appealing sensing modality in hand-held devices to be used at the point-of-care (POC).

  1. Non-instrumented nucleic acid amplification (NINA): instrument-free molecular malaria diagnostics for low-resource settings.

    Science.gov (United States)

    Labarre, Paul; Gerlach, Jay; Wilmoth, Jared; Beddoe, Andrew; Singleton, Jered; Weigl, Bernhard

    2010-01-01

    We have achieved the first complete, non-instrumented nucleic acid amplification test (NAAT) using a calcium oxide heat source thermally linked to an engineered phase change material. These two components alone maintain a thermal profile suitable for the loop-mediated isothermal amplification assay. Starting with computational fluid dynamics analysis, we identified nominal geometry for the exothermic reaction chamber, phase change material chamber, thermal insulation, and packaging. Using this model, we designed and fabricated an alpha prototype assay platform. We have verified the function of this multi-pathogen-capable platform with both fluorescent and visual turbidity indications using samples spiked with malaria DNA. Both the exothermically heated platform samples and samples heated on a Perkin-Elmer GeneAmp9600 thermocycler were first incubated at 62°C for 45 minutes, then heated to 95°C to terminate enzyme activity, then analyzed. Results from the exothermically heated, non-instrumented platform were comparable to those from the thermocycler. These developments will enable point-of-care diagnostics using accurate NAATs which until now have required a well-equipped laboratory. The aim of this research is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where assays such as immunochromatographic strip tests are successfully used but where there is no access to the infrastructure and logistics required to operate and maintain instrument-based diagnostics.

  2. Acid-Breakable Resin-Based Chemical Amplification Positive Resist for Electron-Beam Mastering: Design and Lithographic Performance

    Science.gov (United States)

    Sakamizu, Toshio; Shiraishi, Hiroshi

    2004-07-01

    A positive chemical amplification resist based on acid-catalyzed fragmentation of acetal groups in its main chain has been developed as a means of reducing line-edge roughness. The resist consists of an acid generator, an acid-diffusion controller and an acid-breakable (AB) resin that is synthesized through a co-condensation reaction between polyphenol and aromatic multifunctional vinylether compound. The effects of the fractionation of AB resins on resin properties and line-edge roughness (LER) are evaluated. Although AB resins have wide molecular weight distributions, the density of acetal groups in this AB resin is found to be almost constant except in the lower molecular weight components. The resist with a fractionated resin from which such components are removed provides high-resolution patterns (70-nm-wide pit) with fairly low LER. AFM analysis shows that the surface roughness (SR) of the resist with the fractionated resin is smaller than that of a resist using nonfractionated AB resin, and that the SR value is not altered throughout the range of exposure doses up to just below the start of dissolution. By using the fractionated AB resin, the AB resin-based resist (ABR) is capable of forming sub-100 nm L/S patterns with less than 5 nm of LER (3σ).

  3. Ultrasensitive detection of nucleic acids by template enhanced hybridization followed by rolling circle amplification and catalytic hairpin assembly.

    Science.gov (United States)

    Song, Weiling; Zhang, Qiao; Sun, Wenbo

    2015-02-11

    An ultrasensitive protocol for fluorescent detection of DNA is designed by combining the template enhanced hybridization process (TEHP) with Rolling Circle Amplification (RCA) and Catalytic Hairpin Assembly (CHA), showing a remarkable amplification efficiency.

  4. Implementation of Oral and Rectal Gonococcal and Chlamydial Nucleic Acid Amplification-Based Testing as a Component of Local Health Department Activities.

    Science.gov (United States)

    Nall, Jennifer; Barr, Breona; McNeil, Candice J; Bachmann, Laura H

    2016-10-01

    From January 1, 2014, to May 31, 2015, 452 individuals received extragenital nucleic acid amplification-based Neisseria gonorrhoeae and Chlamydia trachomatis testing through public health venues. Seventy-four individuals (16%) tested positive for Neisseria gonorrhoeae and/or Chlamydia trachomatis at an extragenital site and 40 (54%) would not have been effectively diagnosed and treated in the absence of extragenital testing.

  5. Reliability of nucleic acid amplification methods for detection of Chlamydia trachomatis in urine: results of the first international collaborative quality control study among 96 laboratories

    NARCIS (Netherlands)

    R.P.A.J. Verkooyen (Roel); G.T. Noordhoek; P.E. Klapper; J. Reid; J. Schirm; G.M. Cleator; M. Ieven; G. Hoddevik

    2003-01-01

    textabstractThe first European Quality Control Concerted Action study was organized to assess the ability of laboratories to detect Chlamydia trachomatis in a panel of urine samples by nucleic acid amplification tests (NATs). The panel consisted of lyophilized urine samples, includ

  6. Sugar-assisted kinetic resolution of amino acids and amplification of enantiomeric excess of organic molecules.

    Science.gov (United States)

    Córdova, Armando; Sundén, Henrik; Xu, Yongmei; Ibrahem, Ismail; Zou, Weibiao; Engqvist, Magnus

    2006-07-17

    The origins of biological homochirality have intrigued researchers since Pasteur's discovery of the optical activity of biomolecules. Herein, we propose and demonstrate a novel alternative for the evolution of homochirality that is not based on autocatalysis and forges a direct relationship between the chirality of sugars and amino acids. This process provides a mechanism in which a racemic mixture of an amino acid can catalyze the formation of an optically active organic molecule in the presence of a sugar product of low enantiomeric excess.

  7. Detection of Staphylococcus epidermidis by a Quartz Crystal Microbalance Nucleic Acid Biosensor Array Using Au Nanoparticle Signal Amplification

    Directory of Open Access Journals (Sweden)

    Weiling Fu

    2008-10-01

    Full Text Available Staphylococcus epidermidis is a critical pathogen of nosocomial blood infections, resulting in significant morbidity and mortality. A piezoelectric quartz crystal microbalance (QCM nucleic acid biosensor array using Au nanoparticle signal amplification was developed to rapidly detect S. epidermidis in clinical samples. The synthesized thiolated probes specific targeting S. epidermidis 16S rRNA gene were immobilized on the surface of QCM nucleic acid biosensor arrays. Hybridization was induced by exposing the immobilized probes to the PCR amplified fragments of S. epidermidis, resulting in a mass change and a consequent frequency shift of the QCM biosensor. To further enhance frequency shift results from above described hybridizations, streptavidin coated Au nanoparticles were conjugated to the PCR amplified fragments. The results showed that the lowest detection limit of current QCM system was 1.3×103 CFU/mL. A linear correlation was found when the concentration of S. epidermidis varied from 1.3×103 to 1.3×107 CFU/mL. In addition, 55 clinical samples were detected with both current QCM biosensor system and conventional clinical microbiological method, and the sensitivity and specificity of current QCM biosensor system were 97.14% and 100%, respectively. In conclusion, the current QCM system is a rapid, low-cost and sensitive method that can be used to identify infection of S. epidermidis in clinical samples.

  8. Suitability of an automated nucleic acid extractor (easyMAG) for use with hepatitis C virus and human immunodeficiency virus type 1 nucleic acid amplification testing.

    Science.gov (United States)

    Jarvis, L M; Mulligan, K; Dunsford, T H; McGowan, K; Petrik, J

    2011-02-01

    Serological screening assays have greatly reduced, but not eliminated, the risk of transmission of viral infections by transfusion of blood and blood products. In addition, the 1999 regulation of the European Agency for the Evaluation of Medicinal Products requiring all plasma for fractionation to have tested negative for hepatitis C virus (HCV) RNA (CPMP/BWP/390/97, 1998) led many blood transfusion services to introduce nucleic acid amplification technology (NAT) to screen blood donations for HCV, and in some services for human immunodeficiency virus (HIV) and hepatitis B virus (HBV). BioMérieux's second-generation system, the NucliSENS easyMAG, was evaluated as a suitable platform for the automated extraction of nucleic acids for use with the existing SNBTS NAT assays. Two nucleic acid extraction protocols were examined, either lysis on the easyMAG (on board) or a 30-min pre-incubation of the sample with lysis buffer at 37 °C (off board). Off board lysis was found to extract nucleic acid more efficiently for both HCV and HIV NAT assays although the improvement was more marked with HIV. The 95% limit of detections (LODs) were 10.11 IU/ml (on board) and 7.21 IU/ml (off board) for HCV and 55.11 IU/ml (on board) and 34.13 (off board) for HIV. Using the more sensitive off board lysis, nucleic acid extraction specificity, robustness and reliability of the easyMAG were examined and over 10,000 Scottish blood donations (in 107 pools of 95 donations) were tested for HCV and HIV in parallel with the existing assay. The results indicate that the easyMAG is a suitable and flexible nucleic acid extraction system, providing high quality nucleic acids and a rapid response alternative to commercial, fully automated, approved blood screening platforms. © 2010 Elsevier B.V. All rights reserved.

  9. High Interlaboratory Reprocucibility of DNA Sequence-based Typing of Bacteria in a Multicenter Study

    DEFF Research Database (Denmark)

    Sousa, MA de; Boye, Kit; Lencastre, H de;

    2006-01-01

    Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without...

  10. Quantitative nucleic acid amplification methods and their implications in clinical virology

    Science.gov (United States)

    Singh, Mini P; Galhotra, Shipra; Saigal, Karnika; Kumar, Archit; Ratho, Radha Kanta

    2017-01-01

    Recently, a number of techniques have been approved for quantification of viral nucleic acids in clinical samples. Viral load (VL) tests have considerable importance in the management of patients and are widely used in routine diagnosis. In clinical virology, VL testing are important to monitor the antiviral treatment, to initiate preemptive therapy, to understand pathogenesis, and to evaluate the infectivity. These tests have now become a part of many diagnostic and treatment guidelines. Considering the various challenges for in-house viral testing related to the standardization, validation, and precision; they are gradually being replaced by the United States Food and Drug Administration (US FDA) cleared tests. This review summarizes the various viral quantification methods and also discusses the clinical applicability of these in human immunodeficiency virus, Hepatitis B virus, Hepatitis C virus, Cytomegalovirus, and Epstein Barr virus infected patients. Further the challenges and future perspectives of VL testing have also been discussed.

  11. Improved sensitivity of nucleic acid amplification for rapid diagnosis of tuberculous meningitis

    DEFF Research Database (Denmark)

    Johansen, Isik Somuncu; Lundgren, Bettina; Tabak, Fehmi;

    2004-01-01

    ) for the detection of Mycobacterium tuberculosis complex organisms in parallel with the ProbeTec method with a modified pretreatment procedure with 101 prospectively collected cerebrospinal fluid specimens from 94 patients with suspected TBM. By the modified method, the sample-washing step was omitted. A definitive...... diagnosis was attained by culture. Thirteen specimens from 12 patients were culture positive for M. tuberculosis complex organisms; three specimens (23%) were microscopy positive for acid-fast bacilli. Among the culture-positive specimens, the standard ProbeTec method was positive for 8 (61...... was adjusted from the recommended value of 3,400 to 1,000, the sensitivity of the modified procedure increased to 84.7%, with unchanged specificity. Results were obtained in 3 to 4 h. The new pretreatment procedure with the ProbeTec assay described here provides a rapid, simple, and sensitive tool...

  12. Quantitative nucleic acid amplification methods and their implications in clinical virology.

    Science.gov (United States)

    Singh, Mini P; Galhotra, Shipra; Saigal, Karnika; Kumar, Archit; Ratho, Radha Kanta

    2017-01-01

    Recently, a number of techniques have been approved for quantification of viral nucleic acids in clinical samples. Viral load (VL) tests have considerable importance in the management of patients and are widely used in routine diagnosis. In clinical virology, VL testing are important to monitor the antiviral treatment, to initiate preemptive therapy, to understand pathogenesis, and to evaluate the infectivity. These tests have now become a part of many diagnostic and treatment guidelines. Considering the various challenges for in-house viral testing related to the standardization, validation, and precision; they are gradually being replaced by the United States Food and Drug Administration (US FDA) cleared tests. This review summarizes the various viral quantification methods and also discusses the clinical applicability of these in human immunodeficiency virus, Hepatitis B virus, Hepatitis C virus, Cytomegalovirus, and Epstein Barr virus infected patients. Further the challenges and future perspectives of VL testing have also been discussed.

  13. Nucleic acid amplification tests (NAATs for gonorrhoea diagnosis in women: Experience of a tertiary care hospital in north India

    Directory of Open Access Journals (Sweden)

    Seema Sood

    2014-01-01

    Full Text Available Background & objectives: Gonorrhoea is among the most frequent of the estimated bacterial sexually transmitted infections (STIs and has significant health implications in women. The use of nucleic acid amplification tests (NAATs has been shown to provide enhanced diagnosis of gonorrhoea in female patients. However, it is recommended that an on-going assessment of the test assays should be performed to check for any probable sequence variation occurring in the targeted region. In this study, an in-house PCR targeting opa-gene of Neisseria gonorrhoeae was used in conjunction with 16S ribosomal PCR to determine the presence of gonorrhoea in female patients attending the tertiary care hospitals. Methods: Endocervical samples collected from 250 female patients with complaints of vaginal or cervical discharge or pain in lower abdomen were tested using opa and 16S ribosomal assay. The samples were also processed by conventional methods. Results: Of the 250 female patients included in the study, only one was positive by conventional methods (microscopy and culture whereas 17 patients were found to be positive based on PCR results. Interpretation & conclusions: The clinical sensitivity of conventional methods for the detection of N. gonorrhoeae in female patients was low. The gonococcal detection rates increased when molecular method was used giving 16 additional positives. Studies should be done to find out other gene targets that may be used in the screening assays to detect the presence of gonorrhoea.

  14. Comparison of nucleic acid amplification assays with BD affirm VPIII for diagnosis of vaginitis in symptomatic women.

    Science.gov (United States)

    Cartwright, Charles P; Lembke, Bryndon D; Ramachandran, Kalpana; Body, Barbara A; Nye, Melinda B; Rivers, Charles A; Schwebke, Jane R

    2013-11-01

    A commercially available, nonamplified, nucleic acid probe-based test system (BD Affirm VPIII) was compared with nucleic acid amplification (NAA)-based assays for determining the etiology of vaginitis in a cohort of 323 symptomatic women. First, a semiquantitative, multiplexed PCR assay (BV-PCR) and the Affirm VPIII Gardnerellavaginalis test were compared with a unified bacterial-vaginosis (BV) reference standard incorporating both Nugent Gram stain scores and Amsel clinical criteria. In the evaluable population of 305 patients, BV-PCR was 96.9% (191/197) sensitive and 92.6% specific (100/108) for BV, while Affirm VPIII was 90.1% sensitive (179/197) and 67.6% specific (73/108). Second, a multiplexed PCR assay detecting Candida albicans and Candida glabrata (CAN-PCR) was compared with the Affirm VPIII Candida test using a reference standard for vulvovaginal candidiasis (VVC) of yeast culture plus exclusion of alternate vaginitis etiologies. In the population evaluated (n = 102), CAN-PCR was 97.7% sensitive (42/43) and 93.2% specific (55/59) and Affirm VP III was 58.1% sensitive (25/43) and 100% specific (59/59) for VVC. Finally, the results of a commercial NAA test (GenProbe Aptima Trichomonas vaginalis assay; ATV) for T. vaginalis were compared with the Affirm VPIII Trichomonas vaginalis test. In the absence of an independent reference standard for trichomonal vaginitis (TV), a positive result in either assay was deemed to represent true infection. In the evaluable cohort of 388 patients, the sensitivity of ATV was 98.1% (53/54) versus 46.3% (25/54) for Affirm VPIII. The diagnostic accuracy of the combined NAA-based test construct was approximately 20 to 25% higher than that of the Affirm VPIII when modeled in populations with various prevalences of infectious vaginitis.

  15. Development of Lentivirus-Based Reference Materials for Ebola Virus Nucleic Acid Amplification Technology-Based Assays.

    Science.gov (United States)

    Mattiuzzo, Giada; Ashall, James; Doris, Kathryn S; MacLellan-Gibson, Kirsty; Nicolson, Carolyn; Wilkinson, Dianna E; Harvey, Ruth; Almond, Neil; Anderson, Robert; Efstathiou, Stacey; Minor, Philip D; Page, Mark

    2015-01-01

    The 2013-present Ebola virus outbreak in Western Africa has prompted the production of many diagnostic assays, mostly based on nucleic acid amplification technologies (NAT). The calibration and performance assessment of established assays and those under evaluation requires reference materials that can be used in parallel with the clinical sample to standardise or control for every step of the procedure, from extraction to the final qualitative/quantitative result. We have developed safe and stable Ebola virus RNA reference materials by encapsidating anti sense viral RNA into HIV-1-like particles. The lentiviral particles are replication-deficient and non-infectious due to the lack of HIV-1 genes and Envelope protein. Ebola virus genes were subcloned for encapsidation into two lentiviral preparations, one containing NP-VP35-GP and the other VP40 and L RNA. Each reference material was formulated as a high-titre standard for use as a calibrator for secondary or internal standards, and a 10,000-fold lower titre preparation to serve as an in-run control. The preparations have been freeze-dried to maximise stability. These HIV-Ebola virus RNA reference materials were suitable for use with in-house and commercial quantitative RT-PCR assays and with digital RT-PCR. The HIV-Ebola virus RNA reference materials are stable at up to 37°C for two weeks, allowing the shipment of the material worldwide at ambient temperature. These results support further evaluation of the HIV-Ebola virus RNA reference materials as part of an International collaborative study for the establishment of the 1st International Standard for Ebola virus RNA.

  16. Detection of influenza A and B with the Alere™ i Influenza A & B: a novel isothermal nucleic acid amplification assay

    Science.gov (United States)

    Hazelton, Briony; Gray, Timothy; Ho, Jennifer; Ratnamohan, V Mala; Dwyer, Dominic E; Kok, Jen

    2015-01-01

    Background Rapid influenza diagnostic tests (RIDTs) have an important role in clinical decision-making; however, the performances of currently available assays vary widely. Objectives We evaluated the performance of the Alere™ i Influenza A&B (Alere™ iNAT), a rapid isothermal nucleic acid amplification assay that has recently received FDA clearance, for the detection of influenza A and B viruses during the Australian influenza season of 2013. Results were compared to two other RIDTs tested in parallel; Quidel Sofia® Influenza A+B fluorescent immunoassay (FIA) and Alere™ BinaxNOW® Influenza A & B immunochromatographic (ICT) assay. Methods A total of 202 paired nasopharyngeal swabs collected from patients ≥16 years old with an influenza-like illness (ILI) were eluted in 2 ml of universal transport medium (UTM) that was used to perform all three RIDTs in parallel. Reverse-transcription polymerase chain reaction (RT-PCR) was used as the reference standard. Results Compared to RT-PCR, Alere™ iNAT detected 77·8% influenza A positive samples versus 71·4% and 44·4% for the Quidel Sofia® Influenza A+B FIA and BinaxNOW® Influenza A & B ICT assay, respectively. For influenza B, Alere™ iNAT detected 75% of those positive by RT-PCR, versus 33·3% and 25·0% for Sofia® and BinaxNOW®, respectively. The specificity of Alere™ iNAT was 100% for influenza A and 99% for influenza B. Conclusions Alere™ i Influenza A&B is a promising new rapid influenza diagnostic assay with potential point-of-care applications. PMID:25728758

  17. Detection of virus mRNA within infected host cells using an isothermal nucleic acid amplification assay: marine cyanophage gene expression within Synechococcus sp

    Directory of Open Access Journals (Sweden)

    Hall Matthew J

    2007-06-01

    Full Text Available Abstract Background Signal-Mediated Amplification of RNA Technology (SMART is an isothermal nucleic acid amplification technology, developed for the detection of specific target sequences, either RNA (for expression or DNA. Cyanophages are viruses that infect cyanobacteria. Marine cyanophages are ubiquitous in the surface layers of the ocean where they infect members of the globally important genus Synechococcus. Results Here we report that the SMART assay allowed us to differentiate between infected and non-infected host cultures. Expression of the cyanophage strain S-PM2 portal vertex gene (g20 was detected from infected host Synechococcus sp. WH7803 cells. Using the SMART assay, we demonstrated that g20 mRNA peaked 240 – 360 minutes post-infection, allowing us to characterise this as a mid to late transcript. g20 DNA was also detected, peaking 10 hours post-infection, coinciding with the onset of host lysis. Conclusion The SMART assay is based on isothermal nucleic acid amplification, allowing the detection of specific sequences of DNA or RNA. It was shown to be suitable for differentiating between virus-infected and non-infected host cultures and for the detection of virus gene expression: the first reported use of this technology for such applications.

  18. Isolation and amplification of mRNA within a simple microfluidic lab on a chip.

    Science.gov (United States)

    Reinholt, Sarah J; Behrent, Arne; Greene, Cassandra; Kalfe, Ayten; Baeumner, Antje J

    2014-01-07

    The major modules for realizing molecular biological assays in a micro-total analysis system (μTAS) were developed for the detection of pathogenic organisms. The specific focus was the isolation and amplification of eukaryotic mRNA within a simple, single-channel device for very low RNA concentrations that could then be integrated with detection modules. The hsp70 mRNA from Cryptosporidium parvum was used as a model analyte. Important points of study were surface chemistries within poly(methyl methacrylate) (PMMA) microfluidic channels that enabled specific and sensitive mRNA isolation and amplification reactions for very low mRNA concentrations. Optimal conditions were achieved when the channel surface was carboxylated via UV/ozone treatment followed by the immobilization of polyamidoamine (PAMAM) dendrimers on the surface, thus increasing the immobilization efficiency of the thymidine oligonucleotide, oligo(dT)25, and providing a reliable surface for the amplification reaction, importantly, without the need for blocking agents. Additional chemical modifications of the remaining active surface groups were studied to avoid nonspecific capturing of nucleic acids and hindering of the mRNA amplification at low RNA concentrations. Amplification of the mRNA was accomplished using nucleic acid sequence-based amplification (NASBA), an isothermal, primer-dependent technique. Positive controls consisting of previously generated NASBA amplicons could be diluted 10(15) fold and still result in successful on-chip reamplification. Finally, the successful isolation and amplification of mRNA from as few as 30 C. parvum oocysts was demonstrated directly on-chip and compared to benchtop devices. This is the first proof of successful mRNA isolation and NASBA-based amplification of mRNA within a simple microfluidic device in relevant analytical volumes.

  19. False-positive results and contamination in nucleic acid amplification assays : Suggestions for a prevent and destroy strategy

    NARCIS (Netherlands)

    Borst, A; Box, ATA; Fluit, AC

    Contamination of samples with DNA is still a major problem in microbiology laboratories, despite the wide acceptance of PCR and other amplification techniques for the detection of frequently low amounts of target DNA. This review focuses on the implications of contamination in the diagnosis and

  20. Label-free and sensitive fluorescence detection of nucleic acid, based on combination of a graphene oxid /SYBR green I dye platform and polymerase assisted signal amplification

    Science.gov (United States)

    Zhu, Xiao; Xing, Da

    2012-12-01

    A new label-free isothermal fluorescence amplification detection for nucleic acid has been developed. In this paper, we first developed a novel sensitive and specific detection platform with an unmodified hairpin probe (HP) combination of the graphene oxid (GO)/ SYBR green I dye (SG), which was relied on the selective principle of adsorption and the high quenching efficiency of GO. Then for the application of this new strategy, we used Mirco RNA-21 (Mir-21) as the target to evaluate this working principle of our design. When the target was hybridizing with the HP and inducing its conformation of change, an efficient isothermal circular strand-displacement polymerization reaction was activating to assist the first signal amplification. In this format, the formed complex conformation of DNA would interact with its high affinity dye, then detached from the surface of GO after incubating with the platform of GO/intercalating dye. This reaction would accompany with obvious fluorescence recovery, and accomplish farther signal enhancement by a mass of intercalating dye inserting into the minor groove of the long duplex replication product. By taking advantage of the multiple amplification of signal, this method exerted substantial enhancement in sensitivity and could be used for rapid and selective detection of Mir-21 with attomole range. It is expected that this cost-effective GO based sensor might hold considerable potential to apply in bioanalysis studies.

  1. Sensitive electrochemical detection of telomerase activity using spherical nucleic acids gold nanoparticles triggered mimic-hybridization chain reaction enzyme-free dual signal amplification.

    Science.gov (United States)

    Wang, Wen-Jing; Li, Jing-Jing; Rui, Kai; Gai, Pan-Pan; Zhang, Jian-Rong; Zhu, Jun-Jie

    2015-03-03

    We report an electrochemical sensor for telomerase activity detection based on spherical nucleic acids gold nanoparticles (SNAs AuNPs) triggered mimic-hybridization chain reaction (mimic-HCR) enzyme-free dual signal amplification. In the detection strategy, SNAs AuNPs and two hairpin probes were employed. SNAs AuNPs as the primary amplification element, not only hybridized with the telomeric repeats on the electrode to amplify signal but also initiated the subsequent secondary amplification, mimic-hybridization chain reaction of two hairpin probes. If the cells' extracts were positive for telomerase activity, SNAs AuNPs could be captured on the electrode. The carried initiators could trigger an alternative hybridization reaction of two hairpin probes that yielded nicked double helices. The signal was further amplified enzyme-free by numerous hexaammineruthenium(III) chloride ([Ru(NH3)6](3+), RuHex) inserting into double-helix DNA long chain by electrostatic interaction, each of which could generate an electrochemical signal at appropriate potential. With this method, a detection limit of down to 2 HeLa cells and a dynamic range of 10-10,000 cells were achieved. Telomerase activities of different cell lines were also successfully evaluated.

  2. High Interlaboratory Reprocucibility of DNA Sequence-based Typing of Bacteria in a Multicenter Study

    DEFF Research Database (Denmark)

    Sousa, MA de; Boye, Kit; Lencastre, H de

    2006-01-01

    Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without...... extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature....

  3. Multiplex, rapid and sensitive isothermal detection of nucleic-acid sequence by endonuclease restriction-mediated real-time multiple cross displacement amplification

    Directory of Open Access Journals (Sweden)

    Yi eWang

    2016-05-01

    Full Text Available We have devised a novel isothermal amplification technology, termed endonuclease restriction-mediated real-time multiple cross displacement amplification (ET-MCDA, which facilitated multiplex, rapid, specific and sensitive detection of nucleic-acid sequences at a constant temperature. The ET-MCDA integrated multiple cross displacement amplification strategy, restriction endonuclease cleavage and real-time fluorescence detection technique. In the ET-MCDA system, the functional cross primer E-CP1 or E-CP2 was constructed by adding a short sequence at the 5’ end of CP1 or CP2, respectively, and the new E-CP1 or E-CP2 primer was labelled at the 5’ end with a fluorophore and in the middle with a dark quencher. The restriction endonuclease Nb.BsrDI specifically recognized the short sequence and digested the newly synthesized double-stranded terminal sequences (5’ end short sequences and their complementary sequences, which released the quenching, resulting on a gain of fluorescence signal. Thus, the ET-MCDA allowed real-time detection of single or multiple targets in only a single reaction, and the positive results were observed in as short as 12 minutes, detecting down to 3.125 fg of genomic DNA per tube. Moreover, the analytical specificity and the practical application of the ET-MCDA were also successfully evaluated in this study. Here we provided the details on the novel ET-MCDA technique and expounded the basic ET-MCDA amplification mechanism.

  4. Comparative genomics beyond sequence-based alignments

    DEFF Research Database (Denmark)

    Þórarinsson, Elfar; Yao, Zizhen; Wiklund, Eric D.;

    2008-01-01

    Recent computational scans for non-coding RNAs (ncRNAs) in multiple organisms have relied on existing multiple sequence alignments. However, as sequence similarity drops, a key signal of RNA structure--frequent compensating base changes--is increasingly likely to cause sequence-based alignment me...

  5. Colorimetric sensing by using allosteric-DNAzyme-coupled rolling circle amplification and a peptide nucleic acid-organic dye probe.

    Science.gov (United States)

    Ali, M Monsur; Li, Yingfu

    2009-01-01

    Target detection by the naked eye: The action of an RNA-cleaving allosteric DNAzyme in response to ligand binding was coupled to a rolling circle amplification process to generate long single-stranded DNA molecules for colorimetric sensing (see scheme). Upon hybridization of the resulting DNA with a complementary PNA sequence in the presence of a duplex-binding dye, the color of the dye changed from blue to purple.

  6. Point of care nucleic acid detection of viable pathogenic bacteria with isothermal RNA amplification based paper biosensor

    Science.gov (United States)

    Liu, Hongxing; Xing, Da; Zhou, Xiaoming

    2014-09-01

    Food-borne pathogens such as Listeria monocytogenes have been recognized as a major cause of human infections worldwide, leading to substantial health problems. Food-borne pathogen identification needs to be simpler, cheaper and more reliable than the current traditional methods. Here, we have constructed a low-cost paper biosensor for the detection of viable pathogenic bacteria with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper biosensor to perform a visual test, in which endpoint detection was performed using sandwich hybridization assays. When the RNA products migrated along the paper biosensor by capillary action, the gold nanoparticles accumulated at the designated Test line and Control line. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from Listeria monocytogenes could be detected. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. The developed method is suitable for point-of-care applications to detect food-borne pathogens, as it can effectively overcome the false-positive results caused by amplifying nonviable Listeria monocytogenes.

  7. A simple, inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings.

    Directory of Open Access Journals (Sweden)

    Paul LaBarre

    Full Text Available BACKGROUND: Molecular assays targeted to nucleic acid (NA markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR instrumentation (another is sample preparation. METHODOLOGY/PRINCIPAL FINDINGS: In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. CONCLUSIONS/SIGNIFICANCE: We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes.

  8. Nucleic acid amplification tests for diagnosis of smear-negative TB in a high HIV-prevalence setting: a prospective cohort study.

    Directory of Open Access Journals (Sweden)

    J Lucian Davis

    Full Text Available Nucleic acid amplification tests are sensitive for identifying Mycobacterium tuberculosis in populations with positive sputum smears for acid-fast bacilli, but less sensitive in sputum-smear-negative populations. Few studies have evaluated the clinical impact of these tests in low-income countries with high burdens of TB and HIV.We prospectively enrolled 211 consecutive adults with cough ≥2 weeks and negative sputum smears at Mulago Hospital in Kampala, Uganda. We tested a single early-morning sputum specimen for Mycobacterium tuberculosis DNA using two nucleic acid amplification tests: a novel in-house polymerase chain reaction targeting the mycobacterial secA1 gene, and the commercial Amplified® Mycobacterium tuberculosis Direct (MTD test (Gen-Probe Inc, San Diego, CA. We calculated the diagnostic accuracy of these index tests in reference to a primary microbiologic gold standard (positive mycobacterial culture of sputum or bronchoalveolar lavage fluid, and measured their likely clinical impact on additional tuberculosis cases detected among those not prescribed initial TB treatment.Of 211 patients enrolled, 170 (81% were HIV-seropositive, with median CD4+ T-cell count 78 cells/µL (interquartile range 29-203. Among HIV-seropositive patients, 94 (55% reported taking co-trimoxazole prophylaxis and 29 (17% reported taking antiretroviral therapy. Seventy-five patients (36% had culture-confirmed TB. Sensitivity of MTD was 39% (95% CI 28-51 and that of secA1 was 24% (95% CI 15-35. Both tests had specificities of 95% (95% CI 90-98. The MTD test correctly identified 18 (24% TB patients not treated at discharge and led to a 72% relative increase in the smear-negative case detection rate.The secA1 and MTD nucleic acid amplification tests had moderate sensitivity and high specificity for TB in a predominantly HIV-seropositive population with negative sputum smears. Although newer, more sensitive nucleic acid assays may enhance detection of

  9. Biomaterials in light amplification

    Science.gov (United States)

    Mysliwiec, Jaroslaw; Cyprych, Konrad; Sznitko, Lech; Miniewicz, Andrzej

    2017-03-01

    Biologically produced or inspired materials can serve as optical gain media, i.e. they can exhibit the phenomenon of light amplification. Some of these materials, under suitable dye-doping and optical pumping conditions, show lasing phenomena. The emerging branch of research focused on obtaining lasing action in highly disordered and highly light scattering materials, i.e. research on random lasing, is perfectly suited for biological materials. The use of biomaterials in light amplification has been extensively reported in the literature. In this review we attempt to report on progress in the development of biologically derived systems able to show the phenomena of light amplification and random lasing together with the contribution of our group to this field. The rich world of biopolymers modified with molecular aggregates and nanocrystals, and self-organized at the nanoscale, offers a multitude of possibilities for tailoring luminescent and light scattering properties that are not easily replicated in conventional organic or inorganic materials. Of particular importance and interest are light amplification and lasing, or random lasing studies in biological cells and tissues. In this review we will describe nucleic acids and their complexes employed as gain media due to their favorable optical properties and ease of manipulation. We will report on research conducted on various biomaterials showing structural analogy to nucleic acids such as fluorescent proteins, gelatins in which the first distributed feedback laser was realized, and also amyloids or silks, which, due to their dye-doped fiber-like structure, allow for light amplification. Other materials that were investigated in that respect include polysaccharides, like starch exhibiting favorable photostability in comparison to other biomaterials, and chitosan, which forms photonic crystals or cellulose. Light amplification and random lasing was not only observed in processed biomaterials but also in living

  10. Detection of Vibrio cholerae by isothermal cross-priming amplification combined with nucleic acid detection strip analysis.

    Science.gov (United States)

    Zhang, Xia; Du, Xin-Jun; Guan, Chun; Li, Ping; Zheng, Wen-Jie; Wang, Shuo

    2015-08-01

    Vibrio cholerae is a water- and food-borne human pathogen, and V. cholerae serotypes O1 and O139 have attracted attention because of their severe pathogenesis. However, non-O1, non-O139 cholera vibrios (NCVs) were also recently recognized as having virulence properties. In this study, we developed a cross-priming amplification (CPA) method for the detection of all serotypes of V. cholerae. The specificity of the CPA method was tested using a panel of 60 different bacterial strains. All of the V. cholerae strains showed positive results, and 41 other types of bacteria gave negative results. The limit of detection of the CPA method was 79.28 fg of genomic DNA, 4.2 × 10(2) CFU/ml for bacteria in pure culture, and 5.6 CFU per 25 g of sample with pre-enrichment. This method showed a higher sensitivity than the loop-mediated isothermal amplification (LAMP) method did and was more convenient to perform. These results indicate that the CPA method can be used for the rapid preliminary screening of V. cholerae. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules.

    Science.gov (United States)

    Mayr, Reinhard; Haider, Michaela; Thünauer, Roland; Haselgrübler, Thomas; Schütz, Gerhard J; Sonnleitner, Alois; Hesse, Jan

    2016-04-15

    Here we report the development of a device for the transcription- and amplification-free detection of DNA and RNA molecules down to the zepto-mole range. A microfluidic chip with a built-in microarray was used for manipulation of nano-liter sample volumes. Specific staining and immobilization of the target molecules was achieved via a double hybridization approach thereby avoiding bias due to enzymatic processes like reverse transcription and PCR amplification. Therefore, target molecules were indirectly labeled by pre-hybridization to complementary Cy5-labeled probes. The remaining single-stranded portion of each target molecule could subsequently hybridize to complementary capture probes of a microarray. Thus a target-mediated immobilization of labeled DNA took place. By means of an ultra-sensitive fluorescence readout, all molecules hybridized to the microarray could be detected. The combination of minimized sample volume and single molecule detection yielded a detection limit of 39 fM (831 molecules in 35.4 nl assay volume) for target DNA and 16 fM (338 molecules) for target RNA after 1h on-chip hybridization.

  12. Relative analytical sensitivity of donor nucleic acid amplification technology screening and diagnostic real-time polymerase chain reaction assays for detection of Zika virus RNA.

    Science.gov (United States)

    Stone, Mars; Lanteri, Marion C; Bakkour, Sonia; Deng, Xutao; Galel, Susan A; Linnen, Jeffrey M; Muñoz-Jordán, Jorge L; Lanciotti, Robert S; Rios, Maria; Gallian, Pierre; Musso, Didier; Levi, José E; Sabino, Ester C; Coffey, Lark L; Busch, Michael P

    2017-03-01

    Zika virus (ZIKV) has spread rapidly in the Pacific and throughout the Americas and is associated with severe congenital and adult neurologic outcomes. Nucleic acid amplification technology (NAT) assays were developed for diagnostic applications and for blood donor screening on high-throughput NAT systems. We distributed blinded panels to compare the analytical performance of blood screening relative to diagnostic NAT assays. A 25-member, coded panel (11 half-log dilutions of a 2013 French Polynesia ZIKV isolate and 2015 Brazilian donor plasma implicated in transfusion transmission, and 3 negative controls) was sent to 11 laboratories that performed 17 assays with 2 to 12 replicates per panel member. Results were analyzed for the percentage reactivity at each dilution and by probit analysis to estimate the 50% and 95% limits of detection (LOD50 and LOD95 , respectively). Donor-screening NAT assays that process approximately 500 µL of plasma into amplification reactions were comparable in sensitivity (LOD50 and LOD95 , 2.5 and 15-18 copies/mL) and were approximately 10-fold to 100-fold more sensitive than research laboratory-developed and diagnostic reverse transcriptase-polymerase chain reaction tests that process from 10 to 30 µL of plasma per amplification. Increasing sample input volume assayed with the Centers for Disease Control and Prevention reverse transcriptase-polymerase chain reaction assays increased the LODs by 10-fold to 30-fold. Blood donor-screening ZIKV NAT assays demonstrate similar excellent sensitivities to assays currently used for screening for transfusion-transmitted viruses and are substantially more sensitive than most other laboratory-developed and diagnostic ZIKV reverse transcriptase-polymerase chain reaction assays. Enhancing sensitivities of laboratory-developed and diagnostic assays may be achievable by increasing sample input. © 2017 AABB.

  13. Nucleic acid specific-based amplification and its application in inspection and quarantine%NASBA技术及其在检验检疫中的应用

    Institute of Scientific and Technical Information of China (English)

    王英超; 王宁宁; 吴兴海; 陈长法; 魏晓棠; 封立平; 张成标

    2014-01-01

    Nucleic acid specific-based amplification (NASBA) is a new technology to amplify RNA originated in PCR. As a new research means, NASBA has the characteristics of convenience, good accuracy, high sensitivity and short periods, especially applicable to RNA analysis. The nucleic acid analysis technology is an important means in entry-exit inspection and quarantine, which can be used to detect and identify pathogenic microorganism in food, pests and pathogen in animals and plants. The paper briefly introduced basic principle of NASBA, compared NASBA to RT-PCR, realtime RT-PCR and other isothermal amplification methods and revealed their differences and similarity. According to the usage characteristics of NASBA, the paper reviewed the application and prospect of NASBA in food safety detection and animal and plant quarantine in entry-exit inspection and quarantine.%序列特异性核酸体外扩增技术(nucleic acid specific-based amplification, NASBA)是在PCR基础上发展起来的一种扩增RNA的新技术,作为一种新型研究手段,具有便捷、准确性好、灵敏度高、周期短的特点,尤其适用于RNA的分析研究。核酸分析技术是出入境检验检疫工作的重要手段,可用于食品病原微生物、动植物产品中的有害生物、病原的分析及鉴定。本文简要介绍了NASBA技术的基本原理,在论证对比NASBA技术与普通PCR方法、荧光PCR方法及其他恒温扩增等核酸分析技术的差异及相似性后,根据其使用特点进一步对NASBA在进出境检验检疫的食品安全检测及动植物检疫的应用予以综述及展望。

  14. Nucleotide and Predicted Amino Acid Sequence-Based Analysis of the Avian Metapneumovirus Type C Cell Attachment Glycoprotein Gene: Phylogenetic Analysis and Molecular Epidemiology of U.S. Pneumoviruses

    Science.gov (United States)

    Alvarez, Rene; Lwamba, Humphrey M.; Kapczynski, Darrell R.; Njenga, M. Kariuki; Seal, Bruce S.

    2003-01-01

    A serologically distinct avian metapneumovirus (aMPV) was isolated in the United States after an outbreak of turkey rhinotracheitis (TRT) in February 1997. The newly recognized U.S. virus was subsequently demonstrated to be genetically distinct from European subtypes and was designated aMPV serotype C (aMPV/C). We have determined the nucleotide sequence of the gene encoding the cell attachment glycoprotein (G) of aMPV/C (Colorado strain and three Minnesota isolates) and predicted amino acid sequence by sequencing cloned cDNAs synthesized from intracellular RNA of aMPV/C-infected cells. The nucleotide sequence comprised 1,321 nucleotides with only one predicted open reading frame encoding a protein of 435 amino acids, with a predicted Mr of 48,840. The structural characteristics of the predicted G protein of aMPV/C were similar to those of the human respiratory syncytial virus (hRSV) attachment G protein, including two mucin-like regions (heparin-binding domains) flanking both sides of a CX3C chemokine motif present in a conserved hydrophobic pocket. Comparison of the deduced G-protein amino acid sequence of aMPV/C with those of aMPV serotypes A, B, and D, as well as hRSV revealed overall predicted amino acid sequence identities ranging from 4 to 16.5%, suggesting a distant relationship. However, G-protein sequence identities ranged from 72 to 97% when aMPV/C was compared to other members within the aMPV/C subtype or 21% for the recently identified human MPV (hMPV) G protein. Ratios of nonsynonymous to synonymous nucleotide changes were greater than one in the G gene when comparing the more recent Minnesota isolates to the original Colorado isolate. Epidemiologically, this indicates positive selection among U.S. isolates since the first outbreak of TRT in the United States. PMID:12682171

  15. SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

    Directory of Open Access Journals (Sweden)

    Ricardo Luiz Dantas MACHADO

    1998-09-01

    Full Text Available We report an adaptation of a technique for the blood sample collection (GFM as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.Relatamos a adaptação de uma técnica para coleta de amostras (MFV e outra para extração, amplificação de DNA de parasitas da malária para diagnóstico por PCR/ELISA. O método de coleta de amostras requer menos habilidade e economisa tempo e dinheiro, assim reduzindo a mais da metade o custo. O material é também adequado para análise genética em especimens frescos ou estocados, preparados por este método.

  16. In-house nucleic acid amplification tests for the detection of Mycobacterium tuberculosis in sputum specimens: meta-analysis and meta-regression

    Directory of Open Access Journals (Sweden)

    Pai Madhukar

    2005-10-01

    Full Text Available Abstract Background More than 200 studies related to nucleic acid amplification (NAA tests to detect Mycobacterium tuberculosis directly from clinical specimens have appeared in the world literature since this technology was first introduced. NAA tests come as either commercial kits or as tests designed by the reporting investigators themselves (in-house tests. In-house tests vary widely in their accuracy, and factors that contribute to heterogeneity in test accuracy are not well characterized. Here, we used meta-analytical methods, including meta-regression, to identify factors related to study design and assay protocols that affect test accuracy in order to identify those factors associated with high estimates of accuracy. Results By searching multiple databases and sources, we identified 2520 potentially relevant citations, and analyzed 84 separate studies from 65 publications that dealt with in-house NAA tests to detect M. tuberculosis in sputum samples. Sources of heterogeneity in test accuracy estimates were determined by subgroup and meta-regression analyses. Among 84 studies analyzed, the sensitivity and specificity estimates varied widely; sensitivity varied from 9.4% to 100%, and specificity estimates ranged from 5.6% to 100%. In the meta-regression analysis, the use of IS6110 as a target, and the use of nested PCR methods appeared to be significantly associated with higher diagnostic accuracy. Conclusion Estimates of accuracy of in-house NAA tests for tuberculosis are highly heterogeneous. The use of IS6110 as an amplification target, and the use of nested PCR methods appeared to be associated with higher diagnostic accuracy. However, the substantial heterogeneity in both sensitivity and specificity of the in-house NAA tests rendered clinically useful estimates of test accuracy difficult. Future development of NAA-based tests to detect M. tuberculosis from sputum specimens should take into consideration these findings in improving

  17. Illumina sequencing-based analyses of bacterial communities during short-chain fatty-acid production from food waste and sewage sludge fermentation at different pH values.

    Science.gov (United States)

    Cheng, Weixiao; Chen, Hong; Yan, ShuHai; Su, Jianqiang

    2014-09-01

    Short-chain fatty acids (SCFAs) can be produced by primary and waste activated sludge anaerobic fermentation. The yield and product spectrum distribution of SCFAs can be significantly affected by different initial pH values. However, most studies have focused on the physical and chemical aspects of SCFA production by waste activated sludge fermentation at different pH values. Information on the bacterial community structures during acidogenic fermentation is limited. In this study, comparisons of the bacterial communities during the co-substrate fermentation of food wastes and sewage sludge at different pH values were performed using the barcoded Illumina paired-end sequencing method. The results showed that different pH environments harbored a characteristic bacterial community, including sequences related to Lactobacillus, Prevotella, Mitsuokella, Treponema, Clostridium, and Ureibacillus. The most abundant bacterial operational taxonomic units in the different pH environments were those related to carbohydrate-degrading bacteria, which are associated with constituents of co-substrate fermentation. Further analyses showed that during organic matter fermentation, a core microbiota composed of Firmicutes, Proteobacteria, and Bacteroidetes existed. Comparison analyses revealed that the bacterial community during fermentation was significantly affected by the pH, and that the diverse product distribution was related to the shift in bacterial communities.

  18. Specific detection of DNA and RNA targets using a novel isothermal nucleic acid amplification assay based on the formation of a three-way junction structure.

    Science.gov (United States)

    Wharam, S D; Marsh, P; Lloyd, J S; Ray, T D; Mock, G A; Assenberg, R; McPhee, J E; Brown, P; Weston, A; Cardy, D L

    2001-06-01

    The formation of DNA three-way junction (3WJ) structures has been utilised to develop a novel isothermal nucleic acid amplification assay (SMART) for the detection of specific DNA or RNA targets. The assay consists of two oligonucleotide probes that hybridise to a specific target sequence and, only then, to each other forming a 3WJ structure. One probe (template for the RNA signal) contains a non-functional single-stranded T7 RNA polymerase promoter sequence. This promoter sequence is made double-stranded (hence functional) by DNA polymerase, allowing T7 RNA polymerase to generate a target-dependent RNA signal which is measured by an enzyme-linked oligosorbent assay (ELOSA). The sequence of the RNA signal is always the same, regardless of the original target sequence. The SMART assay was successfully tested in model systems with several single-stranded synthetic targets, both DNA and RNA. The assay could also detect specific target sequences in both genomic DNA and total RNA from Escherichia coli. It was also possible to generate signal from E.coli samples without prior extraction of nucleic acid, showing that for some targets, sample purification may not be required. The assay is simple to perform and easily adaptable to different targets.

  19. Spontaneous formation and amplification of an enantioenriched α-amino nitrile: a chiral precursor for Strecker amino acid synthesis.

    Science.gov (United States)

    Kawasaki, Tsuneomi; Takamatsu, Naoya; Aiba, Shohei; Tokunaga, Yuji

    2015-10-01

    Without the addition of any chiral substances, the spontaneous formation of an enantioenriched α-amino nitrile (up to 96% ee), which is a chiral precursor for Strecker amino acid synthesis, has been achieved in combination with conglomerate formation. The frequency of the formation of enantiomorphs exhibits an approximate stochastic distribution, i.e., L-form occurred 21 times and D-form occurred 22 times, which fulfils the conditions necessary for spontaneous absolute asymmetric synthesis.

  20. Enzymatic electrochemical detection of epidemic-causing Vibrio cholerae with a disposable oligonucleotide-modified screen-printed bisensor coupled to a dry-reagent-based nucleic acid amplification assay.

    Science.gov (United States)

    Yu, Choo Yee; Ang, Geik Yong; Chan, Kok Gan; Banga Singh, Kirnpal Kaur; Chan, Yean Yean

    2015-08-15

    In this study, we developed a nucleic acid-sensing platform in which a simple, dry-reagent-based nucleic acid amplification assay is combined with a portable multiplex electrochemical genosensor. Preparation of an amplification reaction mix targeting multiple DNA regions of interest is greatly simplified because the lyophilized reagents need only be reconstituted with ultrapure water before the DNA sample is added. The presence of single or multiple target DNAs causes the corresponding single-stranded DNA (ssDNA) amplicons to be generated and tagged with a fluorescein label. The fluorescein-labeled ssDNA amplicons are then analyzed using capture probe-modified screen-printed gold electrode bisensors. Enzymatic amplification of the hybridization event is achieved through the catalytic production of electroactive α-naphthol by anti-fluorescein-conjugated alkaline phosphatase. The applicability of this platform as a diagnostic tool is demonstrated with the detection of toxigenic Vibrio cholerae serogroups O1 and O139, which are associated with cholera epidemics and pandemics. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 168 spiked stool samples. The limit of detection was low (10 colony-forming units/ml) for both toxigenic V. cholerae serogroups. A heat stability assay revealed that the dry-reagent amplification reaction mix was stable at temperatures of 4-56 °C, with an estimated shelf life of seven months. The findings of this study highlight the potential of combining a dry-reagent-based nucleic acid amplification assay with an electrochemical genosensor in a more convenient, sensitive, and sequence-specific detection strategy for multiple target nucleic acids.

  1. Selective Adsorption and Chiral Amplification of Amino Acids in Vermiculite Clay -Implications for the origin of biochirality

    CERN Document Server

    Fraser, Donald G; Jakschitz, Thomas; Rode, Bernd M

    2010-01-01

    Smectite clays are hydrated layer silicates that, like micas, occur naturally in abundance. Importantly, they have readily modifiable interlayer spaces that provide excellent sites for nanochemistry. Vermiculite is one such smectite clay and in the presence of small chain-length alkyl-NH3Cl ions, forms sensitive, 1-D ordered model clay systems with expandable nano-pore inter-layer regions. These inter-layers readily adsorb organic molecules. N-propyl NH3Cl vermiculite clay gels were used to determine the adsorption of alanine, lysine and histidine by chiral HPLC. The results show that during reaction with fresh vermiculite interlayers, significant chiral enrichment of either L- and D-enantiomers occurs depending on the amino acid. Chiral enrichment of the supernatant solutions is up to about 1% per pass. In contrast, addition to clay interlayers already reacted with amino acid solutions resulted in little or no change in D/L ratio during the time of the experiment. Adsorption of small amounts of amphiphilic o...

  2. Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

    Directory of Open Access Journals (Sweden)

    Anja Gulliksen

    2012-01-01

    Full Text Available The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n=28 from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

  3. Selective adsorption and chiral amplification of amino acids in vermiculite clay-implications for the origin of biochirality.

    Science.gov (United States)

    Fraser, Donald G; Fitz, Daniel; Jakschitz, T; Rode, Bernd M

    2011-01-21

    Smectite clays are hydrated layer silicates that, like micas, occur naturally in abundance. Importantly, they have readily modifiable interlayer spaces that provide excellent sites for nanochemistry. Vermiculite is one such smectite clay and in the presence of small chain-length alkyl-NH(3)Cl ions forms sensitive, 1-D ordered model clay systems with expandable nano-pore inter-layer regions. These inter-layers readily adsorb organic molecules. n-Propyl NH(3)Cl vermiculite clay gels were used to determine the adsorption of alanine, lysine and histidine by chiral HPLC. The results show that during reaction with fresh vermiculite interlayers, significant chiral enrichment of either L- and D-enantiomers occurs depending on the amino acid. Chiral enrichment of the supernatant solutions is up to about 1% per pass. In contrast, addition to clay interlayers already reacted with amino acid solutions resulted in little or no change in D/L ratio during the time of the experiment. Adsorption of small amounts of amphiphilic organic molecules in clay inter-layers is known to produce Layer-by-Layer or Langmuir-Blodgett films. Moreover atomistic simulations show that self-organization of organic species in clay interlayers is important. These non-centrosymmetric, chirally active nanofilms may cause clays to act subsequently as chiral amplifiers, concentrating organic material from dilute solution and having different adsorption energetics for D- and L-enantiomers. The additional role of clays in RNA oligomerization already postulated by Ferris and others, together with the need for the organization of amphiphilic molecules and lipids noted by Szostak and others, suggests that such chiral separation by clays in lagoonal environments at normal biological temperatures might also have played a significant role in the origin of biochirality.

  4. Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Simple and Rapid Detection of Promyelocytic Leukemia–Retinoic Acid Receptor α mRNA

    Science.gov (United States)

    Hashimoto, Yuki; Hatayama, Yuki; Kojima, Nao; Morishita, Shota; Matsumoto, Satoko; Hosoda, Yuzuru; Hara, Ayako; Motokura, Toru

    2016-01-01

    Background Acute promyelocytic leukemia (APL) is a disease characterized by expression of Promyelocytic Leukemia–Retinoic Acid Receptor α (PML-RARα) chimeric mRNA. Although APL is curable, early death due to hemorrhage is a major problem. Here, we report the development of a simple and rapid diagnostic method for APL based on reverse transcription loop-mediated isothermal amplification (RT-LAMP). Methods An RT-LAMP primer set was designed to detect three types of PML-RARα mRNA in a single reaction. Serial dilutions of plasmid DNA containing bcr1, bcr2, or bcr3 PML-RARα sequences and RNA extracted from bone marrow aspirates of 6 patients with APL were used to compare the results of RT-LAMP and nested PCR assays. Results Plasmid DNA was amplified by RT-LAMP, for which the reaction time was > 4 h shorter and the lower detection limit was higher than for nested RT-PCR. Six of 7 samples tested positive by both methods. Conclusion We developed an RT-LAMP assay for simple and rapid PML-RARα mRNA detection that may be clinically useful for point-of-care testing and APL diagnosis. PMID:28070163

  5. Point-Counterpoint: A Nucleic Acid Amplification Test for Streptococcus pyogenes Should Replace Antigen Detection and Culture for Detection of Bacterial Pharyngitis.

    Science.gov (United States)

    Pritt, Bobbi S; Patel, Robin; Kirn, Thomas J; Thomson, Richard B

    2016-10-01

    Nucleic acid amplification tests (NAATs) have frequently been the standard diagnostic approach when specific infectious agents are sought in a clinic specimen. They can be applied for specific agents such as S. pyogenes, or commercial multiplex NAATs for detection of a variety of pathogens in gastrointestinal, bloodstream, and respiratory infections may be used. NAATs are both rapid and sensitive. For many years, S. pyogenes testing algorithms used a rapid and specific group A streptococcal antigen test to screen throat specimens, followed, in some clinical settings, by a throat culture for S. pyogenes to increase the sensitivity of its detection. Now S. pyogenes NAATs are being used with increasing frequency. Given their accuracy, rapidity, and ease of use, should they replace antigen detection and culture for the detection of bacterial pharyngitis? Bobbi Pritt and Robin Patel of the Mayo Clinic, where S. pyogenes NAATs have been used for well over a decade with great success, will explain the advantages of this approach, while Richard (Tom) Thomson and Tom Kirn of the NorthShore University HealthSystem will discuss their concerns about this approach to diagnosing bacterial pharyngitis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  6. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification.

    Science.gov (United States)

    Silahtaroglu, Asli N; Nolting, Dorrit; Dyrskjøt, Lars; Berezikov, Eugene; Møller, Morten; Tommerup, Niels; Kauppinen, Sakari

    2007-01-01

    The ability to determine spatial and temporal microRNA (miRNA) accumulation at the tissue, cell and subcellular levels is essential for understanding the biological roles of miRNAs and miRNA-associated gene regulatory networks. This protocol describes a method for fast and effective detection of miRNAs in frozen tissue sections using fluorescence in situ hybridization (FISH). The method combines the unique miRNA recognition properties of locked nucleic acid (LNA)-modified oligonucleotide probes with FISH using the tyramide signal amplification (TSA) technology. Although both approaches have previously been shown to increase detection sensitivity in FISH, combining these techniques into one protocol significantly decreases the time needed for miRNA detection in cryosections, while simultaneously retaining high detection sensitivity. Starting with fixation of the tissue sections, this miRNA FISH protocol can be completed within approximately 6 h and allows miRNA detection in a wide variety of animal tissue cryosections as well as in human tumor biopsies at high cellular resolution.

  7. Detection and identification of occult HBV in blood donors in Taiwan using a commercial, multiplex, multi-dye nucleic acid amplification technology screening test.

    Science.gov (United States)

    Lin, K T; Chang, C L; Tsai, M H; Lin, K S; Saldanha, J; Hung, C M

    2014-02-01

    The ability of a new generation commercial, multiplex, multi-dye test from Roche, the cobas TaqScreen MPX test, version 2.0, to detect and identify occult HBV infections was evaluated using routine donor samples from Kaohsiung Blood Bank, Taiwan. A total of 5973 samples were tested by nucleic acid amplification technology (NAT); 5898 in pools of six, 66 in pools of less than six and nine samples individually. NAT-reactive samples were retested with alternative NAT tests, and follow-up samples from the donors were tested individually by NAT and for all the HBV serological markers. Eight NAT-only-reactive donors were identified, and follow-up samples were obtained from six of the donors. The results indicated that all eight donors had an occult HBV infection with viral loads high prevalence of occult HBV infections since the uncertainty associated with identifying samples with very low viremia is removed by the ability of the test to identify the viral target in samples that are reactive with the cobas TaqScreen MPX test, version 2.0. © 2013 International Society of Blood Transfusion.

  8. Evaluation of three enzyme immunoassays and a nucleic acid amplification test for the diagnosis of Clostridium difficile-associated diarrhea at a university hospital in Brazil

    Directory of Open Access Journals (Sweden)

    Rodrigo Otávio Silveira Silva

    2014-07-01

    Full Text Available Introduction Despite the known importance of Clostridium difficile as a nosocomial pathogen, few studies regarding Clostridium difficile infection (CDI in Brazil have been conducted. To date, the diagnostic tests that are available on the Brazilian market for the diagnosis of CDI have not been evaluated. The aim of this study was to compare the performances of four commercial methods for the diagnosis of CDI in patients from a university hospital in Brazil. Methods Three enzyme immunoassays (EIAs and one nucleic acid amplification test (NAAT were evaluated against a cytotoxicity assay (CTA and toxigenic culture (TC. Stool samples from 92 patients with suspected CDI were used in this study. Results Twenty-five (27.2% of 92 samples were positive according to the CTA, and 23 (25% were positive according to the TC. All EIAs and the NAAT test demonstrated sensitivities between 59 and 68% and specificities greater than 91%. Conclusions All four methods exhibited low sensitivities for the diagnosis of CDI, which could lead to a large number of false-negative results, an increased risk of cross-infection to other patients, and overtreatment with empirical antibiotics.

  9. Total tumor load assessed by one-step nucleic acid amplification assay as an intraoperative predictor for non-sentinel lymph node metastasis in breast cancer.

    Science.gov (United States)

    Nabais, Celso; Figueiredo, Joana; Lopes, Paulina; Martins, Manuela; Araújo, António

    2017-04-01

    This study aimed to determine the relationship between CK19 mRNA copy number in sentinel lymph nodes (SLN) assessed by one-step nucleic acid amplification (OSNA) technique, and non-sentinel lymph nodes (NSLN) metastization in invasive breast cancer. A model using total tumor load (TTL) obtained by OSNA technique was also constructed to evaluate its predictability. We conducted an observational retrospective study including 598 patients with clinically T1-T3 and node negative invasive breast cancer. Of the 88 patients with positive SLN, 58 patients fulfill the inclusion criteria. In the analyzed group 25.86% had at least one positive NSLN in axillary lymph node dissection. Univariate analysis showed that tumor size, TTL and number of SLN macrometastases were predictive factors for NSLN metastases. In multivariate analysis just the TTL was predictive for positive NSLN (OR 2.67; 95% CI 1.06-6.70; P = 0.036). The ROC curve for the model using TTL alone was obtained and an AUC of 0.805 (95% CI 0.69-0.92) was achieved. For TTL >1.9 × 10(5) copies/μL we got 73.3% sensitivity, 74.4% specificity and 88.9% negative predictive value to predict NSLN metastases. When using OSNA technique to evaluate SLN, NSLN metastases can be predicted intraoperatively. This prediction tool could help in decision for axillary lymph node dissection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Amplification of electrolyte uptake in the absorptive glass mat (AGM) separator for valve regulated lead acid (VRLA) batteries

    Science.gov (United States)

    Kumar, Vijay; Kameswara Rao, P. V.; Rawal, Amit

    2017-02-01

    Absorptive glass mat (AGM) separators are widely used for valve regulated lead acid (VRLA) batteries due to their remarkable fiber and structural characteristics. Discharge performance and recharge effectiveness of VRLA batteries essentially rely on the distribution and saturation levels of the electrolyte within the AGM separator. Herein, we report an analytical model for predicting the wicking characteristics of AGM battery separators under unconfined and confined states. The model of wicking behavior of AGM is based upon Fries and Dreyer's approach that included the effect of gravity component which was neglected in classic Lucas-Washburn's model. In addition, the predictive model of wicking accounted for realistic structural characteristics of AGM via orientation averaging approach. For wicking under confined state, the structural parameters have been updated under defined level of compressive stresses based upon the constitutive equation derived for a planar network of fibers in AGM under transverse loading conditions. A comparison has been made between the theoretical models and experimental results of wicking behavior under unconfined and confined states. Most importantly, the presented work has highlighted the questionable validity of classic Lucas-Washburn model for predicting the wicking characteristics of AGM separator over longer time duration.

  11. Can mailed swab samples be dry-shipped for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis by nucleic acid amplification tests?

    Science.gov (United States)

    Gaydos, Charlotte A.; Farshy, Carol; Barnes, Mathilda; Quinn, Nicole; Agreda, Patricia; Rivers, Charles A.; Schwebke, Jane; Papp, John

    2012-01-01

    Background Dry-shipped and mailed vaginal swabs collected at home have been used in research studies for the detection of C. trachomatis (CT), N. gonorrhoeae (GC), and Trichomonas vaginalis (TV) by nucleic acid amplification tests (NAATs) in screening programs. A verification study was performed to compare the limit of detection of CT, GC, and TV on swabs that were dry-shipped to paired swabs that were wet-shipped in transport media through the U.S. mail. Methods The Centers for Disease Control and Prevention prepared inocula in sterile water to mock simulated urogenital swabs with high to low concentrations of CT and GC. Replicate swabs were inoculated with 100µl of dilutions, were dry transported or placed into commercial transport media (“wet”) for mailing for NAAT testing. The University of Alabama prepared replicate concentrations of TV, which were similarly shipped and tested by NAAT. Results All paired dry and wet swabs were detectable for CT. For GC, all paired dry and wet swabs were detectable for GC at concentrations ≥103. At 102 and 10 CFU/ml, the 10 replicate GC results were variably positive. For TV, wet and dry shipped concentrations > 102 TV/ml tested positive, while results at 10 TV/ml were negative for dry swabs. Holding replicate dry swabs at 55°C 5 days before testing did not affect results. Conclusion NAATs were able to detect CT, GC, and TV on dry transported swabs. Using NAATs for testing home-collected, urogenital swabs mailed in a dry state to a laboratory may be useful for outreach screening programs. PMID:22578934

  12. Commercial nucleic-acid amplification tests for diagnosis of pulmonary tuberculosis in respiratory specimens: meta-analysis and meta-regression.

    Directory of Open Access Journals (Sweden)

    Daphne I Ling

    Full Text Available BACKGROUND: Hundreds of studies have evaluated the diagnostic accuracy of nucleic-acid amplification tests (NAATs for tuberculosis (TB. Commercial tests have been shown to give more consistent results than in-house assays. Previous meta-analyses have found high specificity but low and highly variable estimates of sensitivity. However, reasons for variability in study results have not been adequately explored. We performed a meta-analysis on the accuracy of commercial NAATs to diagnose pulmonary TB and meta-regression to identify factors that are associated with higher accuracy. METHODOLOGY/PRINCIPAL FINDINGS: We identified 2948 citations from searching the literature. We found 402 articles that met our eligibility criteria. In the final analysis, 125 separate studies from 105 articles that reported NAAT results from respiratory specimens were included. The pooled sensitivity was 0.85 (range 0.36-1.00 and the pooled specificity was 0.97 (range 0.54-1.00. However, both measures were significantly heterogeneous (p<.001. We performed subgroup and meta-regression analyses to identify sources of heterogeneity. Even after stratifying by type of commercial test, we could not account for the variability. In the meta-regression, the threshold effect was significant (p = .01 and the use of other respiratory specimens besides sputum was associated with higher accuracy. CONCLUSIONS/SIGNIFICANCE: The sensitivity and specificity estimates for commercial NAATs in respiratory specimens were highly variable, with sensitivity lower and more inconsistent than specificity. Thus, summary measures of diagnostic accuracy are not clinically meaningful. The use of different cut-off values and the use of specimens other than sputum could explain some of the observed heterogeneity. Based on these observations, commercial NAATs alone cannot be recommended to replace conventional tests for diagnosing pulmonary TB. Improvements in diagnostic accuracy, particularly sensitivity

  13. Concordance study between one-step nucleic acid amplification and morphologic techniques to detect lymph node metastasis in papillary carcinoma of the thyroid.

    Science.gov (United States)

    del Carmen, Sofía; Gatius, Sonia; Franch-Arcas, Guzmán; Baena, José Antonio; Gonzalez, Oscar; Zafon, Carlos; Cuevas, Dolors; Valls, Joan; Pérez, Angustias; Martinez, Mercedes; Ros, Susana; Macías, Carmen García; Iglesias, Carmela; Matías-Guiu, Xavier; de Álava, Enrique

    2016-02-01

    Tumor resection in papillary thyroid carcinoma (PTC) is often accompanied by lymph node (LN) removal of the central and lateral cervical compartments. One-step nucleic acid amplification (OSNA) is a polymerase chain reaction-based technique that quantifies cytokeratin 19 (CK19) messenger RNA copies. Our aim is to assess the value of OSNA in detection of LN metastases in PTC, in comparison with imprints and microscopic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue. A total of 387 LNs from 37 patients were studied. From each half LN, 2 imprints were taken and analyzed with hematoxylin and eosin (H&E) and CK19 immunostaining. One half of the LN was submitted to OSNA and one half to FFPE processing and H&E and CK19 staining. For concordance analysis, every single LN was considered as a case. A group of 11 cases with discordant results between OSNA and H&E/CK19 FFPE sections were subjected to additional FFPE serial sectioning and H&E and CK19 staining. We found a high degree of concordance between the assays used, with sensitivities ranging from 0.81 to 0.95, and specificities ranging from 0.87 and 0.98. OSNA allowed upstaging of patients from pN0 to pN1, in comparison with standard pathologic analysis. Identification of a metastatic LN with more than 15000 CK19 messenger RNA copies predicted the presence of a second LN with macrometastasis (<5000 copies). In summary, the study shows that OSNA application in sentinel or suspicious LN may be helpful in assessing nodal status in PTC patients.

  14. Evaluation of a viral microarray based on simultaneous extraction and amplification of viral nucleotide acid for detecting human herpesviruses and enteroviruses.

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    Yi Liu

    Full Text Available In this study, a viral microarray based assay was developed to detect the human herpesviruses and enteroviruses associated with central nervous system infections, including herpes simplex virus type 1, type 2 (HSV1 and HSV2, Epstein-Barr virus (EBV, cytomegalovirus (CMV, enterovirus 71 (EV71, coxsackievirus A 16 (CA16 and B 5(CB5. The DNA polymerase gene of human herpesviruses and 5'-untranslated region of enteroviruses were selected as the targets to design primers and probes. Human herpesviruses DNA and enteroviruses RNA were extracted simultaneously by using a guanidinium thiocyanate acid buffer, and were subsequently amplified through a biotinylated asymmetry multiplex RT-PCR with the specific primer of enteroviruses. In total, 90 blood samples and 49 cerebrospinal fluids samples with suspected systemic or neurological virus infections were investigated. Out of 139 samples, 66 were identified as positive. The specificities of this multiplex RT-PCR microarray assay were over 96% but the sensitivities were various from 100% for HSV1, HSV2, EV71 and CB5, 95.83% for CMV, 80% for EBV to 71.43% for CA16 in comparison with reference standards of TaqMan qPCR/qRT-PCR. The high Kappa values (>0.90 from HSV1, HSV2, CMV, EV71 and CB5 were obtained, indicating almost perfect agreement in term of the 5 viruses detection. But lower Kappa values for EBV (0.63 and CA16 (0.74 displayed a moderate to substantial agreement. This study provides an innovation of simultaneous extraction, amplification, hybridization and detection of DNA viruses and RNA viruses with simplicity and specificity, and demonstrates a potential clinical utility for a variety of viruses' detection.

  15. Clinical utility of a nested nucleic acid amplification format in comparison to viral culture for the diagnosis of mucosal herpes simplex infection in a genitourinary medicine setting

    Directory of Open Access Journals (Sweden)

    Wyatt Dorothy E

    2001-08-01

    Full Text Available Abstract Background Nested nucleic acid amplification tests are often thought too sensitive or prone to generatingfalse positive results for routine use. The current study investigated the specificity and clinicalutility of a routine multiplex nested assay for mucosal herpetic infections. Methods Ninety patients, categorised into those clinically diagnosed to (a have and (b not haveherpetic infection, were enrolled. Swabs from oral and ano-genital sites were assayed by thenested assay and culture and the results assessed against clinical evaluation for diagnosingherpetic infections; cell content was also recorded. Results Twenty-six and 64 patients were thought to (a have and (b not have mucosal herpeticinfection. Taking the clinical evaluation as indicating the presence of herpetic infection, thenested polymerase chain reaction and culture had respective sensitivities of 19/26 (73% and12/26 (46% (Χ2 p = 0.02. There was no significant difference in specificities between nPCR62/64 (97% and culture 63/64 (98% (Χ2 p = 1.0. Cell content was important for viraldetection by nPCR (Χ2 p = 0.07 but not culture. Nesting was found necessary for sensitivity anddid not reduce specificity. Assay under-performance appeared related to sub-optimal cellcontent (20% but may have reflected clinical over-diagnosis. The results suggest the need forvalidating specimen cell quality. Conclusions This study questions the value of routine laboratory confirmation of mucosal herpetic infection. The adoption of a more discriminatory usage of laboratory diagnostic facilities for genital herpetic infection, taking account of cell content, and restricting it to those cases where it actually affects patient management, may be warranted.

  16. A touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections

    Directory of Open Access Journals (Sweden)

    Feeney Susan A

    2004-10-01

    Full Text Available Abstract Background Immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. To overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 common respiratory viruses. Results Over an 18 month period a total of 222 specimens were tested by both immunofluorescence and the molecular strip. The specimens came from 103 males (median age 3.5 y, 80 females (median age 9 y and 5 quality assurance scheme specimens. Viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. Viral detection by IF was poor in sputa and respiratory swabs. A total of 99 viruses were detected in the study from 79 patients and 4 quality control specimens: 31 by immunofluorescence and 99 using the molecular strip. The strip consistently out-performed immunofluorescence with no loss of diagnostic specificity. Conclusions The touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. Results by immunofluorescence were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting.

  17. Diagnosis of tuberculosis by using a nucleic acid amplification test in an urban population with high HIV prevalence in the United States.

    Directory of Open Access Journals (Sweden)

    Miwako Kobayashi

    Full Text Available BACKGROUND: Use of nucleic acid amplification tests (NAAT for the diagnosis of Mycobacterium tuberculosis (TB has been recommended on respiratory specimens submitted for acid-fast bacilli (AFB testing. It also helps distinguish between TB and non-tuberculous mycobacteria (NTM species in a setting where NTM rates are relatively high. The purposes of this study are to describe the trend and characteristics of all AFB smear-positive respiratory samples that underwent amplified Mycobacterium tuberculosis direct (MTD testing, a type of NAAT, and to evaluate the clinical utility and necessity of the test for diagnosis of TB in a population with high-HIV prevalence. METHODS: Prospective diagnostic testing and retrospective data analyses were conducted on all AFB smear-positive respiratory samples that underwent MTD testing from 2001 to 2011 at Grady Memorial Hospital (GMH, Atlanta, USA. The test performance was compared to culture. RESULTS: A total of 2,240 AFB smear-positive specimens from 1,412 patients were tested and analyzed in the study. The proportion of specimens that were culture-positive for TB was 28.5%. Sensitivity, specificity, positive predictive value, and negative predictive value of the MTD were 99.0%, 98.0%, 95.3% and 99.6%, respectively. A downward trend was observed in the yearly numbers as well as the proportions of MTD-positive specimens during the study period (p<0.01. There were 2,027 (90.5% specimens from patients with known HIV status, of which 70.6% was HIV positive and the majority of them (81.8% had CD4 counts of less than 200 cells/µL. HIV-positives were more likely to have NTM compared to HIV negatives (67.7% vs. 35.4%, p<0.01. CONCLUSION: Despite the decrease in the incidence of TB, NAAT continues to be an accurate and important diagnostic test in a population with high HIV prevalence, and it differentiates TB and NTM organisms.

  18. Microfluidic Digital Chip for Absolute Quantification of Nucleic Acid Amplification%一种可绝对定量核酸的数字PCR微流控芯片

    Institute of Scientific and Technical Information of China (English)

    朱强远; 杨文秀; 高一博; 于丙文; 邱琳; 周超; 金伟; 金钦汉; 牟颖

    2013-01-01

    构建了一种新型的可进行核酸单分子扩增和核酸绝对定量的数字聚合酶链式反应(数字PCR)微流控芯片.应用多层软光刻技术,以聚二甲基硅氧烷(PDMS)作为芯片材料,盖玻片作为基底制作了具有3层结构以及微阀控制功能的微流控芯片.芯片的大小与载玻片相当,可同时检测4个样品,每个样品通入芯片后平均分配到640个反应小室,每个小室的体积为6 nL.以从肺癌细胞A549中提取的18sRNA为样品检测了该芯片的可行性.将样品稀释数倍后通入芯片,核酸分子随机分布在640个小室中并扩增.核酸分子在芯片中的分布符合泊松分布原理,当样品中待测核酸分子平均拷贝数低于0.5个/小室时,则每个反应小室包含0个或1个分子.经过PCR扩增后,有模板分子的小室检测结果为阳性反应,而无模板分子的小室为阴性反应,最后通过计数阳性反应室的个数,可绝对定量原始待测样品中的目标DNA分子拷贝数.实验结果表明,该数字PCR芯片可实现DNA单分子反应和核酸绝对定量,具有成本低、灵敏度高、节省时间和试剂以及操作简单等优点,为数字PCR方法在普通实验室的应用提供了一种新途径,可用于癌症及感染性疾病的早期诊断、单细胞分析、产前诊断以及各种细菌病毒的核酸检验等研究.%A novel microfluidic digital polymerase chain reaction (PCR) chip for single molecule amplification and absolute quantification of nucleic acid was fabricated by multilayer soft lithography technique and composed of three layers with valves controlling liquid, the material of silicone elastomer polydimethylsiloxane (PDMS) and glass coverslip. The microfluidic chip is equal to a piece of glass coverslip in size, which contains 4 separate panels, and each panel contains 640 independent 6 nL-chambers; the chip is capable of detecting 4 samples simultaneously. Digital PCR on the microfluidic chip was tested

  19. Terabit Nyquist PDM-32QAM signal transmission with training sequence based time domain channel estimation.

    Science.gov (United States)

    Zhang, Fan; Wang, Dan; Ding, Rui; Chen, Zhangyuan

    2014-09-22

    We propose a time domain structure of channel estimation for coherent optical communication systems, which employs training sequence based equalizer and is transparent to arbitrary quadrature amplitude modulation (QAM) formats. Enabled with this methodology, 1.02 Tb/s polarization division multiplexed 32 QAM Nyquist pulse shaping signal with a net spectral efficiency of 7.46 b/s/Hz is transmitted over standard single-mode fiber link with Erbium-doped fiber amplifier only amplification. After 1190 km transmission, the average bit-error rate is lower than the 20% hard-decision forward error correction threshold of 1.5 × 10(-2). The transmission distance can be extended to 1428 km by employing intra-subchannel nonlinear compensation with the digital back-propagation method.

  20. Advances in isothermal amplification: novel strategies inspired by biological processes.

    Science.gov (United States)

    Li, Jia; Macdonald, Joanne

    2015-02-15

    Nucleic acid amplification is an essential process in biological systems. The in vitro adoption of this process has resulted in powerful techniques that underpin modern molecular biology. The most common tool is polymerase chain reaction (PCR). However, the requirement for a thermal cycler has somewhat limited applications of this classic nucleic acid amplification technique. Isothermal amplification, on the other hand, obviates the use of a thermal cycler because reactions occur at a single temperature. Isothermal amplification methods are diverse, but all have been developed from an understanding of natural nucleic acid amplification processes. Here we review current isothermal amplification methods as classified by their enzymatic mechanisms. We compare their advantages, disadvantages, efficiencies, and applications. Finally, we mention some new developments associated with this technology, and consider future possibilities in molecular engineering and recombinant technologies that may develop from an appreciation of the molecular biology of natural systems.

  1. Strand Invasion Based Amplification (SIBA®): A Novel Isothermal DNA Amplification Technology Demonstrating High Specificity and Sensitivity for a Single Molecule of Target Analyte

    OpenAIRE

    Mark J Hoser; Mansukoski, Hannu K.; Morrical, Scott W.; Kevin E. Eboigbodin

    2014-01-01

    Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR) in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invas...

  2. Identification of the new HLA-DRB1{sup *}0812 allele detected by sequencing based typing

    Energy Technology Data Exchange (ETDEWEB)

    Versluis, L.F.; Zwan, A.W. van der; Tilanus, M.G.J. [Univ. Hospital Utrecht (Netherlands); Savelkoul, P.H.M.; Berg-Loonen, E.M. van den [Univ. Hospital Maastricht (Netherlands)] [and others

    1996-12-31

    HLA-DRB typing by polymerase chain reaction-sequence specific priming (PCR-SSP) and sequencing based typing (SBT) was studied within the framework of the Antigen and Haplotype Society 11 and the Sequencing Based Typing Component of the Twelfth International HLA workshop. Sequencing was performed as described by McGinnis and co-workers in 1995 on coded samples, including most DR2 subtypes, resulting in high resolution HLA-DR typing. Sequences were compared with a database containing 107 DRB1, four DRB3, and five DRB5 alleles in a similar way as described for HLA-DPB. One sample showed a new DR8 sequence, indicating the presence of a new allele. This individual (4390) is of Indonesian origin. The specific amplification of the DR8 allele and subsequent sequencing resulted in a sequence which did not match the database and new polymorphism was identified. The complementary strand was sequenced and confirmed the presence of a new DRB1 allele. Cloning and subsequent sequencing of the polymerase chain reaction fragment resulted in confirmation of the direct sequence data. Later this variant was officially named DRB1{sup *}0812. The complete nucleotide sequence of exon 2 of this new allele is shown. This allele differs from DRB1{sup *}0810 by one nucleotide at codon 85, resulting in an alanine (GTT), whereas DRB1{sup *}0810 carries a valine (GCT). 5 refs., 1 fig.

  3. Advances in Chemical Amplification Resist Systems

    Science.gov (United States)

    Ito, Hiroshi

    1992-12-01

    The chemical amplification concept proposed in 1982 to boost resist sensitivities is now well accepted by the lithography community, which stems not only from high sensitivities that chemical amplification resist systems can offer but also from additional benefits of high contrasts and unexpectedly high resolution capabilities. The design flexibility and versatility that the use of acid as a catalytic species offers are another attractive feature of chemical amplification, giving rise to a birth of an entire family of advanced resist systems. Manufacture and prototype fabrication of DRAM’s by deep UV lithography have been accomplished with use of chemical amplification resists. However, some process problems uniquely associated with chemical amplification resists have surfaced recently, which include their latent image instability due to their sensitivity toward minute amounts of air-borne contaminants. This paper reviews recent advances made in our laboratory in the field of chemical amplification resist systems and discusses 1) influence of residual casting solvent on absorption of NMP by polymer films, 2) effects of polymer end groups on resist sensitivity, and 3) new imaging mechanisms based on acid-catalyzed dehydration.

  4. Evaluation of a field-portable DNA microarray platform and nucleic acid amplification strategies for the detection of arboviruses, arthropods, and bloodmeals.

    Science.gov (United States)

    Grubaugh, Nathan D; Petz, Lawrence N; Melanson, Vanessa R; McMenamy, Scott S; Turell, Michael J; Long, Lewis S; Pisarcik, Sarah E; Kengluecha, Ampornpan; Jaichapor, Boonsong; O'Guinn, Monica L; Lee, John S

    2013-02-01

    Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors.

  5. Collaborative study for establishment of a European Pharmacopoei Biological Reference Preparation (BRP) for B19 virus DNA testing of plasma pools by nucleic acid amplification technique.

    Science.gov (United States)

    Nübling, C M; Daas, A; Buchheit, K H

    2004-01-01

    The goal of the collaborative study was to calibrate the B19 DNA content of a candidate Biological Reference Preparation (BRP) that is intended to be used for the validation of the analytical procedure, as threshold control and/or as quantitative reference material in the Nucleic Acid Amplification Technique (NAT) test of plasma pools for detection of B19 contamination. The candidate BRP was calibrated against the 1st International Standard for B19 DNA NAT assays. According to the European Pharmacopoeia monograph Human anti-D immunoglobulin, the threshold control needs to have a titre of 10( 4) IU/ml of B19 virus DNA. The lyophilised candidate BRP was prepared from 0.5 ml aliquots of a plasma pool spiked with B19 virus. The B19 virus originated from a "B19 virus window phase" blood donation (anti-B19 negative, B19-DNA high titre positive) and was diluted in a plasma pool tested negative by both serological and NAT assays for Hepatitis B Virus, Hepatitis C Virus and Human Immunodeficiency Virus 1 to obtain a B19-DNA concentration level in the range of 10( 6) copies/ml. The residual water content of the lyophilised candidate BRP was determined as 0.98 +/- 0.65% (mean +/- relative standard deviation). Sixteen laboratories (Official Medicine Control Laboratories, manufacturers of plasma derivatives, NAT test laboratories and NAT kit manufacturers) from nine countries participated. Participants were requested to test the candidate BRP and the International Standard (99/800) in four independent test runs on different days using their in-house qualitative and/or quantitative NAT methods. Sixteen laboratories reported results. Thirteen laboratories reported results from qualitative assays and 5 laboratories reported results from quantitative assays. Two laboratories reported results from both types of assay. For the qualitative assays a weighted combined potency of 5.64 log( 10) IU/ml with 95 per cent confidence limits of +/- 0.17 log( 10) which corresponds to 67 to 150

  6. A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon.

    Science.gov (United States)

    Wu, Dong; Xu, Huo; Shi, Haimei; Li, Weihong; Sun, Mengze; Wu, Zai-Sheng

    2017-03-08

    K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS(2-) by H2O2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Identification of Mycobacterium tuberculosis complex based on amplification and sequencing of the oxyR pseudogene from stored Ziehl-Neelsen-stained sputum smears in Brazil

    Directory of Open Access Journals (Sweden)

    Marcio Roberto Silva

    2011-02-01

    Full Text Available A cross-sectional analysis of stored Ziehl-Neelsen (ZN-stained sputum smear slides (SSS obtained from two public tuberculosis referral laboratories located in Juiz de Fora, Minas Gerais, was carried out to distinguish Mycobacterium bovis from other members of the Mycobacterium tuberculosis complex (MTC. A two-step approach was used to distinguish M. bovis from other members of MTC: (i oxyR pseudogene amplification to detect MTC and, subsequently, (ii allele-specific sequencing based on the polymorphism at position 285 of this gene. The oxyR pseudogene was successfully amplified in 100 of 177 (56.5% SSS available from 99 individuals. No molecular profile of M. bovis was found. Multivariate analysis indicated that acid-fast bacilli (AFB results and the source laboratory were associated (p < 0.05 with oxyR pseudogene amplification. SSS that were AFB++ SSS showed more oxyR pseudogene amplification than those with AFB0, possibly due to the amount of DNA. One of the two source laboratories presented a greater chance of oxyR pseudogene amplification, suggesting that differences in sputum conservation between laboratories could have influenced the preservation of DNA. This study provides evidence that stored ZN-SSS can be used for the molecular detection of MTC.

  8. The Seneca Amplification Construction

    Directory of Open Access Journals (Sweden)

    Wallace Chafe

    2012-01-01

    Full Text Available The polysynthetic morphology of the Northern Iroquoian languages presents a challenge to studies of clause combining. The discussion here focuses on a Seneca construction that may appear within a single clause but may also straddle clause boundaries. It amplifies the information provided by a referent, here called the trigger, that is introduced by the pronominal prefix within a verb or occasionally in some other way. The particle neh signals that further information about that referent will follow. This construction is found at four levels of syntactic complexity. At the first level the trigger and its amplification occur within the same prosodic phrase and the amplification is a noun. At the second level the amplification occurs in a separate prosodic phrase but remains a noun. At the third level the amplification exhibits verb morphology but has been lexicalized with a nominal function. At the fourth level the amplification functions as a full clause and neh serves as a marker of clause combining. Several varieties of amplification are discussed, as are cases in which the speaker judges that no amplification is needed. It is suggested that the typologically similar Caddo language illustrates a situation in which this construction could never arise, simply because Caddo verbs lack the pronominal element that triggers the construction in Seneca.

  9. Amplification of NOON States

    CERN Document Server

    Agarwal, G S; Rai, Amit

    2009-01-01

    We examine the behavior of a Non Gaussian state like NOON state under phase insensitive amplification. We derive analytical result for the density matrix of the NOON state for arbitrary gain of the amplifier. We consider cases of both symmetric and antisymmetric amplification of the two modes of the NOON state. We quantitatively evaluate the loss of entanglement by the amplifier in terms of the logarithmic negativity parameter. We find that NOON states are more robust than their Gaussian counterparts.

  10. Amplification of NOON States

    OpenAIRE

    2009-01-01

    We examine the behavior of a Non Gaussian state like NOON state under phase insensitive amplification. We derive analytical result for the density matrix of the NOON state for arbitrary gain of the amplifier. We consider cases of both symmetric and antisymmetric amplification of the two modes of the NOON state. We quantitatively evaluate the loss of entanglement by the amplifier in terms of the logarithmic negativity parameter. We find that NOON states are more robust than their Gaussian coun...

  11. Sequence-based analysis of the microbial composition of water kefir from multiple sources.

    Science.gov (United States)

    Marsh, Alan J; O'Sullivan, Orla; Hill, Colin; Ross, R Paul; Cotter, Paul D

    2013-11-01

    Water kefir is a water-sucrose-based beverage, fermented by a symbiosis of bacteria and yeast to produce a final product that is lightly carbonated, acidic and that has a low alcohol percentage. The microorganisms present in water kefir are introduced via water kefir grains, which consist of a polysaccharide matrix in which the microorganisms are embedded. We aimed to provide a comprehensive sequencing-based analysis of the bacterial population of water kefir beverages and grains, while providing an initial insight into the corresponding fungal population. To facilitate this objective, four water kefirs were sourced from the UK, Canada and the United States. Culture-independent, high-throughput, sequencing-based analyses revealed that the bacterial fraction of each water kefir and grain was dominated by Zymomonas, an ethanol-producing bacterium, which has not previously been detected at such a scale. The other genera detected were representatives of the lactic acid bacteria and acetic acid bacteria. Our analysis of the fungal component established that it was comprised of the genera Dekkera, Hanseniaspora, Saccharomyces, Zygosaccharomyces, Torulaspora and Lachancea. This information will assist in the ultimate identification of the microorganisms responsible for the potentially health-promoting attributes of these beverages. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  12. Speeding disease gene discovery by sequence based candidate prioritization

    Directory of Open Access Journals (Sweden)

    Porteous David J

    2005-03-01

    Full Text Available Abstract Background Regions of interest identified through genetic linkage studies regularly exceed 30 centimorgans in size and can contain hundreds of genes. Traditionally this number is reduced by matching functional annotation to knowledge of the disease or phenotype in question. However, here we show that disease genes share patterns of sequence-based features that can provide a good basis for automatic prioritization of candidates by machine learning. Results We examined a variety of sequence-based features and found that for many of them there are significant differences between the sets of genes known to be involved in human hereditary disease and those not known to be involved in disease. We have created an automatic classifier called PROSPECTR based on those features using the alternating decision tree algorithm which ranks genes in the order of likelihood of involvement in disease. On average, PROSPECTR enriches lists for disease genes two-fold 77% of the time, five-fold 37% of the time and twenty-fold 11% of the time. Conclusion PROSPECTR is a simple and effective way to identify genes involved in Mendelian and oligogenic disorders. It performs markedly better than the single existing sequence-based classifier on novel data. PROSPECTR could save investigators looking at large regions of interest time and effort by prioritizing positional candidate genes for mutation detection and case-control association studies.

  13. Gene amplification in carcinogenesis

    Directory of Open Access Journals (Sweden)

    Lucimari Bizari

    2006-01-01

    Full Text Available Gene amplification increases the number of genes in a genome and can give rise to karyotype abnormalities called double minutes (DM and homogeneously staining regions (HSR, both of which have been widely observed in human tumors but are also known to play a major role during embryonic development due to the fact that they are responsible for the programmed increase of gene expression. The etiology of gene amplification during carcinogenesis is not yet completely understood but can be considered a result of genetic instability. Gene amplification leads to an increase in protein expression and provides a selective advantage during cell growth. Oncogenes such as CCND1, c-MET, c-MYC, ERBB2, EGFR and MDM2 are amplified in human tumors and can be associated with increased expression of their respective proteins or not. In general, gene amplification is associated with more aggressive tumors, metastases, resistance to chemotherapy and a decrease in the period during which the patient stays free of the disease. This review discusses the major role of gene amplification in the progression of carcinomas, formation of genetic markers and as possible therapeutic targets for the development of drugs for the treatment of some types of tumors.

  14. A modified PCR protocol for consistent amplification of fatty acid desaturase (FAD) alleles in marker-assisted backcross breeding for high oleic trait in peanut

    Science.gov (United States)

    High oleic acid, such as is found in olive oil, is desirable for the healthy cholesterol-lowering benefits. The oxidative stability of the oil with high oleic acid also gives longer “shelve life” for peanut products. These benefits drive the breeding effort toward developing high oleic peanuts worl...

  15. Isothermal nucleic acid amplification technology applied in detection of Salmonella%恒温扩增核酸法检测沙门菌属效果分析

    Institute of Scientific and Technical Information of China (English)

    肖征; 刘秀贞

    2012-01-01

    目的 观察核酸恒温扩增商品试剂盒对沙门菌属的检测效果.方法 对商品销售的两种恒温扩增检测试剂与普通PCR试剂盒进行检测灵敏度、特异性及操作简便性的比较.结果 常规PCR法A、恒温扩增试剂B及C检测沙门菌属的灵敏度分别为约4×103 CFU/ml、4×103 CFU/ml和约4×104 CFU/ml;对21株临床分离的沙门菌属的检测阳性率分别为76.2%、28.6%和100.0%;用11株非沙门肠道菌株直接检测的特异性均为100.0%;检测过程除常规的核酸提取外,核酸扩增常规法、试剂B、C的核酸检测和结果观察的时间约为2、2、2.5h,以常规法及试剂C的结果观察方式比较方便.结论 试剂C操作时间短、使用方便、检测敏感度及特异性比较好,是一种适用于对沙门菌属快速检测的恒温扩增试剂;对待商品试剂应做好试用工作,以选择好真正适用的产品.%OBJECTIVE To evaluate the efficacy of commercially available isothermal nucleic amplification technology reagents in detecting Salmonella spp. METHODS The routine PCR reagent (A) was compared with two commercially available isothermal nucleic amplification reagents (B and C) for their sensitivity, specificity and operation flexibility in detecting Salmonella spp. RESULTS Reagent A, B and C showed sensitivity of detecting 4 X103 CFU/ml, 4 X 103 CFU/ml and 4 X 104 CFU/ml of Salmonella spp, respectively. The positive rates of detection of clinically isolated Salmonella spp were 76. 2%, 28. 6% and 100. 0%, respectively; all reagents showed no reactions with 11 non-Salmonella enteric bacteria strains with the specificity of 100. 0%; the methods A,B and C took 2, 2 and 2. 5 hours respectively to complete the amplification and results reading after the common procedure of DNA extraction. It was more convenient to observe the results with reagents A and C than B. CONCLUSION Reagent C can be used in field test for Salmonella .spp detection. It is suggested that

  16. Label-free and ratiometric detection of nuclei acids based on graphene quantum dots utilizing cascade amplification by nicking endonuclease and catalytic G-quadruplex DNAzyme.

    Science.gov (United States)

    Wang, Guang-Li; Fang, Xin; Wu, Xiu-Ming; Hu, Xue-Lian; Li, Zai-Jun

    2016-07-15

    Herein, we report a ratiometric fluorescence assay based on graphene quantum dots (GQDs) for the ultrasensitive DNA detection by coupling the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for cascade signal amplifications. With o-phenylenediamine acted as the substrate of G-quadruplex/hemin DNAzyme, whose oxidization product (that is, 2,3-diaminophenazine, DAP) quenched the fluorescence intensity of GQDs (at 460nm) obviously, accompanied with the emergence of a new emission of DAP (at 564nm). The ratiometric signal variations at the emission wavelengths of 564 and 460nm (I564/I460) were utilized for label-free, sensitive, and selective detection of target DNA. Utilizing the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for amplified cascade generation of DAP, the proposed bioassay exhibited high sensitivity toward target DNA with a detection limit of 30fM. The method also had additional advantages such as facile preparation and easy operation.

  17. Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant-Associated Fungi.

    Science.gov (United States)

    Ikenaga, Makoto; Tabuchi, Masakazu; Kawauchi, Tomohiro; Sakai, Masao

    2016-09-29

    The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant-associated fungi due to the similar homologies of sequences in primer-annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3' end of the primer-binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant-associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant-associated fungi.

  18. Detection and identification of 32 Escherichia coli by nuclear acid amplification%32株大肠埃希菌核酸检测鉴定分析

    Institute of Scientific and Technical Information of China (English)

    朱水荣; 潘军航; 余昭; 张政; 王志刚

    2010-01-01

    目的:对本实验室保存多年的32株大肠埃希菌进行核酸检测鉴定,同时验证所用引物的特异性及改用改良方法的可行性.方法:应用环介导等温扩增(Loop-mediated isothermal amplification,LAMP)技术,参照最新LAMP改良方法,对32株大肠埃希菌及其它9株非大肠埃希菌实验对照株分别进行大肠埃希菌malB、不耐热性肠毒素I(heat labile I enterotoxin,LTI)和耐热性肠毒素I(heat stable I enterotoxin,STI)基因检测.结果:32株大肠埃希菌均扩增出大肠埃希菌malB基因,其中3株均扩增出LTI和STI基因,14株只扩增出LTI基因,1株只扩增出STI基因.整个检测过程仅需1.5 h,可通过肉眼目测绿色钙锰复合物是否生成判断结果.结论:32株大肠埃希菌从基因水平均得到鉴定;试验再次证实参考文献中设计的引物其特异性好;改用改良LAMP方法目测结果直观可行,可免去电泳、拍照两步,具有更快速、简便、经济等特点,极适合基层实验室人员应用于对可疑大肠埃希菌的鉴定检测.

  19. Study on screening blood donors by nucleic acid amplification technique combined with Enzyme- linked immunosorbent assay%核酸扩增与酶联免疫法联合在血液筛查中的初步应用

    Institute of Scientific and Technical Information of China (English)

    杜勇; 杨亮; 蒋炜; 王佳维; 张哲

    2012-01-01

    Objective;The purpose of this study was to improve security level of clinical blood transfusion and e-valuate the necessity and practicability of the testing methodology based on nucleic acid amplification technique (NAT) in addition to the regular immunoassay test (EIA). Methods; The samples tested as negative by ELISA were screened by NAT with two work flow ( single detection or combined detection). The NAT - positive samples were further tested by Roche COBAS CAP_CTM system and eletro - cheniluminescence(ECL) system to evaluate the virus load and serological properties. Results; 28 NAT-positive samples were detected in the 20,925 ELISA negative donor samples. All samples were HBV DNA positive and 11 among the 28 samples were serology positive. The remaining risk of HBV infection was 0.13% under the routine EIA test. Conclusion; The risk of HBV infection still remain under the current blood donor screening method using repeated ELISA testing. The introduction of NAT test can help to reduce the risk of transfusion - transmitted disease which has a great value to increase the safety of blood.%目的:在酶联免疫法( enzyme immunoassay,EIA)检测的基础上,探讨HBV核酸扩增检测(nucleic acid amplification testing NAT)技术应用于血液筛查的意义.方法:分别使用两种模式(单检或混检)NAT与EIA两遍检测方式同时进行血液筛查,对NAT阳性标本作进一步做鉴别试验和病毒血清标志物.结果:20925份EIA(-)标本共发现28份核酸三项(HBV DNA、HCV RNA、HIV RNA)呈反应性,均为HBV- DNA,即EIA两遍检测合格后的HBV- DNA阳性率0.13%,检测其中11份血清,乙肝标志物均呈阳性.结论:EIA阴性献血者中仍有极少数的HBV感染者,核酸扩增检测和酶联免疫检测互补能够检测出EIA漏检的HBV携带者,对提高HBsAg阴性血液标本中HBV感染检出率具有重要价值.

  20. Amplification of an MFS Transporter Encoding Gene penT Significantly Stimulates Penicillin Production and Enhances the Sensitivity of Penicillium chrysogenum to Phenylacetic Acid

    Institute of Scientific and Technical Information of China (English)

    Jing Yang; Xinxin Xu; Gang Liu

    2012-01-01

    Penicillin is historically important as the first discovered drug against bacterial infections in human.Although the penicillin biosynthetic pathway and regulatory mechanism have been well studied in Penicillium chrysogenum,the compartnentation and molecular transport of penicillin or its precursors are still poorly understood.In search of the genomic database,more than 830 open reading frames (ORFs) were found to encode transmembrane proteins of P.chrysogenum.In order to investigate their roles on penicillin production,one of them (penT) was selected and cloned.The deduced protein of penT belongs to the major facilitator superfamily (MFS) and contains 12transmembrane spanning domains (TMS).During fermentation,the transcription of penT was greatly induced by penicillin precursors phenylacetic acid (PAA) and phenoxyacetic acid (POA).Knock-down of penT resulted in significant decrease of penicillin production,while over-expression of penT under the promoter of trpC enhanced the penicillin production.Introduction of an additional penT in the wild-type strain of P.chrysogenum doubled the penicillin production and enhanced the sensitivity of P.chrysogenum to the penicillin precursors PAA or POA.These results indicate that penT stimulates penicillin production probably through enhancing the translocation of penicillin precursors across fungal cellular membrane.

  1. Amplification of an MFS transporter encoding gene penT significantly stimulates penicillin production and enhances the sensitivity of Penicillium chrysogenum to phenylacetic acid.

    Science.gov (United States)

    Yang, Jing; Xu, Xinxin; Liu, Gang

    2012-11-20

    Penicillin is historically important as the first discovered drug against bacterial infections in human. Although the penicillin biosynthetic pathway and regulatory mechanism have been well studied in Penicillium chrysogenum, the compartmentation and molecular transport of penicillin or its precursors are still poorly understood. In search of the genomic database, more than 830 open reading frames (ORFs) were found to encode transmembrane proteins of P. chrysogenum. In order to investigate their roles on penicillin production, one of them (penT) was selected and cloned. The deduced protein of penT belongs to the major facilitator superfamily (MFS) and contains 12 transmembrane spanning domains (TMS). During fermentation, the transcription of penT was greatly induced by penicillin precursors phenylacetic acid (PAA) and phenoxyacetic acid (POA). Knock-down of penT resulted in significant decrease of penicillin production, while over-expression of penT under the promoter of trpC enhanced the penicillin production. Introduction of an additional penT in the wild-type strain of P. chrysogenum doubled the penicillin production and enhanced the sensitivity of P. chrysogenum to the penicillin precursors PAA or POA. These results indicate that penT stimulates penicillin production probably through enhancing the translocation of penicillin precursors across fungal cellular membrane.

  2. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases

    Directory of Open Access Journals (Sweden)

    Pravas Ranjan Sahoo

    2016-05-01

    Full Text Available India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

  3. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases.

    Science.gov (United States)

    Sahoo, Pravas Ranjan; Sethy, Kamadev; Mohapatra, Swagat; Panda, Debasis

    2016-05-01

    India being a developing country mainly depends on livestock sector for its economy. However, nowadays, there is emergence and reemergence of more transboundary animal diseases. The existing diagnostic techniques are not so quick and with less specificity. To reduce the economy loss, there should be a development of rapid, reliable, robust diagnostic technique, which can work with high degree of sensitivity and specificity. Loop mediated isothermal amplification assay is a rapid gene amplification technique that amplifies nucleic acid under an isothermal condition with a set of designed primers spanning eight distinct sequences of the target. This assay can be used as an emerging powerful, innovative gene amplification diagnostic tool against various pathogens of livestock diseases. This review is to highlight the basic concept and methodology of this assay in livestock disease.

  4. Performance of self-collected penile-meatal swabs compared to clinician-collected urethral swabs for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium by nucleic acid amplification assays.

    Science.gov (United States)

    Dize, Laura; Barnes, Perry; Barnes, Mathilda; Hsieh, Yu-Hsiang; Marsiglia, Vincent; Duncan, Della; Hardick, Justin; Gaydos, Charlotte A

    2016-10-01

    Men were enrolled in a study to assess the performance and acceptability of self-collected penile meatal swabs as compared to clinician-collected urethral swabs for sexually transmitted infections (STIs). We expected penile-meatal swabs to perform favorably to urethral swabs for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) detection by nucleic acid amplification assays (NAATs). Of 203 swab pairs tested; for CT, penile-meatal swab sensitivity was 96.8% and specificity was 98.8%. NG sensitivity and specificity were 100% and 98.9%, respectively. For TV, sensitivity was 85.0% and specificity was 96.7%. For MG sensitivity and specificity were 79.3% and 99.4%, respectively. No significant statistical differences between sample type accuracy (CT: P=0.625; NG: P=0.248; TV: P=0.344; and MG: P=0.070) existed. Most men, 90.1%, reported self-collection of penile-meatal swabs as "Very Easy" or "Easy". Self-collected penile-meatal swabs appeared acceptable for NAAT STI detection and an acceptable collection method by men.

  5. Application of nucleic acid amplification technique for diagnosis of Chlamydophila pneumonia infection%核酸扩增技术在肺炎嗜衣原体感染诊断中的应用

    Institute of Scientific and Technical Information of China (English)

    张军华; 陈丽丽; 吴移谋

    2010-01-01

    Chlamydia pneumoniae (Cpn) is a kind of microorganism parasitizing in eukaryotic cells, causing human respiratory tract infection, and having persistent infection in the body. Rapid diagnosis in the early stage of Cpn infection helps to prevent the spread of disease and the formation of complications. On the Cpn diagnostic methods, domestic and foreign scholars have carried out a series of studys and made great progress. This paper reviews the application of nucleic acid amplification technique for the diagnosis of Cpn infection.%肺炎嗜衣原体(Chlamydophila pneumonia,Cpn)是一类引起人类呼吸道感染的、专营真核细胞内寄生生活的微生物,在人体中存在持续感染.Cpn感染早期的快速诊断有利于阻止疾病的传播和防止并发症的形成.有关Cpn的诊断方法,国内外学者进行了一系列研究并取得了很大的进展.本文就核酸扩增技术在诊断Cpn感染中的应用作一综述.

  6. Integration of isothermal amplification methods in microfluidic devices: Recent advances.

    Science.gov (United States)

    Giuffrida, Maria Chiara; Spoto, Giuseppe

    2017-04-15

    The integration of nucleic acids detection assays in microfluidic devices represents a highly promising approach for the development of convenient, cheap and efficient diagnostic tools for clinical, food safety and environmental monitoring applications. Such tools are expected to operate at the point-of-care and in resource-limited settings. The amplification of the target nucleic acid sequence represents a key step for the development of sensitive detection protocols. The integration in microfluidic devices of the most popular technology for nucleic acids amplifications, polymerase chain reaction (PCR), is significantly limited by the thermal cycling needed to obtain the target sequence amplification. This review provides an overview of recent advances in integration of isothermal amplification methods in microfluidic devices. Isothermal methods, that operate at constant temperature, have emerged as promising alternative to PCR and greatly simplify the implementation of amplification methods in point-of-care diagnostic devices and devices to be used in resource-limited settings. Possibilities offered by isothermal methods for digital droplet amplification are discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Hardness amplification in nondeterministic logspace

    OpenAIRE

    Gupta, Sushmita

    2007-01-01

    A hard problem is one which cannot be easily computed by efficient algorithms. Hardness amplification is a procedure which takes as input a problem of mild hardness and returns a problem of higher hardness. This is closely related to the task of decoding certain error-correcting codes. We show amplification from mild average case hardness to higher average case hardness for nondeterministic logspace and worst-to-average amplification for nondeterministic linspace. Finally we explore possible ...

  8. Camera-based ratiometric fluorescence transduction of nucleic acid hybridization with reagentless signal amplification on a paper-based platform using immobilized quantum dots as donors.

    Science.gov (United States)

    Noor, M Omair; Krull, Ulrich J

    2014-10-21

    Paper-based diagnostic assays are gaining increasing popularity for their potential application in resource-limited settings and for point-of-care screening. Achievement of high sensitivity with precision and accuracy can be challenging when using paper substrates. Herein, we implement the red-green-blue color palette of a digital camera for quantitative ratiometric transduction of nucleic acid hybridization on a paper-based platform using immobilized quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET). A nonenzymatic and reagentless means of signal enhancement for QD-FRET assays on paper substrates is based on the use of dry paper substrates for data acquisition. This approach offered at least a 10-fold higher assay sensitivity and at least a 10-fold lower limit of detection (LOD) as compared to hydrated paper substrates. The surface of paper was modified with imidazole groups to assemble a transduction interface that consisted of immobilized QD-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as an acceptor. A hybridization event that brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs was responsible for a FRET-sensitized emission from the acceptor dye, which served as an analytical signal. A hand-held UV lamp was used as an excitation source and ratiometric analysis using an iPad camera was possible by a relative intensity analysis of the red (Cy3 photoluminescence (PL)) and green (gQD PL) color channels of the digital camera. For digital imaging using an iPad camera, the LOD of the assay in a sandwich format was 450 fmol with a dynamic range spanning 2 orders of magnitude, while an epifluorescence microscope detection platform offered a LOD of 30 fmol and a dynamic range spanning 3 orders of magnitude. The selectivity of the hybridization assay was demonstrated by detection of a single nucleotide polymorphism at a contrast ratio of 60:1. This work provides an

  9. Whole genome sequence-based serogrouping of Listeria monocytogenes isolates.

    Science.gov (United States)

    Hyden, Patrick; Pietzka, Ariane; Lennkh, Anna; Murer, Andrea; Springer, Burkhard; Blaschitz, Marion; Indra, Alexander; Huhulescu, Steliana; Allerberger, Franz; Ruppitsch, Werner; Sensen, Christoph W

    2016-10-10

    Whole genome sequencing (WGS) is currently becoming the method of choice for characterization of Listeria monocytogenes isolates in national reference laboratories (NRLs). WGS is superior with regards to accuracy, resolution and analysis speed in comparison to several other methods including serotyping, PCR, pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable number tandem repeat analysis (MLVA), and multivirulence-locus sequence typing (MVLST), which have been used thus far for the characterization of bacterial isolates (and are still important tools in reference laboratories today) to control and prevent listeriosis, one of the major sources of foodborne diseases for humans. Backward compatibility of WGS to former methods can be maintained by extraction of the respective information from WGS data. Serotyping was the first subtyping method for L. monocytogenes capable of differentiating 12 serovars and national reference laboratories still perform serotyping and PCR-based serogrouping as a first level classification method for Listeria monocytogenes surveillance. Whole genome sequence based core genome MLST analysis of a L. monocytogenes collection comprising 172 isolates spanning all 12 serotypes was performed for serogroup determination. These isolates clustered according to their serotypes and it was possible to group them either into the IIa, IIc, IVb or IIb clusters, respectively, which were generated by minimum spanning tree (MST) and neighbor joining (NJ) tree data analysis, demonstrating the power of the new approach. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Development of Sequence-Based Microsatellite Marker for Phalaenopsis Orchid

    Directory of Open Access Journals (Sweden)

    FATIMAH

    2011-06-01

    Full Text Available Phalaenopsis is one of the most interesting genera of orchids due to the members are often used as parents to produce hybrids. The establishment and development of highly reliable and discriminatory methods for identifying species and cultivars has become increasingly more important to plant breeders and members of the nursery industry. The aim of this research was to develop sequence-based microsatellite (eSSR markers for the Phalaenopsis orchid designed from the sequence of GenBank NCBI. Seventeen primers were designed and thirteen primers pairs could amplify the DNA giving the expected PCR product with polymorphism. A total of 51 alleles, with an average of 3 alleles per locus and polymorphism information content (PIC values at 0.674, were detected at the 16 SSR loci. Therefore, these markers could be used for identification of the Phalaenopsis orchid used in this study. Genetic similarity and principle coordinate analysis identified five major groups of Phalaenopsis sp. the first group consisted of P. amabilis, P. fuscata, P. javanica, and P. zebrine. The second group consisted of P. amabilis, P. amboinensis, P. bellina, P. floresens, and P. mannii. The third group consisted of P. bellina, P. cornucervi, P. cornucervi, P. violaceae sumatra, P. modesta. The forth group consisted of P. cornucervi and P. lueddemanniana, and the fifth group was P. amboinensis.

  11. 16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria

    OpenAIRE

    Subedi, Shradha; Kong, Fanrong; Jelfs, Peter; Gray, Timothy J; Xiao, Meng; Sintchenko, Vitali; Chen, Sharon C-A.

    2016-01-01

    Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 speci...

  12. Quality control for quantitative PCR based on amplification compatibility test.

    Science.gov (United States)

    Tichopad, Ales; Bar, Tzachi; Pecen, Ladislav; Kitchen, Robert R; Kubista, Mikael; Pfaffl, Michael W

    2010-04-01

    Quantitative qPCR is a routinely used method for the accurate quantification of nucleic acids. Yet it may generate erroneous results if the amplification process is obscured by inhibition or generation of aberrant side-products such as primer dimers. Several methods have been established to control for pre-processing performance that rely on the introduction of a co-amplified reference sequence, however there is currently no method to allow for reliable control of the amplification process without directly modifying the sample mix. Herein we present a statistical approach based on multivariate analysis of the amplification response data generated in real-time. The amplification trajectory in its most resolved and dynamic phase is fitted with a suitable model. Two parameters of this model, related to amplification efficiency, are then used for calculation of the Z-score statistics. Each studied sample is compared to a predefined reference set of reactions, typically calibration reactions. A probabilistic decision for each individual Z-score is then used to identify the majority of inhibited reactions in our experiments. We compare this approach to univariate methods using only the sample specific amplification efficiency as reporter of the compatibility. We demonstrate improved identification performance using the multivariate approach compared to the univariate approach. Finally we stress that the performance of the amplification compatibility test as a quality control procedure depends on the quality of the reference set.

  13. Sequence-based classification using discriminatory motif feature selection.

    Directory of Open Access Journals (Sweden)

    Hao Xiong

    Full Text Available Most existing methods for sequence-based classification use exhaustive feature generation, employing, for example, all k-mer patterns. The motivation behind such (enumerative approaches is to minimize the potential for overlooking important features. However, there are shortcomings to this strategy. First, practical constraints limit the scope of exhaustive feature generation to patterns of length ≤ k, such that potentially important, longer (> k predictors are not considered. Second, features so generated exhibit strong dependencies, which can complicate understanding of derived classification rules. Third, and most importantly, numerous irrelevant features are created. These concerns can compromise prediction and interpretation. While remedies have been proposed, they tend to be problem-specific and not broadly applicable. Here, we develop a generally applicable methodology, and an attendant software pipeline, that is predicated on discriminatory motif finding. In addition to the traditional training and validation partitions, our framework entails a third level of data partitioning, a discovery partition. A discriminatory motif finder is used on sequences and associated class labels in the discovery partition to yield a (small set of features. These features are then used as inputs to a classifier in the training partition. Finally, performance assessment occurs on the validation partition. Important attributes of our approach are its modularity (any discriminatory motif finder and any classifier can be deployed and its universality (all data, including sequences that are unaligned and/or of unequal length, can be accommodated. We illustrate our approach on two nucleosome occupancy datasets and a protein solubility dataset, previously analyzed using enumerative feature generation. Our method achieves excellent performance results, with and without optimization of classifier tuning parameters. A Python pipeline implementing the approach is

  14. Antibiotic Selection Pressure Determination through Sequence-Based Metagenomics.

    Science.gov (United States)

    Willmann, Matthias; El-Hadidi, Mohamed; Huson, Daniel H; Schütz, Monika; Weidenmaier, Christopher; Autenrieth, Ingo B; Peter, Silke

    2015-12-01

    The human gut forms a dynamic reservoir of antibiotic resistance genes (ARGs). Treatment with antimicrobial agents has a significant impact on the intestinal resistome and leads to enhanced horizontal transfer and selection of resistance. We have monitored the development of intestinal ARGs over a 6-day course of ciprofloxacin (Cp) treatment in two healthy individuals by using sequenced-based metagenomics and different ARG quantification methods. Fixed- and random-effect models were applied to determine the change in ARG abundance per defined daily dose of Cp as an expression of the respective selection pressure. Among various shifts in the composition of the intestinal resistome, we found in one individual a strong positive selection for class D beta-lactamases which were partly located on a mobile genetic element. Furthermore, a trend to a negative selection has been observed with class A beta-lactamases (-2.66 hits per million sample reads/defined daily dose; P = 0.06). By 4 weeks after the end of treatment, the composition of ARGs returned toward their initial state but to a different degree in both subjects. We present here a novel analysis algorithm for the determination of antibiotic selection pressure which can be applied in clinical settings to compare therapeutic regimens regarding their effect on the intestinal resistome. This information is of critical importance for clinicians to choose antimicrobial agents with a low selective force on their patients' intestinal ARGs, likely resulting in a diminished spread of resistance and a reduced burden of hospital-acquired infections with multidrug-resistant pathogens.

  15. Efficient audio power amplification - challenges

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Michael A.E.

    2005-07-01

    For more than a decade efficient audio power amplification has evolved and today switch-mode audio power amplification in various forms are the state-of-the-art. The technical steps that lead to this evolution are described and in addition many of the challenges still to be faced and where extensive research and development are needed is covered. (au)

  16. Large Brillouin Amplification in Silicon

    CERN Document Server

    Kittlaus, Eric A; Rakich, Peter T

    2015-01-01

    Strong Brillouin coupling has only recently been realized in silicon using a new class of optomechanical waveguides that yield both optical and phononic confinement. Despite these major advances, appreciable Brillouin amplification has yet to be observed in silicon. Using a new membrane-suspended silicon waveguide we report large Brillouin amplification for the first time, reaching levels greater than 5 dB for modest pump powers, and demonstrate a record low (5 mW) threshold for net amplification. This work represents a crucial advance necessary to realize high-performance Brillouin lasers and amplifiers in silicon.

  17. Next generation Chirped Pulse Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Nees, J.; Biswal, S.; Mourou, G. [Univ. Michigan, Center for Ultrafast Optical Science, Ann Arbor, MI (United States); Nishimura, Akihiko; Takuma, Hiroshi

    1998-03-01

    The limiting factors of Chirped Pulse Amplification (CPA) are discussed and experimental results of CPA in Yb:glass regenerative amplifier are given. Scaling of Yb:glass to the petawatt level is briefly discussed. (author)

  18. A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

    Directory of Open Access Journals (Sweden)

    Farkhondeh Saba

    2017-01-01

    Full Text Available Background:  Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR. Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Methods:  According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications. Results:   This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used.Conclusion:   Having the needed flexibility in each step, this protocol is applicable on a very wide range of samples. Hence, various steps can be included depending on the desired quantity and quality.

  19. A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

    Directory of Open Access Journals (Sweden)

    Farkhondeh Saba

    2016-09-01

    Full Text Available Background:  Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR. Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Methods:  According to our results, amplification of various genomic regions including SSU, LSU, ITS, β-tubulin, actin, RPB2, and EF-1 resulted in a reproducible and efficient DNA extraction from a wide range of microorganisms yielding adequate pure genomic material for reproducible PCR-amplifications. Results:   This method relies on a temporary shock of increased concentrations of detergent which can be applied concomitant with multiple freeze-thaws to yield sufficient amount of DNA for PCR amplification of multiple or single fragments(s of the genome. As an advantage, the recipe seems very flexible, thus, various optional steps can be included depending on the samples used.Conclusion:   Having the needed flexibility in each step, this protocol is applicable on a very wide range of samples. Hence, various steps can be included depending on the desired quantity and quality.

  20. Evaluation of two, commercial, multi-dye, nucleic acid amplification technology tests, for HBV/HCV/HIV-1/HIV-2 and B19V/HAV, for screening blood and plasma for further manufacture.

    Science.gov (United States)

    Müller, M M; Fraile, M I G; Hourfar, M K; Peris, L B; Sireis, W; Rubin, M G; López, E M; Rodriguez, G T; Seifried, E; Saldanha, J; Schmidt, M

    2013-01-01

    The cobas TaqScreen MPX Test, version 2.0, a multiplex, multi-dye nucleic acid amplification technology (NAT) test from Roche was evaluated by two European Blood Banks, the German Red Cross Blood Donor Service, Frankfurt, Germany and Centro de Hemoterapia y Hemodonación de Castilla y León, Valladolid, Spain. In addition, the cobas TaqScreen DPX Test was evaluated for the simultaneous detection and quantitation of parvovirus B19 and the detection of hepatitis A virus (HAV). The performances of the two tests were evaluated regarding the analytical sensitivity, the reproducibility of the tests using samples containing low concentrations of each virus and cross-contamination using samples containing high titres of virus. The analytical sensitivity of the MPX Test, version 2.0, obtained by the German Red Cross Blood Donor Service was 1·1, 3·9 and 43·3 IU/ml for HBV, HCV and HIV-1, respectively. The comparable analytical sensitivity at Centro de Hemoterapia y Hemodonación de Castilla y León was 3·5, 17·6 and 50·6 IU/ml for HBV, HCV and HIV-1, respectively. The analytical sensitivity of the DPX test determined by the German Red Cross Blood Donor Service was 0·6 and 3·8 IU/ml for HAV and B19. These multiplex and multi-dye blood screening assays represent a flexible NAT screening system for mini-pools between 6 and 96 samples per pool and fulfil all requirements of the European Pharmacopoeia for HCV and B19V testing of plasma for fractionation. The inclusion of a new multi-dye technology means discriminatory assays are no longer required for either test thus improving workflow, turn-around time and minimize the risk of obtaining a reactive result for which the virus cannot be identified. © 2012 The Author(s). Vox Sanguinis © 2012 International Society of Blood Transfusion.

  1. Uncertainties in Site Amplification Estimation

    Science.gov (United States)

    Cramer, C. H.; Bonilla, F.; Hartzell, S.

    2004-12-01

    Typically geophysical profiles (layer thickness, velocity, density, Q) and dynamic soil properties (modulus and damping versus strain curves) are used with appropriate input ground motions in a soil response computer code to estimate site amplification. Uncertainties in observations can be used to generate a distribution of possible site amplifications. The biggest sources of uncertainty in site amplifications estimates are the uncertainties in (1) input ground motions, (2) shear-wave velocities (Vs), (3) dynamic soil properties, (4) soil response code used, and (5) dynamic pore pressure effects. A study of site amplification was conducted for the 1 km thick Mississippi embayment sediments beneath Memphis, Tennessee (see USGS OFR 04-1294 on the web). In this study, the first three sources of uncertainty resulted in a combined coefficient of variation of 10 to 60 percent. The choice of soil response computer program can lead to uncertainties in median estimates of +/- 50 percent. Dynamic pore pressure effects due to the passing of seismic waves in saturated soft sediments are normally not considered in site-amplification studies and can contribute further large uncertainties in site amplification estimates. The effects may range from dilatancy and high-frequency amplification (such as observed at some sites during the 1993 Kushiro-Oki, Japan and 2001 Nisqually, Washington earthquakes) or general soil failure and deamplification of ground motions (such as observed at Treasure Island during the 1989 Loma Prieta, California earthquake). Examples of two case studies using geotechnical data for downhole arrays in Kushiro, Japan and the Wildlife Refuge, California using one dynamic code, NOAH, will be presented as examples of modeling uncertainties associated with these effects. Additionally, an example of inversion for estimates of in-situ dilatancy-related geotechnical modeling parameters will be presented for the Kushiro, Japan site.

  2. 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.

  3. Comparison of sequence-based and structure-based phylogenetic trees of homologous proteins: Inferences on protein evolution

    Indian Academy of Sciences (India)

    S Balaji; N Srinivasan

    2007-01-01

    Several studies based on the known three-dimensional (3-D) structures of proteins show that two homologous proteins with insignificant sequence similarity could adopt a common fold and may perform same or similar biochemical functions. Hence, it is appropriate to use similarities in 3-D structure of proteins rather than the amino acid sequence similarities in modelling evolution of distantly related proteins. Here we present an assessment of using 3-D structures in modelling evolution of homologous proteins. Using a dataset of 108 protein domain families of known structures with at least 10 members per family we present a comparison of extent of structural and sequence dissimilarities among pairs of proteins which are inputs into the construction of phylogenetic trees. We find that correlation between the structure-based dissimilarity measures and the sequence-based dissimilarity measures is usually good if the sequence similarity among the homologues is about 30% or more. For protein families with low sequence similarity among the members, the correlation coefficient between the sequence-based and the structure-based dissimilarities are poor. In these cases the structure-based dendrogram clusters proteins with most similar biochemical functional properties better than the sequence-similarity based dendrogram. In multi-domain protein families and disulphide-rich protein families the correlation coefficient for the match of sequence-based and structure-based dissimilarity (SDM) measures can be poor though the sequence identity could be higher than 30%. Hence it is suggested that protein evolution is best modelled using 3-D structures if the sequence similarities (SSM) of the homologues are very low.

  4. Method for chemical amplification based on fluid partitioning in an immiscible liquid

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Brian L.; Colston, Bill W.; Elkin, Christopher J.

    2017-02-28

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  5. Apparatus for chemical amplification based on fluid partitioning in an immiscible liquid

    Science.gov (United States)

    Anderson, Brian L [Lodi, CA; Colston, Bill W [San Ramon, CA; Elkin, Christopher J [San Ramon, CA

    2012-05-08

    A system for nucleic acid amplification of a sample comprises partitioning the sample into partitioned sections and performing PCR on the partitioned sections of the sample. Another embodiment of the invention provides a system for nucleic acid amplification and detection of a sample comprising partitioning the sample into partitioned sections, performing PCR on the partitioned sections of the sample, and detecting and analyzing the partitioned sections of the sample.

  6. Complementarity of ELISA and nucleic acid amplification test in blood screening%血筛用酶联免疫吸附试验与核酸检测互补性探讨和研究

    Institute of Scientific and Technical Information of China (English)

    曾劲峰; 叶贤林; 马兰; 张红; 庄乃保; 李活

    2008-01-01

    目的 为了提高临床输血的安全性,探讨核酸检测(NAT)与酶联免疫吸附试验(ELISA)技术在血液筛查工作中的互补特性.方法 对2007年6月至2008年3月采集的无偿献血者标本共计45 022例用ELISA血清学检测方法对血液传染性指标HBsAg、抗-HCV、抗-HIV、梅毒螺旋体、丙氨酸氨基转移酶(ALT)进行检测,各项指标均正常的标本用NAT技术检测,以研究2种检测方法的互补性.结果 45 022例标本中血清学检测及ALT不合格人数共计803例,不合格率为1.98%.对各项检测指标合格的36 806例标本进行核酸检测,结果HBV-DNA呈阳性3例.HBV-RNA、HIV-RNA均未检出.结论 NAT与ELISA的血液筛查检测互补作用主要体现在3个方面:1)病理生理过程互补,检测窗口期的长短主要由检测对象的生理属性来决定,而非检测方法缺陷.2)检测方法学互补,由于检测方法学的不同使得NAT技术的检测灵敏度明显高于ELISA血清学检测方法.3)影响各自实验的错误发生各不相同.%Objective To investigate the complementarity of ELISA and nucleic acid amplification test(NAT)in blood screening,and to improve the security of clinieal blood transfusion.Methods A total of 45 022 blood samples from the blood donors without payment from June 2007to March 2008 were enrolled in the study.ELISA was applied to determining HBsAg,anti-HCV,anti-HIV,anti-treponema pallidum(anti-TP)and ALT,and then the normal samples for the above parameters(serologically negative for HBsAg.anti-HCV, anti-HIV,anti-TP and ALT)were detected with NAT.The complementarity of ELISA and NAT was analyzed.Results Totally 803 cases(1.98%)were unqualified(serologically positive)out of the 45 022 blood samples.The qualified 36 806samples were further detected with NAT.The results showed 3 cases were HBV-DNA positive,but none was positive for HBV-RNA and HIV-RNA.Conclusion The complementary action of ELISA and NAT is due to different window phase for detected

  7. A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acid.

    Science.gov (United States)

    Xue, Qingwang; Lv, Yanqin; Cui, Hui; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

    2015-01-26

    An autonomous DNA nanomachine based on rolling circle amplification (RCA)-bridged two-stage exonuclease III (Exo III)-induced recycling amplification (Exo III-RCA-Exo III) was developed for label-free and highly sensitive homogeneous multi-amplified detection of DNA combined with sensitive fluorescence detection technique. According to the configuration, the analysis of DNA is accomplished by recognizing the target to a unlabeled molecular beacon (UMB) that integrates target-binding and signal transducer within one multifunctional design, followed by the target-binding of UMB in duplex DNA removed stepwise by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and autonomous generation of the primer that initiate RCA process with a rational designed padlock DNA. The RCA products containing thousands of repeated catalytic sequences catalytically hybridize with a hairpin reporter probe that includes a "caged" inactive G-quadruplex sequence (HGP) and were then detected by Exo III-assisted recycling amplification, liberating the active G-quadruplex and generating remarkable ZnPPIX/G-quadruplex fluorescence signals with the help of zinc(II)-protoporphyrin IX (ZnPPIX). The proposed strategy showed a wide dynamic range over 7 orders of magnitude with a low limit of detection of 0.51 aM. In addition, this designed protocol can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases.

  8. A novel method for the purification of DNA by capturing nucleic acid and magnesium complexes on non-woven fabric filters under alkaline conditions for the gene diagnosis of tuberculosis by loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Fukasawa, Tadashi; Oda, Naozumi; Wada, Yasunao; Tamaru, Aki; Fukushima, Yukari; Nakajima, Chie; Suzuki, Yasuhiko

    2010-07-01

    A novel method for purifying DNA from clinical samples based on the complex formation of DNA and magnesium ion (Mg(2+)) was developed for the detection of Mycobacterium tuberculosis. The formation of a DNA-Mg(2+) complex under alkaline conditions was observed by analyzing electrophoretic mobility reduction of DNA on agarose gel. The DNA-Mg(2+) complex increases the efficacy of DNA recovery from the sample solution on polyethylene terephthalate non-woven fabric (PNWF) filters. Among the various divalent metal cations, only Mg(2+) was associated with this effect. The applicability of DNA recovered on the PNWF filter was examined for the gene amplification method; loop-mediated isothermal amplification (LAMP). DNA on the PNWF filter could be used for the amplification of specific DNA fragments without elution from the filter. Using this method, DNA was directly purified from M. tuberculosis spiked sputum and examined by LAMP assay, showing a high sensitivity in comparison to the commercially available DNA extraction kit. These results indicated that the method developed in this study is useful for rapid gene diagnosis of tuberculosis.

  9. Isothermal Amplification of Insect DNA

    Science.gov (United States)

    The loop-mediated isothermal amplification of DNA (LAMP) method can amplify a target DNA sequence at a constant temperature in about one hour. LAMP has broad application in agriculture and medicine because of the need for rapid and inexpensive diagnoses. LAMP eliminates the need for temperature cycl...

  10. Sequencing-based typing reveals new insight in HLA-DPA1 polymorphism.

    Science.gov (United States)

    Rozemuller, E H; Bouwens, A G; van Oort, E; Versluis, L F; Marsh, S G; Bodmer, J G; Tilanus, M G

    1995-01-01

    An HLA-DPA1 sequencing-based typing (SBT) system has been developed to identify DPA1 alleles. Up to now eight DPA1 alleles have been defined. Six can be discriminated based upon exon 2 polymorphism. The three subtypes of DPA1*01: DPA1*0101, DPA1*0102 and DPA1*0103, have identical exon 2 sequences but show differences in exon 4. Exon 4 sequences were known for only the three DPA1*01 subtypes and for DPA1*0201. We now present additional sequence information for exon 4 and the unknown segments at the 3' end of exon 2. Additionally with the use of this sequencing technique it is also possible to identify previously unidentified polymorphism. We have studied the exon 2 and exon 4 polymorphism of DPA1 in 40 samples which include all known DPA1 alleles. A new allele, DPA1*01 new, was identified which differs by one nucleotide in exon 2 from DPA1*0103, resulting in an aspartic acid at codon 28. The DPA1*01 subtypes DPA1*0101 and DPA1*0102 could not be confirmed in samples which previously were used to define these subtypes, and consequently they do not exist. The exon 4 sequence of DPA1*0201 is corrected based on sequence data of DAUDI, the cell line in which DPA1*0202 was originally defined. The exon 4 regions of the remaining four alleles were resolved: the exon 4 regions of the alleles DPA1*02021 and DPA1*02022 were found to be identical to the--corrected--DPA1*0201 whereas the exon 4 region of DPA1*0301 differs by one nucleotide compared to DPA1*0103. The DPA1*0401 exon 4 region differs by one nucleotide compared to the corrected DPA1*0201.(ABSTRACT TRUNCATED AT 250 WORDS)

  11. Secondary Structure Predictions for Long RNA Sequences Based on Inversion Excursions and MapReduce.

    Science.gov (United States)

    Yehdego, Daniel T; Zhang, Boyu; Kodimala, Vikram K R; Johnson, Kyle L; Taufer, Michela; Leung, Ming-Ying

    2013-05-01

    Secondary structures of ribonucleic acid (RNA) molecules play important roles in many biological processes including gene expression and regulation. Experimental observations and computing limitations suggest that we can approach the secondary structure prediction problem for long RNA sequences by segmenting them into shorter chunks, predicting the secondary structures of each chunk individually using existing prediction programs, and then assembling the results to give the structure of the original sequence. The selection of cutting points is a crucial component of the segmenting step. Noting that stem-loops and pseudoknots always contain an inversion, i.e., a stretch of nucleotides followed closely by its inverse complementary sequence, we developed two cutting methods for segmenting long RNA sequences based on inversion excursions: the centered and optimized method. Each step of searching for inversions, chunking, and predictions can be performed in parallel. In this paper we use a MapReduce framework, i.e., Hadoop, to extensively explore meaningful inversion stem lengths and gap sizes for the segmentation and identify correlations between chunking methods and prediction accuracy. We show that for a set of long RNA sequences in the RFAM database, whose secondary structures are known to contain pseudoknots, our approach predicts secondary structures more accurately than methods that do not segment the sequence, when the latter predictions are possible computationally. We also show that, as sequences exceed certain lengths, some programs cannot computationally predict pseudoknots while our chunking methods can. Overall, our predicted structures still retain the accuracy level of the original prediction programs when compared with known experimental secondary structure.

  12. Strand Invasion Based Amplification (SIBA®: a novel isothermal DNA amplification technology demonstrating high specificity and sensitivity for a single molecule of target analyte.

    Directory of Open Access Journals (Sweden)

    Mark J Hoser

    Full Text Available Isothermal nucleic acid amplification technologies offer significant advantages over polymerase chain reaction (PCR in that they do not require thermal cycling or sophisticated laboratory equipment. However, non-target-dependent amplification has limited the sensitivity of isothermal technologies and complex probes are usually required to distinguish between non-specific and target-dependent amplification. Here, we report a novel isothermal nucleic acid amplification technology, Strand Invasion Based Amplification (SIBA. SIBA technology is resistant to non-specific amplification, is able to detect a single molecule of target analyte, and does not require target-specific probes. The technology relies on the recombinase-dependent insertion of an invasion oligonucleotide (IO into the double-stranded target nucleic acid. The duplex regions peripheral to the IO insertion site dissociate, thereby enabling target-specific primers to bind. A polymerase then extends the primers onto the target nucleic acid leading to exponential amplification of the target. The primers are not substrates for the recombinase and are, therefore unable to extend the target template in the absence of the IO. The inclusion of 2'-O-methyl RNA to the IO ensures that it is not extendible and that it does not take part in the extension of the target template. These characteristics ensure that the technology is resistant to non-specific amplification since primer dimers or mis-priming are unable to exponentially amplify. Consequently, SIBA is highly specific and able to distinguish closely-related species with single molecule sensitivity in the absence of complex probes or sophisticated laboratory equipment. Here, we describe this technology in detail and demonstrate its use for the detection of Salmonella.

  13. Controlled Microwave Heating Accelerates Rolling Circle Amplification.

    Science.gov (United States)

    Yoshimura, Takeo; Suzuki, Takamasa; Mineki, Shigeru; Ohuchi, Shokichi

    2015-01-01

    Rolling circle amplification (RCA) generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.

  14. Transgenerational inheritance: Models and mechanisms of non-DNA sequence-based inheritance.

    Science.gov (United States)

    Miska, Eric A; Ferguson-Smith, Anne C

    2016-10-07

    Heritability has traditionally been thought to be a characteristic feature of the genetic material of an organism-notably, its DNA. However, it is now clear that inheritance not based on DNA sequence exists in multiple organisms, with examples found in microbes, plants, and invertebrate and vertebrate animals. In mammals, the molecular mechanisms have been challenging to elucidate, in part due to difficulties in designing robust models and approaches. Here we review some of the evidence, concepts, and potential mechanisms of non-DNA sequence-based transgenerational inheritance. We highlight model systems and discuss whether phenotypes are replicated or reconstructed over successive generations, as well as whether mechanisms operate at transcriptional and/or posttranscriptional levels. Finally, we explore the short- and long-term implications of non-DNA sequence-based inheritance. Understanding the effects of non-DNA sequence-based mechanisms is key to a full appreciation of heritability in health and disease.

  15. Spheromak Impedance and Current Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Fowler, T K; Hua, D D; Stallard, B W

    2002-01-31

    It is shown that high current amplification can be achieved only by injecting helicity on the timescale for reconnection, {tau}{sub REC}, which determines the effective impedance of the spheromak. An approximate equation for current amplification is: dI{sub TOR}{sup 2}/dt {approx} I{sup 2}/{tau}{sub REC} - I{sub TOR}{sup 2}/{tau}{sub closed} where I is the gun current, I{sub TOR} is the spheromak toroidal current and {tau}{sub CLOSED} is the ohmic decay time of the spheromak. Achieving high current amplification, I{sub TOR} >> I, requires {tau}{sub REC} <<{tau}{sub CLOSED}. For resistive reconnection, this requires reconnection in a cold zone feeding helicity into a hot zone. Here we propose an impedance model based on these ideas in a form that can be implemented in the Corsica-based helicity transport code. The most important feature of the model is the possibility that {tau}{sub REC} actually increases as the spheromak temperature increases, perhaps accounting for the ''voltage sag'' observed in some experiments, and a tendency toward a constant ratio of field to current, B {proportional_to} I, or I{sub TOR} {approx} I. Program implications are discussed.

  16. Evaluation of application of pooling nucleic acid amplification testing in men who have sex with men population in China%HIV集合核酸检测在男男性行为人群中的应用评价

    Institute of Scientific and Technical Information of China (English)

    江华洲; 沈圣; 裴丽健; 黄晓婕; 吴昊; 闫红梅; 潘品良; 蒋岩

    2011-01-01

    Objective To evaluate the application of pooling HIV nucleic acid amplification testing (NAAT) among men who had sex with men (MSM) population, and to investigate suitable HIV screening strategy and the feasibility of calculation of HIV incidence using pooling NAAT among MSM population in China.Methods Four thousand eight hundred and fifty-six samples were collected from MSM population from April 2008 to September 2009 among with 4 156 were in Heilongjiang province and 700 were in Beijing in China. After standard testing with an HIV ELISA and WB confirmation testing, HIV antibody-negative samples were pooled and screened for HIV using NAAT.A three-stage pooling strategy was adopted.The HIV positive rate estimated by the four HIV screening strategies was calculated.In addition, 4 156 HIV positive specimens from Heilongjiang province were screened with the BED capture enzyme immunoassay (BED-CEIA).The HIV-1 incidences were estimated by BED-CEIA assay and pooling NAAT individually.ResultsOne hundred and forty-three of 4 856 subjects were HIV infected.130 were 3rd and 4th generation ELISA positive; 13 were antibody-negative but acutely HIV infected.According to the evaluation of four HIV screening strategies, routine HIV screening test together with pooling NAAT was more effective than other strategies for screening out window period generation ELISA+WB+pooling NAAT' were 2.68%(95% confidence interval CI=2.22%-3.14%), 2.82%(95%CI=2.35%-3.29%), 2.94%(95%CI=2.46%-3.42%) and 2.94%(95%CI=2.46%-3.42%), respectively.The differences were not significant (χ2=0.854 3, P=0.836 4).Of the 88 HIV positive samples from Heilongjiang province, 44 participants were tested as recent HIV infections by BED-CEIA assay. The estimated HIV-1 incidence was 2.36% (95%CI=1.63%-3.08%) and 2.92% (95%CI=1.01%-4.83%) based on BED-CEIA assay and pooling NAAT,respectively.Conclusions Pooling NAAT is a effective screening test in HIV negative population to detect window period infection among MSM

  17. 核酸扩增技术在广州市献血员血液筛查中的应用价值%Evaluation of Nucleic Acid Amplification Screening for Blood Donors in Guangzhou

    Institute of Scientific and Technical Information of China (English)

    明凯华; 雷秀霞; 徐邦牢; 罗丽香; 胡洁洁

    2015-01-01

    【目的】评价核酸扩增技术(NAT)在广州市献血员血液筛查的应用价值。【方法】收集22139名无偿献血员血样,采用酶联免疫吸附试验(ELISA)检测乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、梅毒螺旋体(TP)和人免疫缺陷病毒(HIV),并检测谷丙转氨酶(ALT)水平。对四项ELISA检测阴性和ALT≤40U/L者血样,用COBASs201系统进行HBVDNA、HCVRNA、HIVRNA检测。NAT反应性样本、HBV、HCV和HIVELISA检测阳性血样以COBASAmpliScreen试剂盒鉴定。【结果】22139名献血员中,21776例双试剂血清免疫学检测阴性,其中19例为NAT反应阳性,检出率0.087%(19/21776),后经NAT鉴定检测,HBV、HCV和HIV反应阳性检出率分别为0.051%(11/21776)、0.028%(6/21776)和0.009%(2/21776)。126例HBsAg阳性样本中,25例NAT阴性,其中15例HBsAg中和试验阳性,为低水平慢性感染携带者。50例anti‐HCV阳性血样,4例为NAT阴性,补充ELISA检测为anti‐HCV阴性。16例anti‐HIV阳性样本中,7例为NAT阴性,其单样品核酸检测(ID‐NAT)和补充ELISA检测均为anti‐HIV阴性。【结论】NAT血液筛查对HBV、HCV和HIV经ELISA检测阴性样本的检出率较高,在该地开展NAT血液筛查,对于降低输血残余危险有重大意义。少量HBsAg阳性的低水平感染慢性携带者,汇集核酸检测(MP‐NAT)阴性,HBsAg筛查依然是必不可少的筛查手段。%[Objective] To evaluate the application value of nucleic acid amplification technology (NAT) in screening of blood donors in Guangzhou .[Methods] Blood samples from 22 ,139 blood donors in Guangzhou were collected .Enzyme‐linked immunosorbent assay (ELISA) was used to detect the levels of hepatitis B sur‐face antigen (HBsAg ) ,anti‐hepatitis C virus (anti‐HCV ) ,anti‐human immunodeficiency virus (anti‐HIV ) and anti‐Treponemia pallid (anti‐TP) .And the

  18. State of the art and challenges in sequence based T-cell epitope prediction

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Hoof, Ilka; Lund, Ole

    2010-01-01

    Sequence based T-cell epitope predictions have improved immensely in the last decade. From predictions of peptide binding to major histocompatibility complex molecules with moderate accuracy, limited allele coverage, and no good estimates of the other events in the antigen-processing pathway...... to MHC alleles characterized by limited or no peptide binding data. Most of the developed methods are publicly available, and have proven to be very useful as a shortcut in epitope discovery. Here, we will go through some of the history of sequence-based predictions of helper as well as cytotoxic T cell...

  19. Isothermal amplification using a chemical heating device for point-of-care detection of HIV-1.

    Science.gov (United States)

    Curtis, Kelly A; Rudolph, Donna L; Nejad, Irene; Singleton, Jered; Beddoe, Andy; Weigl, Bernhard; LaBarre, Paul; Owen, S Michele

    2012-01-01

    To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.

  20. Functional diversity of microbial communities in pristine aquifers inferred by PLFA- and sequencing-based approaches

    Science.gov (United States)

    Schwab, Valérie F.; Herrmann, Martina; Roth, Vanessa-Nina; Gleixner, Gerd; Lehmann, Robert; Pohnert, Georg; Trumbore, Susan; Küsel, Kirsten; Totsche, Kai U.

    2017-05-01

    anoxic groundwater. Higher relative abundances of sequence reads in the RNA-based datasets affiliated with iron-reducing bacteria in more Fet-rich groundwater supported the occurrence of active dissimilatory iron reduction. The functional diversity of the microbial communities in the biogeochemically distinct groundwater assemblages can be largely attributed to the redox conditions linked to changes in bioavailable substrates and input of substrates with the seepage. Our results demonstrate the power of complementary information derived from PLFA-based and sequencing-based approaches.

  1. Phytophthora-ID.org: A sequence-based Phytophthora identification tool

    Science.gov (United States)

    N.J. Grünwald; F.N. Martin; M.M. Larsen; C.M. Sullivan; C.M. Press; M.D. Coffey; E.M. Hansen; J.L. Parke

    2010-01-01

    Contemporary species identification relies strongly on sequence-based identification, yet resources for identification of many fungal and oomycete pathogens are rare. We developed two web-based, searchable databases for rapid identification of Phytophthora spp. based on sequencing of the internal transcribed spacer (ITS) or the cytochrome oxidase...

  2. Rapid detection of a norovirus pseudo-outbreak by using real-time sequence based information

    NARCIS (Netherlands)

    Rahamat-Langendoen, J. C.; Lokate, M.; Scholvinck, E. H.; Friedrich, A. W.; Niesters, H. G. M.

    2013-01-01

    Background: Sequence based information is increasingly used to study the epidemiology of viruses, not only to provide insight in viral evolution, but also to understand transmission patterns during outbreaks. However, sequence analysis is not yet routinely performed by diagnostic laboratories, limit

  3. Magnetism Teaching Sequences Based on an Inductive Approach for First-Year Thai University Science Students

    Science.gov (United States)

    Narjaikaew, Pattawan; Emarat, Narumon; Arayathanitkul, Kwan; Cowie, Bronwen

    2010-01-01

    The study investigated the impact on student motivation and understanding of magnetism of teaching sequences based on an inductive approach. The study was conducted in large lecture classes. A pre- and post-Conceptual Survey of Electricity and Magnetism was conducted with just fewer than 700 Thai undergraduate science students, before and after…

  4. Microgel Tethering For Microarray-Based Nucleic Acid Diagnostics

    Science.gov (United States)

    Dai, Xiaoguang

    Molecular diagnostics (MDx) have radically changed the process of clinical microbial identification based on identifying genetic information, MDx approaches are both specific and fast. They can identify microbes to the species and strain level over a time scale that can be as short as one hour. With such information clinicians can administer the most effective and appropriate antimicrobial treatment at an early time point with substantial implications both for patient well-being and for easing the burden on the health-care system. Among the different MDx approaches, such as fluorescence in-situ hybridization, microarrays, next-generation sequencing, and mass spectrometry, point-of-care MDx platforms are drawing particular interest due to their low cost, robustness, and wide application. This dissertation develops a novel MDx technology platform capable of high target amplification and detection performance. For nucleic acid target detection, we fabricate an array of electron-beam-patterned microgels on a standard glass microscope slide. The microgels can be as small as a few hundred nanometers. The unique way of energy deposition during electron-beam lithography provides the microgels with a very diffuse water -gel interface that enables them to not only serve as substrates to immobilize DNA probes but do so while preserving them in a highly hydrated environment that optimizes their performance. Benefiting from the high spatial resolution provided by such techniques as position-sensitive microspotting and dip-pen nanolithography, multiple oligonucleotide probes known as molecular beacons (MBs) can be patterned on microgels. Furthermore, nucleic acid target amplification can be conducted in direct contact with the microgel-tethered detection array. Specifically, we use an isothermal RNA amplification reaction - nucleic acid sequence-based amplification (NASBA). ssRNA amplicons of from the NASBA reaction can directly hybridize with microgel-tethered MBs, and the

  5. Dynamics and Control of DNA Sequence Amplification

    CERN Document Server

    Marimuthu, Karthikeyan

    2014-01-01

    DNA amplification is the process of replication of a specified DNA sequence \\emph{in vitro} through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction (PCR) as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal tempe...

  6. Simple system for isothermal DNA amplification coupled to lateral flow detection.

    Directory of Open Access Journals (Sweden)

    Kristina Roskos

    Full Text Available Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP or the Exponential Amplification Reaction (EXPAR, both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.

  7. Dynamics and control of DNA sequence amplification

    Energy Technology Data Exchange (ETDEWEB)

    Marimuthu, Karthikeyan [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Chakrabarti, Raj, E-mail: raj@pmc-group.com, E-mail: rajc@andrew.cmu.edu [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Division of Fundamental Research, PMC Advanced Technology, Mount Laurel, New Jersey 08054 (United States)

    2014-10-28

    DNA amplification is the process of replication of a specified DNA sequence in vitro through time-dependent manipulation of its external environment. A theoretical framework for determination of the optimal dynamic operating conditions of DNA amplification reactions, for any specified amplification objective, is presented based on first-principles biophysical modeling and control theory. Amplification of DNA is formulated as a problem in control theory with optimal solutions that can differ considerably from strategies typically used in practice. Using the Polymerase Chain Reaction as an example, sequence-dependent biophysical models for DNA amplification are cast as control systems, wherein the dynamics of the reaction are controlled by a manipulated input variable. Using these control systems, we demonstrate that there exists an optimal temperature cycling strategy for geometric amplification of any DNA sequence and formulate optimal control problems that can be used to derive the optimal temperature profile. Strategies for the optimal synthesis of the DNA amplification control trajectory are proposed. Analogous methods can be used to formulate control problems for more advanced amplification objectives corresponding to the design of new types of DNA amplification reactions.

  8. Chirality Amplification in Tactoids of Lyotropic Chromonic Liquid Crystals

    Science.gov (United States)

    Peng, Chenhui; Lavrentovich, Oleg

    2014-03-01

    We demonstrate an effective chirality amplification based on the long-range forces, extending over the scales of tens of micrometers, much larger than the single molecule (nanometer) scale. The mechanism is rooted in the long-range elastic nature of orientational order in lyotropic chromonic liquid crystals (LCLCs) that represent water solutions of achiral disc-like molecules. Minute quantities of chiral molecules such as amino acid L-alanine and limonene added to the droplets of LCLC lead to chiral amplification characterized by an increase of optical activity by a factor of 103 - 104. This effect allows one to discriminate and detect the absolute configuration of chiral molecules in an aqueous system, thus opening new possibilities in biosensing and other biological applications.

  9. Clostridium difficile testing algorithms using glutamate dehydrogenase antigen and C. difficile toxin enzyme immunoassays with C. difficile nucleic acid amplification testing increase diagnostic yield in a tertiary pediatric population.

    Science.gov (United States)

    Ota, Kaede V; McGowan, Karin L

    2012-04-01

    We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens.

  10. Identification and evaluation of a new nucleic acid amplification test target for specific detection of Mycobacterium tuberculosis complex%鉴别结核分枝杆菌复合群的新型核酸扩增检测靶标评价

    Institute of Scientific and Technical Information of China (English)

    高诗会; 赵英伟; 胡忠义; 王洁; 陆俊梅; 闫丽萍; 郭琪; 马慧; 秦莲花

    2012-01-01

    Objective To identify and evaluate a new nucleic acid amplification (NAA) test target for specific detection of Mycobacterium tuberculosis (MTB) complex (MTC).Methods MTC-specific fragment was obtained by ISSR genotyping technology.Primer pairs were designed based on the sequences of MTC-specific fragment and tested in 211 mycobacterial strains including 107 MTC strains and 104 nontuberculous mycobacteria (NTM) strains.IS6110 element (specific identification of MTC strains) and 16s rRNA gene (specific identification of Mycobacterium) amplification were used as a control to evaluate the efficacy of the NAA test target in the detection of MTC strains.Results One MTC-specific fragment with the length of 588 bp,located in 315947-316534 of the genome from MTB reference strain H37 Rv,were obtained,cloned and sequenced.MTC-specific primer pairs MTCF/R were designed based on these sequences.All 211 mycobacterial strains accurately produced the genus-specific 16s rRNA amplicon.All MTC strains were positive in the MTCF/R PCR amplification while 99% MTC strains (106/107) were positive in the amplification of IS6110 sequences.All NTM strains were negative in both IS6110 and MTCF/R PCR amplification.Conclusions The MTC-specific fragment developed in this study can be used as a new NAA test target to correctly distinguish MTC from NTM.%目的 寻找新的鉴别诊断MTB复合群(MTC)高特异度和敏感的核酸靶标.方法 利用简单序列重复区间(ISSR)分型技术平台筛选MTC的特征片段,克隆测序获得特征序列并进行序列同源性分析,以该序列为基础设计MTC特征引物,并对211株分枝杆菌菌株(其中MTC107株、非结核分枝杆菌104株)进行鉴别检测.利用分枝杆菌属特征序列16s rRNA和MTC特征序列IS6110的鉴别结果,对MTC特征引物检测结果进行评估.结果 通过ISSR分型获得588 bp的MTC特征片段,该序列为MTC菌株的特征序列,位于MTB标准株H37Rv基因组的315947 ~316534位,以该序

  11. Accuracy Analysis for 6-DOF PKM with Sobol Sequence Based Quasi Monte Carlo Method

    Institute of Scientific and Technical Information of China (English)

    Jianguang Li; Jian Ding; Lijie Guo; Yingxue Yao; Zhaohong Yi; Huaijing Jing; Honggen Fang

    2015-01-01

    To improve the precisions of pose error analysis for 6⁃dof parallel kinematic mechanism ( PKM) during assembly quality control, a Sobol sequence based on Quasi Monte Carlo ( QMC) method is introduced and implemented in pose accuracy analysis for the PKM in this paper. The Sobol sequence based on Quasi Monte Carlo with the regularity and uniformity of samples in high dimensions, can prevail traditional Monte Carlo method with up to 98�59% and 98�25% enhancement for computational precision of pose error statistics. Then a PKM tolerance design system integrating this method is developed and with it pose error distributions of the PKM within a prescribed workspace are finally obtained and analyzed.

  12. PSF : Introduction to R Package for Pattern Sequence Based Forecasting Algorithm

    OpenAIRE

    Bokde, Neeraj; Asencio-Cortés, Gualberto; Martínez-Álvarez, Francisco; Kulat, Kishore

    2016-01-01

    This paper discusses about an R package that implements the Pattern Sequence based Forecasting (PSF) algorithm, which was developed for univariate time series forecasting. This algorithm has been successfully applied to many different fields. The PSF algorithm consists of two major parts: clustering and prediction. The clustering part includes selection of the optimum number of clusters. It labels time series data with reference to such clusters. The prediction part includes functions like op...

  13. Sequence-based characterization of five SLA loci in Asian wild boars.

    Science.gov (United States)

    Jung, W Y; Choi, N R; Seo, D W; Lim, H T; Ho, C S; Lee, J H

    2014-10-01

    Two swine leucocyte antigen (SLA) class I (SLA-1 and SLA-2) and three class II (DRB1, DQB1 and DQA) genes were investigated for their diversity in Asian wild boars using a sequence-based typing method. A total of 15 alleles were detected at these loci, with eleven being novel. The findings provide one of the first glimpses of the SLA allelic diversity and architecture in the wild boar populations. © 2014 John Wiley & Sons Ltd.

  14. Sequence-based characterization of the eight SLA loci in Korean native pigs.

    Science.gov (United States)

    Lee, Y J; Cho, K H; Kim, M J; Smith, D M; Ho, C S; Jung, K C; Jin, D I; Park, C S; Jeon, J T; Lee, J H

    2008-08-01

    Eight swine leucocyte antigen (SLA) gene (SLA-1, SLA-2, SLA-3, SLA-6, DRA, DRB1, DQA, DQB1) alleles were identified using sequence-based typing method in three Korean native pigs used for breeding at the National Institute of Animal Science in Korea. Six new alleles in class I genes and three new alleles in class II genes have been identified in this breed and can give valuable information for xenotransplantation and disease resistance.

  15. Capacitated vehicle routing problem with sequence-based pallet loading and axle weight constraints

    OpenAIRE

    2016-01-01

    In this paper, we introduce and study the capacitated vehicle routing problem with sequence-based pallet loading and axle weight constraints. To the best of our knowledge, it is the first time that axle weight restrictions are incorporated in a vehicle routing model. The aim of this paper is to demonstrate that incorporating axle weight restrictions in a vehicle routing model is possible and necessary for a feasible route planning. Axle weight limits impose a great challenge for transportatio...

  16. Multiscale image contrast amplification (MUSICA)

    Science.gov (United States)

    Vuylsteke, Pieter; Schoeters, Emile P.

    1994-05-01

    This article presents a novel approach to the problem of detail contrast enhancement, based on multiresolution representation of the original image. The image is decomposed into a weighted sum of smooth, localized, 2D basis functions at multiple scales. Each transform coefficient represents the amount of local detail at some specific scale and at a specific position in the image. Detail contrast is enhanced by non-linear amplification of the transform coefficients. An inverse transform is then applied to the modified coefficients. This yields a uniformly contrast- enhanced image without artefacts. The MUSICA-algorithm is being applied routinely to computed radiography images of chest, skull, spine, shoulder, pelvis, extremities, and abdomen examinations, with excellent acceptance. It is useful for a wide range of applications in the medical, graphical, and industrial area.

  17. Risk Perception and Social Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Smith, R.E. [Environment Agency (United Kingdom)

    2001-07-01

    This paper seeks to consider social amplification as it applies to risk perception. Perceptions of the magnitude of a risk are conditioned by issues such as the degree of uncertainty in probability and consequences, the nature of the consequences and the relative weightings placed on probability and consequences. Risk perceptions are also influenced by factors such as confidence in the operator of an industrial process, trust in the regulator and the perceived fairness of regulatory decision-making. Different people may hold different views about these issues and there may also be difficulties in communication. The paper identifies and discusses self-reinforcing mechanisms, which will be labelled 'lock-in' here. They appear to apply in many situations where social amplification is observed. Historically, the term 'lock-in' has been applied mainly in the technological context but, in this paper, four types of lock-in are identified, namely scientific/technological, economic, social and institutional lock-in. One type of lock-in tends to lead to the next and all are buttressed by people's general acceptance of the familiar, fear of the unknown and resistance to change. The regulator seeks to make decisions which achieve the common good rather than supporting or perpetuating any set of vested interests. In this regard the locked-in positions of stakeholders, whether organisations, interest groups, or individual members of the public, are obstacles and challenges. Existing methods of consultation are unsatisfactory in terms of achieving a proper and productive level of dialogue with stakeholders.

  18. By-Product Formation in Repetitive PCR Amplification of DNA Libraries during SELEX

    DEFF Research Database (Denmark)

    Tolle, Fabian; Wilke, Julian; Wengel, Jesper

    2014-01-01

    The selection of nucleic acid aptamers is an increasingly important approach to generate specific ligands binding to virtually any molecule of choice. However, selection-inherent amplification procedures are prone to artificial by-product formation that prohibits the enrichment of target-recogniz......The selection of nucleic acid aptamers is an increasingly important approach to generate specific ligands binding to virtually any molecule of choice. However, selection-inherent amplification procedures are prone to artificial by-product formation that prohibits the enrichment of target......-recognizing aptamers. Little is known about the formation of such by-products when employing nucleic acid libraries as templates. We report on the formation of two different forms of by-products, named ladder- and non-ladder-type observed during repetitive amplification in the course of in vitro selection experiments...

  19. Amplification of cellular oncogenes in solid tumors

    Directory of Open Access Journals (Sweden)

    Ozkan Bagci

    2015-01-01

    Full Text Available The term gene amplification refers to an increase in copy number of a gene. Upregulation of gene expression through amplification is a general mechanism to increase gene dosage. Oncogene amplifications have been shown in solid human cancers and they are often associated with progression of cancer. Defining oncogene amplification is useful since it is used as a prognostic marker in clinical oncology nowadays, especially v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (HER2 targeted agents are used in breast cancer patients with high level of HER2 overexpression as a therapeutic approach. However, patients without HER2 overexpression do not appear to benefit from these agents. We concluded that determination of oncogene amplification in solid tumors is an important factor in treatment of human cancers with many unknowns. We have referred to PubMed and some databases to prepare this article.

  20. Controlled Microwave Heating Accelerates Rolling Circle Amplification.

    Directory of Open Access Journals (Sweden)

    Takeo Yoshimura

    Full Text Available Rolling circle amplification (RCA generates single-stranded DNAs or RNA, and the diverse applications of this isothermal technique range from the sensitive detection of nucleic acids to analysis of single nucleotide polymorphisms. Microwave chemistry is widely applied to increase reaction rate as well as product yield and purity. The objectives of the present research were to apply microwave heating to RCA and indicate factors that contribute to the microwave selective heating effect. The microwave reaction temperature was strictly controlled using a microwave applicator optimized for enzymatic-scale reactions. Here, we showed that microwave-assisted RCA reactions catalyzed by either of the four thermostable DNA polymerases were accelerated over 4-folds compared with conventional RCA. Furthermore, the temperatures of the individual buffer components were specifically influenced by microwave heating. We concluded that microwave heating accelerated isothermal RCA of DNA because of the differential heating mechanisms of microwaves on the temperatures of reaction components, although the overall reaction temperatures were the same.

  1. Pulse Compression And Raman Amplification In Optical Fibres

    Science.gov (United States)

    Byron, Kevin C.

    1988-06-01

    Experimental and theoretical investigations on Raman amplification in fibres have been carried out and simultaneous amplification and pulse compression observed. With a fibre design optimised for amplification high gain may be obtained at practical pump power levels.

  2. Linking Arctic amplification and local feedbacks

    Science.gov (United States)

    Balcerak, Ernie

    2011-11-01

    Climate simulations show that as the Earth warms, the Arctic warms more than the average global warming. However, models differ on how much more the Arctic warms, and although scientists have proposed a variety of mechanisms to explain the Arctic warming amplification, there is no consensus on the main reasons for it. To shed light on this issue, Hwang et al. investigated the relationship between Arctic amplification and poleward energy transport and local Arctic feedbacks, such as changes in cloud cover or ice loss, across a group of models. The researchers noted that differences in atmospheric energy transport did not explain the ranges of polar amplification; rather, models with more amplification showed less energy transport into high latitudes. The authors found that decreasing energy transport is due to a coupled relationship between Arctic amplification and energy transport: Arctic amplification reduces the equator-to-pole temperature gradient, which strongly decreases energy transport. They suggest that this coupled relationship should be taken into account in studies of Arctic amplification. (Geophysical Research Letters, doi:10.1029/2011GL048546, 2011)

  3. Ancient DNA: genomic amplification of Roman and medieval bovine bones

    Directory of Open Access Journals (Sweden)

    A. Valentini

    2010-04-01

    Full Text Available Cattle remains (bones and teeth of both roman and medieval age were collected in the archaeological site of Ferento (Viterbo, Italy with the aim of extracting and characterising nucleic acids. Procedures to minimize contamination with modern DNA and to help ancient DNA (aDNA preservation of the archaeological remains were adopted. Different techniques to extract aDNA (like Phenol/chloroform extraction from bovine bones were tested to identify the method that applies to the peculiar characteristics of the study site. Currently, aDNA investigation is mainly based on mtDNA, due to the ease of amplification of the small and high-copied genome and to its usefulness in evolutionary studies. Preliminary amplification of both mitochondrial and nuclear aDNA fragments from samples of Roman and medieval animals were performed and partial specific sequences of mitochondrial D-loop as well as of nuclear genes were obtained. The innovative amplification of nuclear aDNA could enable the analysis of genes involved in specific animal traits, giving insights of ancient economic and cultural uses, as well as providing information on the origin of modern livestock population.

  4. A Novel Low Temperature PCR Assured High-Fidelity DNA Amplification

    Directory of Open Access Journals (Sweden)

    Shaoxia Zhou

    2013-06-01

    Full Text Available As previously reported, a novel low temperature (LoTemp polymerase chain reaction (PCR catalyzed by a moderately heat-resistant (MHR DNA polymerase with a chemical-assisted denaturation temperature set at 85 °C instead of the conventional 94–96 °C can achieve high-fidelity DNA amplification of a target DNA, even after up to 120 PCR thermal cycles. Furthermore, such accurate amplification is not achievable with conventional PCR. Now, using a well-recognized L1 gene segment of the human papillomavirus (HPV type 52 (HPV-52 as the template for experiments, we demonstrate that the LoTemp high-fidelity DNA amplification is attributed to an unusually high processivity and stability of the MHR DNA polymerase whose high fidelity in template-directed DNA synthesis is independent of non-existent 3'–5' exonuclease activity. Further studies and understanding of the characteristics of the LoTemp PCR technology may facilitate implementation of DNA sequencing-based diagnostics at the point of care in community hospital laboratories.

  5. Advanced backward Raman amplification seeding

    Science.gov (United States)

    Malkin, Vladimir; Fisch, Nathaniel

    2010-11-01

    Next generations of ultrapowerful laser pulses, reaching exawatt and zetawatt powers within reasonably compact facilities, might be based on the backward Raman amplification (BRA) in plasmas. Amplified pulse intensities hundreds times higher than the pump intensity are already observed experimentally. More advanced BRA stages should produce even higher intensities. The largest nonfocused intensity, limited primarily by instabilities associated with the relativistic electron nonlinearity of the amplified laser pulse, is, roughly speaking, 0.1 of the fully relativistic value. It corresponds to the amplified pulse final (and shortest) duration be about the electron plasma wave period. The needed seed pulse should be at least that short then to stay ahead of the amplified pulse, rather than be shadowed by it (which would much reduce the seeding efficiency). However, at earlier BRA stages, when the amplified pulse is longer, the optimal duration of the seed pulse is also longer. This work proposes the use of self-contracting seed pulses for further optimizing the advanced BRA.

  6. Quantum Amplitude Amplification and Estimation

    CERN Document Server

    Brassard, G; Mosca, M; Tapp, A; Brassard, Gilles; Hoyer, Peter; Mosca, Michele; Tapp, Alain

    2000-01-01

    Consider a Boolean function $\\chi: X \\to \\{0,1\\}$ that partitions set $X$ between its good and bad elements, where $x$ is good if $\\chi(x)=1$ and bad otherwise. Consider also a quantum algorithm $\\mathcal A$ such that $A \\ket{0} = \\sum_{x\\in X} \\alpha_x \\ket{x}$ is a quantum superposition of the elements of $X$, and let $a$ denote the probability that a good element is produced if $A \\ket{0}$ is measured. If we repeat the process of running $A$, measuring the output, and using $\\chi$ to check the validity of the result, we shall expect to repeat $1/a$ times on the average before a solution is found. *Amplitude amplification* is a process that allows to find a good $x$ after an expected number of applications of $A$ and its inverse which is proportional to $1/\\sqrt{a}$, assuming algorithm $A$ makes no measurements. This is a generalization of Grover's searching algorithm in which $A$ was restricted to producing an equal superposition of all members of $X$ and we had a promise that a single $x$ existed such tha...

  7. Reproducible analysis of sequencing-based RNA structure probing data with user-friendly tools

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz Jan; Sidiropoulos, Nikos; Vinther, Jeppe

    2015-01-01

    coordinates and vice versa. The collection is implemented as functions in the R statistical environment and as tools in the Galaxy platform, making them easily accessible for the scientific community. We demonstrate the usefulness of the collection by applying it to the analysis of sequencing-based hydroxyl......, we have made a collection of tools, which allow raw sequencing reads to be converted to normalized probing values using different published strategies. In addition, we also provide tools for visualization of the probing data in the UCSC Genome Browser and for converting RNA coordinates to genomic...

  8. Identification of the novel HLA-DPB1*5801 allele detected by sequenced based typing

    Energy Technology Data Exchange (ETDEWEB)

    Versluis, L.F.; Zwan, A.W. van der; Tilanus, M.G.J. [Academic Hospital Utrecht (Netherlands); Daly, L.N.; Degli-Esposti, M.A.; Dawkins, R.L. [Royal Perth Hospital, Perth (Australia)

    1995-01-11

    Within the framework of HLA-DPB typing of the Fourth Asia-Oceanic Histocompatibility workshop (4AOH) we have typed the A, B, and E panels representing 101 samples. Sequenced based typing (SBT) was used as the method for typing described by Versluis and co-workers, but the sequencing chemistry was modified; in this study we have used the Sequenase enzyme instead of the thermal stable Taq enzyme. Sequences obtained were compared to a database containing all known 55 HLA-DPB1 alleles. One sample showed a new heterozygous sequence, indicating the presence of a new allele. 4 refs., 1 fig.

  9. A 2-D graphical representation of protein sequences based on nucleotide triplet codons

    Science.gov (United States)

    Bai, Fenglan; Wang, Tianming

    2005-09-01

    Graphical representation of DNA provides a simple way of viewing, sorting and comparing various gene structures. A 2-D graphical representation of protein sequences based on nucleotide triplet codons has been derived for similarity analysis of protein sequences. This approach is based on a graphical representation of triplets of DNA in which the interior of the left half plane of the complex plane is used to accommodate 64 sites for the 64 codons. We associate a directed curve, numerical value, or matrix with a protein as a descriptor. The approach is illustrated on the Homo sapiens X-linked nuclear protein (ATRX) gene.

  10. Extension of the Legionella pneumophila sequence-based typing scheme to include strains carrying a variant of the N-acylneuraminate cytidylyltransferase gene.

    Science.gov (United States)

    Mentasti, M; Underwood, A; Lück, C; Kozak-Muiznieks, N A; Harrison, T G; Fry, N K

    2014-07-01

    Sequence-based typing (SBT) combined with monoclonal antibody subgrouping of Legionella pneumophila isolates is at present considered to be the reference standard during epidemiological investigation of Legionnaires' disease outbreaks. In some isolates of L. pneumophila, the seventh allele of the standard SBT scheme, neuA, is not amplified, because a homologue that is refractory to amplification with the standard neuA primers is present. Consequently, a complete seven-allele profile, and hence a sequence type, cannot be obtained. Subsequently, primers were designed to amplify both neuA and the homologue, but these yielded suboptimal sequencing results. In this study, novel primers specific for the neuA homologue were designed and internationally validated by members of the ESCMID Study Group for Legionella Infections at national and regional Legionella reference laboratories with a modified version of the online L. pneumophila sequence quality tool. To date, the addition of the neuAh target to the SBT protocol has allowed full typing data to be obtained for 108 isolates of 11 different serogroups, namely 1, 2, 3, 4, 5, 6, 7, 8, 10, 13, and 14, which could not previously be typed with the standard SBT neuA primers. Further studies are necessary to determine why it is still not possible to obtain either a neuA or a neuAh allele from three serogroup 11 isolates.

  11. Can Anomalous Amplification be Attained Without Postselection?

    CERN Document Server

    Martínez-Rincón, Julián; Viza, Gerardo I; Howell, John C

    2015-01-01

    We present a parameter estimation technique based on performing joint measurements of a weak interaction away from the weak-value-amplification approximation. Two detectors are used to collect full statistics of the correlations between two weakly entangled degrees of freedom. Without the need of postselection, the protocol resembles the anomalous amplification of an imaginary-weak-value-like response. The amplification is induced in the difference signal of both detectors allowing robustness to different sources of technical noise, and offering in addition the advantages of balanced signals for precision metrology. All of the Fisher information about the parameter of interest is collected, and a phase controls the amplification response. We experimentally demonstrate the proposed technique by measuring polarization rotations in a linearly polarized laser pulse. The effective sensitivity and precision of a split detector is increased when compared to a conventional continuous-wave balanced detection technique...

  12. Rolling circle amplification of metazoan mitochondrialgenomes

    Energy Technology Data Exchange (ETDEWEB)

    Simison, W. Brian; Lindberg, D.R.; Boore, J.L.

    2005-07-31

    Here we report the successful use of rolling circle amplification (RCA) for the amplification of complete metazoan mt genomes to make a product that is amenable to high-throughput genome sequencing techniques. The benefits of RCA over PCR are many and with further development and refinement of RCA, the sequencing of organellar genomics will require far less time and effort than current long PCR approaches.

  13. PHYLOViZ: phylogenetic inference and data visualization for sequence based typing methods

    Directory of Open Access Journals (Sweden)

    Francisco Alexandre P

    2012-05-01

    Full Text Available Abstract Background With the decrease of DNA sequencing costs, sequence-based typing methods are rapidly becoming the gold standard for epidemiological surveillance. These methods provide reproducible and comparable results needed for a global scale bacterial population analysis, while retaining their usefulness for local epidemiological surveys. Online databases that collect the generated allelic profiles and associated epidemiological data are available but this wealth of data remains underused and are frequently poorly annotated since no user-friendly tool exists to analyze and explore it. Results PHYLOViZ is platform independent Java software that allows the integrated analysis of sequence-based typing methods, including SNP data generated from whole genome sequence approaches, and associated epidemiological data. goeBURST and its Minimum Spanning Tree expansion are used for visualizing the possible evolutionary relationships between isolates. The results can be displayed as an annotated graph overlaying the query results of any other epidemiological data available. Conclusions PHYLOViZ is a user-friendly software that allows the combined analysis of multiple data sources for microbial epidemiological and population studies. It is freely available at http://www.phyloviz.net.

  14. Sequence-based prediction of protein protein interaction using a deep-learning algorithm.

    Science.gov (United States)

    Sun, Tanlin; Zhou, Bo; Lai, Luhua; Pei, Jianfeng

    2017-05-25

    Protein-protein interactions (PPIs) are critical for many biological processes. It is therefore important to develop accurate high-throughput methods for identifying PPI to better understand protein function, disease occurrence, and therapy design. Though various computational methods for predicting PPI have been developed, their robustness for prediction with external datasets is unknown. Deep-learning algorithms have achieved successful results in diverse areas, but their effectiveness for PPI prediction has not been tested. We used a stacked autoencoder, a type of deep-learning algorithm, to study the sequence-based PPI prediction. The best model achieved an average accuracy of 97.19% with 10-fold cross-validation. The prediction accuracies for various external datasets ranged from 87.99% to 99.21%, which are superior to those achieved with previous methods. To our knowledge, this research is the first to apply a deep-learning algorithm to sequence-based PPI prediction, and the results demonstrate its potential in this field.

  15. Comparison of hybridization-based and sequencing-based gene expression technologies on biological replicates

    Directory of Open Access Journals (Sweden)

    Cepko Connie L

    2007-06-01

    Full Text Available Abstract Background High-throughput systems for gene expression profiling have been developed and have matured rapidly through the past decade. Broadly, these can be divided into two categories: hybridization-based and sequencing-based approaches. With data from different technologies being accumulated, concerns and challenges are raised about the level of agreement across technologies. As part of an ongoing large-scale cross-platform data comparison framework, we report here a comparison based on identical samples between one-dye DNA microarray platforms and MPSS (Massively Parallel Signature Sequencing. Results The DNA microarray platforms generally provided highly correlated data, while moderate correlations between microarrays and MPSS were obtained. Disagreements between the two types of technologies can be attributed to limitations inherent to both technologies. The variation found between pooled biological replicates underlines the importance of exercising caution in identification of differential expression, especially for the purposes of biomarker discovery. Conclusion Based on different principles, hybridization-based and sequencing-based technologies should be considered complementary to each other, rather than competitive alternatives for measuring gene expression, and currently, both are important tools for transcriptome profiling.

  16. A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP

    Directory of Open Access Journals (Sweden)

    Yogesh eChander

    2014-08-01

    Full Text Available Meeting the goal of providing point of care (POC tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol is a thermostable viral enzyme that enables true POC use in clinics or in field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations . Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular

  17. Electricity-free amplification and detection for molecular point-of-care diagnosis of HIV-1.

    Science.gov (United States)

    Singleton, Jered; Osborn, Jennifer L; Lillis, Lorraine; Hawkins, Kenneth; Guelig, Dylan; Price, Will; Johns, Rachel; Ebels, Kelly; Boyle, David; Weigl, Bernhard; LaBarre, Paul

    2014-01-01

    In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens.

  18. Heat induces gene amplification in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Bin, E-mail: yanbin@mercyhealth.com [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Mercy Cancer Center, Mercy Medical Center-North Iowa, Mason City, IA 50401 (United States); Ouyang, Ruoyun [Department of Respiratory Medicine, The Second Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410011 (China); Huang, Chenghui [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Department of Oncology, The Third Xiangya Hospital, Xinagya School of Medicine, Central South University, Changsha 410013 (China); Liu, Franklin [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States); Neill, Daniel [Department of Radiation Oncology, University of Mississippi Medical Center, Jackson, MS 39213 (United States); Li, Chuanyuan [Dermatology, Duke University Medical Center, Durham, NC 27710 (United States); Dewhirst, Mark [Department of Radiation Oncology, Duke University Medical Center, Durham, NC 27710 (United States)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer This study discovered that heat exposure (hyperthermia) results in gene amplification in cancer cells. Black-Right-Pointing-Pointer Hyperthermia induces DNA double strand breaks. Black-Right-Pointing-Pointer DNA double strand breaks are considered to be required for the initiation of gene amplification. Black-Right-Pointing-Pointer The underlying mechanism of heat-induced gene amplification is generation of DNA double strand breaks. -- Abstract: Background: Hyperthermia plays an important role in cancer therapy. However, as with radiation, it can cause DNA damage and therefore genetic instability. We studied whether hyperthermia can induce gene amplification in cancer cells and explored potential underlying molecular mechanisms. Materials and methods: (1) Hyperthermia: HCT116 colon cancer cells received water-submerged heating treatment at 42 or 44 Degree-Sign C for 30 min; (2) gene amplification assay using N-(phosphoacetyl)-L-aspartate (PALA) selection of cabamyl-P-synthetase, aspartate transcarbarmylase, dihydro-orotase (cad) gene amplified cells; (3) southern blotting for confirmation of increased cad gene copies in PALA-resistant cells; (4) {gamma}H2AX immunostaining to detect {gamma}H2AX foci as an indication for DNA double strand breaks. Results: (1) Heat exposure at 42 or 44 Degree-Sign C for 30 min induces gene amplification. The frequency of cad gene amplification increased by 2.8 and 6.5 folds respectively; (2) heat exposure at both 42 and 44 Degree-Sign C for 30 min induces DNA double strand breaks in HCT116 cells as shown by {gamma}H2AX immunostaining. Conclusion: This study shows that heat exposure can induce gene amplification in cancer cells, likely through the generation of DNA double strand breaks, which are believed to be required for the initiation of gene amplification. This process may be promoted by heat when cellular proteins that are responsible for checkpoints, DNA replication, DNA repair and

  19. Application of a Non-amplification based Technology to Detect Invasive Fungal Pathogens

    Science.gov (United States)

    Hsu, Joe L.; Binkley, Jon; Clemons, Karl V.; Stevens, David A.; Nicolls, Mark R.; Holodniy, Mark

    2014-01-01

    Current diagnostic techniques for fungal diseases could be improved with respect to sensitivity, specificity and timeliness. To address this clinical need, we adapted a non-amplification based nucleic acid detection technology to identify fungal pathogens. We demonstrate a high-specificity, detection sensitivity, reproducibility and multiplex capacity for detecting fungal strains. PMID:24359934

  20. Application of a Non-amplification based Technology to Detect Invasive Fungal Pathogens

    OpenAIRE

    Hsu, Joe L.; Binkley, Jon; Clemons, Karl V.; Stevens, David A.; Nicolls, Mark R.; Holodniy, Mark

    2013-01-01

    Current diagnostic techniques for fungal diseases could be improved with respect to sensitivity, specificity and timeliness. To address this clinical need, we adapted a non-amplification based nucleic acid detection technology to identify fungal pathogens. We demonstrate a high-specificity, detection sensitivity, reproducibility and multiplex capacity for detecting fungal strains.

  1. A Novel Sequence-Based Feature for the Identification of DNA-Binding Sites in Proteins Using Jensen–Shannon Divergence

    Directory of Open Access Journals (Sweden)

    Truong Khanh Linh Dang

    2016-10-01

    Full Text Available The knowledge of protein-DNA interactions is essential to fully understand the molecular activities of life. Many research groups have developed various tools which are either structure- or sequence-based approaches to predict the DNA-binding residues in proteins. The structure-based methods usually achieve good results, but require the knowledge of the 3D structure of protein; while sequence-based methods can be applied to high-throughput of proteins, but require good features. In this study, we present a new information theoretic feature derived from Jensen–Shannon Divergence (JSD between amino acid distribution of a site and the background distribution of non-binding sites. Our new feature indicates the difference of a certain site from a non-binding site, thus it is informative for detecting binding sites in proteins. We conduct the study with a five-fold cross validation of 263 proteins utilizing the Random Forest classifier. We evaluate the functionality of our new features by combining them with other popular existing features such as position-specific scoring matrix (PSSM, orthogonal binary vector (OBV, and secondary structure (SS. We notice that by adding our features, we can significantly boost the performance of Random Forest classifier, with a clear increment of sensitivity and Matthews correlation coefficient (MCC.

  2. Molecular identification of veterinary yeast isolates by use of sequence-based analysis of the D1/D2 region of the large ribosomal subunit.

    Science.gov (United States)

    Garner, Cherilyn D; Starr, Jennifer K; McDonough, Patrick L; Altier, Craig

    2010-06-01

    Conventional methods of yeast identification are often time-consuming and difficult; however, recent studies of sequence-based identification methods have shown promise. Additionally, little is known about the diversity of yeasts identified from various animal species in veterinary diagnostic laboratories. Therefore, in this study, we examined three methods of identification by using 109 yeast samples isolated during a 1-year period from veterinary clinical samples. Comparison of the three methods-traditional substrate assimilation, fatty acid profile analysis, and sequence-based analysis of the region spanning the D1 and D2 regions (D1/D2) of the large ribosomal subunit-showed that sequence analysis provided the highest percent identification among the three. Sequence analysis identified 87% of isolates to the species level, whereas substrate assimilation and fatty acid profile analysis identified only 54% and 47%, respectively. Less-stringent criteria for identification increased the percentage of isolates identified to 98% for sequence analysis, 62% for substrate assimilation, and 55% for fatty acid profile analysis. We also found that sequence analysis of the internal transcribed spacer 2 (ITS2) region provided further identification for 36% of yeast not identified to the species level by D1/D2 sequence analysis. Additionally, we identified a large variety of yeast from animal sources, with at least 30 different species among the isolates tested, and with the majority not belonging to the common Candida spp., such as C. albicans, C. glabrata, C. tropicalis, and the C. parapsilosis group. Thus, we determined that sequence analysis of the D1/D2 region was the best method for identification of the variety of yeasts found in a veterinary population.

  3. Establishment of a sequence-based typing system for BoLA-DQA1 exon 2.

    Science.gov (United States)

    Takeshima, S; Miki, A; Kado, M; Aida, Y

    2007-02-01

    In cattle, bovine leukocyte antigens (BoLAs) have been extensively used as markers for bovine diseases and immunological traits. Here, we developed a rapid, high-resolution sequence-based typing (SBT) system for BoLA-DQA1. We amplified 355 bp of BoLA-DQA1 by fully nested polymerase chain reaction (PCR) using the newly constructed primers and then performed direct sequencing of each product. Using this method, we investigated the locus in 51 animals whose BoLA haplotypes had been characterized at the Fifth International BoLA Workshop. We identified 15 distinct DQA1 alleles, and there is no conflict between the typing result of PCR-SBT and restriction fragment length polymorphism analysis. Together with the previously developed method for typing BoLA-DRB3, the PCR-SBT for BoLA-DQA1 clearly provides a useful tool for detailed class IIa haplotype analysis.

  4. Fast interactive segmentation algorithm of image sequences based on relative fuzzy connectedness

    Institute of Scientific and Technical Information of China (English)

    Tian Chunna; Gao Xinbo

    2005-01-01

    A fast interactive segmentation algorithm of image-sequences based on relative fuzzy connectedness is presented. In comparison with the original algorithm, the proposed one, with the same accuracy, accelerates the segmentation speed by three times for single image. Meanwhile, this fast segmentation algorithm is extended from single object to multiple objects and from single-image to image-sequences. Thus the segmentation of multiple objects from complex background and batch segmentation of image-sequences can be achieved. In addition, a post-processing scheme is incorporated in this algorithm, which extracts smooth edge with one-pixel-width for each segmented object. The experimental results illustrate that the proposed algorithm can obtain the object regions of interest from medical image or image-sequences as well as man-made images quickly and reliably with only a little interaction.

  5. Study on multiple-hops performance of MOOC sequences-based optical labels for OPS networks

    Science.gov (United States)

    Zhang, Chongfu; Qiu, Kun; Ma, Chunli

    2009-11-01

    In this paper, we utilize a new study method that is under independent case of multiple optical orthogonal codes to derive the probability function of MOOCS-OPS networks, discuss the performance characteristics for a variety of parameters, and compare some characteristics of the system employed by single optical orthogonal code or multiple optical orthogonal codes sequences-based optical labels. The performance of the system is also calculated, and our results verify that the method is effective. Additionally it is found that performance of MOOCS-OPS networks would, negatively, be worsened, compared with single optical orthogonal code-based optical label for optical packet switching (SOOC-OPS); however, MOOCS-OPS networks can greatly enlarge the scalability of optical packet switching networks.

  6. Sequence-Based Introgression Mapping Identifies Candidate White Mold Tolerance Genes in Common Bean

    Directory of Open Access Journals (Sweden)

    Sujan Mamidi

    2016-07-01

    Full Text Available White mold, caused by the necrotrophic fungus (Lib. de Bary, is a major disease of common bean ( L.. WM7.1 and WM8.3 are two quantitative trait loci (QTL with major effects on tolerance to the pathogen. Advanced backcross populations segregating individually for either of the two QTL, and a recombinant inbred (RI population segregating for both QTL were used to fine map and confirm the genetic location of the QTL. The QTL intervals were physically mapped using the reference common bean genome sequence, and the physical intervals for each QTL were further confirmed by sequence-based introgression mapping. Using whole-genome sequence data from susceptible and tolerant DNA pools, introgressed regions were identified as those with significantly higher numbers of single-nucleotide polymorphisms (SNPs relative to the whole genome. By combining the QTL and SNP data, WM7.1 was located to a 660-kb region that contained 41 gene models on the proximal end of chromosome Pv07, while the WM8.3 introgression was narrowed to a 1.36-Mb region containing 70 gene models. The most polymorphic candidate gene in the WM7.1 region encodes a BEACH-domain protein associated with apoptosis. Within the WM8.3 interval, a receptor-like protein with the potential to recognize pathogen effectors was the most polymorphic gene. The use of gene and sequence-based mapping identified two candidate genes whose putative functions are consistent with the current model of pathogenicity.

  7. Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification.

    Directory of Open Access Journals (Sweden)

    David S Boyle

    Full Text Available Improved access to effective tests for diagnosing tuberculosis (TB has been designated a public health priority by the World Health Organisation. In high burden TB countries nucleic acid based TB tests have been restricted to centralised laboratories and specialised research settings. Requirements such as a constant electrical supply, air conditioning and skilled, computer literate operators prevent implementation of such tests in many settings. Isothermal DNA amplification technologies permit the use of simpler, less energy intensive detection platforms more suited to low resource settings that allow the accurate diagnosis of a disease within a short timeframe. Recombinase Polymerase Amplification (RPA is a rapid, low temperature isothermal DNA amplification reaction. We report here RPA-based detection of Mycobacterium tuberculosis complex (MTC DNA in <20 minutes at 39 °C. Assays for two MTC specific targets were investigated, IS6110 and IS1081. When testing purified MTC genomic DNA, limits of detection of 6.25 fg (IS6110 and 20 fg (IS1081were consistently achieved. When testing a convenience sample of pulmonary specimens from suspected TB patients, RPA demonstrated superior accuracy to indirect fluorescence microscopy. Compared to culture, sensitivities for the IS1081 RPA and microscopy were 91.4% (95%CI: 85, 97.9 and 86.1% (95%CI: 78.1, 94.1 respectively (n = 71. Specificities were 100% and 88.6% (95% CI: 80.8, 96.1 respectively. For the IS6110 RPA and microscopy sensitivities of 87.5% (95%CI: 81.7, 93.2 and 70.8% (95%CI: 62.9, 78.7 were obtained (n = 90. Specificities were 95.4 (95% CI: 92.3,98.1 and 88% (95% CI: 83.6, 92.4 respectively. The superior specificity of RPA for detecting tuberculosis was due to the reduced ability of fluorescence microscopy to distinguish Mtb complex from other acid fast bacteria. The rapid nature of the RPA assay and its low energy requirement compared to other amplification technologies suggest RPA-based TB

  8. Time varying arctic climate change amplification

    Energy Technology Data Exchange (ETDEWEB)

    Chylek, Petr [Los Alamos National Laboratory; Dubey, Manvendra K [Los Alamos National Laboratory; Lesins, Glen [DALLHOUSIE U; Wang, Muyin [NOAA/JISAO

    2009-01-01

    During the past 130 years the global mean surface air temperature has risen by about 0.75 K. Due to feedbacks -- including the snow/ice albedo feedback -- the warming in the Arctic is expected to proceed at a faster rate than the global average. Climate model simulations suggest that this Arctic amplification produces warming that is two to three times larger than the global mean. Understanding the Arctic amplification is essential for projections of future Arctic climate including sea ice extent and melting of the Greenland ice sheet. We use the temperature records from the Arctic stations to show that (a) the Arctic amplification is larger at latitudes above 700 N compared to those within 64-70oN belt, and that, surprisingly; (b) the ratio of the Arctic to global rate of temperature change is not constant but varies on the decadal timescale. This time dependence will affect future projections of climate changes in the Arctic.

  9. Amplification, Redundancy, and Quantum Chernoff Information

    Science.gov (United States)

    Zwolak, Michael; Riedel, C. Jess; Zurek, Wojciech H.

    2014-04-01

    Amplification was regarded, since the early days of quantum theory, as a mysterious ingredient that endows quantum microstates with macroscopic consequences, key to the "collapse of the wave packet," and a way to avoid embarrassing problems exemplified by Schrödinger's cat. Such a bridge between the quantum microworld and the classical world of our experience was postulated ad hoc in the Copenhagen interpretation. Quantum Darwinism views amplification as replication, in many copies, of the information about quantum states. We show that such amplification is a natural consequence of a broad class of models of decoherence, including the photon environment we use to obtain most of our information. This leads to objective reality via the presence of robust and widely accessible records of selected quantum states. The resulting redundancy (the number of copies deposited in the environment) follows from the quantum Chernoff information that quantifies the information transmitted by a typical elementary subsystem of the environment.

  10. On Arbitrary Phases in Quantum Amplitude Amplification

    CERN Document Server

    Hoyer, P

    2000-01-01

    We consider the use of arbitrary phases in quantum amplitude amplification which is a generalization of quantum searching. We prove that the phase condition in amplitude amplification is given by $\\tan(\\phi/2)=\\tan(\\phi/2)(1-2a)$, where $\\phi$ and $\\phi$ are the phases used and where $a$ is the success probability of the given algorithm. Thus the choice of phases depends nontrivially and nonlinearly on the success probability. Utilizing this condition, we give methods for constructing quantum algorithms that succeed with certainty and for implementing arbitrary rotations. We also conclude that phase errors of order up to $\\frac{1}{\\sqrt{a}}$ can be tolerated in amplitude amplification.

  11. Continuous phase amplification with a Sagnac interferometer

    CERN Document Server

    Starling, David J; Williams, Nathan S; Jordan, Andrew N; Howell, John C

    2009-01-01

    We describe a weak value inspired phase amplification technique in a Sagnac interferometer. We monitor the relative phase between two paths of a slightly misaligned interferometer by measuring the average position of a split-Gaussian mode in the dark port. Although we monitor only the dark port, we show that the signal varies linearly with phase and that we can obtain similar sensitivity to balanced homodyne detection. We derive the source of the amplification both with classical wave optics and as an inverse weak value.

  12. Effective Privacy Amplification for Secure Classical Communications

    CERN Document Server

    Horvath, Tamas; Scheuer, Jacob

    2011-01-01

    We study the effectiveness of privacy amplification for classical key-distribution schemes. We find that, unlike quantum key distribution schemes, the high fidelity of the raw key in classical systems allow the users to always sift a secure shorter key, given that they have an upper bound of eavesdropper probability to correctly guess the exchanged key-bits. We establish the number of privacy amplification iterations needed to achieve information leak of 10^-8 in several classical systems and highlight the inherent tradeoff between the number of iterations and the security of the raw key.

  13. Parametric Amplification For Detecting Weak Optical Signals

    Science.gov (United States)

    Hemmati, Hamid; Chen, Chien; Chakravarthi, Prakash

    1996-01-01

    Optical-communication receivers of proposed type implement high-sensitivity scheme of optical parametric amplification followed by direct detection for reception of extremely weak signals. Incorporates both optical parametric amplification and direct detection into optimized design enhancing effective signal-to-noise ratios during reception in photon-starved (photon-counting) regime. Eliminates need for complexity of heterodyne detection scheme and partly overcomes limitations imposed on older direct-detection schemes by noise generated in receivers and by limits on quantum efficiencies of photodetectors.

  14. An integrated disposable device for DNA extraction and helicase dependent amplification.

    Science.gov (United States)

    Mahalanabis, Madhumita; Do, Jaephil; ALMuayad, Hussam; Zhang, Jane Y; Klapperich, Catherine M

    2010-04-01

    Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a disposable polymer cartridge format. Smart passive fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE membrane) with a simple on-chip mixer eliminates multiple user operations. The device is able to detect as few as ten colony forming units (CFU) of E. coli in growth medium.

  15. A dual amplification fluorescent strategy for sensitive detection of DNA methyltransferase activity based on strand displacement amplification and DNAzyme amplification.

    Science.gov (United States)

    Cui, Wanling; Wang, Lei; Jiang, Wei

    2016-03-15

    DNA methyltransferase (MTase) plays a critical role in many biological processes and has been regarded as a predictive cancer biomarker and a therapeutic target in cancer treatment. Sensitive detection of DNA MTase activity is essential for early cancer diagnosis and therapeutics. Here, we developed a dual amplification fluorescent strategy for sensitive detection of DNA MTase activity based on strand displacement amplification (SDA) and DNAzyme amplification. A trifunctional double-stranded DNA (dsDNA) probe was designed including a methylation site for DNA MTase recognition, a complementary sequence of 8-17 DNAzyme for synthesizing DNAzyme, and a nicking site for nicking enzyme cleavage. Firstly, the trifunctional dsDNA probe was methylated by DNA MTase to form the methylated dsDNA. Subsequently, HpaII restriction endonuclease specifically cleaved the residue of unmethylated dsDNA. Next, under the action of polymerase and nicking enzyme, the methylared dsDNA initiated SDA, releasing numbers of 8-17 DNAzymes. Finally, the released 8-17 DNAzymes triggered DNAzyme amplification reaction to induce a significant fluorescence enhancement. This strategy could detect DNA MTase activity as low as 0.0082U/mL. Additionally, the strategy was successfully applied for evaluating the inhibitions of DNA MTase using two anticancer drugs, 5-azacytidine and 5-aza-2'-deoxycytidine. The results indicate the proposed strategy has a potential application in early cancer diagnosis and therapeutics.

  16. DNA Extraction and Amplification from Contemporary Polynesian Bark-Cloth

    Science.gov (United States)

    Moncada, Ximena; Payacán, Claudia; Arriaza, Francisco; Lobos, Sergio; Seelenfreund, Daniela; Seelenfreund, Andrea

    2013-01-01

    Background Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before. Methodology We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa) using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker. Conclusions Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials. PMID:23437166

  17. DNA extraction and amplification from contemporary Polynesian bark-cloth.

    Directory of Open Access Journals (Sweden)

    Ximena Moncada

    Full Text Available BACKGROUND: Paper mulberry has been used for thousands of years in Asia and Oceania for making paper and bark-cloth, respectively. Museums around the world hold valuable collections of Polynesian bark-cloth. Genetic analysis of the plant fibers from which the textiles were made may answer a number of questions of interest related to provenance, authenticity or species used in the manufacture of these textiles. Recovery of nucleic acids from paper mulberry bark-cloth has not been reported before. METHODOLOGY: We describe a simple method for the extraction of PCR-amplifiable DNA from small samples of contemporary Polynesian bark-cloth (tapa using two types of nuclear markers. We report the amplification of about 300 bp sequences of the ITS1 region and of a microsatellite marker. CONCLUSIONS: Sufficient DNA was retrieved from all bark-cloth samples to permit successful PCR amplification. This method shows a means of obtaining useful genetic information from modern bark-cloth samples and opens perspectives for the analyses of small fragments derived from ethnographic materials.

  18. Method Of Signal Amplification In Multi-Chromophore Luminescence Sensors

    Energy Technology Data Exchange (ETDEWEB)

    Levitsky, Igor A. (Fall River, MA); Krivoshlykov, Sergei G. (Shrewsbury, MA)

    2004-02-03

    A fluorescence-based method for highly sensitive and selective detection of analyte molecules is proposed. The method employs the energy transfer between two or more fluorescent chromophores in a carefully selected polymer matrix. In one preferred embodiment, signal amplification has been achieved in the fluorescent sensing of dimethyl methylphosphonate (DMMP) using two dyes, 3-aminofluoranthene (AM) and Nile Red (NR), in a hydrogen bond acidic polymer matrix. The selected polymer matrix quenches the fluorescence of both dyes and shifts dye emission and absorption spectra relative to more inert matrices. Upon DMMP sorption, the AM fluorescence shifts to the red at the same time the NR absorption shifts to the blue, resulting in better band overlap and increased energy transfer between chromophores. In another preferred embodiment, the sensitive material is incorporated into an optical fiber system enabling efficient excitation of the dye and collecting the fluorescent signal form the sensitive material on the remote end of the system. The proposed method can be applied to multichromophore luminescence sensor systems incorporating N-chromophores leading to N-fold signal amplification and improved selectivity. The method can be used in all applications where highly sensitive detection of basic gases, such as dimethyl methylphosphonate (DMMP), Sarin, Soman and other chemical warfare agents having basic properties, is required, including environmental monitoring, chemical industry and medicine.

  19. A rapid and robust sequence-based genotyping method for BoLA-DRB3 alleles in large numbers of heterozygous cattle.

    Science.gov (United States)

    Baxter, R; Hastings, N; Law, A; Glass, E J

    2008-10-01

    The BoLA-DRB3 gene is a highly polymorphic major histocompatibility complex class II gene of cattle with over one hundred alleles reported. Most of the polymorphisms are located in exon 2, which encodes the peptide-binding cleft, and these sequence differences play a role in variability of immune responsiveness and disease resistance. However, the high degree of polymorphism in exon 2 leads to difficulty in accurately genotyping cattle, especially heterozygous animals. In this study, we have improved and simplified an earlier sequence-based typing method to easily and reliably genotype cattle for BoLA-DRB3. In contrast to the earlier method, which used a nested primer set to amplify exon 2 followed by sequencing with internal primers, the new method uses only internal primers for both amplification and sequencing, which results in high-quality sequence across the entire exon. The haplofinder software, which assigns alleles from the heterozygous sequence, now has a pre-processing step that uses a consensus of all known alleles and checks for errors in base calling, thus improving the ability to process large numbers of samples. In addition, advances in sequencing technology have reduced the requirement for manual editing and improved the clarity of heterozygous base calls, resulting in longer and clearer sequence reads. Taken together, this has resulted in a rapid and robust method for genotyping large numbers of heterozygous samples for BoLA-DRB3 polymorphisms. Over 400 Holstein-Charolais cattle have now been genotyped for BoLA-DRB3 using this approach.

  20. Ambiguous allele combinations in HLA Class I and Class II sequence-based typing: when precise nucleotide sequencing leads to imprecise allele identification

    Directory of Open Access Journals (Sweden)

    Larsen Paula

    2004-09-01

    Full Text Available Abstract Sequence-based typing (SBT is one of the most comprehensive methods utilized for HLA typing. However, one of the inherent problems with this typing method is the interpretation of ambiguous allele combinations which occur when two or more different allele combinations produce identical sequences. The purpose of this study is to investigate the probability of this occurrence. We performed HLA-A,-B SBT for Exons 2 and 3 on 676 donors. Samples were analyzed with a capillary sequencer. The racial distribution of the donors was as follows: 615-Caucasian, 13-Asian, 23-African American, 17-Hispanic and 8-Unknown. 672 donors were analyzed for HLA-A locus ambiguities and 666 donors were analyzed for HLA-B locus ambiguities. At the HLA-A locus a total of 548 total ambiguous allele combinations were identified (548/1344 = 41%. Most (278/548 = 51% of these ambiguities were due to the fact that Exon 4 analysis was not performed. At the HLA-B locus 322 total ambiguous allele combinations were found (322/1332 = 24%. The HLA-B*07/08/15/27/35/44 antigens, common in Caucasians, produced a large portion of the ambiguities (279/322 = 87%. A large portion of HLA-A and B ambiguous allele combinations can be addressed by utilizing a group-specific primary amplification approach to produce an unambiguous homozygous sequence. Therefore, although the prevalence of ambiguous allele combinations is high, if the resolution of these ambiguities is clinically warranted, methods exist to compensate for this problem.

  1. Intelligence amplification framework for enhancing scheduling processes

    NARCIS (Netherlands)

    Dobrkovic, Andrej; Liu, Luyao; Iacob, Maria-Eugenia; Hillegersberg, van Jos

    2016-01-01

    The scheduling process in a typical business environment consists of predominantly repetitive tasks that have to be completed in limited time and often containing some form of uncertainty. The intelligence amplification is a symbiotic relationship between a human and an intelligent agent. This partn

  2. Desert Amplification in a Warming Climate

    Science.gov (United States)

    Zhou, Liming

    2016-08-01

    Here I analyze the observed and projected surface temperature anomalies over land between 50°S-50°N for the period 1950–2099 by large-scale ecoregion and find strongest warming consistently and persistently seen over driest ecoregions such as the Sahara desert and the Arabian Peninsula during various 30-year periods, pointing to desert amplification in a warming climate. This amplification enhances linearly with the global mean greenhouse gases(GHGs) radiative forcing and is attributable primarily to a stronger GHGs-enhanced downward longwave radiation forcing reaching the surface over drier ecoregions as a consequence of a warmer and thus moister atmosphere in response to increasing GHGs. These results indicate that desert amplification may represent a fundamental pattern of global warming associated with water vapor feedbacks over land in low- and mid- latitudes where surface warming rates depend inversely on ecosystem dryness. It is likely that desert amplification might involve two types of water vapor feedbacks that maximize respectively in the tropical upper troposphere and near the surface over deserts, with both being very dry and thus extremely sensitive to changes of water vapor.

  3. Intelligence amplification framework for enhancing scheduling processes

    NARCIS (Netherlands)

    Dobrkovic, Andrej; Liu, Luyao; Iacob, Maria Eugenia; van Hillegersberg, Jos

    2016-01-01

    The scheduling process in a typical business environment consists of predominantly repetitive tasks that have to be completed in limited time and often containing some form of uncertainty. The intelligence amplification is a symbiotic relationship between a human and an intelligent agent. This

  4. Social amplification of risk: a conceptual framework

    Energy Technology Data Exchange (ETDEWEB)

    Kasperson, R.E.; Renn, O.; Slovic, P.; Brown, H.S.; Emel, J.; Goble, R.; Kasperson, J.X.; Ratick, S.

    1988-06-01

    One of the most perplexing problems in risk analysis is why some relatively minor risks or risk events, as assessed by technical experts, often elicit strong public concerns and result in substantial impacts upon society and economy. This article sets forth a conceptual framework that seeks to link systematically the technical assessment of risk with psychological, sociological, and cultural perspectives of risk perception and risk-related behavior. The main thesis is that hazards interact with psychological, social, institutional, and cultural processes in ways that may amplify or attenuate public responses to the risk or risk event. A structural description of the social amplification of risk is now possible. Amplification occurs at two stages: in the transfer of information about the risk, and in the response mechanisms of society. Signals about risk are processed by individual and social amplification stations, including the scientist who communicates the risk assessment, the news media, cultural groups, interpersonal networks, and others. Key steps of amplifications can be identified at each stage. The amplified risk leads to behavioral responses, which, in turn, result in secondary impacts. Models are presented that portray the elements and linkages in the proposed conceptual framework.

  5. Sequence-based identification of microbial contaminants in non-parenteral products

    Directory of Open Access Journals (Sweden)

    Rajapandi Senthilraj

    Full Text Available ABSTRACT Phenotypic profiles for microbial identification are unusual for rare, slow-growing and fastidious microorganisms. In the last decade, as a result of the widespread use of PCR and DNA sequencing, 16S rRNA sequencing has played a pivotal role in the accurate identification of microorganisms and the discovery of novel isolates in microbiology laboratories. The 16S rRNA region is universally distributed among microorganisms and is species-specific. Accordingly, the aim of our study was the genotypic identification of microorganisms isolated from non-parenteral pharmaceutical formulations. DNA was separated from five isolates obtained from the formulations. The target regions of the rRNA genes were amplified by PCR and sequenced using suitable primers. The sequence data were analyzed and aligned in the order of increasing genetic distance to relevant sequences against a library database to achieve an identity match. The DNA sequences of the phylogenetic tree results confirmed the identity of the isolates as Bacillus tequilensis, B. subtilis, Staphylococcus haemolyticus and B. amyloliqueficians. It can be concluded that 16S rRNA sequence-based identification reduces the time by circumventing biochemical tests and also increases specificity and accuracy.

  6. HLA genes in Madeira Island (Portugal) inferred from sequence-based typing: footprints from different origins.

    Science.gov (United States)

    Spínola, Hélder; Bruges-Armas, Jácome; Mora, Marian Gantes; Middleton, Derek; Brehm, António

    2006-04-01

    Human leukocyte antigen (HLA)-A, HLA-B, and HLA-DRB1 polymorphisms were examined in Madeira Island populations. The data was obtained at high-resolution level, using sequence-based typing (SBT). The most frequent alleles at each loci were: A*020101 (24.6%), B*5101 (9.7%), B*440201 (9.2%), and DRB1*070101 (15.7%). The predominant three-loci haplotypes in Madeira were A*020101-B*510101-DRB1*130101 (2.7%) and A*010101-B*0801-DRB1*030101 (2.4%), previously found in north and central Portugal. The present study corroborates historical sources and other genetic studies that say Madeira were populated not only by Europeans, mostly Portuguese, but also sub-Saharan Africans due to slave trade. Comparison with other populations shows that Madeira experienced a stronger African influence due to slave trade than Portugal mainland and even the Azores archipelago. Despite this African genetic input, haplotype and allele frequencies were predominantly from European origin, mostly common to mainland Portugal.

  7. Application of Sequence-based Methods in Human MicrobialEcology

    Energy Technology Data Exchange (ETDEWEB)

    Weng, Li; Rubin, Edward M.; Bristow, James

    2005-08-29

    Ecologists studying microbial life in the environment have recognized the enormous complexity of microbial diversity for many years, and the development of a variety of culture-independent methods, many of them coupled with high-throughput DNA sequencing, has allowed this diversity to be explored in ever greater detail. Despite the widespread application of these new techniques to the characterization of uncultivated microbes and microbial communities in the environment, their application to human health and disease has lagged behind. Because DNA based-techniques for defining uncultured microbes allow not only cataloging of microbial diversity, but also insight into microbial functions, investigators are beginning to apply these tools to the microbial communities that abound on and within us, in what has aptly been called the second Human Genome Project. In this review we discuss the sequence-based methods for microbial analysis that are currently available and their application to identify novel human pathogens, improve diagnosis of known infectious diseases, and to advance understanding of our relationship with microbial communities that normally reside in and on the human body.

  8. Multitarget Tracking of Pedestrians in Video Sequences Based on Particle Filters

    Directory of Open Access Journals (Sweden)

    Hui Li

    2012-01-01

    Full Text Available Video target tracking is a critical problem in the field of computer vision. Particle filters have been proven to be very useful in target tracking for nonlinear and non-Gaussian estimation problems. Although most existing algorithms are able to track targets well in controlled environments, it is often difficult to achieve automated and robust tracking of pedestrians in video sequences if there are various changes in target appearance or surrounding illumination. To surmount these difficulties, this paper presents multitarget tracking of pedestrians in video sequences based on particle filters. In order to improve the efficiency and accuracy of the detection, the algorithm firstly obtains target regions in training frames by combining the methods of background subtraction and Histogram of Oriented Gradient (HOG and then establishes discriminative appearance model by generating patches and constructing codebooks using superpixel and Local Binary Pattern (LBP features in those target regions. During the process of tracking, the algorithm uses the similarity between candidates and codebooks as observation likelihood function and processes severe occlusion condition to prevent drift and loss phenomenon caused by target occlusion. Experimental results demonstrate that our algorithm improves the tracking performance in complicated real scenarios.

  9. Histoimmunogenetics Markup Language 1.0: Reporting next generation sequencing-based HLA and KIR genotyping.

    Science.gov (United States)

    Milius, Robert P; Heuer, Michael; Valiga, Daniel; Doroschak, Kathryn J; Kennedy, Caleb J; Bolon, Yung-Tsi; Schneider, Joel; Pollack, Jane; Kim, Hwa Ran; Cereb, Nezih; Hollenbach, Jill A; Mack, Steven J; Maiers, Martin

    2015-12-01

    We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines (miring.immunogenomics.org). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  10. M-Sequence-Based Single-Chip UWB-Radar Sensor

    Science.gov (United States)

    Kmec, M.; Helbig, M.; Herrmann, R.; Rauschenbach, P.; Sachs, J.; Schilling, K.

    The article deals with a fully monolithically integrated single-chip M-sequence-based UWB-radar sensor, its architecture, selected design aspects and first measurement results performed on wafer and with packaged IC modules. The discussed chip is equipped with one transmitter and two receivers. The IC was designed and manufactured in commercially available high-performance 0.25 μm SiGe BiCMOS technology (f t = 110 GHz). Due to the combination of fast digital and broadband analogue system blocks in one chip, special emphasis has been placed on the electrical isolation of these functional structures. The manufactured IC is enclosed in a low-cost QFN (quad flat-pack no-leads) package and mounted on a PCB permitting the creation of MIMO-sensor arrays by cascading a number of modules. In spite of its relatively high complexity, the sensor head features a compact design (chip size of 2 × 1 mm2, QFN package size 5 × 5 mm2) and moderate power consumption (below 1 W at -3 V supply). The assembled transceiver chip can handle signals in the frequency range from near DC up to 18 GHz. This leads to an impulse response (IRF) of FWHD ≈ 50 ps (full width at half duration).

  11. Prediction of peptide drift time in ion mobility mass spectrometry from sequence-based features

    KAUST Repository

    Wang, Bing

    2013-05-09

    Background: Ion mobility-mass spectrometry (IMMS), an analytical technique which combines the features of ion mobility spectrometry (IMS) and mass spectrometry (MS), can rapidly separates ions on a millisecond time-scale. IMMS becomes a powerful tool to analyzing complex mixtures, especially for the analysis of peptides in proteomics. The high-throughput nature of this technique provides a challenge for the identification of peptides in complex biological samples. As an important parameter, peptide drift time can be used for enhancing downstream data analysis in IMMS-based proteomics.Results: In this paper, a model is presented based on least square support vectors regression (LS-SVR) method to predict peptide ion drift time in IMMS from the sequence-based features of peptide. Four descriptors were extracted from peptide sequence to represent peptide ions by a 34-component vector. The parameters of LS-SVR were selected by a grid searching strategy, and a 10-fold cross-validation approach was employed for the model training and testing. Our proposed method was tested on three datasets with different charge states. The high prediction performance achieve demonstrate the effectiveness and efficiency of the prediction model.Conclusions: Our proposed LS-SVR model can predict peptide drift time from sequence information in relative high prediction accuracy by a test on a dataset of 595 peptides. This work can enhance the confidence of protein identification by combining with current protein searching techniques. 2013 Wang et al.; licensee BioMed Central Ltd.

  12. Sequence-Based Pronunciation Variation Modeling for Spontaneous ASR Using a Noisy Channel Approach

    Science.gov (United States)

    Hofmann, Hansjörg; Sakti, Sakriani; Hori, Chiori; Kashioka, Hideki; Nakamura, Satoshi; Minker, Wolfgang

    The performance of English automatic speech recognition systems decreases when recognizing spontaneous speech mainly due to multiple pronunciation variants in the utterances. Previous approaches address this problem by modeling the alteration of the pronunciation on a phoneme to phoneme level. However, the phonetic transformation effects induced by the pronunciation of the whole sentence have not yet been considered. In this article, the sequence-based pronunciation variation is modeled using a noisy channel approach where the spontaneous phoneme sequence is considered as a “noisy” string and the goal is to recover the “clean” string of the word sequence. Hereby, the whole word sequence and its effect on the alternation of the phonemes will be taken into consideration. Moreover, the system not only learns the phoneme transformation but also the mapping from the phoneme to the word directly. In this study, first the phonemes will be recognized with the present recognition system and afterwards the pronunciation variation model based on the noisy channel approach will map from the phoneme to the word level. Two well-known natural language processing approaches are adopted and derived from the noisy channel model theory: Joint-sequence models and statistical machine translation. Both of them are applied and various experiments are conducted using microphone and telephone of spontaneous speech.

  13. Improved Bevirimat resistance prediction by combination of structural and sequence-based classifiers

    Directory of Open Access Journals (Sweden)

    Dybowski J Nikolaj

    2011-11-01

    Full Text Available Abstract Background Maturation inhibitors such as Bevirimat are a new class of antiretroviral drugs that hamper the cleavage of HIV-1 proteins into their functional active forms. They bind to these preproteins and inhibit their cleavage by the HIV-1 protease, resulting in non-functional virus particles. Nevertheless, there exist mutations in this region leading to resistance against Bevirimat. Highly specific and accurate tools to predict resistance to maturation inhibitors can help to identify patients, who might benefit from the usage of these new drugs. Results We tested several methods to improve Bevirimat resistance prediction in HIV-1. It turned out that combining structural and sequence-based information in classifier ensembles led to accurate and reliable predictions. Moreover, we were able to identify the most crucial regions for Bevirimat resistance computationally, which are in line with experimental results from other studies. Conclusions Our analysis demonstrated the use of machine learning techniques to predict HIV-1 resistance against maturation inhibitors such as Bevirimat. New maturation inhibitors are already under development and might enlarge the arsenal of antiretroviral drugs in the future. Thus, accurate prediction tools are very useful to enable a personalized therapy.

  14. Comparison of serological and sequence-based methods for typing feline calcivirus isolates from vaccine failures.

    Science.gov (United States)

    Radford, A D; Dawson, S; Wharmby, C; Ryvar, R; Gaskell, R M

    2000-01-29

    Feline calicivirus (FCV) can be typed by exploiting antigenic differences between isolates or, more recently, by the sequence analysis of a hypervariable region of the virus's capsid gene. These two methods were used to characterise FCV isolates from 20 vaccine failures which occurred after the use of a commercial, live-attenuated vaccine. Using virus neutralisation, the isolates showed a spectrum of relatedness to the vaccine; depending on the criterion adopted for identity, 10 to 40 per cent of them appeared to be similar to the vaccine virus. Using sequence analysis, the isolates fell into one of two categories; 20 per cent had a similar sequence to the vaccine (0-67 to 2-67 per cent distant), and the remainder had a dissimilar sequence (21-3 to 36-0 per cent distant). Sequence analysis identified one cat that appeared to be infected with two distinct FCVs. The serological and sequence-based typing methods gave the same result in 80 to 95 per cent of individual cases, depending on the criterion adopted for serological identity. It is suggested that molecular typing is a more definitive method for characterising the relatedness of FCV isolates.

  15. Online Diagnosis System: a webserver for analysis of Sanger sequencing-based genetic testing data.

    Science.gov (United States)

    Sun, Kun; Yuen, Yuet-Ping; Wang, Huating; Sun, Hao

    2014-10-01

    Sanger sequencing is a well-established molecular technique for diagnosis of genetic diseases. In these tests, DNA sequencers produce vast amounts of data that need to be examined and annotated within a short period of time. To achieve this goal, an online bioinformatics platform that can automate the process is essential. However, to date, there is no such integrated bioinformatics platform available. To fulfill this gap, we developed the Online Diagnosis System (ODS), which is a freely available webserver and supports the commonly used file format of Sanger sequencing data. ODS seamlessly integrates base calling, single nucleotide variation (SNV) identification, and SNV annotation into one single platform. It also allows laboratorians to manually inspect the quality of the identified SNVs in the final report. ODS can significantly reduce the data analysis time therefore allows Sanger sequencing-based genetic testing to be finished in a timely manner. ODS is freely available at http://sunlab.lihs.cuhk.edu.hk/ODS/. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Genotyping-by-sequencing-based investigation of the genetic architecture responsible for a ~sevenfold increase in soybean seed stearic acid

    Science.gov (United States)

    Soybean oil is highly unsaturated and oxidatively unstable, rendering it non-ideal for most food applications. Until recently, the majority of soybean oil underwent partial chemical hydrogenation, a process which produces trans fats as an unavoidable consequence. Dietary intake of trans fat and most...

  17. Rapid microfluidic thermal cycler for nucleic acid amplification

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Vafai, Kambiz

    2015-10-27

    A system for thermal cycling a material to be thermal cycled including a microfluidic heat exchanger; a porous medium in the microfluidic heat exchanger; a microfluidic thermal cycling chamber containing the material to be thermal cycled, the microfluidic thermal cycling chamber operatively connected to the microfluidic heat exchanger; a working fluid at first temperature; a first system for transmitting the working fluid at first temperature to the microfluidic heat exchanger; a working fluid at a second temperature, a second system for transmitting the working fluid at second temperature to the microfluidic heat exchanger; a pump for flowing the working fluid at the first temperature from the first system to the microfluidic heat exchanger and through the porous medium; and flowing the working fluid at the second temperature from the second system to the heat exchanger and through the porous medium.

  18. nucleic acid amplification as used in the diagnosis and management ...

    African Journals Online (AJOL)

    DR. AMINU

    2014-06-01

    Jun 1, 2014 ... Bayero Journal of Pure and Applied Sciences, 7(1): 24 – 33 ..... types of HTLV-1 isolates co-exist: so called ... infant using HIV pro-viral DNA detection (Luzuriaga ... evidenced by a rise in the viral load despite ongoing therapy.

  19. A new evolutionary theory deduced mathematically from entropy amplification

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A new evolutionary theory which is able to unite the present evolutionary debates is deduced mathematically from the principle of entropy amplification.It suggests that the extensive evolution is driven by the amplification of entropy,or microscopic diversity,and the biological evolution is driven by the amplification of biodiversity.Forming high hierarchies is the most important way for the amplification and brings out spontaneously three kinds of selection.This theory has some positive cultural meanings.

  20. Amplification and Re-Generation of LNA-Modified Libraries

    DEFF Research Database (Denmark)

    Doessing, Holger; Hansen, Lykke H.; Veedu, Rakesh N.

    2012-01-01

    Locked nucleic acids (LNA) confer high thermal stability and nuclease resistance to oligonucleotides. The discovery of polymerases that accept LNA triphosphates has led us to propose a scheme for the amplification and re-generation of LNA-containing oligonucleotide libraries. Such libraries could...... be used for in vitro selection of e.g., native LNA aptamers. We maintained an oligonucleotide library encoding 40 randomized positions with LNA ATP, GTP, CTP, and TTP for 7 rounds of ‘mock’ in vitro selection in the absence of a target and analyzed the sequence composition after rounds 1, 4 and 7. We...... observed a decrease in LNA-A content from 20.5% in round 1 to 6.6% in round 7. This decrease was accompanied by a substantial bias against successive LNA-As (poly-LNA adenosine tracts) and a relative over-representation of single LNA-As. Maintaining a library with LNA TTP yielded similar results. Together...

  1. Plasmonic Terahertz Amplification in Graphene-Based Asymmetric Hyperbolic Metamaterial

    Directory of Open Access Journals (Sweden)

    Igor Nefedov

    2015-05-01

    Full Text Available We propose and theoretically explore terahertz amplification, based on stimulated generation of plasmons in graphene asymmetric hyperbolic metamaterials (AHMM, strongly coupled to terahertz radiation. In contrast to the terahertz amplification in resonant nanocavities, AHMM provides a wide-band THz amplification without any reflection in optically thin graphene multilayers.

  2. Isothermal DNA amplification in bioanalysis: strategies and applications.

    Science.gov (United States)

    Kim, Joonyul; Easley, Christopher J

    2011-01-01

    Isothermal DNA amplification is an alternative to PCR-based amplification for point-of-care diagnosis. Since the early 1990s, the approach has been refined into a simple, rapid and cost-effective tool by means of several distinct strategies. Input signals have been diversified from DNA to RNA, protein or small organic molecules by translating these signals into input DNA before amplification, thus allowing assays on various classes of biomolecules. In situ detection of single biomolecules has been achieved using an isothermal method, leveraging localized signal amplification in an intact specimen. A few pioneering studies to develop a homogenous isothermal protein assay have successfully translated structure-switching of a probe upon target binding into input DNA for isothermal amplification. In addition to the detection of specific targets, isothermal methods have made whole-genome amplification of single cells possible owing to the unbiased, linear nature of the amplification process as well as the large size of amplified products given by ϕ29 DNA polymerase. These applications have been devised with the four isothermal amplification strategies covered in this review: strand-displacement amplification, rolling circle amplification, helicase-dependent amplification and recombinase polymerase amplification.

  3. Plasmonic Terahertz Amplification in Graphene-Based Asymmetric Hyperbolic Metamaterial

    OpenAIRE

    Igor Nefedov; Leonid Melnikov

    2015-01-01

    We propose and theoretically explore terahertz amplification, based on stimulated generation of plasmons in graphene asymmetric hyperbolic metamaterials (AHMM), strongly coupled to terahertz radiation. In contrast to the terahertz amplification in resonant nanocavities, AHMM provides a wide-band THz amplification without any reflection in optically thin graphene multilayers.

  4. BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes

    DEFF Research Database (Denmark)

    Jespersen, Martin Closter; Peters, Bjoern; Nielsen, Morten

    2017-01-01

    for predicting B-cell epitopes from antigen sequences. BepiPred-2.0 is based on a random forest algorithm trained on epitopes annotated from antibody-antigen protein structures. This new method was found to outperform other available tools for sequence-based epitope prediction both on epitope data derived from...

  5. Post-Fragmentation Whole Genome Amplification-Based Method

    Science.gov (United States)

    Benardini, James; LaDuc, Myron T.; Langmore, John

    2011-01-01

    This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have inherent sample loss issues at

  6. SEQMINER: An R-Package to Facilitate the Functional Interpretation of Sequence-Based Associations.

    Science.gov (United States)

    Zhan, Xiaowei; Liu, Dajiang J

    2015-12-01

    Next-generation sequencing has enabled the study of a comprehensive catalogue of genetic variants for their impact on various complex diseases. Numerous consortia studies of complex traits have publically released their summary association statistics, which have become an invaluable resource for learning the underlying biology, understanding the genetic architecture, and guiding clinical translations. There is great interest in the field in developing novel statistical methods for analyzing and interpreting results from these genotype-phenotype association studies. One popular platform for method development and data analysis is R. In order to enable these analyses in R, it is necessary to develop packages that can efficiently query files of summary association statistics, explore the linkage disequilibrium structure between variants, and integrate various bioinformatics databases. The complexity and scale of sequence datasets and databases pose significant computational challenges for method developers. To address these challenges and facilitate method development, we developed the R package SEQMINER for annotating and querying files of sequence variants (e.g., VCF/BCF files) and summary association statistics (e.g., METAL/RAREMETAL files), and for integrating bioinformatics databases. SEQMINER provides an infrastructure where novel methods can be distributed and applied to analyzing sequence datasets in practice. We illustrate the performance of SEQMINER using datasets from the 1000 Genomes Project. We show that SEQMINER is highly efficient and easy to use. It will greatly accelerate the process of applying statistical innovations to analyze and interpret sequence-based associations. The R package, its source code and documentations are available from http://cran.r-project.org/web/packages/seqminer and http://seqminer.genomic.codes/.

  7. 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.

  8. MICA polymorphism in a population from north Morocco, Metalsa Berbers, using sequence-based typing.

    Science.gov (United States)

    Piancatelli, Daniela; Del Beato, Tiziana; Oumhani, Khadija; El Aouad, Rajae; Adorno, Domenico

    2005-08-01

    The MICA gene encodes a family of nonclassical major histocompatibility complex class I molecules. Data on MICA polymorphism in different populations are still limited. In the present study, MICA allele frequencies (af) were assessed in 82 unrelated healthy individuals from a Moroccan Berber population named Metalsa (ME) by means of sequence-based typing of exons 2, 3, 4, and 5. In consideration of the linkage disequilibrium existing between MICA and human leukocyte antigen (HLA) class I alleles, MICA/HLA-B, MICA/HLA-Cw, and MICA/HLA-A haplotype frequencies (hf) were estimated. A wide allelic distribution including 16 different MICA alleles was found in ME. The most common MICA alleles were MICA*00801 (af = 0.268), *004 (0.232), *00902 (0.140), *00901 (0.085), and *00901 (0.073). The most common MICA/HLA-B haplotypes were MICA*004-B*4403 and MICA*009-B*50 (hf = 0.113 for both these haplotypes). Some known MICA and HLA-B associations were confirmed in this population. Noteworthy was the high frequency of MICA*009 (af = 0.226); the high frequency of B*50 found in ME (af = 0.114) permitted us to evidence the associations of MICA*00902 with B*5001 (hf = 0.068) or *5002 (hf = 0.045), whereas MICA*00901 was mainly associated with B*5101 (hf = 0.038), which corresponds to the previously described association MICA*009/A6-HLA-B*51. This study extends the previous knowledge on MICA polymorphism to a North African white population and may have implications for disease associations and transplantation.

  9. Comparison of two multilocus sequence based genotyping schemes for Leptospira species.

    Directory of Open Access Journals (Sweden)

    Ahmed Ahmed

    2011-11-01

    Full Text Available BACKGROUND: Several sequence based genotyping schemes have been developed for Leptospira spp. The objective of this study was to genotype a collection of clinical and reference isolates using the two most commonly used schemes and compare and contrast the results. METHODS AND FINDINGS: A total of 48 isolates consisting of L. interrogans (n = 40 and L. kirschneri (n = 8 were typed by the 7 locus MLST scheme described by Thaipadungpanit et al., and the 6 locus genotyping scheme described by Ahmed et al., (termed 7L and 6L, respectively. Two L. interrogans isolates were not typed using 6L because of a deletion of three nucleotides in lipL32. The remaining 46 isolates were resolved into 21 sequence types (STs by 7L, and 30 genotypes by 6L. Overall nucleotide diversity (based on concatenated sequence was 3.6% and 2.3% for 7L and 6L, respectively. The D value (discriminatory ability of 7L and 6L were comparable, i.e. 92.0 (95% CI 87.5-96.5 vs. 93.5 (95% CI 88.6-98.4. The dN/dS ratios calculated for each locus indicated that none were under positive selection. Neighbor joining trees were reconstructed based on the concatenated sequences for each scheme. Both trees showed two distinct groups corresponding to L. interrogans and L. kirschneri, and both identified two clones containing 10 and 7 clinical isolates, respectively. There were six instances in which 6L split single STs as defined by 7L into closely related clusters. We noted two discrepancies between the trees in which the genetic relatedness between two pairs of strains were more closely related by 7L than by 6L. CONCLUSIONS: This genetic analysis indicates that the two schemes are comparable. We discuss their practical advantages and disadvantages.

  10. Generation of sequence-based data for pedigree-segregating Mendelian or Complex traits.

    Science.gov (United States)

    Li, Biao; Wang, Gao T; Leal, Suzanne M

    2015-11-15

    There is great interest in analyzing next generation sequence data that has been generated for pedigrees. However, unlike for population-based data there are only a limited number of rare variant methods to analyze pedigree data. One limitation is the ability to evaluate type I and II errors for family-based methods, due to lack of software that can simulate realistic sequence data for pedigrees. We developed RarePedSim (Rare-variant Pedigree-based Simulator), a program to simulate region/gene-level genotype and phenotype data for complex and Mendelian traits for any given pedigree structure. Using a genetic model, sequence variant data can be generated either conditionally or unconditionally on pedigree members' qualitative or quantitative phenotypes. Additionally, qualitative or quantitative traits can be generated conditional on variant data. Sequence data can either be simulated using realistic population demographic models or obtained from sequence-based studies. Variant sites can be annotated with positions, allele frequencies and functionality. For rare variants, RarePedSim is the only program that can efficiently generate both genotypes and phenotypes, regardless of pedigree structure. Data generated by RarePedSim are in standard Linkage file (.ped) and Variant Call (.vcf) formats, ready to be used for a variety of purposes, including evaluation of type I error and power, for association methods including mixed models and linkage analysis methods. bioinformatics.org/simped/rare sleal@bcm.edu. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  11. RNA amplification for successful gene profiling analysis

    Directory of Open Access Journals (Sweden)

    Wang Ena

    2005-07-01

    Full Text Available Abstract The study of clinical samples is often limited by the amount of material available to study. While proteins cannot be multiplied in their natural form, DNA and RNA can be amplified from small specimens and used for high-throughput analyses. Therefore, genetic studies offer the best opportunity to screen for novel insights of human pathology when little material is available. Precise estimates of DNA copy numbers in a given specimen are necessary. However, most studies investigate static variables such as the genetic background of patients or mutations within pathological specimens without a need to assess proportionality of expression among different genes throughout the genome. Comparative genomic hybridization of DNA samples represents a crude exception to this rule since genomic amplification or deletion is compared among different specimens directly. For gene expression analysis, however, it is critical to accurately estimate the proportional expression of distinct RNA transcripts since such proportions directly govern cell function by modulating protein expression. Furthermore, comparative estimates of relative RNA expression at different time points portray the response of cells to environmental stimuli, indirectly informing about broader biological events affecting a particular tissue in physiological or pathological conditions. This cognitive reaction of cells is similar to the detection of electroencephalographic patterns which inform about the status of the brain in response to external stimuli. As our need to understand human pathophysiology at the global level increases, the development and refinement of technologies for high fidelity messenger RNA amplification have become the focus of increasing interest during the past decade. The need to increase the abundance of RNA has been met not only for gene specific amplification, but, most importantly for global transcriptome wide, unbiased amplification. Now gene

  12. Flux variability scanning based on enforced objective flux for identifying gene amplification targets

    Directory of Open Access Journals (Sweden)

    Park Jong

    2012-08-01

    Full Text Available Abstract Background In order to reduce time and efforts to develop microbial strains with better capability of producing desired bioproducts, genome-scale metabolic simulations have proven useful in identifying gene knockout and amplification targets. Constraints-based flux analysis has successfully been employed for such simulation, but is limited in its ability to properly describe the complex nature of biological systems. Gene knockout simulations are relatively straightforward to implement, simply by constraining the flux values of the target reaction to zero, but the identification of reliable gene amplification targets is rather difficult. Here, we report a new algorithm which incorporates physiological data into a model to improve the model’s prediction capabilities and to capitalize on the relationships between genes and metabolic fluxes. Results We developed an algorithm, flux variability scanning based on enforced objective flux (FVSEOF with grouping reaction (GR constraints, in an effort to identify gene amplification targets by considering reactions that co-carry flux values based on physiological omics data via “GR constraints”. This method scans changes in the variabilities of metabolic fluxes in response to an artificially enforced objective flux of product formation. The gene amplification targets predicted using this method were validated by comparing the predicted effects with the previous experimental results obtained for the production of shikimic acid and putrescine in Escherichia coli. Moreover, new gene amplification targets for further enhancing putrescine production were validated through experiments involving the overexpression of each identified targeted gene under condition-controlled batch cultivation. Conclusions FVSEOF with GR constraints allows identification of gene amplification targets for metabolic engineering of microbial strains in order to enhance the production of desired bioproducts. The algorithm

  13. Amplification of postwildfire peak flow by debris

    Science.gov (United States)

    Kean, J. W.; McGuire, L. A.; Rengers, F. K.; Smith, J. B.; Staley, D. M.

    2016-08-01

    In burned steeplands, the peak depth and discharge of postwildfire runoff can substantially increase from the addition of debris. Yet methods to estimate the increase over water flow are lacking. We quantified the potential amplification of peak stage and discharge using video observations of postwildfire runoff, compiled data on postwildfire peak flow (Qp), and a physically based model. Comparison of flood and debris flow data with similar distributions in drainage area (A) and rainfall intensity (I) showed that the median runoff coefficient (C = Qp/AI) of debris flows is 50 times greater than that of floods. The striking increase in Qp can be explained using a fully predictive model that describes the additional flow resistance caused by the emergence of coarse-grained surge fronts. The model provides estimates of the amplification of peak depth, discharge, and shear stress needed for assessing postwildfire hazards and constraining models of bedrock incision.

  14. Parametric nanomechanical amplification at very high frequency.

    Science.gov (United States)

    Karabalin, R B; Feng, X L; Roukes, M L

    2009-09-01

    Parametric resonance and amplification are important in both fundamental physics and technological applications. Here we report very high frequency (VHF) parametric resonators and mechanical-domain amplifiers based on nanoelectromechanical systems (NEMS). Compound mechanical nanostructures patterned by multilayer, top-down nanofabrication are read out by a novel scheme that parametrically modulates longitudinal stress in doubly clamped beam NEMS resonators. Parametric pumping and signal amplification are demonstrated for VHF resonators up to approximately 130 MHz and provide useful enhancement of both resonance signal amplitude and quality factor. We find that Joule heating and reduced thermal conductance in these nanostructures ultimately impose an upper limit to device performance. We develop a theoretical model to account for both the parametric response and nonequilibrium thermal transport in these composite nanostructures. The results closely conform to our experimental observations, elucidate the frequency and threshold-voltage scaling in parametric VHF NEMS resonators and sensors, and establish the ultimate sensitivity limits of this approach.

  15. Gravito-magnetic amplification in cosmology

    CERN Document Server

    Tsagas, Christos G

    2009-01-01

    Magnetic fields interact with gravitational waves in various ways. We consider the coupling between the Weyl and the Maxwell fields in cosmology and study the effects of the former on the latter. The approach is fully analytical and the results are gauge-invariant. We show that the nature and the outcome of the gravito-magnetic interaction depends on the electric properties of the cosmic medium. When the conductivity is high, gravitational waves reduce the standard (adiabatic) decay rate of the B-field, leading to its superadiabatic amplification. In poorly conductive environments, on the other hand, Weyl-curvature distortions can result into the resonant amplification of large-scale cosmological magnetic fields. Driven by the gravitational waves, these B-fields oscillate with an amplitude that is found to diverge when the wavelengths of the two sources coincide. We present technical and physical aspects of the gravito-magnetic interaction and discuss its potential implications.

  16. Internal entanglement amplification by external interactions

    OpenAIRE

    2007-01-01

    We propose a scheme to control the level of entanglement between two fixed spin-1/2 systems by interaction with a third particle. For specific designs, entanglement is shown to be "pumped" into the system from the surroundings even when the spin-spin interaction within the system is small or nonexistent. The effect of the external particle on the system is introduced by including a dynamic spinor in the Hamiltonian. Controlled amplification of the internal entanglement to its maximum value is...

  17. A mechanism for ramified rolling circle amplification

    Directory of Open Access Journals (Sweden)

    Smith James H

    2010-12-01

    Full Text Available Abstract Background Amplification of single-stranded DNA circles has wide utility for a variety of applications. The two-primer ramified rolling circle amplification (RAM reaction provides exponential DNA amplification under isothermal conditions, creating a regular laddered series of double-stranded DNA products. However, the molecular mechanism of the RAM reaction remains unexplained. Results A RAM reaction model predicts exponential accumulation of a double-stranded DNA product size series, and product-size ratios, that are consistent with observed RAM reaction products. The mechanism involves generation of a series of increasing size intermediate templates; those templates produce RAM products and recursively generate smaller intermediate templates. The model allows prediction of the number of rounds of circular template replication. Real-time RAM reaction data are consistent with the model. Analysis of RAM reaction products shows exponential growth limitation consistent with the model's predictions. Conclusions The model provides a rationale for the observed products of the RAM reaction, and the molecular yield among those products. Experimental results are consistent with the model.

  18. IN VITRO SELECTION AND CHARACTERIZATION OF CELLULOSE-BINDING RNA APTAMERS USING ISOTHERMAL AMPLIFICATION

    Science.gov (United States)

    Boese, B. J.; Corbino, K.; Breaker, R. R.

    2017-01-01

    We sought to create new cellulose-binding RNA aptamers for use as modular components in the engineering of complex functional nucleic acids. We designed our in vitro selection strategy to incorporate self-sustained sequence replication (3SR), which is an isothermal nucleic acid amplification protocol that allows for the rapid amplification of RNAs with little manipulation. The best performing aptamer representative was chosen for reselection and further optimization. The aptamer exhibits robust affinity for cellulose in both the powdered and paper form, but did not show any significant affinity for closely related polysaccharides. The minimal cellulose-binding RNA aptamer also can be grafted onto other RNAs to permit the isolation of RNAs from complex biochemical mixtures via cellulose affinity chromatography. This was demonstrated by fusing the aptamer to a glmS ribozyme sequence, and selectively eluting ribozyme cleavage products from cellulose using the glucosamine 6-phosphate to activate glmS ribozyme function. PMID:18696364

  19. A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

    National Research Council Canada - National Science Library

    Farkhondeh Saba; Moslem Papizadeh; Javad Khansha; Mahshid Sedghi; Mehrnoosh Rasooli; Mohammad Ali Amoozegar; Mohammad Reza Soudi; Seyed Abolhassan Shahzadeh Fazeli

    2017-01-01

    Background: Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction...

  20. A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

    National Research Council Canada - National Science Library

    Farkhondeh Saba; Moslem Papizadeh; Javad Khansha; Mahshid Sedghi; Mehrnoosh Rasooli; Mohammad Ali Amoozegar; Mohammad Reza Soudi; Seyed Abolhassan Shahzadeh Fazeli

    2016-01-01

    Background: Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction...

  1. A sequence-based approach to identify reference genes for gene expression analysis

    Directory of Open Access Journals (Sweden)

    Chari Raj

    2010-08-01

    Full Text Available Abstract Background An important consideration when analyzing both microarray and quantitative PCR expression data is the selection of appropriate genes as endogenous controls or reference genes. This step is especially critical when identifying genes differentially expressed between datasets. Moreover, reference genes suitable in one context (e.g. lung cancer may not be suitable in another (e.g. breast cancer. Currently, the main approach to identify reference genes involves the mining of expression microarray data for highly expressed and relatively constant transcripts across a sample set. A caveat here is the requirement for transcript normalization prior to analysis, and measurements obtained are relative, not absolute. Alternatively, as sequencing-based technologies provide digital quantitative output, absolute quantification ensues, and reference gene identification becomes more accurate. Methods Serial analysis of gene expression (SAGE profiles of non-malignant and malignant lung samples were compared using a permutation test to identify the most stably expressed genes across all samples. Subsequently, the specificity of the reference genes was evaluated across multiple tissue types, their constancy of expression was assessed using quantitative RT-PCR (qPCR, and their impact on differential expression analysis of microarray data was evaluated. Results We show that (i conventional references genes such as ACTB and GAPDH are highly variable between cancerous and non-cancerous samples, (ii reference genes identified for lung cancer do not perform well for other cancer types (breast and brain, (iii reference genes identified through SAGE show low variability using qPCR in a different cohort of samples, and (iv normalization of a lung cancer gene expression microarray dataset with or without our reference genes, yields different results for differential gene expression and subsequent analyses. Specifically, key established pathways in lung

  2. Prognostic impact of HER-2 Subclonal Amplification in breast cancer.

    Science.gov (United States)

    Di Oto, Enrico; Brandes, Alba A; Cucchi, Maria C; Foschini, Maria P

    2017-06-02

    The presence of a limited number of cells with HER-2 amplification (Subclonal Amplification) in breast carcinomas is occasionally encountered, but its prognostic impact is poorly known. The purpose of this study is to evaluate the prognostic impact of HER-2 Subclonal Amplification in a retrospective series of breast cancers. Accordingly, 81 consecutive breast carcinomas showing HER-2 Subclonal Amplification were obtained from the histology files (case series). These cases were subdivided into two groups: (a) those cases in which the HER-2 Subclonal Amplification was consonant to the accepted criteria for amplification, showing clusters of amplified cells, and (b) those cases with rare HER-2 Subclonal Amplification that did not reflect the accepted criteria for amplification, showing scattered amplified cells only. The incidence of metastases and late recurrences of the case series was compared with a series composed of 109 consecutive cases, being HER-2 homogeneous (comprising 14 Amplified and 95 Non-Amplified cases), matched for grade and stage (control series). It appeared that cases showing Subclonal Amplification had an incidence of metastases intermediate between the cases Amplified and Non-Amplified. Specifically, Subclonal Amplification with clustered cells had a lower incidence of metastases than Amplified cases (12.9 versus 21.4%). On the contrary, Subclonal Amplification with scattered cells showed an incidence of metastases higher than Non-Amplified cases (14 versus 9.47%). In addition, patients Subclonal Amplification with clustered cells, who were treated with the specific monoclonal antibody, had a lower incidence of metastases than patients showing Subclonal Amplification with scattered cells, who did not receive target therapy. These data, together with those recently published, indicate that Subclonal Amplification has an impact on prognosis and should be taken into consideration to correctly plan the treatment of breast cancer patients.

  3. RNA amplification of bromodeoxyuridine labeled newborn neurons in the monkey hippocampus.

    Science.gov (United States)

    Counts, Scott E; Chen, Er-Yun; Ginsberg, Stephen D; Kordower, Jeffrey H; Mufson, Elliott J

    2005-06-15

    Neurogenesis has been demonstrated in the adult mammalian hippocampus by the immunohistochemical identification of cells co-labeled with the neuronal marker NeuN and bromodeoxyuridine (BrdU), a marker for DNA synthesis. Whether these newly born neurons exhibit a genetic signature similar to that of existing hippocampal cells remains unknown. Recent advances in single cell RNA amplification techniques provide a unique method for profiling the mRNA complement of cells developed during adult neurogenesis. Standard protocols for identifying BrdU-positive cells requires an acid denaturation step that may preclude the amplification of cellular RNA for expression analysis. We first tested whether the BrdU reaction product was visible in monkey hippocampal tissue following treatment with dilutions of HCl (2-0.2 M) or citric acid (1.0-0.1 M). BrdU-labeled cells were visible only in tissue sections treated with 2 M HCl. RNA amplification was not compromised in cells dual-labeled for BrdU and NeuN using the 2 M HCl acid denaturation step. These cells express mRNAs encoding a wide variety of functional protein subclasses including glutamate receptors. The present study demonstrates for the first time that BrdU immunohistochemisty is compatable with gene array technology in the primate hippocampus to evaluate subclasses of genes in newborn neurons.

  4. Characterization of the novel HLA-Cw*0624 allele identified by sequence-based typing.

    Science.gov (United States)

    Deng, Z-H; Wang, D-M; Gao, S-Q; Xu, Y-P

    2010-01-01

    A novel HLA-Cw*0624 variant allele differs from the closest allele Cw*06020101 by single nucleotide change at genomic nt 923 T>C (CDS nt 547 T>C, codon 159 TAC>CAC) in exon 3, which results in an amino acid change Tyr159His.

  5. Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.

    Directory of Open Access Journals (Sweden)

    Ahmed Abd El Wahed

    Full Text Available Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF. Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR are the standard method for molecular detection of the dengue virus (DENV. Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA assays were developed to detect DENV1-4.Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4 to 241 (DENV1-3 RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal and in Bangkok (Thailand. In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31 and 100% (n=23, respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90 and 100%(n=41, respectively.During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

  6. ProteDNA: a sequence-based predictor of sequence-specific DNA-binding residues in transcription factors

    OpenAIRE

    2009-01-01

    This article presents the design of a sequence-based predictor named ProteDNA for identifying the sequence-specific binding residues in a transcription factor (TF). Concerning protein–DNA interactions, there are two types of binding mechanisms involved, namely sequence-specific binding and nonspecific binding. Sequence-specific bindings occur between protein sidechains and nucleotide bases and correspond to sequence-specific recognition of genes. Therefore, sequence-specific bindings are esse...

  7. Whole Genome Amplification from Blood Spot Samples.

    Science.gov (United States)

    Sørensen, Karina Meden

    2015-01-01

    Whole genome amplification is an invaluable technique when working with DNA extracted from blood spots, as the DNA obtained from this source often is too limited for extensive genetic analysis. Two techniques that amplify the entire genome are common. Here, both are described with focus on the benefits and drawbacks of each system. However, in order to obtain the best possible WGA result the quality of input DNA extracted from the blood spot is essential, but also time consumption, flexibility in format and elution volume and price of the technology are factors influencing system choice. Here, three DNA extraction techniques are described and the above aspects are compared between the systems.

  8. Amplification Without Inversion in Semiconductor Quantum Dot

    Science.gov (United States)

    Hajibadali, A.; Abbasian, K.; Rostami, A.

    In this paper, we have realized amplification without inversion (AWI) in quantum dot (QD). A Y-type four-level system of InxGa1-xN quantum dot has been obtained and investigated for AWI. It has been shown that, with proper setting of control fields' amplitude, we can obtain reasonable gain. With proper setting of phase difference of control fields and probe field, we can obtain considerable gain in resonant wavelength. We have designed this system by solving the Schrödinger-Poisson equations for InxGa1-xN quantum dot in GaN substrate, self-consistently.

  9. Amplification of curvature perturbations in cyclic cosmology

    Science.gov (United States)

    Zhang, Jun; Liu, Zhi-Guo; Piao, Yun-Song

    2010-12-01

    We analytically and numerically show that through the cycles with nonsingular bounce, the amplitude of curvature perturbation on a large scale will be amplified and the power spectrum will redden. In some sense, this amplification will eventually destroy the homogeneity of the background, which will lead to the ultimate end of cycles of the global universe. We argue that for the model with increasing cycles, it might be possible that a fissiparous multiverse will emerge after one or several cycles, in which the cycles will continue only at corresponding local regions.

  10. Amplification and characterization of eukaryotic structural genes.

    Science.gov (United States)

    Maniatis, T; Efstratiadis, A; Sim, G K; Kafatos, F

    1978-05-01

    An approach to the study of eukaryotic structural genes which are differentially expressed during development is described. This approach involves the isolation and amplification of mRNA sequences by in vitro conversion of mRNA to double-stranded cDNA followed by molecular cloning in bacterial plasmids. This procedure provides highly specific hybridization probes that can be used to identify genes and their contiguous DNA sequences in genomic DNA, and to detect specific RNA transcripts during development. The nature of the method allows the isolation of individual mRNA sequences from a complex population of molecules at different stages of development.

  11. Amplification Effects and Unconventional Monetary Policies

    Directory of Open Access Journals (Sweden)

    Cécile BASTIDON GILLES

    2012-02-01

    Full Text Available Global financial crises trigger off amplification effects, which allow relatively small shocks to propagate through the whole financial system. For this reason, the range of Central banks policies is now widening beyond conventional monetary policies and lending of last resort. The aim of this paper is to establish a rule for this practice. The model is based on the formalization of funding conditions in various types of markets. We conduct a comprehensive analysis of the “unconventional monetary policies”, and especially quantify government bonds purchases by the Central bank.

  12. Linear molecular beacons for highly sensitive bioanalysis based on cyclic Exo III enzymatic amplification.

    Science.gov (United States)

    Yang, Chaoyong James; Cui, Liang; Huang, Jiahao; Yan, Ling; Lin, Xiaoyan; Wang, Chunming; Zhang, Wei Yun; Kang, Huaizhi

    2011-09-15

    Sensitive analysis or monitoring of biomolecules and small molecules is very important for many biological researches, clinical diagnosis and forensic investigations. As a sequence-independent exonuclease, Exonuclease III (Exo III) has been widely used for amplified detection of proteins and nucleic acids where displacing probes or molecular beacons are used as the signaling probes. However, displacing probes suffer slow hybridization rate and high background signal and molecular beacons are difficult to design and prone to undesired nonspecific interactions. Herein, we report a new type of probes called linear molecular beacons (LMBs) for use in Exo III amplification assays to improve hybridization kinetics and reduce background noises. LMBs are linear oligonucleotide probes with a fluorophore and quencher attached to 3' terminal and penultimate nucleotides, respectively. Compared to conventional molecular beacons and displacing probes, LMBs are easy to design and synthesize. More importantly, LMBs have a much lower background noise and allow faster reaction rates. Using LMBs in cyclic Exo III amplification assay, ultrasensitive nucleic acid detection methods were developed with a detection limit of less than 120fM, which is 2 orders of magnitude lower than that of conventional molecular beacons or displacing probes-based Exo III amplification assays. Furthermore, LMBs can be extended as universal probes for detection of non-nucleic acid molecules such as cocaine with high sensitivity. These results demonstrate that the combination of Exo III amplification and LMB signaling provides a general method for ultrasensitive and selective detection of a wide range of targets. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Mechanism of seasonal Arctic sea ice evolution and Arctic amplification

    OpenAIRE

    Kim, Kwang-Yul; Hamlington, Benjamin D.; Na, Hanna; Kim, Jinju

    2016-01-01

    Sea ice loss is proposed as a primary reason for the Arctic amplification, although the physical mechanism of the Arctic amplification and its connection with sea ice melting is still in debate. In the present study, monthly ERA-Interim reanalysis data are analyzed via cyclostationary empirical orthogonal function analysis to understand the seasonal mechanism of sea ice loss in the Arctic Ocean and the Arctic amplification. While sea ice loss is widespread over much of the p...

  14. Space Optical Communications Using Laser Beam Amplification

    Science.gov (United States)

    Agrawal, Govind

    2015-01-01

    The Space Optical Communications Using Laser Beam Amplification (SOCLBA) project will provide a capability to amplify a laser beam that is received in a modulating retro-reflector (MRR) located in a satellite in low Earth orbit. It will also improve the pointing procedure between Earth and spacecraft terminals. The technology uses laser arrays to strengthen the reflected laser beam from the spacecraft. The results of first year's work (2014) show amplification factors of 60 times the power of the signal beam. MMRs are mirrors that reflect light beams back to the source. In space optical communications, a high-powered laser interrogator beam is directed from the ground to a satellite. Within the satellite, the beam is redirected back to ground using the MMR. In the MMR, the beam passes through modulators, which encode a data signal onto the returning beam. MMRs can be used in small spacecraft for optical communications. The SOCLBA project is significant to NASA and small spacecraft due to its application to CubeSats for optical data transmission to ground stations, as well as possible application to spacecraft for optical data transmission.

  15. Experimental noiseless linear amplification using weak measurements

    Science.gov (United States)

    Ho, Joseph; Boston, Allen; Palsson, Matthew; Pryde, Geoff

    2016-09-01

    The viability of quantum communication schemes rely on sending quantum states of light over long distances. However, transmission loss can degrade the signal strength, adding noise. Heralded noiseless amplification of a quantum signal can provide a solution by enabling longer direct transmission distances and by enabling entanglement distillation. The central idea of heralded noiseless amplification—a conditional modification of the probability distribution over photon number of an optical quantum state—is suggestive of a parallel with weak measurement: in a weak measurement, learning partial information about an observable leads to a conditional back-action of a commensurate size. Here we experimentally investigate the application of weak, or variable-strength, measurements to the task of heralded amplification, by using a quantum logic gate to weakly couple a small single-optical-mode quantum state (the signal) to an ancilla photon (the meter). The weak measurement is carried out by choosing the measurement basis of the meter photon and, by conditioning on the meter outcomes, the signal is amplified. We characterise the gain of the amplifier as a function of the measurement strength, and use interferometric methods to show that the operation preserves the coherence of the signal.

  16. Magnetic Field Amplification in Young Galaxies

    CERN Document Server

    Schober, Jennifer; Klessen, Ralf S

    2013-01-01

    The Universe at present is highly magnetized, with fields of the order of a few 10^-5 G and coherence lengths larger than 10 kpc in typical galaxies like the Milky Way. We propose that the magnetic field was amplified to this values already during the formation and the early evolution of the galaxies. Turbulence in young galaxies is driven by accretion as well as by supernova (SN) explosions of the first generation of stars. The small-scale dynamo can convert the turbulent kinetic energy into magnetic energy and amplify very weak primordial magnetic seed fields on short timescales. The amplification takes place in two phases: in the kinematic phase the magnetic field grows exponentially, with the largest growth on the smallest non-resistive scale. In the following non-linear phase the magnetic energy is shifted towards larger scales until the dynamo saturates on the turbulent forcing scale. To describe the amplification of the magnetic field quantitatively we model the microphysics in the interstellar medium ...

  17. Host suppression and bioinformatics for sequence-based characterization of unknown pathogens.

    Energy Technology Data Exchange (ETDEWEB)

    Branda, Steven S.; Lane, Todd W.; Misra, Milind; Meagher, Robert J.; Patel, Kamlesh D.; Kaiser, Julia N.

    2009-11-01

    Bioweapons and emerging infectious diseases pose formidable and growing threats to our national security. Rapid advances in biotechnology and the increasing efficiency of global transportation networks virtually guarantee that the United States will face potentially devastating infectious disease outbreaks caused by novel ('unknown') pathogens either intentionally or accidentally introduced into the population. Unfortunately, our nation's biodefense and public health infrastructure is primarily designed to handle previously characterized ('known') pathogens. While modern DNA assays can identify known pathogens quickly, identifying unknown pathogens currently depends upon slow, classical microbiological methods of isolation and culture that can take weeks to produce actionable information. In many scenarios that delay would be costly, in terms of casualties and economic damage; indeed, it can mean the difference between a manageable public health incident and a full-blown epidemic. To close this gap in our nation's biodefense capability, we will develop, validate, and optimize a system to extract nucleic acids from unknown pathogens present in clinical samples drawn from infected patients. This system will extract nucleic acids from a clinical sample, amplify pathogen and specific host response nucleic acid sequences. These sequences will then be suitable for ultra-high-throughput sequencing (UHTS) carried out by a third party. The data generated from UHTS will then be processed through a new data assimilation and Bioinformatic analysis pipeline that will allow us to characterize an unknown pathogen in hours to days instead of weeks to months. Our methods will require no a priori knowledge of the pathogen, and no isolation or culturing; therefore it will circumvent many of the major roadblocks confronting a clinical microbiologist or virologist when presented with an unknown or engineered pathogen.

  18. Cofactory: Sequence-based prediction of cofactor specificity of Rossmann folds

    DEFF Research Database (Denmark)

    Geertz-Hansen, Henrik Marcus; Blom, Nikolaj; Feist, Adam;

    2014-01-01

    cofactor specificity using only primary amino acid sequence information. The algorithm identifies potential cofactor binding Rossinann folds and predicts the specificity for the cofactors FAD(H2), NAD(H), and NADP(H) The Rossmann fold sequence search is carried out using hidden Markov models whereas...... artificial neural networks are used for specificity prediction. Training was carried out using experimental data from protein cofactor structure complexes. The overall performance was benchmarked against an independent evaluation set obtaining Matthews correlation coefficients of 0.94, 0.79, and 0.65 for FAD...

  19. Seismic Wave Amplification in 3D Alluvial Basins: 3D/1D Amplification Ratios from Fast Multipole BEM Simulations

    CERN Document Server

    Fajardo, Kristel C Meza; Chaillat, Stéphanie; Lenti, Luca

    2016-01-01

    In this work, we study seismic wave amplification in alluvial basins having 3D standard geometries through the Fast Multipole Boundary Element Method in the frequency domain. We investigate how much 3D amplification differs from the 1D (horizontal layering) case. Considering incident fields of plane harmonic waves, we examine the relationships between the amplification level and the most relevant physical parameters of the problem (impedance contrast, 3D aspect ratio, vertical and oblique incidence of plane waves). The FMBEM results show that the most important parameters for wave amplification are the impedance contrast and the so-called equivalent shape ratio. Using these two parameters, we derive simple rules to compute the fundamental frequency for various 3D basin shapes and the corresponding 3D/1D amplification factor for 5% damping. Effects on amplification due to 3D basin asymmetry are also studied and incorporated in the derived rules.

  20. Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

    Science.gov (United States)

    Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

    2012-04-20

    In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min.

  1. Recursive organizer (ROR): an analytic framework for sequence-based association analysis.

    Science.gov (United States)

    Zhao, Lue Ping; Huang, Xin

    2013-07-01

    The advent of next-generation sequencing technologies affords the ability to sequence thousands of subjects cost-effectively, and is revolutionizing the landscape of genetic research. With the evolving genotyping/sequencing technologies, it is not unrealistic to expect that we will soon obtain a pair of diploidic fully phased genome sequences from each subject in the near future. Here, in light of this potential, we propose an analytic framework called, recursive organizer (ROR), which recursively groups sequence variants based upon sequence similarities and their empirical disease associations, into fewer and potentially more interpretable super sequence variants (SSV). As an illustration, we applied ROR to assess an association between HLA-DRB1 and type 1 diabetes (T1D), discovering SSVs of HLA-DRB1 with sequence data from the Wellcome Trust Case Control Consortium. Specifically, ROR reduces 36 observed unique HLA-DRB1 sequences into 8 SSVs that empirically associate with T1D, a fourfold reduction of sequence complexity. Using HLA-DRB1 data from Type 1 Diabetes Genetics Consortium as cases and data from Fred Hutchinson Cancer Research Center as controls, we are able to validate associations of these SSVs with T1D. Further, SSVs consist of nine nucleotides, and each associates with its corresponding amino acids. Detailed examination of these selected amino acids reveals their potential functional roles in protein structures and possible implication to the mechanism of T1D.

  2. A machine-learning approach for predicting palmitoylation sites from integrated sequence-based features.

    Science.gov (United States)

    Li, Liqi; Luo, Qifa; Xiao, Weidong; Li, Jinhui; Zhou, Shiwen; Li, Yongsheng; Zheng, Xiaoqi; Yang, Hua

    2017-02-01

    Palmitoylation is the covalent attachment of lipids to amino acid residues in proteins. As an important form of protein posttranslational modification, it increases the hydrophobicity of proteins, which contributes to the protein transportation, organelle localization, and functions, therefore plays an important role in a variety of cell biological processes. Identification of palmitoylation sites is necessary for understanding protein-protein interaction, protein stability, and activity. Since conventional experimental techniques to determine palmitoylation sites in proteins are both labor intensive and costly, a fast and accurate computational approach to predict palmitoylation sites from protein sequences is in urgent need. In this study, a support vector machine (SVM)-based method was proposed through integrating PSI-BLAST profile, physicochemical properties, [Formula: see text]-mer amino acid compositions (AACs), and [Formula: see text]-mer pseudo AACs into the principal feature vector. A recursive feature selection scheme was subsequently implemented to single out the most discriminative features. Finally, an SVM method was implemented to predict palmitoylation sites in proteins based on the optimal features. The proposed method achieved an accuracy of 99.41% and Matthews Correlation Coefficient of 0.9773 for a benchmark dataset. The result indicates the efficiency and accuracy of our method in prediction of palmitoylation sites based on protein sequences.

  3. Compared the New Molecular Method for Rapid Detection of Avian Leukemia Virus by Using Denaturing High Performance Liquid Chromatography Combined with Nucleic Acid Amplification and Real-time PCR%PCR结合变性高效液相色谱法与荧光定量PCR法在检测禽白血病中的比较与应用

    Institute of Scientific and Technical Information of China (English)

    孙涛; 张太翔; 徐彪; 梁成珠; 朱来华; 岳志芹

    2011-01-01

    Compared the new molecular method for rapid detection of avian leukemia virus by using denaturing high performance liquid chromatography(DHPLC) combined with nucleic acid amplification and Real-time PCR in this study. According to the sequence of pol gene of ALV, one pair of primers and the TaqMan probe were designed by using Primer Premier 5. 0. The PCR fragment which was amplified by the primers were analysised by DHPLC and the results of Real-time PCR by the primers and the TaqMan probe. They all compared to normal chicken embryo allantoic fluid, duck plague virus(DPV),infectious bronchitis virus(IBV) ,goose parvovirus(GPV) ,avian influenza virus(H5Nl AIV),Newcastle disease virus(NDV), infectious bursal disease virus (IBDV) ,EDSV. There were tested to confirm the specificity of the PCR-DHPLC assay and no positive absorption peaks occurred. The detection limit of ALV AV228 by PCR-DHPLC was 3 pg,10 fold iower than the ordinary Realtime PCR. The results of detcting organ samples from the chickens were tested by PCR-DHPLC and Real-time PCR,showing 100% agreement.%本研究旨在比较PCR结合变性高效液相色谱技术(PCR-DHPLC)与荧光定量PCR (Real-time PCR)两种方法在检测禽白血病中的应用.根据禽白血病pol基因序列,设计1对引物和1条探针,利用引物进行禽白血病模板的RT-PCR扩增,产物经变性高效液相色谱上样处理;利用引物及探针进行荧光定量PCR扩增,结果与PCR-DHPLC进行比对.两种方法同时用正常鸡胚尿囊液、鸭瘟病毒、传染性支气管炎病毒、鹅细小病毒、H5N1亚型禽流感病毒、新城疫病毒、传染性法氏囊病毒、减蛋综合症病毒做特异性检测;以稀释成不同梯度的AV228毒株核酸做敏感性检测.试验结果表明PCR-DHPLC方法只对禽白血病病原有阳性扩增的吸收峰,Real-time PCR也只对禽白血病病原有阳性扩增,两法均对其他禽源病毒核酸无特异性扩增;PCR-DHPLC与Real-time PCR法

  4. Use of signal-mediated amplification of RNA technology (SMART) to detect marine cyanophage DNA.

    Science.gov (United States)

    Hall, M J; Wharam, S D; Weston, A; Cardy, D L N; Wilson, W H

    2002-03-01

    Here, we describe the application of an isothermal nucleic acid amplification assay, signal-mediated amplification of RNA technology (SMART), to detect DNA extracted from marine cyanophages known to infect unicellular cyanobacteria from the genus Synechococcus. The SMART assay is based on the target-dependent production of multiple copies of an RNA signal, which is measured by an enzyme-linked oligosorbent assay. SMART was able to detect both synthetic oligonucleotide targets and genomic cyanophage DNA using probes designed against the portal vertex gene (g20). Specific signals were obtained for each cyanophage strain (S-PM2 and S-BnMI). Nonspecific genomic DNA did not produce false signals or inhibit the detection of a specific target. In addition, we found that extensive purification of target DNA may not be required since signals were obtained from crude cyanophage lysates. This is the first report of the SMART assay being used to discriminate between two similar target sequences.

  5. Amplification of Chirality through Self-Replication of Micellar Aggregates in Water

    KAUST Repository

    Bukhriakov, Konstantin

    2015-03-17

    We describe a system in which the self-replication of micellar aggregates results in a spontaneous amplification of chirality in the reaction products. In this system, amphiphiles are synthesized from two "clickable" fragments: a water-soluble "head" and a hydrophobic "tail". Under biphasic conditions, the reaction is autocatalytic, as aggregates facilitate the transfer of hydrophobic molecules to the aqueous phase. When chiral, partially enantioenriched surfactant heads are used, a strong nonlinear induction of chirality in the reaction products is observed. Preseeding the reaction mixture with an amphiphile of one chirality results in the amplification of this product and therefore information transfer between generations of self-replicating aggregates. Because our amphiphiles are capable of catalysis, information transfer, and self-assembly into bounded structures, they present a plausible model for prenucleic acid "lipid world" entities. © 2015 American Chemical Society.

  6. Optofluidic analysis system for amplification-free, direct detection of Ebola infection

    Science.gov (United States)

    Cai, H.; Parks, J. W.; Wall, T. A.; Stott, M. A.; Stambaugh, A.; Alfson, K.; Griffiths, A.; Mathies, R. A.; Carrion, R.; Patterson, J. L.; Hawkins, A. R.; Schmidt, H.

    2015-09-01

    The massive outbreak of highly lethal Ebola hemorrhagic fever in West Africa illustrates the urgent need for diagnostic instruments that can identify and quantify infections rapidly, accurately, and with low complexity. Here, we report on-chip sample preparation, amplification-free detection and quantification of Ebola virus on clinical samples using hybrid optofluidic integration. Sample preparation and target preconcentration are implemented on a PDMS-based microfluidic chip (automaton), followed by single nucleic acid fluorescence detection in liquid-core optical waveguides on a silicon chip in under ten minutes. We demonstrate excellent specificity, a limit of detection of 0.2 pfu/mL and a dynamic range of thirteen orders of magnitude, far outperforming other amplification-free methods. This chip-scale approach and reduced complexity compared to gold standard RT-PCR methods is ideal for portable instruments that can provide immediate diagnosis and continued monitoring of infectious diseases at the point-of-care.

  7. Current state and future perspectives of loop-mediated isothermal amplification (LAMP)-based diagnosis of filamentous fungi and yeasts.

    Science.gov (United States)

    Niessen, Ludwig

    2015-01-01

    Loop-mediated isothermal amplification is a rather novel method of enzymatic deoxyribonucleic acid amplification which can be applied for the diagnosis of viruses, bacteria, and fungi. Although firmly established in viral and bacterial diagnosis, the technology has only recently been applied to a noteworthy number of species in the filamentous fungi and yeasts. The current review gives an overview of the literature so far published on the topic by discussing the different groups of fungal organisms to which the method has been applied. Moreover, the method is described in detail as well as the different possibilities available for signal detection and quantification and sample preparation. Future perspective of loop-mediated isothermal amplification-based assays is discussed in the light of applicability for fungal diagnostics.

  8. Adaptation of Shift Sequence Based Method for High Number in Shifts Rostering Problem for Health Care Workers

    Directory of Open Access Journals (Sweden)

    Mindaugas Liogys

    2011-08-01

    Full Text Available Purpose—is to investigate a shift sequence-based approach efficiency then problem consisting of a high number of shifts. Research objectives:• Solve health care workers rostering problem using a shift sequence based method.• Measure its efficiency then number of shifts increases. Design/methodology/approach—Usually rostering problems are highly constrained.Constraints are classified to soft and hard constraints. Soft and hard constraints of the problem are additionally classified to: sequence constraints, schedule constraints and roster constraints. Sequence constraints are considered when constructing shift sequences. Schedule constraints are considered when constructing a schedule. Roster constraints are applied, then constructing overall solution, i.e. combining all schedules.Shift sequence based approach consists of two stages:• Shift sequences construction,• The construction of schedules.In the shift sequences construction stage, the shift sequences are constructed for each set of health care workers of different skill, considering sequence constraints. Shifts sequences are ranked by their penalties for easier retrieval in later stage.In schedules construction stage, schedules for each health care worker are constructed iteratively, using the shift sequences produced in stage 1. Shift sequence based method is an adaptive iterative method where health care workers who received the highest schedule penalties in the last iteration are scheduled first at the current iteration. During the roster construction, and after a schedule has been generated for the current health care worker, an improvement method based on an efficient greedy local search is carried out on the partial roster. It simply swaps any pair of shifts between two health care workers in the (partial roster, as long as the swaps satisfy hard constraints and decrease the roster penalty.Findings—Using shift sequence method for solving health care workers rostering

  9. Broadening and Amplification of an Infrared Femtosecond Pulse for Optical Parametric Chirped-Pulse Amplification

    Institute of Scientific and Technical Information of China (English)

    WANG He-Lin; YANG Ai-Jun; LENG Yu-Xin

    2011-01-01

    A high-average-power diode-pumped narrowband regenerative chirped pulse amplifier is developed using the thin-rod Nd:YAG laser architecture for optical parametric chirped-pulse amplification (OPCPA).The effect of the etalons on the amplified pulse in the regenerative cavity is studied experimentally and theoretically.By inserting glass etalons of thickness 1 mm and 5 mm into the regenerative cavity,the pre-stretching pulse from an (O)ffner stretcher is further broadened to above 200ps,which matches the amplification windows of the signal pulses in OPCPA and is suitable for use as a pump source in the OPCPA system.The bandwidth of the amplified pulse is 1.5 nm,and an output energy of 2mJ is achieved at a repetition rate of 10 Hz.Optical parametric chirped pulse amplification (OPCPA)[1-4] has attracted a great deal of attention as the most promising technique for generating ultrashort ultrahigh-peak-power laser pulses because of its very broad gain bandwidth,negligible thermal load on the nonlinear crystal,and extremely high singlepass gain as compared to amplifiers based on laser gain media.For efficient amplification and high fidelity of dispersion compensation in OPCPA,a femtosecond seed pulse is first stretched to several tens of picoseconds with a bulk grating stretcher or a fiber stretcher.%A high-average-power diode-pumped narrowband regenerative chirped pulse amplifier is developed using the thin-rod Nd:YAG laser architecture for optical parametric chirped-pulse amplification (OPCPA). The effect of the etalons on the amplified pulse in the regenerative cavity is studied experimentally and theoretically. By inserting glass etalons of thickness 1 mm and 5 mm into the regenerative cavity, the pre-stretching pulse from an (O)finer stretcher is further broadened to above 200 ps, which matches the amplification windows of the signal pulses in OPCPA and is suitable for use as a pump source in the OPCPA system. The bandwidth of the amplified pulse is 1.5 nm, and an

  10. Zadoff-Chu sequence-based hitless ranging scheme for OFDMA-PON configured 5G fronthaul uplinks

    Science.gov (United States)

    Reza, Ahmed Galib; Rhee, June-Koo Kevin

    2017-05-01

    A Zadoff-Chu (ZC) sequence-based low-complexity hitless upstream time synchronization scheme is proposed for an orthogonal frequency division multiple access passive optical network configured cloud radio access network fronthaul. The algorithm is based on gradual loading of the ZC sequences, where the phase discontinuity due to the cyclic prefix is alleviated by a frequency domain phase precoder, eliminating the requirements of guard bands to mitigate intersymbol interference and inter-carrier interference. Simulation results for uncontrolled-wavelength asynchronous transmissions from four concurrent transmitting optical network units are presented to demonstrate the effectiveness of the proposed scheme.

  11. Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit.

    Science.gov (United States)

    Zahra, Nathalie; Hadi, Sibte; Smith, Judith A; Iyengar, Arati; Goodwin, William

    2011-06-01

    DNA extracted from forensic samples can be degraded and also contain co-extracted contaminants that inhibit PCR. The effects of DNA degradation and PCR inhibition are often indistinguishable when examining a DNA profile. Two internal amplification controls (IACs) were developed to improve quality control of PCR using the AmpFℓSTR® SGM Plus® kit. The co-amplification of these controls with DNA samples was used to monitor amplification efficiency and detect PCR inhibitors. IAC fragments of 90 and 410 bp (IAC₉₀ and IAC₄₁₀) were generated from the plasmid pBR322 using tailed primers and then amplified with ROX-labelled primers. Co-amplification of IAC₉₀ and IAC₄₁₀ was performed with varying amounts of template DNA, degraded DNA and DNA contaminated with humic acid, heme and indigo dye. Both IAC₉₀ and IAC₄₁₀ were successfully amplified with human DNA without significantly affecting the quality of the DNA profile, even with DNA amounts lower than 0.5 ng. In the presence of inhibitors, the IAC₉₀ signal was still present after all human DNA loci fail to amplify; in contrast, the IAC₄₁₀ signal was reduced or absent at low levels of inhibition. Amplification of the two IACs provided an internal PCR control and allowed partial profiles caused by inhibition to be distinguished from degraded DNA profiles.

  12. Control and amplification of cortical neurodynamics

    Science.gov (United States)

    Liljenstroem, Hans; Aronsson, P.

    1999-03-01

    We investigate different mechanisms for the control and amplification of cortical neurodynamics, using a neural network model of a three layered cortical structure. We show that different dynamical states can be obtained by changing a control parameter of the input-output relation, or by changing the noise level. Point attractor, limit cycle, and strange attractor dynamics occur at different values of the control parameter. For certain, optimal noise levels, system performance is maximized, analogous to stochastic resonance phenomena. Noise can also be used to induce different dynamical states. A few noisy network units distributed in a network layer can result in global synchronous oscillations, or waves of activity moving across the network. We further demonstrate that fast synchronization of network activity can be obtained by implementing electromagnetic interactions between network units.

  13. Anisotropic metamaterials with simultaneous attenuation and amplification

    CERN Document Server

    Mackay, Tom G

    2015-01-01

    Anisotropic metamaterials that are neither wholly dissipative nor wholly active at a specific frequency are permitted by classical electromagnetic theory. Well-established formalisms for the homogenization of particulate composite materials indicate that such a metamaterial may be conceptualized quite simply as a random mixture of electrically small spheroidal particles of at least two different isotropic dielectric materials, one of which must be dissipative but the other active. The realization of this metametarial is influenced by the volume fraction, spatial distribution, particle shape and size, and the relative permittivities of the component materials. Metamaterials displaying both dissipation and amplification at the same frequency with more complicated linear as well as nonlinear constitutive properties are possible.

  14. Dispersion compensation in chirped pulse amplification systems

    Science.gov (United States)

    Bayramian, Andrew James; Molander, William A.

    2014-07-15

    A chirped pulse amplification system includes a laser source providing an input laser pulse along an optical path. The input laser pulse is characterized by a first temporal duration. The system also includes a multi-pass pulse stretcher disposed along the optical path. The multi-pass pulse stretcher includes a first set of mirrors operable to receive input light in a first plane and output light in a second plane parallel to the first plane and a first diffraction grating. The pulse stretcher also includes a second set of mirrors operable to receive light diffracted from the first diffraction grating and a second diffraction grating. The pulse stretcher further includes a reflective element operable to reflect light diffracted from the second diffraction grating. The system further includes an amplifier, a pulse compressor, and a passive dispersion compensator disposed along the optical path.

  15. Integrated Amplification Microarrays for Infectious Disease Diagnostics

    Directory of Open Access Journals (Sweden)

    Darrell P. Chandler

    2012-11-01

    Full Text Available This overview describes microarray-based tests that combine solution-phase amplification chemistry and microarray hybridization within a single microfluidic chamber. The integrated biochemical approach improves microarray workflow for diagnostic applications by reducing the number of steps and minimizing the potential for sample or amplicon cross-contamination. Examples described herein illustrate a basic, integrated approach for DNA and RNA genomes, and a simple consumable architecture for incorporating wash steps while retaining an entirely closed system. It is anticipated that integrated microarray biochemistry will provide an opportunity to significantly reduce the complexity and cost of microarray consumables, equipment, and workflow, which in turn will enable a broader spectrum of users to exploit the intrinsic multiplexing power of microarrays for infectious disease diagnostics.

  16. Magnetic field amplification in turbulent astrophysical plasmas

    CERN Document Server

    Federrath, Christoph

    2016-01-01

    Magnetic fields play an important role in astrophysical accretion discs, and in the interstellar and intergalactic medium. They drive jets, suppress fragmentation in star-forming clouds and can have a significant impact on the accretion rate of stars. However, the exact amplification mechanisms of cosmic magnetic fields remain relatively poorly understood. Here I start by reviewing recent advances in the numerical and theoretical modelling of the 'turbulent dynamo', which may explain the origin of galactic and inter-galactic magnetic fields. While dynamo action was previously investigated in great detail for incompressible plasmas, I here place particular emphasis on highly compressible astrophysical plasmas, which are characterised by strong density fluctuations and shocks, such as the interstellar medium. I find that dynamo action works not only in subsonic plasmas, but also in highly supersonic, compressible plasmas, as well as for low and high magnetic Prandtl numbers. I further present new numerical simu...

  17. Amplification sans bruit d'images optiques

    Science.gov (United States)

    Gigan, S.; Delaubert, V.; Lopez, L.; Treps, N.; Maitre, A.; Fabre, C.

    2004-11-01

    Nous utilisons un Oscillateur Paramétrique Optique (OPO) pompé sous le seuil dans le but d'amplifier une image multimode transverse sans dégradation du rapport signal à bruit. Le dispositif expérimental met en œuvre un OPO de type II triplement résonant et semi-confocal pour le faisceau amplifié. L'existence d'effets quantiques lors de l'amplification multimode dans un tel dispositif a été montrée expérimentalement. Plus généralement, ceci nous a amené à étudier les propriétés quantiques transverses des faisceaux lumineux amplifiés. Une telle étude peut trouver des applications non seulement en imagerie, mais également dans le traitement quantique de l'information.

  18. Raman amplification in optical communication systems

    DEFF Research Database (Denmark)

    Kjær, Rasmus

    2008-01-01

    Fiber Raman amplifiers are investigated with the purpose of identifying new applications and limitations for their use in optical communication systems. Three main topics are investigated, namely: New applications of dispersion compensating Raman amplifiers, the use Raman amplification to increase...... fiberbaserede Raman-forstærkere med henblik på at identificere både deres begrænsninger og nye anvendelsesmuligheder i optiske kommunikationssystemer. En numerisk forstærkermodel er blevet udviklet for bedre at forstå forstærkerens dynamik, dens gain- og støjbegrænsninger. Modellen bruges til at forudsige...... forstærkerens statiske og dynamiske egenskaber, og det eftervises at dens resultater er i god overensstemmelse med eksperimentelle forstærkermålinger. Dispersions-kompenserende fiber er på grund af sin store udbredelse og fiberens høje Raman gain effektivitet et meget velegnet Raman gain-medium. Tre nye...

  19. Magnetic Field Amplification and Blazar Flares

    CERN Document Server

    Chen, Xuhui; Fossati, Giovanni; Pohl, Martin

    2013-01-01

    Recent multiwavelength observations of PKS 0208-512 by SMARTS, Fermi, and Swift revealed that gamma-ray and optical light curves of this flat spectrum radio quasars are highly correlated, but with an exception of one large optical flare having no corresponding gamma-ray activity or even detection. On the other hand, recent advances in SNRs observations and plasma simulations both reveal that magnetic field downstream of astrophysical shocks can be largely amplified beyond simple shock compression. These amplifications, along with their associated particle acceleration, might contribute to blazar flares, including the peculiar flare of PKS 0208-512. Using our time dependent multizone blazar emission code, we evaluate several scenarios that may represent such phenomena. This code combines Monte Carlo method that tracks the radiative processes including inverse Compton scattering, and Fokker-Planck equation that follows the cooling and acceleration of particles. It is a comprehensive time dependent code that ful...

  20. Protein Misfolding Cyclic Amplification of Infectious Prions.

    Science.gov (United States)

    Moda, Fabio

    2017-01-01

    Transmissible spongiform encephalopathies, or prion diseases, are a group of incurable disorders caused by the accumulation of an abnormally folded prion protein (PrP(Sc)) in the brain. According to the "protein-only" hypothesis, PrP(Sc) is the infectious agent able to propagate the disease by acting as a template for the conversion of the correctly folded prion protein (PrP(C)) into the pathological isoform. Recently, the mechanism of PrP(C) conversion has been mimicked in vitro using an innovative technique named protein misfolding cyclic amplification (PMCA). This technology represents a great tool for studying diverse aspects of prion biology in the field of basic research and diagnosis. Moreover, PMCA can be expanded for the study of the misfolding process associated to other neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and frontotemporal lobar degeneration. © 2017 Elsevier Inc. All rights reserved.

  1. Targeting HER2 amplifications in gastric cancer

    Directory of Open Access Journals (Sweden)

    Ung L

    2014-01-01

    Full Text Available Lawson Ung, Terence C Chua, Neil D Merrett Department of Surgery, South Western Sydney Upper GI Surgical Unit, Bankstown Hospital, University of Western Sydney, Sydney, NSW, Australia Abstract: While multimodality treatments, including neoadjuvant and adjuvant chemotherapy or chemoradiation, have become the global standard of care in patients with locally advanced and metastatic gastric cancers (GCs, long-term outcomes for patients remain poor. This reflects the aggressive tumor biology of GCs and occult nature of the disease, often presenting in its advanced stages, as well as the challenges of developing effective targeted therapy to treat this disease. The Trastuzumab for Gastric Cancer trial demonstrates that the addition of human epidermal growth factor 2 (HER2 monoclonal antibody trastuzumab to standard chemotherapy regimen consisting of 5-fluorouracil (5-FU or capecitabine with cisplatin results in significant improvement in overall and progression-free survival. Although questions remain regarding the best methods by which to determine HER2 mutation positivity and amplification, through immunohistochemistry or in situ hybridization, and whether trastuzumab is effective for locally advanced, nonmetastatic GC in an adjuvant setting, the trial has led to a surge of clinical trials investigating the potential role of other HER2- and non-HER2-targeted therapies to improve patient outcomes. This review will discuss our current understanding of GC pathogenesis, current available treatments, and the potential impact that targeting HER2 amplifications may have in our efforts to individualize and optimize cancer care in GC individuals. Keywords: Personalized cancer therapy, surgical oncology, gastrectomy, adjuvant treatment, targeted therapies

  2. Mutualism breakdown by amplification of Wolbachia genes.

    Science.gov (United States)

    Chrostek, Ewa; Teixeira, Luis

    2015-02-01

    Most insect species are associated with vertically transmitted endosymbionts. Because of the mode of transmission, the fitness of these symbionts is dependent on the fitness of the hosts. Therefore, these endosymbionts need to control their proliferation in order to minimize their cost for the host. The genetic bases and mechanisms of this regulation remain largely undetermined. The maternally inherited bacteria of the genus Wolbachia are the most common endosymbionts of insects, providing some of them with fitness benefits. In Drosophila melanogaster, Wolbachia wMelPop is a unique virulent variant that proliferates massively in the hosts and shortens their lifespan. The genetic bases of wMelPop virulence are unknown, and their identification would allow a better understanding of how Wolbachia levels are regulated. Here we show that amplification of a region containing eight Wolbachia genes, called Octomom, is responsible for wMelPop virulence. Using Drosophila lines selected for carrying Wolbachia with different Octomom copy numbers, we demonstrate that the number of Octomom copies determines Wolbachia titers and the strength of the lethal phenotype. Octomom amplification is unstable, and reversion of copy number to one reverts all the phenotypes. Our results provide a link between genotype and phenotype in Wolbachia and identify a genomic region regulating Wolbachia proliferation. We also prove that these bacteria can evolve rapidly. Rapid evolution by changes in gene copy number may be common in endosymbionts with a high number of mobile elements and other repeated regions. Understanding wMelPop pathogenicity and variability also allows researchers to better control and predict the outcome of releasing mosquitoes transinfected with this variant to block human vector-borne diseases. Our results show that transition from a mutualist to a pathogen may occur because of a single genomic change in the endosymbiont. This implies that there must be constant selection on

  3. Short-Pulse Amplification by Strongly-Coupled Brillouin Scattering

    CERN Document Server

    Edwards, Matthew R; Mikhailova, Julia M; Fisch, Nathaniel J

    2016-01-01

    We examine the feasibility of strongly-coupled stimulated Brillouin scattering as a mechanism for the plasma-based amplification of sub-picosecond pulses. In particular, we use fluid theory and particle-in-cell simulations to compare the relative advantages of Raman and Brillouin amplification over a broad range of achievable parameters.

  4. A Theoretical Evaluation of Optical Parametric Amplification in BBO Crystal

    Institute of Scientific and Technical Information of China (English)

    邵敏; 薛绍林; 林尊琪

    2005-01-01

    The noncollinear optical parametric amplification in BBO crystal is theoretically investigated. The phase matching angle, gain bandwidth, optimal noncollinear angle and conversion efficiency for both type-Ⅰ and type-Ⅱ BBO are simulated. The numerical simulation results are important to the practical optical parametric amplification experiments with BBO crystal.

  5. Phase Sensitive Amplification using Parametric Processes in Optical Fibers

    DEFF Research Database (Denmark)

    Kang, Ning

    , in specific, the design and optimization of such phase sensitive amplifiers (PSAs). For phase sensitive amplification in highly nonlinear fibers, optima points of operation have been identified for both the standard and the novel high stimulated Brillouin scattering (SBS) threshold highly nonlinear fiber....... Finally, preliminary simulations were carried out to investigate the inline amplification properties of such PSAs, and their pulse shaping capabilities....

  6. Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples.

    Science.gov (United States)

    Marsh, Alan J; O'Sullivan, Orla; Hill, Colin; Ross, R Paul; Cotter, Paul D

    2014-04-01

    Kombucha is a sweetened tea beverage that, as a consequence of fermentation, contains ethanol, carbon dioxide, a high concentration of acid (gluconic, acetic and lactic) as well as a number of other metabolites and is thought to contain a number of health-promoting components. The sucrose-tea solution is fermented by a symbiosis of bacteria and yeast embedded within a cellulosic pellicle, which forms a floating mat in the tea, and generates a new layer with each successful fermentation. The specific identity of the microbial populations present has been the focus of attention but, to date, the majority of studies have relied on culture-based analyses. To gain a more comprehensive insight into the kombucha microbiota we have carried out the first culture-independent, high-throughput sequencing analysis of the bacterial and fungal populations of 5 distinct pellicles as well as the resultant fermented kombucha at two time points. Following the analysis it was established that the major bacterial genus present was Gluconacetobacter, present at >85% in most samples, with only trace populations of Acetobacter detected (95% in the fermented beverage, with a greater fungal diversity present in the cellulosic pellicle, including numerous species not identified in kombucha previously. Ultimately, this study represents the most accurate description of the microbiology of kombucha to date.

  7. Deep sequencing-based identification of small regulatory RNAs in Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Xu, Wen; Chen, Hui; He, Chen-Liu; Wang, Qiang

    2014-01-01

    Synechocystis sp. PCC 6803 is a genetically tractable model organism for photosynthesis research. The genome of Synechocystis sp. PCC 6803 consists of a circular chromosome and seven plasmids. The importance of small regulatory RNAs (sRNAs) as mediators of a number of cellular processes in bacteria has begun to be recognized. However, little is known regarding sRNAs in Synechocystis sp. PCC 6803. To provide a comprehensive overview of sRNAs in this model organism, the sRNAs of Synechocystis sp. PCC 6803 were analyzed using deep sequencing, and 7,951,189 reads were obtained. High quality mapping reads (6,127,890) were mapped onto the genome and assembled into 16,192 transcribed regions (clusters) based on read overlap. A total number of 5211 putative sRNAs were revealed from the genome and the 4 megaplasmids, and 27 of these molecules, including four from plasmids, were confirmed by RT-PCR. In addition, possible target genes regulated by all of the putative sRNAs identified in this study were predicted by IntaRNA and analyzed for functional categorization and biological pathways, which provided evidence that sRNAs are indeed involved in many different metabolic pathways, including basic metabolic pathways, such as glycolysis/gluconeogenesis, the citrate cycle, fatty acid metabolism and adaptations to environmentally stress-induced changes. The information from this study provides a valuable reservoir for understanding the sRNA-mediated regulation of the complex physiology and metabolic processes of cyanobacteria.

  8. PenBase, the shrimp antimicrobial peptide penaeidin database: sequence-based classification and recommended nomenclature.

    Science.gov (United States)

    Gueguen, Yannick; Garnier, Julien; Robert, Lorenne; Lefranc, Marie-Paule; Mougenot, Isabelle; de Lorgeril, Julien; Janech, Michael; Gross, Paul S; Warr, Gregory W; Cuthbertson, Brandon; Barracco, Margherita A; Bulet, Philippe; Aumelas, André; Yang, Yinshan; Bo, Dong; Xiang, Jianhai; Tassanakajon, Anchalee; Piquemal, David; Bachère, Evelyne

    2006-01-01

    Antimicrobial peptides play a major role in innate immunity. The penaeidins, initially characterized from the shrimp Litopenaeus vannamei, are a family of antimicrobial peptides that appear to be expressed in all penaeid shrimps. As of recent, a large number of penaeid nucleotide sequences have been identified from a variety of penaeid shrimp species and these sequences currently reside in several databases under unique identifiers with no nomenclatural continuity. To facilitate research in this field and avoid potential confusion due to a diverse number of nomenclatural designations, we have made a systematic effort to collect, analyse, and classify all the penaeidin sequences available in every database. We have identified a common penaeidin signature and subsequently established a classification based on amino acid sequences. In order to clarify the naming process, we have introduced a 'penaeidin nomenclature' that can be applied to all extant and future penaeidins. A specialized database, PenBase, which is freely available at , has been developed for the penaeidin family of antimicrobial peptides, to provide comprehensive information about their properties, diversity and nomenclature.

  9. Generation of recombinant pestiviruses using a full genome amplification strategy

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Reimann, Ilona; Uttenthal, Åse

    Aim Complete genome amplification of viral RNA provides a new tool for generation of modified pestiviruses. We have recently reported a full genome amplification strategy for direct recovery of infectious pestivirus (Rasmussen et al., 2008). This comprised rescue of BDV strain “Gifhorn” from a full......-length RT-PCR amplicon demonstrating that long RT-PCR can be used for direct generation of an infectious pestivirus. The strategy is not limited to amplification of BDV “Gifhorn”, but can be further utilized for amplification of a diverse selection of pestivirus strains and for the generation of modified...... the amplicons were prepared for cloning into low-copy vectors to produce new infectious cDNA clones. Conclusions Using this full genome amplification strategy the efforts in producing new viral variants can be expedited and focused on a variety of other viral strains and hence is not limited to the availability...

  10. Parametric Analog Signal Amplification Applied to Nanoscale CMOS Technologies

    CERN Document Server

    Oliveira, João P

    2012-01-01

    This book is dedicated to the analysis of parametric amplification with special emphasis on the MOS discrete-time implementation. This implementation is demonstrated by the presentation of several circuits where the MOS parametric amplifier cell is used: small gain amplifier, comparator with embedded pre-amplification, discrete-time mixer/IIR-Filter, and analog-to-digital converter (ADC).  Experimental results are shown to validate the overall design technique. Provides the complete theoretical analysis, supported by electrical simulations, of the parametric amplification technique in both continuous time and discrete time domains; Describes the design flow of an ADC fully based on discrete-time parametric amplification in CMOS technology; Presents a high speed time-interleaved pipeline ADC, based on parametric MOS amplification techniques described, complementing theory discussed with experimental results.

  11. Hendra virus detection using Loop-Mediated Isothermal Amplification.

    Science.gov (United States)

    Foord, Adam J; Middleton, Deborah; Heine, Hans G

    2012-04-01

    Hendra virus (HeV) is a zoonotic paramyxovirus endemic in Australian Pteropus bats (fruit bats or flying foxes). Although bats appear to be unaffected by the virus, HeV can spread from fruit bats to horses, causing severe disease. Human infection results from close contact with the blood, body fluids and tissues of infected horses. HeV is a biosecurity level 4 (BSL-4) pathogen, with a high case-fatality rate in humans and horses. Current assays for HeV detection require complex instrumentation and are generally time consuming. The aim of this study was to develop a Loop-Mediated Isothermal Amplification (LAMP) assay to detect nucleic acid from all known HeV strains in horses without the requirement for complex laboratory equipment. A LAMP assay targeting a conserved region of the HeV P-gene was combined with a Lateral Flow Device (LFD) for detection of amplified product. All HeV isolates, the original HeV isolated in 1994 as well as the most recent isolates from 2011 were detected. Analytical sensitivity and specificity of the HeV-LAMP assay was equal to a TaqMan assay developed previously. Significantly, these assays detected HeV in horses before clinical signs were observed. The combined LAMP-LFD procedure is a sensitive method suitable for HeV diagnosis in a resource-limited situation or where rapid test results are critical.

  12. Amplification and Re-Generation of LNA-Modified Libraries

    Directory of Open Access Journals (Sweden)

    Jesper Wengel

    2012-11-01

    Full Text Available Locked nucleic acids (LNA confer high thermal stability and nuclease resistance to oligonucleotides. The discovery of polymerases that accept LNA triphosphates has led us to propose a scheme for the amplification and re-generation of LNA-containing oligonucleotide libraries. Such libraries could be used for in vitro selection of e.g., native LNA aptamers. We maintained an oligonucleotide library encoding 40 randomized positions with LNA ATP, GTP, CTP, and TTP for 7 rounds of ‘mock’ in vitro selection in the absence of a target and analyzed the sequence composition after rounds 1, 4 and 7. We observed a decrease in LNA-A content from 20.5% in round 1 to 6.6% in round 7. This decrease was accompanied by a substantial bias against successive LNA-As (poly-LNA adenosine tracts and a relative over-representation of single LNA-As. Maintaining a library with LNA TTP yielded similar results. Together, these results suggest that dispersed LNA monomers are tolerated in our in vitro selection protocol, and that LNA-modified libraries can be sustained for up to at least seven selection rounds, albeit at reduced levels. This enables the discovery of native LNA aptamers and similar oligonucleotide structures.

  13. Clinical application of somatosensory amplification in psychosomatic medicine

    Directory of Open Access Journals (Sweden)

    Nakao Mutsuhiro

    2007-10-01

    Full Text Available Abstract Many patients with somatoform disorders are frequently encountered in psychosomatic clinics as well as in primary care clinics. To assess such patients objectively, the concept of somatosensory amplification may be useful. Somatosensory amplification refers to the tendency to experience a somatic sensation as intense, noxious, and disturbing. It may have a role in a variety of medical conditions characterized by somatic symptoms that are disproportionate to demonstrable organ pathology. It may also explain some of the variability in somatic symptomatology found among different patients with the same serious medical disorder. It has been assessed with a self-report questionnaire, the Somatosensory Amplification Scale. This instrument was developed in a clinical setting in the U.S., and the reliability and validity of the Japanese and Turkish versions have been confirmed as well. Many studies have attempted to clarify the specific role of somatosensory amplification as a pathogenic mechanism in somatization. It has been reported that somatosensory amplification does not correlate with heightened sensitivity to bodily sensations and that emotional reactivity exerts its influence on somatization via a negatively biased reporting style. According to our recent electroencephalographic study, somatosensory amplification appears to reflect some aspects of long-latency cognitive processing rather than short-latency interoceptive sensitivity. The concept of somatosensory amplification can be useful as an indicator of somatization in the therapy of a broad range of disorders, from impaired self-awareness to various psychiatric disorders. It also provides useful information for choosing appropriate pharmacological or psychological therapy. While somatosensory amplification has a role in the presentation of somatic symptoms, it is closely associated with other factors, namely, anxiety, depression, and alexithymia that may also influence the same

  14. Sequence-based Association Analysis Reveals an MGST1 eQTL with Pleiotropic Effects on Bovine Milk Composition

    Science.gov (United States)

    Littlejohn, Mathew D.; Tiplady, Kathryn; Fink, Tania A.; Lehnert, Klaus; Lopdell, Thomas; Johnson, Thomas; Couldrey, Christine; Keehan, Mike; Sherlock, Richard G.; Harland, Chad; Scott, Andrew; Snell, Russell G.; Davis, Stephen R.; Spelman, Richard J.

    2016-01-01

    The mammary gland is a prolific lipogenic organ, synthesising copious amounts of triglycerides for secretion into milk. The fat content of milk varies widely both between and within species, and recent independent genome-wide association studies have highlighted a milk fat percentage quantitative trait locus (QTL) of large effect on bovine chromosome 5. Although both EPS8 and MGST1 have been proposed to underlie these signals, the causative status of these genes has not been functionally confirmed. To investigate this QTL in detail, we report genome sequence-based imputation and association mapping in a population of 64,244 taurine cattle. This analysis reveals a cluster of 17 non-coding variants spanning MGST1 that are highly associated with milk fat percentage, and a range of other milk composition traits. Further, we exploit a high-depth mammary RNA sequence dataset to conduct expression QTL (eQTL) mapping in 375 lactating cows, revealing a strong MGST1 eQTL underpinning these effects. These data demonstrate the utility of DNA and RNA sequence-based association mapping, and implicate MGST1, a gene with no obvious mechanistic relationship to milk composition regulation, as causally involved in these processes. PMID:27146958

  15. Exome sequencing-based molecular autopsy of formalin-fixed paraffin-embedded tissue after sudden death.

    Science.gov (United States)

    Bagnall, Richard D; Ingles, Jodie; Yeates, Laura; Berkovic, Samuel F; Semsarian, Christopher

    2017-10-01

    Sudden death in the young is a devastating complication of inherited heart disorders. Finding the precise cause of death is important, but it is often unresolved after postmortem investigation. The addition of postmortem genetic testing, i.e., the molecular autopsy, can identify additional causes of death. We evaluated DNA extracted from formalin-fixed paraffin-embedded postmortem tissue for exome sequencing-based molecular autopsy after sudden death in the young. We collected clinical and postmortem information from patients with sudden death. Exome sequencing was performed on DNA extracted from fixed postmortem tissue. Variants relevant to the cause of death were sought. Five patients with genetically unresolved sudden death were recruited. DNA extracted from fixed postmortem tissue was degraded. Exome sequencing achieved 20-fold coverage of at least 82% of coding regions. A threefold excess of singleton variants was found in the exome sequencing data of one patient. We found a de novo SCN1A frameshift variant in a patient with sudden unexpected death in epilepsy and a LMNA nonsense variant in a patient with dilated cardiomyopathy. DNA extracted from fixed postmortem tissue is applicable to exome sequencing-based molecular autopsy. Fixed postmortem tissues are an untapped resource for exome-based studies of rare causes of sudden death.Genet Med advance online publication 23 March 2017.

  16. Combining sequence-based prediction methods and circular dichroism and infrared spectroscopic data to improve protein secondary structure determinations

    Directory of Open Access Journals (Sweden)

    Lees Jonathan G

    2008-01-01

    Full Text Available Abstract Background A number of sequence-based methods exist for protein secondary structure prediction. Protein secondary structures can also be determined experimentally from circular dichroism, and infrared spectroscopic data using empirical analysis methods. It has been proposed that comparable accuracy can be obtained from sequence-based predictions as from these biophysical measurements. Here we have examined the secondary structure determination accuracies of sequence prediction methods with the empirically determined values from the spectroscopic data on datasets of proteins for which both crystal structures and spectroscopic data are available. Results In this study we show that the sequence prediction methods have accuracies nearly comparable to those of spectroscopic methods. However, we also demonstrate that combining the spectroscopic and sequences techniques produces significant overall improvements in secondary structure determinations. In addition, combining the extra information content available from synchrotron radiation circular dichroism data with sequence methods also shows improvements. Conclusion Combining sequence prediction with experimentally determined spectroscopic methods for protein secondary structure content significantly enhances the accuracy of the overall results obtained.

  17. Deep sequencing-based identification of small regulatory RNAs in Synechocystis sp. PCC 6803.

    Directory of Open Access Journals (Sweden)

    Wen Xu

    Full Text Available Synechocystis sp. PCC 6803 is a genetically tractable model organism for photosynthesis research. The genome of Synechocystis sp. PCC 6803 consists of a circular chromosome and seven plasmids. The importance of small regulatory RNAs (sRNAs as mediators of a number of cellular processes in bacteria has begun to be recognized. However, little is known regarding sRNAs in Synechocystis sp. PCC 6803. To provide a comprehensive overview of sRNAs in this model organism, the sRNAs of Synechocystis sp. PCC 6803 were analyzed using deep sequencing, and 7,951,189 reads were obtained. High quality mapping reads (6,127,890 were mapped onto the genome and assembled into 16,192 transcribed regions (clusters based on read overlap. A total number of 5211 putative sRNAs were revealed from the genome and the 4 megaplasmids, and 27 of these molecules, including four from plasmids, were confirmed by RT-PCR. In addition, possible target genes regulated by all of the putative sRNAs identified in this study were predicted by IntaRNA and analyzed for functional categorization and biological pathways, which provided evidence that sRNAs are indeed involved in many different metabolic pathways, including basic metabolic pathways, such as glycolysis/gluconeogenesis, the citrate cycle, fatty acid metabolism and adaptations to environmentally stress-induced changes. The information from this study provides a valuable reservoir for understanding the sRNA-mediated regulation of the complex physiology and metabolic processes of cyanobacteria.

  18. Single cell genome amplification accelerates identification of the apratoxin biosynthetic pathway from a complex microbial assemblage.

    Directory of Open Access Journals (Sweden)

    Rashel V Grindberg

    Full Text Available Filamentous marine cyanobacteria are extraordinarily rich sources of structurally novel, biomedically relevant natural products. To understand their biosynthetic origins as well as produce increased supplies and analog molecules, access to the clustered biosynthetic genes that encode for the assembly enzymes is necessary. Complicating these efforts is the universal presence of heterotrophic bacteria in the cell wall and sheath material of cyanobacteria obtained from the environment and those grown in uni-cyanobacterial culture. Moreover, the high similarity in genetic elements across disparate secondary metabolite biosynthetic pathways renders imprecise current gene cluster targeting strategies and contributes sequence complexity resulting in partial genome coverage. Thus, it was necessary to use a dual-method approach of single-cell genomic sequencing based on multiple displacement amplification (MDA and metagenomic library screening. Here, we report the identification of the putative apratoxin. A biosynthetic gene cluster, a potent cancer cell cytotoxin with promise for medicinal applications. The roughly 58 kb biosynthetic gene cluster is composed of 12 open reading frames and has a type I modular mixed polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS organization and features loading and off-loading domain architecture never previously described. Moreover, this work represents the first successful isolation of a complete biosynthetic gene cluster from Lyngbya bouillonii, a tropical marine cyanobacterium renowned for its production of diverse bioactive secondary metabolites.

  19. Mechanism of Gene Amplification via Yeast Autonomously Replicating Sequences

    Directory of Open Access Journals (Sweden)

    Shelly Sehgal

    2015-01-01

    Full Text Available The present investigation was aimed at understanding the molecular mechanism of gene amplification. Interplay of fragile sites in promoting gene amplification was also elucidated. The amplification promoting sequences were chosen from the Saccharomyces cerevisiae ARS, 5S rRNA regions of Plantago ovata and P. lagopus, proposed sites of replication pausing at Ste20 gene locus of S. cerevisiae, and the bend DNA sequences within fragile site FRA11A in humans. The gene amplification assays showed that plasmid bearing APS from yeast and human beings led to enhanced protein concentration as compared to the wild type. Both the in silico and in vitro analyses were pointed out at the strong bending potential of these APS. In addition, high mitotic stability and presence of TTTT repeats and SAR amongst these sequences encourage gene amplification. Phylogenetic analysis of S. cerevisiae ARS was also conducted. The combinatorial power of different aspects of APS analyzed in the present investigation was harnessed to reach a consensus about the factors which stimulate gene expression, in presence of these sequences. It was concluded that the mechanism of gene amplification was that AT rich tracts present in fragile sites of yeast serve as binding sites for MAR/SAR and DNA unwinding elements. The DNA protein interactions necessary for ORC activation are facilitated by DNA bending. These specific bindings at ORC promote repeated rounds of DNA replication leading to gene amplification.

  20. Kinetic Hairpin Oligonucleotide Blockers for Selective Amplification of Rare Mutations

    Science.gov (United States)

    Jia, Yanwei; Sanchez, J. Aquiles; Wangh, Lawrence J.

    2014-01-01

    Detection of rare mutant alleles in an excess of wild type alleles is increasingly important in cancer diagnosis. Several methods for selective amplification of a mutant allele via the polymerase chain reaction (PCR) have been reported, but each of these methods has its own limitations. A common problem is that Taq DNA polymerase errors early during amplification generate false positive mutations which also accumulate exponentially. In this paper, we described a novel method using hairpin oligonucleotide blockers that can selectively inhibit the amplification of wild type DNA during LATE-PCR amplification. LATE-PCR generates double-stranded DNA exponentially followed by linear amplification of single-stranded DNA. The efficiency of the blocker is optimized by adjusting the LATE-PCR temperature cycling profile. We also demonstrate that it is possible to minimize false positive signals caused by Taq DNA polymerase errors by using a mismatched excess primer plus a modified PCR profile to preferentially enrich for mutant target sequences prior to the start of the exponential phase of LATE-PCR amplification. In combination these procedures permit amplification of specific KRAS mutations in the presence of more than 10,000 fold excess of wild type DNA without false positive signals. PMID:25082368

  1. Rapid detection of Brucella spp. using loop-mediated isothermal amplification (LAMP).

    Science.gov (United States)

    Chen, Shouyi; Li, Xunde; Li, Juntao; Atwill, Edward R

    2013-01-01

    Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Livestock that are most vulnerable to brucellosis include cattle, goats, and pigs. Brucella spp. cause serious health problems to humans and animals and economic losses to the livestock industry. Traditional methods for detection of Brucella spp. take 48-72 h (Kumar et al., J Commun Dis 29:131-137, 1997; Barrouin-Melo et al., Res Vet Sci 83:340-346, 2007) that do not meet the food industry's need of rapid detection. Therefore, there is an urgent need of fast, specific, sensitive, and inexpensive method for diagnosing of Brucella spp. Loop-mediated isothermal amplification (LAMP) is a method to amplify nucleic acid at constant temperatures. Amplification can be detected by visual detection, fluorescent stain, turbidity, and electrophoresis. We targeted at the Brucella-specific gene omp25 and designed LAMP primers for detection of Brucella spp. Amplification of DNA with Bst DNA polymerase can be completed at 65 °C in 60 min. Amplified products can be detected by SYBR Green I stain and 2.0% agarose gel electrophoresis. The LAMP method is feasible for detection of Brucella spp. from blood and milk samples.

  2. Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2

    Institute of Scientific and Technical Information of China (English)

    Ye-bing Liu; Lei Zhang; Qin-hong Xue; Yi-bao Ning; Zhi-gang Zhang

    2011-01-01

    In this study,the loop-mediated isothermal amplification(LAMP)method was used to develop a rapid and simple detection system for porcine circovirus type 2(PCV2).According to the PCV2 sequences published in GenBank,multiple LAMP primers were designed targeting conserved sequences of PCV2.Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template,LAMP reactions in a PCV2 LAMP system was performed,the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye.The results showed highly-efficient and specific amplification in 30 min at 63℃ with a LAMP real-time turbidimeter.Furthermore,PCV2 DNA templates,with a detection limit of 5.5×10-5ng of nucleic acid,indicated that this assay was highly sensitive.The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter,showing the potential simplicity of interpretation of the assay results.The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples.In addition it offers higher specificity and sensitivity,shorter reaction times and simpler procedures than the currently available methods of PCV2 detection.It is therefore a promising tool for the effective and efficient detection of PCV2.

  3. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays.

    Science.gov (United States)

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao

    2015-06-01

    Isothermal DNA/RNA amplification techniques are the primary methodology for developing on-spot rapid nucleic acid amplification assays, and the loop-mediated isothermal amplification (LAMP) technique has been developed and applied in the detection of foodborne pathogens, plant/animal viruses, and genetically modified (GM) food/feed contents. In this study, one set of LAMP assays targeting on eight frequently used universal elements, marker genes, and exogenous target genes, such as CaMV35S promoter, FMV35S promoter, NOS, bar, cry1Ac, CP4 epsps, pat, and NptII, were developed for visual screening of GM contents in plant-derived food samples with high efficiency and accuracy. For these eight LAMP assays, their specificity was evaluated by testing commercial GM plant events and their limits of detection were also determined, which are 10 haploid genome equivalents (HGE) for FMV35S promoter, cry1Ac, and pat assays, as well as five HGE for CaMV35S promoter, bar, NOS terminator, CP4 epsps, and NptII assays. The screening applicability of these LAMP assays was further validated successfully using practical canola, soybean, and maize samples. The results suggested that the established visual LAMP assays are applicable and cost-effective for GM screening in plant-derived food samples.

  4. A mechanism of gene amplification driven by small DNA fragments.

    Directory of Open Access Journals (Sweden)

    Kuntal Mukherjee

    Full Text Available DNA amplification is a molecular process that increases the copy number of a chromosomal tract and often causes elevated expression of the amplified gene(s. Although gene amplification is frequently observed in cancer and other degenerative disorders, the molecular mechanisms involved in the process of DNA copy number increase remain largely unknown. We hypothesized that small DNA fragments could be the trigger of DNA amplification events. Following our findings that small fragments of DNA in the form of DNA oligonucleotides can be highly recombinogenic, we have developed a system in the yeast Saccharomyces cerevisiae to capture events of chromosomal DNA amplification initiated by small DNA fragments. Here we demonstrate that small DNAs can amplify a chromosomal region, generating either tandem duplications or acentric extrachromosomal DNA circles. Small fragment-driven DNA amplification (SFDA occurs with a frequency that increases with the length of homology between the small DNAs and the target chromosomal regions. SFDA events are triggered even by small single-stranded molecules with as little as 20-nt homology with the genomic target. A double-strand break (DSB external to the chromosomal amplicon region stimulates the amplification event up to a factor of 20 and favors formation of extrachromosomal circles. SFDA is dependent on Rad52 and Rad59, partially dependent on Rad1, Rad10, and Pol32, and independent of Rad51, suggesting a single-strand annealing mechanism. Our results reveal a novel molecular model for gene amplification, in which small DNA fragments drive DNA amplification and define the boundaries of the amplicon region. As DNA fragments are frequently found both inside cells and in the extracellular environment, such as the serum of patients with cancer or other degenerative disorders, we propose that SFDA may be a common mechanism for DNA amplification in cancer cells, as well as a more general cause of DNA copy number variation

  5. Complementary weak-value amplification with concatenated postselections

    CERN Document Server

    Viza, Gerardo I; Liu, Wei-Tao; Howell, John C

    2016-01-01

    We measure a transverse momentum kick in a Sagnac interferometer using weak-value amplification with two postselections. The first postselection is controlled by a polarization dependent phase mismatch between both paths of a Sagnac interferometer and the second postselection is controlled by a polarizer at the exit port. By monitoring the darkport of the interferometer, we study the complementary amplification of the concatenated postselections, where the polarization extinction ratio is greater than the contrast of the spatial interference. In this case, we find an improvement in the amplification of the signal of interest by introducing a second postselection to the system.

  6. Limits for superfocusing with finite evanescent wave amplification

    CERN Document Server

    Gordon, Reuven

    2011-01-01

    Perfect lensing using negative refractive index materials and radiationless electromagnetic interference both provide extreme subwavelength focusing by "amplifying" evanescent wave components that are usually lost. This paper provides a relation between the achievable focus spot size, the amplification available and the focal length. This may be considered as a revised version of Abbe's diffraction limit for focusing systems that have evanescent wave amplification. It is useful in comparing the amplification achieved in various subwavelength focusing implementations, as well as determining when it is better to use existing near-field techniques, such as simple diffraction from an aperture or slit, than to attempt complicated superfocusing.

  7. Fluorescence amplification by electrochemically deposited silver nanowires with fractal architecture.

    Science.gov (United States)

    Goldys, Ewa M; Drozdowicz-Tomsia, Krystyna; Xie, Fang; Shtoyko, Tanya; Matveeva, Eva; Gryczynski, Ignacy; Gryczynski, Zygmunt

    2007-10-10

    Electrochemically deposited silver structures with nanowires 50-100 nm in diameter show high fluorescence amplification and strongly reduced fluorescence lifetimes. Both quantities depend on the structure thickness. With increasing thickness the fluorescence amplification proportionally increases and the fluorescence lifetime decreases. This thickness dependence is caused by fluorophore interaction with a system of plasmon excitations in coupled nanowires extending over micrometer size regions. Thus the amplification is attributed to a combination of extended structure area and strong plasmonic coupling between nanowires which also help to radiatively scatter the fluorescence emission.

  8. Backward Raman amplification in the long-wavelength infrared

    Science.gov (United States)

    Johnson, L. A.; Gordon, D. F.; Palastro, J. P.; Hafizi, B.

    2017-03-01

    The wealth of work in backward Raman amplification in plasma has focused on the extreme intensity limit; however, backward Raman amplification may also provide an effective and practical mechanism for generating intense, broad bandwidth, long-wavelength infrared radiation (LWIR). An electromagnetic simulation coupled with a relativistic cold fluid plasma model is used to demonstrate the generation of picosecond pulses at a wavelength of 10 μm with terawatt powers through backward Raman amplification. The effects of collisional damping, Landau damping, pump depletion, and wave breaking are examined, as well as the resulting design considerations for an LWIR Raman amplifier.

  9. Local Runup Amplification By Resonant Wave Interactions

    CERN Document Server

    Stefanakis, Themistoklis; Dutykh, Denys

    2011-01-01

    Until now the analysis of long wave runup on a plane beach has been focused on finding its maximum value, failing to capture the existence of resonant regimes. One-dimensional numerical simulations in the framework of the Nonlinear Shallow Water Equations (NSWE) are used to investigate the Boundary Value Problem (BVP) for plane and non-trivial beaches. Monochromatic waves, as well as virtual wave-gage recordings from real tsunami simulations, are used as forcing conditions to the BVP. Resonant phenomena between the incident wavelength and the beach slope are found to occur, which result in enhanced runup of non-leading waves. The evolution of energy reveals the existence of a quasi-periodic state for the case of sinusoidal waves, the energy level of which, as well as the time required to reach that state, depend on the incident wavelength for a given beach slope. Dispersion is found to slightly reduce the value of maximum runup, but not to change the overall picture. Runup amplification occurs for both leadin...

  10. Protein misfolding cyclic amplification of infectious prions.

    Science.gov (United States)

    Morales, Rodrigo; Duran-Aniotz, Claudia; Diaz-Espinoza, Rodrigo; Camacho, Manuel V; Soto, Claudio

    2012-06-28

    Prions are proteinaceous infectious agents responsible for the transmission of prion diseases. The lack of a procedure for cultivating prions in the laboratory has been a major limitation to the study of the unorthodox nature of this infectious agent and the molecular mechanism by which the normal prion protein (PrP(C)) is converted into the abnormal isoform (PrP(Sc)). Protein misfolding cyclic amplification (PMCA), described in detail in this protocol, is a simple, fast and efficient methodology to mimic prion replication in the test tube. PMCA involves incubating materials containing minute amounts of infectious prions with an excess of PrP(C) and boosting the conversion by cycles of sonication to fragment the converting units, thereby leading to accelerated prion replication. PMCA is able to detect the equivalent of a single molecule of infectious PrP(Sc) and propagate prions that maintain high infectivity, strain properties and species specificity. A single PMCA assay takes little more than 3 d to replicate a large amount of prions, which could take years in an in vivo situation. Since its invention 10 years ago, PMCA has helped to answer fundamental questions about this intriguing infectious agent and has been broadly applied in research areas that include the food industry, blood bank safety and human and veterinary disease diagnosis.

  11. A PCR amplification method without DNA extraction.

    Science.gov (United States)

    Li, Hongwei; Xu, Haiyue; Zhao, Chunjiang; Sulaiman, Yiming; Wu, Changxin

    2011-02-01

    To develop a simple and inexpensive method for direct PCR amplification of animal DNA from tissues, we optimized different components and their concentration in lysis buffer systems. Finally, we acquired the optimized buffer system composed of 10 mmol tris(hydroxymethyl)aminomethane (Tris)-Cl (pH 8.0), 2 mmol ethylene diamine tetraacetic (EDTA) (pH 8.0), 0.2 mol NaCl and 200 μg/mL Proteinase K. Interestingly, the optimized buffer is also very effective when working with common human sample types, including blood, buccal cells and hair. The direct PCR method requires fewer reagents (Tris-Cl, EDTA, Protease K and NaCl) and less incubation time (only 35 min). The cost of treating every sample is less than $0.02, and all steps can be completed on a thermal cycler in a 96-well format. So, the proposed method will significantly improve high-throughput PCR-based molecular assays in animal systems and in common human sample types.

  12. Kinematic amplification strategies in plants and engineering

    Science.gov (United States)

    Charpentier, Victor; Hannequart, Philippe; Adriaenssens, Sigrid; Baverel, Olivier; Viglino, Emmanuel; Eisenman, Sasha

    2017-06-01

    While plants are primarily sessile at the organismal level, they do exhibit a vast array of movements at the organ or sub-organ level. These movements can occur for reasons as diverse as seed dispersal, nutrition, protection or pollination. Their advanced mechanisms generate a myriad of movement typologies, many of which are not fully understood. In recent years, there has been a renewal of interest in understanding the mechanical behavior of plants from an engineering perspective, with an interest in developing novel applications by up-sizing these mechanisms from the micro- to the macro-scale. This literature review identifies the main strategies used by plants to create and amplify movements and anatomize the most recent mechanical understanding of compliant engineering mechanics. The paper ultimately demonstrates that plant movements, rooted in compliance and multi-functionality, can effectively inspire better kinematic/adaptive structures and materials. In plants, the actuators and the deployment structures are fused into a single system. The understanding of those natural movements therefore starts with an exploration of mechanisms at the origins of movements. Plant movements, whether slow or fast, active or passive, reversible or irreversible, are presented and detailed for their mechanical significance. With a focus on displacement amplification, the most recent promising strategies for actuation and adaptive systems are examined with respect to the mechanical principles of shape morphing plant tissues.

  13. KASER: Knowledge Amplification by Structured Expert Randomization.

    Science.gov (United States)

    Rubin, Stuart H; Murthy, S N Jayaram; Smith, Michael H; Trajković, Ljiljana

    2004-12-01

    In this paper and attached video, we present a third-generation expert system named Knowledge Amplification by Structured Expert Randomization (KASER) for which a patent has been filed by the U.S. Navy's SPAWAR Systems Center, San Diego, CA (SSC SD). KASER is a creative expert system. It is capable of deductive, inductive, and mixed derivations. Its qualitative creativity is realized by using a tree-search mechanism. The system achieves creative reasoning by using a declarative representation of knowledge consisting of object trees and inheritance. KASER computes with words and phrases. It possesses a capability for metaphor-based explanations. This capability is useful in explaining its creative suggestions and serves to augment the capabilities provided by the explanation subsystems of conventional expert systems. KASER also exhibits an accelerated capability to learn. However, this capability depends on the particulars of the selected application domain. For example, application domains such as the game of chess exhibit a high degree of geometric symmetry. Conversely, application domains such as the game of craps played with two dice exhibit no predictable pattern, unless the dice are loaded. More generally, we say that domains whose informative content can be compressed to a significant degree without loss (or with relatively little loss) are symmetric. Incompressible domains are said to be asymmetric or random. The measure of symmetry plus the measure of randomness must always sum to unity.

  14. Small Sample Whole-Genome Amplification

    Energy Technology Data Exchange (ETDEWEB)

    Hara, C A; Nguyen, C P; Wheeler, E K; Sorensen, K J; Arroyo, E S; Vrankovich, G P; Christian, A T

    2005-09-20

    Many challenges arise when trying to amplify and analyze human samples collected in the field due to limitations in sample quantity, and contamination of the starting material. Tests such as DNA fingerprinting and mitochondrial typing require a certain sample size and are carried out in large volume reactions; in cases where insufficient sample is present whole genome amplification (WGA) can be used. WGA allows very small quantities of DNA to be amplified in a way that enables subsequent DNA-based tests to be performed. A limiting step to WGA is sample preparation. To minimize the necessary sample size, we have developed two modifications of WGA: the first allows for an increase in amplified product from small, nanoscale, purified samples with the use of carrier DNA while the second is a single-step method for cleaning and amplifying samples all in one column. Conventional DNA cleanup involves binding the DNA to silica, washing away impurities, and then releasing the DNA for subsequent testing. We have eliminated losses associated with incomplete sample release, thereby decreasing the required amount of starting template for DNA testing. Both techniques address the limitations of sample size by providing ample copies of genomic samples. Carrier DNA, included in our WGA reactions, can be used when amplifying samples with the standard purification method, or can be used in conjunction with our single-step DNA purification technique to potentially further decrease the amount of starting sample necessary for future forensic DNA-based assays.

  15. A novel modification of real-time AS-qPCR by using locked nucleic acid-modified oligonucleotide probe as wild type allele amplification blockers for quantitative detection of the JAK2 V617F mutation%评价AS-LNA-qPCR法检测JAK2 V617F突变率的临床应用价值

    Institute of Scientific and Technical Information of China (English)

    邵冬华; 梁国威; 何美琳; 曹清芸

    2013-01-01

    Objective To develop a novel real-time PCR for sensitively quantitative detection of JAK2 V617F allele burden in peripheral blood.Methods Based on the real-time allele-specific PCR (AS-qPCR),the locked nucleic acid (LNA)-modified oligonucleotide probe was used for selectively blocking amplification of wild-type alleles in AS-qPCR,and then a novel AS-LNA-qPCR method was established.The percentages of sample JAK2 V617F alleles were directly calculated by its threshold cycle (Ct) values according to the standard curve which generated by JAK2 V617F alleles with its Ct values.We validated intra-and inter-assay variability for quantifying JAK2 V617F.We also assayed 623 apparent healthy donors by our method to validate its clinical application value.Results The quantitative lower limit of this method for JAK2 V617F was 0.01%,and the intra-and inter-assay average variability for quantifying percentage of JAK2 V617F in total DNA was 6.3% and 8.6%,respectively.Nineteen JAK2 V617F-positive individuals were identified using AS-LNA-qPCR in blood of 623 apparently healthy donors,and the range of percentages of JAK2 V617F alleles were 0.01%-5.49%.Conclusion The AS-LNA-qPCR with highly sensitive and reproducible quantification of JAK2 V617F mutant burden can be used clinically for diagnosis as well as evaluation of disease prognosis and efficacy of therapy in patients with myeloproliferative neoplasms.%目的 建立一种定量检测外周血细胞酪氨酸激酶2(JAK2)基因V617F突变率的等位基因特异性实时荧光定量PCR(AS-qPCR)方法.方法 在AS-qPCR基础上,引入1条锁核酸(LNA)修饰的寡核苷酸探针,用以选择性抑制AS-qPCR中突变引物对野生等位基因的非特异性扩增,定量检测JAK2 V617F突变率,称之为AS-LNA-qPCR法.通过AS-LNA-qPCR法测定样本的循环阈值(Ct值),根据AS-LNA-qPCR法检测不同JAK2 V617F突变率标准品的Ct值,绘制标准曲线,根据标准曲线直接获得检测样本中JAK2 V617F突变率.

  16. 一步核酸扩增仪术中诊断乳腺癌前哨淋巴结转移的前瞻性、多中心临床观察%Prospective multi-center study of one-step nucleic acid amplification assay for the detection of sentinel lymph nodes metastases in breast cancer patients

    Institute of Scientific and Technical Information of China (English)

    王永胜; 欧阳涛; 吴炅; 刘艳辉; 曹旭晨; 孙晓; 付丽; 廖宁; 杨文涛

    2013-01-01

    Objective To evaluate the roles of Sysmex RD100i one-step nucleic acid amplification (OSNA) assay in the intraoperative assessments of breast cancer sentinel lymph nodes (SLNs).Methods A total of 552 consecutive prospective patients were enrolled from five centers nationwide from February to December 2010.And SLNs were sliced into alternating 2 mm blocks.The odd blocks were tested by the OSNA assay intraoperatively and the even ones assessed by postoperative histology.In addition,intraoperative histological assessments were performed on the even blocks of 211 patients by frozen section and all blocks by touch imprint cytology.Results A total of 1188 SLNs were removed.The mean turnaround time of the assay was 37.3 min.Thcrc was no significant difference of turnaround time at each center (P =0.074).As compared to postoperative histology,the overall performance of the assay had an accuracy of 91.4% (1086/1188),a sensitivity of 83.7% (159/190) and a specificity of 92.9% (927/998).The sensitivity of the assay was higher than frozen section (77.6% (59/76) vs 69.7% (53/76),P =0.286) and was significantly higher than touch imprint cytology (83.6% (158/189) vs 76.2% (144/189),P =0.044).For nodes with micro-metastases,the sensitivity of the assay was higher than frozen section (8/17 vs 4/17,P =0.289) and was significantly higher than touch imprint cytology (62.5% (30/48) vs 35.4% (17/48),P = 0.007).Conclusion As an accurate and rapid intraoperative assay for assessing breast SLNs,the OSNA assay may replace frozen section and touch imprint cytology for clinical applications.%目的 评估应用一步核酸扩增(OSNA)技术进行乳腺癌前哨淋巴结(SLN)术中检测诊断的价值.方法 共入组2010年2至12月全国5个乳腺病中心的552例原发性乳腺癌患者,术中将SLN根据其质量及短轴长度进行切分后,行OSNA检测和印片细胞学检测,术后行逐层切片病理检测.另外,211例患者术中同时行快速冰

  17. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis.

    Science.gov (United States)

    Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K; Klapperich, Catherine M

    2013-01-01

    In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10(-2) pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

  18. In vitro amplification of ovine prions from scrapie-infected sheep from Great Britain reveals distinct patterns of propagation

    Directory of Open Access Journals (Sweden)

    Thorne Leigh

    2012-11-01

    Full Text Available Abstract Background Protein misfolding cyclic amplification (PMCA is a method that facilitates the detection of prions from many sources of transmissible spongiform encephalopathy (TSE. Sheep scrapie represents a unique diversity of prion disease agents in a range of susceptible PRNP genotypes. In this study PMCA was assessed on a range of Great Britain (GB sheep scrapie isolates to determine the applicability to veterinary diagnosis of ovine TSE. Results PrPSc amplification by protein misfolding cyclic amplification (PMCA was assessed as a diagnostic tool for field cases of scrapie. The technique was initially applied to thirty-seven isolates of scrapie from diverse geographical locations around GB, and involved sheep of various breeds and PRNP genotypes. All samples were amplified in either VRQ and/or ARQ PrPC substrate. For PrPSc from sheep with at least one VRQ allele, all samples amplified efficiently in VRQ PrPC but only PrPSc from ARH/VRQ sheep amplified in both substrates. PrPSc from ARQ/ARQ sheep displayed two amplification patterns, one that amplified in both substrates and one that only amplified in ARQ PrPC. These amplification patterns were consistent for a further 14/15 flock/farm mates of these sheep. Furthermore experimental scrapie strains SSBP1, Dawson, CH1641 and MRI were analysed. SSBP1 and Dawson (from VRQ/VRQ sheep amplified in VRQ but not ARQ substrate. MRI scrapie (from ARQ/ARQ sheep nor CH1641 did not amplify in ARQ or VRQ substrate; these strains required an enhanced PMCA method incorporating polyadenylic acid (poly(A to achieve amplification. Conclusions PrPsc from 52 classical scrapie GB field isolates amplified in VRQ or ARQ or both substrates and supports the use of PMCA as a rapid assay for the detection of a wide range of ovine classical scrapie infections involving multiple PRNP genotypes and scrapie strains.

  19. Quantum dot layer-by-layer assemblies as signal amplification labels for ultrasensitive electronic detection of uropathogens.

    Science.gov (United States)

    Xiang, Yun; Zhang, Haixia; Jiang, Bingying; Chai, Yaqin; Yuan, Ruo

    2011-06-01

    The preparation and use of a new class of signal amplification label, quantum dot (QD) layer-by-layer (LBL) assembled polystyrene microsphere composite, for amplified ultrasensitive electronic detection of uropathogen-specific DNA sequences is described. The target DNA is sandwiched between the capture probes immobilized on the magnetic beads and the signaling probes conjugated to the QD LBL assembled polystyrene beads. Because of the dramatic signal amplification by the numerous QDs involved in each single DNA binding event, subfemtomolar level detection of uropathogen-specific DNA sequences is achieved, which makes our strategy among the most sensitive electronic approach for nucleic acid-based monitoring of pathogens. Our signal amplified detection scheme could be readily expanded to monitor other important biomolecules (e.g., proteins, peptides, amino acids, cells, etc.) in ultralow levels and thus holds great potential for early diagnosis of disease biomarkers.

  20. The Amplification in FEL with Inhomogeneous Magnetic Field

    CERN Document Server

    Oganesyan, K B

    2016-01-01

    The gain in a plane wiggler with inhomogeneous magnetic field is calculated.. It is shown, that the account of inhomogenity of the magnetic field leads to appearance of additional peaks in the amplification

  1. Amplification options for patients with mixed hearing loss.

    NARCIS (Netherlands)

    Zwartenkot, J.W.; Snik, A.F.M.; Mylanus, E.A.M.; Mulder, J.J.S.

    2014-01-01

    OBJECTIVES: To compare amplification options for patients with mixed hearing loss. Devices tested include percutaneous and transcutaneous bone conductors (BCDs) and middle ear implants with their actuator directly coupled to the cochlea. SETTING: Tertiary academic medical center. METHOD AND PARTICIP

  2. Nonlinear Zel'dovich effect: Parametric amplification from medium rotation

    CERN Document Server

    Faccio, Daniele

    2016-01-01

    The interaction of light with rotating media has attracted recent interest for both fundamental and applied studies including rotational Doppler shift measurements. It is also possible to obtain amplification through the scattering of light with orbital angular momentum from a rotating and absorbing cylinder, as proposed by Zel'dovich more than 40 years ago. This amplification mechanism has never been observed experimentally yet has connections to other fields such as Penrose superradiance in rotating black holes. Here we propose a nonlinear optics system whereby incident light carrying orbital angular momentum drives parametric interaction in a rotating medium. The crystal rotation is shown to take the phase-mismatched parametric interaction with negligible energy exchange at zero rotation to amplification for sufficiently large rotation rates. The amplification is shown to result from breaking of anti-PT symmetry induced by the medium rotation.

  3. Amplification and flagellin typing of pseudomonas aeruginosa by molecular method

    Directory of Open Access Journals (Sweden)

    gholamreza goudarzi

    2011-08-01

    Conclusion: This method could be utilized to determine flagellated (Motile and non-flagellated strains of P. aeruginosa, genotyping, amplification of full coding sequence of fliC gene in order to clone and express recombinant flagellin protein.

  4. Whole genome amplification: Use of advanced isothermal method

    African Journals Online (AJOL)

    Yomi

    2010-12-29

    Dec 29, 2010 ... 5Department of Animal Science, College of Agriculture & Natural Resources, University of Tehran, Karaj ..... All of these factors produce nonspecific amplification ... PCRs on the same WGA yield, allelic dropout is not locus-.

  5. Studies of nondegenerate, quasi-phase-matched optical parametric amplification

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence Livermore National Laboratory

    2004-03-18

    We have performed extensive numerical studies of quasi-phase-matched optical parametric amplification with the aim to improve its nondegenerate spectral bandwidth. Our multi-section fan-out design calculations indicate a 35-fold increase in spectral bandwidth.

  6. Nonlinear Zel'dovich Effect: Parametric Amplification from Medium Rotation

    Science.gov (United States)

    Faccio, Daniele; Wright, Ewan M.

    2017-03-01

    The interaction of light with rotating media has attracted recent interest for both fundamental and applied studies including rotational Doppler shift measurements. It is also possible to obtain amplification through the scattering of light with orbital angular momentum from a rotating and absorbing cylinder, as proposed by Zel'dovich more than forty years ago. This amplification mechanism has never been observed experimentally yet has connections to other fields such as Penrose superradiance in rotating black holes. Here we propose a nonlinear optics system whereby incident light carrying orbital angular momentum drives parametric interaction in a rotating medium. The crystal rotation is shown to take the phase-mismatched parametric interaction with negligible energy exchange at zero rotation to amplification for sufficiently large rotation rates. The amplification is shown to result from breaking of anti-P T symmetry induced by the medium rotation.

  7. Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.

    Science.gov (United States)

    Rodriguez-Manzano, Jesus; Karymov, Mikhail A; Begolo, Stefano; Selck, David A; Zhukov, Dmitriy V; Jue, Erik; Ismagilov, Rustem F

    2016-03-22

    Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests.

  8. Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.

    Science.gov (United States)

    Moura-Melo, Suely; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Dos Santos Junior, J Ribeiro; da Silva Fonseca, Rosana A; Lobo-Castañón, Maria Jesús

    2015-08-18

    Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas.

  9. Pulse Distortion in Saturated Fiber Optical Parametric Chirped Pulse Amplification

    DEFF Research Database (Denmark)

    Lali-Dastjerdi, Zohreh; Da Ros, Francesco; Rottwitt, Karsten

    2012-01-01

    Fiber optical parametric chirped pulse amplification is experimentally compared for different chirped pulses in the picosecond regime. The amplified chirped pulses show distortion appearing as pedestals after recompression when the amplifier is operated in saturation.......Fiber optical parametric chirped pulse amplification is experimentally compared for different chirped pulses in the picosecond regime. The amplified chirped pulses show distortion appearing as pedestals after recompression when the amplifier is operated in saturation....

  10. Engineering targeted chromosomal amplifications in human breast epithelial cells.

    Science.gov (United States)

    Springer, Simeon; Yi, Kyung H; Park, Jeenah; Rajpurohit, Anandita; Price, Amanda J; Lauring, Josh

    2015-07-01

    Chromosomal amplifications are among the most common genetic alterations found in human cancers. However, experimental systems to study the processes that lead to specific, recurrent amplification events in human cancers are lacking. Moreover, some common amplifications, such as that at 8p11-12 in breast cancer, harbor multiple driver oncogenes, which are poorly modeled by conventional overexpression approaches. We sought to develop an experimental system to model recurrent chromosomal amplification events in human cell lines. Our strategy is to use homologous-recombination-mediated gene targeting to deliver a dominantly selectable, amplifiable marker to a specified chromosomal location. We used adeno-associated virus vectors to target human MCF-7 breast cancer cells at the ZNF703 locus, in the recurrent 8p11-12 amplicon, using the E. coli inosine monophosphate dehydrogenase (IMPDH) enzyme as a marker. We applied selective pressure using IMPDH inhibitors. Surviving clones were found to have increased copy number of ZNF703 (average 2.5-fold increase) by droplet digital PCR and FISH. Genome-wide array comparative genomic hybridization confirmed that amplifications had occurred on the short arm of chromosome 8, without changes on 8q or other chromosomes. Patterns of amplification were variable and similar to those seen in primary human breast cancers, including "sawtooth" patterns, distal copy number loss, and large continuous regions of copy number gain. This system will allow study of the cis- and trans-acting factors that are permissive for chromosomal amplification and provide a model to analyze oncogene cooperativity in amplifications harboring multiple candidate driver genes.

  11. Aerosol Lidar for the Relative Backscatter Amplification Measurements

    Science.gov (United States)

    Razenkov, Igor A.; Banakh, Victor A.; Nadeev, Alexander I.

    2016-06-01

    Backscatter amplification presents only in a turbulent atmosphere, when the laser beam is propagates twice through the same inhomogeneities. We proposed technical solution to detect backscatter amplification. An aerosol micro pulse lidar with a beam expansion via receiving telescope was built to study this effect. Our system allows simultaneous detection of two returns from the same scattering volume: exactly on the axis of the laser beam and off the axis.

  12. Methods for microbial DNA extraction from soil for PCR amplification

    OpenAIRE

    Yeates C; Gillings, MR; Davison AD; Altavilla N; Veal DA

    1998-01-01

    Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1). DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol pr...

  13. The emergence of surface-based Arctic amplification

    Directory of Open Access Journals (Sweden)

    M. C. Serreze

    2008-07-01

    Full Text Available Rises in surface and lower troposphere air temperatures through the 21st century are projected to be especially pronounced over the Arctic Ocean during the cold season. This Arctic amplification is largely driven by loss of the sea ice cover, allowing for strong heat transfers from the ocean to the atmosphere. Consistent with observed reductions in sea ice extent, fields from the NCEP/NCAR reanalysis suggest emergence of surface-based Arctic amplification in the last decade.

  14. Controllable Amplification of Entanglement for Two Qutrits under Decoherence

    Institute of Scientific and Technical Information of China (English)

    ZHENG Qiang; XIE Xiao-Yao; ZHI Qi-Jun; REN Zhong-Zhou

    2011-01-01

    Entanglement dynamics of a two-qutrit Heisenberg spin chain with the external magnetic fields and DM interaction under the intrinsic decoherence is investigated. Depending on whether there is inhomogeneous magnetic field,the entanglement amplification, i.e. the phenomenon that the finally stable entanglement is bigger than that of the initial one, is found for one kind of initial states. The reasons for the controllable entanglement amplification are discussed.

  15. The emergence of surface-based Arctic amplification

    OpenAIRE

    SERREZE, M. C.; A. P. Barrett; J. C. Stroeve; Kindig, D. N.; Holland, M. M.

    2009-01-01

    Rises in surface and lower troposphere air temperatures through the 21st century are projected to be especially pronounced over the Arctic Ocean during the cold season. This Arctic amplification is largely driven by loss of the sea ice cover, allowing for strong heat transfers from the ocean to the atmosphere. Consistent with observed reductions in sea ice extent, fields from both the NCEP/NCAR and JRA-25 reanalyses point to emergence of surface-based Arctic amplification in the last decade.

  16. Rapid ultrasonic isothermal amplification of DNA with multiplexed melting analysis – applications in the clinical diagnosis of sexually transmitted diseases.

    Science.gov (United States)

    Xu, Gaolian; Gunson, Rory N; Cooper, Jonathan M; Reboud, Julien

    2015-02-14

    We describe a nucleic acid testing (NAT) platform for infectious disease diagnostics at the point-of-care, using surface acoustic waves (SAW) to perform a multiplexed loop-mediated isothermal amplification (LAMP) test for sexually transmitted diseases. The ultrasonic actuation not only enables faster NAT reactions but also provides a route towards integrating low-cost, low-power molecular diagnostics into disposable sensors.

  17. The application of loop-mediated isothermal amplification for detection of common pathogenic bacteria in lower respiratory tract infections

    Institute of Scientific and Technical Information of China (English)

    陈愉生

    2014-01-01

    Objective To investigate the spectrum of common pathogenic bacteria of low respiratory tract infection by loop-mediated isothermal amplification(LAMP)of nucleic acid test and to prove the clinical significance of this method.Methods A total of 289 qualified sputum samples from patients with lower respiratory tract infections in Fujian Province were detected by LAMP technique,and then the distribution of pathogenic bacteria was analyzed.The positive cases(the patients whose specific3

  18. Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

    Science.gov (United States)

    Ullrich, Thomas; Ermantraut, Eugen; Schulz, Torsten; Steinmetzer, Katrin

    2012-01-01

    Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a

  19. Competitive reporter monitored amplification (CMA--quantification of molecular targets by real time monitoring of competitive reporter hybridization.

    Directory of Open Access Journals (Sweden)

    Thomas Ullrich

    Full Text Available BACKGROUND: State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. METHODOLOGY AND PRINCIPAL FINDINGS: The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. CONCLUSIONS AND SIGNIFICANCE: The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2, we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls

  20. Human identification from forensic materials by amplification of a human-specific sequence in the myoglobin gene.

    OpenAIRE

    Ono T; Miyaishi S; Yamamoto Y; Yoshitome K; Ishikawa T.; Ishizu H

    2001-01-01

    We developed a method for human identification of forensic biological materials by PCR-based detection of a human-specific sequence in exon 3 of the myoglobin gene. This human-specific DNA sequence was deduced from differences in the amino acid sequences of myoglobins between humans and other animal species. The new method enabled amplification of the target DNA fragment from 30 samples of human DNA, and the amplified sequences were identical with that already reported. Using this method, we ...

  1. Targeting MET Amplification as a New Oncogenic Driver

    Energy Technology Data Exchange (ETDEWEB)

    Kawakami, Hisato [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Okamoto, Isamu, E-mail: okamotoi@kokyu.med.kyushu-u.ac.jp [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Center for Clinical and Translational Research, Kyushu University Hospital, 3-1-1 Maidashi, Higashiku, Fukuoka 812-8582 (Japan); Okamoto, Wataru [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Division of Transrlational Research, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, 6-5-1 Kashiwanoha, Kashiwa, Chiba 277-8577 (Japan); Tanizaki, Junko [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, HIM223, 450 Brookline Avenue, Boston, MA 02215 (United States); Nakagawa, Kazuhiko [Department of Medical Oncology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan); Nishio, Kazuto [Department of Genome Biology, Kinki University Faculty of Medicine, 377-2 Ohno-higashi, Osaka-Sayama, Osaka 589-8511 (Japan)

    2014-07-22

    Certain genetically defined cancers are dependent on a single overactive oncogene for their proliferation and survival, a phenomenon known as “oncogene addiction”. A new generation of drugs that selectively target such “driver oncogenes” manifests a clinical efficacy greater than that of conventional chemotherapy in appropriate genetically defined patients. MET is a proto-oncogene that encodes a receptor tyrosine kinase, and aberrant activation of MET signaling occurs in a subset of advanced cancers as result of various genetic alterations including gene amplification, polysomy, and gene mutation. Our preclinical studies have shown that inhibition of MET signaling either with the small-molecule MET inhibitor crizotinib or by RNA interference targeted to MET mRNA resulted in marked antitumor effects in cancer cell lines with MET amplification both in vitro and in vivo. Furthermore, patients with non-small cell lung cancer or gastric cancer positive for MET amplification have shown a pronounced clinical response to crizotinib. Accumulating preclinical and clinical evidence thus suggests that MET amplification is an “oncogenic driver” and therefore a valid target for treatment. However, the prevalence of MET amplification has not been fully determined, possibly in part because of the difficulty in evaluating gene amplification. In this review, we provide a rationale for targeting this genetic alteration in cancer therapy.

  2. Theory of phase-mixing amplification in an optomechanical system

    Science.gov (United States)

    Ockeloen-Korppi, C. F.; Heikkilä, T. T.; Sillanpää, M. A.; Massel, F.

    2017-09-01

    The investigation of the ultimate limits imposed by quantum mechanics on amplification represents an important topic both on a fundamental level and from the perspective of potential applications. We discuss here a novel regime for bosonic linear amplifiers—beside phase-insensitive and phase-sensitive amplification—which we term here phase-mixing amplification. Furthermore, we show that phase-mixing amplification can be realised in a cavity optomechanical setup, constituted by a mechanical resonator which is dispersively coupled to an optomechanical cavity asymmetrically driven around both mechanical sidebands. While, in general, this amplifier is phase-mixing, for a suitable choice of parameters, the amplifier proposed here operates as a phase-sensitive amplifier. We show that both configurations allow amplification with an added noise below the quantum limit of (phase-insensitive) amplification in a parameter range compatible with current experiments in microwave circuit optomechanics. In particular, we show that introducing phase-mixing amplification typically allows for a significant reduction of the added noise.

  3. Targeting MET Amplification as a New Oncogenic Driver

    Directory of Open Access Journals (Sweden)

    Hisato Kawakami

    2014-07-01

    Full Text Available Certain genetically defined cancers are dependent on a single overactive oncogene for their proliferation and survival, a phenomenon known as “oncogene addiction”. A new generation of drugs that selectively target such “driver oncogenes” manifests a clinical efficacy greater than that of conventional chemotherapy in appropriate genetically defined patients. MET is a proto-oncogene that encodes a receptor tyrosine kinase, and aberrant activation of MET signaling occurs in a subset of advanced cancers as result of various genetic alterations including gene amplification, polysomy, and gene mutation. Our preclinical studies have shown that inhibition of MET signaling either with the small-molecule MET inhibitor crizotinib or by RNA interference targeted to MET mRNA resulted in marked antitumor effects in cancer cell lines with MET amplification both in vitro and in vivo. Furthermore, patients with non-small cell lung cancer or gastric cancer positive for MET amplification have shown a pronounced clinical response to crizotinib. Accumulating preclinical and clinical evidence thus suggests that MET amplification is an “oncogenic driver” and therefore a valid target for treatment. However, the prevalence of MET amplification has not been fully determined, possibly in part because of the difficulty in evaluating gene amplification. In this review, we provide a rationale for targeting this genetic alteration in cancer therapy.

  4. Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications.

    Science.gov (United States)

    Du, Yi-Chen; Zhu, Li-Na; Kong, De-Ming

    2016-12-15

    To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10(-5)U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design.

  5. Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens.

    Science.gov (United States)

    Kersting, Sebastian; Rausch, Valentina; Bier, Frank F; von Nickisch-Rosenegk, Markus

    2014-01-01

    We report on the development of an on-chip RPA (recombinase polymerase amplification) with simultaneous multiplex isothermal amplification and detection on a solid surface. The isothermal RPA was applied to amplify specific target sequences from the pathogens Neisseria gonorrhoeae, Salmonella enterica and methicillin-resistant Staphylococcus aureus (MRSA) using genomic DNA. Additionally, a positive plasmid control was established as an internal control. The four targets were amplified simultaneously in a quadruplex reaction. The amplicon is labeled during on-chip RPA by reverse oligonucleotide primers coupled to a fluorophore. Both amplification and spatially resolved signal generation take place on immobilized forward primers bount to expoxy-silanized glass surfaces in a pump-driven hybridization chamber. The combination of microarray technology and sensitive isothermal nucleic acid amplification at 38 °C allows for a multiparameter analysis on a rather small area. The on-chip RPA was characterized in terms of reaction time, sensitivity and inhibitory conditions. A successful enzymatic reaction is completed in isothermal nucleic acid amplification with RPA and spatially-resolved signal generation on specific immobilized oligonucleotides.

  6. On-chip electrical detection of parallel loop-mediated isothermal amplification with DG-BioFETs for the detection of foodborne bacterial pathogens

    Science.gov (United States)

    The use of field effect transistors (FETs) as the transduction element for the detection of DNA amplification reactions will enable portable and inexpensive nucleic acid analysis. Transistors used as biological sensors,or BioFETs, minimize the cost and size of detection platforms by leveraging fabri...

  7. Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay.

    Science.gov (United States)

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-08-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of a quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear correlations between amplified luminescence and initial ATP concentration were observed. When performing four cycles of continuous-flow ATP amplification, the gradient of amplification was 1.87(N). Whereas the lower quantifiable level was 500 pM without amplification, values as low as 50 pM ATP could be measured after amplification. The sensitivity thus increased 10-fold, with further improvements expected with additional amplification cycles. The continuous-flow system thus effectively increased the sensitivity of the quantitative bioluminescence assay.

  8. ASAP: Amplification, sequencing & annotation of plastomes

    Directory of Open Access Journals (Sweden)

    Folta Kevin M

    2005-12-01

    Full Text Available Abstract Background Availability of DNA sequence information is vital for pursuing structural, functional and comparative genomics studies in plastids. Traditionally, the first step in mining the valuable information within a chloroplast genome requires sequencing a chloroplast plasmid library or BAC clones. These activities involve complicated preparatory procedures like chloroplast DNA isolation or identification of the appropriate BAC clones to be sequenced. Rolling circle amplification (RCA is being used currently to amplify the chloroplast genome from purified chloroplast DNA and the resulting products are sheared and cloned prior to sequencing. Herein we present a universal high-throughput, rapid PCR-based technique to amplify, sequence and assemble plastid genome sequence from diverse species in a short time and at reasonable cost from total plant DNA, using the large inverted repeat region from strawberry and peach as proof of concept. The method exploits the highly conserved coding regions or intergenic regions of plastid genes. Using an informatics approach, chloroplast DNA sequence information from 5 available eudicot plastomes was aligned to identify the most conserved regions. Cognate primer pairs were then designed to generate ~1 – 1.2 kb overlapping amplicons from the inverted repeat region in 14 diverse genera. Results 100% coverage of the inverted repeat region was obtained from Arabidopsis, tobacco, orange, strawberry, peach, lettuce, tomato and Amaranthus. Over 80% coverage was obtained from distant species, including Ginkgo, loblolly pine and Equisetum. Sequence from the inverted repeat region of strawberry and peach plastome was obtained, annotated and analyzed. Additionally, a polymorphic region identified from gel electrophoresis was sequenced from tomato and Amaranthus. Sequence analysis revealed large deletions in these species relative to tobacco plastome thus exhibiting the utility of this method for structural and

  9. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification

    Science.gov (United States)

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei

    2016-01-01

    Sensitive and specific methodologies for detection of pathogenic gene at the point-of-care are still urgent demands in rapid diagnosis of infectious diseases. This work develops a simple and pragmatic electrochemical biosensing strategy for ultrasensitive and specific detection of pathogenic nucleic acids directly by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process in single analysis system. The homogeneous recognition and specific binding of target DNA with the designed hairpin probe triggered circular primer extension reaction to form DNA double-strands which contained T7 RNA polymerase promoter and served as templates for in vitro transcription amplification. The HTITA protocol resulted in numerous single-stranded RNA products which could synchronously hybridized with the detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the biosensor surface. The proposed electrochemical biosensing strategy showed very high sensitivity and selectivity for target DNA with a dynamic response range from 1 fM to 100 pM. Using salmonella as a model, the established strategy was successfully applied to directly detect invA gene from genomic DNA extract. This proposed strategy presented a simple, pragmatic platform toward ultrasensitive nucleic acids detection and would become a versatile and powerful tool for point-of-care pathogen identification.

  10. Amplification of thermostable lipase genes fragment from thermogenic phase of domestic waste composting process

    Science.gov (United States)

    Nurhasanah, Nurbaiti, Santi; Madayanti, Fida; Akhmaloka

    2015-09-01

    Lipases are lipolytic enzymes, catalyze the hydrolysis of fatty acid ester bonds of triglycerides to produce free fatty acids and glycerol. The enzyme is widely used in various fields of biotechnological industry. Hence, lipases with unique properties (e.g.thermostable lipase) are still being explored by variation methods. One of the strategy is by using metagenomic approach to amplify the gene directly from environmental sample. This research was focused on amplification of lipase gene fragment directly from the thermogenic phase of domestic waste composting in aerated trenches. We used domestic waste compost from waste treatment at SABUGA, ITB for the sample. Total chromosomal DNA were directly extracted from several stages at thermogenic phase of compost. The DNA was then directly used as a template for amplification of thermostable lipase gene fragments using a set of internal primers namely Flip-1a and Rlip-1a that has been affixed with a GC clamp in reverse primer. The results showed that the primers amplified the gene from four stages of thermogenic phase with the size of lipase gene fragment of approximately 570 base pairs (bp). These results were further used for Denaturing Gradient Gel Electrophoresis (DGGE) analysis to determine diversity of thermostable lipase gene fragments.

  11. Single-use, electricity-free amplification device for detection of HIV-1.

    Science.gov (United States)

    Curtis, Kelly A; Rudolph, Donna L; Morrison, Daphne; Guelig, Dylan; Diesburg, Steven; McAdams, David; Burton, Robert A; LaBarre, Paul; Owen, Michele

    2016-11-01

    Early and accurate diagnosis of HIV is key for the reduction of transmission and initiation of patient care. The availability of a rapid nucleic acid test (NAT) for use at the point-of-care (POC) will fill a gap in HIV diagnostics, improving the diagnosis of acute infection and HIV in infants born to infected mothers. In this study, we evaluated the performance of non-instrumented nucleic acid amplification, single-use disposable (NINA-SUD) devices for the detection of HIV-1 in whole blood using reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) with lyophilized reagents. The NINA-SUD heating device harnesses the heat from an exothermic chemical reaction initiated by the addition of saline to magnesium iron powder. Reproducibility was demonstrated between NINA-SUD units and comparable, if not superior, performance for detecting clinical specimens was observed as compared to the thermal cycler. The stability of the lyophilized HIV-1 RT-LAMP reagents was also demonstrated following storage at -20, 4, 25, and 30°C for up to one month. The single-use, disposable NAT minimizes hands-on time and has the potential to facilitate HIV-1 testing in resource-limited settings or at the POC. Published by Elsevier B.V.

  12. Establishment of recombinase polymerase amplification assay for five hemorrhagic fever-related viruses

    Directory of Open Access Journals (Sweden)

    Xue-feng CAO

    2017-08-01

    Full Text Available Objective To establish a one-step recombinase polymerase amplification (RPA method for pathogen screening and rapid detection in the field targeting for five hemorrhagic fever related viruses (Zaire ebola virus, Sudan ebola virus, Marburg virus, Lassa virus and Yellow fever virus. Methods The specific nucleic acid (NA fragments of each virus were selected as target genes by genome sequence analysis, and the primers and probes for RPA assays were designed according to the sequence. A series of diluted template genes were used for RPA detection to determine the sensitivity. The hemorrhagic fever-related viral nucleic acids were used for RPA detection to determine the specificity. The amplification experiments were carried out at different temperature ranging from 37℃ to 42℃ to validate the reaction temperature range. Results The RPA reaction systems of the five hemorrhagic fever viruses could effectively amplify the target genes, the sensitivities were between 1.5×102 and 1.5×103 copies. No cross reactions existed with the other hemorrhagic fever-related viral genes. Meanwhile, RPA assay could effectively amplify the target genes at 37-42℃. Conclusion The isothermal RPA assays of five hemorrhagic fever viruses are established, which may amply target genes fast and react at a wide temperature range, and be potentially useful for in field pathogens detection. DOI: 10.11855/j.issn.0577-7402.2017.06.09

  13. A continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay

    OpenAIRE

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji

    2008-01-01

    We constructed a novel ATP amplification reactor using a continuous-flow system, and this allowed us to increase the sensitivity of quantitative bioluminescence assay by controlling the number of ATP amplification cycles. We previously developed a bioluminescence assay coupled with ATP amplification using a batch system. However, it was difficult to control the number of amplification cycles. In this study, ATP amplification was performed using a continuous-flow system, and significant linear...

  14. Regulation of ribosomal DNA amplification by the TOR pathway.

    Science.gov (United States)

    Jack, Carmen V; Cruz, Cristina; Hull, Ryan M; Keller, Markus A; Ralser, Markus; Houseley, Jonathan

    2015-08-01

    Repeated regions are widespread in eukaryotic genomes, and key functional elements such as the ribosomal DNA tend to be formed of high copy repeated sequences organized in tandem arrays. In general, high copy repeats are remarkably stable, but a number of organisms display rapid ribosomal DNA amplification at specific times or under specific conditions. Here we demonstrate that target of rapamycin (TOR) signaling stimulates ribosomal DNA amplification in budding yeast, linking external nutrient availability to ribosomal DNA copy number. We show that ribosomal DNA amplification is regulated by three histone deacetylases: Sir2, Hst3, and Hst4. These enzymes control homologous recombination-dependent and nonhomologous recombination-dependent amplification pathways that act in concert to mediate rapid, directional ribosomal DNA copy number change. Amplification is completely repressed by rapamycin, an inhibitor of the nutrient-responsive TOR pathway; this effect is separable from growth rate and is mediated directly through Sir2, Hst3, and Hst4. Caloric restriction is known to up-regulate expression of nicotinamidase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In contrast, normal glucose concentrations stretch the ribosome synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized transcriptional response to caloric excess by reducing PNC1 expression. PNC1 down-regulation forms a key element in the control of ribosomal DNA amplification as overexpression of PNC1 substantially reduces ribosomal DNA amplification rate. Our results reveal how a signaling pathway can orchestrate specific genome changes and demonstrate that the copy number of repetitive DNA can be altered to suit environmental conditions.

  15. 16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria

    Science.gov (United States)

    Subedi, Shradha; Kong, Fanrong; Jelfs, Peter; Gray, Timothy J.; Xiao, Meng; Sintchenko, Vitali; Chen, Sharon C-A

    2016-01-01

    Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I–V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC. PMID:27749897

  16. 16S-23S Internal Transcribed Spacer Region PCR and Sequencer-Based Capillary Gel Electrophoresis has Potential as an Alternative to High Performance Liquid Chromatography for Identification of Slowly Growing Nontuberculous Mycobacteria.

    Science.gov (United States)

    Subedi, Shradha; Kong, Fanrong; Jelfs, Peter; Gray, Timothy J; Xiao, Meng; Sintchenko, Vitali; Chen, Sharon C-A

    2016-01-01

    Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I-V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC.

  17. Sequence-based Methods in Human Microbial Ecology: A The 2nd HumanGenome Comes of Age

    Energy Technology Data Exchange (ETDEWEB)

    Weng, Li; Rubin, Edward M.; Bristow, James

    2005-06-01

    Ecologists studying microbial life in the environment have recognized the enormous complexity of microbial diversity for more than a decade (Whitman et al. 1998). The development of a variety of culture-independent methods, many of them coupled with high-throughput DNA sequencing, has allowed this diversity to be explored in ever greater detail (Handelsman 2004; Harris et al. 2004; Hugenholtz et al. 1998; Moreira and Lopez-Garcia 2002; Rappe and Giovannoni 2003). Despite the widespread application of these new techniques to the characterization of uncultivated microbes and microbial communities in the environment, their application to human health and disease has lagged behind. Because these techniques now allow not only cataloging of microbial diversity, but also insight into microbial functions, it is time for clinical microbiologists to apply these tools to the microbial communities that abound on and within us, in what has been aptly called ''the second Human Genome Project'' (Relman and Falkow 2001). In this review we will discuss the sequence-based methods for microbial analysis that are currently available and their application to identify novel human pathogens, improve diagnosis and treatment of known infectious diseases, and finally to advance understanding of our relationship with microbial communities that normally reside in and on the human body.

  18. Evaluation of the impact of refrigeration on next generation sequencing-based assessment of the canine and feline fecal microbiota.

    Science.gov (United States)

    Weese, J Scott; Jalali, Mohammad

    2014-09-30

    Evaluation of factors that might impact microbiota assessment is important to avoid spurious results, particularly in field and multicenter studies where sample collection may occur distant from the laboratory. This study evaluated the impact of refrigeration on next generation sequence-based assessment of the canine and feline fecal microbiota. Fecal samples were collected from seven dogs and ten cats, and analysed at baseline and after 3, 7, 10 and 14 days of storage at 4°C. There were no differences in community membership or population structure between timepoints for either dogs or cats, nor were there any differences in richness, diversity and evenness. There were few differences in relative abundance of phyla or predominant genera, with the only differences being significant increases in Actinobacteria between Days 0-14 (P = 0.0184) and 1-14 (P = 0.0182) for canine samples, and a decrease in Erysipelotrichaceae incertae sedis between Day 0 and Day 7 (median 4.9 vs 2.2%, P = 0.046) in feline samples. Storage for at least 14 days at 4°C has limited impact on culture-independent assessment of the canine and feline fecal microbiota, although changes in some individual groups may occur.

  19. [Homologous Analysis Using Repetitive-sequence-based PCR Typing of Exfoliative Toxin-producing Staphylococcus aureus Isolated from Our Hospital].

    Science.gov (United States)

    Miyamoto, Hitoshi; Murakami, Shinobu; Nishimiya, Tatsuya; Suemori, Koichiro; Tauchi, Hisamichi

    2015-05-01

    We examined staphylococcal coagulase types and homologous analysis using the DiversiLab repetitive-sequence-based PCR system in exfoliative toxin (ET)-producing Staphylococcus aureus. Twenty-two isolates (17 methicillin-sensitive Staphylococcus aureus (MSSA) and 5 methicillin-resistant Staphylococcus aureus (MRSA) isolates) obtained in our hospital from January 2012 and December 2013 were used. Three groups were classified according to the coagulase types and serotypes of ET. The first group (4 MSSA) showed coagulase type I and ET-A, and the second group (3 MSSA and 2 MRSA) showed coagulase type I and ET-B. The third group (10 MSSA and 3 MRSA) showed coagulase type V and ET-B. An analysis by DiversiLab demonstrated that homology was high in both the first and second groups. The homogenousness was high among the third group isolates except for the ocular isolates. In our hospital, three important groups were present according to a coagulase type and an ET type, and the homology of ocular isolates could be different from other materials isolates.

  20. ProteDNA: a sequence-based predictor of sequence-specific DNA-binding residues in transcription factors.

    Science.gov (United States)

    Chu, Wen-Yi; Huang, Yu-Feng; Huang, Chun-Chin; Cheng, Yi-Sheng; Huang, Chien-Kang; Oyang, Yen-Jen

    2009-07-01

    This article presents the design of a sequence-based predictor named ProteDNA for identifying the sequence-specific binding residues in a transcription factor (TF). Concerning protein-DNA interactions, there are two types of binding mechanisms involved, namely sequence-specific binding and nonspecific binding. Sequence-specific bindings occur between protein sidechains and nucleotide bases and correspond to sequence-specific recognition of genes. Therefore, sequence-specific bindings are essential for correct gene regulation. In this respect, ProteDNA is distinctive since it has been designed to identify sequence-specific binding residues. In order to accommodate users with different application needs, ProteDNA has been designed to operate under two modes, namely, the high-precision mode and the balanced mode. According to the experiments reported in this article, under the high-precision mode, ProteDNA has been able to deliver precision of 82.3%, specificity of 99.3%, sensitivity of 49.8% and accuracy of 96.5%. Meanwhile, under the balanced mode, ProteDNA has been able to deliver precision of 60.8%, specificity of 97.6%, sensitivity of 60.7% and accuracy of 95.4%. ProteDNA is available at the following websites: http://protedna.csbb.ntu.edu.tw/, http://protedna.csie.ntu.edu.tw/, http://bio222.esoe.ntu.edu.tw/ProteDNA/.

  1. Directional Amplification of Horizontal Motion at Rock Sites

    Science.gov (United States)

    Pischiutta, M.; Cianfarra, P.; Salvini, F.; Rovelli, A.

    2016-12-01

    Directional amplifications of horizontal ground motion along site-dependent azimuths are often unexpectedly found in rocky environments and in stiff-rock conditions. Directional amplification has been studied by several authors at sites with fractured rocks across fault damage zones (e.g. Pischiutta et al., 2013), or close to gravitational instabilities (e.g. Burjanek et al, 2012). In the majority of the cases, a transversal relation between the maximum amplification and the orientation of the predominant fracture field is recognized, interpreted as the effect of the stiffness anisotropy. Cracks in fault damage zones also cause the reduction of rock velocity (especially near-surface Vs). Studies performed on high number of rocky sites revealed that directional amplification effects are much more diffuse than expected. A systematic study involving 258 seismological stations of the Italian Seismic Network points out that 56 of stations (20%) are clearly affected by directional site amplification effects. This station sample was studied by analyzing the surface topography in order to unravel the role of topographic irregularities on ground motion amplifications. Models proposed in literature predict amplification at wavelengths comparable to the mountain width (e.g. Géli et al, 1988) and the scatter of wavefield that is polarized in the direction orthogonal to the relief elongation (e.g. Spudich et al., 1996). Since some bias exists in the objective quantification morphological parameters, we use an original methodology that combines morphometric analysis of digital elevation models and principal component analysis to define the dimension and elongation orientation of pronounced ridges. This study showed that the expected relations between the shape/dimension of relief and ground motion were found only at 5 stations (out of 56) , suggesting a major of local morphology, geological complexities and subsurface properties on directional amplification, consistently with

  2. Optimizing direct amplification of forensic commercial kits for STR determination.

    Science.gov (United States)

    Caputo, M; Bobillo, M C; Sala, A; Corach, D

    2017-04-01

    Direct DNA amplification in forensic genotyping reduces analytical time when large sample sets are being analyzed. The amplification success depends mainly upon two factors: on one hand, the PCR chemistry and, on the other, the type of solid substrate where the samples are deposited. We developed a workflow strategy aiming to optimize times and cost when starting from blood samples spotted onto diverse absorbent substrates. A set of 770 blood samples spotted onto Blood cards, Whatman(®) 3 MM paper, FTA™ Classic cards, and Whatman(®) Grade 1 was analyzed by a unified working strategy including a low-cost pre-treatment, a PCR amplification volume scale-down, and the use of the 3500 Genetic Analyzer as the analytical platform. Samples were analyzed using three different commercial multiplex STR direct amplification kits. The efficiency of the strategy was evidenced by a higher percentage of high-quality profiles obtained (over 94%), a reduced number of re-injections (average 3.2%), and a reduced amplification failure rate (lower than 5%). Average peak height ratio among different commercial kits was 0.91, and the intra-locus balance showed values ranging from 0.92 to 0.94. A comparison with previously reported results was performed demonstrating the efficiency of the proposed modifications. The protocol described herein showed high performance, producing optimal quality profiles, and being both time and cost effective. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  3. Adaptive base-isolation of civil structures using variable amplification

    Institute of Scientific and Technical Information of China (English)

    Kenneth K. Walsh; Makola M. Abdullah

    2006-01-01

    Semi-active dampers are used in base-isolation to reduce the seismic response of civil engineering structures.In the present study, a new semi-active damping system using variable amplification will be investigated for adaptive baseisolation. It uses a novel variable amplification device (VAD) connected in series with a passive damper. The VAD is capable of producing multiple amplification factors, each corresponding to a different amplification state. Forces from the damper are amplified to the structure according to the current amplification state, which is selected via a semi-active control algorithm specifically tailored to the system's unique damping characteristics. To demonstrate the effectiveness of the VAD-damper system for adaptive base-isolation, numerical simulations are conducted for three and seven-story base-isolated buildings subject to both far and near-field ground motions. The results indicate that the system can achieve significant reductions in response compared to the base-isolated buildings with no damper. The proposed system is also found to perform well compared to a typical semi-active damper.

  4. Evaluating the displacement amplification factors of concentrically braced steel frames

    Science.gov (United States)

    Mahmoudi, Mussa; Zaree, Mahdi

    2013-12-01

    According to seismic design codes, nonlinear performance of structures is considered during strong earthquakes. Seismic design provisions estimate the maximum roof and story drifts occurring during major earthquakes by amplifying the drifts computed from elastic analysis at the prescribed seismic force level with a displacement amplification factor. The present study tries to evaluate the displacement amplification factors of conventional concentric braced frames (CBFs) and buckling restrained braced frames (BRBFs). As such, static nonlinear (pushover) analysis and nonlinear dynamic time history analysis have been performed on the model buildings with single and double bracing bays, and different stories and brace configurations (chevron V, invert V, and X bracing). It is observed that the displacement amplification factors for BRBFs are higher than that of CBFs. Also, the number of bracing bays and height of buildings have a profound effect on the displacement amplification factors. The evaluated ratios between displacement amplification factors and response modification factors are from 1 to 1.12 for CBFs and from 1 to 1.4 for BRBFs.

  5. Local seismic site amplification: effects of obliquely incident antiplane motions

    Science.gov (United States)

    Cherid, D.; Hammoutene, M.; Tiliouine, B.; Berrah, M. K.

    2016-11-01

    Seismic site amplification studies are generally used to assess the effects of local geology and soil conditions on ground motion characteristics. Although extensive reviews on site amplification phenomena associated with stratigraphic effects can be found in the specialized literature, it should be pointed out that most of the practical applications have been limited to the study of vertically propagating shear horizontal (SH) waves, i.e., to the 1-D soil amplification problem. Furthermore, little attention, if any, has been devoted to the study of the effects of non-vertically incident SH waves on surface accelerograms and on the earthquake response of structures. In the present work, the study is extended to an investigation of 2-D site amplification of non-vertically propagating seismic shear waves in multilayered viscoelastic soil deposits. Sensitivity analyses of the effects of non-vertical incidence on site amplification functions are performed based on site geotechnical data collected from post-seismic investigations of the 1980 El-Asnam earthquake. Analytical results are discussed in terms of seismic site transfer functions, spectral ratios, surface acceleration time histories, and structural response spectra for different values of wave incidence angle. Both bedrock and rock outcropping cases are examined.

  6. Magnetic Amplification by Magnetized Cosmic Rays in SNR Shocks

    CERN Document Server

    Riquelme, Mario A

    2009-01-01

    (Abridged) X-ray observations of synchrotron rims in supernova remnant (SNR) shocks show evidence of strong magnetic field amplification (a factor of ~100 between the upstream and downstream medium). This amplification may be due to plasma instabilities driven by shock-accelerated cosmic rays (CRs). One candidate is the cosmic ray current-driven (CRCD) instability (Bell 2004), caused by the electric current of large Larmor radii CRs propagating parallel to the upstream magnetic field. Particle-in-cell (PIC) simulations have shown that the back-reaction of the amplified field on CRs would limit the amplification factor of this instability to less than ~10 in galactic SNRs. In this paper, we study the possibility of further amplification driven near shocks by "magnetized" CRs, whose Larmor radii are smaller than the length scale of the field that was previously amplified by the CRCD instability. We find that additional amplification can occur due to a new instability, driven by the CR current perpendicular to t...

  7. Amplification of Information by Photons and the Quantum Chernoff Bound

    Science.gov (United States)

    Zwolak, Michael; Riedel, C. Jess; Zurek, Wojciech H.

    2014-03-01

    Amplification was regarded, since the early days of quantum theory, as a mysterious ingredient that endows quantum microstates with macroscopic consequences, key to the ``collapse of the wavepacket,'' and a way to avoid embarrassing problems exemplified by Schrödinger's cat. This bridge between the quantum microworld and the classical world of our experience was postulated ad hoc in the Copenhagen Interpretation. Quantum Darwinism views amplification as replication, in many copies, of information about quantum states. We show that such amplification is a natural consequence of a broad class of models of decoherence, including the photon environment we use to obtain most of our information. The resultant amplification is huge, proportional to # ξQCB . Here, #  is the environment size and ξQCB is the ``typical'' Quantum Chernoff Information, which quantifies the efficiency of the amplification. The information communicated though the environment is imprinted in the states of individual environment subsystems, e.g., in single photons, which document the transfer of information into the environment and result in the emergence of the classical world. See, http://mike.zwolak.org

  8. Processes and impacts of Arctic amplification: A research synthesis

    Science.gov (United States)

    Serreze, Mark C.; Barry, Roger G.

    2011-05-01

    The past decade has seen substantial advances in understanding Arctic amplification — that trends and variability in surface air temperature tend to be larger in the Arctic region than for the Northern Hemisphere or globe as a whole. We provide a synthesis of research on Arctic amplification, starting with a historical context and then addressing recent insights into processes and key impacts, based on analysis of the instrumental record, modeling studies, and paleoclimate reconstructions. Arctic amplification is now recognized as an inherent characteristic of the global climate system, with multiple intertwined causes operating on a spectrum of spatial and temporal scales. These include, but are not limited to, changes in sea ice extent that impact heat fluxes between the ocean and the atmosphere, atmospheric and oceanic heat transports, cloud cover and water vapor that alter the longwave radiation flux to the surface, soot on snow and heightened black carbon aerosol concentrations. Strong warming over the Arctic Ocean during the past decade in autumn and winter, clearly associated with reduced sea ice extent, is but the most recent manifestation of the phenomenon. Indeed, periods of Arctic amplification are evident from analysis of both warm and cool periods over at least the past three million years. Arctic amplification being observed today is expected to become stronger in coming decades, invoking changes in atmospheric circulation, vegetation and the carbon cycle, with impacts both within and beyond the Arctic.

  9. Modeling the amplification dynamics of human Alu retrotransposons.

    Directory of Open Access Journals (Sweden)

    Dale J Hedges

    2005-09-01

    Full Text Available Retrotransposons have had a considerable impact on the overall architecture of the human genome. Currently, there are three lineages of retrotransposons (Alu, L1, and SVA that are believed to be actively replicating in humans. While estimates of their copy number, sequence diversity, and levels of insertion polymorphism can readily be obtained from existing genomic sequence data and population sampling, a detailed understanding of the temporal pattern of retrotransposon amplification remains elusive. Here we pose the question of whether, using genomic sequence and population frequency data from extant taxa, one can adequately reconstruct historical amplification patterns. To this end, we developed a computer simulation that incorporates several known aspects of primate Alu retrotransposon biology and accommodates sampling effects resulting from the methods by which mobile elements are typically discovered and characterized. By modeling a number of amplification scenarios and comparing simulation-generated expectations to empirical data gathered from existing Alu subfamilies, we were able to statistically reject a number of amplification scenarios for individual subfamilies, including that of a rapid expansion or explosion of Alu amplification at the time of human-chimpanzee divergence.

  10. Primer design versus PCR bias in methylation independent PCR amplifications.

    Science.gov (United States)

    Wojdacz, Tomasz K; Borgbo, Tanni; Hansen, Lise Lotte

    2009-05-16

    Many protocols in methylation studies utilize one primer set to generate a PCR product from bisulfite modified template regardless of its methylation status (methylation independent amplification MIP). However, proportional amplification of methylated and unmethylated alleles is hard to achieve due to PCR bias favoring amplification of unmethylated relatively GC poor sequence. Two primer design systems have been proposed to overcome PCR bias in methylation independent amplifications. The first advises against including any CpG dinucleoteides into the primer sequence (CpG-free primers) and the second, recently published by us, is based on inclusion of a limited number of CpG sites into the primer sequence. Here we used the Methylation Sensitive High Resolution Melting (MS-HRM) technology to investigate the ability of primers designed according to both of the above mentioned primer design systems to proportionally amplify methylated and unmethylated templates. Ten "CpG-free" primer pairs and twenty primers containing limited number of CpGs were tested. In reconstruction experiments the "CpG-free" primers showed primer specific sensitivity and allowed us to detect methylation levels in the range from 5 to 50%. Whereas while using primers containing limited number of CpG sites we were able to consistently detect 1-0.1% methylation levels and effectively control PCR amplification bias. In conclusion, the primers with limited number of CpG sites are able to effectively reverse PCR bias and therefore detect methylated templates with significantly higher sensitivity than CpG free primers.

  11. Somatic recombination, gene amplification and cancer.

    Science.gov (United States)

    Ramel, C; Cederberg, H; Magnusson, J; Vogel, E; Natarajan, A T; Mullender, L H; Nivard, J M; Parry, J M; Leyson, A; Comendador, M A; Sierra, L M; Ferreiro, J A; Consuegra, S

    1996-06-12

    The principle objective of this research programme, to analyse chemical induction of somatic recombination and related endpoints, i.e., mobilization of transposing elements and gene amplification, has been approached by means of several assay systems. These have included Drosophila, Saccharomyces and mammalian cell cultures. 6.1. Screening assays for mitotic recombination. A large number of chemicals have been investigated in the three Drosophila assay systems employed--the multiple wing hair/flare wing spot system developed by Graf et al., 1984, the white-ivory system developed by Green et al., 1986 and the white/white+ eye spot assay developed by Vogel (Vogel and Nivard, 1993). Particularly the screening of 181 chemicals, covering a wide array of chemical classes, by the last mentioned assay has shown that measurement of somatic recombination in Drosophila constitutes a sensitive and efficient short-term test which shows a remarkably good correlation with the agent score of 83 short-term tests analysed by ICPEMC (Mendelsohn et al., 1992; Table 2) as well as the assay performance in international collaborative programmes measuring carcinogen/non-carcinogens (de Serres and Ashby, 1981; Ashby et al., 1985, 1988). Also the wing spot assay has gained wide international recognition as a similarly sensitive test. These two assay systems in Drosophila measure both intrachromosomal events and interchromosomal recombination. The white-ivory system on the other hand is based on the loss of a tandem duplication in the white locus, the mechanism of which is less known, but probably involves intrachromosomal recombination. The difference in the mechanism between this assay and the former two was indicated by the lack of response to methotrexate in the white-ivory assay, while this compound was strongly recombinogenic in both the wing spot and white/white+ assays. The use of different strains of Drosophila with the white/white+ assay demonstrated the importance of the

  12. Modeling Loss Amplification After Devastating Disasters

    Science.gov (United States)

    Boissonnade, A. C.; Muir Wood, R.

    2008-05-01

    With the catastrophic events that occurred in 2004 and 2005 came the realization that Catastrophic (Cat) loss models were not properly modeling insured losses and their associated uncertainty. One reason was that major catastrophes were generally characterized by losses caused by the primary initiating events. Such approaches are not adequate when losses can result from the compounded impacts of scenarios of secondary cascading events (physical, economic, social and political) that can have much larger impacts than those due to the primary events themselves. Situations where more and more cascading events can occur will result in different outcomes, some leading to extreme loss events, generally referred as Super Cats. These situations occurred in December 2004 with the Sumatra earthquake and tsunami and in August 2005 with hurricane Katrina and resulting New Orleans flooding. A review of historical events shows that these events are not exceptions. Modeling such scenarios adds new levels of complexity and different perspectives in the understanding of characterizing and assessing impacts of catastrophic events. Modeling economic consequences of extreme events can be improved by developing scenarios of cascades of secondary events triggered by the primary event(s). The likelihood of each scenario should be modeled, along with the hazards of primary and secondary events and resulting losses with their impacts to the different stakeholders. In addition, it is also important to model the impacts of the hazards on the infrastructure and the resulting disruption to the residents and the local economy because these can result in additional losses. This paper describes current work with the goals of better modeling the full economic impacts from catastrophic events, and of a more comprehensive treatment of uncertainty. We will present approaches for modeling loss amplification that account for all the ways in which the cost incurred for a certain level of damage due to a

  13. Identification by sequencing based typing and complete coding region analysis of three new HLA class II alleles: DRB3*0210, DRB3*0211 and DQB1*0310.

    Science.gov (United States)

    Balas, A; Santos, S; Aviles, M J; Garcia-Sanchez, F; Lillo, R; Vicario, J L

    2000-10-01

    The study of HLA class II polymorphism by direct exon 2 DNA sequencing analysis has been established to be a reliable and accurate high-resolution typing procedure. This approach shows some advantages in relation to previous methods, polymerase chain reaction using sequence-specific oligonucleotides (PCR-SSO) and sequence-specific primers (PCR-SSP), basically due to the capability of analysis for the complete sequenced genomic region, including non-polymorphic motifs. DRB3 and DQB1 sequencing based typing (SBT) in unrelated bone marrow donor searching allowed us to detect three new alleles. The complete coding region sequences were characterised from cDNA. Two new DRB3 alleles, DRB3*0210 and DRB3*0211, were described in two Caucasian bone marrow donors. Both sequences showed single point mutations regarding DRB3*0202, producing amino acid replacements at positions 51 (Asp to Thr) and 67 (Leu to Ile), respectively. These two point mutations can be found in other DRB alleles, and suggest that gene conversion would be involved in the origin of both alleles. A new DQB1 sequence was found in a Spanish patient that showed two nucleotide differences, positions 134 and 141, with regard to its close similar DQB1*03011 allele. Only substitution at position 134 provoked amino acid replacement at residue 45, Glu to Gly. This single amino acid change would be involved in the lack of serologic recognition of this new molecule by DQ7-specific reagents.

  14. Amplification of complex fields in Nd:YAG amplifiers

    Science.gov (United States)

    Chen, Xudong; Chang, Chengcheng; Pu, Jixiong

    2017-04-01

    High energy nanosecond vortex beams and cylindrically polarized beams are generated in Nd:YAG amplifiers. Vortex seed beams and cylindrically polarized seed beams are converted from a conventional Nd:YAG laser by spiral phase plate and polarization converter, respectively. Maximum output energy of optical vortex up to 995 mJ and cylindrically polarized beams up to 772 mJ have been achieved at 10 Hz in a 10-ns pulse, respectively. The amplification efficiency, the beam quality and pulse width of the amplification output are studied. Both the topological charge of the vortex seed beams and polarization state of cylindrically polarized beams are confirmed to be conserved during the amplification. The generation of high energy vortex beams and cylindrically polarized beams would be beneficial to laser material processing.

  15. Linear Amplification of Optical Signal in Coupled Photonic Crystal Waveguides

    CERN Document Server

    Jandieri, Vakhtang

    2015-01-01

    We introduce a weakly coupled photonic crystal waveguide as a promising and realistic model for all-optical amplification. A symmetric pillar type coupled photonic crystal waveguide consisting of dielectric rods periodically distributed in a free space is proposed as all-optical amplifier. Using the unique features of the photonic crystals to control and guide the light, we have properly chosen the frequency at which only one mode (odd mode) becomes the propagating mode in the coupled photonic crystal waveguide, whereas another mode (even mode) is completely reflected from the guiding structure. Under this condition, the all-optical amplification is fully realized. The amplification coefficient for the continuous signal and the Gaussian pulse is calculated.

  16. Measurement-based noiseless linear amplification for quantum communication

    Science.gov (United States)

    Chrzanowski, H. M.; Walk, N.; Haw, J. Y.; Thearle, O.; Assad, S. M.; Janousek, J.; Hosseini, S.; Ralph, T. C.; Symul, T.; Lam, P. K.

    2014-11-01

    Entanglement distillation is an indispensable ingredient in extended quantum communication networks. Distillation protocols are necessarily non-deterministic and require non-trivial experimental techniques such as noiseless amplification. We show that noiseless amplification could be achieved by performing a post-selective filtering of measurement outcomes. We termed this protocol measurement-based noiseless linear amplification (MBNLA). We apply this protocol to entanglement that suffers transmission loss of up to the equivalent of 100km of optical fibre and show that it is capable of distilling entanglement to a level stronger than that achievable by transmitting a maximally entangled state through the same channel. We also provide a proof-of-principle demonstration of secret key extraction from an otherwise insecure regime via MBNLA. Compared to its physical counterpart, MBNLA not only is easier in term of implementation, but also allows one to achieve near optimal probability of success.

  17. Determining Parameters for Images Amplification by Pulses Interpolation

    Directory of Open Access Journals (Sweden)

    Morera-Delfín Leandro

    2015-01-01

    Full Text Available This paper presents the implementation of a method for image samples interpolation based on a physical scanning model. It uses the theory to take digital image samples and to perform an implementation of such mechanism through software. This allows us to get the appropriate parameters for the images amplification using a truncated sampler arrangement. The shown process copies the physical model of image acquisition in order to incorporate the required samples for the amplification. This process is useful in the reconstruction of details in low resolution images and for images compression. The proposed method studies the conservation of high frequency in the high resolution plane for the generation of the amplification kernel. A new way of direct application of the physical model for scanning images in analytic mode is presented.

  18. Current Amplification Characteristics of BJT on Fast Neutron Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Sung Ho; Sun, Gwang Min; Baek, Hani [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2016-10-15

    BJT (Bipolar Junction Transistor) is a three-terminal device with an important feature in that the current through two terminals can be controlled by small changes we make in the current or voltage at the third terminal. This control feature allows us to amplify small AC signals or to switch the device from an on state and off state and back. Fast neutron irradiation incurs lattice damage in bulk Si. The recombination rate of minority carriers and register are increased by the lattice damage. This study will investigate the current amplification characteristics of a pnp Si BJT through fast neutron irradiation experiments. In this paper, the current amplification characteristics of a pnp Si BJT were investigated for fast neutron irradiation. The experimental results show that base-tocollector current amplification ratio is decreased with an increase in the fast neutron irradiation. These indicate that the lattice damage caused by fast neutron irradiation increases the recombination rate of minority carriers and resistor.

  19. An Intrinsically Digital Amplification Scheme for Hearing Aids

    Directory of Open Access Journals (Sweden)

    Brenton R. Steele

    2005-11-01

    Full Text Available Results for linear and wide-dynamic range compression were compared with a new 64-channel digital amplification strategy in three separate studies. The new strategy addresses the requirements of the hearing aid user with efficient computations on an open-platform digital signal processor (DSP. The new amplification strategy is not modeled on prior analog strategies like compression and linear amplification, but uses statistical analysis of the signal to optimize the output dynamic range in each frequency band independently. Using the open-platform DSP processor also provided the opportunity for blind trial comparisons of the different processing schemes in BTE and ITE devices of a high commercial standard. The speech perception scores and questionnaire results show that it is possible to provide improved audibility for sound in many narrow frequency bands while simultaneously improving comfort, speech intelligibility in noise, and sound quality.

  20. Genomic DNA pooling strategy for next-generation sequencing-based rare variant discovery in abdominal aortic aneurysm regions of interest-challenges and limitations

    NARCIS (Netherlands)

    Harakalova, M.; Nijman, I.J.; Medic, J.; Mokry, M.; Renkens, I.; Blankensteijn, J.D.; Kloosterman, W.P.; Baas, A.F.; Cuppen, E.

    2011-01-01

    The costs and efforts for sample preparation of hundreds of individuals, their genomic enrichment for regions of interest, and sufficient deep sequencing bring a significant burden to next-generation sequencing-based experiments. We investigated whether pooling of samples at the level of genomic DNA

  1. Identification of genetic elements associated with EPSPs gene amplification.

    Directory of Open Access Journals (Sweden)

    Todd A Gaines

    Full Text Available Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS gene evolved in the weed species Amaranthus palmeri to confer resistance to glyphosate, the world's most important herbicide. However, the gene amplification mechanism is unknown. We sequenced the EPSPS gene and genomic regions flanking EPSPS loci in A. palmeri, and searched for mobile genetic elements or repetitive sequences. The EPSPS gene was 10,229 bp, containing 8 exons and 7 introns. The gene amplification likely proceeded through a DNA-mediated mechanism, as introns exist in the amplified gene copies and the entire amplified sequence is at least 30 kb in length. Our data support the presence of two EPSPS loci in susceptible (S A. palmeri, and that only one of these was amplified in glyphosate-resistant (R A. palmeri. The EPSPS gene amplification event likely occurred recently, as no sequence polymorphisms were found within introns of amplified EPSPS copies from R individuals. Sequences with homology to miniature inverted-repeat transposable elements (MITEs were identified next to EPSPS gene copies only in R individuals. Additionally, a putative Activator (Ac transposase and a repetitive sequence region were associated with amplified EPSPS genes. The mechanism controlling this DNA-mediated amplification remains unknown. Further investigation is necessary to determine if the gene amplification may have proceeded via DNA transposon-mediated replication, and/or unequal recombination between different genomic regions resulting in replication of the EPSPS gene.

  2. The efficiency of magnetic field amplification at shocks by turbulence

    Science.gov (United States)

    Ji (), Suoqing; Oh, S. Peng; Ruszkowski, M.; Markevitch, M.

    2016-12-01

    Turbulent dynamo field amplification has often been invoked to explain the strong field strengths in thin rims in supernova shocks ( ˜ 100 μG) and in radio relics in galaxy clusters ( ˜ μG). We present high-resolution magnetohydrodynamic simulations of the interaction between pre-shock turbulence, clumping and shocks, to quantify the conditions under which turbulent dynamo amplification can be significant. We demonstrate numerically converged field amplification which scales with Alfvén Mach number, B/B_0 ∝ M_A, up to M_A ˜ 150. This implies that the post-shock field strength is relatively independent of the seed field. Amplification is dominated by compression at low M_A, and stretching (turbulent amplification) at high M_A. For high M_A, the B-field grows exponentially and saturates at equipartition with turbulence, while the vorticity jumps sharply at the shock and subsequently decays; the resulting field is orientated predominately along the shock normal (an effect only apparent in 3D and not 2D). This agrees with the radial field bias seen in supernova remnants. By contrast, for low M_A, field amplification is mostly compressional, relatively modest, and results in a predominantly perpendicular field. The latter is consistent with the polarization seen in radio relics. Our results are relatively robust to the assumed level of gas clumping. Our results imply that the turbulent dynamo may be important for supernovae, but is only consistent with the field strength, and not geometry, for cluster radio relics. For the latter, this implies strong pre-existing B-fields in the ambient cluster outskirts.

  3. Identification of cancer predisposition variants in apparently healthy individuals using a next-generation sequencing-based family genomics approach.

    Science.gov (United States)

    Karageorgos, Ioannis; Mizzi, Clint; Giannopoulou, Efstathia; Pavlidis, Cristiana; Peters, Brock A; Zagoriti, Zoi; Stenson, Peter D; Mitropoulos, Konstantinos; Borg, Joseph; Kalofonos, Haralabos P; Drmanac, Radoje; Stubbs, Andrew; van der Spek, Peter; Cooper, David N; Katsila, Theodora; Patrinos, George P

    2015-06-20

    Cancer, like many common disorders, has a complex etiology, often with a strong genetic component and with multiple environmental factors contributing to susceptibility. A considerable number of genomic variants have been previously reported to be causative of, or associated with, an increased risk for various types of cancer. Here, we adopted a next-generation sequencing approach in 11 members of two families of Greek descent to identify all genomic variants with the potential to predispose family members to cancer. Cross-comparison with data from the Human Gene Mutation Database identified a total of 571 variants, from which 47 % were disease-associated polymorphisms, 26 % disease-associated polymorphisms with additional supporting functional evidence, 19 % functional polymorphisms with in vitro/laboratory or in vivo supporting evidence but no known disease association, 4 % putative disease-causing mutations but with some residual doubt as to their pathological significance, and 3 % disease-causing mutations. Subsequent analysis, focused on the latter variant class most likely to be involved in cancer predisposition, revealed two variants of prime interest, namely MSH2 c.2732T>A (p.L911R) and BRCA1 c.2955delC, the first of which is novel. KMT2D c.13895delC and c.1940C>A variants are additionally reported as incidental findings. The next-generation sequencing-based family genomics approach described herein has the potential to be applied to other types of complex genetic disorder in order to identify variants of potential pathological significance.

  4. Monitoring transmission routes of Listeria spp. in smoked salmon production with repetitive element sequence-based PCR techniques.

    Science.gov (United States)

    Zunabovic, M; Domig, K J; Pichler, I; Kneifel, W

    2012-03-01

    Various techniques have been used for tracing the transmission routes of Listeria species and for the assessment of hygiene standards in food processing plants. The potential of repetitive element sequence-based PCR (Rep-PCR) methods (GTG₅ and REPI + II) for the typing of Listeria isolates (n = 116), including Listeria monocytogenes (n = 46), was evaluated in a particular situation arising from the relocation of a company producing cold-smoked salmon. Pulsed-field gel electrophoresis (PFGE) using three restriction enzymes (ApaI, AscI, and SmaI) was used for comparison. Identical transmission scenarios among two companies could be identified by cluster analysis of L. monocytogenes isolates that were indistinguishable by both Rep-PCR and PFGE. The calculated diversity index (DI) indicates that Rep-PCR subtyping of Listeria species with primer sets GTG₅ and REPI + II has a lower discrimination power than does PFGE. When concatenated Rep-PCR cluster analysis was used, the DI increased from 0.934 (REPI + II) and 0.923 (GTG₅) to 0.956. The discrimination power of this method was similar to that of PFGE typing based on restriction enzyme Apa I (DI = 0.955). Listeria welshimeri may be useful as an indicator for monitoring smoked salmon processing environments. Rep-PCR meets the expectations of a reasonable, fast, and low-cost molecular subtyping method for the routine monitoring of Listeria species. The discriminatory power as characterized by the DI sufficiently quantifies the probability of unrelated isolates being characterized as different subtypes. Therefore, Rep-PCR typing based on two primer systems (GTG₅ and REPI + II) may be a useful tool for monitoring industrial hygiene.

  5. Sequence-based genotyping clarifies conflicting historical morphometric and biological data for 5 Eimeria species infecting turkeys.

    Science.gov (United States)

    El-Sherry, S; Ogedengbe, M E; Hafeez, M A; Sayf-Al-Din, M; Gad, N; Barta, J R

    2015-02-01

    Unlike with Eimeria species infecting chickens, specific identification and nomenclature of Eimeria species infecting turkeys is complicated, and in the absence of molecular data, imprecise. In an attempt to reconcile contradictory data reported on oocyst morphometrics and biological descriptions of various Eimeria species infecting turkey, we established single oocyst derived lines of 5 important Eimeria species infecting turkeys, Eimeria meleagrimitis (USMN08-01 strain), Eimeria adenoeides (Guelph strain), Eimeria gallopavonis (Weybridge strain), Eimeria meleagridis (USAR97-01 strain), and Eimeria dispersa (Briston strain). Short portions (514 bp) of mitochondrial cytochrome c oxidase subunit I gene (mt COI) from each were amplified and sequenced. Comparison of these sequences showed sufficient species-specific sequence variation to recommend these short mt COI sequences as species-specific markers. Uniformity of oocyst features (dimensions and oocyst structure) of each pure line was observed. Additional morphological features of the oocysts of these species are described as useful for the microscopic differentiation of these Eimeria species. Combined molecular and morphometric data on these single species lines compared with the original species descriptions and more recent data have helped to clarify some confusing, and sometimes conflicting, features associated with these Eimeria spp. For example, these new data suggest that the KCH and KR strains of E. adenoeides reported previously represent 2 distinct species, E. adenoeides and E. meleagridis, respectively. Likewise, analysis of the Weybridge strain of E. adenoeides, which has long been used as a reference strain in various studies conducted on the pathogenicity of E. adenoeides, indicates that this coccidium is actually a strain of E. gallopavonis. We highly recommend mt COI sequence-based genotyping be incorporated into all studies using Eimeria spp. of turkeys to confirm species identifications and so

  6. Next generation sequencing-based expression profiling identifies signatures from benign stromal proliferations that define stromal components of breast cancer

    Science.gov (United States)

    2013-01-01

    Introduction Multiple studies have shown that the tumor microenvironment (TME) of carcinomas can play an important role in the initiation, progression, and metastasis of cancer. Here we test the hypothesis that specific benign fibrous soft tissue tumor gene expression profiles may represent distinct stromal fibroblastic reaction types that occur in different breast cancers. The discovered stromal profiles could classify breast cancer based on the type of stromal reaction patterns in the TME. Methods Next generation sequencing-based gene expression profiling (3SEQ) was performed on formalin fixed, paraffin embedded (FFPE) samples of 10 types of fibrous soft tissue tumors. We determined the extent to which these signatures could identify distinct subsets of breast cancers in four publicly available breast cancer datasets. Results A total of 53 fibrous tumors were sequenced by 3SEQ with an average of 29 million reads per sample. Both the gene signatures derived from elastofibroma (EF) and fibroma of tendon sheath (FOTS) demonstrated robust outcome results for survival in the four breast cancer datasets. The breast cancers positive for the EF signature (20-33% of the cohort) demonstrated significantly better outcome for survival. In contrast, the FOTS signature-positive breast cancers (11-35% of the cohort) had a worse outcome. Conclusions We defined and validated two new stromal signatures in breast cancer (EF and FOTS), which are significantly associated with prognosis. Our group has previously identified novel cancer stromal gene expression signatures associated with outcome differences in breast cancer by gene expression profiling of three soft tissue tumors, desmoid-type fibromatosis (DTF), solitary fibrous tumor (SFT), and tenosynovial giant cell tumor (TGCT/CSF1), as surrogates for stromal expression patterns. By combining the stromal signatures of EF and FOTS, with our previously identified DTF and TGCT/CSF1 signatures we can now characterize clinically

  7. HLA-A Gene Polymorphism Defined by High-Resolution Sequence Based Typing in 161 Northern Chinese Han People

    Institute of Scientific and Technical Information of China (English)

    Chunxia Yan; Haiyan Sun; Xiuqing Zhang; Jian Wang; Huanming Yang; Shengbin Li; Ruilin Wang; Jingxiang Li; Yajun Deng; Dongying Wu; Hongbo Zhang; Hongxing Zhang; Lidong Wang; Chunrong Zhang

    2003-01-01

    Human leukocyte antigen (HLA) system is the most polymorphic region known in the human genome. In the present study, we analyzed for the first time the HLA-A gene polymorphisms defined by the high-resolution typing methods--sequence-based typing (SBT) in 161 Northern Chinese Han people. A total of 74 different HLA-A gene types and 36 alleles were detected. The most frequent alleles were A*110101 (GF=0.2360), A*24020101 (GF=0.1646), and A*020101 (GF=0.1553); followed by A*3303 (GF=0.1180), A*3001 (GF=0.0590),and A*310102 (GF=0.0404). The frequencies of following alleles, A*0203, A*0205,A*0206, A*0207, A*030101, A*2423, A*2601, A*3201, and A*3301, are all higher than 0.0093. The homozygous alleles include A*020101, A*110101, A*24020101 and A*310102. Heterozygosity (H), polymorphism information content (PIC), discrimination power (DP) and probability of paternity exclusion (PPE) of HLA-A in the samples were calculated and their values were 0.8705, 0.8491, 0.6014, and 0.9475, respectively. These results by SBT analysis of HLA-A polymorphism in Northern Chinese Han population, especially the allele subtypes character, will be of great interest for clinical transplantation, disease-associated study and forensic identification. Implementation of high-resolution typing methods allows a significantly wider spectrum of HLA variation including rare alleles. This spectrum will further be extensively utilized in many fields.

  8. Amplification of maximally-path-entangled number states

    Science.gov (United States)

    Agarwal, G. S.; Chaturvedi, S.; Rai, Amit

    2010-04-01

    We examine the behavior of a non-Gaussian state like the maximally path-entangled number state commonly known as a N00N state under phase-insensitive amplification. We derive an analytical result for the density matrix of the N00N state for arbitrary gain of the amplifier. We consider cases of both symmetric and antisymmetric amplification of the two modes of the N00N state. We quantitatively evaluate the loss of entanglement by the amplifier in terms of the logarithmic negativity parameter. We find that N00N states are more robust than their Gaussian counterparts.

  9. Raman amplification in the broken-wave regime

    CERN Document Server

    Farmer, John P

    2015-01-01

    In regimes far beyond the wavebreaking theshold of Raman amplification, we show that significant amplifcation can occur after the onset of wavebreaking, before phase mixing destroys the coupling between pump and probe. The amplification efficiency in this regime is therefore strongly dependent on the energy-transfer rate when wavebreaking occurs, and is, as such, sensitive to both the probe amplitude and profile. In order to access the higher-efficiency broken-wave regime, a short, intense probe is required. Parameter scans show the marked difference in behaviour compared to below wavebreaking, where longer, more energetic pulses lead to improved efficiencies.

  10. Methods for microbial DNA extraction from soil for PCR amplification

    Directory of Open Access Journals (Sweden)

    Yeates C

    1998-01-01

    Full Text Available Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1. DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.

  11. Wavelength division and subcarrier system based on Brillouin amplification

    Science.gov (United States)

    Lee, Yang-Han; Wu, Jingshown; Kao, Ming-Seng; Tsao, Hen-Wai

    1991-10-01

    This paper analyzes an optical wavelength division multiplexing system (WDM) with subcarrier multiplexing (SCM). The pump laser is tuned to amplify the corresponding optical carrier by fiber Brillouin amplification (FBA) in WDM for the desired group of SCM signals and then a microwave tuner is used to select the desired channel in this group. This system has the benefits of eliminating the need of polarization control, the ability of phase noise cancelling due to the 'squaring' photodetection process of the selected optical carrier together with its SCM channels, and enhancement of optical receiver sensitivities by amplification of the carrier.

  12. Ultra-broad bandwidth parametric amplification at degeneracy.

    Science.gov (United States)

    Limpert, J; Aguergaray, C; Montant, S; Manek-Hönninger, I; Petit, S; Descamps, D; Cormier, E; Salin, F

    2005-09-19

    We report on a novel approach of ultra-broad bandwidth parametric amplification around degeneracy. A bandwidth of up to 400 nm centered around 800 nm is amplified in a BBO crystal by using chirped pump pulses with a bandwitdth as broad as 10 nm. A supercontinuum signal is generated in a microstructured fiber, having to first order a quadratic chirp, which is necessary to ensure temporal overlap of the interacting waves over this broad bandwidth. Furthermore, we discuss the potential of this approach for an octave-spanning parametric amplification.

  13. Exponential quadruplex priming amplification for DNA-based isothermal diagnostics.

    Science.gov (United States)

    Partskhaladze, Tamar; Taylor, Adam; Lomidze, Levan; Gvarjaladze, David; Kankia, Besik

    2015-02-01

    Polymerase chain reaction (PCR) is a method of choice for molecular diagnostics. However, PCR relies on thermal cycling, which is not compatible with the goals of point-of-care diagnostics. A simple strategy to turn PCR into an isothermal method would be to use specific primers, which upon polymerase elongation can self-dissociate from the primer-binding sites. We recently demonstrated that a monomolecular DNA quadruplex, GGGTGGGTGGGTGGG, meets these requirements, which led to the development of the linear versions of quadruplex priming amplification (QPA). Here we demonstrate exponential version of isothermal QPA, which allows an unprecedented 10(10)-fold amplification of DNA signal in less than 40 min.

  14. The emergence of surface-based Arctic amplification

    Directory of Open Access Journals (Sweden)

    M. C. Serreze

    2009-02-01

    Full Text Available Rises in surface and lower troposphere air temperatures through the 21st century are projected to be especially pronounced over the Arctic Ocean during the cold season. This Arctic amplification is largely driven by loss of the sea ice cover, allowing for strong heat transfers from the ocean to the atmosphere. Consistent with observed reductions in sea ice extent, fields from both the NCEP/NCAR and JRA-25 reanalyses point to emergence of surface-based Arctic amplification in the last decade.

  15. Influence of environmental noise on the weak value amplification

    Science.gov (United States)

    Zhu, Xuannmin; Zhang, Yu-Xiang

    2016-08-01

    Quantum systems are always disturbed by environmental noise. We have investigated the influence of the environmental noise on the amplification in weak measurements. Three typical quantum noise processes are discussed in this article. The maximum expectation values of the observables of the measuring device decrease sharply with the strength of the depolarizing and phase damping channels, while the amplification effect of weak measurement is immune to the amplitude damping noise. To obtain significantly amplified signals, we must ensure that the preselection quantum systems are kept away from the depolarizing and phase damping processes.

  16. High-frequency electric field amplification in a magnetized plasma

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, Aleksandr V [Russian Research Centre ' Kurchatov Institute' , Moscow (Russian Federation)

    2006-11-30

    In the investigation of cyclotron ion heating in systems designed for plasma isotope separation, the high-frequency (HF) electric field amplification effect was found to occur in equilibrium plasma. In the present article this effect is treated as a result of the interaction of the plasma placed in a constant external magnetic field with the HF modes of the vacuum chamber. Consistent elaboration of this approach allowed obtaining a clear interpretation of the HF electric field amplification effect and constructing a simple model of HF field excitation in a plasma column embedded in the external magnetic field. (methodological notes)

  17. Influence of amplification on pulse shaping for coherent control applications

    CSIR Research Space (South Africa)

    Du Plessis, A

    2011-07-01

    Full Text Available of using low seed laser powers for amplification of shaped pulses in a typical setup for coherent control experiments. An acousto-optic programmable dispersive filter (Dazzler from FastLite) is used to shape 130 fs pulses before amplification... measured as such) for low and high seed powers. Clearly, at lower seed powers as in (a), the measured trace corresponds to approximately the 4:1 ratio expected, but at high seed powers this ratio changes towards 2:1, indicating the smaller of the two...

  18. Amplification of Short Pulse High Power UV Laser

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    At recent year, with the development of CPA and other amplification technology, laser intensity achieves great increase and laser power can be high to PW(105) now, this ultrashort pulse lasers offer scientists a route to investigate laser-matter interaction in an absolute new regime.So far the researches on ultrashort pulse laser-matter interaction concentrated on infrared regime, yet ultraviolet laser has the advantage in intense field physics and ICF researches for its short wavelength and less nonlinear effects. KrF excimer is the best medium in UV ultrashort pulse amplification for its small saturation energy and high contrast ratio accessible.

  19. High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

    Directory of Open Access Journals (Sweden)

    Du Yuefen

    2003-05-01

    Full Text Available Abstract Background Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. Results A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA. Fluorescent signal output was measured in real time and as an end point. Conclusions Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample.

  20. Performance comparison of genetic markers for high-throughput sequencing-based biodiversity assessment in complex communities.

    Science.gov (United States)

    Zhan, Aibin; Bailey, Sarah A; Heath, Daniel D; Macisaac, Hugh J

    2014-09-01

    Metabarcode surveys of DNA extracted from environmental samples are increasingly popular for biodiversity assessment in natural communities. Such surveys rely heavily on robust genetic markers. Therefore, analysis of PCR efficiency and subsequent biodiversity estimation for different types of genetic markers and their corresponding primers is important. Here, we test the PCR efficiency and biodiversity recovery potential of three commonly used genetic markers - nuclear small subunit ribosomal DNA (18S), mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (mt16S) - using 454 pyrosequencing of a zooplankton community collected from Hamilton Harbour, Ontario. We found that biodiversity detection power and PCR efficiency varied widely among these markers. All tested primers for COI failed to provide high-quality PCR products for pyrosequencing, but newly designed primers for 18S and 16S passed all tests. Furthermore, multiple analyses based on large-scale pyrosequencing (i.e. 1/2 PicoTiter plate for each marker) showed that primers for 18S recover more (38 orders) groups than 16S (10 orders) across all taxa, and four vs. two orders and nine vs. six families for Crustacea. Our results showed that 18S, using newly designed primers, is an efficient and powerful tool for profiling biodiversity in largely unexplored communities, especially when amplification difficulties exist for mitochondrial markers such as COI. Universal primers for higher resolution markers such as COI are still needed to address the possible low resolution of 18S for species-level identification.

  1. Rapid detection of sacbrood virus (SBV by one-step reverse transcription loop-mediated isothermal amplification assay

    Directory of Open Access Journals (Sweden)

    Jin-Long Yang

    2012-02-01

    Full Text Available Abstract Background Sacbrood virus (SBV primarily infects honeybee broods, and in order to deal with the problem cost effective detection methods are required. Findings A one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP assay was developed for the rapid identification of SBV. The data demonstrated that, in a simple water bath, SBV RNA could be detected as early as 20 min at 65°C, and a positive amplification reaction was visible to the naked eye due to a color change brought on by the addition of nucleic acid stain SYBR Green. Conclusions The current study presents a method for the rapid and simple detection of SBV by RT-LAMP with high sensitivity and analytic specificity.

  2. A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

    Directory of Open Access Journals (Sweden)

    Gavin J. Nixon

    2014-12-01

    Full Text Available Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR. There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT, akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

  3. A combined sequence-based and fragment-based characterization of microbial eukaryote assemblages provides taxonomic context for the Terminal Restriction Fragment Length Polymorphism (T-RFLP) method.

    Science.gov (United States)

    Kim, Diane Y; Countway, Peter D; Yamashita, Warren; Caron, David A

    2012-12-01

    Microbial eukaryotes in seawater samples collected from two depths (5 m and 500 m) at the USC Microbial Observatory off the coast of Southern California, USA, were characterized by cloning and sequencing of 18S rRNA genes, as well as DNA fragment analysis of these genes. The sequenced genes were assigned to operational taxonomic units (OTUs), and taxonomic information for the sequence-based OTUs was obtained by comparison to public sequence databases. The sequences were then subjected to in silico digestion to predict fragment sizes, and that information was compared to the results of the T-RFLP method applied to the same samples in order to provide taxonomic context for the environmental T-RFLP fragments. A total of 663 and 678 sequences were analyzed for the 5m and 500 m samples, respectively, which clustered into 157 OTUs and 183 OTUs. The sequences yielded substantially fewer taxonomic units as in silico fragment lengths (i.e., following in silico digestion), and the environmental T-RFLP resulted in the fewest unique OTUs (unique fragments). Bray-Curtis similarity analysis of protistan assemblages was greater using the T-RFLP dataset compared to the sequence-based OTU dataset, presumably due to the inability of the fragment method to differentiate some taxa and an inability to detect many rare taxa relative to the sequence-based approach. Nonetheless, fragments in our analysis generally represented the dominant sequence-based OTUs and putative identifications could be assigned to a majority of the fragments in the environmental T-RFLP results. Our empirical examination of the T-RFLP method identified limitations relative to sequence-based community analysis, but the relative ease and low cost of fragment analysis make this method a useful approach for characterizing the dominant taxa within complex assemblages of microbial eukaryotes in large datasets.

  4. Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer.

    Science.gov (United States)

    Purnomosari, D; Aryandono, T; Setiaji, K; Nugraha, S B; Pals, G; van Diest, P J

    2006-01-01

    The HER-2/neu transmembrane tyrosine kinase receptor is both a prognostic marker and a therapeutic target for breast cancer. Accurate determination of HER-2/neu status is a prerequisite for selecting breast tumors for HER-2/neu immunotherapy or for taxan based chemotherapy. Unfortunately, there is no consensus concerning how this determination should be reached. We compared assessment of HER-2/neu status using Multiplex ligation-dependent probe amplification (MLPA) and immunohistochemistry (IHC). The patient group comprised 60 Indonesian breast cancers patients. IHC was performed on paraffin sections using the CB11 antibody from Novocastra. Results were scored according to the Hercept test. For MLPA, DNA was extracted from frozen samples, PCR amplified with a probe set containing three hemi-primer sets for the HER-2 locus and another nine control probes spread over chromosome 17 and other chromosomes, and analyzed on a gene scanner. A ratio above two for at least two HER-2 locus probes compared to the control probes was regarded as amplification. IHC for HER-2/neu was negative in 36 cases, and 24 cases (40%) showed expression. Seven, eight and nine of the latter cases were 1+, 2+ and 3+ positive, respectively. Forty-seven cases showed no amplification by MLPA, and 13 cases (22%) were amplified. Comparison of IHC and MPLA showed that none of the 36 IHC-negative or seven IHC 1+ cases was amplified. Five of the eight (63%) 2+ cases were amplified, and eight of nine (89%) of the IHC 3+ tumors showed gene amplification by MLPA assay. For HER-2/neu, there is a good correlation between gene amplification detected by MLPA and overexpression by IHC in invasive breast cancer. It appears that MLPA can detect the HER-2 amplified cases in the IHC 2+ class. Because MLPA is quick and inexpensive, it is an attractive method for detecting HER-2/neu amplification in daily laboratory practice.

  5. Highly efficient amplification of chronic wasting disease agent by protein misfolding cyclic amplification with beads (PMCAb.

    Directory of Open Access Journals (Sweden)

    Chad J Johnson

    Full Text Available Protein misfolding cyclic amplification (PMCA has emerged as an important technique for detecting low levels of pathogenic prion protein in biological samples. The method exploits the ability of the pathogenic prion protein to convert the normal prion protein to a proteinase K-resistant conformation. Inclusion of Teflon® beads in the PMCA reaction (PMCAb has been previously shown to increase the sensitivity and robustness of detection for the 263 K and SSLOW strains of hamster-adapted prions. Here, we demonstrate that PMCAb with saponin dramatically increases the sensitivity of detection for chronic wasting disease (CWD agent without compromising the specificity of the assay (i.e., no false positive results. Addition of Teflon® beads increased the robustness of the PMCA reaction, resulting in a decrease in the variability of PMCA results. Three rounds of serial PMCAb allowed detection of CWD agent from a 6.7 × 10(-13 dilution of 10% brain homogenate (1.3 fg of source brain. Titration of the same brain homogenate in transgenic mice expressing cervid prion protein (Tg(CerPrP1536(+/- mice allowed detection of CWD agent from the 10(-6 dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 10(5. Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having Prnp alleles associated with reduced disease susceptibility.

  6. Highly efficient amplification of chronic wasting disease agent by protein misfolding cyclical amplification with beads (PMCAb)

    Science.gov (United States)

    Johnson, Chad J.; Aiken, Judd M.; McKenzie, Debbie; Samuel, Michael D.; Pedersen, Joel A.

    2012-01-01

    Protein misfolding cyclic amplification (PMCA) has emerged as an important technique for detecting low levels of pathogenic prion protein in biological samples. The method exploits the ability of the pathogenic prion protein to convert the normal prion protein to a proteinase K-resistant conformation. Inclusion of Teflon® beads in the PMCA reaction (PMCAb) has been previously shown to increase the sensitivity and robustness of detection for the 263 K and SSLOW strains of hamster-adapted prions. Here, we demonstrate that PMCAb with saponin dramatically increases the sensitivity of detection for chronic wasting disease (CWD) agent without compromising the specificity of the assay (i.e., no false positive results). Addition of Teflon® beads increased the robustness of the PMCA reaction, resulting in a decrease in the variability of PMCA results. Three rounds of serial PMCAb allowed detection of CWD agent from a 6.7×10−13 dilution of 10% brain homogenate (1.3 fg of source brain). Titration of the same brain homogenate in transgenic mice expressing cervid prion protein (Tg(CerPrP)1536+/−mice) allowed detection of CWD agent from the 10−6 dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 105. Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having Prnp alleles associated with reduced disease susceptibility.

  7. Rapid DNA amplification using a battery-powered thin-film resistive thermocycler.

    Science.gov (United States)

    Herold, Keith E; Sergeev, Nikolay; Matviyenko, Andriy; Rasooly, Avraham

    2009-01-01

    A prototype handheld, compact, rapid thermocycler was developed for multiplex analysis of nucleic acids in an inexpensive, portable configuration. Instead of the commonly used Peltier heating/cooling element, electric thin-film resistive heater and a miniature fan enable rapid heating and cooling of glass capillaries leading to a simple, low-cost Thin-Film Resistive Thermocycler (TFRT). Computer-based pulse width modulation control yields heating rates of 6-7 K/s and cooling rates of 5 K/s. The four capillaries are closely coupled to the heater, resulting in low power consumption. The energy required by a nominal PCR cycle (20 s at each temperature) was found to be 57+/-2 J yielding an average power of approximately 1.0 W (not including the computer and the control system). Thus the device can be powered by a standard 9 V alkaline battery (or other 9 V power supply). The prototype TFRT was demonstrated (in a benchtop configuration) for detection of three important food pathogens (E. coli ETEC, Shigella dysenteriae, and Salmonella enterica). PCR amplicons were analyzed by gel electrophoresis. The 35 cycle PCR protocol using a single channel was completed in less then 18 min. Simple and efficient heating/cooling, low cost, rapid amplification, and low power consumption make the device suitable for portable DNA amplification applications including clinical point of care diagnostics and field use.

  8. Genotyping of the CCR5 chemokine receptor by isothermal NASBA amplification and differential probe hybridization.

    Science.gov (United States)

    Romano, J W; Tetali, S; Lee, E M; Shurtliff, R N; Wang, X P; Pahwa, S; Kaplan, M H; Ginocchio, C C

    1999-11-01

    The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectability of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems.

  9. DNA quantification via ICP-MS using lanthanide-labeled probes and ligation-mediated amplification.

    Science.gov (United States)

    Brückner, Kathrin; Schwarz, Kathleen; Beck, Sebastian; Linscheid, Michael W

    2014-01-07

    The combination of lanthanide-tagged oligonucleotide probes with inductively coupled plasma mass spectrometry (ICP-MS) as the detection technique is a novel labeling and analysis strategy for heterogeneous nucleic acid quantification assays. We describe a hybridization assay based on biotin-streptavidin affinity using lanthanide-labeled reporter probes and biotinylated capture probes. For the basic sandwich type assay, performed in streptavidin-coated microtitration wells, the limit of detection (LOD) was 7.2 fmol of DNA target, corresponding to a final concentration of 6 pM terbium-labeled probes detectable by ICP-MS after elution from the solid support. To improve the sensitivity and sequence specificity of the approach, it was combined with established molecular biological techniques, i.e., elution with a restriction endonuclease and signal and target amplification by the ligase detection reaction (LDR) and ligase chain reaction (LCR), respectively. Initial experiments showed that the enzymes facilitated the discrimination of single-base mismatches within the recognition or ligation site. Furthermore, LCR as a target amplification step resulted in a 6000-fold increase of sensitivity, and finally an LOD of 2.6 amol was achieved with an artificial double-stranded DNA target.

  10. Distinguishing mechanisms of plasma-based amplification for short laser pulses

    Science.gov (United States)

    Jia, Qing; Edwards, Matthew; Barth, Ido; Mikhailova, Julia; Fisch, Nathaniel

    2016-10-01

    Several plasma-based amplification mechanisms have been proposed to obtain short laser pulses with ultrahigh intensities beyond the damage threshold of solid-state devices, including Compton-like superradiant amplification, backward Raman amplification and strongly-coupled Brillouin amplification. These three mechanisms are all based on the periodic structure of particle (electrons for the former two and ions for Brillouin amplification) density fluctuations that function as a grating. By turning off the ion motion in particle-in-cell simulations, we can distinguish Brillouin from Raman, and show that Raman amplification is responsible for the main leading spike amplification of ultrashort pulses. By artificially turning off the longitudinal electric field (Ex) in simulations, we can distinguish Raman from Compton-like superradiant amplification. Interestingly, we find that the superradiant amplification in Ex-off simulation is similar to the amplification in pair plasmas, with roughly half amplification efficiency of the latter due to absence of equal contribution from positrons. In addition, we also discuss the competition between Brillouin amplification and superradiant amplification in pair plasmas by comparing the dominance of thermal pressure and ponderomotive force.

  11. Reversible Gating of Plasmonic Coupling for Optical Signal Amplification.

    Science.gov (United States)

    Khoury, Christopher G; Fales, Andrew M; Vo-Dinh, Tuan

    2016-07-20

    Amplification of optical signals is useful for a wide variety of applications, ranging from data signal transmission to chemical sensing and biomedical diagnostics. One such application in chemical sensing is surface-enhanced Raman scattering (SERS), an important technique for increasing the Raman signal using the plasmonic effect of enhanced electromagnetic fields associated with metallic nanostructures. One of the most important limitations of SERS-based amplification is the difficulty to reproducibly control the SERS signal. Here, we describe the design and implementation of a unique hybrid system capable of producing reversible gating of plasmonic coupling for Raman signal amplification. The hybrid system is composed of two subsystems: (1) colloidal magneto-plasmonic nanoparticles for SERS enhancement and (2) a micromagnet substrate with an externally applied magnetic field to modulate the colloidal nanoparticles. For this proof of concept demonstration, the nanoparticles were labeled with a Raman-active dye, and it was shown that the detected SERS signal could be reproducibly modulated by controlling the externally applied magnetic field. The developed system provides a simple, robust, inexpensive, and reusable device for SERS signal modulation. These properties will open up new possibilities for optical signal amplification and gating as well for high-throughput, reproducible SERS detection.

  12. Factors Affecting the Benefits of High-Frequency Amplification

    Science.gov (United States)

    Horwitz, Amy R.; Ahlstrom, Jayne B.; Dubno, Judy R.

    2008-01-01

    Purpose: This study was designed to determine the extent to which high-frequency amplification helped or hindered speech recognition as a function of hearing loss, gain-frequency response, and background noise. Method: Speech recognition was measured monaurally under headphones for nonsense syllables low-pass filtered in one-third-octave steps…

  13. Controlling the amplification of chirality in hydrogen-bonded assemblies

    NARCIS (Netherlands)

    Mateos-Timoneda, Miguel A.; Crego-Calama, Mercedes; Reinhoudt, David N.

    2005-01-01

    The amplification of chirality (a high enantiomeric or diastereomeric excess induced by a small initial amount of chiral bias) on hydrogen-bonded assemblies has been studied using “sergeants-and-soldiers” experiments under thermodynamically controlled conditions. Here it is shown that different subs

  14. Whole genome amplification and its impact on CGH array profiles

    Directory of Open Access Journals (Sweden)

    Meldrum Cliff

    2008-07-01

    Full Text Available Abstract Background Some array comparative genomic hybridisation (array CGH platforms require a minimum of micrograms of DNA for the generation of reliable and reproducible data. For studies where there are limited amounts of genetic material, whole genome amplification (WGA is an attractive method for generating sufficient quantities of genomic material from miniscule amounts of starting material. A range of WGA methods are available and the multiple displacement amplification (MDA approach has been shown to be highly accurate, although amplification bias has been reported. In the current study, WGA was used to amplify DNA extracted from whole blood. In total, six array CGH experiments were performed to investigate whether the use of whole genome amplified DNA (wgaDNA produces reliable and reproducible results. Four experiments were conducted on amplified DNA compared to unamplified DNA and two experiments on unamplified DNA compared to unamplified DNA. Findings All the experiments involving wgaDNA resulted in a high proportion of losses and gains of genomic material. Previously, amplification bias has been overcome by using amplified DNA in both the test and reference DNA. Our data suggests that this approach may not be effective, as the gains and losses introduced by WGA appears to be random and are not reproducible between different experiments using the same DNA. Conclusion In light of these findings, the use of both amplified test and reference DNA on CGH arrays may not provide an accurate representation of copy number variation in the DNA.

  15. Fiber amplification of radially and azimuthally polarized laser light

    CERN Document Server

    Fridman, Moti; Dubinskiy, Mark; Friesem, Asher A; Davidson, Nir

    2010-01-01

    The results on amplifying either radially or azimuthally polarized light with a fiber amplifier are presented. Experimental results reveal that more than 85% polarization purity can be retained at the output even with 40dB amplification, and that efficient conversion of the amplified light to linear polarization can be obtained.

  16. Static Generalized Brans-Dicke Universe and Gravitational Waves Amplification

    CERN Document Server

    Berman, M S; Berman, Marcelo S.; Trevisan, Luis A.

    2001-01-01

    We find a static solution for the scale-factor in a Brans-Dicke generalized theory where the scalar field and the coupling constant vary with time. We find also that in the early Universe there may be amplification of gravitational waves.

  17. Sexing Bovine Embryos Using PCR Amplification of Bovine SRY Sequence

    Institute of Scientific and Technical Information of China (English)

    曾溢滔; 张美兰; 陈美珏; 周霞娣; 黄英; 任兆瑞; 黄淑帧; 胡明信; 吴学清; 高建明; 张斌; 徐慧如

    1994-01-01

    This study analyses the bovine SRY DNA sequence by direct sequencing procedure, followed by the designation of the PCR primers specific for bovine SRY. Using PCR amplification of bovine SRY gene, the embryo sex was determined. The results of the embryo sex identification were confirmed after the embryo transfer and pregnancies.

  18. Identification and Characterization of Genomic Amplifications in Ovarian Serous Carcinoma

    Science.gov (United States)

    2008-01-01

    targets in the Notch pathway are the Notch receptors, in which ;-secretase inhibitors prevent the generation of the oncogenic (intracellular) domain of...mutations, and chromosomal amplification at the Notch receptor loci, are the known mechanisms for constitutive activation of Notch pathway . Despite the

  19. Parametric amplification in a micro Coriolis mass flow sensor

    NARCIS (Netherlands)

    Groenesteijn, J.; Droogendijk, H.; Wiegerink, R.J.; Lammerink, T.S.J.; Lötters, J.C.; Sanders, R.G.P.; Krijnen, G.J.M.

    2014-01-01

    We report on the application of parametric amplification to a micro Coriolis mass flow sensor. We demonstrate that this mechanism allows for reduction of the system's power dissipation while retaining sensitivity to flow. By reducing this power dissipation, less heat will be transferred to the fluid

  20. Identification of genetic elements associated with EPSPS gene amplification

    Science.gov (United States)

    Weed populations can have high genetic plasticity and rapid responses to environmental selection pressures. For example, 100-fold amplification of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene evolved to confer resistance to glyphosate, the world's most important herbicide, in the wee...