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


    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. (United States)

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


    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

    Directory of Open Access Journals (Sweden)

    Kager Piet A


    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. (United States)

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


    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 (United States)

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


    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


    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. 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. (United States)

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


    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.

  8. 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


    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.

  9. 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. (United States)

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


    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.

  10. Miniaturized isothermal nucleic acid amplification, a review. (United States)

    Asiello, Peter J; Baeumner, Antje J


    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.

  11. Bioanalytical applications of isothermal nucleic acid amplification techniques. (United States)

    Deng, Huimin; Gao, Zhiqiang


    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.

  12. Isothermal Amplification of Nucleic Acids. (United States)

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


    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.

  13. Enrichment, Amplification, and Sequence-Based Typing of Salmonella enterica and Other Foodborne Pathogens. (United States)

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


    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

  14. Nucleic acid amplification: Alternative methods of polymerase chain reaction. (United States)

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


    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.

  15. 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


    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

  16. A method for amplification of unknown flanking sequences based on touchdown PCR and suppression-PCR. (United States)

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


    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.

  17. A multicenter international evaluation of single-tube amplification protocols for sequencing-based typing of HLA-DRB1 and HLA-DRB3,4,5. (United States)

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


    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.

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

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    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

  19. 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


    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.

  20. Next-generation sequencing-based 5' rapid amplification of cDNA ends for alternative promoters. (United States)

    Perera, Bambarendage P U; Kim, Joomyeong


    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.

  1. 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


    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.

  2. Comparison of Three Nucleic Acid Amplification Assays of Cerebrospinal Fluid for Diagnosis of Cytomegalovirus Encephalitis (United States)

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


    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

  3. Loop-mediated isothermal amplification for detection of nucleic acids. (United States)

    Tanner, Nathan A; Evans, Thomas C


    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.

  4. 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


    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

  5. 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


    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

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


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


    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...

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

    NARCIS (Netherlands)

    Niesters, H G


    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

  8. An integrated portable hand-held analyser for real-time isothermal nucleic acid amplification. (United States)

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


    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.

  9. 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


    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).

  10. Polyethersulfone improves isothermal nucleic acid amplification compared to current paper-based diagnostics. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Michael G. Mauk


    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.

  12. Shortening distance of forward and reverse primers for nucleic acid isothermal amplification. (United States)

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


    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.

  13. 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


    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

  14. Integrated Microfluidic Nucleic Acid Isolation, Isothermal Amplification, and Amplicon Quantification. (United States)

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


    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.

  15. Isothermal amplification detection of nucleic acids by a double-nicked beacon. (United States)

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


    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.

  16. Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges. (United States)

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


    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.

  17. Powerful Amplification Cascades of FRET-Based Two-Layer Nonenzymatic Nucleic Acid Circuits. (United States)

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


    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.

  18. Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids. (United States)

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


    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.

  19. 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

  20. 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


    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.

  1. Nuclemeter: A Reaction-Diffusion Column for Quantifying Nucleic Acids Undergoing Enzymatic Amplification (United States)

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


    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.

  2. Effect of nucleic acid binding dyes on DNA extraction, amplification, and STR typing. (United States)

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


    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.

  3. 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.

  4. 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. (United States)

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


    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.

  5. 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


    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

  6. 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.


    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

  7. 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. (United States)

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


    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.

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

    DEFF Research Database (Denmark)

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


    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...

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

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


    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.

  10. Review of 2005 Public Health Laboratory Network Neisseria gonorrhoeae nucleic acid amplification tests guidelines. (United States)

    Whiley, David M; Lahra, Monica M


    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.

  11. 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.


    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

  12. 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


    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....

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

    Directory of Open Access Journals (Sweden)

    Madhu Mati Goel


    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

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

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


    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.

  15. Entropy Beacon: A Hairpin-Free DNA Amplification Strategy for Efficient Detection of Nucleic Acids. (United States)

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


    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.

  16. Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids (United States)

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


    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

  17. Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic Acids. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Pragati Chigurupati


    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.

  19. Early detection of Mycobacterium tuberculosis complex in BACTEC MGIT cultures using nucleic acid amplification. (United States)

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


    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.

  20. 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


    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.

  1. Successful Combination of Nucleic Acid Amplification Test Diagnostics and Targeted Deferred Neisseria gonorrhoeae Culture. (United States)

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


    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.

  2. A sensitive SERS assay for detecting proteins and nucleic acids using a triple-helix molecular switch for cascade signal amplification. (United States)

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


    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.

  3. Genotyping-by-Sequencing-Based Investigation of the Genetic Architecture Responsible for a ∼Sevenfold Increase in Soybean Seed Stearic Acid. (United States)

    Heim, Crystal B; Gillman, Jason D


    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.

  4. 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


    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.

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

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


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


    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.

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

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Gülnur Tarhan


    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

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

    DEFF Research Database (Denmark)

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


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

  10. 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)



    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.

  11. Two-stage sample-to-answer system based on nucleic acid amplification approach for detection of malaria parasites. (United States)

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


    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.

  12. 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

  13. Label-free electrochemical nucleic acid biosensing by tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme amplification. (United States)

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


    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.

  14. 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.


    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.

  15. On-Chip Isothermal Nucleic Acid Amplification on Flow-Based Chemiluminescence Microarray Analysis Platform for the Detection of Viruses and Bacteria. (United States)

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


    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.

  16. 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


    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

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

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


    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.

  18. A novel, sensitive and label-free loop-mediated isothermal amplification detection method for nucleic acids using luminophore dyes. (United States)

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


    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).

  19. Acid-Breakable Resin-Based Chemical Amplification Positive Resist for Electron-Beam Mastering: Design and Lithographic Performance (United States)

    Sakamizu, Toshio; Shiraishi, Hiroshi


    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).

  20. Ultrasensitive detection of nucleic acids by template enhanced hybridization followed by rolling circle amplification and catalytic hairpin assembly. (United States)

    Song, Weiling; Zhang, Qiao; Sun, Wenbo


    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.

  1. 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


    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

  2. Implementation of Oral and Rectal Gonococcal and Chlamydial Nucleic Acid Amplification-Based Testing as a Component of Local Health Department Activities. (United States)

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


    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.

  3. Sugar-assisted kinetic resolution of amino acids and amplification of enantiomeric excess of organic molecules. (United States)

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


    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.

  4. 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


    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.

  5. 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;


    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...

  6. 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


    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.

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

    DEFF Research Database (Denmark)

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


    ) 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...

  8. Quantitative nucleic acid amplification methods and their implications in clinical virology. (United States)

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


    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.

  9. Quantitative nucleic acid amplification methods and their implications in clinical virology (United States)

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


    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.

  10. Detection of influenza A and B with the Alere™ i Influenza A & B: a novel isothermal nucleic acid amplification assay (United States)

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


    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

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


    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.

  12. 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


    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.

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

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


    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.

  14. 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 (United States)

    Zhu, Xiao; Xing, Da


    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.

  15. Comparative genomics beyond sequence-based alignments

    DEFF Research Database (Denmark)

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


    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...

  16. 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


    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.

  17. Point of care nucleic acid detection of viable pathogenic bacteria with isothermal RNA amplification based paper biosensor (United States)

    Liu, Hongxing; Xing, Da; Zhou, Xiaoming


    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.

  18. 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.

  19. 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

  20. Biomaterials in light amplification (United States)

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


    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

  1. Detection of Vibrio cholerae by isothermal cross-priming amplification combined with nucleic acid detection strip analysis. (United States)

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


    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.

  2. A microfluidic platform for transcription- and amplification-free detection of zepto-mole amounts of nucleic acid molecules. (United States)

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


    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.

  3. 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


    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。

  4. 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 (United States)

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


    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


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    Ricardo Luiz Dantas MACHADO


    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.

  6. 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. (United States)

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


    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.

  7. 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


    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...

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

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    Anja Gulliksen


    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.

  9. 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

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    Rodrigo Otávio Silveira Silva


    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.

  10. Detection of microRNAs in frozen tissue sections by fluorescence in situ hybridization using locked nucleic acid probes and tyramide signal amplification. (United States)

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


    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.

  11. Development of Reverse Transcription Loop-Mediated Isothermal Amplification for Simple and Rapid Detection of Promyelocytic Leukemia–Retinoic Acid Receptor α mRNA (United States)

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


    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

  12. Selective adsorption and chiral amplification of amino acids in vermiculite clay-implications for the origin of biochirality. (United States)

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


    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.

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

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


    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.

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


    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.

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

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


    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

  17. 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

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    Wyatt Dorothy E


    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.

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

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    Feeney Susan A


    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.

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

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    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.

  20. 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


    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

  1. Terabit Nyquist PDM-32QAM signal transmission with training sequence based time domain channel estimation. (United States)

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


    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.

  2. Advances in isothermal amplification: novel strategies inspired by biological processes. (United States)

    Li, Jia; Macdonald, Joanne


    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.

  3. 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


    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.

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


    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. The Seneca Amplification Construction

    Directory of Open Access Journals (Sweden)

    Wallace Chafe


    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.

  6. Amplification of NOON States

    CERN Document Server

    Agarwal, G S; Rai, Amit


    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.

  7. Amplification of NOON States



    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...

  8. Speeding disease gene discovery by sequence based candidate prioritization

    Directory of Open Access Journals (Sweden)

    Porteous David J


    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.

  9. High-Throughput Sequencing Based Methods of RNA Structure Investigation

    DEFF Research Database (Denmark)

    Kielpinski, Lukasz Jan

    describe several computational methods. One that alleviates PCR bias by estimating number of unique molecules existing before the amplification, and two methods for data normalization: one applicable when the paired end sequencing is performed, and the other that works with the single read sequencing...

  10. Gene amplification in carcinogenesis

    Directory of Open Access Journals (Sweden)

    Lucimari Bizari


    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.

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

  12. 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


    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

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

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


    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.

  14. 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. (United States)

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


    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.

  15. 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


    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.

  16. 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


    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.

  17. Loop mediated isothermal amplification: An innovative gene amplification technique for animal diseases. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Pravas Ranjan Sahoo


    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.

  19. 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


    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.

  20. Amplification of an MFS transporter encoding gene penT significantly stimulates penicillin production and enhances the sensitivity of Penicillium chrysogenum to phenylacetic acid. (United States)

    Yang, Jing; Xu, Xinxin; Liu, Gang


    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.

  1. 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. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)



    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.

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

    Noor, M Omair; Krull, Ulrich J


    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

  4. Hardness amplification in nondeterministic logspace


    Gupta, Sushmita


    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 ...

  5. 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


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


    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...

  6. Antibiotic Selection Pressure Determination through Sequence-Based Metagenomics. (United States)

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


    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.

  7. 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

  8. Quality control for quantitative PCR based on amplification compatibility test. (United States)

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


    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.

  9. Efficient audio power amplification - challenges

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Michael A.E.


    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)

  10. 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


    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.

  11. 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


    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)

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


    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.

  13. Uncertainties in Site Amplification Estimation (United States)

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


    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.

  14. 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


    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.

  15. 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.


    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.

  16. 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


    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

  17. 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. (United States)

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


    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.

  18. Secondary Structure Predictions for Long RNA Sequences Based on Inversion Excursions and MapReduce. (United States)

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


    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.

  19. Sequencing-based typing reveals new insight in HLA-DPA1 polymorphism. (United States)

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


    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)

  20. Isothermal Amplification of Insect DNA (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...

  1. 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.

  2. Controlled Microwave Heating Accelerates Rolling Circle Amplification. (United States)

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


    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.

  3. Transgenerational inheritance: Models and mechanisms of non-DNA sequence-based inheritance. (United States)

    Miska, Eric A; Ferguson-Smith, Anne C


    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.

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

    DEFF Research Database (Denmark)

    Lundegaard, Claus; Hoof, Ilka; Lund, Ole


    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...

  5. Spheromak Impedance and Current Amplification

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  6. 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


    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

  7. 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


    【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

  8. Magnetism Teaching Sequences Based on an Inductive Approach for First-Year Thai University Science Students (United States)

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


    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…

  9. 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.


    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

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

    DEFF Research Database (Denmark)

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


    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...

  11. 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.

  12. Dynamics and Control of DNA Sequence Amplification

    CERN Document Server

    Marimuthu, Karthikeyan


    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...

  13. 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:, E-mail: [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)


    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.

  14. 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


    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.

  15. Sequence-based characterization of five SLA loci in Asian wild boars. (United States)

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


    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.

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


    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.

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


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


    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...

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



    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...

  19. Chirality Amplification in Tactoids of Lyotropic Chromonic Liquid Crystals (United States)

    Peng, Chenhui; Lavrentovich, Oleg


    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.

  20. 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. (United States)

    Ota, Kaede V; McGowan, Karin L


    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.

  1. 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


    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

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

    DEFF Research Database (Denmark)

    Tolle, Fabian; Wilke, Julian; Wengel, Jesper


    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...

  3. Multiscale image contrast amplification (MUSICA) (United States)

    Vuylsteke, Pieter; Schoeters, Emile P.


    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.

  4. Amplification of cellular oncogenes in solid tumors

    Directory of Open Access Journals (Sweden)

    Ozkan Bagci


    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.

  5. Pulse Compression And Raman Amplification In Optical Fibres (United States)

    Byron, Kevin C.


    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.

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

    Directory of Open Access Journals (Sweden)

    A. Valentini


    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.

  7. Linking Arctic amplification and local feedbacks (United States)

    Balcerak, Ernie


    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)

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

    Directory of Open Access Journals (Sweden)

    Shaoxia Zhou


    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.

  9. 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)


    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.

  10. A 2-D graphical representation of protein sequences based on nucleotide triplet codons (United States)

    Bai, Fenglan; Wang, Tianming


    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.

  11. Extension of the Legionella pneumophila sequence-based typing scheme to include strains carrying a variant of the N-acylneuraminate cytidylyltransferase gene. (United States)

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


    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.

  12. Quantum Amplitude Amplification and Estimation

    CERN Document Server

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


    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...

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

    Directory of Open Access Journals (Sweden)

    Francisco Alexandre P


    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

  14. Raman amplification in optical communication systems

    DEFF Research Database (Denmark)

    Kjær, Rasmus


    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...

  15. Can Anomalous Amplification be Attained Without Postselection?

    CERN Document Server

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


    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...

  16. Rolling circle amplification of metazoan mitochondrialgenomes

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Yogesh eChander


    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

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


    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.

  19. Application of a Non-amplification based Technology to Detect Invasive Fungal Pathogens (United States)

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


    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. 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


    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.

  1. Heat induces gene amplification in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Bin, E-mail: [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)


    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

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

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


    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. 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


    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.

  4. Study on multiple-hops performance of MOOC sequences-based optical labels for OPS networks (United States)

    Zhang, Chongfu; Qiu, Kun; Ma, Chunli


    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.

  5. 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


    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.

  6. Amplification, Redundancy, and Quantum Chernoff Information (United States)

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


    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.

  7. On Arbitrary Phases in Quantum Amplitude Amplification

    CERN Document Server

    Hoyer, P


    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.

  8. Continuous phase amplification with a Sagnac interferometer

    CERN Document Server

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


    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.

  9. Effective Privacy Amplification for Secure Classical Communications

    CERN Document Server

    Horvath, Tamas; Scheuer, Jacob


    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.

  10. Parametric Amplification For Detecting Weak Optical Signals (United States)

    Hemmati, Hamid; Chen, Chien; Chakravarthi, Prakash


    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.

  11. A dual amplification fluorescent strategy for sensitive detection of DNA methyltransferase activity based on strand displacement amplification and DNAzyme amplification. (United States)

    Cui, Wanling; Wang, Lei; Jiang, Wei


    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.

  12. A rapid and robust sequence-based genotyping method for BoLA-DRB3 alleles in large numbers of heterozygous cattle. (United States)

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


    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.

  13. 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


    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.

  14. M-Sequence-Based Single-Chip UWB-Radar Sensor (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).

  15. Comparison of serological and sequence-based methods for typing feline calcivirus isolates from vaccine failures. (United States)

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


    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.

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

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

    KAUST Repository

    Wang, Bing


    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.

  18. 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.

  19. Application of Sequence-based Methods in Human MicrobialEcology

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Dybowski J Nikolaj


    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.

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

    Directory of Open Access Journals (Sweden)

    Hui Li


    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.

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

  3. 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)


    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.

  4. DNA Extraction and Amplification from Contemporary Polynesian Bark-Cloth (United States)

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


    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

  5. 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.

  6. Intelligence amplification framework for enhancing scheduling processes

    NARCIS (Netherlands)

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


    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

  7. 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.


    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.

  8. Desert Amplification in a Warming Climate (United States)

    Zhou, Liming


    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.

  9. Rapid microfluidic thermal cycler for nucleic acid amplification

    Energy Technology Data Exchange (ETDEWEB)

    Beer, Neil Reginald; Vafai, Kambiz


    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.

  10. A new evolutionary theory deduced mathematically from entropy amplification

    Institute of Scientific and Technical Information of China (English)


    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.

  11. MICA polymorphism in a population from north Morocco, Metalsa Berbers, using sequence-based typing. (United States)

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


    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.

  12. A new trilocus sequence-based multiplex-PCR to detect major Acinetobacter baumannii clones. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Ahmed Ahmed


    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.

  14. SEQMINER: An R-Package to Facilitate the Functional Interpretation of Sequence-Based Associations. (United States)

    Zhan, Xiaowei; Liu, Dajiang J


    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 and

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

    DEFF Research Database (Denmark)

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


    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...

  16. Isothermal DNA amplification in bioanalysis: strategies and applications. (United States)

    Kim, Joonyul; Easley, Christopher J


    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.

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

    Directory of Open Access Journals (Sweden)

    Igor Nefedov


    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.

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


    Igor Nefedov; Leonid Melnikov


    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.

  19. Post-Fragmentation Whole Genome Amplification-Based Method (United States)

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


    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 (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 practically every step, particularly nucleic acid extraction. In engineering a molecular means of amplifying nucleic acids directly from single cells in their native state within the sample matrix, this innovation has circumvented entirely the need for DNA extraction regimes in the sample processing scheme.

  20. RNA amplification for successful gene profiling analysis

    Directory of Open Access Journals (Sweden)

    Wang Ena


    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

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

    Directory of Open Access Journals (Sweden)

    Park Jong


    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

  2. Gravito-magnetic amplification in cosmology

    CERN Document Server

    Tsagas, Christos G


    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.

  3. Parametric nanomechanical amplification at very high frequency. (United States)

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


    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.

  4. Amplification of postwildfire peak flow by debris (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Chari Raj


    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

  6. Internal entanglement amplification by external interactions



    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...


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


    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

  8. Characterization of the novel HLA-Cw*0624 allele identified by sequence-based typing. (United States)

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


    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.

  9. RNA amplification of bromodeoxyuridine labeled newborn neurons in the monkey hippocampus. (United States)

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


    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.

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



    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...

  11. Amplification Without Inversion in Semiconductor Quantum Dot (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.

  12. Amplification Effects and Unconventional Monetary Policies

    Directory of Open Access Journals (Sweden)



    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.

  13. Amplification and characterization of eukaryotic structural genes. (United States)

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


    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.

  14. 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.


    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.

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

    DEFF Research Database (Denmark)

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


    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...

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


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


    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...

  17. Recursive organizer (ROR): an analytic framework for sequence-based association analysis. (United States)

    Zhao, Lue Ping; Huang, Xin


    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.

  18. A machine-learning approach for predicting palmitoylation sites from integrated sequence-based features. (United States)

    Li, Liqi; Luo, Qifa; Xiao, Weidong; Li, Jinhui; Zhou, Shiwen; Li, Yongsheng; Zheng, Xiaoqi; Yang, Hua


    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.

  19. Magnetic Field Amplification in Young Galaxies

    CERN Document Server

    Schober, Jennifer; Klessen, Ralf S


    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 ...

  20. Experimental noiseless linear amplification using weak measurements (United States)

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


    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.

  1. 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


    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.

  2. Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay. (United States)

    Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan


    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.

  3. 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


    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

  4. 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


    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

  5. Use of signal-mediated amplification of RNA technology (SMART) to detect marine cyanophage DNA. (United States)

    Hall, M J; Wharam, S D; Weston, A; Cardy, D L N; Wilson, W H


    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.

  6. Amplification of Chirality through Self-Replication of Micellar Aggregates in Water

    KAUST Repository

    Bukhriakov, Konstantin


    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.

  7. Optofluidic analysis system for amplification-free, direct detection of Ebola infection (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.


    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.

  8. 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


    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

  9. Development of internal amplification controls for DNA profiling with the AmpFℓSTR(®) SGM Plus(®) kit. (United States)

    Zahra, Nathalie; Hadi, Sibte; Smith, Judith A; Iyengar, Arati; Goodwin, William


    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.

  10. Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples. (United States)

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


    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.

  11. Control and amplification of cortical neurodynamics (United States)

    Liljenstroem, Hans; Aronsson, P.


    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.

  12. Amplification sans bruit d'images optiques (United States)

    Gigan, S.; Delaubert, V.; Lopez, L.; Treps, N.; Maitre, A.; Fabre, C.


    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.

  13. Dispersion compensation in chirped pulse amplification systems (United States)

    Bayramian, Andrew James; Molander, William A.


    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.

  14. Magnetic field amplification in turbulent astrophysical plasmas

    CERN Document Server

    Federrath, Christoph


    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...

  15. Anisotropic metamaterials with simultaneous attenuation and amplification

    CERN Document Server

    Mackay, Tom G


    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.

  16. Integrated Amplification Microarrays for Infectious Disease Diagnostics

    Directory of Open Access Journals (Sweden)

    Darrell P. Chandler


    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.

  17. Magnetic Field Amplification and Blazar Flares

    CERN Document Server

    Chen, Xuhui; Fossati, Giovanni; Pohl, Martin


    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...

  18. Short-Pulse Amplification by Strongly-Coupled Brillouin Scattering

    CERN Document Server

    Edwards, Matthew R; Mikhailova, Julia M; Fisch, Nathaniel J


    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.

  19. 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


    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.

  20. Sequence-based Association Analysis Reveals an MGST1 eQTL with Pleiotropic Effects on Bovine Milk Composition (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.


    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

  1. Targeting HER2 amplifications in gastric cancer

    Directory of Open Access Journals (Sweden)

    Ung L


    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. (United States)

    Chrostek, Ewa; Teixeira, Luis


    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. 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.

  4. Parametric Analog Signal Amplification Applied to Nanoscale CMOS Technologies

    CERN Document Server

    Oliveira, João P


    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.

  5. Hendra virus detection using Loop-Mediated Isothermal Amplification. (United States)

    Foord, Adam J; Middleton, Deborah; Heine, Hans G


    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.

  6. 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.

  7. Rapid detection of Brucella spp. using loop-mediated isothermal amplification (LAMP). (United States)

    Chen, Shouyi; Li, Xunde; Li, Juntao; Atwill, Edward R


    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.

  8. 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


    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.

  9. GMO detection in food and feed through screening by visual loop-mediated isothermal amplification assays. (United States)

    Wang, Cong; Li, Rong; Quan, Sheng; Shen, Ping; Zhang, Dabing; Shi, Jianxin; Yang, Litao


    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.

  10. Kinetic Hairpin Oligonucleotide Blockers for Selective Amplification of Rare Mutations (United States)

    Jia, Yanwei; Sanchez, J. Aquiles; Wangh, Lawrence J.


    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

  11. 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

  12. 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


    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.

  13. 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


    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

  14. Limits for superfocusing with finite evanescent wave amplification

    CERN Document Server

    Gordon, Reuven


    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.

  15. Complementary weak-value amplification with concatenated postselections

    CERN Document Server

    Viza, Gerardo I; Liu, Wei-Tao; Howell, John C


    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.

  16. Fluorescence amplification by electrochemically deposited silver nanowires with fractal architecture. (United States)

    Goldys, Ewa M; Drozdowicz-Tomsia, Krystyna; Xie, Fang; Shtoyko, Tanya; Matveeva, Eva; Gryczynski, Ignacy; Gryczynski, Zygmunt


    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.

  17. Backward Raman amplification in the long-wavelength infrared (United States)

    Johnson, L. A.; Gordon, D. F.; Palastro, J. P.; Hafizi, B.


    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.

  18. Low cost extraction and isothermal amplification of DNA for infectious diarrhea diagnosis. (United States)

    Huang, Shichu; Do, Jaephil; Mahalanabis, Madhumita; Fan, Andy; Zhao, Lei; Jepeal, Lisa; Singh, Satish K; Klapperich, Catherine M


    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.

  19. 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


    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.

  20. KASER: Knowledge Amplification by Structured Expert Randomization. (United States)

    Rubin, Stuart H; Murthy, S N Jayaram; Smith, Michael H; Trajković, Ljiljana


    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.

  1. Local Runup Amplification By Resonant Wave Interactions

    CERN Document Server

    Stefanakis, Themistoklis; Dutykh, Denys


    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...

  2. Protein misfolding cyclic amplification of infectious prions. (United States)

    Morales, Rodrigo; Duran-Aniotz, Claudia; Diaz-Espinoza, Rodrigo; Camacho, Manuel V; Soto, Claudio


    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.

  3. A PCR amplification method without DNA extraction. (United States)

    Li, Hongwei; Xu, Haiyue; Zhao, Chunjiang; Sulaiman, Yiming; Wu, Changxin


    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.

  4. 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


    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.

  5. Quantum dot layer-by-layer assemblies as signal amplification labels for ultrasensitive electronic detection of uropathogens. (United States)

    Xiang, Yun; Zhang, Haixia; Jiang, Bingying; Chai, Yaqin; Yuan, Ruo


    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.

  6. 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.


    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

  7. Nonlinear Zel'dovich effect: Parametric amplification from medium rotation

    CERN Document Server

    Faccio, Daniele


    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.

  8. Nonlinear Zel'dovich Effect: Parametric Amplification from Medium Rotation (United States)

    Faccio, Daniele; Wright, Ewan M.


    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.

  9. The Amplification in FEL with Inhomogeneous Magnetic Field

    CERN Document Server

    Oganesyan, K B


    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

  10. Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones. (United States)

    Rodriguez-Manzano, Jesus; Karymov, Mikhail A; Begolo, Stefano; Selck, David A; Zhukov, Dmitriy V; Jue, Erik; Ismagilov, Rustem F


    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.

  11. Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms. (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


    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.

  12. 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

  13. 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)



    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

  14. Rapid ultrasonic isothermal amplification of DNA with multiplexed melting analysis – applications in the clinical diagnosis of sexually transmitted diseases. (United States)

    Xu, Gaolian; Gunson, Rory N; Cooper, Jonathan M; Reboud, Julien


    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.

  15. Human identification from forensic materials by amplification of a human-specific sequence in the myoglobin gene.


    Ono T; Miyaishi S; Yamamoto Y; Yoshitome K; Ishikawa T.; Ishizu H


    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 ...

  16. Methods for microbial DNA extraction from soil for PCR amplification


    Yeates C; Gillings, MR; Davison AD; Altavilla N; Veal DA


    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...

  17. Pulse Distortion in Saturated Fiber Optical Parametric Chirped Pulse Amplification

    DEFF Research Database (Denmark)

    Lali-Dastjerdi, Zohreh; Da Ros, Francesco; Rottwitt, Karsten


    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....

  18. Aerosol Lidar for the Relative Backscatter Amplification Measurements (United States)

    Razenkov, Igor A.; Banakh, Victor A.; Nadeev, Alexander I.


    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.

  19. The emergence of surface-based Arctic amplification

    Directory of Open Access Journals (Sweden)

    M. C. Serreze


    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.

  20. 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


    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.

  1. Engineering targeted chromosomal amplifications in human breast epithelial cells. (United States)

    Springer, Simeon; Yi, Kyung H; Park, Jeenah; Rajpurohit, Anandita; Price, Amanda J; Lauring, Josh


    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.

  2. The emergence of surface-based Arctic amplification


    SERREZE, M. C.; A. P. Barrett; J. C. Stroeve; Kindig, D. N.; Holland, M. M.


    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.

  3. 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: [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)


    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. Targeting MET Amplification as a New Oncogenic Driver

    Directory of Open Access Journals (Sweden)

    Hisato Kawakami


    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.

  5. Multiplex isothermal solid-phase recombinase polymerase amplification for the specific and fast DNA-based detection of three bacterial pathogens. (United States)

    Kersting, Sebastian; Rausch, Valentina; Bier, Frank F; von Nickisch-Rosenegk, Markus


    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. Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications. (United States)

    Du, Yi-Chen; Zhu, Li-Na; Kong, De-Ming


    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.

  7. 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 (United States)

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


    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

  8. 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. (United States)

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


    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.

  9. Continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay. (United States)

    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji


    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.

  10. Amplification of thermostable lipase genes fragment from thermogenic phase of domestic waste composting process (United States)

    Nurhasanah, Nurbaiti, Santi; Madayanti, Fida; Akhmaloka


    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. Direct ultrasensitive electrochemical biosensing of pathogenic DNA using homogeneous target-initiated transcription amplification (United States)

    Yan, Yurong; Ding, Shijia; Zhao, Dan; Yuan, Rui; Zhang, Yuhong; Cheng, Wei


    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.

  12. ProteDNA: a sequence-based predictor of sequence-specific DNA-binding residues in transcription factors. (United States)

    Chu, Wen-Yi; Huang, Yu-Feng; Huang, Chun-Chin; Cheng, Yi-Sheng; Huang, Chien-Kang; Oyang, Yen-Jen


    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:,,

  13. 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


    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.

  14. [Homologous Analysis Using Repetitive-sequence-based PCR Typing of Exfoliative Toxin-producing Staphylococcus aureus Isolated from Our Hospital]. (United States)

    Miyamoto, Hitoshi; Murakami, Shinobu; Nishimiya, Tatsuya; Suemori, Koichiro; Tauchi, Hisamichi


    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.

  15. ASAP: Amplification, sequencing & annotation of plastomes

    Directory of Open Access Journals (Sweden)

    Folta Kevin M


    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

  16. A continuous-flow ATP amplification system for increasing the sensitivity of quantitative bioluminescence assay


    Satoh, Tetsuya; Shinoda, Yasuharu; Alexandrov, Maxym; Kuroda, Akio; Murakami, Yuji


    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...

  17. Regulation of ribosomal DNA amplification by the TOR pathway. (United States)

    Jack, Carmen V; Cruz, Cristina; Hull, Ryan M; Keller, Markus A; Ralser, Markus; Houseley, Jonathan


    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.

  18. Identification by sequencing based typing and complete coding region analysis of three new HLA class II alleles: DRB3*0210, DRB3*0211 and DQB1*0310. (United States)

    Balas, A; Santos, S; Aviles, M J; Garcia-Sanchez, F; Lillo, R; Vicario, J L


    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.

  19. Optical Parametric Amplification for High Peak and Average Power

    Energy Technology Data Exchange (ETDEWEB)

    Jovanovic, I


    Optical parametric amplification is an established broadband amplification technology based on a second-order nonlinear process of difference-frequency generation (DFG). When used in chirped pulse amplification (CPA), the technology has been termed optical parametric chirped pulse amplification (OPCPA). OPCPA holds a potential for producing unprecedented levels of peak and average power in optical pulses through its scalable ultrashort pulse amplification capability and the absence of quantum defect, respectively. The theory of three-wave parametric interactions is presented, followed by a description of the numerical model developed for nanosecond pulses. Spectral, temperature and angular characteristics of OPCPA are calculated, with an estimate of pulse contrast. An OPCPA system centered at 1054 nm, based on a commercial tabletop Q-switched pump laser, was developed as the front end for a large Nd-glass petawatt-class short-pulse laser. The system does not utilize electro-optic modulators or multi-pass amplification. The obtained overall 6% efficiency is the highest to date in OPCPA that uses a tabletop commercial pump laser. The first compression of pulses amplified in highly nondegenerate OPCPA is reported, with the obtained pulse width of 60 fs. This represents the shortest pulse to date produced in OPCPA. Optical parametric amplification in {beta}-barium borate was combined with laser amplification in Ti:sapphire to produce the first hybrid CPA system, with an overall conversion efficiency of 15%. Hybrid CPA combines the benefits of high gain in OPCPA with high conversion efficiency in Ti:sapphire to allow significant simplification of future tabletop multi-terawatt sources. Preliminary modeling of average power limits in OPCPA and pump laser design are presented, and an approach based on cascaded DFG is proposed to increase the average power beyond the single-crystal limit. Angular and beam quality effects in optical parametric amplification are modeled

  20. Magnetic Amplification by Magnetized Cosmic Rays in SNR Shocks

    CERN Document Server

    Riquelme, Mario A


    (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...

  1. Processes and impacts of Arctic amplification: A research synthesis (United States)

    Serreze, Mark C.; Barry, Roger G.


    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.

  2. Amplification of Information by Photons and the Quantum Chernoff Bound (United States)

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


    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,

  3. Local seismic site amplification: effects of obliquely incident antiplane motions (United States)

    Cherid, D.; Hammoutene, M.; Tiliouine, B.; Berrah, M. K.


    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.

  4. Adaptive base-isolation of civil structures using variable amplification

    Institute of Scientific and Technical Information of China (English)

    Kenneth K. Walsh; Makola M. Abdullah


    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.

  5. Evaluating the displacement amplification factors of concentrically braced steel frames (United States)

    Mahmoudi, Mussa; Zaree, Mahdi


    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.

  6. Modeling the amplification dynamics of human Alu retrotransposons.

    Directory of Open Access Journals (Sweden)

    Dale J Hedges


    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.

  7. 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.


    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

  8. Somatic recombination, gene amplification and cancer. (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


    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

  9. 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


    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.

  10. Identification of cancer predisposition variants in apparently healthy individuals using a next-generation sequencing-based family genomics approach. (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


    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.

  11. Sequence-based genotyping clarifies conflicting historical morphometric and biological data for 5 Eimeria species infecting turkeys. (United States)

    El-Sherry, S; Ogedengbe, M E; Hafeez, M A; Sayf-Al-Din, M; Gad, N; Barta, J R


    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

  12. Monitoring transmission routes of Listeria spp. in smoked salmon production with repetitive element sequence-based PCR techniques. (United States)

    Zunabovic, M; Domig, K J; Pichler, I; Kneifel, W


    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.

  13. Measurement-based noiseless linear amplification for quantum communication (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.


    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.

  14. Linear Amplification of Optical Signal in Coupled Photonic Crystal Waveguides

    CERN Document Server

    Jandieri, Vakhtang


    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.

  15. Determining Parameters for Images Amplification by Pulses Interpolation

    Directory of Open Access Journals (Sweden)

    Morera-Delfín Leandro


    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.

  16. An Intrinsically Digital Amplification Scheme for Hearing Aids

    Directory of Open Access Journals (Sweden)

    Brenton R. Steele


    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.

  17. 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. (United States)

    Kim, Diane Y; Countway, Peter D; Yamashita, Warren; Caron, David A


    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.

  18. Performance comparison of genetic markers for high-throughput sequencing-based biodiversity assessment in complex communities. (United States)

    Zhan, Aibin; Bailey, Sarah A; Heath, Daniel D; Macisaac, Hugh J


    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.

  19. The efficiency of magnetic field amplification at shocks by turbulence (United States)

    Ji (), Suoqing; Oh, S. Peng; Ruszkowski, M.; Markevitch, M.


    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.

  20. 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.

  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


    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. High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

    Directory of Open Access Journals (Sweden)

    Du Yuefen


    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.

  3. Theory of light amplification in active fishnet metamaterials

    CERN Document Server

    Hamm, Joachim M; Tsakmakidis, Kosmas L; Hess, Ortwin


    We establish a theory that traces light amplification in an active double-fishnet metamaterial back to its microscopic origins. Based on ab initio calculations of the light/plasmon fields we extract energy rates and conversion efficiencies associated with gain/loss channels directly from Poynting's theorem. We find that for the negative refactive index mode both radiative loss and gain outweigh resistive loss by more than a factor of two, opening a broad window of steady-state amplification (free of instabilities) accessible even when a gain reduction close to the metal is taken into account.

  4. 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)


    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)

  5. Exponential quadruplex priming amplification for DNA-based isothermal diagnostics. (United States)

    Partskhaladze, Tamar; Taylor, Adam; Lomidze, Levan; Gvarjaladze, David; Kankia, Besik


    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.

  6. Methods for microbial DNA extraction from soil for PCR amplification

    Directory of Open Access Journals (Sweden)

    Yeates C


    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.

  7. The emergence of surface-based Arctic amplification

    Directory of Open Access Journals (Sweden)

    M. C. Serreze


    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.

  8. Amplification of Short Pulse High Power UV Laser

    Institute of Scientific and Technical Information of China (English)


    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.

  9. Influence of environmental noise on the weak value amplification (United States)

    Zhu, Xuannmin; Zhang, Yu-Xiang


    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.

  10. Ultra-broad bandwidth parametric amplification at degeneracy. (United States)

    Limpert, J; Aguergaray, C; Montant, S; Manek-Hönninger, I; Petit, S; Descamps, D; Cormier, E; Salin, F


    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.

  11. Raman amplification in the broken-wave regime

    CERN Document Server

    Farmer, John P


    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.

  12. Amplification of maximally-path-entangled number states (United States)

    Agarwal, G. S.; Chaturvedi, S.; Rai, Amit


    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.

  13. Comparison of multiplex ligation dependent probe amplification to immunohistochemistry for assessing HER-2/neu amplification in invasive breast cancer. (United States)

    Purnomosari, D; Aryandono, T; Setiaji, K; Nugraha, S B; Pals, G; van Diest, P J


    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.

  14. Classification of Cryptosporidium species from patients with sporadic cryptosporidiosis by use of sequence-based multilocus analysis following mutation scanning. (United States)

    Jex, Aaron R; Pangasa, Aradhana; Campbell, Bronwyn E; Whipp, Margaret; Hogg, Geoff; Sinclair, Martha I; Stevens, Melita; Gasser, Robin B


    In the present study, we analyzed genetic variation in Cryptosporidium species from humans (n = 62) with clinical cryptosporidiosis in South Australia. Sequence variation was assessed in regions within the small subunit of nuclear rRNA (p-SSU), the 70-kDa heat shock protein (p-hsp70), and the 60-kDa glycoprotein (p-gp60) genes by employing single-strand conformation polymorphism analysis and sequencing. Based on the analyses of p-SSU and p-hsp70, Cryptosporidium hominis (n = 38) and Cryptosporidium parvum (n = 24) were identified. The analysis of p-gp60 revealed eight distinct subgenotypes, classified as C. hominis IaA17R1 (n = 3), IbA9G3R2 (n = 14), IbA10G2R2 (n = 20), and IfA12G1R1 (n = 1), as well as C. parvum IIaA18G3R1 (n = 15), IIaA20G3R1 (n = 6), IIaA22G4R1 (n = 2), and IIcA5G3R2 (n = 1). Subgenotypes IaA17R1 and IIaA22G4R1 are new. Of the six other subgenotypes, IbA10G2R2, IIaA18G3R1, IIaA20G3R1, and IIcA5G3R2 were reported previously from the state of Victoria. This is the fourth record in Australia of C. parvum subgenotype IIaA18G3R1 from humans, which, to date, has been isolated only from cattle in other countries. This subgenotype might be a significant contributor to sporadic human cryptosporidiosis and may indicate a greater zoonotic contribution to the infection of humans in the area of study. Comparative analyses revealed, for the first time, the differences in the genetic makeup of Cryptosporidium populations between two relatively close, major metropolitan cities.

  15. Functional continuity: did field-induced oriented aperiodic constraints at Life's origin aid its sequence-based evolution? (United States)

    Mitra-Delmotte, G.; Mitra, A. N.


    A non-biological analog undergoing Darwinian-like evolution could have enhanced the probability of many crucial independent bottom-up emergent steps, engendered within its premises, and smoothen the inanimate-animate transition. Now, the higher-level environment-mutable DNA sequences influence the lower-level pattern of oriented templates (enzymes, lipid membranes, RNA) in the organized cell matrix and hence their associated substrate-dynamics; note how templates are akin to local fields, kinetically constraining reactant orientations. Since the lowerlevel is likely the more primitive of the two (rather than Cairns-Smith's "readily available" rigid clay crystal sequence-based replicators as a memory-like basis for slowly mutating predecessor-patterns enroute to complex RNA-based Darwinian evolution), a gradual thermodynamic-to-kinetic transition in an isotropic medium, is proposed as driven by some order-parameter --via "available" field-responsive dipolar colloid networks, as apart from bio-organics, mineral colloids also can display liquid crystal (LC) phases (see [1]). An access to solid-like orientational order in a fluid matrix suggests how aperiodic patterns can be influenced and sustained (a la homeostasis) via external inhomogeneous fields (e.g. magnetic rocks); this renders these cooperative networks with potential as confining host-media, whose environment-sensitivity can not only influence their sterically-coupled guest-substrates but also their network properties (the latter can enable 'functions' like spontaneous transport under non-equilibrium suggesting a natural basis for their selection by the environment). In turn LC systems could have been preceded by even simpler anisotropic fluid hosts, viz., external field-induced mineral magnetic nanoparticle (MNP) aggregates. Indeed, the capacity of an MNP to couple its magnetic and rotational d.o.f.s suggests how an environment-sensitive field-influenced network of interacting dipolar colloids close to

  16. 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.

  17. Highly efficient amplification of chronic wasting disease agent by protein misfolding cyclical amplification with beads (PMCAb) (United States)

    Johnson, Chad J.; Aiken, Judd M.; McKenzie, Debbie; Samuel, Michael D.; Pedersen, Joel A.


    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.

  18. Rapid DNA amplification using a battery-powered thin-film resistive thermocycler. (United States)

    Herold, Keith E; Sergeev, Nikolay; Matviyenko, Andriy; Rasooly, Avraham


    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.

  19. Genotyping of the CCR5 chemokine receptor by isothermal NASBA amplification and differential probe hybridization. (United States)

    Romano, J W; Tetali, S; Lee, E M; Shurtliff, R N; Wang, X P; Pahwa, S; Kaplan, M H; Ginocchio, C C


    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.

  20. Distinguishing mechanisms of plasma-based amplification for short laser pulses (United States)

    Jia, Qing; Edwards, Matthew; Barth, Ido; Mikhailova, Julia; Fisch, Nathaniel


    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.

  1. Direct Extraction and Amplification of DNA from Soil. (United States)

    Trevors, Jack T.; Leung, K.


    Presents an exercise that describes the direct extraction and purification of DNA from a small soil sample. Also discusses the subsequent amplification of a 343-bp Tn7 transposate A gene fragment (tnsA) from a strain of Pseudomonas aureofaciens 3732RNL11. Contains 21 references. (DDR)

  2. Ultrafast double-pulse parametric amplification for precision Ramsey metrology

    NARCIS (Netherlands)

    Kandula, D.Z.; Renault, A.A.L.; Gohle, C.; Wolf, A.L.; Witte, S.; Hogervorst, W.; Ubachs, W.M.G.; Eikema, K.S.E.


    We demonstrate phase stable, mJ-level parametric amplification of pulse pairs originating from a Ti: Sapphire frequency comb laser. The amplifier-induced phase shift between the pulses has been determined interferometrically with an accuracy of approximate to 10 mrad. Typical phase shifts are on the

  3. Barcoded Primers Used in Multiplex Amplicon Pyrosequencing Bias Amplification



    “Barcode-tagged” PCR primers used for multiplex amplicon sequencing generate a thus-far-overlooked amplification bias that produces variable terminal restriction fragment length polymorphism (T-RFLP) and pyrosequencing data from the same environmental DNA template. We propose a simple two-step PCR approach that increases reproducibility and consistently recovers higher genetic diversity in pyrosequencing libraries.

  4. 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.


    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

  5. Identification and Characterization of Genomic Amplifications in Ovarian Serous Carcinoma (United States)


    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

  6. Static Generalized Brans-Dicke Universe and Gravitational Waves Amplification

    CERN Document Server

    Berman, M S; Berman, Marcelo S.; Trevisan, Luis A.


    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.

  7. Loop-mediated isothermal amplification of single pollen grains

    Institute of Scientific and Technical Information of China (English)

    Ali Bektaş; Ignacio Chapela


    The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental require-ments of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplifica-tion methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pol en cloud of an ecosystem would provide very useful ecological information, yet would require a field-enabled, high-throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop-mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pol en analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pol en cloud, and that it can amplify successful y with sensitivity down to single pol en grains, thus opening the possibility of field-based, high-throughput analysis.

  8. Identification of genetic elements associated with EPSPS gene amplification (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...

  9. Macromolecular amplification of binding response in superaptamer hydrogels. (United States)

    Bai, Wei; Gariano, Nicholas A; Spivak, David A


    It is becoming more important to detect ultralow concentrations of analytes for biomedical, environmental, and national security applications. Equally important is that new methods should be easy to use, inexpensive, portable, and if possible allow detection by the naked eye. By and large, detection of low concentrations of analytes cannot be achieved directly but requires signal amplification by catalysts, macromolecules, metal surfaces, or supramolecular aggregates. The rapidly progressing field of macromolecular signal amplification has been advanced using conjugated polymers, chirality in polymers, solvating polymers, and polymerization/depolymerization strategies. A new type of aptamer-based hydrogel with specific response to target proteins presented in this report demonstrates an additional category of macromolecular signal amplification. This superaptamer assembly provides the first example of using protein-specific aptamers to create volume-changing hydrogels with amplified response to the target protein. A remarkable aspect of these superaptamer hydrogels is that volume shrinking is visible to the naked eye down to femtomolar concentrations of protein. This extraordinary macromolecular amplification is attributed to a complex interplay between protein-aptamer supramolecular cross-links and the consequential reduction of excluded volume in the hydrogel. Specific recognition is even maintained in biological matrices such as urine and tears. Furthermore, the gels can be dried for long-term storage and regenerated for use without loss of activity. In practice, the ease of this biomarker detection method offers an alternative to traditional analytical techniques that require sophisticated instrumentation and highly trained personnel.

  10. 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


    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.

  11. Reversible Gating of Plasmonic Coupling for Optical Signal Amplification. (United States)

    Khoury, Christopher G; Fales, Andrew M; Vo-Dinh, Tuan


    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. Whole genome amplification and its impact on CGH array profiles

    Directory of Open Access Journals (Sweden)

    Meldrum Cliff


    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.

  13. Controlling the amplification of chirality in hydrogen-bonded assemblies

    NARCIS (Netherlands)

    Mateos-Timoneda, Miguel A.; Crego-Calama, Mercedes; Reinhoudt, David N.


    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. Differential transimpedance amplifier circuit for correlated differential amplification (United States)

    Gresham, Christopher A.; Denton, M. Bonner; Sperline, Roger P.


    A differential transimpedance amplifier circuit for correlated differential amplification. The amplifier circuit increase electronic signal-to-noise ratios in charge detection circuits designed for the detection of very small quantities of electrical charge and/or very weak electromagnetic waves. A differential, integrating capacitive transimpedance amplifier integrated circuit comprising capacitor feedback loops performs time-correlated subtraction of noise.

  15. Soil amplification with a strong impedance contrast: Boston, Massachusetts (United States)

    Baise, Laurie G.; Kaklamanos, James; Berry, Bradford M; Thompson, Eric


    In this study, we evaluate the effect of strong sediment/bedrock impedance contrasts on soil amplification in Boston, Massachusetts, for typical sites along the Charles and Mystic Rivers. These sites can be characterized by artificial fill overlying marine sediments overlying glacial till and bedrock, where the depth to bedrock ranges from 20 to 80 m. The marine sediments generally consist of organic silts, sand, and Boston Blue Clay. We chose these sites because they represent typical foundation conditions in the city of Boston, and the soil conditions are similar to other high impedance contrast environments. The sediment/bedrock interface in this region results in an impedance ratio on the order of ten, which in turn results in a significant amplification of the ground motion. Using stratigraphic information derived from numerous boreholes across the region paired with geologic and geomorphologic constraints, we develop a depth-to-bedrock model for the greater Boston region. Using shear-wave velocity profiles from 30 locations, we develop average velocity profiles for sites mapped as artificial fill, glaciofluvial deposits, and bedrock. By pairing the depth-to-bedrock model with the surficial geology and the average shear-wave velocity profiles, we can predict soil amplification in Boston. We compare linear and equivalent-linear site response predictions for a soil layer of varying thickness over bedrock, and assess the effects of varying the bedrock shear-wave velocity (VSb) and quality factor (Q). In a moderate seismicity region like Boston, many earthquakes will result in ground motions that can be modeled with linear site response methods. We also assess the effect of bedrock depth on soil amplification for a generic soil profile in artificial fill, using both linear and equivalent-linear site response models. Finally, we assess the accuracy of the model results by comparing the predicted (linear site response) and observed site response at the Northeastern

  16. Amplification biases: possible differences among deviating gene expressions

    Directory of Open Access Journals (Sweden)

    Piumi Francois


    Full Text Available Abstract Background Gene expression profiling has become a tool of choice to study pathological or developmental questions but in most cases the material is scarce and requires sample amplification. Two main procedures have been used: in vitro transcription (IVT and polymerase chain reaction (PCR, the former known as linear and the latter as exponential. Previous reports identified enzymatic pitfalls in PCR and IVT protocols; however the possible differences between the sequences affected by these amplification defaults were only rarely explored. Results Screening a bovine cDNA array dedicated to embryonic stages with embryonic (n = 3 and somatic tissues (n = 2, we proceeded to moderate amplifications starting from 1 μg of total RNA (global PCR or IVT one round. Whatever the tissue, 16% of the probes were involved in deviating gene expressions due to amplification defaults. These distortions were likely due to the molecular features of the affected sequences (position within a gene, GC content, hairpin number but also to the relative abundance of these transcripts within the tissues. These deviating genes mainly encoded housekeeping genes from physiological or cellular processes (70% and constituted 2 subsets which did not overlap (molecular features, signal intensities, gene ID. However, the differential expressions identified between embryonic stages were both reliable (minor intersect with biased expressions and relevant (biologically validated. In addition, the relative expression levels of those genes were biologically similar between amplified and unamplified samples. Conclusion Conversely to the most recent reports which challenged the use of intense amplification procedures on minute amounts of RNA, we chose moderate PCR and IVT amplifications for our gene profiling study. Conclusively, it appeared that systematic biases arose even with moderate amplification procedures, independently of (i the sample used: brain, ovary or embryos, (ii

  17. Identification of SPRED1 deletions using RT-PCR, multiplex ligation-dependent probe amplification and quantitative PCR. (United States)

    Spencer, Emily; Davis, Julia; Mikhail, Fady; Fu, Chuanhua; Vijzelaar, Raymon; Zackai, Elaine H; Feret, Holly; Meyn, M Stephen; Shugar, Andrea; Bellus, Gary; Kocsis, Kristina; Kivirikko, Sirpa; Pöyhönen, Minna; Messiaen, Ludwine


    Legius syndrome, is a recently identified autosomal dominant disorder caused by loss of function mutations in the SPRED1 gene, with individuals mainly presenting with multiple café-au-lait macules (CALM), freckling and macrocephaly. So far, only SPRED1 point mutations have been identified as the cause of this syndrome. To determine if copy number changes (CNCs) are a cause of Legius syndrome, we have used a Multiplex Ligation-dependent Probe Amplification (MLPA) assay covering all SPRED1 exons in a cohort of 510 NF1-negative patients presenting with multiple CALMs with or without freckling, but no other NF1 diagnostic signs. Four different deletions were identified by MLPA and confirmed by quantitative PCR, reverse transcriptase PCR and/or array CGH: a deletion of exon 1 and the SPRED1 promoter region in a proband and two first-degree relatives; a deletion of the entire SPRED1 gene in a sporadic patient; a deletion of exon 2-6 in a proband and her father; and an ∼6.6 Mb deletion on chromosome 15 that spans SPRED1 in a sporadic patient. Deletions account for ∼10% of the 40 detected SPRED1 mutations in this cohort of 510 individuals. These results indicate the need for dosage analysis to complement sequencing-based SPRED1 mutation analyses.

  18. Differentiation of Candida parapsilosis, C. orthopsilosis, and C. metapsilosis by specific PCR amplification of the RPS0 intron. (United States)

    del Pilar Vercher, M; García Martínez, José M; Cantón, Emilia; Pemán, Javier; Gómez García, M Micaela; Gómez, Eulogio Valentín; del Castillo Agudo, Lucas


    Although Candida parapsilosis is the most prevalent among the 3 species of the *psilosis group, studies applying DNA-based diagnostic techniques with isolates previously identified as C. parapsilosis have revealed that both C. orthopsilosis and C. metapsilosis account for 0-10% of all these isolates, depending on the geographical area. Differences in the degrees of antifungal susceptibility and virulence have been found, so a more precise identification is required. In a first approach, we reidentified 38 randomly chosen clinical isolates, previously identified as C. parapsilosis, using the RPO2 (CA2) RAPD marker. Among them, we reclassified 4 as C. metapsilosis and 5 as C. orthopsilosis. We previously developed a method to identify different pathogen yeast species, including C. parapsilosis, based on the amplification of the RPS0 gene intron. In this work, we extend this approach to the new *psilosis species by partially sequencing their RPS0 gene, including the intron sequence. Based on intron sequences, we designed specific primers capable of identifying C. orthopsilosis and C. metapsilosis species, and we reidentified species among the initial isolates. These new primers have allowed a specific and rapid identification of C. orthopsilosis and C. metapsilosis.

  19. Bacteriophage Amplification-Coupled Detection and Identification of Bacterial Pathogens (United States)

    Cox, Christopher R.; Voorhees, Kent J.

    Current methods of species-specific bacterial detection and identification are complex, time-consuming, and often require expensive specialized equipment and highly trained personnel. Numerous biochemical and genotypic identification methods have been applied to bacterial characterization, but all rely on tedious microbiological culturing practices and/or costly sequencing protocols which render them impractical for deployment as rapid, cost-effective point-of-care or field detection and identification methods. With a view towards addressing these shortcomings, we have exploited the evolutionarily conserved interactions between a bacteriophage (phage) and its bacterial host to develop species-specific detection methods. Phage amplification-coupled matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) was utilized to rapidly detect phage propagation resulting from species-specific in vitro bacterial infection. This novel signal amplification method allowed for bacterial detection and identification in as little as 2 h, and when combined with disulfide bond reduction methods developed in our laboratory to enhance MALDI-TOF-MS resolution, was observed to lower the limit of detection by several orders of magnitude over conventional spectroscopy and phage typing methods. Phage amplification has been combined with lateral flow immunochromatography (LFI) to develop rapid, easy-to-operate, portable, species-specific point-of-care (POC) detection devices. Prototype LFI detectors have been developed and characterized for Yersinia pestis and Bacillus anthracis, the etiologic agents of plague and anthrax, respectively. Comparable sensitivity and rapidity was observed when phage amplification was adapted to a species-specific handheld LFI detector, thus allowing for rapid, simple, POC bacterial detection and identification while eliminating the need for bacterial culturing or DNA isolation and amplification techniques.

  20. Transcriptome dynamics of transgene amplification in Chinese hamster ovary cells. (United States)

    Vishwanathan, Nandita; Le, Huong; Jacob, Nitya M; Tsao, Yung-Shyeng; Ng, Sze-Wai; Loo, Bernard; Liu, Zhong; Kantardjieff, Anne; Hu, Wei-Shou


    Dihydrofolate reductase (DHFR) system is used to amplify the product gene to multiple copies in Chinese Hamster Ovary (CHO) cells for generating cell lines which produce the recombinant protein at high levels. The physiological changes accompanying the transformation of the non-protein secreting host cells to a high producing cell line is not well characterized. We performed transcriptome analysis on CHO cells undergoing the selection and amplification processes. A host CHO cell line was transfected with a vector containing genes encoding the mouse DHFR (mDHFR) and a recombinant human IgG (hIgG). Clones were isolated following selection and subcloned following amplification. Control cells were transfected with a control plasmid which did not have the hIgG genes. Although methotrexate (MTX) amplification increased the transcript level of the mDHFR gene significantly, its effect on both hIgG heavy and light chain genes was more modest. The subclones appeared to retain the transcriptome signatures of their parental clones, however, their productivity varied among those derived from the same clone. The transcript levels of hIgG transgenes of all subclones fall in a narrower range than the product titer, alluding to the role of many functional attributes, other than transgene transcript, on productivity. We cross examined functional class enrichment during selection and amplification as well as between high and low producers and discerned common features among them. We hypothesize that the role of amplification is not merely increasing transcript levels, but also enriching survivors which have developed the cellular machinery for secreting proteins, leading to an increased frequency of isolating high-producing clones. We put forward the possibility of assembling a hyper-productivity gene set through comparative transcriptome analysis of a wide range of samples.

  1. Rapid isothermal detection of Phytophthora species on plant samples using recombinase polymerase amplification (United States)

    Recently several isothermal amplification techniques have been developed that are extremely tolerant towards inhibitors present in many plant extracts. Recombinase polymerase amplification (RPA) assays for the genus Phytophthora have been developed which provide a simple and rapid method to macerate...

  2. Systematic evaluation of bias in microbial community profiles induced by whole genome amplification

    NARCIS (Netherlands)

    Direito, S.O.L.; Zaura, E.; Little, M.; Ehrenfreund, P.; Röling, W.F.M.


    Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplific

  3. New perspectives on microbial community distortion after whole-genome amplification (United States)

    Whole-genome amplification (WGA) has become an important tool to explore the genomic information of microorganisms in an environmental sample with limited biomass, however potential selective biases during the amplification processes are poorly understood. Here, we describe the e...

  4. A novel sequence-based antigenic distance measure for H1N1, with application to vaccine effectiveness and the selection of vaccine strains (United States)

    Pan, Keyao; Subieta, Krystina C.; Deem, Michael W.


    H1N1 influenza causes substantial seasonal illness and was the subtype of the 2009 influenza pandemic. Precise measures of antigenic distance between the vaccine and circulating virus strains help researchers design influenza vaccines with high vaccine effectiveness. We here introduce a sequence-based method to predict vaccine effectiveness in humans. Historical epidemiological data show that this sequence-based method is as predictive of vaccine effectiveness as hemagglutination inhibition assay data from ferret animal model studies. Interestingly, the expected vaccine effectiveness is greater against H1N1 than H3N2, suggesting a stronger immune response against H1N1 than H3N2. The evolution rate of hemagglutinin in H1N1 is also shown to be greater than that in H3N2, presumably due to greater immune selection pressure. PMID:21123189

  5. Molecular Identification of Veterinary Yeast Isolates by Use of Sequence-Based Analysis of the D1/D2 Region of the Large Ribosomal Subunit▿


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


    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 me...


    Directory of Open Access Journals (Sweden)

    Ronja OCKER


    Full Text Available The loop-mediated isothermal amplification method (LAMP is a recently developed molecular technique that amplifies nucleic acid under isothermal conditions. For malaria diagnosis, 150 blood samples from consecutive febrile malaria patients, and healthy subjects were screened in Thailand. Each sample was diagnosed by LAMP, microscopy and nested polymerase chain reaction (nPCR, using nPCR as the gold standard. Malaria LAMP was performed using Plasmodiumgenus and Plasmodium falciparum specific assays in parallel. For the genus Plasmodium, microscopy showed a sensitivity and specificity of 100%, while LAMP presented 99% of sensitivity and 93% of specificity. For P. falciparum, microscopy had a sensitivity of 95%, and LAMP of 90%, regarding the specificity; and microscopy presented 93% and LAMP 97% of specificity. The results of the genus-specific LAMP technique were highly consistent with those of nPCR and the sensitivity of P. falciparum detection was only marginally lower.

  7. Sensor Systems with Magnetic and Optomagnetic Readout of Rolling Circle Amplification Products

    DEFF Research Database (Denmark)

    Hansen, Mikkel Fougt; Donolato, Marco; Fock, Jeppe


    We are developing robust biosensors for homogeneous detection of rolling cirle amplification (RCA) products with magnetic and/or optomagnetic readouts based on surface-functionalized magnetic nanoparticles. Binding of RCA amplicons to nanoparticles modifies their ability to rotate in response...... to an applied oscillating magnetic field. As a result, magnetic or optical measurements of these changes in the rotational response of nanoparticles vs. frequency of the magnetic field can be used to quantitate the number of amplicons, and, hence, the concentration of target nucleic acid analytes. After...... describing the basic principles of this approach, we present the current status of the development of compact and portable sensing devices used to measure the dynamic response of magnetic particle suspensions. Then, we give examples and results of different RCA detection strategies designed by us, and we...

  8. Tm3+-doped ion-exchanged aluminum germanate glass waveguide for S-band amplification (United States)

    Yang, D. L.; Pun, E. Y. B.; Lin, H.


    K+-Na+ ion-exchanged channel waveguide amplifiers have been fabricated in Tm3+-doped acid-resistant aluminum germanate glasses. The optical and relative gains of a 3.15-cm-long waveguide channel were achieved to be 4.05 and 2.29 dB at 1.482 μm wavelength under 110 mW 793 nm laser excitation, respectively. After compensating the propagation loss, an internal gain of 1.50 dB and a remarkable gain coefficient of 0.48 dB/cm were obtained, which reveals a definite S-band signal amplification in the low phonon energy glass waveguide. As an expectation, UV-radiation-sensitive glass waveguide should promote the developments of gain-flatten S-band waveguide amplifiers, infrared UV-writing grating waveguide lasers, and compact multifunctional integrated optical devices.


    Ocker, Ronja; Prompunjai, Yongyut; Chutipongvivate, Salakchit; Karanis, Panagiotis


    The loop-mediated isothermal amplification method (LAMP) is a recently developed molecular technique that amplifies nucleic acid under isothermal conditions. For malaria diagnosis, 150 blood samples from consecutive febrile malaria patients, and healthy subjects were screened in Thailand. Each sample was diagnosed by LAMP, microscopy and nested polymerase chain reaction (nPCR), using nPCR as the gold standard. Malaria LAMP was performed using Plasmodiumgenus and Plasmodium falciparum specific assays in parallel. For the genus Plasmodium, microscopy showed a sensitivity and specificity of 100%, while LAMP presented 99% of sensitivity and 93% of specificity. For P. falciparum, microscopy had a sensitivity of 95%, and LAMP of 90%, regarding the specificity; and microscopy presented 93% and LAMP 97% of specificity. The results of the genus-specific LAMP technique were highly consistent with those of nPCR and the sensitivity of P. falciparum detection was only marginally lower.

  10. Fiber-Optical Parametric Amplification of Sub-Picosecond Pulses for High-Speed Optical Communications

    DEFF Research Database (Denmark)

    Lali-Dastjerdi, Zohreh; Cristofori, Valentina; Rottwitt, Karsten;


    This article reviews recent results of amplification of short optical pulses using fiber-optical parametric amplifiers. This includes chirped-pulse amplification of 400 fs pulses, error-free amplification of a 640-Gbit/s optical time-division multiplexed signal with less than a 1-dB power penalty...

  11. Loop-mediated isothermal amplification assay for the detection of Ehrlichia canis DNA in blood samples from dogs

    Directory of Open Access Journals (Sweden)

    SA Faggion


    Full Text Available The rickettsial bacterium Ehrlichia canis is the etiological agent of canine monocytic ehrlichiosis, one of the most important canine tick-borne diseases in the world. In this study, a loop-mediated isothermal amplification (LAMP assay was developed for detection of E. canis DNA using LAMP primers targeting the groESL operon. Reactions were performed at 60°C for 60 min and the results were visualized by gel electrophoresis. Successful amplification was obtained using plasmid DNA containing a fragment of the groESL operon and DNA extracted from blood samples that tested positive for E. canis by real-time PCR. The specificity of amplification was confirmed by EcoRI restriction of internal sites in the LAMP primers and no cross-reactivity with blood samples positive for Babesia spp., another common tick-borne pathogen, was observed. The high cost of nucleic acid tests (NAT is one of the disadvantages for their large-scale use as routine diagnostic tests. The E. canis LAMP assay developed here is an interesting alternative to PCR since it does not require a thermocycler, thus reducing costs for the veterinary clinical laboratory.

  12. Sensitive and rapid detection of Paragonimus westermani infection in humans and animals by loop-mediated isothermal amplification (LAMP). (United States)

    Chen, M X; Ai, L; Zhang, R L; Xia, J J; Wang, K; Chen, S H; Zhang, Y N; Xu, M J; Li, X; Zhu, X Q; Chen, J X


    In the present study, a loop-mediated isothermal amplification (LAMP) assay was developed and validated for the detection of Paragonimus westermani adults, metacercariae, and eggs in human and animal samples. The LAMP amplification can be finished in 45 min under isothermal condition at 60°C by employing a set of four species-specific primer mixtures and the results can be checked by naked-eye visualization. No amplification products were detected with deoxyribunucleic acid (DNA) of related trematode species including Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Opisthorchis viverrini, Schistosoma mansoni, and Schistosoma japonicum. The method was further validated by examining P. westermani DNA in intermediate hosts including freshwater crabs and crayfish, as well as in sputum and pleural fluid samples from patients of paragonimiasis. These results indicated that the LAMP assay was highly specific, sensitive, and rapid, and it was approximately 100 times more sensitive than conventional specific PCR. The LAMP assay established in this study provides a rapid and sensitive tool for the detection of P. westermani DNA in freshwater crabs, crayfish, sputum, and pleural fluid samples, which has important implications for effective control of human paragonimiasis.

  13. A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens. (United States)

    Wang, Tiantian; Kim, Sanghyo; An, Jeong Ho


    Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids integrated with a disposable complementary-metal-oxide semiconductor (CMOS) image sensor. This novel system works as an amplification-coupled detection platform, relying on a CMOS image sensor, with the aid of a computerized circuitry controller for the temperature and light sources. The CMOS image sensor captures the light which is passing through the sensor surface and converts into digital units using an analog-to-digital converter (ADC). This new system monitors the real-time photon variation, caused by the color changes during amplification. Escherichia coli O157 was used as a proof-of-concept target for quantitative analysis, and compared with the results for Staphylococcus aureus and Salmonella enterica to confirm the efficiency of the system. The system detected various DNA concentrations of E. coli O157 in a short time (45min), with a detection limit of 10fg/μL. The low-cost, simple, and compact design, with low power consumption, represents a significant advance in the development of a portable, sensitive, user-friendly, real-time, and quantitative analytic tools for point-of-care diagnosis.

  14. Nucleic acid detection system and method for detecting influenza

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Hong; Song, Jian


    The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

  15. Nucleic acid detection using MNAzymes. (United States)

    Gerasimova, Yulia V; Kolpashchikov, Dmitry M


    Deoxyribozymes are promising biotechnological tools. In a recent JACS article, Mokany et al. reported on the design of multi-component deoxyribozyme (MNAzyme) sensors based on 10-23 and 8-17 DNA enzymes. The sensors can detect down to 5 pM of a specific nucleic acid. The versatility of MNAzyme platform allows the design of catalytic cascades for signal amplification. This work is a step forward to PCR-free molecular diagnostics.

  16. [Detection of the Zaire Subtype of the Ebola Virus by Isothermal Multiple Self-matching Initiated Amplification]. (United States)

    Li, Xinna; Nie, Kai; Wang, Ji; Zhang, Dan; Guan, Li; Liu, Jun; Ke, Yuehua; Zhou, Hangyu; Ma, Xuejun


    Given the Ebola outbreak in West Africa and the risks of spread to other regions, a rapid, sensitive and simple method for the detection of the Ebola virus (EBOV) is of great significance for the prevention and control of Ebola. We developed a simple colorimetric isothermal multiple self-matching initiated amplification (IMSA) for rapid detection of the Zaire subtype of the Ebola virus (EBOV-Z). This method employed six primers that recognized seven sites of the EBOV-Z nucleoprotein gene for amplification of nucleic acids under isothermal conditions at 63 degrees C for 1 h. Amplification products were detected through visual inspection of color change by pre-addition of hydroxyl naphthol blue dye. Relative sensitivity was validated by detection of serial tenfold dilutions of virus-like particles containing the partial EBOV-Z nucleoprotein gene and mock clinical sample. Specificity of IMSA was validated by detection of the plasma of 30 healthy volunteers, the dengue virus, and Japanese encephalitis virus. IMSA had comparable sensitivity to Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and cross-reaction with human plasma or other viruses was not observed. Reverse transcription-isothermal multiple self-matching initiated amplification (RT-IMSA) was also evaluated and compared in parallel with the commercial RT-qPCR kit for detection of EBOV-suspected samples of human blood in Sierra Leone. Sensitivity and specificity of the RT-IMSA was 91.4% and 100%, respectively. These data suggest that RT-IMSA is a valuable tool for the detection of the EBOV with the distinct advantages of simplicity and low cost compared with RT-qPCR.

  17. Soliton-induced relativistic-scattering and amplification

    CERN Document Server

    Rubino, E; Belgiorno, F; Cacciatori, S L; Couairon, A; Leonhardt, U; Faccio, D


    Solitons are of fundamental importance in photonics due to applications in optical data transmission and also as a tool for investigating novel phenomena ranging from light generation at new frequencies and wave-trapping to rogue waves. Solitons are also relativistic scatterers: they generate refractive-index perturbations moving at the speed of light. Here we found that such perturbations scatter light in an unusual way: they amplify light by the mixing of positive and negative frequencies, as we describe using a first Born approximation and numerical simulations. The simplest scenario in which these effects may be observed is within the initial stages of optical soliton propagation: a steep shock front develops that may efficiently scatter a second, weaker probe pulse into relatively intense positive and negative frequency modes with amplification at the expense of the soliton. Our results show a novel all-optical amplification scheme that relies on relativistic scattering.

  18. An enzymatic signal amplification system for calorimetric studies of cellobiohydrolases

    DEFF Research Database (Denmark)

    Murphy, Leigh; Baumann, Martin Johannes; Borch, Kim


    is heat production. This can be converted to the rate of reaction and allows direct and continuous monitoring of the hydrolysis of complex substrates. To overcome the low molar enthalpy of the hydrolysis of the glycosidic bond, which is typically on the order of −2.5 kJ mol−1, an enzymatic signal......The study of cellulolytic enzymes has traditionally been carried out using endpoint measurements by quantitation of reaction products using high-performance liquid chromatography (HPLC) or overall determination of produced reducing ends. To measure catalytic activity, model substrates...... amplification method has been developed to measure even slow hydrolytically active enzymes such as cellobiohydrolases. This method is explained in detail for the amplification of the heat signal by more than 130 times by using glucose oxidase and catalase. The kinetics of this complex coupled reaction system...

  19. Quantum Privacy Amplification for a Sequence of Single Qubits

    Institute of Scientific and Technical Information of China (English)

    DENG Fu-Guo; LONG Gui-Lu


    We present a scheme for quantum privacy amplification (QPA) for a sequence of single qubits. The QPA procedure uses a unitary operation with two controlled-not gates and a Hadamard gate. Every two qubits are performed with the unitary gate operation, and a measurement is made on one photon and the other one is retained.The retained qubit carries the state information of the discarded one. In this way, the information leakage is reduced.The procedure can be performed repeatedly so that the information leakage is reduced to any arbitrarily low level. With this QPA scheme, the quantum secure direct communication with single qubits can be implemented with arbitrarily high security. We also exploit this scheme to do privacy amplification on the single qubits in quantum information sharing for long-distance communication with quantum repeaters.

  20. Assessing Linearity of the Parasite Varroa destructor DNA Amplification

    Directory of Open Access Journals (Sweden)

    ODAGIU Antonia


    Full Text Available The importance of honeybee products make of disease prevention and control in honeybees one of the mainconcerns of beekeepers in the world. The PCR – RT reaction represents an alternative for amplification performed inorder to realize the Varroa destructor O. genotypization, very important stage in haoneybee resistance to parasitedescription and also in management of the treatments. The linearity data is a very important parameter and very usefulin determination of the amplification of the parasite DNA and success of the genotypization process. The amplificationefficiency was very satisfactory, fact revealed by the value of the regression line y = - 2.3103 * 26.552 together withcoefficient of determination equal (r2 = 0.9691, meaning that more than 96% of the reaction efficiency may beexplained by the process liniarity. The implementation of the RT-PCR method was successful and it represents apremise for validation process evolution.

  1. Whole genome amplification - Review of applications and advances

    Energy Technology Data Exchange (ETDEWEB)

    Hawkins, Trevor L.; Detter, J.C.; Richardson, Paul


    The concept of Whole Genome Amplification is something that has arisen in the past few years as modifications to the polymerase chain reaction (PCR) have been adapted to replicate regions of genomes which are of biological interest. The applications here are many--forensics, embryonic disease diagnosis, bio terrorism genome detection, ''imoralization'' of clinical samples, microbial diversity, and genotyping. The key question is if DNA can be replicated a genome at a time without bias or non random distribution of the target. Several papers published in the last year and currently in preparation may lead to the conclusion that whole genome amplification may indeed be possible and therefore open up a new avenue to molecular biology.

  2. Rapid amplification of cDNA ends (RACE). (United States)

    Yeku, Oladapo; Frohman, Michael A


    Rapid Amplification of cDNA ends (RACE) provides an inexpensive and powerful tool to quickly obtain full-length cDNA when the sequence is only partially known. Starting with an mRNA mixture, gene-specific primers generated from the known regions of the gene and non-specific anchors, full-length sequences can be identified in as little as 3 days. RACE can also be used to identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5(') and 3(') cDNA ends using the "new RACE" technique.

  3. DC-driven thermoelectric Peltier device for precise DNA amplification (United States)

    Yamaguchi, Shigeo; Suzuki, Tadzunu; Inoue, Kazuhito; Azumi, Yoshitaka


    Using a DC-driven Peltier device, we fabricated a DNA amplification system [polymerase chain reaction (PCR) system] with the aim of increasing its speed and precision. The Peltier device had a well block sandwiched by Bi2Se0.37Te2.36 as an N-type thermoelectric material and Bi0.59Sb1.30Te3 as a P-type material. The well block was directly controlled by the electric current, leading to a high thermal response. Using the Peltier device with the well block, we performed thermal cycles of a PCR, and we demonstrated that our PCR system produces a smaller amount of nonspecific products for the genome DNA (gDNA) of Arabidopsis thaliana, leading to a more precise DNA amplification system.

  4. Resonant Amplification of Turbulence by the Blast Wawes

    CERN Document Server

    Zankovich, A M


    We discuss an idea whether spherical blast waves can amplify by a non-local resonant hydrodynamic mechanism inhomogeneities formed by turbulence or phase segregation in the interstellar medium. We consider the problem of a blast-wave-turbulence interaction in the Linear Interaction Approximation. Mathematically, this is an eigenvalue problem for finding the structure and amplitude of eigenfunctions describing the response of the shock-wave flow to forced oscillations by external perturbations in the ambient interstellar medium. Linear analysis shows that the blast wave can amplify density and vorticity perturbations for a wide range of length scales with amplification coefficients of up to 20, with amplification the greater, the larger the length. There also exist resonant harmonics for which the gain becomes formally infinite in the linear approximation. Their orbital wavenumbers are within the range of macro- ($l \\sim 1$), meso- ($l \\sim 20$) and microscopic ($l > 200$) scales. Since the resonance width is ...

  5. Amplification, Decoherence, and the Acquisition of Information by Spin Environments (United States)

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


    Quantum Darwinism recognizes the role of the environment as a communication channel: Decoherence can selectively amplify information about the pointer states of a system of interest (preventing access to complementary information about their superpositions) and can make records of this information accessible to many observers. This redundancy explains the emergence of objective, classical reality in our quantum Universe. Here, we demonstrate that the amplification of information in realistic spin environments can be quantified by the quantum Chernoff information, which characterizes the distinguishability of partial records in individual environment subsystems. We show that, except for a set of initial states of measure zero, the environment always acquires redundant information. Moreover, the Chernoff information captures the rich behavior of amplification in both finite and infinite spin environments, from quadratic growth of the redundancy to oscillatory behavior. These results will considerably simplify experimental testing of quantum Darwinism, e.g., using nitrogen vacancies in diamond.

  6. Phase Sensitive Amplification using Parametric Processes in Optical Fibers

    DEFF Research Database (Denmark)

    Kang, Ning

    Phase sensitive amplification using the parametric processes in fiber has the potential of delivering high gain and broadband operation with ultralow noise. It is able to regenerate both amplitude and phase modulated signals, simultaneously, with the appropriate design. This thesis concerns...... types. The regeneration capability of PSAs on phase encoded signal in an optical link has been optimized. Flat-top phase sensitive profile has been synthesized. It is able to provide simultaneous amplitude and phase noise squeezing, with enhanced phase noise margin compared to conventional designs......, 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...

  7. Retrieval and Amplification of DNA from Unstained Histopathological Sections

    Institute of Scientific and Technical Information of China (English)

    DonnaC.MONTAGUE; BeverlyD.LYN-COOK; 等


    Testing of compounds for carcinogenic potential in vivo involves various experimental designs.A few of these techniques are directed to demonstrate the genotoxicity and mutagenicity of the compound by histopathology.These changes shown by histochemical means include monoclonal antibody directed cellular markers.Development of the polymerase chain reaction technique(PCR)for amplification of DNA has facilitated the investigation of molecular events related to the formation of malignant neoplasms.We describe here a method for screening tissues for mutations of the H-ras gene using monoclonal antibodies directed toward normal and mutant p21 proteins.Formalin-fixed,paraffinembedded tissue sections are used to subsequently confirm the gene mutation by PCR amplification of the H-ras gene.The results indicated a successful application of this technique to demonstrate the presence of p21 oncoprotein in the tissues tested.

  8. Weak value amplification is suboptimal for estimation and detection. (United States)

    Ferrie, Christopher; Combes, Joshua


    We show by using statistically rigorous arguments that the technique of weak value amplification does not perform better than standard statistical techniques for the tasks of single parameter estimation and signal detection. Specifically, we prove that postselection, a necessary ingredient for weak value amplification, decreases estimation accuracy and, moreover, arranging for anomalously large weak values is a suboptimal strategy. In doing so, we explicitly provide the optimal estimator, which in turn allows us to identify the optimal experimental arrangement to be the one in which all outcomes have equal weak values (all as small as possible) and the initial state of the meter is the maximal eigenvalue of the square of the system observable. Finally, we give precise quantitative conditions for when weak measurement (measurements without postselection or anomalously large weak values) can mitigate the effect of uncharacterized technical noise in estimation.

  9. Surface plasmon polariton amplification in metal-semiconductor structures. (United States)

    Fedyanin, Dmitry Yu; Arsenin, Aleksey V


    We propose a novel scheme of surface plasmon polariton (SPP) amplification that is based on a minority carrier injection in a Schottky diode. This scheme uses compact electrical pumping instead of bulky optical pumping. Compact size and a planar structure of the proposed amplifier allow one to utilize it in integrated plasmonic circuits and couple it easily to passive plasmonic devices. Moreover, this technique can be used to obtain surface plasmon lasing.

  10. Generation and Amplification of Terahertz Radiation in Carbon Nanotubes


    Abukari, S. S.; Mensah, S. Y.; Mensah, N. G.; Adu, K. W.; Rabiu, M; Dompreh, K. A.; Twum, A.


    We investigate theoretically the feasibility of generation and amplification of terahertz radiation in aligned achiral carbon nanotubes (zigzag and armchair) in comparison with a superlattice in the presence of a constant (dc) and high-frequency (ac) electric fields. The electric current density expression is derived using the semiclassical Boltzmann transport equation with a constant relaxation time with the electric field applied along the nanotube axis. Our analysis on the current density ...

  11. Adiabatic Amplification of Plasmons and Demons in 2D Systems. (United States)

    Sun, Zhiyuan; Basov, D N; Fogler, M M


    We theoretically investigate charged collective modes in a two-dimensional conductor with hot electrons where the instantaneous mode frequencies gradually increase or decrease with time. We show that the loss compensation or even amplification of the modes may occur. We apply our theory to two types of collective modes in graphene, the plasmons and the energy waves, which can be probed in optical pump-probe experiments.

  12. Amplification of acoustic waves in laminated piezoelectric semiconductor plates

    Energy Technology Data Exchange (ETDEWEB)

    Yang, J.S.; Yang, X.M.; Turner, J.A. [University of Nebraska, Department of Engineering Mechanics, Lincoln, NE (United States)


    Two-dimensional equations for coupled extensional, flexural and thickness-shear motions of laminated plates of piezoelectric semiconductors are obtained systematically from the three-dimensional equations by retaining lower order terms in power series expansions in the plate thickness coordinate. The equations are used to analyze extensional waves in a composite plate of piezoelectric ceramics and semiconductors. Dispersion and dissipation due to semiconduction as well as wave amplification by a dc electric field are discussed. (orig.)

  13. Complete genome amplification of Equine influenza virus subtype 2


    Sguazza, G. H.; Fuentealba, N. A.; Tizzano, Marco Antonio; Galosi, Cecilia Mónica; Pecoraro, M. R.


    This work reports a method for rapid amplification of the complete genome of equine influenza virus subtype 2 (H3N8). A ThermoScriptTM reverse transcriptase instead of the avian myeloblastosis virus reverse transcriptase or Moloney murine leukemia virus reverse transcriptase was used. This enzyme has demonstrated higher thermal stability and is described as suitable to make long cDNA with a complex secondary structure. The product obtained by this method can be cloned, used in later...

  14. Making an Effort to Listen: Mechanical Amplification in the Ear


    Hudspeth, A. J.


    The inner ear’s performance is greatly enhanced by an active process defined by four features: amplification, frequency selectivity, compressive nonlinearity, and spontaneous otoacoustic emission. These characteristics emerge naturally if the mechanoelectrical transduction process operates near a dynamical instability, the Hopf bifurcation, whose mathematical properties account for specific aspects of our hearing. The active process of non-mammalian tetrapods depends upon active hair-bundle m...

  15. Phase sensitive amplification in silicon photonic crystal waveguides

    CERN Document Server

    Yanbing,; Husko, Chad; Schroder, Jochen; Lefrancois, Simon; Rey, Isabella H; Krauss, Thomas F; Eggleton, Benjamin J


    We experimentally demonstrate phase sensitive amplification (PSA) in a silicon photonic crystal waveguide based on pump-degenerate four-wave mixing. An 11 dB phase extinction ratio is obtained in a record compact 196 {\\mu}m nanophotonic device due to broadband slow-light, in spite of the presence of two-photon absorption and free-carriers. Numerical calculations show good agreement with the experimental results.

  16. Phase-sensitive amplification in silicon photonic crystal waveguides. (United States)

    Zhang, Yanbing; Husko, Chad; Schröder, Jochen; Lefrancois, Simon; Rey, Isabella H; Krauss, Thomas F; Eggleton, Benjamin J


    We experimentally demonstrate phase-sensitive amplification in a silicon photonic crystal waveguide based on pump-degenerate four-wave mixing. An 11 dB phase-extinction ratio is obtained in a record compact 196 μm nanophotonic device due to broadband slow light, in spite of the presence of two-photon absorption and free carriers. Numerical calculations show good agreement with the experimental results.

  17. Metrology with Weak Value Amplification and Related Topics (United States)


    common optical telecom networks. More recently, the amplification properties of this weak value effect have been exploited in similar optical systems to...applications). The light in one port was measured with a photodiode and used to lock the power at 2 mW with an acousto- optic modulator before the fiber ...We examine a sequence of polarized laser pulses effectively trapped inside an interferometer using a Pockels cell and polarization optics . In

  18. Purely nonlinear disorder-induced localizations and their parametric amplification

    CERN Document Server

    Folli, Viola; Conti, Claudio


    We investigate spatial localization in a quadratic nonlinear medium in the presence of randomness. By means of numerical simulations and theoretical analyses we show that, in the down conversion regime, the transverse random modulation of the nonlinear susceptibility generates localizations of the fundamental wave that grow exponentially in propagation. The localization length is optically controlled by the pump intensity which determines the amplification rate. The results also apply to cubic nonlinearities.

  19. Resonant amplification of quantum fluctuations in a spinor gas

    DEFF Research Database (Denmark)

    Topic, O.; Scherer, M.; Gebreyesus, G.;


    Bose-Einstein condensates of atoms with non-zero spin are known to constitute an ideal system to investigate fundamental properties of magnetic superfluids. More recently it was realized that they also provide the fascinating opportunity to investigate the macroscopic amplification of quantum and...... of seed atoms is triggered purely by quantum fluctuations and thus the system acts as a matter-wave amplifier for the vacuum state....

  20. Hyper dispersion pulse compressor for chirped pulse amplification systems (United States)

    Barty, Christopher P. J.


    A grating pulse compressor configuration is introduced for increasing the optical dispersion for a given footprint and to make practical the application for chirped pulse amplification (CPA) to quasi-narrow bandwidth materials, such as Nd:YAG. The grating configurations often use cascaded pairs of gratings to increase angular dispersion an order of magnitude or more. Increased angular dispersion allows for decreased grating separation and a smaller compressor footprint.

  1. Weak Value Amplification of a Post-Selected Single Photon (United States)

    Hallaji, Matin

    Weak value amplification (WVA) is a measurement technique in which the effect of a pre- and post-selected system on a weakly interacting probe is magnified. In this thesis, I present the first experimental observation of WVA of a single photon. We observed that a signal photon --- sent through a polarization interferometer and post-selected by photodetection in the almost-dark port --- can act like eight photons. The effect of this single photon is measured as a nonlinear phase shift on a separate laser beam. The interaction between the two is mediated by a sample of laser- cooled 85Rb atoms. Electromagnetically induced transparency (EIT) is used to enhance the nonlinearity and overcome resonant absorption. I believe this work to be the first demonstration of WVA where a deterministic interaction is used to entangle two distinct optical systems. In WVA, the amplification is contingent on discarding a large portion of the original data set. While amplification increases measurement sensitivity, discarding data worsens it. Questioning whether these competing effects conspire to improve or diminish measurement accuracy has resulted recently in controversy. I address this question by calculating the maximum amount of information achievable with the WVA technique. By comparing this information to that achievable by the standard technique, where no post-selection is employed, I show that the WVA technique can be advantageous under a certain class of noise models. Finally, I propose a way to optimally apply the WVA technique.

  2. Disturbance amplification in boundary layers over thin wall films (United States)

    Saha, Sandeep; Page, Jacob; Zaki, Tamer A.


    In single-fluid boundary layers, streaks can amplify at sub-critical Reynolds numbers and initiate early transition to turbulence. Introducing a wall film of different viscosities can appreciably alter the stability of the base flow and, in particular, the transient growth of the perturbation streaks. The formalism of seminorms is used to identify optimal disturbances which maximize the kinetic energy in the two-fluid flow. An examination of optimal growth over a range of viscosity ratios of the film relative to the outer flow reveals three distinct regimes of amplification, each associated with a particular combination of the eigenfunctions. In order to elucidate the underlying amplification mechanisms, a model problem is formulated: An initial value problem is solved using an eigenfunction expansion and is used to compute the evolution of pairs of eigenfunctions. By appropriately selecting the pair, the initial value problem qualitatively reproduces the temporal evolution of the optimal disturbance, and provides an unambiguous explanation of the dynamics. Two regimes of transient growth are attributed to the evolution of the interface mode along with free-stream vortical modes; the third regime is due to the evolution of the interface and a discrete mode. The results demonstrate that a lower-viscosity film can effectively reduce the efficacy of the lift-up mechanism and, as a result, transient growth of disturbances. However, another mechanism of amplification of wall-normal vorticity arises due to the deformation of the two-fluid interface and becomes dominant below a critical viscosity ratio.

  3. EGFR Amplification and Glioblastoma Stem-Like Cells

    Directory of Open Access Journals (Sweden)

    Katrin Liffers


    Full Text Available Glioblastoma (GBM, the most common malignant brain tumor in adults, contains a subpopulation of cells with a stem-like phenotype (GS-cells. GS-cells can be maintained in vitro using serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor-2, and heparin. However, this method does not conserve amplification of the Epidermal Growth Factor Receptor (EGFR gene, which is present in over 50% of all newly diagnosed GBM cases. GS-cells with retained EGFR amplification could overcome the limitations of current in vitro model systems and contribute significantly to preclinical research on EGFR-targeted therapy. This review recapitulates recent methodological approaches to expand stem-like cells from GBM with different EGFR status in order to maintain EGFR-dependent intratumoral heterogeneity in vitro. Further, it will summarize the current knowledge about the impact of EGFR amplification and overexpression on the stem-like phenotype of GBM-derived GS-cells and different approaches to target the EGFR-dependent GS-cell compartment of GBM.

  4. Enhanced sequencing coverage with digital droplet multiple displacement amplification. (United States)

    Sidore, Angus M; Lan, Freeman; Lim, Shaun W; Abate, Adam R


    Sequencing small quantities of DNA is important for applications ranging from the assembly of uncultivable microbial genomes to the identification of cancer-associated mutations. To obtain sufficient quantities of DNA for sequencing, the small amount of starting material must be amplified significantly. However, existing methods often yield errors or non-uniform coverage, reducing sequencing data quality. Here, we describe digital droplet multiple displacement amplification, a method that enables massive amplification of low-input material while maintaining sequence accuracy and uniformity. The low-input material is compartmentalized as single molecules in millions of picoliter droplets. Because the molecules are isolated in compartments, they amplify to saturation without competing for resources; this yields uniform representation of all sequences in the final product and, in turn, enhances the quality of the sequence data. We demonstrate the ability to uniformly amplify the genomes of single Escherichia coli cells, comprising just 4.7 fg of starting DNA, and obtain sequencing coverage distributions that rival that of unamplified material. Digital droplet multiple displacement amplification provides a simple and effective method for amplifying minute amounts of DNA for accurate and uniform sequencing.

  5. Diagnosis of brugian filariasis by loop-mediated isothermal amplification.

    Directory of Open Access Journals (Sweden)

    Catherine B Poole

    Full Text Available In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.

  6. Diagnosis of brugian filariasis by loop-mediated isothermal amplification. (United States)

    Poole, Catherine B; Tanner, Nathan A; Zhang, Yinhua; Evans, Thomas C; Carlow, Clotilde K S


    In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.

  7. Field and Current Amplification in the SSPX Spheromak

    Energy Technology Data Exchange (ETDEWEB)

    Hill, D N; Blumer, R H; Cohen, B I; Hooper, E B; McLean, H S; Moller, J; Pearlstein, L D; Ryutov, D D; Stallard, B W; Wood, R D; Woodruff, S; Holcomb, C T; Jarboe, T; Bellan, P; Romero-Talamas, C


    Results are presented from experiments relating to magnetic field generation and current amplification in the SSPX spheromak. The SSPX spheromak plasma is driven by DC coaxial helicity injection using a 2MJ capacitor bank. Peak toroidal plasma currents of up to 0.7MA and peak edge poloidal fields of 0.3T are produced; lower current discharges can be sustained up to 3.5msec. When edge magnetic fluctuations are reduced below 1% by driving the plasma near threshold, it is possible to produce plasmas with Te > 150eV, <{beta}{sub e}>-4% and core {chi}{sub e} {approx} 30m{sup 2}/s. Helicity balance for these plasmas suggests that sheath dissipation can be significant, pointing to the importance of maximizing the voltage on the coaxial injector. For most operational modes we find a stiff relationship between peak spheromak field and injector current, and little correlation with plasma temperature, which suggests that other processes than ohmic dissipation may limit field amplification. However, slowing spheromak buildup by limiting the initial current pulse increases the ratio of toroidal current to injected current and points to new operating regimes with more favorable current amplification.

  8. Arctic amplification: does it impact the polar jet stream?

    Directory of Open Access Journals (Sweden)

    Valentin P. Meleshko


    Full Text Available It has been hypothesised that the Arctic amplification of temperature changes causes a decrease in the northward temperature gradient in the troposphere, thereby enhancing the oscillation of planetary waves leading to extreme weather in mid-latitudes. To test this hypothesis, we study the response of the atmosphere to Arctic amplification for a projected summer sea-ice-free period using an atmospheric model with prescribed surface boundary conditions from a state-of-the-art Earth system model. Besides a standard global warming simulation, we also conducted a sensitivity experiment with sea ice and sea surface temperature anomalies in the Arctic. We show that when global climate warms, enhancement of the northward heat transport provides the major contribution to decrease the northward temperature gradient in the polar troposphere in cold seasons, causing more oscillation of the planetary waves. However, while Arctic amplification significantly enhances near-surface air temperature in the polar region, it is not large enough to invoke an increased oscillation of the planetary waves.

  9. Mechanism of seasonal Arctic sea ice evolution and Arctic amplification (United States)

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


    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 perimeter of the Arctic Ocean in summer, sea ice remains thin in winter only in the Barents-Kara seas. Excessive turbulent heat flux through the sea surface exposed to air due to sea ice reduction warms the atmospheric column. Warmer air increases the downward longwave radiation and subsequently surface air temperature, which facilitates sea surface remains to be free of ice. This positive feedback mechanism is not clearly observed in the Laptev, East Siberian, Chukchi, and Beaufort seas, since sea ice refreezes in late fall (November) before excessive turbulent heat flux is available for warming the atmospheric column in winter. A detailed seasonal heat budget is presented in order to understand specific differences between the Barents-Kara seas and Laptev, East Siberian, Chukchi, and Beaufort seas.

  10. Comparison between NuGEN's WT-Ovation Pico and one-direct amplification systems. (United States)

    Morse, Alison M; Carballo, Valentina; Baldwin, Donald A; Taylor, Christopher G; McIntyre, Lauren M


    Differential gene expression between groups of homogenous cell types is a biological question whose time has come. RNA can be extracted from small numbers of cells, such as those isolated by laser-capture microdissection, but the small amounts obtained often require amplification to enable whole genome transcriptome profiling by technologies such as microarray analysis and RNA-seq. Recently, advances in amplification procedures make amplification directly from whole cell lysates possible. The aim of this study was to compare two amplification systems for variations in observed RNA abundance attributable to the amplification procedure for use with small quantities of cells isolated by laser-capture microdissection. Arabidopsis root cells undergoing giant cell formation as a result of nematode infestation and uninfested control root cells were laser-captured and used to evaluate two amplification systems. One, NuGEN's WT-Ovation Pico (Pico) amplification system, uses total RNA as starting material, and the other, NuGEN's WT-One-Direct (One-Direct) amplification system, uses lysate containing the captured cells. The reproducibility of whole genome transcript profiling and correlations of both systems were investigated after microarray analysis. The One-Direct system was less reproducible and more variable than the Pico system. The Pico amplification kit resulted in the detection of thousands of differentially expressed genes between giant cells and control cells. This is in marked contrast to the relatively few genes detected after amplification with the One-Direct amplification kit.

  11. Amplification of seismic ground motion in the Tunis basin: Numerical BEM simulations vs experimental evidences

    CERN Document Server

    Kham, Marc; Bouden-Romdhane, Nejla


    This paper aims at the analysis of seismic wave amplification in a deep alluvial basin in the city of Tunis in Tunisia. This sedimentary basin is 3000m wide and 350m deep. Since the seismic hazard is significant in this area, the depth of the basin and the strong impedance ratio raise the need for an accurate estimation of seismic motion amplification. Various experimental investigations were performed in previous studies to characterize site effects. The Boundary Element Method is considered herein to assess the parameter sensitivity of the amplification process and analyse the prevailing phenomena. The various frequencies of maximum amplification are correctly estimated by the BEM simulations. The maximum amplification level observed in the field is also well retrieved by the numerical simulations but, due to the sensitivity of the location of maximum amplification in space, the overall maximum amplification has to be considered. The influence of the wave-field incidence and material damping is also discuss...

  12. The N-acylneuraminate cytidyltransferase gene, neuA, is heterogenous in Legionella pneumophila strains but can be used as a marker for epidemiological typing in the consensus sequence-based typing scheme. (United States)

    Farhat, Claudia; Mentasti, Massimo; Jacobs, Enno; Fry, Norman K; Lück, Christian


    Sequence-based typing (SBT) is the internationally recognized standard method for genotyping Legionella pneumophila. To date all strains of serogroup 1 (SG1) and some of SGs 2 to 14 yield a seven-allele profile and can be assigned a sequence type (ST). However, for some strains belonging to SGs 2 to 14, the targeted region of the neuA gene could not be amplified using the published standard primers. We determined the DNA sequence of a neuA gene homolog located in the lipopolysaccharide synthesis locus of strain Dallas-1E. By using newly designed degenerate consensus primers based on the neuA homolog in strains Dallas-1E, Philadelphia-1, Paris, Lens, and Corby, we were able to obtain DNA sequences for all 48 non-SG1 strains which were untypeable by the standard method. Our data show that the neuA gene is present in all L. pneumophila strains but differs significantly in some non-SG1 strains at both the DNA and amino acid levels. The new primers can be used to amplify and sequence the neuA gene in all strains and can substitute for the standard primers. This offers the possibility of assigning an ST to all strains of L. pneumophila.

  13. Sequence-based prediction of protein-binding sites in DNA: comparative study of two SVM models. (United States)

    Park, Byungkyu; Im, Jinyong; Tuvshinjargal, Narankhuu; Lee, Wook; Han, Kyungsook


    As many structures of protein-DNA complexes have been known in the past years, several computational methods have been developed to predict DNA-binding sites in proteins. However, its inverse problem (i.e., predicting protein-binding sites in DNA) has received much less attention. One of the reasons is that the differences between the interaction propensities of nucleotides are much smaller than those between amino acids. Another reason is that DNA exhibits less diverse sequence patterns than protein. Therefore, predicting protein-binding DNA nucleotides is much harder than predicting DNA-binding amino acids. We computed the interaction propensity (IP) of nucleotide triplets with amino acids using an extensive dataset of protein-DNA complexes, and developed two support vector machine (SVM) models that predict protein-binding nucleotides from sequence data alone. One SVM model predicts protein-binding nucleotides using DNA sequence data alone, and the other SVM model predicts protein-binding nucleotides using both DNA and protein sequences. In a 10-fold cross-validation with 1519 DNA sequences, the SVM model that uses DNA sequence data only predicted protein-binding nucleotides with an accuracy of 67.0%, an F-measure of 67.1%, and a Matthews correlation coefficient (MCC) of 0.340. With an independent dataset of 181 DNAs that were not used in training, it achieved an accuracy of 66.2%, an F-measure 66.3% and a MCC of 0.324. Another SVM model that uses both DNA and protein sequences achieved an accuracy of 69.6%, an F-measure of 69.6%, and a MCC of 0.383 in a 10-fold cross-validation with 1519 DNA sequences and 859 protein sequences. With an independent dataset of 181 DNAs and 143 proteins, it showed an accuracy of 67.3%, an F-measure of 66.5% and a MCC of 0.329. Both in cross-validation and independent testing, the second SVM model that used both DNA and protein sequence data showed better performance than the first model that used DNA sequence data. To the best of

  14. Aspartic acid (United States)

    Aspartic acid is a nonessential amino acids . Amino acids are building blocks of proteins. "Nonessential" means that our ... this amino acid from the food we eat. Aspartic acid is also called asparaginic acid. Aspartic acid helps ...

  15. Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

    Directory of Open Access Journals (Sweden)

    Masahiro Itonaga

    Full Text Available Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP, for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.

  16. G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection. (United States)

    Jiang, Hong-Xin; Liang, Zhen-Zhen; Ma, Yan-Hong; Kong, De-Ming; Hong, Zhang-Yong


    Real-time PCR has revolutionized PCR from qualitative to quantitative. As an isothermal DNA amplification technique, rolling circular amplification (RCA) has been demonstrated to be a versatile tool in many fields. Development of a simple, highly sensitive, and specific strategy for real-time monitoring of RCA will increase its usefulness in many fields. The strategy reported here utilized the specific fluorescence response of thioflavin T (ThT) to G-quadruplexes formed by RCA products. Such a real-time monitoring strategy works well in both traditional RCA with linear amplification efficiency and modified RCA proceeded in an exponential manner, and can be readily performed in commercially available real-time PCR instruments, thereby achieving high-throughput detection and making the proposed technique more suitable for biosensing applications. As examples, real-time RCA-based sensing platforms were designed and successfully used for quantitation of microRNA over broad linear ranges (8 orders of magnitude) with a detection limit of 4 aM (or 0.12 zmol). The feasibility of microRNA analysis in human lung cancer cells was also demonstrated. This work provides a new method for real-time monitoring of RCA by using unique nucleic acid secondary structures and their specific fluorescent probes. It has the potential to be extended to other isothermal single-stranded DNA amplification techniques.

  17. Application of Isothermal Amplification Technology in Molecular Diagnosis%恒温扩增技术在分子诊断中的应用

    Institute of Scientific and Technical Information of China (English)



    聚合酶链反应(PCR)技术是近年来广泛应用于科学研究和临床检验的一项技术.目前传统的PCR技术中核酸的扩增存在效率不高且需要专用的仪器设备等缺陷,而恒温扩增技术由于引物种类多样,检测方法多变,从而克服了传统PCR核酸扩增技术的缺点,使其越来越多地被应用于分子诊断中.经国内外学者的研究证实,恒温扩增技术有着广泛的应用前景,现着重对恒温扩增技术予以综述.%Polymerase chain reaction( PCR ) is a technology which is widely applied in scientific research and Clinical examination. At present, the conventional PCR employed by nucleic acid amplification has some shortcomings , surh as low efficiency , needing of special instrument ,etc. On the contrary, . the isothermal amplification methods have overcome these disadvantages by utilizing a rich variety of primers and testing methods. It is confirmed by domestic and oversea scholars that a wide range of medical applications of isothermal amplification technology is expected. Here is to provide an overview of isothermal amplification technology.

  18. KRAS and MAPK1 Gene Amplification in Type II Ovarian Carcinomas

    Directory of Open Access Journals (Sweden)

    Noriyuki Ishikawa


    Full Text Available In this study, we examined the clinical significance of KRAS and MAPK1 amplification and assessed whether these amplified genes were potential therapeutic targets in type II ovarian carcinoma. Using fluorescence in situ hybridization, immunohistochemistry, and retrospectively collected clinical data, KRAS and MAPK1 amplifications were identified in 9 (13.2% and 5 (7.4% of 68 type II ovarian carcinoma tissue samples, respectively. Interestingly, co-amplification of KRAS and MAPK1 seemed to be absent in the type II ovarian carcinomas tested, except one case. Active phospho-ERK1/2 was identified in 26 (38.2% out of 68 type II ovarian carcinomas and did not correlate with KRAS or MAPK1 amplification. There was no significant relationship between KRAS amplification and overall or progression-free survival in patients with type II ovarian carcinoma. However, patients with MAPK1 amplification had significantly poorer progression-free survival than patients without MAPK1 amplification. Moreover, type II ovarian carcinoma cells with concomitant KRAS amplification and mutation exhibited dramatic growth reduction following treatment with the MEK inhibitor PD0325901. These findings indicate that KRAS/MAPK1 amplification is critical for the growth of a subset of type II ovarian carcinomas. Additionally, RAS/RAF/MEK/ERK pathway-targeted therapy may benefit selected patients with type II ovarian carcinoma harboring KRAS/MAPK1 amplifications.

  19. Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology. (United States)

    Daher, Rana K; Stewart, Gale; Boissinot, Maurice; Boudreau, Dominique K; Bergeron, Michel G


    Recombinase polymerase amplification (RPA) technology relies on three major proteins, recombinase proteins, single-strand binding proteins, and polymerases, to specifically amplify nucleic acid sequences in an isothermal format. The performance of RPA with respect to sequence mismatches of closely-related non-target molecules is not well documented and the influence of the number and distribution of mismatches in DNA sequences on RPA amplification reaction is not well understood. We investigated the specificity of RPA by testing closely-related species bearing naturally occurring mismatches for the tuf gene sequence of Pseudomonas aeruginosa and/or Mycobacterium tuberculosis and for the cfb gene sequence of Streptococcus agalactiae. In addition, the impact of the number and distribution of mismatches on RPA efficiency was assessed by synthetically generating 14 types of mismatched forward primers for detecting five bacterial species of high diagnostic relevance such as Clostridium difficile, Staphylococcus aureus, S. agalactiae, P. aeruginosa, and M. tuberculosis as well as Bacillus atropheus subsp. globigii for which we use the spores as internal control in diagnostic assays. A total of 87 mismatched primers were tested in this study. We observed that target specific RPA primers with mismatches (n > 1) at their 3'extrimity hampered RPA reaction. In addition, 3 mismatches covering both extremities and the center of the primer sequence negatively affected RPA yield. We demonstrated that the specificity of RPA was multifactorial. Therefore its application in clinical settings must be selected and validated a priori. We recommend that the selection of a target gene must consider the presence of closely-related non-target genes. It is advisable to choose target regions with a high number of mismatches (≥36%, relative to the size of amplicon) with respect to closely-related species and the best case scenario would be by choosing a unique target gene.

  20. PredSL: A Tool for the N-terminal Sequence-based Prediction of Protein Subcellular Localization

    Institute of Scientific and Technical Information of China (English)

    Evangelia I. Petsalaki; Pantelis G. Bagos; Zoi I. Litou; Stavros J. Hamodrakas


    The ability to predict the subcellular localization of a protein from its sequence is of great importance, as it provides information about the protein's function.We present a computational tool, PredSL, which utilizes neural networks, Markov chains, profile hidden Markov models, and scoring matrices for the prediction of the subcellular localization of proteins in eukaryotic cells from the N-terminal amino acid sequence. It aims to classify proteins into five groups: chloroplast,thylakoid, mitochondrion, secretory pathway, and "other". When tested in a fivefold cross-validation procedure, PredSL demonstrates 86.7% and 87.1% overall accuracy for the plant and non-plant datasets, respectively. Compared with TargetP, which is the most widely used method to date, and LumenP, the results of PredSL are comparable in most cases. When tested on the experimentally verified proteins of the Saccharomyces cerevisiae genome, PredSL performs comparably if not better than any available algorithm for the same task. Furthermore, PredSL is the only method capable for the prediction of these subcellular localizations that is available as a stand-alone application through the URL:

  1. Sequence-Based Prediction of RNA-Binding Proteins Using Random Forest with Minimum Redundancy Maximum Relevance Feature Selection

    Directory of Open Access Journals (Sweden)

    Xin Ma


    Full Text Available The prediction of RNA-binding proteins is one of the most challenging problems in computation biology. Although some studies have investigated this problem, the accuracy of prediction is still not sufficient. In this study, a highly accurate method was developed to predict RNA-binding proteins from amino acid sequences using random forests with the minimum redundancy maximum relevance (mRMR method, followed by incremental feature selection (IFS. We incorporated features of conjoint triad features and three novel features: binding propensity (BP, nonbinding propensity (NBP, and evolutionary information combined with physicochemical properties (EIPP. The results showed that these novel features have important roles in improving the performance of the predictor. Using the mRMR-IFS method, our predictor achieved the best performance (86.62% accuracy and 0.737 Matthews correlation coefficient. High prediction accuracy and successful prediction performance suggested that our method can be a useful approach to identify RNA-binding proteins from sequence information.

  2. New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes for radiation molecular cytogenetics

    Directory of Open Access Journals (Sweden)

    Repin Mikhail V


    Full Text Available Abstract Background The objective of this work is to obtain the correct relative DNA contents of chromosomes in the normal male and female human diploid genomes for the use at FISH analysis of radiation-induced chromosome aberrations. Results The relative DNA contents of chromosomes in the male and female human diploid genomes have been calculated from the publicly available international Human Genome Project data. New sequence-based data on the relative DNA contents of human chromosomes were compared with the data recommended by the International Atomic Energy Agency in 2001. The differences in the values of the relative DNA contents of chromosomes obtained by using different approaches for 15 human chromosomes, mainly for large chromosomes, were below 2%. For the chromosomes 13, 17, 20 and 22 the differences were above 5%. Conclusion New sequence-based data on the relative DNA contents of chromosomes in the normal male and female human diploid genomes were obtained. This approach, based on the genome sequence, can be recommended for the use in radiation molecular cytogenetics.

  3. Long-Distance Communication and Signal Amplification in Systemic Acquired Resistance

    Directory of Open Access Journals (Sweden)

    Jyoti eShah


    Full Text Available Systemic acquired resistance (SAR is an inducible defense mechanism in plants that confers enhanced resistance against a variety of pathogens. SAR is activated in the uninfected systemic (distal organs in response to a prior (primary infection elsewhere in the plant. SAR is associated with the activation of salicylic acid (SA signaling and the priming of defense responses for robust activation in response to subsequent infections. The activation of SAR requires communication by the primary infected tissues with the distal organs. The vasculature functions as a conduit for the translocation of factors that facilitate long-distance intra-plant communication. In recent years, several metabolites putatively involved in long-distance signaling have been identified. These include the methyl ester of SA (MeSA, the abietane diterpenoid dehydroabietinal (DA, the dicarboxylic acid azelaic acid (AzA, and a glycerol-3-phosphate (G3P-dependent factor. Long-distance signaling by some of these metabolites also requires the lipid-transfer protein DIR1 (DEFECTIVE IN INDUCED RESISTANCE 1. The relative contribution of these factors in long-distance signaling is likely influenced by environmental conditions, for example light. In the systemic leaves, the AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1-dependent production of the lysine catabolite pipecolic acid (Pip, FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1 signaling, as well as SA synthesis and downstream signaling are required for the activation of SAR. This review summarizes the involvement and interaction between long-distance SAR signals and details the recently discovered role of Pip in defense amplification and priming that allows plants to acquire immunity at the systemic level. Recent advances in SA signaling and perception are also highlighted.

  4. Phonon amplification using evaporation and adsorption of helium

    Energy Technology Data Exchange (ETDEWEB)

    More, T.; Adams, J.S.; Bandler, S.R.; Broueer, S.M.; Lanou, R.E.; Maris, H.J.; Seidel, G.M. [Department of Physics, Brown University, Providence, Rhode Island 02912 (United States)


    We report the results of experiments designed to investigate the feasibility of amplifying a phonon signal using the evaporation of helium from a superfluid film and its subsequent readsorption onto a helium-free surface. We envision a multistage amplifier in which helium is evaporated from a wafer with a helium film only on one side and then adsorbed onto the film-free surface of a similar wafer. The phonons created by the adsorption reach the film on the opposite side of the wafer and potentially desorb more helium than was evaporated by the first wafer. The amplification would come from the high ratio of the binding energy of a helium atom to a film-free surface relative to the binding energy to the liquid. A number of experiments are reported that investigate the efficiencies of the individual steps of the process. The gain per stage is found to be about 3 for high-energy densities in which multiphonon processes are possible. At low-energy densities, the energy deposited into a film-free wafer is found to be less than the original input energy, with the ratio of output to input energy 0.2. Since in applications requiring amplification the phonon density produced by the adsorption of helium on a wafer will be low, the configuration we have studied{emdash}phonons produced in silicon coated with a saturated {sup 4}He film{emdash}will not result in amplification. However, other configurations might improve the efficiency enough to make an amplifier possible. {copyright} {ital 1996 The American Physical Society.}

  5. Randomness Amplification under Minimal Fundamental Assumptions on the Devices (United States)

    Ramanathan, Ravishankar; Brandão, Fernando G. S. L.; Horodecki, Karol; Horodecki, Michał; Horodecki, Paweł; Wojewódka, Hanna


    Recently, the physically realistic protocol amplifying the randomness of Santha-Vazirani sources producing cryptographically secure random bits was proposed; however, for reasons of practical relevance, the crucial question remained open regarding whether this can be accomplished under the minimal conditions necessary for the task. Namely, is it possible to achieve randomness amplification using only two no-signaling components and in a situation where the violation of a Bell inequality only guarantees that some outcomes of the device for specific inputs exhibit randomness? Here, we solve this question and present a device-independent protocol for randomness amplification of Santha-Vazirani sources using a device consisting of two nonsignaling components. We show that the protocol can amplify any such source that is not fully deterministic into a fully random source while tolerating a constant noise rate and prove the composable security of the protocol against general no-signaling adversaries. Our main innovation is the proof that even the partial randomness certified by the two-party Bell test [a single input-output pair (u* , x* ) for which the conditional probability P (x*|u*) is bounded away from 1 for all no-signaling strategies that optimally violate the Bell inequality] can be used for amplification. We introduce the methodology of a partial tomographic procedure on the empirical statistics obtained in the Bell test that ensures that the outputs constitute a linear min-entropy source of randomness. As a technical novelty that may be of independent interest, we prove that the Santha-Vazirani source satisfies an exponential concentration property given by a recently discovered generalized Chernoff bound.

  6. Extensive gene amplification and concerted evolution within the CPR family of cuticular proteins in mosquitoes. (United States)

    Cornman, R Scott; Willis, Judith H


    Annotation of the Anopheles gambiae genome has revealed a large increase in the number of genes encoding cuticular proteins with the Rebers and Riddiford Consensus (the CPR gene family) relative to Drosophila melanogaster. This increase reflects an expansion of the RR-2 group of CPR genes, particularly the amplification of sets of highly similar paralogs. Patterns of nucleotide variation indicate that extensive concerted evolution is occurring within these clusters. The pattern of concerted evolution is complex, however, as sequence similarity within clusters is uncorrelated with gene order and orientation, and no comparable clusters occur within similarly compact arrays of the RR-1 group in mosquitoes or in either group in D. melanogaster. The dearth of pseudogenes suggests that sequence clusters are maintained by selection for high gene-copy number, perhaps due to selection for high expression rates. This hypothesis is consistent with the apparently parallel evolution of compact gene architectures within sequence clusters relative to single-copy genes. We show that RR-2 proteins from sequence-cluster genes have complex repeats and extreme amino-acid compositions relative to single-copy CPR proteins in An. gambiae, and that the amino-acid composition of the N-terminal and C-terminal sequence flanking the chitin-binding consensus region evolves in a correlated fashion.

  7. Rapid detection of European orthobunyaviruses by reverse transcription loop-mediated isothermal amplification assays. (United States)

    Camp, Jeremy V; Nowotny, Norbert


    The development of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assays are described herein for the detection of two orthobunyaviruses (Bunyaviridae), which represent the two main serogroups found in mosquitoes in Central Europe. The RT-LAMP assays were optimized for the detection of Ťahyňa virus (a California encephalitis group virus found in Aedes sp or Ochlerotatus sp mosquitoes) and Batai virus (also called Čalovo virus, a Bunyamwera group virus found in Anopheles maculipennis s.l. mosquitoes) nucleic acid using endemic European virus isolates. The sensitivity of the RT-LAMP assays was determined to be comparable to that of conventional tests, with a limit of detectionisothermal conditions using very simple equipment. Furthermore, it was possible to proceed with the assays without nucleic acid extraction, albeit at a 100-fold loss of sensitivity. The RT-LAMP assays are a sensitive, cost-efficient method for both arbovirus surveillance as well as diagnostic laboratories to detect the presence of these endemic orthobunyaviruses.

  8. Limits of Femtosecond Fiber Amplification by Parabolic Pre-Shaping

    CERN Document Server

    Fu, Walter; McComb, Timothy S; Lowder, Tyson L; Wise, Frank W


    We explore parabolic pre-shaping as a means of generating and amplifying ultrashort pulses. We develop a theoretical framework for modeling the technique and use its conclusions to design a femtosecond fiber amplifier. Starting from 9 ps pulses, we obtain 4.3 $\\mu$J, nearly transform-limited pulses 275 fs in duration, simultaneously achieving over 40 dB gain and 33-fold compression. Finally, we show that this amplification scheme is limited by Raman scattering, and outline a method by which the pulse duration and energy may be further improved and tailored for a given application.

  9. Astigmatism transfer phenomena in the optical parametric amplification process (United States)

    Li, Wenkai; Chen, Yun; Li, Yanyan; Xu, Yi; Guo, Xiaoyang; Lu, Jun; Leng, Yuxin


    We numerically and experimentally investigate the astigmatism transfer phenomena in femtosecond optical parametric amplification (OPA). We model the OPA process based on the coupled second-order three-wave nonlinear propagation equations. The numerical and experimental results support that the input pump pulse astigmatism can be transferred into the idler pulse but not the signal pulse, and the idler pulse astigmatism originating from spatial walk-off is less than the idler pulse astigmatism received from the pump. Thus, we can provide a clear understanding of astigmatism transfer mechanisms in the OPA process, and make better use of broadband tunable OPA sources.

  10. Precision phase estimation based on weak-value amplification (United States)

    Qiu, Xiaodong; Xie, Linguo; Liu, Xiong; Luo, Lan; Li, Zhaoxue; Zhang, Zhiyou; Du, Jinglei


    In this letter, we propose a precision method for phase estimation based on the weak-value amplification (WVA) technique using a monochromatic light source. The anomalous WVA significantly suppresses the technical noise with respect to the intensity difference signal induced by the phase delay when the post-selection procedure comes into play. The phase measured precision of this method is proportional to the weak-value of a polarization operator in the experimental range. Our results compete well with the wide spectrum light phase weak measurements and outperform the standard homodyne phase detection technique.

  11. Weak value amplification in a shot-noise limited interferometer

    CERN Document Server

    Nishizawa, Atsushi; Fujimoto, Masa-Katsu


    We study the weak-value amplification (WVA) in a phase measurement with an optical interferometer in which shot noise limits the sensitivity. We compute the signal and the shot noise including the full-order interaction terms of the WVA, and show that the shot-noise contribution to a phase shift in a pointer variable is always larger than the final variance of the pointer variable. To clarify an advantage for practical uses of the WVA, we discuss signal-to-noise ratio and its optimization in the presence of the shot noise.

  12. Direct field measurement of the dynamic amplification in a bridge (United States)

    Carey, Ciarán; OBrien, Eugene J.; Malekjafarian, Abdollah; Lydon, Myra; Taylor, Su


    In this paper, the level of dynamics, as described by the Assessment Dynamic Ratio (ADR), is measured directly through a field test on a bridge in the United Kingdom. The bridge was instrumented using fiber optic strain sensors and piezo-polymer weigh-in-motion sensors were installed in the pavement on the approach road. Field measurements of static and static-plus-dynamic strains were taken over 45 days. The results show that, while dynamic amplification is large for many loading events, these tend not to be the critical events. ADR, the allowance that should be made for dynamics in an assessment of safety, is small.

  13. Clinical performance of automated real-time nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance assay in diagnosis of pulmonary tuberculosis in 214 cases%利福平耐药结核分枝杆菌实时荧光定量核酸扩增检测技术在214例诊断肺结核患者中的临床应用评价

    Institute of Scientific and Technical Information of China (English)

    何贵清; 李涛; 施伎蝉; 宁洪叶; 吴祥兵; 蔡明明; 吴正兴; 胡陈婵; 蒋贤高


    目的:评估利福平耐药结核分枝杆菌实时荧光定量核酸扩增检测技术(Xpert M TB/RIF)在温州地区诊断肺结核及利福平耐药的临床应用价值。方法纳入可疑肺结核、临床诊断肺结核和可疑耐药肺结核患者214例,采集痰标本同时送检抗酸染色涂片、液体培养和 Xpert M TB/RIF 检测。以临床最终诊断结果为金标准,比较三种方法检测结核分枝杆菌的敏感度、特异度。以液体药物敏感试验结果为金标准,Xpert M TB/RIF 检测利福平耐药的敏感度和特异度。率的比较采取卡方检验。结果以临床最终诊断结果作为金标准,Xpert M TB/RIF 检测结核分枝杆菌的敏感度高于抗酸染色涂片(69.5%比44.1%,χ2=23.31,P <0.01),而与液体培养比较敏感度差异无统计学意义(69.5%比62.1%,χ2=2.15,P>0.05);Xpert M TB/RIF 在痰涂片阳性与阴性的标本中检测结核分枝杆菌的敏感度分别为97.4%和47.5%,在痰涂片阳性与阴性的标本中检测结核分枝杆菌的特异度均为100.0%。以液体药物敏感试验结果为金标准,Xpert M TB/RIF 检测痰标本利福平耐药的敏感度和特异度分别为92.9%和98.8%。结论 Xpert M TB/RIF 能够快速、准确地检测结核分枝杆菌及其利福平耐药性,且敏感度较高,具有很好的应用价值。%Objective To evaluate the clinical application of the automated nucleic acid amplification technology for rapid and simultaneous detection of tuberculosis and rifampicin resistance (Xpert M TB/RIF) in diagnosis of pulmonary tuberculosis in Wenzhou .Methods A total of 214 patients with suspected pulmonary tuberculosis , clinical diagnosed tuberculosis or suspected drug‐resistant tuberculosis were enrolled in this study .The patients′ sputum specimens were collected for acid‐fast smear ,liquid culture and Xpert M TB/RIF assay .The sensitivity

  14. Recovery of community genomes to assess subsurface metabolic potential: exploiting the capacity of next generation sequencing-based metagenomics (United States)

    Wrighton, K. C.; Thomas, B.; Miller, C. S.; Sharon, I.; Wilkins, M. J.; VerBerkmoes, N. C.; Handley, K. M.; Lipton, M. S.; Hettich, R. L.; Williams, K. H.; Long, P. E.; Banfield, J. F.


    , the capacity to oxidize complex organic carbon, as well as lack of membrane bound electron transport chains and an incomplete citric acid cycle. We propose that these organisms grow cryptically on residual biomass from previous biostimulation experiments and thus demonstrate that resource utilization and turnover in the aquifer can be decoupled from existing acetate amendment and external terminal electron accepting processes. In addition to the first recovery of multiple genomes from these novel candidate divisions, our community genomic approach uncovered viral diversity not yet observed at the site, with the reconstruction of six phage genomes and the presence of CRISPR loci detected in bacterial genomes from diverse lineages. These findings have implications for predictive ecosystem modeling, highlighting the importance of integrating the response, adaptation, as well as biological and geochemical feedback mechanisms existing within complex subsurface communities to long term organic carbon amendment.

  15. One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method



    [EN] An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning wit...

  16. A cascade amplification strategy based on rolling circle amplification and hydroxylamine amplified gold nanoparticles enables chemiluminescence detection of adenosine triphosphate. (United States)

    Wang, Ping; Zhang, Tonghuan; Yang, Taoyi; Jin, Nan; Zhao, Yanjun; Fan, Aiping


    A highly sensitive and selective chemiluminescent (CL) biosensor for adenosine triphosphate (ATP) was developed by taking advantage of the ATP-dependent enzymatic reaction (ATP-DER), the powerful signal amplification capability of rolling circle amplification (RCA), and hydroxylamine-amplified gold nanoparticles (Au NPs). The strategy relies on the ability of ATP, a cofactor of T4 DNA ligase, to trigger the ligation-RCA reaction. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction between the two ends of the padlock probe, producing a closed circular DNA template that initiates the RCA reaction with phi29 DNA polymerase and dNTP. Therein, many complementary copies of the circular template can be generated. The ATP-DER is eventually converted into a detectable CL signal after a series of processes, including gold probe hybridization, hydroxylamine amplification, and oxidative gold metal dissolution coupled with a simple and sensitive luminol CL reaction. The CL signal is directly proportional to the ATP level. The results showed that the detection limit of the assay is 100 pM of ATP, which compares favorably with those of other ATP detection techniques. In addition, by taking advantage of ATP-DER, the proposed CL sensing system exhibits extraordinary specificity towards ATP and could distinguish the target molecule ATP from its analogues. The proposed method provides a new and versatile platform for the design of novel DNA ligation reaction-based CL sensing systems for other cofactors. This novel ATP-DER based CL sensing system may find wide applications in clinical diagnosis as well as in environmental and biomedical fields.

  17. Development of loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Penicillium nordicum in dry-cured meat products. (United States)

    Ferrara, M; Perrone, G; Gallo, A; Epifani, F; Visconti, A; Susca, A


    The need of powerful diagnostic tools for rapid, simple, and cost-effective detection of food-borne fungi has become very important in the area of food safety. Currently, several isothermal nucleic acid amplification methods have been developed as an alternative to PCR-based analyses. Loop-mediated isothermal amplification (LAMP) is one of these innovative methods; it requires neither gel electrophoresis to separate and visualize the products nor expensive laboratory equipment and it has been applied already for detection of pathogenic organisms. In the current study, we developed a LAMP assay for the specific detection of Penicillium nordicum, the major causative agent of ochratoxin A contamination in protein-rich food, especially dry-cured meat products. The assay was based on targeting otapksPN gene, a key gene in the biosynthesis of ochratoxin A (OTA) in P. nordicum. Amplification of DNA during the reaction was detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue, visible to the naked eye, avoiding further post amplification analyses. Only DNAs isolated from several P. nordicum strains led to positive results and no amplification was observed from non-target OTA and non OTA-producing strains. The assay was able to detect down to 100 fg of purified targeted genomic DNA or 10(2) conidia/reaction within 60 min. The LAMP assay for detection and identification of P. nordicum was combined with a rapid DNA extraction method set up on serially diluted conidia, providing an alternative rapid, specific and sensitive DNA-based method suitable for application directly "on-site", notably in key steps of dry-cured meat production.

  18. Parametric dispersion and amplification of acoustohelicon waves in piezoelectric semiconductors (United States)

    Neogi, A.; Ghosh, S.


    Assuming that the origin of the nonlinear interaction lies in the second-order optical susceptibility arising from the nonlinear induced current density and using the coupled-mode theory, the parametric dispersion and amplification of acoustohelicon waves is analytically investigated in a longitudinally magnetized piezoelectric semiconductor of noncentrosymmetric nature. The relevant experiments have not been reported. The threshold value of the pump electric field E0th and its corresponding excitation intensity is obtained. The longitudinal magnetic field decreases the required magnitude of E0th for the excitation of parametric amplification. The phenomenon of self-defocusing of the signal in the prevailing case is found to be a consequence of the negative dispersive characteristics exhibited by the acoustohelicon waves. Numerical analyses are performed for an InSb crystal at 77 K, duly irradiated by frequency-doubled pulsed 10.6-μm CO2 lasers. The parametric gain constant is observed to be maximum when the cyclotron frequency ωc attains the magnitude equal to that of ω0, the incident laser frequency (=1.78×1014 s-1 ).

  19. Generalized modulational instability in multimode fibers: wideband multimode parametric amplification

    CERN Document Server

    Guasoni, M


    In this paper intermodal modulational instability (IM-MI) is analyzed in a multimode fiber where several spatial and polarization modes propagate. The coupled nonlinear Schr\\"{o}dinger equations describing the modal evolution in the fiber are linearized and reduced to an eigenvalue problem. As a result, the amplification of each mode can be described by means of the eigenvalues and eigenvectors of a matrix that stores the information about the dispersion properties of the modes and the modal power distribution of the pump. Some useful analytical formulas are also provided that estimate the modal amplification as function of the system parameters. Finally, the impact of third-order dispersion and of absorbtion losses is evaluated, which reveals some surprising phenomena into the IM-MI dynamics. These outcomes generalize previous studies on bimodal-MI, related to the interaction between 2 spatial or polarization modes, to the most general case of $N>2$ interacting modes. Moreover, they pave the way towards the ...

  20. Radiopolymerization of {beta}(-)pinene: A case of chiral amplification

    Energy Technology Data Exchange (ETDEWEB)

    Cataldo, Franco [Soc. Lupi Chemical Research, Via Casilina 1626/A, 00133 Rome (Italy)]. E-mail:; Keheyan, Yeghis [CNR, Istituto per lo studio dei Materiali Nanostrutturati, Department of Chemistry, University ' La Sapienza' , P.le Aldo Moro 1, Rome (Italy)


    {beta}(-)Pinene was treated with {gamma} radiation at three dose levels: 150, 300 and 600 kGy. The expected effect of radiation at these high doses was the partial racemization of the substrate as already observed in the case of other terpene monomers. Unexpectedly {beta}(-)pinene underwent a radiopolymerization reaction into a solid resin and into a dimer. The structure of the products was studied by FT-IR spectroscopy also in comparison to a reference {beta}(-)pinene resin prepared by cationic polymerization. A highly ordered structure was found in the case of the radiopolymer in comparison to the resin from cationic polymerization. Polarimetric measurements have shown astonishing enhancement in the optical activity of the radiopolymer and radiodimer in comparison to the starting optical activity of the {beta}(-)pinene monomer. The results have been discussed in terms of amplification of chirality caused by {gamma} radiation and the implications of this fact on the mechanism of chiral amplification on prebiotic molecules.

  1. Development and Application of Surface Plasmon Polaritons on Optical Amplification

    Directory of Open Access Journals (Sweden)

    Tong Zhang


    Full Text Available Propagation of surface plasmon polaritons (SPPs along the interface between a metal and a dielectric has attracted significant attention due to its unique optical properties, which has inspired a plethora of fascinating applications in photonics and optoelectronics. However, SPPs suffer from large attenuation because of the ohmic losses in the metal layer. It has become the main bottom-neck problem for the development of high performance plasmonic devices. This limitation can be overcome by providing the material adjacent to the metal with optical gain. In this paper, a review of gain compensation to SPPs is presented. We focus on the spontaneous radiation amplification and simulated radiation amplification. The ohmic loss of metal was greatly improved by introducing optical gain. Then we introduce several gain mediums of dye doped, quantum dots, erbium ion, and semiconductor to compensate optical loss of SPPs. Using gain medium mentioned above can compensate losses and achieve many potential applications, for example, laser, amplifier, and LRSPP discussed.

  2. Health Risk Information Engagement and Amplification on Social Media. (United States)

    Strekalova, Yulia A


    Emerging pandemics call for unique health communication and education strategies in which public health agencies need to satisfy the public's information needs about possible risks while preventing risk exaggeration and dramatization. As a route to providing a framework for understanding public information behaviors in response to an emerging pandemic, this study examined the characteristics of communicative behaviors of social media audiences in response to Ebola outbreak news. Grounded in the social amplification of risks framework, this study adds to an understanding of information behaviors of online audiences by showing empirical differences in audience engagement with online health information. The data were collected from the Centers for Disease Control and Prevention (CDC) Facebook channel. The final data set included 809 CDC posts and 35,916 audience comments. The analysis identified the differences in audience information behaviors in response to an emerging pandemic, Ebola, and health promotion posts. While the CDC had fewer posts on Ebola than health promotion topics, the former received more attention from active page users. Furthermore, audience members who actively engaged with Ebola news had a small overlap with those who engaged with non-Ebola information during the same period. Overall, this study demonstrated that information behavior and audience engagement is topic dependent. Furthermore, audiences who commented on news about an emerging pandemic were homogenous and varied in their degree of information amplification.

  3. Social amplification of risk in the Internet environment. (United States)

    Chung, Ik Jae


    This article analyzes the dynamic process of risk amplification in the Internet environment with special emphasis on public concern for environmental risks from a high-speed railway tunnel construction project in South Korea. Environmental organizations and activists serving as social stations collected information about the project and its ecological impact, and communicated this with the general public, social groups, and institutions. The Internet provides social stations and the public with an efficient means for interactive communication and an open space for active information sharing and public participation. For example, while the website of an organization such as an environmental activist group can initially trigger local interest, the Internet allows this information to be disseminated to a much wider audience in a manner unavailable to the traditional media. Interaction among social stations demonstrates an amplifying process of public attention to the risk. Analyses of the volume of readers' comments to online newspaper articles and public opinions posted on message board of public and nonprofit organizations show the ripple effects of the amplification process as measured along temporal, geographical, and sectoral dimensions. Public attention is also influenced by the symbolic connotations of risk information. Interpretations of risk in religious, political, or legal terms intensify public concern for the environmental risk.

  4. Linkage mechanics and power amplification of the mantis shrimp's strike. (United States)

    Patek, S N; Nowroozi, B N; Baio, J E; Caldwell, R L; Summers, A P


    Mantis shrimp (Stomatopoda) generate extremely rapid and forceful predatory strikes through a suite of structural modifications of their raptorial appendages. Here we examine the key morphological and kinematic components of the raptorial strike that amplify the power output of the underlying muscle contractions. Morphological analyses of joint mechanics are integrated with CT scans of mineralization patterns and kinematic analyses toward the goal of understanding the mechanical basis of linkage dynamics and strike performance. We test whether a four-bar linkage mechanism amplifies rotation in this system and find that the rotational amplification is approximately two times the input rotation, thereby amplifying the velocity and acceleration of the strike. The four-bar model is generally supported, although the observed kinematic transmission is lower than predicted by the four-bar model. The results of the morphological, kinematic and mechanical analyses suggest a multi-faceted mechanical system that integrates latches, linkages and lever arms and is powered by multiple sites of cuticular energy storage. Through reorganization of joint architecture and asymmetric distribution of mineralized cuticle, the mantis shrimp's raptorial appendage offers a remarkable example of how structural and mechanical modifications can yield power amplification sufficient to produce speeds and forces at the outer known limits of biological systems.

  5. Static and Dynamic Amplification Using Strong Mechanical Coupling

    KAUST Repository

    Ilyas, Saad


    Amplifying the signal-to-noise ratio of resonant sensors is vital toward the effort to miniaturize devices into the sub-micro and nano regimes. In this paper, we demonstrate theoretically and experimentally, amplification through mechanically coupled microbeams. The device is composed of two identical clamped-clamped beams, made of polyimide, connected at their middle through a third beam, which acts as a mechanical coupler. Each of the clamped-clamped microbeams and the coupler are designed to be actuated separately, hence providing various possibilities of actuation and sensing. The coupled resonator is driven into resonance near its first resonance mode and its dynamic behavior is explored via frequency sweeps. The results show significant amplification in the resonator amplitude when the signal is measured at the midpoint of the coupler compared with the response of the individual uncoupled beams. The static pull-in characteristics of the resonator are also studied. It is shown that the compliant mechanical coupler can serve as a low-power radio frequency switch actuated at low voltage loads. [2016-0100

  6. CDK4 amplification predicts recurrence of well-differentiated liposarcoma of the abdomen.

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    Sanghoon Lee

    Full Text Available The absence of CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD and dedifferentiated (DD liposarcomas.From 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. MDM2 and CDK4 gene amplification were analyzed by quantitative real-time polymerase chain reaction (Q-PCR.There were 31 WD and 17 DD liposarcomas. Locoregional recurrence was observed in 11 WD and 3 DD liposarcomas. WD liposarcomas showed better patient survival compared to DD liposarcomas (P<0.05. Q-PCR analysis of the liposarcomas revealed the presence of CDK4 amplification in 44 cases (91.7% and MDM2 amplification in 46 cases (95.8%. WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence (P = 0.041. High level of CDK4 amplification (cases with CDK4 amplification higher than the median 7.54 was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P = 0.012 and multivariate analyses (P = 0.020.Level of CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas after surgical resection.

  7. Principle and Application of Loop-mediated Isothermal Amplification%环介导等温扩增反应的原理及应用

    Institute of Scientific and Technical Information of China (English)

    黄思佳; 解庭波; 严家新


    环介导等温扩增反应(Loop-mediated isothermal amplification,LAMP)是日本学者Notomi于2000年在NucleicAcids Res杂志上公开的一种新的基因诊断技术,该法操作简便,扩增效率及灵敏度高,已广泛应用于食品、临床、农业等领域,有望成为主流的基因诊断方法.本文对LAMP的反应特征及原理、优缺点、临床应用作一综述.%Loop-mediated isothermal amplification (LAMP) is a novel technique for genetic diagnosis, which was published on Nucleic Acids Res in 2000 by Notomi, a Japanese scientist. It is a simple, efficient and sensitive DNA amplification method which has been widely used in various fields including food, clinic and agriculture, and might be a mainstream method for genetic diagnosis. This paper reviews the reaction characters, principle, advantage, disadvantage as well as clinical application of LAMP.

  8. Assessing the ability of sequence-based methods to provide functional insight within membrane integral proteins: a case study analyzing the neurotransmitter/Na+ symporter family

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    Eskandari Sepehr


    Full Text Available Abstract Background Efforts to predict functional sites from globular proteins is increasingly common; however, the most successful of these methods generally require structural insight. Unfortunately, despite several recent technological advances, structural coverage of membrane integral proteins continues to be sparse. ConSequently, sequence-based methods represent an important alternative to illuminate functional roles. In this report, we critically examine the ability of several computational methods to provide functional insight within two specific areas. First, can phylogenomic methods accurately describe the functional diversity across a membrane integral protein family? And second, can sequence-based strategies accurately predict key functional sites? Due to the presence of a recently solved structure and a vast amount of experimental mutagenesis data, the neurotransmitter/Na+ symporter (NSS family is an ideal model system to assess the quality of our predictions. Results The raw NSS sequence dataset contains 181 sequences, which have been aligned by various methods. The resultant phylogenetic trees always contain six major subfamilies are consistent with the functional diversity across the family. Moreover, in well-represented subfamilies, phylogenetic clustering recapitulates several nuanced functional distinctions. Functional sites are predicted using six different methods (phylogenetic motifs, two methods that identify subfamily-specific positions, and three different conservation scores. A canonical set of 34 functional sites identified by Yamashita et al. within the recently solved LeuTAa structure is used to assess the quality of the predictions, most of which are predicted by the bioinformatic methods. Remarkably, the importance of these sites is largely confirmed by experimental mutagenesis. Furthermore, the collective set of functional site predictions qualitatively clusters along the proposed transport pathway, further

  9. A new method for typing bovine major histocompatibility complex class II DRB3 alleles by combining two established PCR sequence-based techniques. (United States)

    Takeshima, S-N; Matsumoto, Y; Miyasaka, T; Arainga-Ramirez, M; Saito, H; Onuma, M; Aida, Y


    Recently, two polymerase chain reaction sequence-based typing (PCR-SBT) methods were reported for the genotyping of the bovine leukocyte antigen (BoLA)-DRB3. One technique is a single PCR-SBT (sPCR-SBT) method that generates heterozygous sequences that are subsequently analyzed by the haplofinder program, while the other technique is a nested PCR-SBT (nPCR-SBT) method that allows the analysis of heterozygous sequences using the assign 400ATF software. In this study, these techniques were compared and then integrated to produce an improved genotyping method. The primer set used for sPCR-SBT was more accurate than those used for nPCR-SBT. Combining sPCR-SBT with the assign 400ATF software previously reported for nPCR-SBT enables rapid and accurate genotyping of a large number of DNA samples.

  10. Benefit-of-doubt (BOD) scoring: a sequencing-based method for SNP candidate assessment from high to medium read number data sets. (United States)

    Sedlazeck, Fritz Joachim; Talloji, Prabhavathi; von Haeseler, Arndt; Bachmair, Andreas


    Identification of single nucleotide polymorphisms (SNPs) is a key element in sequence-based genetic analysis. Next generation sequencing offers a cost-effective basis to generate the necessary, large sequence data sets, and bioinformatic methods are being developed to process sequencing machine readouts. We were interested in detection of SNPs in a 350 kb region of an EMS-mutagenized Arabidopsis chromosome 3. The region was selectively analyzed using PCR-generated, overlapping fragments for Solexa sequencing. The ensuing reads provided a high coverage and were processed bioinformatically. In order to assess the SNP candidates obtained with a frequently used alignment program and SNP caller, we developed an additional method that allows the identification of high confidence SNP loci. The method can easily be applied to complete genome sequence data of sufficient coverage.

  11. Utility of a next-generation sequencing-based gene panel investigation in German patients with genetically unclassified limb-girdle muscular dystrophy. (United States)

    Kuhn, Marius; Gläser, Dieter; Joshi, Pushpa Raj; Zierz, Stephan; Wenninger, Stephan; Schoser, Benedikt; Deschauer, Marcus


    Limb-girdle muscular dystrophies (LGMDs) are genetically heterogeneous and the diagnostic work-up including conventional genetic testing using Sanger sequencing remains complex and often unsatisfactory. We performed targeted sequencing of 23 LGMD-related genes and 15 genes in which alterations result in a similar phenotype in 58 patients with genetically unclassified LGMDs. A genetic diagnosis was possible in 19 of 58 patients (33 %). LGMD2A was the most common form, followed by LGMD2L and LGMD2I. In two patients, pathogenic mutations were identified in genes that are not classified as LGMD genes (glycogen branching enzyme and valosin-containing protein). Thus, a focused next-generation sequencing-based gene panel is a rather satisfactory tool for the diagnosis in unclassified LGMDs.

  12. GMO detection using a bioluminescent real time reporter (BART of loop mediated isothermal amplification (LAMP suitable for field use

    Directory of Open Access Journals (Sweden)

    Kiddle Guy


    Full Text Available Abstract Background There is an increasing need for quantitative technologies suitable for molecular detection in a variety of settings for applications including food traceability and monitoring of genetically modified (GM crops and their products through the food processing chain. Conventional molecular diagnostics utilising real-time polymerase chain reaction (RT-PCR and fluorescence-based determination of amplification require temperature cycling and relatively complex optics. In contrast, isothermal amplification coupled to a bioluminescent output produced in real-time (BART occurs at a constant temperature and only requires a simple light detection and integration device. Results Loop mediated isothermal amplification (LAMP shows robustness to sample-derived inhibitors. Here we show the applicability of coupled LAMP and BART reactions (LAMP-BART for determination of genetically modified (GM maize target DNA at low levels of contamination (0.1-5.0% GM using certified reference material, and compare this to RT-PCR. Results show that conventional DNA extraction methods developed for PCR may not be optimal for LAMP-BART quantification. Additionally, we demonstrate that LAMP is more tolerant to plant sample-derived inhibitors, and show this can be exploited to develop rapid extraction techniques suitable for simple field-based qualitative tests for GM status determination. We also assess the effect of total DNA assay load on LAMP-BART quantitation. Conclusions LAMP-BART is an effective and sensitive technique for GM detection with significant potential for quantification even at low levels of contamination and in samples derived from crops such as maize with a large genome size. The resilience of LAMP-BART to acidic polysaccharides makes it well suited to rapid sample preparation techniques and hence to both high throughput laboratory settings and to portable GM detection applications. The impact of the plant sample matrix and genome loading

  13. Trigeminal ganglion neurons of mice show intracellular chloride accumulation and chloride-dependent amplification of capsaicin-induced responses.

    Directory of Open Access Journals (Sweden)

    Nicole Schöbel

    Full Text Available Intracellular Cl(- concentrations ([Cl(-](i of sensory neurons regulate signal transmission and signal amplification. In dorsal root ganglion (DRG and olfactory sensory neurons (OSNs, Cl(- is accumulated by the Na(+-K(+-2Cl(- cotransporter 1 (NKCC1, resulting in a [Cl(-](i above electrochemical equilibrium and a depolarizing Cl(- efflux upon Cl(- channel opening. Here, we investigate the [Cl(-](i and function of Cl(- in primary sensory neurons of trigeminal ganglia (TG of wild type (WT and NKCC1(-/- mice using pharmacological and imaging approaches, patch-clamping, as well as behavioral testing. The [Cl(-](i of WT TG neurons indicated active NKCC1-dependent Cl(- accumulation. Gamma-aminobutyric acid (GABA(A receptor activation induced a reduction of [Cl(-](i as well as Ca(2+ transients in a corresponding fraction of TG neurons. Ca(2+ transients were sensitive to inhibition of NKCC1 and voltage-gated Ca(2+ channels (VGCCs. Ca(2+ responses induced by capsaicin, a prototypical stimulus of transient receptor potential vanilloid subfamily member-1 (TRPV1 were diminished in NKCC1(-/- TG neurons, but elevated under conditions of a lowered [Cl(-](o suggesting a Cl(--dependent amplification of capsaicin-induced responses. Using next generation sequencing (NGS, we found expression of different Ca(2+-activated Cl(- channels (CaCCs in TGs of mice. Pharmacological inhibition of CaCCs reduced the amplitude of capsaicin-induced responses of TG neurons in Ca(2+ imaging and electrophysiological recordings. In a behavioral paradigm, NKCC1(-/- mice showed less avoidance of the aversive stimulus capsaicin. In summary, our results strongly argue for a Ca(2+-activated Cl(--dependent signal amplification mechanism in TG neurons that requires intracellular Cl(- accumulation by NKCC1 and the activation of CaCCs.

  14. Short communication: Establishment of a new polymerase chain reaction-sequence-based typing method for genotyping cattle major histocompatibility complex class II DRB3. (United States)

    Takeshima, S-N; Matsumoto, Y; Aida, Y


    Sequence-based typing (SBT) is the most comprehensive method for characterizing major histocompatibility complex (MHC) gene polymorphisms. We report here a new PCR-SBT method for genotyping cattle MHC (BoLA) class II DRB3 using the Assign 400ATF ver. software (Conexio Genomics, Fremantle, Australia), which detects alleles in a semiautomated manner. We examined 12 sets of PCR reactions for their ability to amplify BoLA-DRB3 exon 2 and selected an optimal primer set, which used ERB3N-HL031 for first-round PCR and ALL-DRB3B for second-round PCR. Next, we constructed a BoLA-DRB3 allele database using the reference sequences of the Assign 400ATF software and successfully assigned heterozygous samples (including those with deletion alleles) using bidirectional sequencing, unlike our previously described method, which used unidirectional sequencing for detecting of deletion alleles. Next, blood samples of 128 Holstein cattle were used to correlate the results of our modified PCR-SBT method with those of our previously described PCR-SBT method. Each new PCR-SBT result corresponded completely with the DRB3 allele that was genotyped by our previously described PCR-SBT method. Moreover, we confirmed the accuracy of our modified PCR-SBT method by genotyping 7 sire cattle and their 22 calves using Japanese Black cattle. This new method will contribute to high-throughput genotyping of BoLA-DRB3 by sequence-based typing.

  15. Sequence based polymorphic (SBP marker technology for targeted genomic regions: its application in generating a molecular map of the Arabidopsis thaliana genome

    Directory of Open Access Journals (Sweden)

    Sahu Binod B


    Full Text Available Abstract Background Molecular markers facilitate both genotype identification, essential for modern animal and plant breeding, and the isolation of genes based on their map positions. Advancements in sequencing technology have made possible the identification of single nucleotide polymorphisms (SNPs for any genomic regions. Here a sequence based polymorphic (SBP marker technology for generating molecular markers for targeted genomic regions in Arabidopsis is described. Results A ~3X genome coverage sequence of the Arabidopsis thaliana ecotype, Niederzenz (Nd-0 was obtained by applying Illumina's sequencing by synthesis (Solexa technology. Comparison of the Nd-0 genome sequence with the assembled Columbia-0 (Col-0 genome sequence identified putative single nucleotide polymorphisms (SNPs throughout the entire genome. Multiple 75 base pair Nd-0 sequence reads containing SNPs and originating from individual genomic DNA molecules were the basis for developing co-dominant SBP markers. SNPs containing Col-0 sequences, supported by transcript sequences or sequences from multiple BAC clones, were compared to the respective Nd-0 sequences to identify possible restriction endonuclease enzyme site variations. Small amplicons, PCR amplified from both ecotypes, were digested with suitable restriction enzymes and resolved on a gel to reveal the sequence based polymorphisms. By applying this technology, 21 SBP markers for the marker poor regions of the Arabidopsis map representing polymorphisms between Col-0 and Nd-0 ecotypes were generated. Conclusions The SBP marker technology described here allowed the development of molecular markers for targeted genomic regions of Arabidopsis. It should facilitate isolation of co-dominant molecular markers for targeted genomic regions of any animal or plant species, whose genomic sequences have been assembled. This technology will particularly facilitate the development of high density molecular marker maps, essential for

  16. Clinical characteristics and outcome of patients with neuroblastoma presenting genomic amplification of loci other than MYCN.

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    Anne Guimier

    Full Text Available BACKGROUND: Somatically acquired genomic alterations with MYCN amplification (MNA are key features of neuroblastoma (NB, the most common extra-cranial malignant tumour of childhood. Little is known about the frequency, clinical characteristics and outcome of NBs harbouring genomic amplification(s distinct from MYCN. METHODS: Genomic profiles of 1100 NBs from French centres studied by array-CGH were re-examined specifically to identify regional amplifications. Patients were included if amplifications distinct from the MYCN locus were seen. A subset of NBs treated at Institut Curie and harbouring MNA as determined by array-CGH without other amplification was also studied. Clinical and histology data were retrospectively collected. RESULTS: In total, 56 patients were included and categorised into 3 groups. Group 1 (n = 8 presented regional amplification(s without MNA. Locus 12q13-14 was a recurrent amplified region (4/8 cases. This group was heterogeneous in terms of INSS stages, primary localisations and histology, with atypical clinical features. Group 2 (n = 26 had MNA as well as other regional amplifications. These patients shared clinical features of those of a group of NBs MYCN amplified (Group 3, n = 22. Overall survival for group 1 was better than that of groups 2 and 3 (5 year OS: 87.5%±11% vs 34.9%±7%, log-rank p<0.05. CONCLUSION: NBs harbouring regional amplification(s without MNA are rare and seem to show atypical features in clinical presentation and genomic profile. Further high resolution genetic explorations are justified in this heterogeneous group, especially when considering these alterations as predictive markers for targeted therapy.

  17. Social Amplification of Risk and Crisis Communication Planing - Case Study (United States)

    Stanciugelu, I.; Frunzaru, V.; Armas, I.; Duntzer, A.; Stan, S.


    Risk management has become a dominant concern of public policy and the ability of government to anticipate the strength and focus of public concerns remains weak. The Social Amplification of Risk Framework (SARF) was designed to assist in this endeavor. It aims to facilitate a greater understanding of the social processes that can mediate between a hazard event and its consequences. SARF identifies categories of mediator/moderator that intervene between risk event and its consequences and suggests a causal and temporal sequence in which they act. Information flows first through various sources and then channels, triggering social stations of amplification, initiating individual station of amplification and precipitating behavioral reactions. The International Risk Governance Council Framework is an interdisciplinary and multilevel approach, linking risk management and risk assessment sphere through communication. This study aims to identify categories of mediator/moderator that intervene between the risk event and its consequences, using a survey on earthquake risk perception addressing population of Bucharest city. Romania has a unique seismic profile in Europe, being the country with the biggest surface affected in case of a serious earthquake. Considering the development of the urban area that took place in the last two decades and the growing number of inhabitants, Bucharest is the largest city in Romania and is exposed to extensive damages in case of an earthquake. The sociological survey has been conducted in December 2009 on a representative sample of the Bucharest population aged 18 and over (N=1376) using one stage sampling design. We used a stratified sample method shearing the investigated populations in six layers according to the six sectors of Bucharest. The respondents were selected using random digit dialling method (RDD) and the questionnaires were administered by research staff with computer assisted telephone interviewing method (CATI). The

  18. Chip-based sequencing nucleic acids (United States)

    Beer, Neil Reginald


    A system for fast DNA sequencing by amplification of genetic material within microreactors, denaturing, demulsifying, and then sequencing the material, while retaining it in a PCR/sequencing zone by a magnetic field. One embodiment includes sequencing nucleic acids on a microchip that includes a microchannel flow channel in the microchip. The nucleic acids are isolated and hybridized to magnetic nanoparticles or to magnetic polystyrene-coated beads. Microreactor droplets are formed in the microchannel flow channel. The microreactor droplets containing the nucleic acids and the magnetic nanoparticles are retained in a magnetic trap in the microchannel flow channel and sequenced.

  19. Degradation of fungal DNA in formalin-fixed paraffin-embedded sinus fungal balls hampers reliable sequence-based identification of fungi. (United States)

    Cabaret, Odile; Toussain, Guillaume; Abermil, Nassera; Alsamad, Issam Abd; Botterel, Françoise; Costa, Jean-Marc; Papon, Jean-François; Bretagne, Stéphane


    Identification of the etiologic agent responsible for sinus fungal ball (SFB) is rarely obtained due to either the culture of patient specimens not being ordered or if cultures were inoculated they proved to be negative. Obviously, this has a significant impact on the design of appropriate therapeutic strategies. We investigated whether paraffin-embedded (PE) tissues, the only materials often available, were suitable for the correct identification of the responsible fungi. We obtained PE tissues of SFB from 16 different patients who had risk factors for invasive fungal infections. DNA was extracted using an automated extractor and the internal transcribed spacer (ITS) sequenced following amplification with two sets of primers designed to amplify >300 bp fragments. This was attempted in parallel with a real-time quantitative PCR assay targeting Aspergillus spp. mitochondrial DNA designed to amplify <150 bp fragments. ITS sequencing succeeded in appropriately identifying the etiologic agents in 10 of the 16 samples (nine Aspergillus fumigatus, one Lewia spp.). In contrast, the <150 bp PCR assay amplified all specimens correctly except the one involving Lewia spp. If fungal identification is warranted to understand the pathophysiology of SFB and guide clinicians, we cannot rely only on ITS sequencing of the DNA obtained from PE tissues. The main reason is probably due to the fact that formalin prevents amplification of long DNA fragments and consequently, frozen or fresh tissues should be employed.

  20. "Social Laser": Action Amplification by Stimulated Emission of Social Energy

    CERN Document Server

    Khrennikov, Andrei


    The problem of the "explanation" of recent social explosions, especially in the Middle East, but also in Southern Europe and the USA, have been debated actively in the social and political literature. We can mention the contributions of P. Mason, F. Fukuyama, E. Schmidt and J. Cohen, I. Krastev to this debate. We point out that the diversity of opinions and conclusions is really amazing. At the moment, there is no consistent and commonly acceptable theory of these phenomena. We present a model of social explosions based on a novel approach for the description of social processes, namely, the quantum-like approach. Here quantum theory is treated simply as an operational formalism - without any direct relation to physics. We explore the quantum-like laser model to describe the possibility of Action Amplification by Stimulated Emission of Social Energy (ASE).